U.S. patent application number 10/472595 was filed with the patent office on 2004-07-29 for assays, methods and means.
Invention is credited to Maxwell, Patrick Henry, Pugh, Christopher William, Ratcliffe, Peter John, Schofield, Christopher Joseph.
Application Number | 20040146964 10/472595 |
Document ID | / |
Family ID | 26245874 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040146964 |
Kind Code |
A1 |
Maxwell, Patrick Henry ; et
al. |
July 29, 2004 |
Assays, methods and means
Abstract
A novel class of hydroxylases is described having the amino acid
sequence of SEQ ID NO: 2, 4, 6 and 8, and variants and fragments
thereof having HIF hydroxylation activity. The polypeptides of the
invention have in particular prolyl hydroxylase activity. An assay
method monitors the interaction of the HIF hydroxylase with a
substrate. Modulators of HIF hydroxylase are provided for use in
the treatment of a condition associated with increased or decreased
HIF levels or activity or for the treatment of a condition where it
is desirable to modulate HIF levels or activity.
Inventors: |
Maxwell, Patrick Henry;
(Oxford, GB) ; Pugh, Christopher William; (Oxford,
GB) ; Ratcliffe, Peter John; (Oxford, GB) ;
Schofield, Christopher Joseph; (Oxford, GB) |
Correspondence
Address: |
Hamilton Brook
Smith & Reynolds
530 Virginia Road
PO Box 9133
Concord
MA
01742-9133
US
|
Family ID: |
26245874 |
Appl. No.: |
10/472595 |
Filed: |
January 20, 2004 |
PCT Filed: |
March 21, 2002 |
PCT NO: |
PCT/GB02/01381 |
Current U.S.
Class: |
435/25 |
Current CPC
Class: |
C07K 2317/30 20130101;
C07C 235/80 20130101; A61K 31/198 20130101; A61P 29/00 20180101;
G01N 2333/90245 20130101; A61K 31/455 20130101; C07D 213/82
20130101; A61P 17/02 20180101; A61K 31/225 20130101; A61P 37/06
20180101; G01N 2500/04 20130101; A61K 31/24 20130101; A61P 9/12
20180101; A61K 31/265 20130101; C07K 14/475 20130101; A61K 31/195
20130101; A61K 31/137 20130101; A61K 31/327 20130101; A61P 9/10
20180101; G01N 2500/20 20130101; A61K 31/194 20130101; A61K 38/005
20130101; A61P 43/00 20180101; A61K 31/185 20130101; C07C 323/60
20130101; A61P 35/00 20180101; A61K 31/14 20130101; A61K 31/221
20130101; A61K 31/192 20130101; A61K 31/223 20130101; A61K 31/235
20130101; A61K 39/3955 20130101; C07D 213/81 20130101; C12Q 1/26
20130101; C07K 16/40 20130101; A61K 31/166 20130101; C07K 14/4702
20130101; C12Q 1/34 20130101; A61K 31/165 20130101; A61K 31/197
20130101; A61K 38/00 20130101; C12N 9/0071 20130101; C07D 213/80
20130101; A61K 31/21 20130101; A61K 31/44 20130101; A61K 31/4412
20130101; C07C 327/32 20130101; G01N 33/573 20130101; A01K 2217/05
20130101 |
Class at
Publication: |
435/025 |
International
Class: |
C12Q 001/26 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 21, 2001 |
GB |
0107123.2 |
Aug 2, 2001 |
GB |
0118952.1 |
Claims
1. An assay method for identifying an agent which modulates the
interaction of a hypoxia inducible factor (HIF) hydroxylase with a
substrate of the hydroxylase the method comprising: contacting a
HIF hydroxylase and a test substance in the presence of a substrate
of the hydroxylase under conditions in which the hydroxylase
interacts with the substrate in the absence of the test substance;
and determining the interaction, or lack of interaction of, the
hydroxylase and the substrate.
2. An assay method according to claim 1, wherein the interaction of
the hydroxylase with the substrate in the presence of the test
substance is determined by measuring the hydroxylase activity of
the hydroxylase.
3. An assay method according to claim 2, wherein the hydroxylase
and the test substance are contacted in the presence of
2-oxoglutarate and the hydroxylase activity is determined by
determining the turnover of said 2-oxoglutarate to succinate and
CO.sub.2.
4. An assay method according to claim 2 or 3, wherein the
hydroxylase activity is determined by determining the hydroxylation
of one or more proline residues in the substrate.
5. An assay method according to any one of the preceding claims,
wherein the substrate is a HIF polypeptide.
6. An assay method according to claim 5, wherein the HIF
polypeptide comprises HIF-1.alpha., a fragment thereof comprising a
proline equivalent to Pro 564 and/or Pro 402 of HIF-1.alpha. or a
peptide analogue of HIF-1.alpha. or fragment thereof comprising a
proline equivalent to Pro 564 and/or Pro 402 of HIF-1.alpha., and
wherein hydroxylation of Pro 564 and/or Pro 402 o or HIF-1.alpha.
or of a said equivalent proline is determined.
7. An assay method according to any one of the preceding claims,
wherein the substrate comprises a polypeptide sequence comprising
residues 556-574, such as 549 to 582 of the human HIF-1.alpha.
sequence.
8. An assay method according to any one of the preceding claims,
wherein the HIF hydroxylase is a prolyl hydroxylase.
9. An assay method according to any of the preceding claims,
wherein the HIF hydroxylase comprises: (a) the amino acid sequence
of SEQ ID NO: 2, 4, 6 or 8; (b) a variant thereof having at least
60% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8
and having HIF hydroxylase activity; or (c) a fragment of either
thereof having HIF hydroxylase activity.
10. An assay method according to any one of the preceding claims,
wherein the HIF hydroxylase is selected from SEQ ID NO: 2, 4, 6 or
8, FLJ21620 (BAB105101) or Clorf12(NP071334).
11. An assay method according to any one of claims 5 to 10,
comprising providing a cell expressing a HIF polypeptide;
expressing the HIF hydroxylase recombinantly in the cell;
contacting the cell with a test substance and monitoring for HIF
polypeptide abundance or stability or HIF-associated activity in
the cell to determine the effect of the test substance on HIF
hydroxylase activity.
12. An assay method according to claim 11, wherein the HIP
associated activity is selected from interaction with von
Hippel-Lindau protein (VHL), HIF-mediated transcription or
expression of a reporter gene driven by a HIF regulated
promoter.
13. An assay method according to any one of the preceding claims,
wherein the HIF hydroxylase is a human HIF hydroxylase.
14. An assay method according to any one of the preceding claims,
wherein the HIF polypeptide is human HIF-1.alpha. or
HIF-2.alpha..
15. A method for providing a pharmaceutical composition, which
method comprises: (a) identifying an agent by an assay method as
defined in any one of claims 1 to 14; and (b) formulating the agent
thus identified with a pharmaceutically acceptable excipient.
16. An agent identified by an assay method according to any one of
claims 1 to 14.
17. A polypeptide comprising: (a) the amino acid sequence of SEQ ID
NO: 2, 4, 6 or 8; (b) a variant thereof having at least 60%
identity to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 and
having HIF hydroxylase activity; or (c) a fragment of either
thereof having HIP hydroxylase activity.
18. A polynucleotide comprising a sequence which encodes a
polypeptide as defined in claim 17.
19. A polynucleotide according to claim 18 which comprises (i) SEQ
ID NO: 1, 3, 5 or 7 or a complementary sequence thereto; (ii) a
sequence which hybridises under stringent conditions to the
sequence defined in (i); (iii) a sequence which is degenerate as a
result of the genetic code to a sequence as defined in (i) or (ii);
(iv) a sequence having at least 60% identity to a sequence as
defined in (i); or (v) a fragment of any of the sequences (i),
(ii), (iii) or (iv), and which encodes a polypeptide having
hydroxylase activity or capable of generating antibodies specific
for a HIF hydroxylase.
20. A polynucleotide according to claim 18 or 19 which is DNA.
21. An expression vector, comprising a polynucleotide as defined in
any one of claims 18 to 20.
22. An antibody or a fragment thereof capable of specifically
binding a polypeptide as defined in claim 17.
23. An agent according to claim 16, a polypeptide according to
claim 17, a polynucleotide according to any one of claims 18 to 20,
a vector according to claim 21 or an antibody according to claim 22
for use in a method of treatment of the human or animal body by
therapy.
24. A pharmaceutical composition comprising an agent according to
claim 16, a polypeptide according to claim 17, a polynucleotide
according to any one of claims 18 to 20, a vector according to
claim 21 or an antibody according to claim 22 and a
pharmaceutically acceptable excipient.
25. Use of a modulator of HIF hydroxylase activity, an agent
according to claim 16, a polypeptide according to claim 17, a
polynucleotide according to any one of claims 18 to 20, a vector
according to claim 21 or an antibody according to claim 22 in the
manufacture of a medicament for the treatment of a condition
associated with increased or decreased HIF levels or activity or
the treatment of a condition where it is desirable to modulate HIF
levels or activity.
26. Use according to claim 25, wherein said condition is selected
from the group consisting of ischaemia, wound healing, auto-,
allo-, and xeno-transplantation, systemic high blood pressure,
cancer, and inflammatory disorders.
27. Use according to claim 25 or 26, wherein the modulator of HIF
hydroxylase activity is a collagen prolyl hydroxylase (CPH)
inhibitor which inhibits the biological activity of a HIF
hydroxylase and wherein the condition is one associated with
reduced HIF levels or activity.
28. Use according to claim 27, wherein said inhibitor has the
following formula: R.sup.1-A*B*C*D(R.sup.2).sub.y (A) where the
group R.sup.1 is capable of forming an electrostatic interaction
with the sidechain of the arginine which binds the 5-carboxylate of
2-oxoglutarate during catalysis; A*B is a chain of two atoms which
are, independently, carbon, oxygen, nitrogen or sulphur, which
chain can be functionalised; A, B, C and D being linked to one
another by single and/or double and/or triple bonds, y is 0 or 1;
and C*D is a chain of two atoms which are, independently, carbon,
oxygen, nitrogen, or sulphur, which chain C-D can be
functionalised, such that when y is 0 or 1 at least one of the
atoms of said chain C-D is capable of chelating with a metal group
and when y is 1 said chain C-D is attached to R.sup.2 which is
capable of chelating with a metal group.
29. Use according to claim 28, wherein R.sup.1 is --CO.sub.2H,
--SO.sub.3H, --B(OH).sub.2 or --PO.sub.3H.sub.2.
30. Use according to claim 28 or 29, wherein y is 0 and C and/or D
is attached to .dbd.S, .dbd.O, --SH or --OH.
31. Use according to claim 28 or 29, wherein y is 1 and R.sup.2 is
--SH, --OH, --CO.sub.2H, --SO.sub.3H, --B(OH).sub.2,
--PO.sub.3H.sub.2, --NHOH, --CONHR.sup.3, --CONHOR.sup.3,
--CONR.sup.3OH or CONR.sup.3OR.sup.3 where in R.sup.3 is a branched
or straight chain alkyl group of 1 to 6 carbon atoms which can be
functionalised.
32. Use according to any one of claims 28 to 31, wherein said
inhibitor has the following formula: 77where R.sup.1 and R may
independently be H, a branched or straight C.sub.1 to C.sub.6 alkyl
chain which can be functionalised, any natural amino acid side
chain, a 4 to 7 membered heterocyclic ring optionally containing 1
or 2 N, S, O or P atoms or a 5 or 6 membered aromatic ring,
optionally containing 1 or 2 N, O or S atoms, which can be fused to
another ring; and X is NH, NR" where R" is OH, a branched or
straight C.sub.1 to C.sub.6 alkyl chain which can be functionalised
or O; and Y is O or S.
33. Use according to claim 32, wherein said inhibitor is
dimethyloxalylglycine.
34. Use according to any one of claims 28 to 31, wherein said
inhibitor has the following formula: 78where R.sup.1 and R may
independently be H, a branched or straight C.sub.1 to C.sub.6 alkyl
chain, any natural amino acid side chain, a 4 to 7 membered
heterocyclic ring optionally containing 1 or 2 N, S, O or P atoms
or a 5 or 6 membered aromatic ring, optionally containing 1 or 2 N,
O or S atoms, which can be fused to another ring; and Y is O or
S.
35. Use according to any one of claims 28 to 31, wherein said
inhibitor has the following formula: 79where R.sup.I to R.sup.V may
independently be H, a branched or straight chain alkyl of from 1 to
6 C atoms, a halogen group (i.e. fluoro-, chloro-, bromo-or iodo-),
a carboxylate group, a 4 to 7 membered heterocyclic ring optionally
containing 1 or more N, S, O or P atoms, a 5 or 6 membered aromatic
ring, optionally containing 1 or more N, O or S atoms, which can be
fused to another ring or a C(.dbd.O)XR group in which: X is O, NH,
NR, where R is H, OH, a branched or straight chain alkyl of from 1
to 6 C atoms, which can be functionalised, alkoxy containing a
branched or straight chain alkyl of from 1 to 6 C atoms which can
be functionalised, a 4 to 7 membered heterocyclic ring containing i
or 2 N, S, O or P atoms, a 5 or 6 membered aromatic ring,
optionally containing 1 or 2 N, O or S atoms, which can be fused to
another ring or O, and mis 0or 1.
36. Use according to any one of claims 28 to 31, wherein said
inhibitor has the following formula: 80where R.sup.III to R.sup.V
may independently be H, a branched or straight C.sub.1 to C.sub.6
alkyl chain, optionally with 1, 2, 3, 4 or 5 halo substitutions, a
4 to 7 membered heterocyclic ring optionally containing 1 or 2 N,
S, O or P atoms, or a 5 or 6 membered aromatic ring, optionally
containing 1 or 2 N, O or S atoms, which can be fused to another
ring.
37. Use according to anyone of claims 28 to 31, wherein said
inhibitor has the following formula: 81where R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 may independently be H, a branched or straight
C.sub.1 to C.sub.6 alkyl chain, OH, 0-alkyl having a branched or
straight C.sub.1 to C.sub.6 alkyl chain optionally containing 1 or
2 N, O or S atoms, COOH, a branched or straight C.sub.1 to C.sub.6
alkyl ester, a 4 to 7 membered heterocyclic ring optionally
containing 1 or 2 N, S, O or P atoms or a 5 or 6 membered aromatic
ring, optionally containing 1 or more N, O or S atoms, which can be
fused to another ring.
38. Use according to any one of claims 28 to 31, wherein said
inhibitor has the following formula; 82where R.sup.I to R.sup.V may
independently be H, a branched or straight C.sub.1 to C.sub.6 alkyl
chain, optionally with 1, 2, 3, 4 or 5 halo substitutions, a 4 to 7
membered heterocyclic ring optionally containing 1 or 2 N, S, O or
P atoms, or a 5 or 6 membered aromatic ring, optionally containing
1 or 2 N, O or S atoms, which can be fused to another ring.
39. Use according to any one of claims 28 to 31, wherein said
inhibitor has the formula: 83where A*B*C*D are as defined in claim
28 and R.sup.21 is hydrogen or acyl.
40. Use according to claim 25, wherein the modulator of HIF
hydroxylase is a selective inhibitor of the HIF hydroxylase
polypeptide and the condition is one associated with reduced HIF
levels or activity.
41. A method of treating a condition associated with increased or
reduced HIF activity or a condition in which is desired to modulate
HIF activity comprising administering a therapeutically effective
amount of an agent according to claim 16, a polypeptide according
to claim 17, a modulator of the activity of a polypeptide according
to claim 17, a poly,nucleotide according to any one of claims 18 to
20, a vector according to claim 21 or an antibody according to
claim 22 to an individual in need thereof.
42. A method of modulating the amount of HIF polypeptide in a cell
comprising contacting the cell with a substance which inhibits the
prolyl hydroxylase activity of a HIF hydroxylase.
43. A method according to claim 41 or 42, wherein the modulator is
an agent according to claim 16.
44. A method according to any one of claims 41 to 43, wherein the
modulator inhibits the biological activity of a HIF hydroxylase but
does not inhibit biological activity of a collagen prolyl
hydroxylase.
45. A method of making a pharmaceutical composition comprising
admixing an agent according to claim 16, a polypeptide according to
claim 17, a modulator of the activity of a polypeptide according to
claim 17, a polynucleotide according to any one of claims 18 to 20,
a vector according to claim 21 or an antibody according to claim 22
with a pharmaceutically acceptable excipient.
46. A method of producing a HIF hydroxylase comprising: (a) causing
expression from nucleic acid which encodes a HIF hydroxylase in a
suitable expression system to produce the polypeptide
recombinantly; and (b) determining the hydroxylation of a HIF
polypeptide by said recombinantly produced polypeptide.
47. An assay method for identifying a HIF hydroxylase comprising,
(a) providing a test polypeptide, (b) bringing into contact an HIF
polypeptide and the test polypeptide under conditions in which the
HIF polypeptide is hydroxylated by a HIF hydroxylase; and (c)
determining hydroxylation of the HIF polypeptide.
48. Use of an polypeptide according to claim 17 for the prolyl
hydroxylation of a HIF polypeptide.
49. An isolated polypeptide of SEQ ID NO: 9. DLDLEMLAP*YIPMDDDFQL
(SEQ ID NO: 9.) wherein P* is 4-hydroxy proline.
50. An isolated polypeptide which is a variant of SEQ ID NO: 9,
wherein such variants comprises from 1 to 4 amino acid
substitutions of any amino acid apart from P*, said variant
retaining the ability to antagonise the interaction of a
HIF-.alpha. subunit with VHL.
51. An assay for a modulator of the interaction between VHL and
HIF-.alpha. comprising contacting a polypeptide according to claim
49 or claim 50 with a HIF-1.alpha. binding domain of VHL in the
presence of a test substance and determining the interaction of the
polypeptides.
52. An assay for an inhibitor of VHL-mediated HIF-.alpha.
destruction, which comprises providing a HIF-.alpha., or fragment
thereof which includes a VHL-binding portion, together with its
cognate prolyl-hydroxylase under conditions suitable for the
hydroxylation of a proline residue in the HIF-.alpha. VHL-binding
domain; providing a putative modulator of hydroxylation; and
determining whether the amount of hydroxylation of said proline
residue has been modulated by said putative modulator.
53. An assay for a promoter of hydroxylation of a proline residue
in HIF-.alpha., which comprises: providing HIF-.alpha., or fragment
thereof which includes a VHL-binding portion, under hypoxic
conditions, said HIF-.alpha. or fragment thereof containing a
proline residue in the VHL-binding domain; providing a putative
hydroxylation promoting agent; and determining whether said agent
provides for hydroxylation of said proline.
54. A method of identifying a substrate of a polypeptide as defined
in claim 17, comprising contacting a test polypeptide substrate
with a HIF hydroxylase according to claim 17, and monitoring for
hydroxylation of said test polypeptide substrate.
55. A polynucleotide comprising: (a) an antisense polynucleotide to
the polynucleotide of claim 19 or (b) an RNAi to the polynucleotide
of claim 19.
Description
FIELD OF INVENTION
[0001] The present invention relates to hydroxylases which act on
hypoxia inducible factor alpha (HIF-.alpha.) and which are involved
in the regulation of the cellular turnover of HIF. Compounds,
methods and means for the modulation of the activity of these
enzymes are provided.
BACKGROUND OF INVENTION
[0002] The transcription factor HIF (hypoxia inducible factor)
system is a key regulator of responses to hypoxia, occupying a
central position in oxygen homeostasis in a wide range of organisms
(1). A large number of transcriptional targets have been
identified, with critical roles in angiogenesis, erythropoiesis,
energy metabolism, inflammation, vasomotor function, and
apoptotic/proliferative responses (1). The system is essential for
normal development (2, 3), and plays a key role in
pathophysiological responses to ischaemia/hypoxia (1). HIF is also
important in cancer, in which it is commonly upregulated, and has
major effects on tumour growth and angiogenesis (1). The WAIF DNA
binding complex consists of a heterodimer of .alpha. and .beta.
subunits (4) Regulation by oxygen occurs through hydroxylation of
the .alpha.-subunits, which are rapidly destroyed by the proteasome
in oxygenated cells (5, 6, 7). This involves binding of HIF-.alpha.
subunits by the von Hippel-Lindau tumour suppressor protein (pVHL).
(8), with pVHL acting as the, or part of the, recognition component
for a ubiquitin ligase that promotes ubiquitin dependent
proteolysis through interaction with a specific sequence or
sequences in HIF-.alpha. subunits (11, 12, 13, 14). In hypoxia this
process is suppressed, so stabilizing HIF-.alpha. and permitting
transcriptional activation via the HIF.alpha.-.beta..
DISCLOSURE OF THE INVENTION
[0003] Investigations by the present inventors have revealed that
the interaction between HIF-.alpha. and VHL is controlled by
oxidation of critical proline residues in the HIF-.alpha. protein.
In the human HIF-1.alpha. protein these are Pro402 and Pro564,
though the equivalent residue(s) exists in other HIF-.alpha. forms
and are conserved in C. elegans, indicating that these are critical
components which have been conserved through evolution.
[0004] The data herein demonstrates that hydroxylation of proline
residues such as Pro564 in HIF-1.alpha. is mediated by a family of
specific prolyl-hydroxylases, referred to here as the HIF
hydroxylases, which include the C. elegans protein EGL-9 and the
human proteins PHD1-3. These enzymes recognise a conserved core
LXXLAP motif for prolyl hydroxylation. Different members of the
family act differentially on hydroxylation sites within HIF-.alpha.
and the activity of the recombinant enzymes is directly modulated
by oxygen tension, iron availability and cobaltous ions.
[0005] The activity of HIF hydroxylases represents a novel target
for the control of HIF.alpha.. By blocking activity, hydroxylation
of HIF.alpha. will be reduced, leading to the accumulation of
HIF-.alpha.in cells. This, in turn, will lead to the promotion or
modulation of angiogenesis, erythropoiesis, energy metabolism,
inflammation, vasomotor function, and will also affect
apoptotic/proliferative responses. Thus, mechanisms which either
block, inhibit, reduce or decrease the activity of the HIF
hydroxylase, and in particular its prolyl-hydroxylase activity,
have therapeutic applications in certain target cells.
[0006] Conversely, in hypoxic conditions such as those commonly
found in tumours, the lack of hydroxylation may lead to the
accumulation of HIF.alpha. and the concomitant promotion of
angiogenesis and other growth promoting events. Thus mechanisms
which either rescue, stimulate, enhance or increase the activity of
the HIF hydroxylase, and in particular the prolyl-hydroxylase
activity of the enzyme, have different therapeutic applications
with respect to certain target cells.
[0007] One aspect of the present invention therefore provides an
assay method for identifying an agent which modulates the
interaction of a HIF hydroxylase with a substrate of the
hydroxylase, the method including contacting a HIF hydroxylase and
a substrate of the hydroxylase in the presence of a test substance;
and, determining the interaction or lack of interaction of the HIP
hydroxylase and the substrate.
[0008] The HIF hydroxylase and the test substance may be contacted
under conditions in which the HIF hydroxylase normally interacts
with the substrate of the hydroxylase.
[0009] Interaction, or lack of interaction, between the HIF
hydroxylase and the substrate may be determined in the presence
and/or absence of the test substance. A change, i.e. an increase or
decrease in interaction in the presence relative to the absence of
test substance being indicative of the test substance being a
modulator of said interaction.
[0010] Interaction may be determined according to any one of a
range of conventional techniques and may include determining the
prolyl hydroxylation of the substrate as described below.
Interaction in such assays may be any functional interrelation.
[0011] Accordingly, the present invention provides an assay method
for identifying an agent which modulates the interaction of a
hypoxia inducible factor (HIF) hydroxylase with a substrate of the
hydroxylase, the method comprising:
[0012] contacting a HIF hydroxylase and a test substance in the
presence of a substrate of the hydroxylase under conditions in
which the hydroxylase interacts with the substrate in the absence
of the test substance; and
[0013] determining the interaction, or lack of interaction, of the
hydroxylase and the substrate.
[0014] The present invention also provides the HIF hydroxylases
themselves. Thus in accordance with the present invention, there is
provided a polypeptide comprising:
[0015] (a) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8;
[0016] (b) a variant thereof having at least 60% identity to the
amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 and having HIF
hydroxylase activity; or
[0017] (c) a fragment of either thereof having HIF hydroxylase
activity.
[0018] Preferably, a polypeptide of the invention has prolyl
hydroxylase activity.
[0019] The present invention also relates to polynucleotides which
encode a polypeptide of the invention. Thus, in accordance with
another aspect of the invention, a polynucleotide comprises:
[0020] (i) SEQ ID NO: 1, 3, 5 or 7 or a complementary sequence
thereto;
[0021] (ii) a sequence which hybridises under stringent conditions
to the sequence defined in (i);
[0022] (iii) a sequence which is degenerate as a result of the
genetic code to a sequence as defined in (i) or (ii);
[0023] (iv) a sequence having at least 60% identity to a sequence
as defined in (i); or
[0024] (v) a fragment of any of the sequences (i), (ii), (iii) or
(iv), and which encodes a polypeptide having hydroxylase activity
or capable of generating antibodies specific for a HIF
hydroxylase.
[0025] The invention also relates to expression vectors comprising
a polynucleotide of the invention and antibodies capable of
specifically binding a polypeptide of the invention.
[0026] The invention also relates to the use of the substances
identified in accordance with the assays of the present invention
and to the use of inhibitors of the activity of the peptides of the
invention in the treatment of a condition or disease associated
with altered HIF levels with respect to healthy (or normal) levels,
or a condition in which it is desirable to alter HIF activity.
DETAILED DESCRIPTION OF THE INVENTION
[0027] SEQ ID NO: 1 comprises the nucleotide and amino acid
sequence for PHD1.
[0028] SEQ ID NO: 2 comprises the amino acid sequence for PHD1.
[0029] SEQ ID NO: 3 comprises the nucleotide and amino acid
sequence for PHD2.
[0030] SEQ ID NO: 4 comprises the amino acid sequence alone for
PHD2.
[0031] SEQ ID NO: 5 comprises the nucleotide and amino acid
sequence for PHD3.
[0032] SEQ ID NO: 6 comprises the amino acid sequence alone for
PHD3
[0033] SEQ ID NO: 7 comprises the nucleotide and amino acid
sequence for EGL-9.
[0034] SEQ ID NO: 8 comprises the amino acid sequence alone for
EGL-9.
[0035] SEQ ID NOs: 9 to 16 represent a number of polypeptides which
antagonise the interaction of a HIF.alpha. subunit with VHL.
[0036] SEQ ID NOs: 17 to 22 represent a number of HIF hydroxylase
sequence motifs.
[0037] SEQ ID NO: 23 provides the amino acid sequence of pVHL
minimal binding domain of HIF-1.alpha..
[0038] SEQ ID NO: 24 provides the amino acid sequence of pVHL
minimal binding domain of HIF-2.alpha..
[0039] SEQ ID NO: 25 provides the amino acid sequence of pVHL
minimal binding domain of HIF-.alpha. from X. laevis.
[0040] SEQ ID NO: 26 provides the amino acid sequence of pVHL
minimal binding domain of HIF-.alpha. from D. melanogaster.
[0041] SEQ ID NO: 27 provides the amino acid sequence of pVHL
minimal binding domain of HIF-.alpha. from C. elegans.
[0042] SEQ ID NOs: 28 to 34 represent the amino acid sequence of a
number of synthetic peptides assessed for their ability to block
HIF-1.alpha./pVHL interaction.
[0043] SEQ ID NO: 35 comprises the amino acid-sequence alone for
human HIF-.alpha..
[0044] SEQ ID NO: 36 comprises the amino acid sequence alone for C.
elegans HIF-.alpha..
[0045] SEQ ID NO: 37 comprises the amino acid sequence of a portion
of HIF-1.alpha. which is involved in VHL dependent ubiquitylation
and contains an LxxLAP motif.
[0046] SEQ ID NO: 38 comprises the amino acid sequence of a portion
of HIF-2.alpha. which is involved in VHL dependent ubiquitylation
and contains an LxxLAP motif.
[0047] SEQ ID NO: 39 comprises the amino acid sequence of a second
portion of HIF-1.alpha. which is involved in VHL dependent
ubiquitylation and contains an LxxLAP motif.
[0048] SEQ ID NO: 40 comprises the amino acid sequence of the
predicted jelly roll core of the C. elegans HIF hydroxylase
EGL-9.
[0049] SEQ ID NO: 41 comprises the amino acid sequence of the
predicted jelly roll core of PHD 1.
[0050] SEQ ID NO: 42 comprises the amino acid sequence of the
predicted jelly roll core of PHD 2.
[0051] SEQ ID NO: 43 comprises the amino acid sequence of the
predicted jelly roll core of PHD 3.
[0052] SEQ ID NO: 44 comprises the amino acid sequence of the
predicted jelly roll core of rat SM20.
[0053] SEQ ID NO: 45 comprises the amino acid sequence of the
prolyl-3-hydroylase from Streptomyces.
[0054] SEQ ID NOs: 46 and 47 provide the nucleotide sequences of
two primers used to generate a mutagenised ceHIF.
[0055] SEQ ID No: 48 provides the amino acid sequence of a possible
HIF hydroxylase motif.
[0056] HIF Hydroxylases
[0057] The present invention relates to a family of novel
hydroxylases, referred to herein as HIF hydroxylases, functional
variants thereof and functional fragments of HIF hydroxylases or of
variants thereof. Sequence information for three human HIF
hydroxylases termed PHD polypeptides (PHD 1, 2 and 3) are provided
in SEQ ID NOS: 1, 3 and 5 (nucleotide and amino acid) and in SEQ ID
NOS: 2, 4 and 6 comprising the corresponding, amino acid sequence.
Sequence information for a C. elegans HIF hydroxylase, EGL-9, is
provided in SEQ ID NO: 7 (nucleotide and amino acid) and in SEQ ID
NO: 8 comprising the corresponding amino acid sequence. A
polypeptide of the invention thus consists essentially of the amino
acid sequence of SEQ ID NO: 2, 4, 6 or 8 or a variant of any one of
these sequences or of a fragment of any one of these sequences or
variants of the fragments.
[0058] PHD1, 2 and 3 are 2-oxoglutarate dependent non-haem
iron-dependent dioxygenases. These dioxygenases have hydroxylase
activity and in particular they mediate hydroxylation HIF 1.alpha..
They are related by sequence to non-haem oxygenases for which
crystal structures are known such as proline-3-hydroxylase (Clifton
et al., Eur. J. Biochem., 2001, 268, 6625-6636). PHD 1, 2 and 3 are
related to EGL9 of C. elegans and may also be referred to herein as
EGLN 2, 1 and 3 respectively. The PHD 1, 2 and 3 and EGL9
hydroxylases are all considered to be HIF hydroxylases of the
invention. The HIF hydroxylases of the invention, and in particular
the human HIF hydroxylases, may also be referred to as EGLN
polypeptides.
[0059] In a preferred embodiment the HIF hydroxylase of the
invention is a prolyl-hydroxylase. Typically the HIF hydroxylase is
a human HIF hydroxylase and in particular it is PHD 1, 2 or 3. In a
preferred embodiment the various assays, methods, medicaments and
other embodiments of the invention employ, or are concerned, with a
human HIF hydroxylase and in particular PHD 1. 2 or 3.
[0060] A polypeptide of the invention may be in isolated and/or
purified form, free or substantially free of material with which it
is naturally associated, such as other polypeptides or such as
human polypeptides other than that for which the amino acid
sequence is encoded by the gene encoding the HIF hydroxylase and in
particular the PHD-1, -2 or -3 gene or (for example if produced by
expression in a prokaryotic cell) lacking in native glycosylation,
e.g. unglycosylated.
[0061] It will be understood that the polypeptide may be mixed with
carriers or diluents which will not interfere with the intended
purpose of the polypeptide and still be regarded as substantially
isolated. A polypeptide in substantially purified form will
generally comprise the polypeptide in a preparation in which more
than 50% e.g. more than 80%, 90%, 95% or 99%, by weight of the
polypeptide in the preparation is a polypeptide of the invention.
Routine methods can be employed to purify and/or synthesize the
proteins according to the invention. Such methods are well
understood by persons. skilled in the art and, include techniques
such as those disclosed in Sambrook et al, Molecular Cloning: A
Laboratory Manual, Second Edition, CSH Laboratory Press, 1989, the
disclosure of which is included herein in its entirety by w,ay of
reference.
[0062] The term "variant" refers to a polypeptide which shares at
least one property or function with the HIF hydroxylases of SEQ ID
NOS: 2, 4, 6 or 8 and in particular those of SEQ ID NOS: 2, 4 or 6.
A "fragment" of the invention also possesses at least one function
or property of the HIF hydroxylase of SEQ ID NO: 2; 4, 6 or 8 and
in particular of SEQ D NOS: 2, 4 or 6. The HIF hydroxylases of the
invention are hydroxylases, that is they have the ability to
hydroxylate an amino acid residue in a peptide. Preferably, a
polypeptide of the invention is capable of hydroxylating one or
more prolyl residues of a peptide substrate. In preferred aspects
of the invention, a HIF hydroxylase, variant or fragment in
accordance with the invention has the ability to hydroxylate one or
more residues of HIF-1.alpha., preferably a prolyl residue of HIF
and in particular Pro 564 and/or Pro 402 of HIF-1.alpha. or a
peptide analogue of HIF-1.alpha. or fragment thereof incorporating
such a prolyl. Preferably, a variant of a HIF hydroxylase in
accordance with the present invention has at least 60% sequence
identity with the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8
and in particular with that of SEQ ID NO: 2, 4 or 6.
[0063] The present invention also includes active portions,
fragments, derivatives and functional mimetics of the polypeptides
of the invention. An "active portion" of a polypeptide means a
peptide which is less than said full length polypeptide, but which
retains hydroxylase activity and in particular maintains HIF
hydroxylase activity, preferably HIF prolyl hydroxylase activity.
Such an active fragment may be included as part of a fusion
protein, e.g. including a binding portion for a different i.e.
heterologous ligand.
[0064] A "fragment" of a polypeptide generally means a stretch of
amino acid residues of at least about five contiguous amino acids,
often at least about seven contiguous amino acids, typically at
least about nine contiguous amino acids, more preferably at least
about 13 contiguous amino acids, and, more preferably, at least
about 20 to 30 or more contiguous amino acids. Fragments of the HIF
hydroxylase sequence may include antigenic determinants or epitopes
useful for raising antibodies to a portion of the amino acid
sequence. Alanine scans are commonly used to find and refine
peptide motifs within polypeptides, this involving the systematic
replacement of each residue in turn with the amino acid alanine,
followed by an assessment of biological activity. Such scans may
therefore be used in the identification of preferred fragments of
the invention.
[0065] The polypeptides of the present invention generally have
hydroxylase activity, preferably prolyl hydroxylase activity. Thus,
the invention also relates to such polypeptides, in particular, for
use in assays of hydroxylase activity on substrates such as HIF.
The polypeptides may also be used for hydroxylation of suitable
substrates and in particular prolyl hydroxylation of such
substrates. A variant or an active fragment of a HIF hydroxylase of
the invention may typically be identified by monitoring for
hydroxylase activity as described in more detail below. In
preferred embodiments the HIF hydroxylase has prolyl hydroxylase
activity such as prolyl-4-hydroxylase activity.
[0066] Such HIF hydroxylases may be a eukaryotic polypeptide,
preferably a mammalian polypeptide, more preferably a human
polypeptide.
[0067] A HIF hydroxylase preferably has HIF prolyl hydroxylase
(HPH) activity and preferably recognises and/or has specificity for
the substrate amino acid sequence motif LXXLXP, in particular
LXXLAP, or LXXLRP where X is any amino acid i.e. hydroxylates the
proline residue of the LXXLXP or LXXLAP motif of a polypeptide
which comprises this sequence.
[0068] A HIF hydroxylase preferably contains a .beta.-barrel jelly
roll structure consisting of a minimum of eight strands. Typically,
the jelly roll structure may have eight strands. FIG. 9 shows an
alignment of various HIF hydroxylases with the locations of the
eight .beta.-barrel strands of the jelly roll motif indicated. A
diagram of the jelly roll structure is shown in FIG. 10.
[0069] Preferred HIF hydroxylases comprise the sequence;
HXD[X].sub.nH
[0070] where X is any amino acid and n is between 1 and 200, 20 and
150 or 30 and 100 amino acids, for example 10, 20, 30, 40, 50, 60,
70, 80, 90 or 100 amino acids.
[0071] In especially preferred embodiments, the HXD portion of the
motif is located on the second strand of the jelly roll motif of
the HIF hydroxylase and the remaining H is on or close to the
second strand of the motif.
[0072] In some enzymes related to the PHD 1, 2 and 3 enzymes
isolated, such as clavaminic acid synthase, the HXD motif is
replaced by a HXE motif. Thus the invention also encompasses HIF
hydroxylases which have in place of a HXD motif a HXE motif. This
may be because the HIF hydroxylase normally has such a motif, or
alternatively, because the HXD motif originally present has been
replaced by a HXE motif. Thus for any of the HXD motifs described
herein, the invention also encompasses enzymes with a motif where
the Aspartic acid residue has been replaced with a Glutamic acid
residue.
[0073] A suitable HXD[X].sub.nH motif may comprise the residues
His487, Asp489 and His 548 with reference of the Egl-9 sequence. A
suitable HIF hydroxylase may thus comprise or include the
sequence;
HXD[X].sub.58H
[0074] Amino acid residues described herein are numbered according
to the EGL-9 sequence (GI5923812), unless otherwise stated.
Sequences of the catalytic regions of EGL-9 and other HIF
hydroxylases are shown in FIG. 9. It will be appreciated that
because of variations in sequence, the equivalent or corresponding
residues in other HIF hydroxylase sequences may have different
numbers. Reference herein to a residue numbered according to the
EGL-9 sequence is understood to include the equivalent residue in
other HIF hydroxylases.
[0075] Preferred HIF hydroxylases may comprise one or more of the
following residues; Met473, Asp494, Tyr502, Leu517, Pro532, Asp543,
Va-1550, Arg557.
[0076] Such a preferred polypeptide may comprise the following
amino acid sequence;
M(X).sub.13HXD(X).sub.4D(X).sub.7Y(X).sub.14L(X).sub.14P(X).sub.10D(X).sub-
.4HXV(X).sub.6R
[0077] where X is any amino acid residue.
[0078] Especially preferred HIF hydroxylases may additionally
comprise one or more of the following residues;
[0079] Arg469, Tyr477, Pro478, Gly479, Asn480, Gly481, Tyr584,
Val585, Val488, Asn490, Pro491, Gly495, Arg496, Cys497, Thr499,
I1e501, Tyr503, Asn505, Trp505, Asp509, Gly514, Gly515, Phe520,
Pro521, Glu522, Asp535, Arg536, Leu537, Phe539, Trp541, Ser542,
Arg544, Arc545, Asn546, Pro547, Glu549, Pro552, Ala559, Thr561,
Val562, Trp563, Tyr564, Asp566, Glu569, Arg570, Ala573, Ala575,
Lys576, Lys578.
[0080] Such an especially preferred polypeptide may, for example,
comprise the following amino acid sequence;
1 RXXXMXXXYP GNGXXYVXHV DNPXXDGRCX TXIYYXNXXW D(X).sub.4GGXLX
XFPE(X).sub.9PX XDRLXFXWSD RRNPHEVXP(X).sub.4 RXAXTVWYXD XXERXXAXAK
XX
[0081] where X is any amino acid residue.
[0082] In other preferred embodiments one or more of the following
variations of the above sequence may be present. Residue 478 may be
Asn, residue 485 may be Ile, residue 496 may be Lys, residue 497
may be Val, residue 515 may be Ser, residue 520 may be Tyr, residue
530 may be Ile, Val or Met, residue 536 may be Lys, residue 537 may
be Ile, residue 539 may be Ile, residue 546 may be Thr, residue 559
may be Ser, residue 560 may be Ile, Met or Leu, residue 561 may be
Cys and/or residue 564 may be Phe.
[0083] A suitable HIF hydroxylase may comprise a polypeptide
sequence selected from the group consisting of SM20 (NCBI Ace No:
NP071334), EGL-9 (GI5923812), CGI1114 (AAF52-050), Clorfl2
(NP071334), EGLN1/PHD2 (gi1457146), EGLN2/PHD1 (gi1457148),
EGLN3/PHD3 (gi14547150), FALKOR (gi13649965), and FLJ21620
(BAB15101) as shown in Table 1.
[0084] A polypeptide of the invention may further comprise an amino
acid sequence which shares greater than about 60% sequence identity
with one of the above amino acid sequences, preferably greater than
about 70%, more preferably greater than about 80%, more preferably
greater than about 90%, most preferably greater than about 95%.
Suitable sequences have prolyl hydroxylase and in particular HIF
prolyl hydroxylase activity.
[0085] In one embodiment the invention provides a polypeptide
having a least 60% sequence identity with the amino acid sequence
encoded by the PHD2 (EGLN1) gene.
[0086] Further aspects of the present invention relate to methods
for identifying HIF hydroxylases. Such a method may comprise;
[0087] screening a database for an open reading frame encoding a
polypeptide comprising the sequence;
M(X).sub.13HXD(X).sub.4D(X).sub.7Y(X).sub.14L(X).sub.14P(X).sub.10D(X).sub-
.4HXV(X).sub.6R
[0088] expressing said open reading frame to produce said
polypeptide; and,
[0089] determining the ability of said polypeptide to hydroxylate a
prolyl or other residue of HIF polypeptide as described herein.
Crystallographic information may also be used to identify other HIF
hydroxylases, for use in subsequent assays of HIF hydroxylase
activity.
[0090] In an alternative aspect of the present invention, a HIF
hydroxylase of the invention may be a variant which does not show
the same activity as the HIF hydroxylase, but is one which inhibits
a function of the wild type polypeptide. For example, a modified or
variant HIF hydroxylase may be one which competes for HIF
hydroxylase substrates but which does not lead to prolyl
hydroxylation of such substrate. Such variants may be used in the
various embodiments of the invention.
[0091] Amino Acid Sequence Identity
[0092] A polypeptide may comprise an amino acid sequence which
shares greater than about 60% sequence identity with a polypeptide
sequence described or referenced herein, greater than about 70%,
greater than about 80% greater than about 90%, greater than about
95%, or greater than about 98%.
[0093] For amino acid "homology", this may be understood to be
identity e.g. as determined using the algorithm GAP (as described
below).
[0094] Amino acid identity is generally defined with reference to
the algorithnm GAP (Genetics Computer Group, Madison, Wis.). GAP
uses the Needleman and Wunsch algorithm to align two complete
sequences that maximizes the number of matches and minimizes the
number of gaps. Generally, the default parameters are used, with a
gap creation penalty=12 and gap extension penalty=4. Use of GAP may
be preferred but other algorithms may be used, e.g BLAST, (which
uses the method of Altschul et al. (1990) J. Mol. Biol. 215:
405-410), gapped BLAST, PSI-BLAST, (Altshul S. (1997) Nucleic Acid
Res.17 3389-33402), FASTA (which uses the method of Pearson and
Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman
algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197).
Generally, the default parameters are used, with a gap creation
penalty=12 and gap extension penalty=4.
[0095] Sequence comparison may be made over the full-length of the
relevant sequence shown herein, or may more preferably be over a
contiguous sequence of about or greater than about 20, 25, 30, 33,
40, 50, 67, 133, 167, 200, 233, 267, 300, 333, 400 or more amino
acids, compared with the relevant amino acid sequence.
[0096] Where default parameters or other features of these programs
are subject to revision, it is to be understood that reference to
the programs and their parameters are as of the priority date of
the instant application.
[0097] Substitutions made to polypeptides of the invention may
include conserved substitutions, for example according to the
following table, where amino acids on the same block in the second
column and preferably in the same line in the third column may be
substituted for each other:
2 ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q
Polar - charged D E K R H AROMATIC F W Y
[0098] Alternatively, any amino acid may be replaced by a small
aliphatic amino acid, preferably glycine or alanine.
[0099] In addition, deletions and insertions (e.g. from 1 to 5
subject to a maximum of 40% of the amino acids) may also be made.
Insertions are preferably insertions of small aliphatic amino
acids, such as glycine or alanine, although other insertions are
not excluded.
[0100] Variant polypeptides-may also modified in any of the ways
described herein for polypeptides of the invention. This includes
for example "reverse" C-terminal to N-terminal sequences, synthetic
amino acids, glycosylated peptides, phosphorylated peptides,
addition of metal ions such as ions of calcium, zinc, iron or
manganese modified side chains and labelling. Polypeptides may be
provided in the form of molecules which contain multiple copies of
the polypeptide (or mixtures of polypeptides). For example, the
amino group of the side chain of lysine may be used as an
attachment point for the carboxy terminus of an amino acid. Thus
two amino acids may be joined to lysine via carbonyl linkages,
leading to a branched structure which may in turn be branched one
or more times. By way of example, four copies of a polypeptide of
the invention may be joined to such a multiple antigen peptide
(MLAP), such as a MAP of the structure Pep.sub.4-Lys.sub.2-Lys-X,
where Pep is a polypeptide from the HIF hydroxylase or variant
thereof (optionally in the form of a heterologous fusion), Lys is
lysine and X is a terminal group such as .beta.-alanine which
provides for joining of the MAP core to a solid support such as a
resin for synthesis of the Pep.sub.4-MAP peptide and which may be
removed from the support once synthesis is complete.
[0101] Other multiple polypeptide structures may be obtained using
the MAP cores described in: Lu et al, 1991, Mol Immunol, 28,
623-30: Briand et al, 1992, J Immunol Methods, 156, 255-65;
Ahlborg, 1995, J Immunol Methods. 179, 269-75.
[0102] Where multimers of the invention are provided, they may
comprise different polypeptides of the invention or be multimers of
the same polypeptide.
[0103] Except where specified to the contrary, the polypeptide
sequences described herein are shown in the conventional t-letter
code and in the N-terminal to C-terminal orientation. The amino
acid sequence of polypeptides of the invention may also be further
modified to include non-naturally-occurring amino acids or to
increase the stability of the compound in vivo. When the compounds
are produced by synthetic means, such amino acids may be introduced
during production. The compound may also be modified following
either synthetic or recombinant production.
[0104] Polypeptides of the invention may also be made synthetically
using D-amino acids. In such cases, the amino acids may be linked
in a reverse sequence in the C to N orientation. .beta.-amino acids
(or higher homologues) may also be used.
[0105] A number of side-chain modifications for amino acids are
known in the art and may be made to the side chains of polypeptides
of the present invention. Such modifications include for example,
modifications of amino groups by reductive alkylation by reaction
with an aldehyde followed by reduction with NaBH.sub.4, amidination
with methylacetimidate or acylation with acetic anhydride.
[0106] The guanidino groups of arginine residues may be modified by
the formation of heterocyclic condensation products with reagents
such as 2,3-butanedione or glyoxal. Sulphydryl groups may be
modified by methods such as carboxymethylation, tryptophan residues
may be modified by oxidation or alkylation of the indole ring and
the imidazole ring of histidine residues may be modified by
alkylation.
[0107] The carboxy terminus and any other carboxy side chains may
be blocked in the form of an ester group, e.g. a C.sub.1-6alkyl
ester.
[0108] The above examples of modifications to amino acids are not
exhaustive. Those of skill in the art may modify amino acid side
chains where desired using chemistry known per se in the art.
[0109] Polypeptides may be made synthetically or recombinantly,
using techniques which are widely available in the art. Synthetic
production generally involves step-wise addition of individual
amino acid residues to a reaction vessel in which a polypeptide of
a desired sequence is being made.
[0110] Polynucleotides
[0111] The invention also includes nucleotide sequences that encode
for a HIF hydroxylase or a variant or fragment thereof as well as
nucleotide sequences which are complementary thereto. In
particular, the invention provides nucleotide sequences which
encode a human HIF hydroxylase or a fragment or variant of a human
HIF hydroxylase as well as nucleotide sequences complementary to
any of these sequences. The nucleotide sequence may be RNA or DNA
including genomic DNA, synthetic DNA or cDNA. Preferably the
nucleotide sequence is a DNA sequence and most preferably, a cDNA
sequence. The invention also encompasses PNA (protein nucleic acid)
molecules comprising the sequences of the invention. Nucleotide
sequence information for human PHD 1, 2 and 3 is provided in SEQ ID
NOs: 1, 3 and 5 respectively. Nucleotide sequence information is
provided in SEQ ID NO: 7, for the EGL-9 polypeptide of C. elegans.
Such nucleotides can be isolated from cells or synthesised
according to methods well known in the art, as described by way of
example in Sambrook et al, 1989.
[0112] Typically a polynucleotide of the invention comprises a
contiguous sequence of nucleotides which is capable of hybridizing
under selective conditions to the coding sequence or the complement
of the coding sequence of SEQ ID NO: 1, 3, 5 or 7 and in particular
to the coding sequence or the complement of SEQ ID NO: 1, 3 or
5.
[0113] A polynucleotide of the invention can hybridize to the
coding sequence or the complement of the coding sequence of SEQ ID
NO: 1, 3, 5 or 7, and in particular to the coding sequence or the
complement of SEQ ID NO: 1, 3 or 5, at a level significantly above
background. Background hybridization may occur, for example,
because of other cDNAs present in a cDNA library. The signal level
generated by the interaction between a polynucleotide of the
invention and the coding sequence or complement of the coding
sequence of SEQ ID NO: 1, 3, 5 or 7 is typically at least 10 fold,
preferably at least 100 fold, as intense as interactions between
other polynucleotides and the coding sequence of SEQ ID NO: 1, 3, 5
or 7. The intensity of interaction may be measured, for example, by
radiolabelling the probe, e.g. with .sup.32P. Selective
hybridisation may typically be achieved using conditions of medium
to high stringency. However, such hybridisation may be carried out
under any suitable conditions, known in the art (see Sambrook et
al, 1989. For example, if high stringency is required suitable
conditions include from 0.1 to 0.2.times.SSC at 60.degree. C. up to
65.degree. C. If lower stringency is required suitable conditions
include 2.times.SSC at 60.degree. C.
[0114] The coding sequence of SEQ ID NO: 1, 3, 5 or 7 may be
modified by nucleotide substitutions, for example from 1, 2 or 3 to
10, 25, 50 or 100 substitutions. The polynucleotide of SEQ ID NO:
1, 3, 5 or 7 may alternatively or additionally be modified by one
or more insertions and/or deletions and/or by an extension at
either or both ends. A polynucleotide may include one or more
introns, for example may comprise genomic DNA. The modified
polynucleotide generally encodes a polypeptide which has HIF
hydroxylase activity, typically which has hydroxylase activity and
in particular prolyl hydroxylase activity. Alternatively, a
polynucleotide encodes a ligand-binding portion of a polypeptide or
a polypeptide which modulates HIF hydroxylase activity. Degenerate
substitutions may be made and/or substitutions may be made which
would result in a conservative amino acid substitution when the
modified sequence is translated, for example as shown in the Table
above.
[0115] A nucleotide sequence which is capable of selectively
hybridizing to the complement of the DNA coding sequence of SEQ ID
NO: 1, 3, 5 or 7 will generally have at least 60%, at least 70%, at
least 80%, at least 88%, at least 90%, at least 95%, at least 98%
or at least 99% sequence identity to the coding sequence of SEQ ID
NO: 1, 3, 5 or 7 over a region of at least 20, preferably at least
30, for instance at least 40, at least 60, more preferably at least
100 contiguous nucleotides or most preferably over the full length
of SEQ ID NO: 1, 3, 5 or 7. Preferably the nucleotide sequence
encodes a polypeptide which has the same domain structure as a HIF
hydroxylase as described in more detail above.
[0116] For example the UWGCG Package provides the BESTFIT program
which can be used to calculate homology (for example used on its
default settings) (Devereux et al (1984) Nucleic Acids Research 12,
p387-395). The PILEUP and BLAST algorithms can be used to calculate
homology or line up sequences (typically on their default
settings), for example as described in Altschul (1993) J. Mol.
Evol. 36: 90-300; Altschul et al (1990) J. Mol. Biol.
215:403-10.
[0117] Software for performing BLAST analyses is publicly available
through the National Centre for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pair (HPSs) by identifying short
words of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighbourhood word score threshold (Altschul et al, 1990).
These initial neighbourhood word hits act as seeds for initiating
searches to find HSPs containing them. The word hits are extended
in both directions along each sequence for as far as the cumulative
alignment score can be increased. Extensions for the word hits in
each direction are halted when: the cumulative alignment score
falls off by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the accumulation of
one or more negative-scoring residue alignments; or the end of
either sequence is reached. The BLAST algorithm parameters W, T and
X determine the sensitivity and speed of the alignment. The BLAST
program uses as defaults a word length (W) of 11, the BLOSUM462
scoring matrix (see Henikoff and Henikoff(1992) Proc. Natl. Acad.
Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of
10, N=5, N=4, and a comparison of both strands.
[0118] The BLAST algorithm performs a statistical analysis of the
similarity between two sequences; see e.g., Karlin and Altschul
(1993) Proc. Natl. Acad Sci. USA 90: 5873-5787 and Altschul and
Gish (1996) Methods Enzymol. 266: 460-480. One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a sequence is considered
similar to another sequence if the smallest sum probability in
comparison of the first sequence to the second sequence is less
than about 1, preferably less than about 0.1, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0119] Any combination of the above mentioned degrees of sequence
identity and minimum sizes may be used to define polynucleotides of
the invention, with the more stringent combinations (i.e. higher
sequence identity over longer lengths) being preferred. Thus, for
example a polynucleotide which has at least 90% sequence identity
over 25, preferably over 30 nucleotides forms one aspect of the
invention, as does a polynucleotide which has at least 95% sequence
identity over 40 nucleotides.
[0120] The nucleotides of the invention may comprise a label for
example, they may be radiolabelled or fluorescently labelled. The
label may be such that it is only visualised on hybridisation to a
complementary nucleic acid. For example, the label may be quenched
until hybridisation. The nucleotides of the invention may be
immobilised to a support such as a membrane or as a microarray.
[0121] The nucleotides according to the invention have utility in
production of the proteins according to the invention, which may
take place in vitro, in in vivo or ex vivo. Accordingly, the
invention provides a polypeptide encoded by a polynucleotide of the
invention and in particular encoded by SEQ ID NO: 1, 3, 5 or 7. The
invention includes a PHD polypeptide encoded by PHD gene, in
particular by the PHD 1, 2 or 3 genes. The invention also provides
fragments of such polypeptides which have HIF hydroxylase activity
and in particular prolyl hydroxylase activity.
[0122] The nucleotides may be involved in recombinant protein
synthesis or indeed as therapeutic agents in their own right,
utilised in gene therapy techniques. Nucleotides complementary to
those encoding HIF hydroxylase, or antisense sequences, or
interfering RNA may also be used in gene therapy.
[0123] The present invention also includes expression vectors that
comprise nucleotide sequences encoding the proteins of the
invention. Such expression vectors are routinely constructed in the
art of molecular biology and may for example involve the use of
plasmid DNA and appropriate initiators, promoters, enhancers and
other elements, such as for example polyadenylation signals which
may be necessary, and which are positioned in the correct
orientation, in order to allow for protein expression. Other
suitable vectors would be apparent to persons skilled in the art.
By way of further example in this regard we refer to Sambrook et
al. 1989.
[0124] Polynucleotides according to the invention may also be
inserted into the vectors described above in, an antisense
orientation in order to provide for the production of antisense
RNA. Antisense RNA or other antisense polynucleotides may also be
produced by synthetic means. Such antisense polynucleotides may be
used as test compounds in the assays of the invention or may be
useful in a method of treatment of the human or animal body by
therapy.
[0125] Polynucleotides of the invention may also be used to design
double stranded RNAs for use in RNA interference. Such RNA
comprises short stretches of double stranded RNA having the same
sequence as a target mRNA. Such sequences can be used to inhibit
translation of the mRNA. Alternatively, small fragments of the gene
encoding a HIF hydroxylase may be provided, cloned back to back in
a plasmid. Expression leads to production of the desired double
stranded RNA. Such short interfering RNA (siRNA) may be used for
example to reduce or inhibit expression of a HIF hydroxylase of the
invention, in assays or in a method of therapy. The invention also
relates to such siRNAs. Such siRNAs may be designed to inhibit
groups of HIF hydroxylases of the invention by basing their
sequences on regions of conserved sequence in the encoding genes of
the hydroxylases. Alternatively, the siRNAs may be made specific to
a particular HIF hydroxylase by choosing a sequence unique to the
encoding gene of the particular hydroxylase gene to be
inhibited.
[0126] Preferably, a polynucleotide of the invention in a vector is
operably linked to a control sequence which is capable of providing
for the expression of the coding sequence by the host cell, i.e.
the vector is an expression vector. The term "operably linked"
refers to a juxtaposition wherein the components described are in a
relationship permitting them to function in their intended manner.
A regulatory sequence, such as a promoter, "operably linked" to a
coding sequence is positioned in such a way that expression of the
coding sequence is achieved under conditions compatible with the
regulatory sequence.
[0127] The vectors may be for example, plasmid, virus or phage
vectors provided with a origin of replication, optionally a
promoter for the expression of the said polynucleotide and
optionally a regulator of the promoter. The vector may be an
artificial chromosome. The vectors may contain one or more
selectable marker genes, for example an ampicillin resistance gene
in the case of a bacterial plasmid or a resistance gene for a
fungal vector. Vectors may be used in vitro for example for the
production of[DNA or RNA or used to transfect or transform a host
cell, for example, a mammalian host cell. The vectors may also be
adapted to be used in vivo, for example in a method of gene
therapy.
[0128] Promoters and other expression regulation signals may be
selected to be compatible with the host cell for which expression
is designed. For example, yeast promoters include S. cerevisiae
GAL4 and ADH promoters, S. pombe nmt1 and adh promoter. Mammalian
promoters include the metallothionein promoter which can be induced
in response to heavy metals such as cadmium. Viral promoters such
as the SV40 large T antigen promoter or adenovirus promoters may
also be used. An IRES promoter may also be used. All these
promoters are readily available in the art.
[0129] Mammalian promoters, such as .beta.-actin promoters, may be
used. Tissue-specific promoters are especially preferred. Inducible
promoters are also preferred. Promoters inducible by hypoxic
conditions may, for example, be employed. Viral promoters may also
be used, for example the Moloney murine leukaemia virus long
terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR
promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE
promoter, adenovirus, HSV promoters (such as the HSV IE promoters),
or HPV promoters, particularly the HPV upstream regulatory region
(URR). Viral promoters are readily available in the art.
[0130] The vector may further include sequences flanking the
polynucleotide giving rise to polynucleotides which comprise
sequences homologous to eukaryotic genomic sequences, preferably
mammalian genomic sequences, or viral genomic sequences. This will
allow the introduction of the polynucleotides of the invention into
the genome of eukaryotic cells or viruses by homologous
recombination. In particular, a plasmid vector comprising the
expression cassette flanked by viral sequences can be used to
prepare a viral vector suitable for delivering the polynucleotides
of the invention to a mammalian cell. Homologous recombination may
also be used to disrupt or mutate endogenous sequences in cells
encoding HIF hydroxylases. Other examples of suitable viral vectors
include herpes simplex viral vectors and retroviruses, including
lentiviruses, adenoviruses, adeno-associated viruses and HPV
viruses. Gene transfer techniques using these viruses are known to
those skilled in the art. Retrovirus vectors for example may be
used to stably integrate the polynucleotide giving rise to the
polynucleotide into the host genome. Replication-defective
adenovirus vectors by contrast remain episomal and therefore allow
transient expression.
[0131] The invention also includes cells that have been modified to
express a HIF hydroxylase of the invention. Such cells include
transient, or preferably stable higher eukaryotic cell lines, such
as mammalian cells or insect cells, using for example a baculovirus
expression system, lower eukaryotic cells, such as yeast or
prokaryotic cells such as bacterial cells. Particular examples of
cells which may be modified by insertion of vectors encoding for a
polypeptide according to the invention include mammalian thymic
epithelial cells, fibroblasts, HEK293T, U20S, CHO, HeLa, BHK, 3T3
and COS cells.
[0132] A polypeptide of the invention may be expressed in cells of
a transgenic non-human animal, typically a mammal, preferably a
rodent, more preferably a mouse. The animal may be a larger animal
such as a pig or sheep. A transgenic non-human animal expressing a
polypeptide of the invention is included within the scope of the
invention. Also included are transgenic animals expressing an
antisense RNA, siRNA or ribozyme designed to inhibit expressions of
a polypeptide of the invention. The transgenic animals of the
invention may have a gene encoding an endogenous HIF hydroxylase
disrupted or mutated. For example, the endogenous HIF hydroxylase
may be rendered inactive and lack hydroxylase activity.
[0133] Antibodies
[0134] According to another aspect, the present invention also
relates to antibodies, specific for a polypeptide of the invention.
Such antibodies are for example useful in purification, isolation
or screening methods involving immunoprecipitation techniques or,
indeed, as therapeutic agents in their own right. Antibodies may be
raised against specific epitopes of the polypeptides according to
the invention.
[0135] Antibodies may be used to impair HIF hydroxylase function.
An antibody, or other compound, "specifically binds" to a protein
when it binds with preferential or high affinity to the protein for
which it is specific but does not substantially bind or binds with
only low affinity to other proteins. A variety of protocols for
competitive binding or immunoradiometric assays to determine the
specific binding capability of an antibody are well known in the
art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226,
1993). Such immunoassays typically involve the formation of
complexes between the specific protein and its-antibody and the
measurement of complex formation.
[0136] Antibodies of the invention may be antibodies to human
polypeptides or fragments thereof. For the purposes of this
invention, the term "antibody", unless specified to the contrary,
includes fragments which bind a polypeptide of the invention. Such
fragments include Fv, F(ab') and F(ab').sub.2 fragments, as well as
single chain antibodies. Furthermore, the antibodies and fragment
thereof may be chimeric antibodies, CDR-grafted antibodies or
humanised antibodies.
[0137] Antibodies may be used in a method for detecting
polypeptides of the invention in a biological sample, which method
comprises:
[0138] I providing an antibody of the invention;
[0139] II incubating a biological sample with said antibody under
conditions which allow for the formation of an antibody-antigen
complex; and
[0140] III determining whether antibody-antigen complex comprising
said antibody is formed.
[0141] A sample may be for example a tissue extract, blood, serum
and saliva. Antibodies of the invention may be bound to a solid
support and/or packaged into kits in a suitable container along
with suitable reagents, controls, instructions, etc. Antibodies may
be linked to a revealing label and thus may be suitable for use in
methods of in vivo HIF hydroxylase imaging.
[0142] Antibodies of the invention can be produced by any suitable
method. Means for preparing and characterising antibodies are well
known in the art, see for example Harlow and Lane (1988)
"Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. For example, an antibody may be
produced by raising antibody in a host animal against the whole
polypeptide or a fragment thereof, for example an antigenic epitope
thereof, herein after the "immunogen".
[0143] A method for producing a polyclonal antibody comprises
immunising a suitable host animal, for example an experimental
animal, with the immunogen and isolating immunoglobulins from the
animal's serum. The animal may therefore be inoculated with the
immunogen, blood subsequently removed from the animal and the IgG
fraction purified.
[0144] A method for producing a monoclonal antibody comprises
immortalising cells which produce the desired antibody; Hybridoma
cells may be produced by fusing spleen cells from an inoculated
experimental animal with tumour cells (Kohler and Milstein (1975)
Nature 256, 495-497).
[0145] An immortalized cell producing the desired antibody may be
selected by a conventional procedure. The hybridomas may be grown
in culture or injected intraperitoneally for formation of ascites
fluid or into the blood stream of an allogenic host or
immunocompromised host. Human antibody may be prepared by in vitro
immunisation of human lymphocytes, followed by transformation of
the lymphocytes with Epstein-Barr virus and in transgenic mice
enabling production of human antibodies.
[0146] For the production of both monoclonal and polyclonal
antibodies, the experimental animal is suitably a goat, rabbit, rat
or mouse. If desired, the immunogen may be administered as a
conjugate in which the immunogen is coupled, for example via a side
chain of one of the amino acid residues, to a suitable carrier. The
carrier molecule is typically a physiologically acceptable carrier.
The immunogen may, for example, be administered with an adjuvant.
The antibody obtained may be isolated and, if desired,
purified.
[0147] Assays
[0148] Our data show s that hydroxylation of HIF-.alpha. is
mediated by a hydroxylase enzyme which has specificity or
selectivity for HIF-.alpha.. The enzyme responsible are referred to
as HIF hydroxylases and include EGL-9 and PHD1, 2 and 3. The action
of HIF hydroxylases, and in particular human HIF hydroxylases,
represent a novel target for the control of HIF.alpha.. By blocking
HIF hydroxylase activity, this will reduce hydroxylation of
HIF-.alpha. and thus lead to the accumulation of HIF-.alpha. in
cells. This in turn will lead to the activation of systemic local
defences against hypoxia or ischaemia that may include the
promotion of angiogenesis, erythropiesis, energy metabolism,
inflammation, vasomotor function, and will also affect
apoptotic/proliferative responses.
[0149] We describe below in more detail a number of different
assays that may be carried out to identify modulators of HIF
hydroxylase activity and in particular of prolyl hydroxylase
activity, or which affect regulation of HIF-.alpha. levels in a
cell and hence which affect HIF mediated activity. Some of these
assays utilise HIF polypeptides and VHL polypeptides, and in
particular HIF hydroxylases in accordance with the present
invention. Typically, the assays may utilise a human HIF
hydroxylase such as PHD 1, 2 or 3 or a fragment or variant of a
human HIF hydroxylase in a preferred embodiment an enzyme with HIF
prolyl-hydroxylase activity may be used. These components are
described in more detail below. Each of these components, where
required may be provided either in purified or unpurified form, for
example, as cellular extracts or by purification of the relevant
component from such extracts. Alternatively, the relevant component
can be expressed using recombinant expression techniques and
purified for use in the assay. Alternatively, the components may be
expressed recombinantly in a cell for use in cell based assays.
[0150] Typically, a polynucleotide encoding the relevant component
is provided within an expression vector. Such expression vectors
are routinely constructed in the art and may for example involve
the use of plasmid DNA and appropriate initiators, promoters,
enhancers and other elements, such as for example polyadenylation
signals which may be necessary and which are positioned in the
correct orientation in order to allow full protein expression.
Suitable vectors would be very readily apparent to those of skill
in the art, such as those described in more detail herein with
reference to the HIF hydroxylases. Promoter sequences may be
inducible or constitutive promoters depending on the selected assay
format. The promoter may be tissue specific. Examples of promoters
and other flanking sequences for use in the expression vectors are
described in more detail herein with reference to the HIF
hydroxylases of the invention and in particular to the human HIF
hydroxylases of the invention.
[0151] HIF Polypeptides and Peptide Analogues
[0152] The assays of the present invention may use a substrate of a
HIF hydroxylase and in particular a prolyl containing substrate of
the enzyme. In particular, such substrates may be used in assays to
monitor for the activity of a modulator of HIF hydroxylase
activity. The substrate may be a HIF polypeptides or peptide
analogue thereof. Typically, a HIF polypeptide will be used as the
substrate.
[0153] Any suitable substrate in which a residue, preferably a
proline residue, is hydroxylated by a HIF hydroxylase of SEQ ID NO:
2, 4, 6 or 8 may be used and in particular one which is
hydroxylated by the HIF hydroxylase of SEQ ID NO: 2, 4 or 6. In
preferred embodiments of the invention, such a substrate is a HIF
polypeptide such as a HIF-1.alpha. or HIF-2.alpha.-subunit protein
or fragment of either or peptide analogue of the subunit or
fragment. Preferably, the HIF-.alpha. peptide conveys an oxygen
regulated response. More preferably, the HIF-.alpha. peptide is
capable of oxygen regulated binding to pVHL. Preferably, such HIF
polypeptides, fragments or peptide analogues incorporate a proline
residue equivalent to Pro 564 and/or Pro 402 as defined with
reference to HIF-1.alpha.. The proline equivalent to Pro 564 and/or
Pro 402 of HIF-1.alpha. may be determined by aligning the HIF
variant, fragment or analogue to the sequence of HIF-1.alpha. to
obtain the best sequence alignment and identifying thereby the
proline equivalent to Pro 564 and/or Pro 402 of HIF-1.alpha.. In
the assays of the invention the hydroxylation of one or both of
these prolines may be determined.
[0154] A HIF poly,peptide may be of eukaryotic origin, in
particular a human or other mammalian, HIF-.alpha. subunit protein
or fragment thereof. Alternatively, the polypeptide may be of C.
elegans origin. In those assays which monitor for hydroxylation of
HIF-.alpha. through its interaction with and subsequent destruction
by VHL, the HIF polypeptide has the ability to bind to a wild type
full length pVHL protein, such that the binding, is able, in a
normoxic cellular environment, to target the HIF-.alpha. subunit
for destruction i.e. the polypeptide comprises a pVHL binding
domain.
[0155] A number of HIF.alpha. subunit proteins have been cloned.
These include HIF-1.alpha., the sequence of which is available as
Genbank accession number U2243 1, HIF-2.alpha., available as
Genbank accession number U81984 and HIP-3.alpha., available as
Genbank accession numbers AC007193 and AC079154. These are all
human HIF .alpha. subunit proteins and all may be used in the
invention. HIF-.alpha. subunit proteins from other species,
including murine HIF-1.alpha. (accession numbers AF003695, U59496
and X95580), rat HIF-1.alpha. (accession number Y09507), murine
HIF-2.alpha. (accession numbers U81983 and D89787) and murine
HIF-3.alpha. (accession number AF060194) may also be used in the
invention. Other mammalian, vertebrate, invertebrate or other
homologues may be obtained by techniques similar to those described
above for obtaining pVHL homologues.
[0156] One HIF-.alpha. protein of particular interest is the C.
elegans HIF-.alpha. subunit protein. The HMF-.alpha./VHL system of
regulation is conserved in C. elegans, so that the C. elegans
system may be used in assays of the present invention.
[0157] There are a number of common structural features found in
the two HIF-.alpha. subunit proteins identified to date. Some of
these features are identified in O'Rourke et al (1999, J. Biol.
Chem., 274; 2060-2071) and may be involved in the trans-activation
functions of the HIF-.alpha. subunit proteins. One or more of these
common structural features are preferred features of the HIF
polypeptides.
[0158] Fragments of HIF-1.alpha. or peptide analogues preferably
include proline residue 402 and/or 564 (U22431), which are
hydroxylated by HIF prolyl hydroxylases. Suitable fragments may
include or consist of residues 344-698, particularly residues
364-678, more particularly residues 364-638 or 384-638 and still
more particularly residues 364-598 or 394-598. Other suitable
fragments may include or consist of residues 549-652 and even more
particularly the N-terminal region thereof which interacts with the
VHL protein. C-terminal fragments may include residues 549 to 582
and in particular residues 556-574. Other suitable fragments
comprise or consist of residues 344-417, more preferably 380-417.
Such a region, or its equivalent in other HIF-.alpha. subunit
proteins, is desirably present in the HIF.alpha. polypeptides
described herein. The substrates used in the assays of the
invention may typically comprise residues 549 to 582 of the human
HIF-1.alpha. sequence.
[0159] Variants of the above HIF-.alpha. subunits may be used, such
as synthetic variants which have at least 45% amino acid identity
to a naturally occurring HIF-.alpha. subunit (particularly to a
human HIF-.alpha. subunit such as, for example HIF-1.alpha.),
preferably at least 50%, 60%, 70%, 80%, 90%, 95% or 98% identity.
Such variants may include substitutions or modifications as
described above with respect to HIF hydroxylases. Amino acid
activity may also be calculated as described above with reference
to HIF hydroxylases.
[0160] HIF fragments may also include non-peptidyl functionalities
and may be optimised for assay purposes such that the level of
identity is lowered. Such functionalities may be covalently bound
such as sugars or non-covalently bound such as metal ions.
[0161] HIF.alpha. polypeptides as described herein may be fragments
of the HIF-.alpha. subunit protein or variants as described above,
provided that said fragments retain the ability to interact with a
wild-type pVHL, preferably wild-type human pVHL. When using
proteinogenic amino acid residues, such fragments are desirably at
least 20, preferably at least 40, 50, 75, 100, 200, 250 or 400
amino acids in size. Desirably, such fragments include proline
residue 402 and/or 564. Some preferred fragments include the region
556-574 found in human HIF-1.alpha. or its equivalent regions in
other HIF-.alpha. subunit proteins, e.g. 517-542 of HIF-2.alpha..
Optionally, the fragments also include one or more domains of the
protein responsible for trans-activation. Reference herein to a
HIF-.alpha. polypeptide or HIF-.alpha. subunit protein includes the
above mentioned mutants and fragments or other HIF-.alpha.
fragments which are functionally able to bind VHL protein unless
the context is explicitly to the contrary.
[0162] Cell based assays of the present invention may involve
upregulation of an endogenous HIF-.alpha. or expression of a
HIF-.alpha. by recombinant techniques and in particular of
HIF-.alpha..
[0163] VHL
[0164] Some assays in accordance with the present invention utilise
VHL and in particular monitor the interaction between hydroxylated
HIF and VHL and the subsequent destruction of HIF-.alpha.. The VHL
may be any suitable mammalian VHL, particularly human VHL. It may
be a C. elegans VHL. Human VHL has been cloned and sources of the
gene can be readily identified by those of skill in the art. Its
sequence is available as Genbank accession numbers AF010238 and
L15409. Other mammalian VHLs are also available, such as murine VHL
(accession number U12570) and rat (accession numbers U14746 and
S80345). Non-mammalian homologues include the VHL-like protein of
C. elegans, accession number F08G12.4. VHL gene sequences may also
be obtained by routine cloning techniques, for example by using all
or part of the human VHL gene sequence as a probe to recover and to
determine the sequence of the VHL gene in other species. A wide
variety of techniques are available for this, for example PCR
amplification and cloning of the gene using a suitable source of
mRNA (e.g. from an embryo or a liver cell), obtaining a cDNA
library from a mammalian, vertebrate, invertebrate or other source,
e.g a cDNA library from one of the above-mentioned sources, probing
said library with a polynucleotide of the invention under stringent
conditions, and recovering a cDNA encoding all or part of the VHL
protein of that mammal. Suitable stringent conditions include
hybridization on a solid support (filter) overnight incubation at
42.degree. C. in a solution containing 50% formamide, 5.times.SSC
(750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate
(pH7.6), 5.times.Denhardt's solution, 10% dextran sulphate and 20
.mu.g/ml salmon sperm DNA, followed by washing in 0.2.times.SSC at
from about 50.degree. C. to about 60.degree. C.). Where a partial
cDNA is obtained, the full length coding sequence may be determined
by primer extension techniques.
[0165] A further approach is to use the above-identified sequences
as query sequences to search databases for homologous gene
sequences or partial gene sequences (particularly ESTs). Matches
identified may be examined and where an actual or putative VHL
sequence is found, the gene recovered by physical cloning using,
for example PCR and RACE-PCR based on the sequence of the
match.
[0166] Although wild-type VHL is preferred, mutants and variants of
VHL which still retain the ability to interact directly with the
HIF-.alpha. subunit may also be used. Examples of VHL mutants are
well known in the art and include mutants described by Stebbins et
al (Science, 1999, 284; 55-61) which have changes to the Elongin C
interacting interface.
[0167] Mutants and other variants will generally be based on
wild-type mammalian VHLs and have a degree of amino acid identity
which is desirably at least 70%, preferably at least 80%, 90%, 95%
or even 98% homologous to a wild type mammalian VHL, preferably to
human VHL.
[0168] It is not necessary to use the entire VHL proteins
(including their mutants and other variants) for assays of the
invention. Fragments of the VHL may be used provided such fragments
retain the ability to interact with the target domain of the
HIF.alpha. subunit. Optionally, the fragment may include the
Elongin C interacting interface domain. Fragments of VHL may be
venerated in any suitable way known to those of skill in the art.
Suitable ways include, but are not limited to, recombinant
expression of a fragment of the DNA encoding the VHL. Such
fragments may be generated by taking DNA encoding the VHL,
identifying suitable restriction enzyme recognition sites either
side of the portion to be expressed, and cutting out said portion
from the DNA. The portion may then be operably linked to a suitable
promoter in a standard commercially available expression system.
Another recombinant approach is to amplify the relevant portion of
the DNA with suitable PCR primers. Small fragments of the VHL (up
to about 20 or 30 amino acids) may also be generated using peptide
synthesis methods which are well known in the art. Generally
fragments will be at least 40, preferably at least.50, 60, 70, 80
or 100 amino acids in size.
[0169] Particularly preferred fragments include those which are
based upon the beta domain located within the fragment 63-156 of
the 213 amino acid human VHL protein, or the equivalent domain in
other variants. In a preferred embodiment, such domains will have
at least 70%, preferably 80%, 90%, 95% or even 98% degree of
sequence identity to the 64-156 fragment of human VHL. Fragments of
this region and its variants may be used. These fragments may be
15-80 amino acids in length, for example from 20 to 80, such as
30-60 amino acids in length. Fragments may include the regions
71-90 or 90-109 of human VHL or their equivalents in the above
described variants. Desirably, the wild-type sequence of the beta
domain is retained.
[0170] One fragment which may be used is that in which up to 53 of
the N-terminal residues, e.g. from 1 to n wherein n is an integer
of from 2 to 53, have been deleted, the rest of the protein being
wild-type.
[0171] The ability of suitable fragments to bind to the HIF.alpha.
subunit (or fragment thereof) may be tested using routine
procedures such as those described in the accompanying Examples
relating to intact VHL. Reference herein to a VHL protein includes
the above mentioned mutants and fragments which are functionally
able to bind the HIF .alpha. subunit unless the context is
explicitly to the contrary.
[0172] Hydroxylases
[0173] In a number of the assays in accordance with the present
invention, hydroxylase enzyme is provided. In preferred
embodiments, the hydroxylase enzyme is a HIF hydroxylase in
accordance with the present invention. The enzyme is preferably a
prolyl hydroxylase. In a particularly preferred embodiment of the
invention the HIF hydroxylase used comprises:
[0174] (a) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8;
[0175] (b) a variant thereof having at least 60% identity to the
amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 and having HIF
hydroxylase activity; or
[0176] (c) a fragment of either thereof having HIF hydroxylase
activity.
[0177] Such hydroxylase enzymes,, and in particular
prolyl-hydroxylases such as for example 4-prolyl hyrdroxylase, are
obtainable from extracts of mammalian cells, including immortalised
mammalian cells in culture such as HeLa, RCC or CHO--K1 cells,
primary-cells, tissues or primary cell lysates (e.g. rabbit
reticulocyte or human placental lysates). Cell extracts may be
prepared in accordance with standard techniques available in the
art by reference to the accompanying examples. Assays may
alternatively be carried out as cell based assays in which
hydroxylase enzyme is expressed endogenously.
[0178] In a preferred embodiment of any one of the assays in
accordance with the present invention, the assay utilises a HIF
hydroxylase, typically a human HIF hydroxylase, and in particular a
PHD hydroxylase of the present invention. Such hydroxylases may be
upregulated before or during the course of the assay.
Alternatively, the enzyme may be expressed recombinantly, and the
HIF hydroxylase of the invention isolated from such recombinant
expression systems in purified or unpurified form for use in the
assays. Alternatively, cells may be provided which have been
transformed or transfected with expression vectors expressing a HIF
hydroxylase in accordance with the present invention. Such methods
provide assays for substances that inhibit, promote or otherwise
modulate the individual activities of HIF hydroxylases in either a
specific or a general manner. The methods may also be used to
identify substances that inhibit, promote or otherwise modulate the
activities of a group of HIF hydroxylases such as, for example, all
of PHD 1, 2 and 3 or any two of the three enzymes.
[0179] The assays of the invention may use an EGLN polypeptide such
as EGLN1. (gi1457146), EGLN2 (gi145714S), EGLN-3 (gi14547150),
FTLJ21620 (BkB15101) or Clorfl2 (NP071334).
[0180] In general, the HIF hydroxylases of the invention are iron
dependent, that is they typically require ferrous (FeII) ions for
activity. Accordingly, the assays of the invention will typically
include ferrous compounds, unless the purpose of the assay is to
determine the effect of the absence of ferrous ions or it is
desired to carry out control reactions where no ferrous ions are
present.
[0181] Assay Methods
[0182] The present invention provides an assay method for
identifying an agent which modulates the interaction of a hypoxia
inducible factor (HIF) hydroxylase with a substrate of the
hydroxylase, the method comprising:
[0183] contacting a HIF hydroxylase and a test substance in the
presence of a substrate of the hydroxylase under conditions in
which the hydroxylase interacts with the substrate in the absence
of the test substance; and
[0184] determining the interaction, or lack of interaction, of the
hydroxylase and the substrate. The interaction of the hydroxylase
with the substrate may be determined by measuring the hydroxylase
activity of the hydroxylase.
[0185] The interaction between hydroxylase and substrate refers to
physical interaction or to functional interaction. The interaction
may therefore be measured by any suitable method, including binding
of the hydroxylase to a substrate, the activity of the hydroxylase
on the substrate, or any activity related to the action of the
hydroxylase on the substrate, such as the levels of co-factors or
by-products used or produced in the hydroxylation reaction, or
downstream effects mediated through hydroxylation of the
substrate.
[0186] In another aspect of the present invention, there is
provided an assay for an inhibitor of HIF-.alpha. destruction or
HIF-.alpha. transcription inactivation comprising providing
HIF-.alpha. or a peptide analogue thereof, incubating HIF-.alpha.
or the peptide analogue with a test substance under conditions
which allow for hydroxylation of HIF-.alpha. in the absence of the
test substance, and monitoring for hydroxylation of HIF-.alpha..
Preferably, HIF-.alpha. or the peptide analogue thereof includes a
prolyl residue such as Pro 564 and/or Pro 402 of HIF-.alpha. or an
equivalent prolyl in a peptide analogue, and said assay is carried
out under conditions which allow for hydroxylation of Pro 564
and/or Pro 402 in the absence of the test substance, and monitoring
for hydroxylation of Pro 564 and/or Pro 402.
[0187] In a further aspect, there is provided an assay for an
inhibitor of VHL-mediated HIF-.alpha. destruction, which
comprises
[0188] providing a HIF-.alpha., or fragment thereof which includes
a VHL-binding portion, together with its cognate prolyl-hydroxylase
under conditions suitable for the hydroxylation of a proline
residue in the HIF-.alpha. VHL-binding domain;
[0189] providing a putative modulator of hydroxylation; and
[0190] determining whether the amount of hydroxylation of said
proline residue has been modulated by said putative modulator. In
one embodiment of the invention the cognate prolyl-hydroxylase is a
prolyl-4-hydroxylase.
[0191] Conversely, in hypoxic conditions such as those commonly
found in tumours, the lack of hydroxylation of HIF, and in
particular of proline hydroxylation, may lead to the accumulation
of HIF.alpha. and the concomitant promotion of angiogenesis and
other growth promoting events. Alternatively, in ischaemic/hypoxic
conditions in which normal levels of HIF activity are present, it
may also be desirable to increase existing HIF activity. Thus
mechanisms which either upregulate a HIF hydroxylase, increase the
activity of a HIF hydroxylase, rescue or bypass the hydroxylase are
a target in such cells.
[0192] The invention also provides an assay for a promoter of
hydroxylation of HIF-.alpha., for example prolyl hydroxylation at
Pro 564 and/or Pro 402 which comprises providing HIF-.alpha. or a
peptide analogue; incubating HIF-.alpha. or the peptide analogue
under hypoxic conditions or conditions under which hydroxylation of
HIF-.alpha. does not occur in the absence of the test substance,
and monitoring for hydroxylation of HIF-.alpha. or the peptide
analogue thereof, such as at Pro 564 and/or Pro 402.
[0193] Accordingly, there is provided an assay for a promoter of
hydroxylation of a proline residue in HIF-.alpha., which
comprises;
[0194] providing HIF-.alpha., or fragment thereof which includes a
VHL-binding portion, under hypoxic conditions, said HIF-.alpha. or
fragment thereof containing a proline residue in the VHL-binding
domain;
[0195] providing a putative hydroxylation promoting agent; and
[0196] determining whether said agent provides for hydroxylation of
said proline.
[0197] In the experiments described herein, HIF hydroxylases, and
in particular PHD polypeptides, have been found to hydroxylate
HIF-.alpha. at one or more proline residues within the pVHL binding
domain. This hydroxylation mediates pVHL binding.
[0198] Accordingly, the present invention provides an assay for a
modulator of HIF hydroxylase activity comprising contacting a HIF
hydroxylase and a substrate of the hydroxylase, preferably a
prolyl-containing substrate, in the presence of a test substance;
and, determining the hydroxylase activity of the HIF hydroxylase,
and in particular the prolyl hydroxylase thereof.
[0199] Such an assay may be used to identify inhibitors of HIF
hydroxylase activity and are thus preferably carried out under
conditions under which hydroxylation, and in particular prolyl
hydroxylation, takes place in the absence of the test substance. As
an alternative, the assays may be used to look for promoters of
hydroxylase activity, for example, by looking for increased
hydroxylation of the proline substrate compared to an assay carried
out in the absence of a test substance. Alternatively, the assays
may be carried out under conditions in which hydroxylation is
reduced or absent, such as under hypoxic conditions and monitoring
for the presence of or increased hydroxylation under such
conditions.
[0200] Such an assay method may by virtue of using a specific HIF
hydroxylase polypeptide be specific for inhibitors or promoters of
the activity of that polypeptide and may by way of comparison be
used to define inhibitors or activators that are specific for that
HIF hydroxylase and not active or less active on a different HIF
hydroxylase. In particular, such assays may be used to identify
inhibitors or activators specific for a particular human HIF
hydroxylase such as PHD 1, 2 or 3.
[0201] An assay method for obtaining an agent which modulates the
activity of a HIF hydroxylase may include:
[0202] contacting an HIF hydroxylase polypeptide and a substrate
thereof, such as an HIF.alpha. polypeptide in the presence of a
test substance; and,
[0203] determining the hydroxylase activity of said HIF
hydroxylase, preferably the prolyl hydroxylase activity, or
HIF-.alpha. hydroxylase activity thereof.
[0204] In one embodiment the assay method may be for obtaining an
agent which modulates the activity of an EGLN polypeptide and
comprise:
[0205] contacting an EGLN polypeptide and a test compound in the
presence of an HIF polypeptide under conditions in which the EGLN
polypeptide normally catalyses prolyl hydroxylation of said HIF
polypeptide; and
[0206] determining the HIF prolyl hydroxylase activity of said EGLN
polypeptide.
[0207] Such assays may be performed under conditions in which the
HIF hydroxylase/ELGN polypeptide normally catalyses hydroxylation
and, in particular prolyl hydroxylation of a HIF.alpha.
polypeptide. Suitable conditions may include pH 6.6 to 8.5 in an
appropriate buffer (for example, Tris HCl or MOPS) in the presence
of 2-oxoglutarate, dioxygen and preferably ascorbate and ferrous
iron.
[0208] Reducing agents such as dithiothreitol or
tris(carboxyethyl)phosphi- ne may also be present to optimise
activity. Other enzymes such as catalase and protein disulphide
isomerase may be used for the optimisation of activity. The
enzymes, such as protein disulphide isomerase, may be added in
purified or unpurified form. Further components capable of
promoting or facilitating the activity of protein disulphide
isomerase may also be added.
[0209] In an alternative embodiment, the invention provides an
assay method for identifying an agent which modulates the
interaction of a EGLN polypeptide and a HIF polypeptide
comprising:
[0210] contacting an EGLN polypeptide and a test compound in the
presence of an HIF polypeptide, under conditions in which the EGLN
polypeptide normally interacts with the HIF polypeptide; and
[0211] determining the interaction of said HIF polypeptide and said
EGLN polypeptide.
[0212] The present invention also provides an assay method for the
identification of a HIF hydroxylase and in particular for the
identification of a HIF prolyl hydroxylase. The method typically
comprising:
[0213] (a) providing a test polypeptide;
[0214] (b) bringing into contact a HIF polypeptide and the test
polypeptide under conditions in which the HIF polypeptide is
hydroxylated by a HIF hydroxylase; and
[0215] (c) determining whether or not the HIF polypeptide is
hydroxylated.
[0216] In one embodiment the assay method may, in step (b), bring
the HIF polypeptide and test polypeptide into contact under
conditions in which the HIF polypeptide is hydroxylated by a PHD
(EGLN) polypeptide.
[0217] Typically, libraries of test polypeptides may be screened to
identify a HIF hydroxylase, for example an expression library from
a particular species or tissue may be screened or one produced
under a particular set of conditions. Alternatively, candidate HIF
hydroxylases identified on the basis of criteria such as sequence
homology or a particular protein structure may be assessed and
their hydroxylase activity confirmed.
[0218] The HIF polypeptide used in the screening may be any of
those described herein it may be a human HIF polypeptide or a
homolog from another species such as, for example, ceHIF. It may be
from the species the library of test polypeptides is derived from.
The hydroxylation of the HIF polypeptide, and in particular the
hydroxylation of proline, may be identified by any of the methods
discussed herein. For example hydroxylation may be confirmed by
using a functional assay based on the effect of the hydroxylation
on the HIF polypeptide, such as its decreased stability.
[0219] Once a HIF hydroxylase has been identified it may be further
characterised by, for example, assessing whether or not it is
inhibited by compounds such as dimethyloxalolylglycine (DMOG) a
precursor or pro-drug for oxalolylglycine. The effect of the HIF
hydroxylase on HIF stability in the organism or tissue the
hydroxylase is identified from may be assessed. The identified
hydroxylase may be used in the same way as the other hydroxylases
of the invention and in particular in the assays and therapeutic
applications of the invention.
[0220] The present invention also provides an assay method for
identifying alternative substrates of a HIF hydoxylase of the
invention. Thus polypeptides or polypeptide analogues which can be
hydroxylated and in particular have proline residues hydroxylated
may be identified. The assay method typically comprises:
[0221] (b) contacting a test polypeptide with a HIF hydroxylase of
the invention under conditions which HIF would normally be
hydroxylated by the hydroxylase;
[0222] (c) determining whether the polypeptide is hydroxylated.
[0223] Typically, hydroxylation and in particular prolyl
hydroxyaltion of the test substance may be confirmed using any of
the methods discussed herein.
[0224] The present invention also provides an assay method for
identifying a polypeptide or polypeptide analogue capable of
specifically interacting with a HIF hydroxylase of the invention
and in particular which is capable of specifically binding to the
active site of the HIF hydroxylase in a manner which mimics or
resembles the binding of the normal substrate of the enzyme. The
method typically comprises:
[0225] (a) contacting a test polypeptide with a HIF hydroxylase of
the invention under conditions which HIF would bind to the
hydroxylase;
[0226] (b) determining whether the test polypeptide or analogue
binds the hydroxylase.
[0227] The binding of the test polypeptide to the hydroxylase may
be confirmed by any of the techniques discussed herein. In one
embodiment binding of the polypeptide to a HIF hydroxylase may
identified by looking for co-immunoprecipitation of the test
polypeptide with the hydroxylase. The ability of the test
polypeptide to inhibit binding of HIF to the hydroxylase may also
be used.
[0228] The alternative polypeptide substrates and the polypeptides
identified as being capable of specifically binding the
hydroxylases of the invention may be used in the assay methods of
the invention, for example, to identify modulators. Those
polypeptides capable of preventing the normal interaction of HIF
with a hydroxylase of the invention may also be used
therapeutically.
[0229] The assays of the invention may also comprise modifying the
agent identified. The assays may also comprise formulating the
identified agent into a pharmaceutical composition. Typically, such
pharmaceutical compositions may be for the treatment of a condition
associated with increased or decreased HIF levels or activity.
[0230] Methods for Monitoring Modulation
[0231] The precise format of any of the screening or assay methods
of the present invention may be varied by those of skill in the art
using routine skill and knowledge. The skilled person is well aware
of the need to additionally employ appropriate controlled
experiments. The assays of the present invention may involve
monitoring for hydroxylation of a suitable substrate (in particular
monitoring for prolyl hydroxylation), monitoring for the
utilisation of substrates and co-substrates, monitoring for the
production of the expected products between the enzyme and its
substrate. Assay methods of the-present invention may also involve
screening for the direct interaction between components in the
system. Alternatively, assays may be carried out which monitor for
downstream effects such as binding and subsequent destruction of
HIF by VHL, alterations to the levels of HIF in the system and
downstream effects mediated by HIF such as HIF mediated
transcription using suitable reporter constructs or by monitoring
for the upregulation of genes or alterations in the expression
patterns of genes know to be regulated directly or indirectly by
HIF.
[0232] Various methods for determining hydroxylation are known in
the art and are described and exemplified herein. Any suitable
method may be used for determining activity of the HIF hydroxylase
such as by substrate or co-substrate utilization, product
appearance such as peptide hydroxylation or down-stream effects
mediated by hydroxylated or non-hydroxylated products.
[0233] Our finding that the Pro564 residue of HIF-1.alpha. is
hydroxylated by a prolyl-hydroxylase provides the basis for assay
methods designed to screen for inhibitors or promoters of this
process. Any suitable method may be used to monitor for
hydroxylation of HIF-1.alpha. or a HIF polypeptide or analogue
thereof. Assays may be carried out to monitor directly for
hydroxylation of the relevant proline residue or another position.
Alternatively, assays may be carried out to monitor for depletion
of co-factors and co-substrates. Alternatively, such assays may
monitor the downstream effects of hydroxylation of HIF or indeed
inhibition of hydroxylation of HIF, for example, by monitoring the
interaction between HIF and VHL levels of HIF protein or HIF
mediated transcription. Alternatively, reporter gene constructs
driven by HIF regulated promoters may be used. Assays are also
provided for the identification of enhancers of the activity of the
HIF hydroxylase and in particular of the HIF prolyl hydroxylase
activity of these enzymes. The assay may be used to identify an
enhancer a human HIF hydroxylases and, in particular, of PHD 1,2 or
3. Such enhancers may be used to reduce HIF.alpha. activity.
[0234] In one embodiment, to perform an assay for an inhibitor of
VHL-mediated HIF-.alpha. destruction a suitable substrate of the
HIF hydroxylase is provided. This may be HIF-.alpha. or a fragment
thereof which includes a VHL binding portion and which included the
Pro564 and/or Pro 402 residue is provided. The substrate may not be
hydroxylated at the Pro564 and/or Pro 402 position. This may be
achieved by providing synthetic polypeptide substrates, or by
producing HIF-.alpha. polypeptides in bacterial cells, insect cells
or mammalian cells or in in vitro transcription and translation
systems. Alternatively, assays may be carried out over a selected
time course such that the substrate is produced during the course
of the assay, initially in un-hydroxylated form.
[0235] The substrate, enzyme and potential inhibitor compound may
be incubated together under conditions which in the absence of
inhibitor provide for hydroxylation of Pro564 and/or Pro 402, and
the effect of the inhibitor may be determined by determining
hydroxylation of the substrate. This may be accomplished by any
suitable means. Small polypeptide substrates may be recovered and
subject to physical analysis, such as mass spectrometry or
chromatography, or to functional analysis, such as the ability to
bind to VHL (or displace a reporter molecule from VHL) and be
targeted for destruction. Such methods are known as such in the art
and may be practiced using routine skill and knowledge.
Determination may be quantitative or qualitative. In both cases,
but particularly in the latter, qualitative determination may be
carried out in comparison to a suitable control, e.g. a substrate
incubated without the potential inhibitor.
[0236] Inhibitor compounds which are identified in this manner may
be recovered and formulated as described above for polypeptides of
the invention.
[0237] Another assay of the invention is for a promoter of
hydroxylation of HIF-.alpha. subunits. Typically, a HIF-.alpha.
subunit or portion thereof is prepared as described above, and
incubated under hypoxic conditions. By "hypoxic", it is meant less
than 5%, preferably less than 3%, more preferably less than 1%, end
preferably less than 0.5%, such as less than 0.1% O.sub.2. The
HIF-.alpha. subunit is incubated with a cell extract which includes
the HIF hydroxylase as described above, optionally further in the
presence of a source of ferrous (FeII) ions and/or ascorbate. A
suitable concentration of ferrous ions is in the range of from 1 to
500 .mu.M, such as from 25 to 250 .mu.M and in particular from
50-200 .mu.M. Ferrous ions may be supplied in the form of ferrous
chloride, ferrous sulphate, and the like. Ascorbate may be provided
in the form of a salt, such as sodium ascorbate, and in a
concentration range of from 0.1 to 10 mM, such as from 1 to 5 mM.
Another cofactor is .alpha.-ketoglutarate, which may also be
supplied in the form of a salt at a range of from 0.1 to 5 mM, such
as from 1 to 5 mM.
[0238] In this embodiment of the invention, the substrate will be
incubated in the presence of a potential hydroxylation promoting
agent, and the effect of the agent determined, by determining the
hydroxylation of the Pro564 and/or Pro 402. As with the assay of
the other aspect of the invention described above, determination
may be quantitative or qualitative, and in either case determined
relative to a suitable control.
[0239] The interaction between the polypeptides may be studied in
vitro by labelling one with a detectable label and bringing it into
contact with the other which has been immobilised on a solid
support. Suitable detectable labels include .sup.35S, which may be
incorporated into recombinantly produced peptides and polypeptides.
Recombinantly produced peptides and polypeptides may also be
expressed as a fusion protein containing an epitope which can be
labelled with an antibody.
[0240] Fusion proteins may, for example, incorporate six histidine
residues at either the N-terminus or C-terminus of the recombinant
protein. Such a histidine tag may be used for purification of the
protein by using commercially available columns which contain a
metal ion, either nickel or cobalt (Clontech, Palo Alto, Calif.,
USA). These tags also serve for detecting the protein using
commercially available monoclonal antibodies directed against the
six histidine residues (Clontech, Palo Alto, Calif., USA).
[0241] The protein which is immobilized on a solid support may be
immobilized using an antibody against that protein bound to a solid
support or via other technologies which are known per se. A
preferred in vitro interaction may utilise a fusion protein
including glutathione-S-transferase (GST). This may be immobilized
on glutathione agarose beads. In an in vitro assay format of the
type described above, a test compound can be assayed by determining
its ability to diminish the amount of labelled peptide or
polypeptide which binds to the immobilized GST-fusion polypeptide.
This may be determined by fractionating the glutathione-agarose
beads by SDS-polyacrylamide gel electrophoresis. Alternatively, the
beads may be rinsed to remove unbound protein and the amount of
protein which has bound can be determined for example, by counting
the amount of label present in a suitable scintillation
counter.
[0242] Thus, assays in accordance with the present invention may
involve monitoring for the interaction between VHL and HIF. The
interaction between HIF and VHL is mediated by hydroxylation of
HIF. The VHL-HIF interaction leads to ubiquitylation of HIF. Assays
to monitor for test substances which interfere with the interaction
between HIF and VHL, and in particular which interfere with
hydroxylation of HIF may be monitored by any suitable method using
HIF associated regulation. For example, in assay systems making use
of recombinant HIF hydroxylase in accordance with the present
invention or in which HIF hydroxylase expression is upregulated
within a cell, the effect of test substances can be monitored
through monitoring the levels of HIF in the cell. Alternatively,
transcription and expression of genes known to be upregulated or
down regulated by the presence of HIF could be monitored. In
particular, upregulation of HIF regulated genes would demonstrate
inhibition of prolyl hydroxylation whereas down regulation would
suggest enhancement or promotion of prolyl hydroxylation.
[0243] In alternative embodiments, reporter constructs may be
provided in which promoters mediated by HIF are provided operably
linked to a reporter gene. Any suitable reporter gene could be
used, such as for example enzymes which may then be used in
colorometric, fluorometric, fluorescence resonance or spectrometric
assays.
[0244] HIF hydroxlase is a 2OG dependent oxygenase which catalyses
the following reaction, in which R is HIF.alpha. and ROH singly is
hydroxylated HIF.alpha.; 1
[0245] Typically, the hydroxylation is prolyl hydroxylation. The
hydroxylase may catalyse more than one hydroxylation of
HIF-.alpha..
[0246] In the assay methods described herein, typically the HIF
hydroxylase and the substrate of the hydroxylase are contacted in
the presence of a co-substrate, such as 2-oxoglutarate (2OG). The
hydroxylase activity of the HIF hydroxylase may be determined by
determining the turnover of the co-substrate. This may be achieved
by determining the presence and/or amount of reaction products,
such as hydroxylated substrate or succinic acid. The amount of
product may be determined relative to the amount of substrate.
Typically, in such embodiments the substrate may be an HIF-.alpha.
polypeptide and, for example, the product measured may be
hydroxylated HIF-.alpha. polypeptide.
[0247] HIF.alpha. prolyl hydroxylase activity may be determined by
determining the turnover of said 2OG to succinate and CO.sub.2, as
described in Myllyharju J. et al EMBO J. 16 (6): 1173-1180 (199])
or as in Cunliffe C. J. et al Biochem. J. 240 617-619 (1986), or
other suitable assays for CO.sub.2, bicarbonate or succinate
production. These methods may be used in the assays of the
invention and in particular to assess the HIF-.alpha. prolyl
hydroxylase activity of the HIF hydroxylase of the invention,
including that of human HIF hydroxylases and in particular of PHD
or EGLN polypeptides of the invention. Such assays can be modified
to high throughput format and the invention encompasses such high
throughput assays for hydroxylase activity.
[0248] Alternatively, the end-point determination may be based on
conversion of HIF.alpha. or peptide fragments (including synthetic
and recombinant peptides) derived from HIF.alpha. into detectable
products. Peptides may be modified to facilitate the assays so that
they can be rapidly carried out and may be suitable for high
throughput screening.
[0249] For example, reverse phase HPLC (C-18 octadecylsilane
column), as exemplified herein, may be used to separate starting
synthetic peptide substrates for HIF hydroxylase from the
hydroxylated products, as the latter have a shorter retention time
in the column. Modifications of this assay or alternative assays
for HIF hydroxylase activity may employ, for example, mass
spectrometric, spectroscopic, and/or fluorescence techniques as are
well known in the art (Masimirembwa C. et al Combinatorial
Chemistry & High Throughput Screening (2001) 4 (3) 245-263,
Owicki J. (2000) J. Biomol. Screen. 5 (5) 297-305, Gerslikovich A
et al (1996) J. Biochem. & Biophys. Meths. 33 (3) 135-162,
Kraaft G. et al (1994) Meths. Enzymol. 241 70-86). Fluorescent
techniques may employ versions of the substrate modified in such as
way as to carry out or optimise spectroscopic or fluorescence
assays.
[0250] For example, HIF.alpha. polypeptide may be immobilised e.g.
on a bead or plate, and hydroxylation of the appropriate residue
detected using an antibody or other binding molecule which binds
the pVHL binding domain of HIF.alpha. with a different affinity
when a proline residue such as proline 402 or proline 564 is
hydroxylated from when the residue is not hydroxylated. Such
antibodies may be obtained by means of standard techniques which
are well known in the art, e.g. using a hydroxylated HIF.alpha.
peptide.
[0251] Binding of a molecule which discriminates between the
hydroxylated and non-hydroxylated form of a HIF.alpha. polypeptide
may be assessed using any technique available to those skilled in
the art, which may involve determination of the presence of a
suitable label.
[0252] Assays may be used to screen for inhibitors of HIF
hydroxylase and in particular for inhibitors of HIF prolyl
hydroxylase (HPH) activity in a similar way to that described for
the human prolyl hydroxylase involved in collagen biosynthesis
(CPH) (Cunliffe et al. Biochemical J. 240 611-619 (1986); Cunliffe
C J et al. Biochem. J. 239 311-315 (1986), Franklin T J and Hitchen
M Biochem. J. 261: 127-130 (1989) Franklin T J. et al. Biochemical
Society Transactions 19 (4): 812-815 (1991)).
[0253] HIF prolyl-hydroxylase activity of a HIF hydroxylase
polypeptide may be determined by determining the hydroxylation of
one or more proline residues of the substrate of the HIF
hydroxylase used, which will typically be a HIF.alpha. polypeptide.
Preferably, the hydroxylation of one or more proline residues
within the pVHL binding domain of the HIF-.alpha. polypeptide, for
example, proline 402 and/or proline 564. For convenience, these
proline residues are referred to herein as Pro402 and Pro564 or
position 402 and position 564. It will be understood that this
terminology is also applied to polypeptides which contain far fewer
than 564 residues, and to other HIF-.alpha. isoforms where the
equivalent proline residue may occur at a slightly different
position.
[0254] Assay methods of the present invention may also take the
form of an in vivo assay. The in vivo assay may be performed in a
cell line such as a yeast strain in which the relevant polypeptides
or peptides are expressed from one or more vectors introduced into
the cell.
[0255] C. elevans Assay Systems
[0256] Our finding that the HIF-VHL interaction is conserved in C.
elegans provides a system to study the interaction in an in vivo
environment, and its consequences.
[0257] Thus in a further aspect, the invention provides an assay
for a modulator of HIF-VHL interaction, said method comprising:
[0258] providing a C. elegans which has wild-type HIF and VHL genes
in normoxic or hypoxic conditions (wherein hypoxic conditions are
as defined above);
[0259] exposing said C elegans to a-potential modulator of the
HIF-VHL interaction; and
[0260] determining the extent to which the modulator promotes or
decreases the interaction between HIF and VHL in said C.
elegans.
[0261] The determining may comprise immunoprecipitating one or
other of the HIF and VHL components and then determining, e.g. by
antibody detection, the amount of the other of the HIF and VHL
component which is associated with the immunoprecipitated protein.
Radiolabelling of one of the two proteins may allow determination
of the amount of VHL or HIF captured. Alternatively, a reporter
gene may be linked to a HIF-responsive promoter, and the amount of
HIF in the subject C. elegans determined by-measuring the activity
of a reporter gene product, such as green fluorescent protein,
luciferase, chloramphenicol acetyl transferase, beta-galactosidase,
and the like.
[0262] In alternative aspects of the invention, the source of
2-oxoglutarate dependent dioxygenase and in particular the
prolyl-hydroxylase of the invention is a HIF hydroxylase according
to the invention. Such polypeptides may be introduced using
recombinant systems so as to allow for the assay of inhibitory,
augmenting, blocking or other modulating activities that show
differential effects among the said HIF hydroxylase.
[0263] In Vivo Assays
[0264] The assays may be carried out using cell based, organ based
or whole animal assays conducted in vivo. Such assays may utilize
the endogenous expression of the HIF hydroxylase nucleotides and/or
polypeptides. In other forms of the invention, upregulation of
specific endogenous HIF hydroxylases may be achieved by stimulators
of the expression thereof. Such stimulators may be growth factor
such as platelet derived growth factor or angiotensin II, or
chemicals such as phorbol esters that are known to upregulate
specific HIF hydroxylases. In another form of the invention,
nucleotide constructs may be introduced into cells or transgenic
animals to increase production of one or more specific HIF
hydroxylases. Alternatively nucleotide constructs may be introduced
into cells so as reduce or abrogate expression of one or more
specific HIF hydroxylases. Appropriate methods that include but are
not limited to homologous recombination, antisense expression,
ribozyme expression and RNA interference are outlined herein and
known by those skilled in the art.
[0265] Tissue culture cells, organs, animals and other biological
systems, obtained by the aforementioned forms of the invention, may
be used to provide a further source of a HIF hydroxylase, or may be
used for the assay, or especially comparative assay, of the
activity of test substances may inhibit, augment, block or
otherwise modulate the activity of specific HIF hydroxylases.
[0266] The activity of the HIF hydroxylases may be assayed by any
of the aforementioned methods or by cell, tissue, or other assays
conducted in vivo that measure the effects of altered activity of
the HIF hydroxylases. A preferred form of these assays measures the
level of a HIF-.alpha. polypeptide or the level of activity of a
HIF-.alpha. polypeptide that is a substrate for the PHD
polypeptide.
[0267] The level of a HIF-.alpha. peptide may measured by such
methods as immunoblotting, immunoprecipitation, or other
immunological methods using, specific antibodies and methods that
are known to those skilled in the art. The amounts and the activity
of HIF-.alpha. peptides can be related to each other but are not
necessarily related to each other. Therefore in a further form of
the invention, the activity of a HIF-.alpha. peptide that is a
target for a HIF hydroxylase is assayed by measurement of
transcriptional activity or another property of the said
HIF-.alpha. polypeptide.
[0268] HIF-.alpha. polypeptides are known to form complexes with
other molecules that include other HIF subunits and co-activator
molecules such p300. In this form HIF complexes activate hypoxia
response elements that are found in the promoters and/or enhancers
of endogenous genes that are regulated by the said HIF complexes.
Such hypoxia response elements may also be isolated and
operationally linked to reporter genes so as to assay the activity
of the HIF complex through detection and/or quantitation of the
reporter gene or its product. Therefore in a further form of the
invention the activity of a HIF-.alpha. polypeptide that is
regulated by its cognate HIF hydroxylase will be assayed by
measuring the effects of the HIF complex on the expression of an
endogenous gene or reporter gene that is functionally linked to a
HIF binding hypoxia response element. Examples of endogenous genes
that are regulated in this way are to be found in the role of the
aryl hydrocarbon nuclear translocator (ARNT) in hypoxic induction
of gene expression, see for example, Studies in ARNT-deficient
cells. S. M. Wood, J. M. Gleadle, C. W. Pugh, O. Hankinson, P. J.
Ratcliffe. Journal of Biological Chemistry 271 (1996) 15117-15123,
and Hypoxia inducible expression of tumor-associated carbonic
anyhydrases, C. C. Wykoff, N. J. P. Beasley, K. J. Turner, J.
Pastorek, A. Sibtain. G. D. Wilson, H. Turley, K. Talks, P. H.
Maxwell, C. W. Pugh, P. J. Ratcliffe, A. L. Harris. Cancer Research
60 (2000) 7075-7083. Examples include but are not limited to
glucose transporter isoform 1, phosphoglycerate kinase-1, carbon
anhydrase isoform 9, vascularendothelial growth factor. Each of
said genes contains one or hypoxia response elements that may be
isolated and operationally linked as single or multiple copies to a
reporter gene for the measurement of activity of a HIF-.alpha.
polypeptide that varies in accordance with the activity of a HIF
hydroxylase.
[0269] The activity of genes or gene products that are regulated by
a HIF-.alpha. polypeptide in accordance with the activity of a HIF
hydroxylase affects cellular, organ, and animal physiology in a
manner that provide further aspects of the invention. Thus a
further embodiment of the invention provides for assays that
utilise a specific functional response that is regulated in
accordance with the activity of a HIF-.alpha. polypeptide in
accordance with the activity of a HIF hydroxylase. Such responses
include the uptake rate of glucose or glucose analogues that are
not metabolized, the ingrowth of blood vessels by angiogenesis, the
activity of a carbonic anhydrase enzyme. It is recognised that many
other responses that operate at a cellular or systemic level are
controlled by the activity of a HIF-.alpha. polypeptide in
accordance with the activity of a HIF hydroxylase and may be
utilized as assays of the said HIF hydroxylase activity in further
aspects of the invention.
[0270] A HIF-.alpha. polypeptide that is a substrate for a HIF
hydroxylase may be fused to a further polypeptide so as to cause
the activity of the said HIF hydroxylase to regulate the activity
of the fusion peptide. Accordingly a further form of the invention
provides for the assay of the activity of a fusion polypeptide. In
the preferred form such a fusion polypeptide may contain the whole
of part of a HIF-.alpha. polypeptide, for example human
HIF-1.alpha. residues 344-698, 344-417, 554-698, 652-826, or
775-826 linked to a heterologous transcription factor and expressed
together with its cognate DNA response element. The Gal4 DNA
binding domain including the amino acids 1-143 together with the
Gal binding upstream activating sequence (UAS) is an example of
such a transcription factor and cognate DNA response element whose
operation can be assayed by those skilled in the art.
[0271] In a preferred embodiment, the assays discussed herein
relate to, or utilise, human HIF hydroxylases and in particular PHD
1, 2 or 3. These may also be referred to as EGLN polypeptides.
[0272] Test Compounds
[0273] Compounds which may be screened using the assay methods
described herein may be natural or synthetic chemical compounds
used in drug screening programmes. Extracts of plants, microbes or
other organisms, which contain several characterised or
uncharacterised components may also be used.
[0274] Combinatorial library technology (including solid phase
synthesis and parallel synthesis methodologies) provides an
efficient way of testing a potentially vast number of different
substances for ability to modulate an interaction. Such libraries
and their use are known in the art, for all manner of natural
products, small molecules and peptides, among others. The use of
peptide libraries may be preferred in certain circumstances.
[0275] Potential inhibitor compounds may be polypeptides, small
molecules such as molecules from commercially available
combinatorial libraries, or the like, Small molecule compounds
which may be used include 2-oxoglutarate analogues, or HIF-.alpha.
analogues, or those that incorporate features of both
2-oxoglutarate and affect HIF-.alpha., which inhibit the action of
the enzyme. We have found in particular that compounds
dimethyl-oxalylglycine, N-oxalylglycine and N-oxalyl-2S-alanine and
certain thiols all act as inhibitors of the HIF-.alpha.
hydroxylase. N-oxalyl-2R-alanine, an enantiomer of
N-oxalyl-2S-alanine, is also an inhibitor of HIF hydroxylase and
may be used in the invention. Thus the invention provides the use
of a compound which acts as a hydroxylase inhibitor, and in
particular prolyl hydroxylase inhibitor for the manufacture of a
medicament for the treatment of a condition in a patient which
requires the promotion of cell growth, such as angiogenesis. The
invention also provides a method of treatment of a patient
suffering from a condition which is treatable by promoting cell
growth, which comprises administering to said patient an effective
amount of a HIF hydroxylase inhibitor. Such inhibitors include
dimethyl-oxalylglycine, N-oxalylglycine and N-oxalyl-2S-alanine,
and salts thereof. More generally, such inhibitors include other
N-oxalyl-amino acid compounds and salts thereof, wherein the amino
acids are either naturally occurring amino acids or synthetic amino
acids with a side chain which provides for the compound to act as
an inhibitor. Such side chains include hydrocarbyl side chains
containing a carbon chain which may be straight or branched,
optionally including one or two heteroatoms such as N, O or S, and
optionally substituted by a group such as halogen (particularly
fluoro, chloro, bromo or iodo), thiol, hydroxy, methoxy, amino,
mono- or di- C1-3 alkyl amino or nitro. Salts include
pharmaceutically acceptable salts such as sodium, potassium,
magnesium and the like.
[0276] Potential promoting agents may be screened from a wide
variety of sources, particularly from libraries of small compounds
which are commercially available. Oxygen-containing compounds may
be included in candidate compounds to be screened, for example
2-oxoglutarate analogues.
[0277] CPH Inhibitors
[0278] Inhibitors of the 2-OG dependent enzyme collagen prolyl
hydroxylase (CPH) are well known in the art and have been
previously proposed for use in the treatment of lung fibrosis, skin
fibrosis (scleroderma), atherosclerosis and other conditions
associated with collagen biosynthesis. Inhibitors of
parahydroxyphenylpyruvate oxygenase (a non-haem oxygenase employing
ferrous iron as a co-factor) such as triketones are used as
herbicides (Lee D. et al (1998) Pestic. Sci. 54(4) 377-384).
[0279] The present inventors have now found that certain of these
CPH inhibitors also inhibit the biological activity of HIF
hydroxylases and in particular the ability of the HIF hydroxylase
to catalyse prolyl hydroxylation of HIF (HPH activity).
[0280] Another aspect of the present invention therefore provides
the use of a CPH inhibitor or modified CPH inhibitor which inhibits
the biological activity of a HIF hydroxylase, and in particular its
HPH activity, in the manufacture of a medicament for use in the
treatment of a condition associated with reduced or suboptimal HIF
levels or activity, for example ischaemia, wound healing, auto-,
allo-, and xeno-transplantation, systemic high blood pressure,
cancer, and inflammatory disorders. In one embodiment a CPH
inhibitor which inhibits the biological activity of a human HIF
hydroxylase such as a PED polypeptide (EGLN polypeptide) may be
used.
[0281] CPH inhibitors which inhibit HIF hydroxylases, and in
particular the prolyl hydroxylase activity (HPH activity) of a HIF
hydroxylase, may be modified to generate selective inhibitors of
HIF hydroxylases and in particular of HPH activity. Further, the
discovery allows for the development of collagen prolyl hydroxylase
inhibitors that do not inhibit HIF hydroxylases, and in particular
HPH, by the use of comparative screening assays.
[0282] Another aspect of the present invention therefore provides
the use of a modulator of a HIF hydroxylase in the manufacture of a
medicament for the treatment of a condition associated with reduced
HIF levels or activity as described above and below.
[0283] Such an modulator may be a selective inhibitor. A selective
inhibitor is an inhibitor which shows a greater level of inhibition
of a HIF hydroxylase than on other enzymes including collagen proly
hydroxylase. In particular, a selective inhibitor is one which
inhibits HPH activity relative to CPH activity as described
above.
[0284] HPH Modulators
[0285] Various methods and uses of modulators which inhibit,
potentiate, increase or stimulate hydroxylation of HIF-.alpha. by
HIF hydroxylase are provided as further aspects of the present
invention.
[0286] The purpose of disruption, interference with or modulation
of the hydroxylation of HIF-1.alpha. by a HIF hydroxylase may be to
modulate cellular functions such as angiogenesis, erythropoiesis,
energy metabolism, inflammation, matrix metabolism, vasomotor
function, and apoptotic/proliferative responses and
pathophysiological responses to ischaemia/hypoxia, all of which are
mediated by HIF.alpha., as discussed above and further below.
[0287] A test compound which increases, potentiates, stimulates,
disrupts, reduces, interferes with or wholly or partially abolishes
hydroxylation of HIF-.alpha. polypeptide and which may thereby
modulate HIF hydroxylation activity, may be identified and/or
obtained using the assay methods described herein.
[0288] Agents which increase or potentiate hydroxylation, and in
particular prolyl hydroxylation of HIF, may be identified and/or
obtained under conditions which, in the absence of a
positively-testing agent, limit or prevent hydroxylation. Such
agents may be used to potentiate, increase, enhance or stimulate
the function of a HIF hydroxylase, and may have an effect on cells
under hypoxic conditions such as those found in tumours, in which
the lack of hydroxylation leads to the accumulation of HIF.alpha.
and the concomitant promotion of angiogenesis and other growth
promoting events.
[0289] The term `agent` includes a compound having one of the
formulae I to XXVIII as described herein in particular a compound
shown in Table 3.
[0290] Methods of determining the presence of, and optionally
quantifying the amount of HIF hydroxylase in a test sample may have
a diagnostic or prognostic purpose, e.g. in the diagnosis or
prognosis of any medical condition discussed herein (e.g. a
proliferative disorder such as cancer) or in the evaluation of a
therapy to treat such a condition.
[0291] In various aspects, the present invention provides an agent
or compound identified by a screening method of the invention to be
a modulator of HIF.alpha. hydroxylation e.g. a substance which
inhibits or reduces, increases or potentiates the hydroxylase
activity of a HIF hydroxylase.
[0292] Following identification of a modulator, the substance may
be purified and/or investigated further (e.g. modified) and/or
manufactured. A modulator may be used to obtain peptidyl or
non-peptidyl mimetics, e.g. by methods well known to those skilled
in the art and discussed herein. A modulator may be modified, for
example to increase selectively, as described herein. It may be
used in a therapeutic context as discussed below.
[0293] Agents according to the present invention, which are useful
in modulating the hydroxylation of HIF-.alpha. and therefore the
modulation of HIF's intracellular levels and hence one or more of
its cellular functions, may modulate the hydroxylase activity of
the HIF hydroxylase. Such agents may specifically inhibit the
ability of the HIF hydroxylase, and in particular of a PHD
polypeptide, to hydroxylate the appropriate residue of HIF-.alpha..
Assays and screens for such agents are provided as described above
in accordance with the present invention, along with the agents
themselves and their use in modulating the hydroxylation and
thereby the function of HIF-.alpha..
[0294] An agent able to inhibit hydroxylation of HIF-.alpha. by a
HIF hydroxylase may include a substance able to affect the
catalytic properties of the enzymatically active site of the
hydroxylase. An inhibitor of hydroxylation may interact with the
HIF hydroxylase within the active prolyl hydroxylase domain, for
example within the HXD[X].sub.nH or jelly roll motifs described
herein or in the HXE[X].sub.nH motifs. Residues within this domain
are involved in interaction with HIF-.alpha. and catalysis of the
hydroxylation. An inhibitor may, for example, interact with His358
or ARG 557 of the EGLN2/PHD1 sequence using the EGL9 numbering
system or the equivalent residue of other HIF hydroxylases, or may
interact with residues in the region between residues 369 and 389
or other residues of the jelly roll motif of the PHD1 sequence or
the equivalent residues of other HIF hydroxylases. Residues outside
of the domain may also be involved in interacting with HIF-.alpha.
and agents which interfere with such interaction may also affect
the hydroxylation as discussed elsewhere herein. Alternatively or
additionally, the inhibitor may bind in such a way as to inhibit
dioxygen binding. For example, it is appreciated that dioxygen may
approach the iron in the HIF hydroxylase from a different direction
to the HIF-.alpha. substrate and in particular through a tunnel
through the centre of the jelly roll motif. Inhibitors may bind to
residues within or at the entrance to this tunnel.
[0295] Further aspects of the present invention relate to methods
for modulating the amount of HIF polypeptide in a cell. Such a
method may comprise contacting the cell with a substance which
inhibits the hydroxylase activity of a HIF hydroxylase and in
particular its prolyl hydroxylase activity.
[0296] A suitable substance is an agent as described herein. A
substance which is a selective HIF hydroxylase inhibitor may be
used in such a method. A selective HIF hydroxylase inhibitor is an
inhibitor which inhibits the biological activity of a HIF
hydroxylase but does not inhibit biological activity of a collagen
prolyl hydroxylase, as described herein and in particular one which
inhibits HPH activity of a HIF hydroxylase but not CPH activity of
a collagen prolyl hydroxylase.
[0297] Examples of HPH Modulators
[0298] Compounds which modulate 2OG oxygenases, in particular
collagen 4-prolyl hydroxylase, may be useful as modulators of HIF
prolyl hydroxylase, or may be used as `lead` compounds which may be
modified and/or optimised to develop modulators of HIF prolyl
hydroxylase, in particular selective modulators.
[0299] Some of these compounds generally possess the formula:
R.sup.1-A*B*C*D(R.sup.2).sub.y (A)
[0300] where the group R.sup.1 is capable of forming an
electrostatic interaction with the side chain of the arginine which
together with other residues binds the 5-carboxylate of
2-oxoglutarate during catalysis, A*B is a chain of two atoms which
are, independently, carbon, oxygen, nitrogen or sulphur, which
chain can be functionalised, y is 0 or 1 and C*D is a chain of two
atoms which are, independently, carbon, oxygen, nitrogen, or
sulphur, which chain can be functionalised, A, B, C and D being,
linked to one another by single and/or double and/or triple bonds,
such that when y is 0 or 1 at least one of the atoms of which is
capable of chelating with a metal group and when y is 1 said chain
is attached to R.sup.2 which is capable of chelating with a metal
group. Generally at least one of A, B, C and D is not carbon.
Typical chains include C--N--C--C, C--C--C.dbd.O and C--O--C--C.
The chain atoms can form part of a ring, such as pyrolidine and
tetrahydro-pyran, and unsaturated derivatives thereof including
pyridine and pyran or partially hydrogenated pyrans. The rings can
be fused. When y=0 C and/or D is attached to, for example, .dbd.S,
.dbd.O, --SH or --OH. Typically R' is an acid group such as
carboxylate, --SO.sub.3H, --B(OH).sub.2 or --PO3H.sub.2. Typical
values for R.sup.2 include --SH, --OH, --CO.sub.2H, --SO.sub.3H,
--B(OH).sub.2 or --PO.sub.3H.sub.2, --NHOH, --CONHR.sup.3,
--CONHOR.sup.3, --CONR.sup.3OH and --CONR.sup.3OR.sup.3 where
R.sup.3 is a branched or straight chain alkyl group of 1 to 6
carbon atoms which can be functionalised. Preferred compounds will
have typical values for more than one group, for example for all
groups.
[0301] One class of compounds which have been found to modulate the
activity of HIF.alpha. hydroxylases and in particular their prolyl
hydroxylase activity are oxalo-amino acid derivatives, including
oxalo derivatives having one of the following general formulae (I
to IV), for example compounds having the formula V, VI or VII;
[0302] I: N-oxalo Inhibitors for In Vitro Use 2
[0303] III: N-oxalo Inhibitors for In Vivo Use 3
[0304] IV: 4
[0305] V: (N-oxalyl glycine) 5
[0306] VI: (N-oxalyl alanine) 6
[0307] such as oxalyl-L-alanine (IS70) as well as oxalyl-D-alanine
(IS71)
[0308] VII: (N-oxalyl valine) 7
[0309] where R.sup.1 and R may independently be H, a branched or
straight C.sub.1 to C.sub.6 alkyl chain, especially methyl, which
can be functionalised, e.g. as
--C.sub.2H.sub.4CO.sub.2C.sub.2H.sub.5, any natural amino acid side
chain such as alanine, valine and glutamic acid, a 4 to 7 membered
heterocyclic ring optionally containing 1 or 2 N, S, O or P atoms
or a 5 or 6 membered aromatic ring, optionally containing 1 or 2 N,
O or S atoms, such as phenyl or naphthyl which may be fused to
another ring or a said alkyl chain substituted by a said aromatic
ring;
[0310] R.sup.2 is C1 to C6 alkyl chain which may be functionalised
such that R.sup.2 is (CR.sup.1R.sup.1).sub.n where n=1 to 6 and
where the R.sup.1 groups-may be the same or different and are as
defined above or R.sup.2 is absent;
[0311] X is NH, NR", where R" is OH, a branched or straight C.sub.1
to C.sub.6 alkyl chain which can be functionalised, or O i.e. XR is
O-alkyl having a branched or straight C.sub.1 to C.sub.6 alkyl
chain, especially MeO, which can be functionalised; and,
[0312] Y is O or S.
[0313] The said alkyl groups and chains are typically
functionalised by fluorine, alcohol, thiol, a carboxylic acid,
phosphonic or phosphinic acid, sulphonic acid or other chelating
group, in the case of the chains typically via an alkyl group.
[0314] Formula I is further exemplified by compound IS3 and
oxalylglycine (IS2), Formula II is by oxaloylamino-L-alanine, and
Formula III by compounds IS1 and dimethyloxaloylglycine (MMOG) in
Table 3 as well as the methyl esters of methyloxalyl-L-alanine
(IS80) and methyloxalyl-D-alamine (IS81), methyloxalyl-L-alanine
(IS68) and methoxalyl-D-alanine (IS69), diethyl
N-methoxyoxalyl-L-glutamate (IS12), -oxalyl-L-glutamate (IS13),
oxalyl S-alanine (IS70) and oxalyl R-alanine (IS71).
[0315] These compounds may obtained by synthesis as described in
Cunliffe et al (1992) J. Med. Chem. 35 2652-2658.
[0316] The present inventors have found that N-oxalo derivatives of
amino acids, which are known to inhibit collagen prolyl-hydroxylase
(CPH), inhibit the modification of HIF-.alpha. by HIF hydroxylase
and in particular by inhibiting their prolyl hydroxylase activity
(HPH). This leads to reduced pVHL binding and increased cellular
HIF levels.
[0317] N-oxaloglycine has been found to be an inhibitor of both CPH
and HPH. However, (RS)-N-oxaloalanine is a poor inhibitor of CPH
compared to N-oxaloglycine and (S) oxaloalanine is a preferred
inhibitor of HIF hydroxylase, and in particular of HPH activity, as
described below. (S)-oxalovaline has little or no activity as an
HPH inhibitor.
[0318] Selectivity for particular HIF hydroxylase may be increased
or enhanced by modification of the oxaloglycine backbone. N-Oxalo
amino acid derivatives may be converted into methyl or ethyl ester
form for use as a `pro drug` as described in E. Baeder et al.
(1994) Biochem. J. 300.525-530 and exemplified in Table 2.
[0319] Another class of compounds of interest for use in the
modulation of HIF hydroxylases, and in particular of HPH activity,
are hydroxamic acid derivatives of the general formulae (VIII-XII):
8
[0320] where R.sup.I to R.sup.VIII may independently be H, OH, a
branched or straight C1 to C.sub.6 alkyl chain, optionally with 1,
2, 3, 4 or 5 halo substitutions, which can be functionalised, a 4
to 7 membered heterocyclic ring optionally containing 1 or 2 N, S,
O or P atoms, or a 5 or 6 membered aromatic ring, optionally
containing 1 or 2 N, O or S atoms which may be fused to another
ring or a said alkyl chain substituted by a said aromatic ring such
that R.sup.III can also be NH.sub.2 or a salt thereof such as HCl,
or NHR.sup.IX where R.sup.IX is acyl, such as unsubstituted or
substituted alkanoyl such as acetyl or phenoxyacetyl; and,
[0321] XR is OH, NH.sub.2 or NHR.sup.X, where R.sup.X is OH, a
branched or straight C.sub.1 to C.sub.6 alkyl chain, or O-alkyl
having a branched or straight C.sub.1 to C.sub.6 alkyl chain,
[0322] Preferred compounds are those where R is methyl or benzyl
and/or R.sup.II is hydrogen and/or R.sup.III is NH.sub.2 or
NHCOCH.sub.2O.sub.6H.sub.5.
[0323] Hydroxamic acids may be obtained as described in Walter M.
W. et al (1999) Bioorg. Chem. 27 (1): 35-40.
[0324] Hydroxamic acids, including cyclic and natural products such
as alahopcin (Higashide E, et al (1995) J. Antibiotics 38:
285-295), are known to be inhibitors of CPH and are therefore of
interest for the inhibition of HIF hydroxylases and in particular
of HPH. Modulators based on these compounds, which are specific for
HPH, may be developed using the methods described within.
[0325] Compounds of formula X are exemplified by benzo-hydroxamic
acid and compounds of formula XI, which are a sub-class of formula
X, are exemplified by compounds NK45, NK46, NK47 and NK84 in Table
3.
[0326] Another class of compounds of interest for use in the
modulation of HIF hydroxylases, and in particular of HPH activity,
are hydroxylated aromatic compounds including catechols,
phenanthrolines and hydroxanthroquinones, including
hydroxyanthroquinones of the formulae; 9
[0327] where R.sup.1 to R.sup.11 may independently be H, a branched
or straight C.sub.1 to C.sub.6 alkyl chain, OH, O-alkyl having a
branched or straight C.sub.1 to C.sub.6 alkyl chain optionally
containing 1 or 2 N, O or S atoms, COOH, a branched or straight
C.sub.1 to C.sub.6 alkyl ester (alkoxycarbonyl), a 4 to 7 membered
heterocyclic ring optionally containing 1 or 2 N, S, O or P atoms
or
[0328] a 5 or 6 membered aromatic ring, optionally containing 1 or
more N, O or S atoms, which can be fused to another ring, or a said
alkyl chain substituted by a said aromatic ring, and 10
[0329] Dihydroxybenzoate (EDB), as shown in Table 3, and
3,4-dihydroxybenzoic acid (protocatechuic acid) are examples of a
compound of formula XV. These compounds of formula XIV are known
CPH inhibitors (Franklin et al.(2001) Biochem. J. 353: 333-33S,
Cunliffe et al Biochem J. (1986) 239(2) 311-315, Franklin et al
(1989) Biochem. J. 261 (1) 127-130). Modulators based on these
compounds which are specific for HPH may be developed using the
methods described within. Specific compounds of formula XVA
include: 11
[0330] Thus typically R.sup.1, R.sup.3, R.sup.7, R.sup.8 and
R.sup.11 are hydrogen and R.sup.2, R.sup.4, R.sup.9 and R.sup.10
are OH. Another class of compounds of interest for use in the
modulation of HIF hydroxylases, and in particular of HPH activity
are N-containing heterocyclic compounds which have one of the
following general formulae:
[0331] XVI: 3-hydroxyquinolone-2-carboximide Derivatives 12
[0332] where R.sup.1 to R.sup.5 may be H, a branched or straight
C.sub.1 to C.sub.6 alkyl chain such as Me, a 4 to 7 membered
heterocyclic ring optionally containing 1 or more N, S, O or P
atoms, or a 5 or 6 membered aromatic ring, optionally containing 1
or more N, O or S atoms, which can be fused to another ring, or a
said alkyl chain substituted by a said chromatic (group,
A=substituted alkylene, B.dbd.CO.sub.2H, NHSO.sub.2CF.sub.3,
tetrazolyl, imidazolyl or 3-hydroxyisoxazolyl, and m is 0 or 1.
[0333] XVII: Pyridine and Pyridine N-oxide Derivatives 13
[0334] where R.sup.I to R.sup.IV may independently be H, a branched
or straight chain alkyl of from 1 to 6 C atoms, a halogen group
(i.e. fluoro-, chloro-, bromo- or iodo-), a carboxylate group, a 4
to 7 membered heterocyclic ring optionally containing 1 or more N,
S, O or P atoms, a 5 or 6 membered aromatic ring, optionally
containing 1 or more N, O or S atoms which can be fused to another
ring or a said alkyl chain substituted by a said aromatic ring, or
a C(.dbd.O)XR group as defined below,
[0335] X is O, NH, NR, where R is H, OH, a branched or straight
chain alkyl of from 1 to 6 C atoms which can be functionalised,
alkoxy containing a branched or straight chain alkyl of from 1 to 6
C atoms which can be functionalised, a 4 to 7 membered heterocyclic
ring optionally containing 1 or 2 N, S, O or P atoms, a 5 or 6
membered aromatic ring, optionally containing 1 or 2 N, O or S
atoms which can be fused to another ring, such that RX is typically
straight or branched C.sub.1 to C.sub.6 alkoxy, and m is 0 or 1.
14
[0336] where R.sup.1 is as defined in EP0114031: i.e. C.sub.1 to
C.sub.4 alkyl chain, such as C.sub.2H.sub.4, which may be
substituted with an alkoxy group with a C.sub.1 to C.sub.4 alkyl
chain such as methyl and the CONHR.sub.1 groups are typically in
the 2 and 4 positions. Thus R' is typically methoxyethyl.
[0337] Compounds of formula XVII and XVIII are exemplified by
2,5-(C8), 2,4-(C9), 2,3-(C10) and 3,4-(C11)-pyridine dicarboxylic
acids, and compounds of the formula XIX are exemplified by the
compounds IS4, IS5, IS6, IS7, IS8 and IS9 in Table 3.
[0338] Another suitable N containing heterocycle may have the
formula; XX: 15
[0339] XXI: Pyridylcarbonyl Glycines and Derivatives 16
[0340] where X.dbd.O, Y.dbd.N or CR.sub.3, m=0 or 1, A=substituted
alkylene, B.dbd.CO.sub.2H, NHSO.sub.2CF.sub.3, tetrazolyl,
imidazolyl or 3-hydroxyisoxazolyl, R1, R2 and R3 may independently
be H, OH, halo, cyano, CF.sub.3, NO.sub.2, CO.sub.2H, alkyl,
cycloalkyl, cycloalkoxy, aryl, aralkynyl, alkynylcarbonyl,
alkylcarbonyloxy, carbamoyl, alkynyloxyalkyl, alkenyloxy,
alkoxyalkoxy, alkynyl, retinyloxycarbonyl, alkenyloxycarbonyloxy,
where R.sup.1 and R.sup.2 or R.sup.2 and
R.sup.3.dbd.(CH.sub.2).sub.o in which 1-2 CH.sub.2 groups of the
saturated or C:C unsaturated chain may be replaced by O, S, SO, SO,
or imino, o=3-5, R4.dbd.H.
[0341] XXII: 3-Alkoxypyridine-2-carboxamide Derivatives 17
[0342] where A, B and R.sup.4 as defined in WO97/41103:
A=(substituted alkylene), B=(modified) carboxy, tetrazolyl,
imidazolyl, 3-hydroxyisoxazolyl, R4.dbd.H, OH, halo, cyano,
CF.sub.3, NO.sub.2, CO.sub.2H, alkyl (e.g. branched or straight
chain C1-C6 alkyl), cycloalkyl, cycloalkylalkyl, cycloalkylalkoxy,
cycloalkoxyalkyl, aryl, aralklyl, aralkoxy, hydroxyalkyl, alkenyl,
alkynyl, alkynyloxyalkyl, alkoxycarbonyl, alkylcarbonyloxy,
arylcarbonyloxy, cinnamoyl, alkenylcarbonyl, arylcarbamoyl or
aralkoxycarbonyloxy.
[0343] Various--containing heterocycles and derivatives are known
to be inhibitors of CPH. Their mechanism of action is believed to
be via bidentate chelation of the aromatic nitrogen (or its
N-oxide) and a carbonyl group located at the 2-position on the
heterocyclic ring. Suitable compounds are described in Bickel et al
Hepatology 28(2) 404-411, DE-A-1974628, EP-A-0846685, WO-A-9741103,
EP07865871, DE-A-19504226, EP-A-0673932, EP-A-0673931 and
EP-A-0673930.
[0344] Analogues of these compounds, appropriately derivatised for
pharmaceutical use, may be made selective for HIF hydroxylases, and
in particular for HPH activity, using the methods described
herein.
[0345] 2,4-diethylpyridine dicarboxylate is a known CPH inhibitor
(Friedman L. et al (2000) PNAS 97 (9) 4736-4741) which was found.
not to inhibit HPH. This provides indication that selective HIF
hydroxylase inhibitors, and in particular selective HPH inhibitors,
are possible.
[0346] Another class of compounds of interest for use in the
modulation of HIF hydroxylases, and in particular of HPH activity,
have the general formulae (XXV-XXVIII, XXVIIIA and XXVIIIB): 18
[0347] where R, R.sup.I to R.sup.VI may independently be H, a
branched or straight C.sub.1 to C.sub.6 alkyl chain, a 4 to 7
membered heterocyclic ring optionally containing 1 or 2 N, S, O or
P atoms, a 5 or 6 membered aromatic ring, optionally containing 1
or 2 N, O or S atoms, which can be fused to another ring, or a said
alkyl chain substituted by a said aromatic ring, preferably H or
methyl, R.sup.20 is hydrogen or acyl typically aromatic acyl such
as benzoyl.
[0348] X is NH, NR", where R" is OH, Me, alkyl, OMe, Oalkyl with a
C.sub.1 to C.sub.6 alkyl chain, and
[0349] Y is O or S. 19
[0350] where A*B*C*D* are as defined for formula (A), R.sup.21 s
hydrogen or acyl, typically aromatic acyl such as benzoyl, R is as
defined for formula III, n is from 1 to 5, x is from 1 to 5 and y
is from 1 to 5, such that the resulting methylene chains can be
functionalised by one or more groups as defined for R or by
NH.sub.2, such as glutathione (gamma-glutamyl-cysteinyl-glycine)
(C16) and cysteinyl-glycine (CO166).
[0351] These compounds may be obtained from commercial sources (for
example, Sigma Chemical Co.)or prepared using standard
methodology.
[0352] N-(Mercaptopropionyl)glycine and glutathione have been found
to be inhibitors of HPH. This provides indication that
appropriately functionalised thiols may be inhibitors of 2OG
dependent oxyoenases. N-(Mercaptopropionyl)glycine was not an
inhibitor of clavaminate synthase from Streptomyces clavligerus
under standard assay conditions, demonstrating that this family of
compounds can be selective for different 2OG oxygenases.
Modification of N-(mercaptopropionyl)glycine may improve
selectivity for HPH. These compounds may be made into useful
pharmaceutical agents by conversion into their ester `pro drug`
forms. These are commonly methyl or ethyl esters although others
are possible.
[0353] N-(Mercaptopropionyl)glycine and glutathione are
structurally related to
L-.delta.(.alpha.-aminoadipoyl)-L-cysteinyl-D-valine (ACV) which is
the substrate of isoepenicillin-N-synthase (IPNS). IPNS is an
oxidase which is closely related to the 2OG dependent oxygenases by
sequence and structure, although it does not use a 2OG
co-substrate. Glutathione is important in maintaining the correct
redox potential inside cells and the observation that it is an
inhibitor of HPH may have physiological relevance.
[0354] Compounds of Formula XXVIII include
N-(3-mercaptopropanoyl)-L-alani- ne (IS37) and the corresponding D
isomer (IS38) as well as N-(3-benzoylthiopropionoyl)-L-alanine
(IS20) and the corresponding D isomer (IS21). Other compounds
include: 20
[0355] where R' is H, a branched or straight C.sub.1 to C.sub.6
alkyl chain which can be functionalised, any natural amino acid
side chain for example of glutamic acid, a 4 to 7 membered
heterocyclic ring optionally containing 1 or 2 N, S, O or P atoms
or a 5 or 6 membered aromatic ring, optionally containing 1 or 2 N,
O or S atoms which may be fused to another ring or a said alkyl
chain substituted by a said aromatic ring and each of R.sup.2 to
R.sup.6, which may be the same or different, is as defined for
R.sup.1 or is NH.sub.2 or OR.sup.7 where R.sup.7 is as defined for
R' and E-represents a monocyclic ring system such as thiophene or
pyran and E' is absent or forms with E a bicyclic ring system such
as naphthalene or indole, E' typically being benzene. The ring or
rings can be functionalised at any of their carbon atoms with a
group as defined for R'. Typical compounds include
2-hydroxy-hippuric acid, N(2-hydroxy-benzoyl)-glycine (C14) and
N-benzoyl-glutamic acid (C15).
[0356] Where appropriate the acids can be in the form of salts, eg.
sodium salts.
[0357] Peptide fragments derived from the sequence of a HIF
hydroxylase or an HIF-.alpha. polypeptide form another class of
compounds which may have modulating activity. Nucleic acid encoding
such peptides, vectors and host cells containing such nucleic acid
and methods of expressing nucleic acid encoding such peptides are
further aspects of the present invention. In a preferred embodiment
such fragments are fragments of human HIF hydroxylases and in
particular of any of PHD 1, 2 or 3.
[0358] A suitable modulator may be an analogue of HIF.alpha., the
prime substrate for HIF prolyl hydroxylation by HIF hydroxylase,
and may act, for example, as a competitive inhibitor of HIF.alpha.,
or through another mechanism, such as by irreversibly modifying the
HIF hydroxylase. Uncoupled oxidation of the co-substrate 2OG may
still occur in the event of competitive inhibition of
HIF-.alpha..
[0359] A suitable fragment of an HIF.alpha. polypeptide may
comprise a proline residue which is hydroxylated by a HIF
hydroxylase in the full-length polypeptide, and which is, itself,
capable of being hydroxylated by a HIF hydroxylase, for example a
fragment containing a proline residue which corresponds to proline
residue 402 or 564 of the human HIF-1.alpha. sequence. Smaller
fragments, and analogues and variants of these fragments may
similarly be employed, e.g. as identified using techniques such as
deletion analysis or alanine scanning.
[0360] Knowledge of the HIF.alpha. sequence, in particular the
identity of the residues which are hydroxylated, therefore allows
an inhibitor to be designed with a proline analogue in the position
of hydroxylation to inhibit the hydroxylation reaction.
[0361] In particular, a modulator, such as an-inhibitor,.may
include a peptide fragment of HIF.alpha. or analogue thereof in
which the proline residue which undergoes hydroxylation, e.g.
proline 564, is replaced by a proline analogue which is not a HIF
hydroxylase substrate, such as 5-oxaproline, 3,4-dehydroproline and
4-thiaproline (Wu et al (1999) J. Am. Chem. Soc. 121(3)587-588,
DE-A-3818850). The proline inhibitor analogues may be modified such
that they also bind to the 2-oxoglutarate binding residues of the
hydroxylase.
[0362] Examples of proline analogues are; 21
[0363] where one of Y or X.dbd.O (oxaprolines) and the other is
--CR'R"- or Y.dbd.S (4-thiaprolines) where one of X and Y is
C.dbd.O or --SO.sub.2 or --P(.dbd.O)O(H) and the other is
--CR'R"--, and R is a peptide or peptide analogue and R, R.sup.I,
R.sup.II are H, a branched or straight C.sub.1 to C.sub.6 alkyl
chain, a 4 to 7 membered heterocyclic ring optionally containing 1
or more F,N, S, O or P atoms, a 5 or 6 membered aromatic ring,
optionally containing 1 or more N, O or S atoms, which can be fused
to another ring.
[0364] Peptides or peptide analogues suitable for use in accordance
with the present invention tend to be short, and may be about 40
amino acids in length or less, preferably about 35 amino acids in
length or less, more preferably about 30 amino acids in length, or
less, more preferably about 25 amino acids or less, more preferably
about 20 amino acids or less, more preferably about 15 amino acids
or less, more preferably about 10 amino acids or less, or 9, 8, 7,
6, 5 or less in length. Peptides according to the present invention
may be about 10-40 amino acids in length, about 5-10, about 10-15,
about 10-20, about 10-30, about 20-30, or about 30-40 amino acids
in length. Peptides which are HIF.alpha. fragments generally
include one or more of the relevant proline residues.
[0365] A peptide modulator which is a derivative of a peptide for
which the specific sequence is disclosed herein may be in certain
embodiments the same length or shorter than the specific peptide in
other embodiments the peptide sequence or a variant thereof may be
included in a larger peptide, as discussed above, which may or may
not include an additional portion of HIF hydroxylase or HIF-1
polypeptide. 1, 2, 3, 4 or 5 or more additional amino acids,
adjacent to the relevant specific peptide fragment of the HIF
hydroxylase or HIF-1 polypeptide, or heterologous thereto may be
included at one end or both ends of the peptide.
[0366] Peptides may be modified, for example, for use as
pharmaceuticals, for example by replacing amide bonds and/or using
D rather than L.alpha.-amino acids or .beta.-amino acids or by
using conformational restraints.
[0367] Examples of potential HIF hydroxylase inhibitors, and in
particular of HPH activity, of the classes described above are
shown in Table 3.
[0368] In the formulae described herein, a branched or straight
C.sub.1 to C.sub.6 alkyl chain may be a methyl, ethyl, propyl,
butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, neopentyl
tert-pentyl or a primary, secondary or tertiary hexyl group.
Preferably the alkyl group is methyl or ethyl while the preferred
heterocyclic rings are pyrolidine or tetrahydzopyrane and the
aromatic rings are benzene, naphthalene or pyridine.
[0369] Without being limited to any particular mechanism, analysis
of the structure of IPNS complexed to Fe(II) and ACV, together with
the structural relationship between N-(mercaptopropionyl) glycine,
2OG, and N-oxaloglycine, provides indication that the former
inhibits HIF hydroxylase and in particular HPH activity via a
complex in its thiol binds to the Fe(II) and the carboxylate to the
same residues as the S-carboxylate of 2OG.
[0370] HPH Selectivity
[0371] A number of distinct HIF hydroxylases with HIF prolyl
hydroxylase activity exist in humans and it may be also be
advantageous to modulate these selectively, as single targets, or
in selected groups as well as an entire family. Agents which
modulate HIF hydroxylase activity and in particular HIF prolyl
hydroxylase activity are therefore preferably specific i.e. they
have an increased or enhanced effect on a HIF hydroxylase relative
to other 2OG dependent oxygenases as defined below, in particular
collagen prolyl hydroxylases (CPH). Such agents may be specific for
a particular human HIF hydroxylases and in particular PHD 1, 2 or
3.
[0372] Assay methods as described herein may therefore further
comprise contacting the test compound with one or more 2OG
dependent oxygenases under conditions in which said 2OG dependent
oxygenases are normally active and determining activity of said
oxygenases
[0373] A difference in activity in the presence relative to the
absence of test compound is indicative of the test compound
modulating the activity of the one or more 2OG dependent
oxygenases.
[0374] A test compound which provides increased or enhanced
modulation of a HIF hydroxylase, relative to the one or more 2OG
dependent oxygenases shows selectivity or specificity for the HIF
hydroxylase.
[0375] 2OG dependent oxygenases may include for example, claviminte
synthase, deacetoxycephalosporin C synthase,
collagen-prolyl-4-hydroxylas- e,collagen prolyl-3-hydroxylase,
lysyl hydroxylase, aspartyl hydroxylase, phytandyl coenzyme A
hydroxylase or gamma-butyrobetaine hydroxylase. 2OG dependent
oxygenases may be mammalian, preferably human polypeptides.
[0376] The structures of various 2-OG oxygenase enzymes have been
reported; oxygenase clavaminic acid synthase (Zhang Z. et al (2000)
Nature Structural Biol. 7 127-133), deacetoxycephalosporin C
synthase (Lloyd et al (1999) J. Mol. Biol. 287 943-960),
cephalosporin synthase (Valegard K. et al (1998) Nature 394
805-809), isopenicillin N-synthase (Roach P. (1995) Nature 375
700-704) and 200 dependent oxygenases (Schofield, C. & Zhang,
Z. (1999) Curr. Opin. Struct. Biol. 9 722-731).
[0377] The assays of the invention may also be used to identify
agents which are: selective for HIF hydroxylases, and in particular
HPH activity, but not for other 2OG dependent oxygenases; agents
which are selective for HIF hydroxylase, and in particular HIF
prolyl hydroxylase activity, compared to other hydroxylases and
prolyl hydroxylases; and agents which are specific for a particular
HIF hydroxylase. The assays may be used to identify agents
selective for a particular human HIF hydroxylase, such as for
example specific for an enzyme with the amino acid sequence of SEQ
ID NO: 2, or SEQ ID NO; 4 or SEQ ID NO: 6.
[0378] The invention provides for the use of such selective
inhibitors of HIF hydroxylases in the manufacture of a medicament
for the treatment of a condition associated with reduced HIF levels
of activity.
[0379] In alternative aspects of the present invention, the assays
can be used to establish whether agents which have been identified
as inhibitors or activators of other 2OG dependent oxygenases are
specific for such oxygenases, or at least do not affect HIF
hydroxylase and in particular HIF prolyl hydroxylase activity of
the polypeptides of the present invention. In particular, the
assays may be used to establish that such agents do not affect
human HIF hydroxylases. Thus, the assays may be carried out using
agents which have been identified as inhibitors of a 2OG dependent
oxygenase, such as collagen prolyl hydroxylase to identify whether
such an agent is specific for collagen prolyl hydroxylase and is
not active or shows reduced activity against HIF hydroxylases and
in particular their prolyl hydroxylase activity.
[0380] Assay Formats
[0381] A screening or assay method may include purifying and/or
isolating a test compound, agent, or substance of interest from a
mixture or extract, i.e. reducing the content of at least one
component of the mixture or extract, e.g. a component with which
the test substance is naturally associated. The screening or assay
method may include determining the ability of one or more fractions
of a test mixture or extract to modulate the hydroxylase and
in-particular the prolyl activity of the HIF hydroxylase, and
typically these activities in relation to HIF-.alpha..
[0382] The purification and/or isolation may employ any method
known to those skilled in the art. An agent or compound obtained
and/or identified using an assay method described herein may be
modified, for example to increase selectivity for a HIF hydroxylase
relative to other 2OG dependent oxygenases such as CPH or to
increase selectivity for a particular HIF hydroxylase relative to
other HIF hydroxylases.
[0383] The approach of modifying a class of compounds containing
specific functional groups to be selective for particular enzymes
is well-known, for example the inhibition of specific serine
proteases by differently modified trifluoroketones,
chloromethylketones, beta lactams, or other generic serine protease
inhibitors (Walker B. & Lynas J. (2001) Cell. Mol. Life Sci.
58(4) 596-624, Rai R. (2001) et al Curr. Med. Chem. 8 (2) 101-119,
Marquis R. (2000) Ann. Rep. Med. Chem. 35 309-320, Lebon, F. &
Ledecq, M. (2000) Curr. Med. Chem. 7 (4) 455-477).
[0384] Selectivity for a particular HIF hydroxylase can be achieved
by performing assays as described herein with a plurality of
different HIF hydroxylases. The preferential or selective
modulation of the HIF hydroxylase activity, and in particular the
prolyl hydroxylase activity, of one or more HIF hydroxylases
relative to the other HIF hydroxylase by a test compound may
thereby be determined. Similarly, selectivity for the PHD family of
HIF hydroxylases can be achieved by performing assays as described
herein with a plurality of different 2-oxoglutarate dependent
oxygenases i.e. a panel of related enzymes. The preferential or
selective modulation of the HIF prolyl hydroxylase activity of one
or more PHD polypeptides relative to the activity of other
2-oxoglutarate dependent oxygenases by a test compound may thereby
be determined.
[0385] Structural information, including primary sequence data and
3D information such as crystallographic data, may also be used to
identify structural differences between HIF hydroxylases and other
2-oxoglutarate dependent oxygenases. These differences may be used
to design compounds which selectively or preferentially modulate
HIF hydroxylases as described herein relative to other
2-oxoglutarate dependent oxygenases.
[0386] Structural Analysis and Rational Drug Design
[0387] Secondary structural analysis predicts that the HIF
hydroxylases fold to produce a common jelly roll structure that
positions a non-haem iron co-ordinating HXD[X].sub.nH motif at the
catalytic site.
[0388] Kinetic and time resolved crystallographic studies of the
catalytic mechanism among members of this class of oxygenase have
indicated ordered binding of Iron (II), 2-oxoglutarate, and prime
substrate (Zhang et al., (2000) supra; Zhou et al (1998) J. Am.
Chem. Soc. 120 13539-13540).
[0389] Binding of the latter `primes` the enzyme for reversible
binding of dioxygen, probably by displacing a water molecule from
the iron. Weak binding at the iron centre is associated with a
cofactor requirement for iron (II). The activity of the recombinant
enzyme requires iron, and is directly inhibited by cobaltous ions
by substitution at the catalytic center. A mutation (EGLN2/PHD1;
H358A) that is predicted to abrogate iron binding, but not
otherwise alter the 3-dimensional structure was observed to
completely ablate enzyme activity.
[0390] Structural analysis and sequence studies show that SM20
comprises an iron atom at its active site in complex with residues
His313 and Asp315 (Acc No: af29245 or gi11320937). Related enzymes
clavaminate synthase (CAS) and deacetoxycephalosporin C synthase
(DAOCS) also show iron coordination at the active site as shown in
FIG. 8.
[0391] An inhibitor may form a mono-, di-, or tri-dentate complex
with the coordinate iron atom to inhibit the activity of the
enzyme. Examples of such inhibitors include the hydroxamates and
hydroxyanthroquinones described herein. The coordination of an
inhibitor to the iron atom may be determined by spectroscopic
analysis or crystallography.
[0392] As noted above, an agent may be peptidyl, e.g. a peptide
which includes a sequence as recited above, or may be a functional
analogue of such a peptide.
[0393] As used herein, the expression "functional analogue" relates
to peptide variants or organic compounds having the same functional
activity as the peptide in question, which may interfere with the
hydroxylation and in particular the prolyl hydroxylation of
HIF.alpha. by a HIF hydroxylase. Examples of such analogues include
chemical compounds which are modelled to resemble the three
dimensional structure of the substrate of the HIF hydroxylase
(HIF.alpha.) in the contact area or in the pVHL binding domain, and
in particular the arrangement of the key amino acid residues,
including proline 564.
[0394] In a further aspect, the present invention provides the use
of a HIF.alpha. polypeptide, in particular a peptide fragment which
undergoes hydroxylation by a HIF hydroxylase, in a method of
designing a peptide or non-peptidyl mimetic, which mimetic is able
to interact with the HIF hydroxylase active site and modulate the
hydroxylation of a proline residue i.e. proline 402 or 564 of
HIF.alpha., by the HIF hydroxylase.
[0395] Accordingly, the present invention provides a method of
designing, a mimetic, for example of a HIF.alpha. polypeptide,
which modulates the hydroxylation of a proline residue by a HIF
hydroxylase, said method comprising:
[0396] (i) analysing a substance to determine the amino acid
residues essential and important for biological activity to define
a pharmacophore; and,
[0397] (ii) modelling the pharmacophore to design and/or screen
candidate mimetics which modulate the hydroxylation as
described.
[0398] Suitable modelling techniques are known in the art. This
includes the study of the bonding between a HIF hydroxylase and
HIF-.alpha. and the design of compounds which contain corresponding
functional groups arranged in such a manner that they could
reproduce that bonding.
[0399] The iron atom at the HIF hydroxylase active site is normally
hexacoordinate (but can be pentacoordinate during catalysis). The
amino acid sequence provides three ligands so there are three
positions vacant for binding to inhibitors. Inhibitor molecules may
therefore be designed to coordinate with the iron atom at these
three positions.
[0400] The designing of mimetics to a known pharmaceutically active
compound is a known approach to the development of pharmaceuticals
based on a "lead" compound, for example a compound as described
herein. This might be desirable where the active compound is
difficult or expensive to synthesise or where it is unsuitable for
a particular method of administration, for instance, HIF-.alpha. or
HIF hydroxylase and in particular peptides derived from them may
not be well suited as active agents for oral compositions as they
tend to be quickly degraded by proteases in the alimentary
canal.
[0401] There are several steps commonly taken in the design of a
mimetic from a compound having a given target property. Firstly,
the particular parts of the compound that are critical and/or
important in determining the target property are determined. In the
case-of a peptide, this can be done by systematically varying the
amino acid residues in the peptide, e.g. by substituting each
residue in turn. These parts or residues constituting the active
region of the compound are known as its "pharmacophore".
[0402] Once the pharmacophore has been found, its structure is
modelled according to its physical properties, e.g.
stereochemistry, bonding, size and/or charge, using data from a
range of sources, e.g. spectroscopic techniques, X-ray diffraction
data and NMR. Computational analysis, similarity mapping (which
models the charge and/or volume of a pharmacophore, rather than the
bonding between atoms) and other techniques can be used in this
modelling process.
[0403] In a variant of the above approach, the three-dimensional
structure of a ligand and its binding partner are modelled. This
can be especially useful where the ligand and/or binding partner
change conformation on binding, allowing the model to take account
of this in the design of the mimetic.
[0404] Polypeptide Inhibitors of the Invention
[0405] The invention also provides a polypeptide of SEQ ID NO: 9
DLDLEMLAP*YIPMDDDFQL wherein P* is 4-hydroxy proline. Such peptides
may be provided in an isolated form. The invention also provides
variants of SEQ ID NO:9, as defined above. Variants will retain the
ability to antagonise the interaction of a HIF-.alpha. subunit with
VHL. This means that the presence of the variant in a cell will
lead to an increase in HIF-.alpha. subunit protein compared to the
amount of HIF-.alpha. subunit protein present in the absence of the
polypeptide.
[0406] Particular examples of such substitutions include the
following:
3 DLDLEMLAP*YISMDDDFQL; SEQ ID NO: 10 DLDLEMLLP*YIPMDDDFQL; SEQ ID
NO: 11 DLDLEMLVP*YIPMDDDFQL; SEQ ID NO: 12 DLDLEMIAP*YIPMDDDFQL;
SEQ ID NO: 13 DLDLEMIAP*YIPMEDDFQL; SEQ ID NO: 14, and
DLDLEMLVP*YISMDDDFQL. SEQ ID NO: 15
[0407] The invention also provides an isolated polypeptide which is
a variant of SEQ ID NO: 9, wherein such variants comprise from 1 to
4 amino acid substitutions of any amino acid apart from P*, said
variant retaining the ability to antagonise the interaction of a
HIF-.alpha. subunit with VHL.
[0408] A particular polypeptide of the latter type is:
[0409] PFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSP (SEQ ID NO:16);
[0410] or variants thereof as defined for SEQ ID NO:9 above;
and
[0411] polypeptides consisting of from 35 to 50 amino acids which
contain SEQ ID NO:16.
[0412] Fragments of these polypeptides which retain the P* residue,
and which are at least 6, preferably at least 10, such as at least
12 or at least 15 amino acids in length are also a further aspect
of the invention, and are also referred to herein as polypeptides
of the invention. Preferably these fragments retain the motif LXP*.
e.g. LAP*, and more preferably the fragments retain the motif
LXXLXP*, e.g. LXXLAP*. "X" means any amino acid. In particular,
such a polypeptide has or includes the sequence LAP*YIP, Thus the
invention also relates to a peptide comprising or having the
sequence LAP*YIP and having the ability to antagonise the
interaction between HIF and VHL. Such peptides may be formulated or
used as described for peptides of SEQ ID NO:9.
[0413] We have also found that a second proline residue in
HIF-1.alpha. is subject to hydroxylation. Our findings indicate
that residue 402 which shares a common motif of "LXXLAP" with the
site of hydroxylation at position 564. Our findings indicate that
this site. Polypeptides of at least 8, e.g. at least 10, at least
12, at least 15 or at least 18 amino acids, up to no more than 50,
such as no more than 35 or no more than 20 amino acids, based upon
the sequences of this region also form a further aspect of the
invention. Such polypeptides include those in which proline at 402
is hydroxylated. Substitutions, modifications, purification,
isolation and/or synthesis may be carried out as described for HIF
hydroxylases described above.
[0414] The hydroxylated peptides in accordance with the present
invention can be used in assays to monitor for agents which inhibit
the interaction between VHL and HIF. In accordance with this aspect
of the invention, a hydroxylated polypeptide as described above is
incubated with VHL or a HIF binding region thereof in the presence
of a test substance and the interaction between the hydroxylated
polypeptide and the VHL polypeptide is monitored. The polypeptides
may also be used as displacement probes in high throughput assays
for inhibitors.
[0415] These polypeptides of the present invention may be prepared
as a pharmaceutical preparation. Such preparations will comprise
the polypeptide together with suitable carriers, diluents and
excipients. Typically, they will comprise the polypeptide together
with a pharmaceutically acceptable polypeptide. Such formulations
form a further aspect of the present invention.
[0416] Formulations may be prepared suitable for any desired route
of administration, including oral, buccal, topical, intramuscular,
intravenous, subcutaneous and the like.
[0417] Formulations for topical administration to the skin may
include ingredients which enhance the permeability of the skin to
the polypeptides. Such formulations may be in the form of
ointments, creams, transdermal patches and the like.
[0418] Formulations for administration by injection (i.m., i.v.,
subcutaneous and the like) will include sterile carriers such as
physiological saline, optionally together with agents which
preserve or stabilise the polypeptide. Albumin may be a suitable
agent.
[0419] Formulations of polypeptides in particular may be used in
methods of treatment ischaemic conditions, such as organ ischaemia,
such as is manifest in coronary, cerebrovascular and peripheral
vascular insufficiency. Any ischaemia is a therapeutic target. The
therapy may be applied in two ways; following declared tissue
damage, e.g. myocardial infarction (in order to limit tissue
damage), or prophylactically to prevent ischaemia, e.g. promotion
of coronary collaterals in the treatment of angina. Additionally,
vasomotor control is subject to regulation by HIF. Activation of
HIF might affect systemic vascular resistance and hence systemic
blood pressure.
[0420] Polypeptides may also be used in combination with promoters
of angiogenesis. These include vascular endothelial growth factor
and other angiogenic growth factors such as basic fibroblast growth
factors and thymidine phosphorylase and pro-angiogenic and might be
used in combination therapy. Other compounds which might
conceivably be used in combination are 2-deoxy ribose and
prostaglandin E.
[0421] In administering polypeptides of the invention to a subject,
the doses will be determined at the discretion of the physician,
taking into account the needs of the patient and condition to be
treated. Generally, doses will be provided to achieve
concentrations at a desired site of action that are from 0.1 .mu.M
to 1 mM, for example in the 1-10 .mu.M range.
[0422] Therapeutic Applications
[0423] A compound, substance or agent which is found to have the
ability to affect the hydroxylase activity of a HIF hydroxylase,
and in particular its prolyl hydroxylase activity, has therapeutic
and other potential in a number of contexts, as discussed. For
therapeutic treatment, such a compound may be used in combination
with any other active substance, e.g. for anti-tumour therapy
another anti-tumour compound or therapy, such as radiotherapy or
chemotherapy.
[0424] An agent identified using one or more primary screens (e.g.
in a cell-free system) as having ability to modulate the HIF.alpha.
hydroxylation activity of a HIF hydroxylase may be assessed further
using one or more secondary screens. A secondary screen may involve
testing for an increase or decrease in the amount of HIF-.alpha. or
HIF activity, for example as manifest by the level of a HIF target
gene or process present in a cell in the presence of the agent
relative to the absence of the agent.
[0425] A HIF hydroxylase or a HIF polypeptide may be used in
therapies which include treatment with full length polypeptides or
fragments thereof, or otherwise modified polypeptides (e.g. to
enhance stability or ensure targeting, including in conjunction
with other active agents such as antibodies.
[0426] Generally, an agent, compound or substance which is a
modulator according to the present invention is provided in an
isolated and/or purified form, i.e. substantially pure. This may
include being in a composition where it represents at least about
90% active ingredient, more preferably at least about 95%, more
preferably at least about 98%. Any such composition may, however,
include inert carrier materials or other pharmaceutically and
physiologically acceptable excipients, such as those required for
correct delivery, release and/or stabilisation of the active agent.
As noted below, a composition according to the present invention
may include in addition to an modulator compound as disclosed, one
or more other molecules of therapeutic use, such as an anti-tumour
agent.
[0427] Products Obtained by Assays of the Invention
[0428] The invention further provides compounds obtained by assay
methods of the present invention, and compositions comprising said
compounds, such as pharmaceutical compositions wherein the compound
is in a mixture with a pharmaceutically acceptable carrier or
diluent. The carrier may be liquid, e.g. saline, ethanol, glycerol
and mixtures thereof, or solid, e.g. in the form of a tablet, or in
a semi-solid form such as a gel formulated as a depot formulation
or in a transdermally administerable vehicle, such as a transdermal
patch.
[0429] The invention further provides a method of treatment which
includes administering to a patient an agent which interferes with
the hydroxylation of the target residue of an HIF.alpha.
polypeptide by a HIF hydroxylase. Such agents may include
inhibitors of hydroxylase activity, typically of prolyl hydroxylase
activity and in particular these activities in relation to HIF.
Examples of inhibitors of HIF prolyl hydroxylase activity include,
for example compounds of structures I to XXVIII as described
herein. Exemplary purposes of such treatment are discussed
elsewhere herein.
[0430] The invention further provides various therapeutic methods
and uses of one or more substances selected from (i) a HIF
hydroxylase which is able to bind to HIF-1; (ii) a modulator
identified by a screening method of the present invention; (iii) a
mimetic of any of the above substances which can bind to HIF-I or a
HIF hydroxylase, or the polypeptide inhibitors of the
invention.
[0431] The therapeutic/prophylactic purpose of such a method or use
may be the modulation of the level of HIF.alpha. in a cell by
modulation, e.g. disruption or interference, of the hydroxylation
of HIF.alpha., which may occur for example at proline 402, 564 or
other proline residue. Hydroxylation of HIF.alpha. promotes pVHL
binding which leads to ubiquitin dependent proteolysis of
HIF.alpha. as described above.
[0432] The therapeutic/prophylactic purpose may be related to the
treatment of a condition associated with reduced or suboptimal or
increased HIF levels or activity, or conditions in which have
normal HIF levels, but where an modulation in HIF levels such as an
increase or decrease in HIF levels is desirable such as:
[0433] (i) ischaemic conditions, for example organ ischaemia,
including coronary, cerebrovascular and peripheral vascular
insufficiency. The therapy may be applied in two ways; following
declared tissue damage, e.g. myocardial infarction (in order to
limit tissue damage), or prophylactically to prevent ischaemia, e.g
promotion of coronary collaterals in the treatment of angina.
[0434] (ii) wound healing and organ regeneration
[0435] (iii) auto-, allo-, and xeno-transplantation.
[0436] (iv) systemic blood pressure
[0437] (v) cancer; HIF.alpha. is commonly up-regulated in tumour
cells and has major effects on tumour growth and angiogenesis.
[0438] (vi) inflammatory disorders.
[0439] (vii) pulmonary arterial blood pressure, neurodegenerative
disease.
[0440] Pharmaceutical Compositions
[0441] In various further aspects, the present invention thus
provides a pharmaceutical composition, medicament, drug or other
composition for such a purpose, the composition comprising one or
more agents, compounds or substances as described herein, including
HIF hydroxylase inhibitors and in particular inhibitors of their
HIF prolyl hydroxylase (HPH) activity such as compounds of formulae
I to XXVIII, the use of such an composition in a method of medical
treatment, a method comprising administration of such a composition
to a patient, e.g. for treatment(which may include preventative
treatment) of a medical condition as described above, use of such
an agent compound or substance in the manufacture of a composition,
medicament or drug for administration for any such purpose, e.g.
for treatment of a condition as described herein, and a method of
making a pharmaceutical composition comprising admixing such an
agent, compound or substance with a pharmaceutically acceptable
excipient, vehicle or carrier, and optionally other
ingredients.
[0442] In one embodiment the method for providing a pharmaceutical
composition may typically comprise:
[0443] (a) identifying an agent by an assay method of the
invention; and
[0444] (b) formulating the agent thus identified with a
pharmaceutically acceptable excipient.
[0445] The pharmaceutical compositions of the invention may
comprise an agent, polypeptide, polynucleotide, vector or antibody
according to the invention and a pharmaceutically acceptable
excipient.
[0446] The agent may be used as sole active agent or in combination
with one another or with any-other active substance, e.g. for
anti-tumour therapy another anti-tumour compound or therapy, such
as radiotherapy or chemotherapy.
[0447] Whatever the agent used in a method of medical treatment of
the present invention, administration is preferably in a
"prophylactically effective amount" or a "therapeutically effective
amount" (as the case may be, although prophylaxis may be considered
therapy), this being sufficient to show benefit to the individual.
The actual amount administered, and rate and time-course of
administration, will depend on the nature and severity of what is
being treated. Prescription of treatment, e.g. decisions on dosage
etc, is within the responsibility of general practitioners and
other medical doctors.
[0448] An agent or composition may be administered alone or in
combination with other treatments, either simultaneously or
sequentially dependent upon the condition to be treated, e.g. as
described above.
[0449] Pharmaceutical compositions according to the present
invention, and for use in accordance with the present invention,
may include, in addition to active ingredient, a pharmaceutically
acceptable excipient, carrier, buffer, stabiliser or other
materials well known to those skilled in the art. In particular
they may include a pharmaceutically acceptable excipient. Such
materials should be non-toxic and should not interfere with the
efficacy of the active ingredient. The precise nature of the
carrier or other material will depend on the route of
administration, which may be oral, or by injection, e.g. cutaneous,
subcutaneous or intravenous.
[0450] Pharmaceutical compositions for oral administration may be
in tablet, capsule, powder or liquid form. A tablet may include a
solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical
compositions generally include a liquid carrier such as water,
petroleum, animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included.
[0451] For intravenous, cutaneous or subcutaneous injection, or
injection at the site of affliction, the active ingredient will be
in the form of a parenterally acceptable aqueous solution which is
pyrogen-free and has suitable pH, isotonicity and stability. Those
of relevant skill in the art are well able to prepare suitable
solutions using, for example, isotonic vehicles such as Sodium
Chloride Injection, Ringer's Injection, Lactated Ringer's
Injection. Preservatives, stabilisers, buffers, antioxidants and/or
other additives may be included, as required.
[0452] Liposomes, particularly cationic liposomes, may be used in
carrier formulations. Examples of techniques and protocols
mentioned above can be found in Remington's Pharmaceutical
Sciences, 16th edition, Osol, A. (ed), 1980.
[0453] The substance or composition may be administered in a
localised manner to a particular site or may be delivered in a
manner in which it targets particular cells or tissues, for example
using intra-arterial stent based delivery.
[0454] Targeting therapies may be used to deliver the active
substance more specifically to certain types of cell, by the use of
targeting systems such as antibody or cell specific ligands.
Targeting may be desirable for a variety of reasons, for example if
the agent is unacceptably toxic, or if it would otherwise require
too high a dosage, or if it would not otherwise be able to enter
the target cells.
[0455] In a further embodiment the invention provides for the use
of an agent of the invention in the manufacture of a medicament for
the treatment of a condition associated with increased or decreased
HIF levels or activity. The condition may, for example, be selected
from the group consisting of ischaemia, wound healing, auto-,
allo-, and xeno-transplantation, systemic high blood pressure,
cancer, and inflammatory disorders.
[0456] Gene Therapy
[0457] The HIF hydroxylases of the present invention can be used to
promote or enhance hydroxylation of HIF-.alpha. in target cells.
Such promotion of hydroxylation may therefor facilitate
ubiquitilation and subsequent destruction of HIF-.alpha. and thus
reduce accumulation of HIF-.alpha. in cells. This will be of
assistance in reducing angeogenesis and effect other apoptotic and
proliferative responses in target cells. Thus, in accordance with
this aspect of the invention a nucleic acid encoding a HIF
hydroxylase may be provided to target cells in need thereof.
[0458] Where the substances are peptides or polypeptides, they may
be produced in the target cells by expression from an encoding
nucleic acid introduced into the cells, e.g. from a viral vector.
The vector may be targeted to the specific cells to be treated, or
it may contain regulatory elements which are switched on more or
less selectively by the target cells.
[0459] Nucleic acid encoding a substance e.g. a peptide able to
modulate, e.g. interfere with, prolyl hydroxylation of HIF.alpha.
by a HIF hydroxylase, may be used in methods of gene therapy, for
instance in treatment of individuals, e.g. with the aim of
preventing or curing (wholly or partially) a disorder.
[0460] Nucleic acid encoding a HIF hydroxylase as described herein
may also be used in the anti-sense regulation of the HIF
hydroxylase activity and in particular, of the HIF prolyl
hydroxylase activity within a cell.
[0461] Down-regulation of expression of a gene encoding a HIF
hydroxylase may be achieved using anti-sense technology, or RNA
interference.
[0462] In using anti-sense genes or partial gene sequences to
down-regulate gene expression, a nucleotide sequence is placed
under the control of a promoter in a "reverse orientation" such
that transcription yields RNA which is complementary to normal mRNA
transcribed from the "sense" strand of the target gene. See, for
example, Smith et al,(1988) Nature 334, 724-726;. Antisense
technology is also reviewed in Flavell, (1994) PNAS USA 91,
3490-3496.
[0463] The complete sequence corresponding to the reverse
orientation of the coding sequence need not be used. For example,
fragments of sufficient length may be used. It is a routine matter
for the person skilled in the art to screen fragments of various
sizes and from various parts of the coding sequence to optimise the
level of anti-sense inhibition. It may-be advantageous to include
the initiating methionine ATG codon, and perhaps one or more
nucleotides upstream of the initiating codon. A further possibility
is to target a conserved sequence of a gene, e.g. a sequence that
is characteristic of one or more genes, such as a regulatory
sequence.
[0464] The sequence employed maybe 500 nucleotides or less,
possibly about 400 nucleotides, about 300 nucleotides, about 200
nucleotides, or about 100 nucleotides. It may be possible to use
oligonucleotides of much shorter lengths, 14-23 nucleotides,
although longer fragments, and generally even longer than 500
nucleotides are preferable where possible.
[0465] Anti-sense oligonucleotides may be designed to hybridise to
the complementary sequence of nucleic acid, pre-mRNA or mature
mRNA, interfering with the production of a HIF hydroxylase encoded
by a given DNA sequence (e.g. either native polypeptide or a mutant
form thereof), so that its expression is reduce or prevented
altogether. Anti-sense techniques may be used to target a coding
sequence, a control sequence of a gene, e.g. in the 5' flanking
sequence, whereby the anti-sense oligonucleotides can interfere
with control sequences. Anti-sense oligonucleotides may be DNA or
RNA and may be of around 14-23 nucleotides, particularly around
15-18 nucleotides, in length. The construction of antisense
sequences and their use is described in Peyman and Ulman, Chemical
Reviews, 90:543-584, (1990), and Crooke, Ann. Rev. Pharmacol.
Toxicol., 32:329-376, (1992).
[0466] It may be preferable that there is complete sequence
identity in the sequence used for down-regulation of expression of
a target sequence, and the target sequence, though total
complementarity or similarity of sequence is not essential. One or
more nucleotides may differ in the sequence used from the target
gene. Thus, a sequence employed in a down-regulation of gene
expression in accordance with the present invention may be a
wild-type sequence (e.g. gene) selected from those available, or a
mutant, derivative, variant or allele, by way of insertion,
addition, deletion or substitution of one or more nucleotides, of
such a sequence.
[0467] The sequence need not include an open reading frame or
specify an RNA that would be translatable. It may be preferred for
there to be sufficient homology for the respective sense RNA
molecules to hybridise. There may be down regulation of gene
expression even where there is about 5%, 10%, 15% or 20% or more
mismatch between the sequence used and the target gene.
[0468] Other approaches to specific down-regLulation of genes which
may be used to modulate HIF hydroxylase expression are well known,
including the use of ribozymes designed to cleave specific nucleic
acid sequences. Ribozymes are nucleic acid molecules, actually RNA,
which specifically cleave single-stranded RNA, such as mRNA, at
defined sequences, and their specificity can be engineered.
Hammerhead ribozymes may be preferred because they recognise base
sequences of about 11-18 bases in length, and so have greater
specificity than ribozymes of the Tetrahymena type which recognise
sequences of about 4 bases in length, though the latter type of
ribozymes are useful in certain circumstances. References on the
use of ribozymes include Marschall, et al. Cellular and Molecular
Neurobiology, 1994. 14(5): 523; Hasselhoff, Nature 334: 585 (1988)
and Cech, J. Amer. Med. Assn., 260: 3030 (1988).
[0469] Vectors such as viral vectors have been used in the prior
art to introduce nucleic acid into a wide variety of different
target cells. Typically the vectors are exposed to the target cells
so that transfection can take place in a sufficient proportion of
the cells to provide a useful therapeutic or prophylactic effect
from the expression of the desired peptide. The transfected nucleic
acid may be permanently incorporated into the genome of each of the
targeted cells, providing long lasting effect, or alternatively the
treatment may have to be repeated periodically.
[0470] A variety of vectors, both viral vectors and plasmid
vectors, are known in the art, see U.S. Pat. No. 5,252,479 and
WO93/07282. In particular, a number of viruses have been used as
gene transfer vectors, including papovaviruses, such as SV40,
vaccinia virus, herpesviruses, including HSV and EBV, and
retroviruses. Many gene therapy protocols in the prior art have
used disabled murine retroviruses.
[0471] As an alternative to the use of viral vectors in gene
therapy other known methods of introducing nucleic acid into cells
includes mechanical techniques such as microinjection, transfer
mediated by liposomes and receptor-mediated DNA transfer.
[0472] Receptor-mediated gene transfer, in which- the nucleic acid
is linked to a protein ligand via polylysine, with the ligand being
specific for a receptor present on the surface of the target cells,
is an example of a technique for specifically targeting nucleic
acid to particular cells.
[0473] In various further aspects, the present invention thus
provides a pharmaceutical composition, medicament, drug or other
composition for use in a method of treating a medical condition
described above, the composition comprising an isolated nucleic
acid molecule as described herein, the use of such an composition
in a method of medical treatment, a method comprising
administration of such a composition to a patient, e.g. for
treatment (which may include preventative treatment) of a medical
condition as described above, use of such an agent compound or
substance in the manufacture of a composition, medicament or drug
for administration for any such purpose, e.g. for treatment of a
condition as described herein, and a method of making a
pharmaceutical composition comprising admixing such an agent,
compound or substance with a pharmaceutically acceptable excipient,
vehicle or carrier, and optionally other ingredients.
[0474] A peptide or other substance having an ability to modulate
or interfere with the prolyl hydroxylation of the residue of
HIF-.alpha. by a polypeptide,or a nucleic acid molecule which
encodes a peptide having that ability, may be provided in a kit,
e.g. scaled in a suitable container which protects its contents
from the external environment. Such a kit may include instructions
for use.
[0475] Use of Polypeptides
[0476] Another aspect of the present invention provides the use of
a HIF hydroxylase as described herein or a fragment thereof for the
hydroxylation, and in particular the prolyl hydroxylation, of an
HIF polypeptide, or a proline-containing substrate of HIF
hydroxylation.
[0477] Another aspect of the present invention provides a method of
producing a HIF hydroxylase comprising:
[0478] (a) causing expression from nucleic acid which encodes a HIF
hydroxylase in a suitable expression system to produce the
polypeptide recombinantly;
[0479] (b) determining the prolyl hydroxylation of an HIF.alpha.
polypeptide by said recombinantly produced polypeptide. The
polypeptide expressed by the method may be a PHD (EGLN)
polypeptide.
[0480] Suitable expression systems are well-known in the art. HIF
hydroxylases may be expressed in a prokaryote, such as E. coli,
lower eukaryote such as S. cerevisiae or a higher eukaryotic cell,
such as a mammalian cell e.g. a CHO or COS cell.
[0481] Prolyl hydroxylation of an HIF.alpha. polypeptide, in
particular within the pVHL binding domain, such as residue 402 or
564, may be determined as described herein.
[0482] Another aspect of the present invention provides an assay
method for identifying/obtaining a HIF hydroxylase, and in
particular HIF.alpha. prolyl hydroxylase, comprising,
[0483] (b) bringing into contact an HIF.alpha. polypeptide and the
test polypeptide under conditions in which the HIF.alpha.
polypeptide is hydroxylated by a HIF hydroxylase; and
[0484] (c) determining hydroxylation and in particular the prolyl
hydroxylation of the HIF.alpha. polypeptide.
[0485] A HIF hydroxylase polypeptide according to the present
invention can also be used to identify additional substrates of HIF
hydroxylases. For example, peptides which have either previously
been demonstrated to be hydroxylated by other hydroxylases, or
other peptides may be brought into contact with a HIF hydroxylase
according to the present invention and monitoring for hydroxylation
of such peptides. Any suitable conditions may be selected including
the provision of agents and co-factors known to enhance
hydroxylation by the hydroxylases of the present invention. In a
preferred aspect, prolyl containing substrates are contacted with a
HIF hydroxylase of the present invention, and hydroxylation of the
prolyl residue is monitored. Hydroxylation of the substrate may be
monitored by any suitable method including monitoring levels of
co-factors or by products of hydroxylation.
[0486] The invention also provides a method of modulating the
amount of HIF polypeptide in a cell comprising contacting the cell
with a substance which inhibits the 4-prolyl hydroxylase activity
of a HIF hydroxylase such as, for example, an EGLN polypeptide. The
substance may, for example, be an agent of the invention. In a
preferred embodiment, the substance inhibits the biological
activity of HIF hydroxylase, such as, for example an EGLN
polypeptide, but does not inhibit biological activity of a collagen
prolyl hydroxylase.
[0487] Various further aspects and embodiments of the present
invention will be apparent to those skilled in the art in view of
the present disclosure.
[0488] Certain aspects and embodiments of the invention will now be
illustrated by way of example and with reference to the figures
described below.
FIGURES
[0489] FIG. 1. Analysis of the minimal pVHL binding domain of
HIF-1.alpha.. A. Left panel; sequence alignment of the minimal pVHL
binding domain from HIF-1.alpha. and HIF-2.alpha., with HIF-.alpha.
genes from other organisms. B. Summary of the ability of synthetic
polypeptides to block the HIF-1.alpha./pVHL interaction before and
after exposure to reticulocyte lysate supplemented with Fe(II).
Treated and untreated polypeptides were added to a mixture of
HIF-1.alpha. and pVHL.HA IVTTs that was then assayed for
interaction by anti-HA immunoprecipitation. Substituted residues
are underlined. Y* denotes phosphotyrosine.
[0490] FIG. 2. Amino acid sequences of human HIF-1.alpha. and C.
elegans HIF. Identical amino acids are boxed in black. The PAS
domains are indicated. The VHL minimal binding region defined in
studies of human HIF-1.alpha. is indicated.
[0491] FIG. 3A Line up of the indicated amino acids of the
indicated HIF alpha chains showing conservation of the LxxLAP motif
in all three domains demonstrated to be involved in VHL dependent
ubiquitylation.
[0492] FIG. 3B U20S cells were transiently co-transfected with
plasmids encoding the indicated Gal-Hif-1 alpha-VP16 sequences in
combination with pUAS-tk-luc (a Gal 4 upstream activating sequence
dependent luciferase reporter gene plasmid). Luciferase activities
were determined in extracts made from transfected cells maintained
for 48 hours, either entirely in normoxia (white bars) or with
hypoxic stimulation for the last 16 hours. (black bars). Low
normoxic activity (explicable by rapid destruction of the fusion
protein) is seen when the Gal-Hif-VP16 fusion protein contains wild
type Hif-1 alpha amino acids 344-417 but not when the sequence
bears the P402A mutation or is truncated to amino acid 400.
[0493] FIG. 4 The CHO mutant cell line Kal3 (deficient in Hif alpha
subunit activity) was co-transfected with pcDNA3 expression
plasmids encoding wild type full length Hif-1 alpha (HIF-1), full
length Hif-1 alpha bearing the P402A mutation (P402A), full length
Hif-1 alpha bearing the P564G mutation (P564G) or full length Hif-1
alpha bearing the double mutation (P402A+P564G) in combination with
a hypoxia response element dependent luciferase reporter gene
construct. Luciferase activities were determined in extracts made
from transfected cells maintained for 48 hours, either entirely in
normoxia (white bars) or with hypoxic stimulation for the last 16
hours (black bars). The individual mutations showed showed slightly
enhanced normoxic activity compared with the wild type sequence but
the combined mutant showed constitutive activity in normoxia with
no further induction by hypoxia.
[0494] FIG. 5 shows a schematic of the on-bead modification assay
used to assay for HIF prolyl hydroxylase activity,
[0495] FIG. 6 shows the effect of dimethyl oxalyl glycine on HIF
activity in Hep3B and U2OS cell lines.
[0496] FIG. 7 shows the effects of forced expression of EGLN2 (PHD
1) or a naturally occurring splice variant lacking amino acids
369-389 (PHD4) on the action of HIF in cells incubated in
atmospheres containing 20% or 2% oxygen.
[0497] FIG. 8 shows views derived from the active sites of
clavaminate synthase and deacetoxycephalosporin C synthase.
[0498] FIG. 9 shows sequence alignments of the predicted jelly roll
cores of HIF-PHs. Sequences shown are C elegans EGL-9 (462-550),
Human EGLN1(288-403), Human EGLN2 (462-580), Human EGLN3 (111-225),
rat SM20 (226-341). Also shown is the Streptomyces sp.
Prolyl-3-hydroxylase (P30H). The residues of the 2-His-1-Asp motif
are indicated by arrows, as is the arginine proposed to bind the
5-carboxylate of 2-oxoglutarate (Mukherji et al (2001) Chem. Comm.
11 972-973).
[0499] FIG. 10 shows a topographical diagram of the conserved jelly
roll core (strands 1 to 8 ) of 2-oxoglutarate dependent, showing
the approximate location of the conserved 2-histidine-1 carboxylate
iron binding ligands and 2 oxogiutarate binding basic residue (Arg
557) used to identify candidate HIF-PHs. Numbering refers to the
positions in the EGL-9 sequence.
[0500] FIG. 11 shows an HPLC elution profile of absorbance at 218
nm of the hydroxylation of a synthetic peptide by purified PHD1.
Synthetic HIF-1.alpha. (B19Pro) peptide was treated with MBP/PHD1
in the presence or absence of 2-oxoglutarate and the products
analysed by HPLC as described. Top curve shows results of
incubation in the absence of iron(II), upper middle curve shows
incubation in the absence of .alpha.KG, lower middle shows
incubation with all cofactors present. Positions of peaks
corresponding to hydroxylated and unhydroxylated B19Pro standards
are shown in the bottom panel. Appearance of hydroxlated peptide
coincides with the ability to interact with pVHL.
[0501] FIG. 12 shows an HPLC analysis of the hydroxylation of
proline 621 of HIF-1 by EGL-9. E. coli were co-transformed with a
plasmid expressing either His.sub.6GalHIF-1(590-713) or a mutant
(P621G) derivative and plasmids expressing MBP/.DELTA..EGL-9 or MBP
alone. Retrieved His.sub.6GalHIF-1 was analysed by HPLC for
4-hydroxyproline as shown.
[0502] FIG. 13 shows NODD and CODD expression plasmids enhance HRE
reporter gene activity. (a) Proposed model for peptide effects on
HIF.alpha.-VHL interaction. Degradation is prevented by the NODD
and CODD polypeptides competing for prolyl hydroxylation and/or VHL
binding, thereby blocking subsequent ubiquitination. (b)
Transfections of the N-terminal or C-terminal ODD (HIF-1.alpha.
aa343-41 7 or aa549-82) led to increased HRE-dependent luciferase
activity comparable to hypoxic levels. In contrast, no induction
was seen following transfections with corresponding sequences
bearing P402A or P564G mutations. RLU: Relative Light Units. (c)
Use of a CHO cell line, lacking HIF.alpha. chains (KA-13), or its
stable HIF-1.alpha. transfectant (KH-1) shows dependence of the
observed HRE response on HIF-1.alpha. expression. (d) and (e),
Amino acids 390-410 for the NODD and amino acids 556-72 for the
CODD were the shortest active domains defined. (f), Sequence
alignment of human and mouse HIP-1.alpha. NODD and CODD
domains.
[0503] FIGS. 14 A and B show dose-response curves for oxalyglycine
as well as oxalyl R and S-alanine, FIGS. 15 to 20 show the relative
HIP-PH activity for a variety of inhibitors.
EXAMPLES
Example 1
Oxygen Regulated Modification by Enzymatic Prolyl Hydroxylation
Targets HIF-.alpha. to the Von Hippel-Lindau Ubiquitylation
Complex
[0504] In this example it is shown that the interaction between
pVHL and a specific domain of the HIF-1.alpha. subunit is regulated
by enzymatic hydroxylation of a proline residue (HIF-1.alpha. P564)
in a manner that is dependent on oxygen and iron. An absolute
requirement of the enzyme for dioxygen as a co-substrate and iron
as a co-factor suggests a direct mechanism of cellular oxygen
sensing.
[0505] In previous studies of the HIF-.alpha./pVHL interaction we
found that treatment of cells with cobaltous ions and iron
chelators prevented the HIF-.alpha./pVHL association suggesting
that the oxygen sensing mechanism might impinge directly on this
protein interaction (8). Surprisingly, these studies indicated that
the HIF-.alpha./pVHL complex could be retrieved intact from hypoxic
cells. Given the rapidity of pVHL dependent proteolysis of
HIF-.alpha. in oxygenated cells, we considered that re-oxygenation
of cell extracts during cell lysis might have promoted the observed
HIF-.alpha./pVHL interaction in vitro. To test this we repeated
pVHL co-immunoprecipitation experiments in extracts of
.sup.35S-methionine/cysteine labelled cells exposed to hypoxia and
harvested in a hypoxia work station using deoxygenated buffers, or
exposed to hypoxia and hanrested conventionally (18).
[0506] Experiments were performed on stably transfected renal
carcinoma cells expressing haemagglutinin (HA) tagged VHL
(RCC4/VHL.HA)(11)). RCC4 cells, which lack pVHL, were used as a
control. RCC4/VHL.HA cells were labelled with .sup.35S-amino acids
in the presence of the proteasomal inhibitor MG132, either in
normoxia or hypoxia for 4 hrs. Cells were lysed on ice, either in
the hypoxic workstation or on the bench. RCC4 cells were also
similarly labelled and lysed and both labelling and lysis were
carried out under normoxic conditions. pVHL and associated proteins
were captured with anti-HA antibody. As reported previously(8),
anti-HA immunoprecipitates captured HIF-.alpha. subunits
(HIF-1.alpha. and HIF-2.alpha.) efficiently from the proteasomally
blocked normoxic cells. However, when hypoxic RCC4/VHL.HA cells
were lysed under hypoxic conditions, HIF-.alpha. subunits were not
co-precipitated with pVHL, despite abundance in the lysate as
demonstrated by a HIF-1.alpha. immunoblot. This contrasted with the
result using conventional buffers, which had not been deoxygenated,
where HIF-.alpha. subunits were captured very efficiently. Capture
of pVHL and elongins B&C was found to be similar in all
RCC4VHL.HA samples.
[0507] Taken together with previously published data, these results
indicate that the classical features of regulation by oxygen and
iron availability (and interference by cobaltous ions) are
reflected in the HIF-.alpha./pVHL interaction in vitro, and that
promotion of the interaction mediated by oxygen can occur rapidly
during the preparation of a cell extract.
[0508] 1.1: Oxygen Sensitivity of the HIF-.alpha./pVHL
Interaction
[0509] .sup.35S-Methionine labelled HIF-1.alpha. subunits and
pVHL.HA were produced separately by IVTT (in vitro transcription
and translation) in reticulocyte lysates. IVTTs were performed
under different conditions, mixed, and assayed for interaction by
anti-HA co-immunoprecipitation. These in vitro assays allowed
analysis of the HIF.alpha./pVHL interaction using the recombinant
proteins.
[0510] Labelled HIF-1.alpha. and pVHL.HA were generated separately
in reticulocyte lysates (IVTT), in the presence or absence of Co
(II), desferrioxamine, or Fe(II). Lysates were mixed in various
combinations, and interactions assayed by anti-HA
immunoprecipitation. We found that supplementary Fe(II) (ferrous
chloride, 100 .mu.M) in the HIF-1.alpha. IVTT greatly enhanced
capture by pVHL.HA, whereas addition of Co(II) (cobaltous chloride,
100 .mu.M) or desferrioxamine, 100 .mu.M (DFO) to the HIF-1.alpha.
IVTT greatly diminished capture. In contrast, pVHL IVTTs performed
under these different conditions were all equally effective in
supporting the HIF-1.alpha. interaction.
[0511] Further experiments were carried out to determine the effect
of producing the IVTTs under hypoxic conditions. Labelled
HIF-1.alpha. was generated in IVTT reactions in the presence or
absence of Fe(II) either under ambient conditions or in a hypoxic
workstation. Samples were then diluted in buffer in the hypoxic
workstation and purified recombinant GST-VHL-elonginC-elonginB was
added. VHL and associated proteins were captured using
glutathione-agarose. The results showed that when HIF-1.alpha.
IVTTs were prepared under hypoxic conditions, hypoxia reduced their
ability to interact with pVHL, irrespective of whether the latter
was produced either as a normoxic or hypoxic IVTT, or as a
bacterially expressed complex of pVHL and elongins B and C.
[0512] Next, we used reticulocyte IVTTs of Gal4/VP16 fusion
proteins bearing specific HIF subsequences to show that the
regulated interaction with pVHL was supported even by a minimal
pVHL binding HIF-1.alpha. subsequence comprising residues 556-574.
Fusions bearing amino acid residues 556-574, 549-574, 556-582 or
549-582 of HIF-1.alpha. were expressed in reticulocyte lysates with
or without added Fe(II). The fusion proteins included the
HIF-1.alpha. sequence between a Gal4 DNA binding domain -and VP16
transactivation domain. As a control a fusion containing no
HIF-1.alpha. sequences was also assessed; Aliquots were assessed
for co-immunoprecipitation with pVHL.HA by anti-HA
immunoprecipitation. All of the fusions bearing HIF-1.alpha.
subsequences displayed iron-dependent recognition by pVHL including
the fusion comprising the shortest region of HIF-1.alpha.
subsequence tested comprising residues 556-574. The control fusion,
lacking HIF-1.alpha. sequences, did not recognise pVHL either in
the presence or absence of iron.
[0513] To better understand these findings we surveyed the ability
of a series of recombinant pVHL and HIF-1.alpha. products produced
in different prokaryotic and eukaryotic expression systems (20) to
interact. All pVHL products could interact with HIF-1.alpha. that
was derived from mammalian expression systems. In contrast,
HIF-1.alpha. could interact only if produced in vivo in tissue
culture cells, or in reticulocyte IVTT, and not if produced in
bacteria, wheat germ lysates, or insect cells. Together, these
results indicate that a factor in mammalian cell extracts was
necessary to promote the interaction with specific HIF-1.alpha.
sequences and that this factor operated in an iron and oxygen
dependent manner.
[0514] 1.2: Modifying Activity which Promotes Interaction of HIF
and VHL
[0515] To analyse this further we immunopurified a
Gal/HIF-1.alpha./VP16 fusion protein expressing HIF-1.alpha.
residues 549-582, from IVTT reactions prepared in the presence of
DFO, using anti-Gal antibodies. The unlabelled HIF-1.alpha.
substrate was immunopurified on beads, washed, and aliquots
incubated under different test conditions in buffer or cell
extract. After further washing, the beads were assayed for ability
to interact with 35-S labelled pVHL IVTT (21) which was then
visualised by fluorography. Increased ability to capture pVHL was
seen after exposure of die HIF fusion protein to cell extract in
the presence off(II) but not after exposure to Fe(II) without cell
extract. The increased ability to capture pVHL after exposure to
cell extract and Fe(II) was also found to be oxygen dependent. In
analogous experiments it was found that the modifying activity was
present in extracts prepared from a variety of mammalian cells,
(Hela, RCC, CHO-K1 and rabbit reticulocyte lysate), but that insect
cell lysates were essentially inactive on the mammalian HIF fusion
protein. The Fe(II) dependent activity of the cell extract was
reduced by cooling and was abrogated by pre-heating at 60.degree.
C. for 10 minutes. The modifying activity did not pass through a 5
kDa ultrafilter. Titration of Fe(II) supplementation indicated full
activation at 5 .mu.M. Pre-incubation of the cell extract with
hexokinase (50 U/ml) and glucose (50 mM) to deplete ATP did not
alter activity, though this treatment abrogated the ability of the
cell extracts to phosphorylate a control target. Pretreatment of
extracts with clotrimazole (10 .mu.M), methyl-viologen (1 mM), or
NADase (20 mU/ml), did not significantly affect activity
[0516] Experiments were also performed using PK epitope tagged
HIF-1.alpha. (PK.HIF) expressed in insect cells as a HIF substrate.
Both RCC4 cell extract, and reticulocyte lysate, in the presence of
Fe(II) promoted the ability of HIF to capture wild type but not
mutant (Pro86His) pVHL. Thus human HIF-1.alpha. produced in insect
cells required treatment with mammalian extract to promote
interaction with wild type but not mutant pVHL (22). Addition of
NaCl to the RCC4 cell extract (to 1M final concentration) abrogated
the modifying activity, whereas incubation of the PK-HIF in NaCl
(1M) after exposure to the cell extract did not alter its
subsequent ability to capture pVHL. Likewise treatment of the HIF
fusion protein after modification by exposure to extract, with
phosphatase or DFO did not prevent pVHL capture. Overall this
suggested an enzyme-mediated modification of HIF-1.alpha. that was
not phosphorylation.
[0517] 1.3: Study of the HIF-1.alpha. Recognition.
[0518] FIG. 1A shows alignment of the known or putative pVHL
binding domains amongst HIF-.alpha. homologues. The effects of
selected point mutations in human HIF-1.alpha. on the ability to
interact with pVHL were also tested. Wild type (WT) and modified
full length HIF-1.alpha. molecules bearing the mutations D556N,
D558N, D560Q, P564G, Y565A, P567G, M568R, D569N and D570N were
generated in reticulocyte lysate and examined for interaction with
VHL.HA by anti-HA immunoprecipitation. Among the tested
substitutions, mutation of conserved proline, Pro564Gly totally
abrogated interaction and Tyr565Ala reduced the interaction,
whereas other mutations had little effect or even enhanced
interaction.
[0519] Further studies were performed using synthetic polypeptides
as inhibitors of the HIF-1.alpha./pVHL interaction(23). When added
to an interaction mix of pVHL.HA and HIF-1.alpha. IVTTs the 34
residue sequence encompassing amino acids 549-582 was unable to
block interaction. However, blocking activity was strikingly
induced by exposure to cell extract supplemented with Fe(II) (FIG.
1B). Induction of blocking activity showed precisely the same
characteristics as had been determined for promotion of interaction
between pVHL and recombinant HIF proteins. Results for a series of
polypeptides derived from this domain are summarized in FIG. 1B and
implicate a similar minimal interaction domain. One of the
polypeptides shown to have blocking activity following, exposure to
Fe(II) supplemented cell extract was 19 WT. This polypeptide
comprises HIF-1.alpha. polypeptide residues 556 to 574 and its
ability to block binding following exposure to a variety of direct
oxidation conditions was assessed. Exposure of polypeptide to
Fe(II) (100 .mu.M) with hydrogen peroxide (1 mM), NADH oxidase (1
U/.mu.l) with NADH (1 mM), NADH-FMN oxidoreductase (7 mU/.mu.l)
with NADH (1 mM) or metachlorobenzoic acid (1 mM) did not promote
the blocking activity whereas exposure to cell extract plus iron
did.
[0520] In summary, no polypeptide could block the interaction
without prior enzymatic modification, blocking activity could not
be induced by a variety of direct oxidation systems,
phosphorylation of Tyr565 had no effect on the ability of extract
to promote blocking activity, and the mutant sequence Pro564Gly did
not block the HIF-1.alpha./pVHL interaction, even after exposure to
extract.
[0521] Mass spectrometric analyses (24) (MALDI-Tof) of
extract-treated synthetic polypeptide, and recombinant HIF
(expressed in insect cells and subsequently treated with mammalian
extracts to promote pVHL binding ability) implied several
oxidations as evidenced by +16 Da mass shifts in ions derived from
this sequence. Further analyses by MS/MS (ESI-QTof) indicated
oxidation affecting Pro564 and the nearby methionine residues.
Since the methionine residues are either non-conserved or could be
mutated without effect, and direct oxidation methods known to
oxidize methionine efficiently could not mimic the enzymatic
activity, we postulated that the enzymatic oxidation that promoted
interaction of this HIF-1.alpha. sequence with pVHL was the
oxidation of Pro564.
[0522] 1.4 Hydroxyproline Incorporation into a Synthetic
Polypeptide.
[0523] We synthesized a polypeptide (HIF-1.alpha. residues
556-574), containing a trans-4-hydroxy-S-proline residue at
position 564 (19:Pro564Hyp), since the trans-4-hydroxylation is the
commonest enzymatic proline oxidation (25). A polypeptide blocking
assay was carried out using the 19:Pro564Hyp modified polypeptide
and, as a control, the unmodified polypeptide without the
trans-4-hydroxylation (19:WT). The 19:WT polypeptide was either
incubated with cell extract before the binding assay or was
untreated. The 19:Pro564Hyp polypeptide was added to a mixture of
HIF-1.alpha. and pVHL-HA IVTTs at a concentration of 1, 0.25, 0.05
or 0.01 .mu.M and the cell extract treated or untreated 19:WT
polypeptide at a concentration of 0.5 .mu.M. Interaction of
HIF-1.alpha. with pVHL was then assayed by anti-HA
immunoprecipitation. The hydroxyproline substituted polypeptide
(19:Pro564Hyp) was highly effective at inhibiting the
HIF-1.alpha./pVHL interaction without the need for modification by
cell extract. The 19:WT control unsubstituted equivalent
polypeptide showed the expected requirement for cell extract in
order to inhibit interaction. Control reactions carried out with no
blocking polypeptide showed the expected binding of HIF-1.alpha. to
pVHL-HA. A pVHL capture assay was then carried out using
biotinylated synthetic polypeptides. The same polypeptides,
19:Pro564Hyp and 19:WT were assessed for the ability to capture
wild type or mutant (Pro86His) pVHL. 19:WT control polypeptide
captured pVHL only after incubation with cell extract, whereas
19:Pro564Hyp captured wild type pVHL without pre-treatment. In both
cases capture was specific for wild type, as opposed to mutant,
pVHL.
[0524] In summary, in striking contrast with previously tested
polypeptides, 19:Pro564Hyp blocked the HIF-1.alpha./pVHL
interaction without the need for exposure to cell extract.
Moreover, a biotinylated version of 19:Pro564Hyp specifically
captured wild type but not mutant pVHL, and its ability to capture
pVHL was not increased further by incubation with cell extract
(26). In comparison, the equivalent unmodified synthetic
polypeptide 19:WT could not interact without prior incubation with
cell extract.
[0525] These results reveal that the enzymatic activity promoting
interaction of HIF-1.alpha. with pVHL is a prolyl-4-hydroxylase,
which we term HIF-.alpha. prolyl-hydroxylase (HIF-PH). All
previously described prolyl-4-hydroxylases are members of the
superfamily of 2-oxoglutarate-dependent, and related, dioxygenases
(27). Consistent with the data presented above none of these
enzymes have an absolute requirement for ATP or NAD(P) but they do
have an absolute requirement for Fe(II) as a co-factor and dioxygen
as a co-substrate (27). Structural studies within the class have
defined a non-haem iron centre co-ordinated by an HXD/E . . . H
motif (28). Interestingly, and consistent with our findings, the
Fe(II) is not firmly bound and can be readily removed by chelating
agents, and enzyme inhibition occurs following substitution of
Fe(II) with Co(II) or Ni(II)(25).
[0526] 1.5: Effect of Ascorbate Supplementation and Various
Inhibitors of HIF-PH Activity.
[0527] The capture of labelled pVHL by different HIF substrates was
monitored after exposure to various test conditions.
[0528] The effect of ascorbate on pVHL capture by a
Gal/HIF-1.alpha.549-582/VP16 fusion protein substrate was monitored
and ascorbate (2 mM) was found to enhance the modifying activity of
cell extract, but have no effect in the absence of cell extract.
Ascorbate therefore enhances the activity of HIF-PH.
[0529] We went on to test a series of 2-oxoglutarate analogues
which act as competitive inhibitors of this class of enzyme (29)
for ability to inhibit HIF-PH as assessed by the ability of cell
extracts to modify either HIF polypeptide (19:WT) or a HIF fusion
protein (Gal/HIF-1.alpha.549-582/VP16) so as to promote pVHL
capture. Concordant results were obtained with both sources of HIF
sequence. In one such experiment the effect of N-oxalylglycine on
pVHL capture by a biotinylated HIF polypeptides as substrate was
monitored. The WT:19 and 19:Pro564Hyp polypeptides described above
were used as substrates. N-Oxalylglycine (0.2-1 mM) was found to
completely inhibit the modifying activity of cell extract on 19:WT.
Inhibition by N-Oxalylglycine was overcome by addition of 5 mM
2-oxoglutarate. As previously, 19:Pro564Hyp captured pVHL
efficiently without modification by cell extract, and this was not
influenced by exposure to N-oxalylglycine. Similar inhibition, also
competed by 2-oxoglutarate, was observed with N-oxalyl-2S-alanine
but not the enantiomer N-oxalyl-2R-alanine, demonstrating that the
effect was not due to simple Fe(II) chelation in solution. We also
used a 2-oxoglutarate dependent dioxygenase, phytanovl-CoA
.alpha.-hydroxylase(30), together with a readily available
unnatural substrate (isovaleryl CoA) (31) to deplete the cell
extract of 2-oxoglutarate produced by the citric acid cycle; as
predicted, this prevented the subsequent modification of HIF
polypeptide. The effect of dimethyl-oxalyglycine on HIF-1.alpha.
expression was also studied. HIF-1.alpha. immunoblot analysis of
extracts of Hep3B and U2OS cells exposed to dimethyl-oxalylgylcine
(0, 0.1 or 1.0 mM) for 6 hours was carried out. HIF-1.alpha. was
seen to be strongly induced under normoxic culture conditions.
[0530] Prolyl-4-hydroxylases have been identified in many
organisms. In mammalian cells these form .alpha..sub.2.beta..sub.2
tetramers in which the .beta.-subunit is identical with the
multifunctional protein disuphide isomerase(27). These enzymes
function in collagen modification in the endoplasmic reticulum, and
are reported to have a strict substrate specificity for prolyl
residues in collagen repeat sequences, typically
(Pro-Pro-Gly).sub.n(27). When tested as substrate for recombinant
[.alpha.1 or .alpha.2] human prolyl-4-hydroxylase, the HIF
polypeptide showed no activity(32). Taken together these findings
lead us to postulate that HIF-PH is a novel prolyl-4-hydroxylase
which marks HIF promoting recognition by the pVHL ubiquitination
ligase system. Since such enzymes utilise molecular oxygen as a
co-substrate this predicts a mechanism for direct sensing of
oxygen. To test this we examined the effect of hypoxia in the
presence of supplements of other co-factors, on HIF-PH activity as
assessed by ability to modify the Gal/HIF-1.alpha.549-582/VP16
fusion protein so as to promote pVHL capture. HIF substrate was
incubated with cell extract (supplemented with 2 mM ascorbate and
10 .mu.M Fe(II)) for 1, 2, 5 or 10 mins at 30.degree. C. under
ambient conditions or in the hypoxic workstation. The reaction was
stopped by washing with DFO, and the HIF substrate assayed for
ability to interact with pVHL. A time-dependent increase in capture
was seen in normoxia and a marked suppression of activity by
hypoxia.
[0531] 1.6: Summary of Example 1
[0532] Our findings therefore demonstrate a novel method of protein
modification that regulates interaction with pVHL ubiquitylation
complexes and indicate that enzymatic prolyl hydroxylation may act
directly as a sensor of molecular oxygen. The known properties of
2-oxoglutarate dependent oxygenases readily explain the classical
features of mimicry of hypoxia by iron chelators or cobaltous ions.
Two explanations have been advanced previously for these findings.
First, it has been proposed that cobaltous ions might substitute
for ferrous ions at an oxygen sensing iron centre (15). Since most
iron centres (e.g. haem and the large majority of iron sulphur
clusters) do not exchange in this way it was proposed that such a
protein must be turning over rapidly. Second, it has been
postulated that cobaltous ions and iron chelators might act by
interfering with Fenton chemistry and signalling through reactive
oxygen species(17, 33). For instance non-enzymatic `metal catalysed
oxidation` systems that oxidatively modify specific amino acids by
local Fenton chemistry can also be inhibited by iron chelators and
non-iron transition metal ions (34) providing an alternative
hypothesis for effects of these substances on the HIF system.
Clearly the labile iron centres associated with
prolyl-4-hydroxylases can accommodate the original iron centre
substitution hypothesis without the need to propose rapid turnover
of the sensor. In contrast we were repeatedly unable to promote
specific interactions of HIF-.alpha. sequences with pVHL by a
variety of non-enzymatic oxidation systems and our-evidence clearly
indicates an enzymatic mechanism of proline hydroxylation. Our
findings do not exclude direct oxidation processes or other oxygen
sensing systems impinging on HIF at other sites, on other molecules
involved in HIF signal transduction, or indeed on components of the
enzymatic prolyl hydroxylation complex. Though our evidence
indicates that HIF-PH is distinct from the [.alpha.1 and .alpha.2]
prolyl-4 hydroxylases associated with collagen modification, it is
interesting that these enzymes employ protein disulfide isomerase
as .beta. subunit, thus providing a potential link to sulfhydryl
redox chemistry. Also of interest, P4HA1 has recently been shown to
be HIF responsive(35), suggesting that similar hypoxic induction of
HIF-PH activity could down-regulate HIF in prolonged hypoxia,
contributing to accommodation of the HIF response.
[0533] The pVHL multi-protein complex belongs to the SCF class of
ubiquitin ligases, with pVHL acting as the F-box like substrate
recognition component (36, 37). To date, characterised examples of
recognition by F-box proteins have been regulated by
phosphorylation of the target sequence. Furthermore, HIF-1.alpha.
is a phosphoprotein, and phosphorylation has ben implicated in HIP
regulation(38, 39). While our findings do not exclude the
possibility that HIF-.alpha. phosphorylation could influence pVHL
recognition, they demonstrate that the key event in recognition of
the minimal interaction domain studied here is enzymatic
hydroxylation of Pro564. This defines a novel mechanism of
regulating substrate-recognition for the F-box class of ubiquitin
ligases. Furthermore, it is of interest that evolutionarily
conserved proline residues are observed at a number of other sites
in HIF-.alpha. subunits, and that other internal regions of
HIF-1.alpha. can convey oxygen-dependent destruction(6). In other
studies we have defined a second subdomain within the N-terminal
portion of the HIF-1.alpha. oxygen dependent degradation domain
that supports pVHL dependent ubiquitylation and contains a
functionally critical proline residue. Furthermore, we have
established the existence of a functionally conserved pVHL/HIF
system in C. elegans (see below) and demonstrated the critical
importance of a conserved proline residue in the ceVHL/ceHIF
interaction (indicated in FIG. 1A).
[0534] Overall this suggests that similar marking modifications may
occur elsewhere in HIF-.alpha. molecules and could contribute to
the oxygen sensitive properties of other domains. Whether proline
hydroxylation occurs in other molecules on residues that form part
of so-called "PEST" domains that are associated with rapid turnover
is also clearly now of interest(40). Equally, if the prolyl
modification is relatively specific to pVHL-mediated ubiquitylation
then the new findings may help define other substrates that are
important in pVHL tumor suppressor function.
[0535] References & Notes for Example 1
[0536] 1. G. L. Semenza, Genes Dev 14, 1983-91. (9000).
[0537] 2. N. V. Iyer, et al., Genes Dev. 12, 149-162 (1997).
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M. C. Simon, Nature 386, 403-4.07 (1997).
[0539] 4. G. L. Wang, B.-H. Jiang, E. A. Rue, G. L. Semenza, Proc.
Natl. Acad. Sci. USA 92, 5510-5514 (1995).
[0540] 5. S. Salceda, J. Caro, J. Biol. Chem. 272, 22642-22647
(1997).
[0541] 6. L. E. Huang, J. Gu, M. Schau, H. F. Bunn, Proc. Natl.
Acad. Sci. USA 95, 7987-7992 (1998).
[0542] 7. M. S. Wiesener, et al., Blood 92, 2260-2268 (1998).
[0543] 8. P. H. Maxwell, et al., Nature 399, 271-275 (1999).
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12436-12441 (1999).
[0545] 10. J. Lisztwan, G. Imbert, C. Wirbelauer, M, Gstaiger, W.
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[0546] 11. M. E. Cockman, et al., J Biol Chem (2000).
[0547] 12. M. Ohh, et al., Nat Cell Biol 2, 423-7. (2000).
[0548] 13. T. Kamura, et al., Proc Natl Acad Sci USA 97, 10430-5.
(2000).
[0549] 14. K. Tanimoto, Y. Makino, T. Pereira, L. Poellinger, Embo
J 19, 4298-309. (2000).
[0550] 15. M. A. Goldberg, S. P. Dunning, H. F. Bunn, Science 242,
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[0551] 16. G. L. Wang, G. L. Semenza, Blood 82, 3610-3615
(1993).
[0552] 17. G. L. Semenza, Cell 98, 281-284 (1999).
[0553] 18. Hypoxia (<0.1% oxygen) was obtained in a workstation
with O.sub.2, CO.sub.2 and temperature control (Ruskinn
Technologies, Leeds, UK). For hypoxic harvest, buffers were
preincubated in the chamber overnight. RCC4-VHL.HA, labelling
conditions and co-immunoprecipitation assays have been described
previously (11); in the current study 12.5 .mu.M MG132 was used for
proteasomal inhibition. For standard harvest, the cells were
removed from the chamber after hypoxic exposure, prior to cell
lysis. Co-immunoprecipitation assays on all lysates were performed
at 4.degree. C. under ambient oxygen conditions. Parallel
experiments established that adding desferrioxamine (100 .mu.M) to
the lysis and immunoprecipitation buffers did not alter the protein
species co-precipitated with pVHL.
[0554] 19. pcDNA3.VHL.HA and pcDNA3.HIF-1.alpha..PK were used to
program TNT reticulocyte lysate (Promega). When programming in
hypoxia, reaction mix was preincubated in the workstation for 10
minutes before addition of the DNA template. An aliquot was removed
from the workstation for transcription/translation under ambient
oxygenation. Interaction assays were as described previously
(11).
[0555] 20. Protein expression systems used were wheatgerm lysate
(Promega) programmed with pcDNA3 based vectors, insect cell
expression using recombinant baculovirus (pFastBacl, (GibcoBRL)
encoding PK.HIF-1.alpha.(344-698) and PK.HIF-1.alpha.(1-826))
bacterial expression as glutathione-S-transferase (GST-VBC complex)
and maltose binding protein fusions (pMAL-HIF-1.alpha.(344-698)).
For insect cell expression, Sf9 cells (GibcoBRL) were infected 60
hours prior to harvest.
[0556] 21. pGal/HIF-1.alpha.549-582/VP16 was used to program
reticulocyte lysate in the presence of unlabelled methionine. The
fusion protein product was immunopurified with beads pre-coated
with anti-Gal4 antibody RK5C1 (Santa Cruz). After washing with NETN
buffer, experimental exposures were to hypotonic extraction buffer
(HEB: 20 mM Tris pH7.5, 5 mM KCl, 1.5 mM MgCl2, 1 mM DTT) or cell
lysate prepared in HEB. Incubations were for 60 minutes at
22.degree. C. unless otherwise stated, following which the beads
were washed with NETN containing DFO, and incubated for 2 hours at
4.degree. C. in NETN+DFO with 5 .mu.l rabbit reticulocyte lysate
programmed with pcDNA3.VHL.HA.
[0557] 22. Baculoviral PK.HIF-1.alpha. (1-826) or PK.HIF-1.alpha.
(344-698) were immunoprecipitated with anti-PK antibody (Serotec).
Bead bound immunoprecipitates were washed, then incubated with test
cell lysates, following which the immunoprecipitates were washed
again with NTEN containing DFO, incubated with pVHL; and assayed
for interaction.
[0558] 23. For polypeptide inhibition assays, polypeptides were
added to NETN buffer containing a mixture of HIF-1.alpha. and
pVHL.HA. Final concentration of polypeptide was 1 .mu.M unless
otherwise stated. Pre-incubation of polypeptide in cell extract or
other conditions was for 60 minutes at 30.degree. C.
[0559] 24. Samples for mass spectroscopic analyses were either
biotinylated synthetic polypeptides 19:WT (residues 556-574), or
34:WT (residues 549-582), or PK-tagged HIF-1.alpha. retrieved from
insect cell lysates. After modification by mammalian cell lysates
the material was purified either by streptavidin/biotin capture
(synthetic polypeptides) or anti-PK immunoprecipitation and
SDS-PAGE. Proteolytic digestion was performed either on the beads
or in-gel with trypsin and V8 protease at pH7.8, or V8 protease at
pH4.5. Samples were lyophilised, and dissolved in aqueous 0.1% TFA.
Polypeptides were concentrated, desalted on a 300 .mu.m ID/5 mm
length C18 PepMap column (LC Packings, San Francisco, Calif., USA)
and eluted with 80% acetonitrile. The HPLC (CapLC, Waters, Milford,
Mass., USA) was coupled via a Nano-LC inlet to a Q-Tof mass
spectrometer (Micromass, Manchester, UK) equipped with a
nanoelectrospray Z-spray source. The eluted polypeptide mixture was
analysed by tandem mass spectrometric sequencing with an automated
MS-to-MS/MS switching protocol. Online determination of
precursor-ion masses was performed over the m/z range from 300 to
1200 atomic mass units in the positive charge detection mode with a
cone voltage of 30 V. The collision induced dissociation for
polypeptide sequencing by MS/MS was performed with argon gas at
20-40 eV and a 3 Da quadrupole resolution.
[0560] 25. K. I. Kivirikko, R. Myllyla, in The Enzymology of
Post-translational Modification of Proteins R. B. Freeman, H. C.
Hawkins, Eds. (Academic Press, London, 1980) pp. 53-104.
[0561] 26. For pVHL capture assays using biotinylated polypeptides,
the polypeptide was interacted with VHL.HA for 30 minutes at
4.degree. C., and precipitated with streptavidin beads.
Pre-incubation with cell extract or buffer under test conditions
was for 30 minutes at 30.degree. C.
[0562] 27. K. I. Kivirikko, J. Myllyharju, Matrix Biol 16, 357-68.
(1998).
[0563] 28. C. J. Schofield, Z. Zhang, Curr Opin Struct Biol 9,
722-31. (1999).
[0564] 29. C. J. Cunliffe, T. J. Franklin, N. J. Hales, G. B. Hill,
J Med Chem 35, 2652-8. (1992).
[0565] 30. G. A. Jansen, et al., J Lipid Res 40, 2244-54,
(1999).
[0566] 31. M. Mukherji, M. D. Lloyd et al. unpublished
observations.
[0567] 32. Prolyl 4-hydroxylase activity was assayed by a method
based on the hydroxylation-coupled decarboxylation of
2-oxo[1-.sup.14C]glutarate (Kivirikko, K. I., Mylly, R.:
Posttranslational enzymes in the biosynthesis of collagen:
intracellular enzymes. Methods Enzymol., 82, 245-304, 1982) using
recombinant human type I and II prolyl 4-hydroxylases expressed in
insect cells (26). 0.5 or 1.0 mg of polypeptide was used in each
reaction. The assay was performed by Dr. Johanna Myllyharju at the
Collagen Research Unit, Department of Medical Biochemistry,
University of Oulu, Finland.
[0568] 33. W. Ehleben, T. Porwol, J. Fandrey, W. Kurnier, H. Acker,
Kidney Int. 51, 483-491 (1997).
[0569] 34. E. R. Stadtman, Annu. Rev. Biochem. 62, 797-821
(1993).
[0570] 35, Y. Takahashi, S. Takahashi, Y. Shiga, T. Yoshimi, T.
Miura, J Biol Chem 275, 14139-46. (2000).
[0571] 36. D. Skowyra, K. L. Craig, M. Tyers, S. J. Elledge, J. W.
Harper, Cell 91, 209-219 (1997).
[0572] 37. E. E. Patton, A. R. Willems, M. Tyers, Trends Genet. 14,
236-243 (1998).
[0573] 38. D. E. Richard, E. Berra, E. Gothie, D. Roux, J.
Pouyssgur, J. Biol. Chem. 274 32631-32637(1999).
[0574] 39. P. W. Conrad, T. L. Freeman, D. Beitner-Johnson, D. E.
Millhorn, J. Biol. Chem. 274, 33709-33713 (1999).
[0575] 40. M. Rechsteiner, S. W. Rogers, Trends Biol. Sci. 21,
267-27l (1996).
Example 2
Identification of Hypoxia Inducible Factor and Von Hippel-Lindau
Tumour Suppressor Homologues in C. elegans
[0576] In this Example we define a HIF homologue in c. elegans and
demonstrate that both the transcriptional response to hypoxia, and
an important mode of regulation through interaction with the von
Hippel-Lindau tumour suppressor are conserved.
[0577] 2.1 Identification of a Homologue.
[0578] We sought homologues to HIF-.alpha. subunits in the c.
elegans EST database using an tBLASTn enquiry with the human
sequence. Prior to completion of the c. elegans sequencing
programme an EST was found with significant homology to HIF-.alpha.
in the basic-helix-loop-helix region, and we assembled a contig of
ESTs covering the putative homologue. Complete determination of the
c. elegans sequence revealed a further six predicted PAS proteins
but no closer matches to mammalian HIF-.alpha.. The EST contig we
had identified corresponds to a predicted open reading frame (ORF)
on chromosome V (F38A6.3) that is identical except for a 104 amino
acid amino terminal extension in the latter. Extensive searching of
the EST database has not revealed any cDNAs that map to this
putative 5' extension. No PCR products corresponding to the
extension could be identified and RACE-PCR products did contain a
putative trans spliced leader sequence. These Findings argue
against the predicted N-terminal extension and support the presence
of a 719 amino acid protein encoded by 9 exons. FIG. 2 shows an
alignment of the human and c. elegans sequences.
[0579] 2.2: Regulation of HIF in C.elegans.
[0580] To characterize the putative c.elegans HIF homologue
(ceHIF), we constructed a riboprobe encompassing nucleotides 1366
to 1496 of the predicted open reading frame, and raised antisera to
a bacterially expression recombinant protein containing amino acids
360 to 497 of the putative protein. The antisera recognised a
single species of the appropriate mobility in Cos7 cells
transfected with an expression vector expressing the full length
cDNA. Total RNA and protein extracts were prepared from populations
of worms exposed to normobaric hypoxia by incubation in bell jars
flushed with premixed gases of specified oxygen content balanced
with nitrogen. Immunoblotting of worm extracts showed a striking
induction of ceHIF under hypoxia.
[0581] Immunoblots of ceHIF levels in extracts of c. elegans were
carried out to monitor regulation by hypoxia and iron chelation.
Firstly, the oxygen dependence of protein induction was analysed.
Worms were grown on plates in bell jars flushed with air (N), or
with oxygen/balance nitrogen having an oxygen concentration of 5%,
1%, 0.5% or 0.1% for 18. hrs. A graded increase in protein level
was seen as the oxygen level was reduced below 5% with the highest
level of induction at 0.5 and 0.1% oxygen concentration. The time
course of protein induction was then studied. Worms were grown in
bell jars flushed with a 0.1% oxygen/balance nitrogen mixture for
0, 4, 8, 16 or 24 hrs before preparation of extracts. The results
showed strong induction within 4 hours which was sustained over a
24-hour period. The time course of protein decay on re-oxygenation
was then assessed. Worms were grown-in bell jars flushed with air
(N) or a 0.1% oxygen/balance nitrogen mixture. Extracts were made
either immediately, or after 4 and 8 minutes of re-oxygenation.
Decay of ceHIF protein was very rapid on re-oxygenation. Protein
levels were clearly reduced after 4 minutes and undetectable after
8 minutes of re-oxygenation of the culture. A time course RNAse
protection assay showing cehif mRNA levels in worms exposed to 0.1%
oxygen/balance nitrogen for 0, 4. 8, 16 and 24 hrs was carried out.
No induction of ceHIF mRNA by hypoxia was seen. Thus ceHIF
expression was strongly induced by hypoxia at the protein level,
but not at the mRNA level, in a manner very similar to that
described for mammalian HIF-.alpha. subunits. In mammalian cells
HIF-.alpha. protein is also strongly induced by iron chelating
agents as well as hypoxia, a characteristic that has suggested that
an interaction of iron and oxygen is central to the underlying
mechanism of oxygen sensing. Induction by iron chelation was also
studied in C. elegans. Worms were cultured in liquid media in the
presence or absence of the penetrant bidentate iron chelator 2', 2'
dipyrridyl (200 .mu.M) for 6 or 16 hrs. A striking Induction of
ceHIF by iron chelation was observed at both 6 and 16 hours and the
level of induction was equivalent to that observed in severe
hypoxia. ceHIF was not induced in the absence of iron
chelation.
[0582] 2.3 Conserved Role for VHL.
[0583] Regulation of mammalian HIF-.alpha. subunit protein levels
occurs though a one or more systems of ubiquitin mediated, oxygen
regulated proteolysis. To date the most clearly defined of these
involves the von Hippel-Lindau tumour suppressor protein (pVHL),
which physically interacts with specific HIF-.alpha. residues,
acting as the recognition component of an E3 ubiquitin ligase. In
VHL defective renal carcinoma cells HIF-.alpha. subunits are
constitutively stabilised leading to greatly increased steady-state
levels in normoxia. Recently a putative pVHL homolgue in c.elegans
has been proposed on the basis of database analysis and a sequence
alignment showing 23% amino acid identity. The analysis of HIF
regulation in c.elegans performed here shows a conserved role for
pVHL.
[0584] To determine whether pVHL function in HIF regulation might
also be conserved we first tested for interaction. 35-S labelled
ceHIF and HA tagged pVHL were synthesised by IVTT in rabbit
reticulocyte lysate, the ceHIF and/or tagged pVHL were then added
to EBC buffer with or without worn extract, prior to
immunoprecipitation with an anti-HA antibody.
Co-immunoprecipitation of ceHIF with pVHL was observed, but only
when the recombinant ceHIF IVTT was preincubated with worm extract.
Interestingly, though mammalian HIF-.alpha. produced in
reticulocyte lystaes will interact with pVHL, we have found that
this is dependent on a factor in the reticulocyte lysate that can
be substituted by other mammalian cell extracts, but not the
c.elegans extract. Though the human and c.elegans systems appear
homologous this suggests the existence of a species specific
modifying factor that promotes the HIF/pVHL interaction. Mammalian
pVHL recognises HIF-.alpha. through a subsequence within a
transferrable oxygen dependent degradation domain (ODDD) that shows
short regions of conservation with ceHIF. To test the functional
importance of this we mutated a conserved proline residue that is
critical for the mammalian interaction and replaced it with
glycine. Whilst wild type ceHIF could interact with tagged pVHL,
die ceHIF Pro621-Gly mutant form was unable to interact with pVHL
mirroring the findings with mammalian HIF.
[0585] To pursue the functional importance of the interaction
between ceVHL and ceHIF, we next employed a viable homozygous
deletion mutant worm lacking ceVHL, and assayed worm extracts for
ceHIF by immunoblotting. In normoxic ceVHL worms ceHIF levels were
strikingly upregulated and were essentially unregulated by oxygen,
being similar in hyperoxia (80% O2), air, and hypoxia (0.1% O2).
Thus a critical function for pVHL in the response to oxygen appears
also to be conserved. Surprisingly, ceVHL deficient worms are
phenotypically relatively normal, with only slightly slower growth
rates and mildly reduced reproductive capacity compared to wild
type.
[0586] This tight conservation of the HIF/pVHL system indicates
that c.elegans provides a new model for analysis of the oxygen
sensing and signalling pathways that regulate HIF, and for the
analysis of downstream effects on patterns of gene expression. As a
first step in exploring this potential we assessed ceHIF induction
by hypoxia in a mutant worms selected to test candidate molecules
in the sensing/signalling pathway. A number of studies support the
involvement of oxygen radicals though the source and mode of
interaction with the HIF/pVHL complex is unclear. Other studies
have suggested the involvement of particular growth factor
signalling pathways in HIF regulation, but the relation of these
findings to the oxygen sensitive signal is uncertain. In one line
of investigation it has been found that insulin and insulin-like
growth factors can activate HIF in normoxic cells, that the tumour
suppressor PTEN acts as a negative regulator of HIF, and that the
downstream target of PTEN, Akt shows oxygen dependent
phosphorylation, suggesting the involvement of an insulin
receptor/PI3-kinase pathway in HIF regulation. This pathway is
conserved in c.elegans and interestingly has been implicated in ROS
metabolism. We therefore tested several mutants to determine their
effect on the interaction of ceHIF with VHL.
[0587] The level of ceHIF in wild type and a series of mutant worms
was determined by immunoblotting. Worms were grown on plates in
bell jars flushed with normoxic (21% oxygen) or hypoxic (0.1%
oxygen) gas mixtures for 6 hrs. As expected the vhl mutant worms
were found to have high levels of ceHIF protein regardless of
oxygen tension. The mutants daf-18 (encoding a PTEN homologue),
daf-2 (encoding an insulin receptor homologue) and age-1 (encoding
a P13-kinase homolgue), in contrast with the vhl mutant worms, all
showed regulation of ceHIF by oxygen that was similar to wild type.
The other mutants screened for effects on ceHIF were selected oil
the basis of known effects on ROS metabolism, or altered phenotypic
sensitivity to oxidant stress included several mutants affecting
mitochondrial proteins (mev-1, clk-1, gas-1), a ctl-1 mutant that
affects cytosolic catalase activity and others (mev-2, mev-3) where
the product is not yet characterized and again regulation was
similar to wild type suggesting that this a distinct oxygen sensing
system that in c.elegans is not tightly linked to general systems
of oxidant defence.
[0588] In view of the data presented here demonstrating a critical
role for enzymatic hydroxylation of prolyl residues within HIF in
its normoxic recognition by pVHL and subsequent ubiquitylation and
destruction by the proteasome in the mammalian system we also
tested worms bearing mutations in known prolyl hydroxylases (dpy-18
and phy-2), and genes containing sequence motifs compatible with a
function as a prolyl hydroxylase (egl-9--located at F22E 2.4). The
effect of prolyl hydroxylase mutants on HIF activity was studied by
blotting. Extracts were made from wild type and mutant worms grown
in normoxic (21% oxygen) and h),oxic (0.1% oxygen) conditions.
Immunoblots for ceHIF were performed after separation on SDS/PAGE.
The band representing ceHIF was identified. No detectable ceHIF was
seen in an extract from normoxic wild type worms. In contrast in
normoxic extracts from egl-9 deficient worms ceHIF is easily
detected (allele MT 1201; allele MT 1216 grown at 25 decrees C.),
at levels comparable with those seen in extracts from these strains
grown in hypoxic conditions. As the egl-9 deficient worms have high
normoxic levels of ceHIF, this suggests that this gene product is
involved in the normal degradation of ceHIF. The dpy-18 and phy-2
deficient worms showed normal ceHIF levels.
[0589] We also used dimethyloxalylglycine (a cell permeant alpha
ketoglutarate analogue known to block this family of dioxygenases)
and demonstrated an increased abundance of ceHIF in normoxia in the
present of the inhibitor. In these experiments extracts were made
from wild type worms grown in normoxic (21% oxygen) conditions in
the presence and absence of dimethyloxalylglycine (1 mM).
Immunoblots for ceHIF were performed after separation on SDS/PAGE.
The band representing ceHIF was identified and it could clearly be
seen that inhibitor treatment clearly results in a substantial
increase in the -amount of immunodetectable ceHIF in normoxia.
[0590] 2.4: Expression of HIF Target Genes.
[0591] We wished to test directly for effects of the HIF/pVHL
system on patterns of gene expression in c.elegans. First we tested
for hypoxia inducible expression amongst a set of c.elegans
homologues of mammalian genes that are known HIF targets, and
compared the upregulation of mRNA upon hypoxic exposure of wild
type worms with that observed in the vhl mutant worm. The results
obtained are shown in Tables A and B below.
[0592] Table A summarises results for a subset of genes selected
for analysis on the basis of putative homology to mammalian HIF
target genes and tested for regulation by hypoxia and VHL in
c.elegans. Table B summarises results for a subset of genes
confirmed as regulated by vhl by RNAse protection after
identification in comparative array screening of wild type and vhl
mutant worms, and subsequently tested for regulation by hypoxia.
The full gene array dataset from which these genes were identified
are available at http://genomoe-www4.stanford.edu/cgi-bin/SMD/-
login.pl.
4TABLE A Sequence Regulated Regulated Name Gene Description by
Hypoxia by VHL F13D12.2 Lactate de hydrogenase + + F54D8.4 Putative
carbonic anhydrase - - T28F2.3 Putative carbonic anhydrase - -
R01E6.3 Putative carbonic anhydrase, strong + + similarity to human
CA2 R173.1 Putative carbonic anhydrase - - K05G3.3 Putative
carbonic anhydrase, strong - - similarity to human CA7 B0412.2
daf-7/member of the TGF.beta. - - superfamily C14F5.1 nip 3/bcl-2 -
- B0432.5 Putative tyrosine hydroxylase - -
[0593]
5TABLE B Sequence Regulated Regulated Name Gene Description by
Hypoxia by VHL F22B5.4 Protein of unknown function + + F35G2.4
Prolyl 4-hydroxylase alpha subunit + + C55B7.4 Member of the
acyl-CoA + + dehydrogenase protein family K09E4.4 Strong similarity
to human alpha - - T-acetylglucosaminidase T05B4.2 Member of the
nuclear hormone + + receptor/Zinc finger protein family H14N18.4
Member of the gamma- - - glutamyltransferase (tentative) protein
family C16C10.3 Piwi related protein + +
[0594] Clear induction by hypoxia was observed for mRNA encoding
lactate dehydrogenase-A and an isoform of carbonic anhydrase, and
in each case the mRNA was strikingly upregulated in vhl worms.
[0595] Second we tested for induction by hypoxia among a subset of
pVHL dependent differentially expressed gene defined by array
screening. Of eight genes demonstrated by RNAse to be upregulated
in vhl worms five were strongly inducible by hypoxia in wild type
worms.
[0596] Oxygen homeostasis is a fundamental physiological problem in
all organisms that can live in an aerobic environment, and genetic
studies in bacteria and yeast have defined specific sensing systems
that regulate gene expression in accordance with oxygen
availability. However efforts to link these systems to responses in
mammalian cells have so far been unsuccessful, and database
analysis has not reveal a HIF homologue in the s. cerevisiae genome
or sequenced prokaryotic genomes. The current work therefore
provides the clearest analysis to date of homology with a primitive
organism that has been developed for genetic analysis. Given recent
advances in large scale analysis of gene expression gene-function
in c.elegans the findings provide important new opportunities to
understand cellular responses to oxygen availability.
[0597] In mammalian cells transcriptional activation of HIF is
believed to be a multi-step process involving separate regulatory
steps in nuclear localization, DNA binding, and co-activator
recruitment as well as different systems of ubiquitin mediated
proteolysis. Somewhat surprisingly, in VHL defective renal
carcinoma cell lines HIF-.alpha. subunits are constitutively
stabilised and hypoxia inducible mRNAs are constitutively
upregulated in normoxic cells, indicating that at least in this
cell background pVHL has a dominant non-redundant function in the
regulation of the HIF transcriptional response. Both ceHIF protein
and its transcriptional target mRNAs also showed striking
up-regulation in normoxic vhl mutant worms. Importantly this
indicates that a critical non-redundant function of VHL in
regulation of HIF extends outside the cell background of VHL
associated tumours, and most likely operates generally in higher
eukaryotes.
[0598] In mammalian systems the HIF/pVHL system has important
functions in the regulation of oxygen delivery through effects on
angiogenesis, vasomotor control and erythropoiesis. Conservation in
c. elegans indicates that the HIF/pVHL system of oxygen regulated
gene expression antedates the development of these complex oxygen
delivery systems and that the system must have a critical function
in other responses to oxygen availability. The effects observed
already on the expression of metabolic enzymes may provide clues to
such functions. However though the viablity of both vhl mutant and
hif mutant worms in the laboratory suggests that the critical
functions that have directed the evolution of this system are
likely to be observed under other, presumably more stressful,
conditions.
[0599] 2.5: Methods
[0600] Identification of c.elegans hif cDNA. C.elegans EST database
searches were performed using the tBLASTn programme and the human
HIF-1.alpha. sequence as a probe. The putative C.elegans hif cDNA
was assembled from 4 overlapping cDNA clones, yk510h7, yk4a2,
yk383g1, and yk272d11 (kindly provided by Yuji Kohara, National
Institute of Genetics, Mishima, Japan), and inserted into the
polylinker of pcDNA 1 AMP (invitrogen) to create pcDNA1 cehif using
standard methods.
[0601] Antibody Generation and Immunoblotting. DNA encoding amino
acids 360 to 497 of ceHIF was inserted into pGEX-4t-1 and the
corresponding GST/ceHIF fusion protein was expressed in E. coli.
The protein was purified using glutathione agarose and used to
raise antisera in rabbits. Antisera were tested for reactivity
using extracts of Cos7 cells transfected with pcDNA1cehif, and
purified by ammonium sulphate precipitation. Worm extracts used in
immunoblotting were prepared from washed worms by homogenisation in
4 volumes extraction buffer (150mM NaCl, 1 mM EDTA, 50 mM Tris pH
7.5, 1% NP-40 1% sodium deoxycholate) using all Ultraturax T20
homogeniser.
[0602] Riboprobes and RNAse Protection Riboprobe templates were
generated from total c.elegans RNA using RT-PCR. Details of the
primers, and sequences are provided in supplementary information.
RNAse protection assays were performed as described in (ref) using
10-50 mg total RNA prepared from a mixed population of worms using
Tri-Reagent (Sigma).
[0603] Protein expression and interaction assays. 35S labelled
proteins were generated in reticulocyte lysates (Promega)
programmed with plamids encoding wild type ceHIF (pcDNA1cehif),
mutant ceHIF (pcDNA1cehif.P621G) or c-terminal HA tagged ceVHL
(pcDNA3ceVHL-HA). pcDNA1cehif.PxxxG was generated from pcDNA1cehif
using a site directed mutagenesis system (Stratagene) and die
following forward and reverse primers:
6 5'GATTTATCGTGCTTGGCAGGATTCGTTGACACTTATG (forward)
5'GTGTCAACGAATCCTGCCAAGCACGATAAATCAGGC (reverse).
[0604] pcDNA3ceVHL.HA was obtained by RT-PCR amplification of
nucleotides 1 to 525 of the predicted ORF of sequence F08G12.4 from
c.elegans RNA, and-exchange for human VHL sequence in
pcDNA3-VHL.HA. For interaction assays 1 .mu.l of each programmed
lysate was mixed in EBC buffer at 4.degree. C. for 1 hr before
anti-HA immunoprecipitation as described in Cockman et al.
Pretreatment of ceHIF with worm extract was for 30 min at
25.degree. C. with 10 .mu.I of extract derived by hypotonic
extraction of a worm homogenate in 20 mM Tris pH7.5, 5 mMKCl, 1.5
MgCl2, 1 mMDTT.
[0605] Worm strains and experimental conditions C. elegans strains
were cultured as described by Brenner[Brenner, 1974 #1]. Exposure
to hypoxia was in bell jars gassed with humidified air or
certificated nitrogen/oxygen mixes (British Oxygen Company).
Exposure to iron chelators worms was by growth in a liquid medium
as described previously [Lewis, 1997 #2] with or without 200 .mu.M
2,2 Dipyridyl. Wild type worms were Bristol strain (N2). okl6l was
generated by Dr. Robert Barstead, Oklahoma Medical Foundation,
using ultraviolet and psoralen mediated utagenesis. PCR using
oligonucleotides from the flanking genomic sequence was used to
select worms bearing a deletion at the FO8G12.4 (vhl) locus.
Confounding mutations in ok161 were removed by backcross selection
using visible markers that flank the VHL locus (dpy-6 unc-9).
Example 3
The VHL E3 Ligase Complex Interacts with Two Independent Regions of
HIF-1.alpha.
[0606] In this Example we show that two independent regions of the
HIF-1.alpha. a ODDD are targeted for ubiquitylation by VHL E3 in a
manner dependent upon proline hydroxylation. However these two VHL
E3 target sites differ in their overall sequence, their ability to
bind VHL directly and their requirement for other cellular factors.
These data reinforce the critical role for pVHL in HIF-.alpha.
regulation, but implicate a more complex model for pVHL/HIF-.alpha.
interactions.
[0607] Immunoprecipitation and band shift assays show that VHL and
HIF-.alpha. subunits are physically associated in a wide range of
cell types, consistent with a general role for VHL in
oxygen-dependent regulation of HIF-.alpha. subunits. At the same
time biochemical studies show that VHL exists as a multiprotein
complex with elongins B and C, CUL-2 and RBX1. This complex is
homologous to the SCF (Skp-1-Cdc53/Cullin-F-box) class of E3
ubiquitin ligases. Like SCF E3, the VHL complex has inherent
ubiquitin ligase activity. VHL itself is thought to play a role
analagous to the F-box substrate recognition component. HIF-.alpha.
subunits are therefore clear candidate substrates for VHL E3 and
have since been shown to be ubiquitylated in a VHL-dependent manner
in vitro.
[0608] Example 1 above demonstrates that degradation of
HIF-1.alpha. mediated by the VHL binding site occurs through
oxygen-dependent hydroxylation at proline 564. It is currently
unclear whether oxygen-dependent degradation of HIF-.alpha.
subunits is solely VHL-dependent. In renal cell carcinoma lines and
in CHO cells VHL appears to be the critical mediator. However only
one VHL binding site has been identified in HIF-1.alpha. and
regions outside this site can confer oxygen-dependent regulation in
vivo. To investigate the mechanisms underlying this we have
employed in vitro ubiquitylation assays which provide evidence of
functional interaction with the VHL E3 ligase. We find that two
independent regions of the HIF-1.alpha. ODDD are targeted for
ubiquitylation by VHL E3 in a manner dependent upon proline
hydroxylation. However these two VHL E3 target sites differ in
their overall sequence their ability to bind VHL directly and their
requirement for other cellular factors. These data reinforce the
critical role for pVHL in HIF-.alpha. regulation, but implicate a
more complex model for pVHL/HIF-.alpha. interactions.
[0609] Materials and Methods
[0610] Plasmid constructs--His.sub.6-E1-tagged mouse E1 cDNA in
pRSET was a kind gift of T.Hunt. pcDNA3-VHLHA has been previously
described Cockman et al. pGAL. 344-417VP16 has been previously
described (O'Rourke). Plasmids bearing mutations were generated
using a site-directed mutagenesis kit (QuickChange; Stratagene) and
mutagenic oligonucleotides designed according to the manufacturer's
recommendations. All PCRs were performed using pfu DNA polymerase
(Stratagene).
[0611] Cell culture and transient transfection--7860, U2OS and RCC4
cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, glutamine (2mM), penicillin
(50 IU/ml) and streptomycin sulfate (50 .mu.g/ml). Ka13 cells (Wood
et al) were grown in Ham's F12 medium with the same
supplements.
[0612] Cell extract preparation and Western Blotting--Cytoplasmic
extract for ubiquitylation assays was prepared as previously
described (Cockman et al). S100 extract was obtained by an
additional ultracentrifugation step at 100,000 g at 4.degree. C.
for 4 h. Extracts for Western blotting were prepared by
resuspending cell pellets in 7M urea, 10%. glycerol, 1% SDS, 10 mM
Tris pH6.8, containing 50 .mu.M phcnylmethylsulfonyl fluoride and
leupeptin, pepstatin and aprotinin all at 0.1 .mu.g/ml, followed by
disruption using a hand-held homogenizer (Ultra-Turrax T8 with 5G
dispersing tool; Janke & Kunkel GmbH). Following. SDS-PAGE
proteins were transferred onto Immobilon-P membrane (Millipore) and
processed for western blotting using the indicated antibody.
[0613] Antibodies--Anti-HA antibody (12CA5) was from Roche
Molecular Biochemicals, anti-GAL4(DBD) (RK5C1) agrose conjugate
from Santa Cruz Biotechnology and anti-HIF-1.alpha. clone 54)
antibody from Transduction Laboratories.
[0614] Ubiquitylation enzymes and assays--The E1 activating enzyme
used in ubiquitylation assays was either obtained from Affinit
Research (Exeter, UK) or purified from BL21 (DE3) E.coli
transfected with plasmid expressing His.sub.6-tagged mouse E1.
His.sub.6-E1 was purified by Ni.sup.2+-agarose affinity
chromatography. After dialysis against phosphate buffered saline,
glycerol was added to 10% (vol/vol) and 25 ng/.mu.l aliquots stored
at -80.degree. C. Human CDC34 recombinant E2 enzyme was from
Arffiniti Research (Exeter, UK). VHL E3 was obtained by anti-HA
immunoprecipitation from stably transfected 7860-VHLHA cell lysates
(Iliopoulos et al). GAL-HIF-1.alpha. substrate was prepared by
anti-GAL immunoprecipitation from [.sup.35S] methionine-labeled TnT
rabbit reticulocyte (Promega) translates. Each 40 .mu.l
ubiquitylation reaction consisted of 4 .mu.l of 5 mg/ml ubiquitin,
4 .mu.l of 10.times.ATP regenerating system (20 mM Tris pH7.5, 10
mM ATP, 10 mM magnesium acetate, 300 mM creatine phosphate, 0.5
mg/ml creatine phosphokinase), 2 .mu.l E1, 3 .mu.l E2, 6 .mu.l VHL
E3 immunopurified on protein G sepharose, 6 .mu.l GAL-HIF-1.alpha.
substrate immunopurified on agarose beads. Reactions were incubated
at 30.degree. C. for 2 h with occasional mixing, stopped by the
addition of SDS sample buffer and analysed by SDS-PAGE and
autoradiography. Cytoplasmic extract-based ubiquitylation assays
have been previously described (Cockman).
[0615] In vitro interaction assays--TnT rabbit reticulocyte
(Promega) translates (4 .mu.l [.sup.35S] methionine-labeled) were
mixed either in 70 ul hypotonic extraction buffer (20 mM Tris
ph7.5; 5 mMKCl; 1.5 mMMgCl2; 1 mM DTT) or RCC4 cytoplasmic extract
at 30 degrees C. for 1 hour. Samples were then cooled and incubated
with 400 .mu.l extract from 786-0 cells stably transfected with
pcDNA3 VHL.HA for 90 minutes on ice prior to immunoprecipitation
with excess anti-HA antibodies and protein G beads. Input samples
of the GAL-HIF-1 alpha fusion proteins and retrieved
immunoprecipitates were analysed by SDS/PAGE and
autoradiography.
[0616] Luciferase and beta-Galactosidase Assays--Luciferase
activities in cell extracts were determined using a commercially
available luciferase assay system (Promega) and a TD-20e
luminometer (Turner Designs). Relative beta-galactosidase activity
in extracts were measured using
o-nitrophenyl-beta-D-galactopyranoside (0.67 mg/ml) as substrate in
a 0.1 M phosphate buffer (pH 7.0) containing 10 mM KCl, 1 mM
MgSO.sub.4 and 30 mM beta-mercaptoethanol incubated at 30.degree.
C. for 15-45 min The A.sub.420 was determined after stopping the
reaction by the addition of 0.4. sodium carbonate (final
concentration).
[0617] Cell extract preparation and Western Blotting--Cytoplasmic
extract for ubiquitylation assays was prepared as previously
described (Cockman et al). S100 extract was obtained by an
additional ultracentrifugation step at 100,000 g at 4.degree. C.
for 4 h. Extracts for Western blotting were prepared by
resuspending cell pellets in 7M urea, 10% glycerol, 1% SDS, 10 mM
Tris pH6.8, containing 50 .mu.M phenylmethylsulfonyl fluoride and
leupeptin, pepstatin and aprotinin all at 0.1 .mu.g/ml, followed by
disruption using a hand-held homogenizer (Ultra-Turrax T8 with 5G
dispersing tool; Janke & Kunkel GmbH). Following SDS-PAGE.
proteins were transferred onto Immobilon-P membrane (Millipore) and
processed for western blotting using the indicated antibody.
[0618] 3.1 The VHL E3 Ligase can Interact Functionally with Two
Distinct Regions of the HIF-1.alpha. ODDD in vitro.
[0619] In order to understand more about the interactions of pVHL
with HIF-1.alpha. we analysed VHL-dependent ubiquitylation of the
HIF-1.alpha. OCDD in an in vitro assay using cytoplasmic extracts
as a source of ubiquitylation enzymes. 35S-methionine labelled
GAL-HIF-1 alpha fusion proteins containing the amino acids 344 to
698, 344 to 553, 554 to 698 or 504 to 554 of HIF-1.alpha. were
generated by IVTT and subjected to iii vitro ubiquitylation in
cytoplasmic extracts from RCC4 cells, which lack pVHL (RCC4), or
RCC4 cells stably transfected with. pcDNA3 VHL.HA (RCC4/VHL) in the
presence or absence of exogenous ubiquitin. PVHL dependent
ubiquitylation, resulting in a strong signal of decreased mobility
at the top of the lane, was clearly observed when the substrate
contained HIF-1 alpha amino acids 344-698, 344-553 and 554-698, but
not amino acids 504-554. HIF-.alpha. residues 344-553 and 554-698
are both capable of oxygen-dependent regulation in vivo (O'Rouke et
al) and when analysed in vitro here both regions exhibit
VHL-dependent ubiquitylation. This indicates that the VHL E3 ligase
can interact functionally with at least two sites in
HIF-1.alpha..
[0620] 3.2 Requirements for Functional Interactions.
[0621] To investigate this further, it was necessary to develop a
ubiquitylation assay using purified components. 35S-methionine
labelled GAL-HIF-1.alpha. amino acids 344-698 fusion protein was
generated by IVTT, immunopurified with anti-Gal antibody conjugated
agarose and subjected to in vitro ubiquitylation with purified
components. This resulted in the production of high molecular
weight GAL344-698-related species in a ubiquitin and ATP-dependent
manner. These high molecular weight species correspond to
ubiquitylated forms of GAL344-698 as their production is E1-, E2-
and VHLE3-dependent.
[0622] In vitro ubiquitylation was then performed on a variety of
GAL-HIF-1.alpha. fusions. 35S-methionine labelled immunopurified
GAL-HIF-1.alpha. fusions comprising amino acid residues 344 to 698,
344 to 553, 554 to 698 or 652 to 826 of HIF-1.alpha. were used as
substrates using reaction mixtures containing E1, E2, VHL E3
ligase, ubiquitin and ATP or reaction mixtures where ubiquitin or
VHL E3 ligase were omitted to act as controls. VHL E3 ligase
dependent ubiquitylation was clearly seen when the substrate
contained HIF-1.alpha. amino acids 344-698, 344-553 or 554-698 but
not for substrates containing residues 652-826 of HIF-1.alpha.. The
fusion containing residues 652-826 of HIF-1.alpha. acted as a
control as residues 652-826 of HIF-1.alpha. show no
oxygen-dependent regulation at the protein level in vivo (O'Rouke)
and do not interact with pVHL in vitro (Cockman et al). As the GAL
344-553 and GAL 554-698 substrates were both found to be targets
for the VHL E3 but GAL652-826 are not, the results obtained using
the purified component assay concurs with the cytoplasmic extract
assay in identifying HIF-1.alpha. residues 344-553 and 554-698 as
independent VHL E3 targets in vitro.
[0623] 3.3 Cytoplasmic Extract Enhances Functional Interaction of
the VHL E3 Ligase with the 5' Target Site in HIF-1.alpha..
[0624] It was noted however that VHL-dependent ubiquitylation of
the GAL 344-553 substrate differed greatly between the two assays.
In the cytoplasmic extract assay GAL 344-553 is a much better
substrate for VHL-dependent ubiquitylation than GAL 554-698, but in
the purified component assay the position is reversed with GAL
344-553 an extremely weak substrate. We wondered whether
cytoplasmic extract was important for recognition of the 344-553
region by VHL E3. To test this 35S-methionine labelled GAL 344-553
substrate was generated by IVTT, incubated in buffer, cytoplasmic
extract or nuclear extract prior to in vitro ubiquitylation in the
purified component assay in the presence or absence of the VHL E3
ligase. Treatment with cytoplasmic extract dramatically enhanced
the VHL dependent ubiquitylation of the substrate. Thus, whilst the
buffer-treated substrate remains an extremely weak target for VHL
E3, pre-treatment with cytoplasmic extract has a dramatic effect,
converting the GAL 344-553 substrate into a strong target for
VHL-dependent ubiquitylation. Accompanying this effect a marked
mobility shift of the GAL 344-553 substrate was seen due to
phosphorylation in the cytoplasmic extract.
[0625] Phosphorylation is known to play an important role in
regulating recognition of substrates by the SCF E3 ligase.
HIF-1.alpha. is known to be a phosphoprotein, although an
oxygen-dependent phosphorylation event has not been identified. A
potential link between HIF-1.alpha. phosphorylation and
ubiquitylation was therefore of interest. Pre-incubation of the GAL
344-553 substrate with nuclear extract also resulted in a
phosphorylation-induced mobility shift, but this was not
accompanied by increased VHL-dependent ubiquitylation.
[0626] To clarify the role of phosphorylation in the cytoplasmic
extract effect, hexokinase treatment was used. 35S-methionine
labelled GAL-HIF-1 alpha amino acids 344-553 fusion protein
substrate was generated by IVTT, incubated in buffer, cytoplasmic
extract, cytoplasmic extract that had been depleted of ATP by
pre-incubation with hexokinase or cytoplasmic extract which had
been heat denatured. Enhanced VHL dependent substrate
ubiquitylation was found to persist in the absence of ATP (and
consequent absence of phosphorylation) but not following heat
denaturation. As the ATP-depleted extract can no longer support GAL
344-553 phosphorylation but is still capable of supporting enhanced
VHL-dependent ubiquitylation, phosphorylation of GAL 344-553 is
therefore not the key event mediating interaction with VHL E3. As
heat-treated cytoplasmic extract was unable to support enhanced
VHL-dependent ubiquitylation this suggests that a protein factor
may be involved either in binding to, or modifying the GAL 344-553
substrate.
[0627] The demonstration in Example 1 above that interaction of VHL
with the VHL binding site in HIF-1.alpha. is promoted by
cytoplasmic extract and iron led us to test the effect of
cytoplasmic extract on ubiquitylation of the GAL 554-698 substrate
and to test the effect of iron on ubiquitylation of GAL 344-417.
35S-methionine labelled GAL-HIF-1.alpha. fusions comprising amino
acids 344-417 or 554-698 of HIF-1.alpha. substrates were generated
by IVTT, incubated in buffer, cytoplasmic extract, cytoplasmic
extract supplemented with 100 .mu.M iron chloride prior to in vitro
ubiquitylation in the purified component assay in the presence or
absence of the VHL E3 ligase. Iron was found to enhance the
ubiquitylation of Gal-HIF-1.alpha. 344-417 fusions in the presence
of cytoplasmic extract. Cytoplasmic extract enhanced the
ubiquitylation of Gal-HIF-1 alpha 554-698 although the effect was
less pronounced than that of GAL344-417. These data suggested that
the two independent VHL E3 ligase target sites may be regulated by
a similar mechanism.
[0628] 3.4 Mapping of 380-417 as a Minimal Domain Targeted by
Cytoplasmic Extract and VHL E3.
[0629] To begin to understand the mechanism it was necessary to
define a minimal functional domain. Residues 344-55-3 correspond to
exons 9-11 of HIF-1.alpha. and so an exon-based deletional strategy
was used. 35S-methionine labelled GAL-HIF-1 alpha amino acids
344-553 fusion protein substrate was generated by IVTT, incubated
in buffer or cytoplasmic extract prior to in vitro ubiquitylation
in the purified component assay in the presence or absence of the
VHL E3 ligase. The GAL 344-503 fusion (corresponding to exons 9 and
10 of HIF-1.alpha.) still displayed enhanced VHL-dependent
ubiquitylation following cytoplasmic extract pre-treatment. Exons 9
and 10 were then assayed individually by generating fusions
carrying GAL-HIF-1.alpha. amino acid residues 344 to 503, 344 to
417 or 418 to 503. The only fusion which was not ubiquitylated was
that carrying residues 418 to 503. Thus both ubiquitylation and the
cytoplasmic extract effect were found to localise to exon 9,
represented by GAL 344-417. VHL dependent extract enhanced
ubiquitylation therefore clearly depends on HIF-1.alpha. amino
acids 344-417.
[0630] The corresponding exon in HIF-2.alpha. was then assayed. The
substrates used were Gal-HIF-2.alpha. fusion comprising amino acids
344-417 or 345-416. Residues 345-416 of HIF-2.alpha. were also
found to be a target for VHL-dependent ubiquitylation and also
exhibited enhanced ubiquitylation following cytoplasmic extract
pre-treatment. The function of this region is therefore conserved
between HIF-1 a and HIF-2.alpha. and sequence comparisons will help
to identify critical residues.
[0631] Deletional analysis was further extended to screen the
HIF-1.alpha. 344-417 region. Gal-HIF-1.alpha. fusions comprising
amino acids 344 to 417, 344 to 400, 344 to 379, 360 to 417 or 380
to 417 of HIF-1.alpha. were individually assessed as above.
Deletions made at the C-terminus completely ablated VHL-dependent
ubiquitylation (GAL 344-400 and GAL 344-379), whereas deletions
made at the N-terminus retained activity. The minimal functional
domain defined by this analysis was HIF-1.alpha. residues 380-417.
Although the output ubiquitylation signal was reduced, GAL 380-417
was still a target for VHL-dependent ubiquitylation and still
displayed enhanced ubiquitylation following cytoplasmic extract
pre-treatment.
[0632] 3.5 Identification of a Potential Functional Motif Conserved
Between the 5' and 3' VHL E3 Target Sites.
[0633] The HIF-1.alpha. 380-417 sequence was analysed in an attempt
to identify residues critical to the functional effect. The
HIF-1.alpha. sequence was aligned with the corresponding region of
HIF-2.alpha. and the VHL-binding site. Within the VHL-binding site,
hydroxylation at proline 564 is identified in Example 1 above as a
key regulatory event. Interestingly, a potential conserved motif
encompassing this proline can be identified between the two VHL E3
ligase target sites (FIG. 3A). Mutations of this potential motif
were assayed in the context of GAL 344-417. 35S-methionine labelled
GAL-HIF-1 alpha amino acids 344-417 wild type and mutant substrates
(comprising the mutation P402A or the double mutation LL397, 400A)
were generated by IVTT, incubated in buffer or cytoplasmic extract
prior to in vitro ubiquitylation in the presence or absence of the
VHL E3 ligase. The double mutation of leucines 397 and 400 to
alanine (LL 397,400 AA) was found to ablate VHL-dependent
ubiquitylation. The-point mutation of praline 402 to alanine (P 402
A) also ablated VHL-dependent ubiquitylation.
[0634] Mutations of the 344-417 region were then tested for their
effects on oxygen-dependent regulation in vivo. The HIF-1.alpha.
344-417 region is known to confer oxygen-dependent regulation on a
GAL-VP16 fusion (O'Rouke). The C-terminal deletion (344-400) and
the P 402 A mutation were tested in this context and both were
found to abolish oxygen-dependent regulation in vivo (FIG. 3B).
[0635] 3.6 identification of Critical Point Mutations.
[0636] Identification of critical point mutations allows these two
VHL E3 target sites to be assayed within the full-length
HIF-1.alpha. molecule. The P 402 A mutation was introduced to
ablate activity of the 5' VHL E3 target site and the P 564 G
mutation to ablate activity of the 3' VHL E3 target site.
35S-methionine labelled full length HIF-1.alpha. wild type and
mutant substrates were generated by IVTT and subjected to in vitro
ubiquitylation in cytoplasmic extracts from RCC4 cells, which lack
pVHL (RCC4), or RCC4 cells stably transfected with pcDNA3 VHL.HA
(RCC4/VHL) in the presence or absence of exogenous ubiquitin. The
double mutant P402A+P564G was found to show no VHL dependent
ubiquitylation, but isolated mutations of the critical prolines at
each individual VHL E3 target site did not ablate ubiquitylation.
Thus when these mutations are introduced individually the mutant
HIF-1.alpha. proteins still remain targets for VHL-dependent
ubiquitylation (presumably because each retains an active VHL E3
target site) HIF-1.alpha. therefore appears to contain two, and
only two target sites for VHL-dependent ubiquitylation. To assay
importance in vivo, the single and double VHL E3 target site
mutants were transfected into the HIF-1.alpha. deficient cell line
KA13 (Wood et al) and tested for their ability to mediate
oxygen-dependent transcriptional regulation (FIG. 4). The
transfected wild-type HIF-1.alpha. protein displayed
oxygen-dependent regulation. However the P 402 A, and P 564G point
mutants were transcriptionally active under normoxic conditions and
showed very little upregulation in hypoxia (FIG. 4). The P 402 A +P
564G double mutant was essentially constitutive under normoxic
conditions (FIG. 4).
[0637] 3.7 The 5' and 3' VHL E3 Target Sites Differ in their
Functional Requirements.
[0638] The ability of pVHL to interact directly with both the 5'
and 3' E3 target sites was tested in vitro. The 35S-methionine
labelled GAL-HIF-1.alpha. fusion proteins GAL 344-553 P402A, GAL
344-553, GAL 652-826, GAL 554-698 were made by IVTT, incubated in
buffer or cell extract from RCC4 cells lacking pVHL at 30 degrees
C. for 1 hour. Samples were then cooled and incubated with extract
from 786-0 cells stably transfected with pcDNA3 VHL.HA for 90
minutes on ice prior to immunoprecipitation with anti-HA antibodies
and protein G beads. Input samples of the GAL-HIF-1 alpha fusion
proteins and retrieved immunoprecipitates were analysed by SDS/PAGE
and autoradiography. The 3' VHL E3 target site is already known to
bind VHL in an in vitro interaction assay (Cockman et al) and the
results obtained confirmed this. In contrast the 5'VHL E3 target
site (represented by GAL 344-553) does not appear to bind pVHL in
this assay. Either the interaction of pVHL with the 5' E3 target
site is transient and too weak to be detected, or the interaction
is not direct. After treatment of the Gal-Hif-1 alpha fusion
proteins with cytoplasmic extract both the 5' and 3' VHL E3 target
sites can be captured by the anti-HA immunoprecipitation.
Interaction is not seen when the Gal-Hif-1 alpha fusion protein
contains the P402A mutation known to disrupt function of the 5'
site.
[0639] In a previous domain analysis of HIF-1.alpha. the 5' VHL E3
target site was not detected (Ohh et cl). We wondered whether this
was due to the use of S100 extract. VHL-dependent ubiquitylation of
both the 5' and 3' E3 target sites was compared using the standard
cytoplasmic extract or S100. 35S-methionine labelled GAL-HIF-1
alpha amino acids 344-553 fusion protein and GAL-HIF-1 alpha amino
acids 554-698 fusion protein substrates were generated by IVTT.
Ubiquitylation was performed in fresh cytoplasmic extract,
cytoplasmic extract which had been left at 4 degrees C. for 4 hours
or the S100 supernatant of cytoplasmic extract from RCC4 cells,
which lack pVHL or RCC4 cells stably transfected with pcDNA3
VHL.HA. The S100 extracts clearly enabled VHL dependent
ubiquitylation of GAL-HIF-1 alpha amino acids 554-698 fusion
protein but not GAL-HIF-1 alpha amino acids 344-553 fusion protein.
A factor specifically required for recognition of the 5' VHL E3
target site is either lost or inactivated during S100
preparation.
[0640] 3.8 The 5' VHL E3 Target Site is also Regulated by Proline
Hydroxylation.
[0641] It has been shown above that the 3' VHL E3 target site
responds to oxygen level via hydroxylation at proline residue 564.
This proline residue forms part of a potential motif conserved
between the 5' and 3' target sites. Mutation of the corresponding
proline residue (P402A) in the 5' target site also results in
functional inactivation. It was possible therefore that proline
residue 402 was also a target for regulatory hydroxylation. To test
this we asked whether polypeptides corresponding to the 3' VHL E3
target site could interfere with the cytoplasmic extract-dependent
modification of the 5' VHL E3 target site. 35S-methionine labelled
GAL-HIF-1 alpha amino acids 344-553 fusion protein substrate was
generated by IVTT and incubated in vitro in buffer or cytoplasmic
extracts from RCC4 cells in the presence of wild-type 19mer peptide
representing HIF-1 alpha amino acids 556-574 (12.5 .mu.M); a
polypeptide where the critical proline is mutated to glycine
(P564G); or a polypetide where the proline is modified to a
hydroxy-proline (P--OH). The products of this reaction were then
used as substrates in an in vitro ubiquitylation assay in the
presence or absence of VHL E3 ligase. The 19mer wild-type
polypeptide (P) was found to completely ablate the cytoplasmic
extract effect. In contrast a polypeptide in which the critical
proline is mutated to glycine (P-G) was found to have no effect.
The 3' VHL E3 target site polypeptide can therefore compete the
cytoplasmic extract-dependent modification at the 5' site in a
maimer dependent upon integrity of proline 564. Pre-hydroxylation
of proline 564 rendered the polypeptide unable to compete for
modification at the 5'VHL E3 target site presumably because it is
no longer a substrate for the enzymatic modification which is
occurring at the 5' VHL E3 target site. Thus proline hydroxylation
appears to be involved in regulating VHL-dependent ubiquitylation
at both the 5' and 3' E3 target sites.
[0642] 3.9 Discussion.
[0643] Through the use of in vitro ubiquitylation assays we have
identified 2 independent regions of HIF-1.alpha. targeted by the
VHL E3 ligase. Both target sites are located within the ODDD and
are functional in vivo. Identification of the two VHL E3 target
sites is consistent with published data which implied the existence
of more than one oxygen-dependent degradation domain within
HIF-1.alpha.. Residues 532-585 of HIF-1.alpha. encompassing the 3'
VHL E3 target site has previously been shown to be a target for
VHL-dependent ubiquitylation. Identification of a second VHL E3
target site provides further evidence of the critical role played
by VHL in HIF-1-mediated oxygen-sensing.
[0644] Although HIF-1.alpha. possesses two target sites for VHL E3,
they appear to be functionally different. The 3' VHL E3 target site
corresponds to the previously identified VHL-binding site. This
region of HIF-1.alpha. appears to be targeted directly by VHL
acting as the recognition component of the VHL E3 ligase. In
contrast we have no evidence that the 5' VHL E3 site can bind VHL
directly although it can interact with the complete VHL E3 ligase
complex. This may be because the interaction of VHL with the 5'
site is indirect or weak compared to the 3' site and difficult to
detect by the in vitro binding assay used. Both target sites
contain a potential consensus motif "LXXLAP" but differ in the
sequences surrounding the motif. Since the sites also differ
functionally (i.e. in their ability to interact with VHL and their
ability to be ubiquitylated by VHL E3 in S100 extract), this
indicates that determinants other than the conserved core residues
are important. It is important to understand the key determinants
both for oxygen-dependent proline hydroxylation and for subsequent
interaction with VHL E3. Particularly since database searches
identify "LXXLAP" motifs in a wide variety of cellular
proteins.
[0645] Although the two sites have functional differences, they
both seem to be regulated by the same enzymatic modification.
Hydroxylation at proline 564 is the key modification controlling
activity of the 3' VHL E3 site. The corresponding proline in the 5'
VHL E3 site is also critical for function and polypeptide
competition experiments implicate regulatory hydroxylation. Direct
evidence of this will come from mass spectrometric analysis. Also
of interest is whether the same enzyme is responsible for
oxygen-dependent proline hydroxylation at both sites. Sequence
differences in the target sites may allow recruitment of different
enzymes which in turn may allow graded or cell-type specific
differences in the oxygen response. S100 extract was found to be
incapable of supporting VHL-dependent ubiquitylation at the 5'
site. This may be due to removal of a 5' site-specific enzyme.
Attentively it may be due to removal of a bridging protein proposed
to act between the 5' VHL E3 target site and VHL E3. The bridging
protein may be an unknown protein or an already identified
component of the VHL E3 ligase.
[0646] References
[0647] Cockman, M. E., et al., Hypoxia inducible factor-alpha
binding and ubiquitylation by the von Hippel-Lindau tumor
suppressor protein. J Biol Chem, 2000. 275: p. 25733-41.
[0648] 2. Brenner, S., The genetics of Caenorhabditis elegans.
Genetics, 1 974. 77: p. 71-94.
[0649] 3. Wood, S. M., el al., Selection and analysis of a mutant
cell line defective in the hypoxia-inducible factor-1alpha-subunit
(HIF-1alpha). Journal of Biological Chemistry; 1998, 273: p.
8360-8368.
[0650] 4. Huang, L. E., et al., Regulation of hypoxia-inducible
factor 1.alpha. is mediated by an oxygen-dependent domain via the
ubiquitin-proteasome pathway. Proceedings of the National Academy
of Sciences, USA, 1998. 95: p. 7987-7992.
[0651] 5. Iliopoulos, O., et al, Negative regulation of
hypoxia-inducible genes by the von Hippel-Lindau protein.
Proceedings of the National Academy of Sciences, USA, 1996. 93: p.
10595-10599.
[0652] 6. Ohh, M., et al., Ubiquitination of hypoxia-inducible
factor requires direct binding to the beta-domain of the von
Hippel-Lindau protein. Nat Cell Biol, 2000. 2(7): p. 423-427.
[0653] 7. O'Rourke, J. F., et al., Oxygen-regulated and
transactivating domains in endothelial PAS protein 1: comparison
with hypoxia inducible factor-1 alpha. Journal of Biological
Chemistry, 1999. 274: p. 2060-2071.
[0654] 8. Lewis, J. A. and Fleming J. T. Basic culture methods
(1995) In Methods in Cell Biology, Vol 48 (ed. H. F. Epstein and D.
C. Shakes) p.3 Academic Press, San Diego, Calif.
Experimental for Example 4
[0655] Materials and Methods
[0656] C. elegans Culture Strains and Extract Preparation.
[0657] Worms were cultured using standard methods. Exposure to
hypoxia was in bell jars gassed with humidified air or certificated
nitrogen/oxygen mixes (British Oxygen Company). Exposure to 2,2
dipyridyl (200 .mu.m), or dimethyl-oxalylglycine (1 mM) was
performed during growth in a liquid medium. Wild type worms were
Bristol strain (N2). Mutant strains were obtained from the
Caenorhabdltis Genetics Centre and are as indicated in table 5. A
deletion mutant in the vhl-1 gene (ok1610) was generated using
trimethylpsoralen. The vhl-1 strain CB5603 was constructed by
backcrossing ok161 twice against wildtype (N2), then constructing a
triple mutant with markers on either side of vhl-1 (genotype: dpy-6
(e2062) vhl-1 (ok161) unc-9 (8101), and then removing these markers
by further crosses against N2. Worm extracts were prepared by
homogenisation (Ultraturax T20, 1KA Labortechnlk) in 4 volumes
extraction buffer (100 mM NaCI, 1 mm EDTA, 50 mM Tris pH7.5, 1%
NP-40, 1% sodium deoxycholate) for immunoblotting or in 2 volumes
of hypotonic extraction buffer, HEB (20 mM Tris pH7.5 5 mM KCl,
MgCl.sub.2 1 mM DTT) for modification reactions.
[0658] Mammalian Cells and Extract Preparation
[0659] HeLa and RCC4 cells were cultured in DMEM. Cell extracts
were prepared in HEB.
[0660] Antibodies for Immunoblotting and Immunoprecipitation.
[0661] For detection of native C.elegans HIF-1 and VHL-1 proteins,
antisera were produced in rabbits immunised with either a
glutathione-S-transferase fusion protein expressing amino acids
360-467 of HIF-1, or a maltose binding protein fusion linked to
full length (1-174) VHL-1. Recombinant proteins were expressed in
E.coli. Antisera were tested for reactivity using extracts of
appropriately transfected Cos7 cells, and purified by ammonium
sulphate precipitation. Mouse anti-HA antibody was 12CAS (Roche),
and mouse anti-Gal4 antibody was RK5C1 (Santa Cruz).
[0662] Riboprobes and RNAse Protection.
[0663] Details of riboprobe templates are provided in table 4.
RNAse protection assays were performed as described (Wiesener et
al., (1998) Blood 92 2260-2268) using total RNA prepared from a
mixed population of worms using Tri-Reagent (Sigma), or total RNA
prepared from HeLa cells using RNAzolB (Biogenesis).
[0664] Plasmid C. elegans cDNAs.
[0665] The hif-1 cDNA was assembled from 4 overlapping cDNA clones,
yk510h7, yk4a2, yk383g1 and yk272d11 (Yuji Kohara, National
Institute of Genetics, Japan), and inserted into pcDNA1AMP
(Invitrogen). The vhl-1 cDNA and the cDNA encoding the predicted
ORF of T20B3.7 were obtained by RT -PCR of worm RNA and inserted
into pcDNA3 (with linkers that encoded an N-terminal HA tag), and
pSP72 (Promega) respectively. The egl-9 cDNA was subcloned into
pcDNA1 from yk130h5 (Yuji Kohara). Phy-1 and phy-2 cDNAs were
subcloned in pCR-Script (Winter and Page, (2000) Mol. Cell. Biol.
20 4084-4093) Gal 4/HIF-1 fusion proteins were generated by PCR and
inserted into pcDNA3 Gal (O'Rourke et al., (1999) J. Biol. Chem.
274 2060-2071).
[0666] For insect cell expression, sequences encoding Gal4/HIF-1
(289-790) and EGL-9 (1-723) were subcloned into pFastBac1 (Gibco
BRL). For bacterial expression, sequences encoding Gal4/HIF-1
(590-790) and EGL-9 (359-723) were subcloned into pET-28a
(Novagen), and pMAL-p2X (NEB) respectively.
[0667] Mammalian cDNAs
[0668] The cDNAs encoding the human polypeptides designated EGLN-2
(PHD1), EGLN1 (PHD2), and EGLN3 (PHD3) were obtained by PCR
amplification and/or restriction endonuclease digestion from
publicly available cDNA banks (The I.M.A.G.E consortium, end NEDO
human cDNA sequencing project) or a human colonic cDNA library.
Products were ligated into pcDNA3 for expression in reticulocyte
lysate IVTTs, or into pMAL-c2X for expression in E. coli as maltose
binding protein fusions. pPDS15 (Lipscomb et .al. (1999) J.
Neurochem. 73 429-432) was used for expression of rat SM-20 in
reticulocyte lysate IVTT; sequences encoding amino acids 60-355
were subcloned into pTYB11 (NEB) for expression in E. coli.
[0669] For bacterial expression, human HIF-1.alpha. sequences
encoding amino acids 344-503 or 530-698 were subcloned into
pET28a.
[0670] Mutations were generated using a site directed mutagenesis
system (Stratagene). All plasmid sequences were verified by DNA
sequencing.
[0671] Protein Expression.
[0672] .sup.35S-labelled or unlabelled proteins were generated in
TNT reticulocyte lysate or wheat germ lysate (Promega). Protein
expression in insect cells was performed using the Bac-to-BacI/Sf9
system (Gibco BRL). Bacterially expressed proteins were produced in
E. coli strain BL21 (DE3). Proteins were used in lysates or
purified using amylase resin, DEAE-Sepharose, nickel affinity
chromatography, or anti-Gal antibodies, as appropriate.
[0673] Interaction Assays
[0674] Assays for interaction between recombinant VHL and HIF
polypeptides conformed to the following experimental design.
Recombinant VHL and HIF polypeptides were produced separately in
vitro. The HIF polypeptide was then pre-incubated with extract or a
recombinant enzyme as described below, then mixed with VHL and
incubated in EBC buffer (50 mM Tris pH 7.5, 150 M NaCI, 0.5% v/v
Igepal, 0.5 mM EDTA) at 4.degree. C. for 1 hour, before
immunoprecipitation with anti HA antibodies (for HA tagged VHL) or
anti Gal antibodies (for Gal4HIF fusions) and analysis by PAGE
(Jaakkola et al. (2001) supra).
[0675] A schematic of the on bead modification assay is shown in
FIG. 5.
[0676] Preincubation of C.elegans HIF-1 with worm extract or
recombinant EGL-9 was for 30 min at 25.degree. C. Preincubation of
mammalian HIF-A polypeptides with cell extract-or recombinant
enzymes was at 37.degree. C. for 10-30min unless otherwise stated.
For assays of recombinant enzymes, 2-oxoglutarate (2 mM), iron (100
.mu.M), and ascorbate (2 mM) were added to the reaction buffer
unless otherwise indicated. Reactions performed in hypoxia were in
the stated atmospheric oxygen concentration (balance nitrogen)
obtained using a controlled environment. Invivo.sub.2 400 hypoxia
work-station (Ruskinn Technologies) and buffers pre-equilibrated
with the appropriate atmosphere. Reactions (50 .mu.l) were
performed in open Eppendorf tubes with mixing and stopped by the
addition of 20 volumes desferrioxamine (100 .mu.M}.
[0677] For peptide blocking experiments peptides (final conc. 1
.mu.M) were pre-incubated with VHL-1 for 15 min before addition to
the interaction.
[0678] For VHL capture assays using synthetic biotinylated
HIF1-.alpha. peptides, peptide was preincubated as indicated for 30
min at 37.degree. C., then bound to strepavidin beads, washed,
mixed with recombinant VHL or extract, re-captured using beads, and
bound VHL analysed by PAGE.
[0679] For HIF-1.alpha. capture assays using 786-0/VHL cell
extract, HIF-1.alpha. polypeptides were produced by IVTT,
pre-incubated with enzyme, then interacted with cell extract under
conditions (10 mM Tris pH7.5, 0.25M NaCI, 0.5% NP40, at 4.degree.
C.) that do not permit modification of HIF1-.alpha. (Masson et
al.(2001) EMBO), then immunoprecipitated with anti-HA and analysed
by PAGE.
[0680] Details of the capture assay protocol are provided
below.
[0681] HPLC Analyses
[0682] Hydroxylation of the HIF-1.alpha. peptide B19Pro (residues
556-574) was analysed by reverse phase HPLC using a Phenomenex
Hypersil 5.mu. C18(octadecylsilane) 250.times.4.6 mm column and a
5% to 95% acetonitrile gradient in 0.1% TFA at 1 ml/min as the
mobile phase. A Gilson HPLC system using 306 pumps and 115
UV-detector controlled by Gilson 715 software was used. Standards
were unmodified B19Pro and a synthetic peptide (B19Hyp) bearing a
hydroxyproline substitution at Pro564.
[0683] Assays were performed with 2.5 mM ascorbate, 1.25 mM DTT, 50
.mu.M .alpha.KG, 1.25 mM Fe(II), 25 .mu.M peptide, 0.66 mg/ml
catalase, 1.75 mg/ml EGLN2pMAL, in 50 mM Tris/HCl, 1.5 mM
MgCl.sub.2 5 mM KCl. All cofactors were mixed simultaneously by the
addition of enzyme to separate drops and incubation was at
37.degree. C. for 30 mins. Assays stopped with methanol (70 .mu.l)
and frozen on dry-ice before centrifugation and injection.
[0684] For analysis of hydroxyproline, peptides or proteins were
subject to acid hydrolysis, derivatisation with
phenylisothiocyanate and HPLC using standard methods.
[0685] Decarboxylation assays were performed using
1-[.sup.14C]-2-oxogluta- rate purified polypeptide substrates at
approximately 25 .mu.M, and a purified EGLN2 (PHD1) fusion as
described in Mukherji et al. (2001) supra.
[0686] On Bead Modification
[0687] Gal/549-582/VP16 In vitro transcription translation (IVTT)
was prepared using 20 .mu.l Promega TnT Quick Coupled Retic lysate
(Promega, Madison, USA) 1 .mu.l DNA (1 .mu.g/ul), 2 .mu.l 1 mM
desferrioxamine (DFO) and 2 .mu.l cold methionine (supplied with
IVTT kit). For a positive control, the 2 .mu.l DFO was replaced
with 2 .mu.l 1 mM FeCl.sub.2 (freshly made). The IVTT reaction was
incubated at 30.degree. C. for 90 min
[0688] Beads were prepared using 20 .mu.l gal beads (Santa Cruz no.
sc-S10 AC), 5 .mu.l IVTT, & 100 .mu.l EBC+100 .mu.M DFO and
incubated in an End-Over-End rotator for 30-60 min. The beads were
then spun at 2,000 rpm for 1 minute, the supernatant removed and
the beads washed in 1 ml of EBC (no EDTA or DFO). This was repeated
three times.
[0689] The beads were then re-suspended in 1000 .mu.l HEB
(hypotonic extraction buffer: 20 mM Tris pH7.5, 5 mM KCl, 1.5 mM
MgCl.sub.2, 1 mM dithiothreitol) for each reaction. 100 .mu.l of
the re-suspended beads were transferred into fresh microfuge tubes
containing 500 .mu.l of HEB+DTT. The tubes were spun at 2000 rpm
for 1 minute and the supernatant removed. Beads were then incubated
at room temperature for ten minutes in an end-over-end rotator with
a lysate sample under modification conditions as described below
then spun at 2000 rpm for 1 minute.
[0690] Supernatant was removed and the beads washed three times in
500 .mu.l EBC (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% v/v Igepal, 0.5
mM EDTA+DFO (In the case of incubation with neat retic lysate,
removal of supernatant was facilitated by addition of 500 ul of
EBC+DFO prior to the first spin). The supernatant was then removed
from the final wash and the beads used for pull down assays.
[0691] VHL Capture Assays
[0692] VHL capture or `pull-down` assays on the Gal/549-582/VP16
beads modified as described above were performed on ice VHL-HA
(T2.1) IVTT performed by mixing 20 .mu.l Promega TnT Quick Coupled
Retic lysate (Promega) with 3 .mu.l H.sub.2O, 1 .mu.l DNA (1
.mu.g/.mu.l) and 1 .mu.l (0.37 MBq) .sup.35S-methionine (Aemrsham
Redivue no. AG1094) and incubating at 30.degree. C. for 90 minutes.
VHL-HA IVTT was then diluted in 100 .mu.l of EBC+100 .mu.M DFO, for
each set of beads to be assayed. To the modified, washed
gal/ODD/PI6 beads, 100 .mu.l of the VHL IVTT (T2.1)were added in
EBC buffer+100 .mu.M DFO.
[0693] The reaction was incubated in an end-over-end rotator for 2
hours in cold room, then spun at 2,000 rpm for 1 minute and the
supernatant removed(radioactive liquid waste). The beads were
washed with 500 .mu.l of EBC buffer and 100 .mu.M DFO and spun
again at 2,000 rpm for 1 minute. The wash steps were repeated a
total of 5 times. The supernatant was removed from the final wash
and eluted in 15 .mu.l of 2.times. SDS sample buffer.
[0694] Samples were stored at -20.degree. C. and examined by
SDS-PAGE.
[0695] DNA and Protein Manipulation
[0696] DNA manipulation and cloning and protein expression and
analysis by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)were
performed according to standard techniques which are well known to
those of skill in the art and described in detail in Molecular
Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989,
Cold Spring Harbor Laboratory Press and CurTent Protocols in
Molecular Biology, Ausubel et al. eds., John Wiley & Sons,
1992.
[0697] 4.1: Identification and Characterization of a HIF-1.alpha.
Homologue in C.elegans
[0698] A tBLASTn enquiry with the HIF-.alpha. human sequence was
used to identify HIF-.alpha. subunit homologues in the C.elegans
EST database. An EST contig was identified which was identical to
an open reading frame (ORF: F38A6.3). This ORF was predicted
following determination of the C.elegans genome sequence, with the
exception of a 104 amino acid amino terminal extension in the
latter. No further ESTs or PCR products corresponding to the
extension were identified and RACE-PCR products contained a
putative trans spliced leader sequence. These findings predict that
F38A8.3 encodes a 119 amino acid polypeptide that lacks the
proposed amino terminal extension.
[0699] To characterize the regulation of the putative HIF-.alpha.
homologue (HIF-1), we raised antisera to a recombinant polypeptide
and immunoblotted worm extracts. Extracts were prepared from worms
exposed to hypoxia, or the cell penetrating iron chelator 2, 2'
dipyridyl.
[0700] Immunoblotting showed a striking induction of HIF-1 by both
stimuli. Induction by hypoxia was progressively below 5% oxygen and
maximal at the lowest tested concentrations of 0.5% and 0.1%
oxygen. In 0.1% oxygen, HIF-1 protein level was strongly induced
within 4 hrs, and sustained over 24 hrs, but disappeared within
minutes following re-oxygenation. In contrast, hif-1 mRNA levels
were unchanged by hypoxia. Thus, these-experiments confirmed
up-regulation of HIF-1 by hypoxia, and suggested a mode of
regulation at die protein level similar to that described for
mammalian HIF-.alpha. subunits.
[0701] 4.2 Critical Function of a pVHL Homologue VHL-1 in the
Regulation of HIF-1 in C.elegans.
[0702] We compared HIF-1 expression in wild type and a series of
mutant worms that were selected because of potential relevance to
previously proposed models for oxygen sensing and signal
transduction processes in the mammalian HIF system (Chandel et al.,
(2000) J. Biol. Chem. (August) 1-37; Ehleben et al. (1997) Kidney
Int. 51 483-49w; Zundel et al. (2000) Genes & Dev. 14 391-396)
for review see (Semenza, (1999) Cell 98 281-284). These included
mutants in the PTEN/insulin receptor/PI-3-kinase pathway (daf-18,
daf-1, age-1), a mutant in a putative homologue of VHL (vhl-1),
mutants affecting mitochondrial proteins (mev-1, clk-1, gas-1), a
mutant that affects cytosolic catalase activity ctl-1, and others
selected for resistance or sensitivity to oxidant stresses but
where the mutant gene is not yet characterized (mev-2, mev-3).
[0703] With the exception of vhl-1 all mutant worms showed
preserved regulation of HIF-1 protein levels. In contrast, the
vhl-1 worms showed high levels of HIF-1 in normoxia that were
essentially unregulated by oxygen. These results confirmed that
proposed homology for vhl-1 (Woodward et al.(2000) Genomics 65
253-265), and indicated a conserved role for C.elegans VHL-1
protein in the response to hypoxia.
[0704] 4.3 Interaction of HIF-1 with VHL-1 is Regulated by Prolyl
Hydroxylation.
[0705] To address the mechanism of regulation of HIF-1 by VHL-1,
interaction between the two proteins was tested.
.sup.35S-Methionine labelled haemagglutinin (HA) tagged
VHL-1(HA.VHL-1), and HIF-1 were produced separately in vitro in
coupled transcription translation reactions (IVTT) in reticulocyte
lysate. IVTTs were then mixed and assayed for interaction by
anti-HA immunoprecipitation. When produced this way, the proteins
did not interact. However, when recombinant HIF-1 was pre-incubated
with worm extract, a clear interaction was observed.
[0706] A series of N-terminal truncations of HIF-1 linked to a Gal4
DNA binding domain was constructed. The Gal/HIF-1 fusion proteins
were expressed in reticulocyte lysates, pre-incubated with worm
extracts and then tested for interaction with HA. VHL-1. These
experiments demonstrated that whilst N terminal truncations up to
and including Gal4/HIF-1(590-719) were captured efficiently by HA.
VHL-1, Gal4/HIF-1(641-719) was not, implicating HIF-1 amino acids
590-641 in the interaction.
[0707] Inspection of this region revealed homology to pVHL-binding
domains in human HIF-1.alpha. that have recently been shown to
contain sites of prolyl hydroxylation (Ivan et al., (2001) Science
292 464-468; Jaalckola et al. Science (2001) 292 468-472). We
therefore mutated the homologous prolyl residue in C. elegans HIF-1
(P621 to G) and found that this mutation ablated interaction with
HA. VHL-1.
[0708] The demonstration of a critical conserved prolyl residue in
C.elegans HIF-1, together with the need for pre-incubation with
worm extract provided indication that the mechanism regulating the
HIF-1/VHL-1 interaction through enzymatic prolyl hydroxylation
might also be conserved in C. elegans. To verify this,
N-oxalyl-2S-alanine, a 2-oxoglutarate analogue that inhibits this
class of enzymes (Cunliffe et al.(l1992) supra) was added to the
worm extract during pre-incubation with HIF-1. This strongly
inhibited activity in a manner that was competed by excess
2-oxoglutarate, as inhibition was antagonized by 5 mM 2-OG.
[0709] To test whether hydroxylation of the critical P621 residue
in C. elegans HIF-1 could indeed promote binding to VHI-1, we
synthesised N-terminal biotinylated peptides corresponding to
residues 607-634 of C. elegans HIF-1 that contained either a
proline (B28Pro) or a (2S.4R)-trans-hydroxyproline residue (B28Hyp)
at position 621. We found that B28Hyp but not B28Pro blocked
capture of pretreated HIF-1 by HA.VHL-1, when added to the
interaction mix.
[0710] Furthermore. B28Hyp but not B28Pro captured immunodetectable
native VHL-1 when mixed with extracts from wild type but not vhl-1
mutant worms. Finally, to test the importance of prolyl
hydroxylation in regulating C.elegans HIF-1 in vivo we exposed
worms to the cell-penetrating prolyl hydroxylase inhibitor,
dimethyloxalylglycine. This strongly induced HIF-1 in normoxic
worms. These results demonstrated that conservation of the HIF/pVHL
system in C. elegans extends to the mode of regulation by prolyl
hydroxylation.
[0711] 4.4 The C. elegeans egl-9 Gene Product is a Prototype
HIF-PH.
[0712] The best characterised prolyl hydroxylases are the
procollagen-modifying enzymes (Kivirikko and Myllyharju, (1998)
Matrix Biol. 16 357-368). However, worms containing inactivating
mutations in each of two isoforms of the catalytic subunits, dpy-1B
(also termed phy-1) and phy-2 (Friedman et al., 2000 supra: Winter
and Page, 2000 supra) showed normal HIF-1 regulation, consistent
with HIF-PH being distinct from the collagen modifying enzymes.
[0713] We searched C. elegans and mammalian databases for
additional HIF-PH candidate genes that were well conserved between
these species and possessed a common .beta.-barrel jelly roll
motif.
[0714] Of particular interest was a family of genes related to the
C. elegans gene egl-9, a gene of previously unknown function that
was first identified on the basis of an egg-laying abnormal (egl)
phenotype (Trent et al., (1983) Genetics 104 619-647).
[0715] Sequence analyses coupled with secondary structure
predictions in the light of crystallographic data (Valegard et al.
(1998) supra: Zhang et al, (2000) Nature Structural Biology 7
127-133) predicted that these genes would encode a family of
enzymes conserved in C.elegans and mammals. The predictions
suggested that the enzymes would contain not only the jelly roll
motif, but also conserved iron and 2-oxoglutarate binding residues
in the same relationship that they occur in crystallographically
characterised enzymes e.g. the IXD . . . H iron binding motif on
the second and seventh strands of the jelly roll motif.
[0716] Mutants worms containing defective egl-9 alleles were
therefore assessed for regulation of HIF-1 by immunoblotting. Three
strains bearing inactivating mutant alleles of egl-9, (sa307,
sa330, and n571) (Darby et al., (1999) PNAS 96 15202-15207; Trent
et al.(1983) Genetics 104 619-647) all showed sinking constitutive
up-regulation of HIF-1 in normoxia and loss of induction by
hypoxia. Moreover, a further temperature sensitive egl-9 mutant,
n586 showed enhanced normoxic HIF-1 level at the non-permissive
temperature.
[0717] To determine the effect of EGL-9 on the HIF-1
transcriptional response, we measured mRNA levels of a range of
hypoxia inducible transcripts and found striking up-regulation in
egl-9 worms. A strongly inducible mRNA of unknown function
(F22B6.4) was also identified. These findings demonstrated a
critical function for EGL-9 in the regulation of HIF-1 and provided
further indication that EGL-9 functions as a HIF.PH that targets
HIF-1 to VHL-1.
[0718] We produced recombinant EGL-9 and assessed its ability to
catalyse the post-translational modification of HIF-1. HIF-1 was
captured-efficiently by HA. VHL-1 after incubation with EGL-9
programmed reticulocyte or wheat germ lysates, but not unprogrammed
lysate.
[0719] In contrast, IVTTs expressing recombinant C.elegans PHY-1,
PHY-2 and the gene product of the predicted ORF T20B3.7 that also
has significant homology to known prolyl hydroxylases, had no
activity in these assays.
[0720] To test whether EGL-9 could act directly on HIF-1, further
preparations were made by baculoviral expression in insect cells
and by expression as maltose binding protein (Map) fusion proteins
in E. coli. Since full length MBP/EGL-9 protein was insoluble when
expressed in E. coli we prepared an N-terminal truncation
containing residues 359-723 (MBP/.DELTA.N.EGL-9) that preserved the
predicted catalytic domain and had HIF.1 modifying activity when
expressed as an IVTT. C.elegans HIF-1 substrates were made as
N-terminal Gal4 fusion proteins in either insect cells or E.coli
and purified by anti-Gal immunoprecipitation. These substrates were
incubated with lysates of insects cell expressing full length EGL-9
or purified MBP/.DELTA.N.EGL.9, and tested for ability to capture
VHL-1.
[0721] Both forms of recombinant EGL-9 efficiently promoted
modification of HIF.1 as indicated by HA.VHL-1 capture. Moreover
analysis of this activity demonstrated 2-oxoglutarate, iron, and
oxygen dependence, and direct inhibition by cobaltous ions.
[0722] To demonstrate that activity in the HA.VHL-1 capture assays
corresponded to hydroxylation of the critical HIF-1 residue P621,
we assayed modified HIF-1 polypeptides for 4-hydroxyproline content
by HPLC. To provide larger quantities of protein for this analysis,
we co-transformed E.coli with wild type or the P621 to G mutant
form of a His.sub.6/Gal4/HIF-1(590-719) (HGH) fusion protein and
either MBP/.DELTA.N.EGL-9 or MBP. His.sub.6/Gal4/HIF-1 substrates
were retrieved by nickel affinity chromatography and aliquots
assayed for ability to capture 35S-methionine labelled HA.VHL-1
using anti-Gal immunoprecipitation, or subjected to acid hydrolysis
and HPLC analysis for the presence of phenylisothiocyanate
derivatised 4-hydroxproline.
[0723] In concordance with the HA.VHL-I capture assay results,
4-hydroxyproline was produced in the wild type but not the mutant
HiS.sub.6/Gal4/HIF-1 substrate following exposure to active enzyme
(FIG. 12) These results therefore demonstrated the activity of
EGL-9 as a prolyl hydroxylase that targets HIF-1 to VHL-1 in
C.elegans.
[0724] 4.5 Identification of a Series of Mammalian HIF-PH
Isoforms.
[0725] Sequence similarity between EGL-9 and a rat gene product
termed SM-20 (Wax et-al., (1994) J. Biol. Chem. 269 13041.-13047)
has been noted previously though no functional connection was
recognised (Darby et at.(l999) supra). Our sequence-structure
search identified a larger series of homologies and predicted three
closely related genes in each of the human and rodent genomes that
bore striking homology to egl-9, in particular over the core
putative catalytic domain.
[0726] FIG. 9 illustrates sequence alignment of EGL-9 (acc. no.
AAD56365) SM.20 (acc. no, AAA 19321) and the predicted human
proteins defined by acc. nos. XP.sub.--040482, AAG33965, and
NP.sub.--071356 (EGLN1-3), corresponding to Unigene clusters
Hs.324277, Hs.6523, and Hs. 18878.
[0727] The human protein products have been termed EGLN1, 2, and 3
or `Prolyl Hydroxylase Domain containing` (PHD) 2, 1 and 3
respectively. Note that the gene termed EGLN3 or PHD3 has
previously been identified as human homologue of rat SM-20 (Dupuy
et al. (2000) Genomics 69 348-354).
[0728] To test the role of these gene products in regulating the
interaction between human HIF.alpha. sub-units and the VHL E3
ligase complex, we first produced the proteins by reticulocyte
IVTT. Since unprogrammed lysate has a low level of HIF-PH activity
(Jaakkola. et al., 2001 supra) we tested for enhanced ability of
the programmed Iysates to promote the HIF.alpha../VHL E3
interaction. After incubation with relevant enzyme, HIF-.alpha.
substrates were mixed with extracts from 786-0/VHL cells that
stably express HA tagged human pVHL, and tested for interaction by
anti-HA immunoprecipitation.
[0729] With full length wild-type HIF-1.alpha., striking activity
was observed with rat SM-20 and all three human gene products, but
not a mutant EGLN1 bearing an H358A substitution at the predicted
catalytic site, and not a different human 2-oxoglutarate dependent
oxygenase (phytanoyl coenzyme A hydroxylase (Mukherji et al. 2001)
that was tested as a negative control. Similar results were
obtained with HIF-2.alpha..
[0730] Examination of HIF.1.alpha. mutants bearing missense
substitutions at the critical prolyl residues in the: HIF-1.alpha.
ODDD (Masson et al 2001) showed that that enzymes were
differentially efficient at promoting interaction via the
C-terminal (P564) and N-terminal (P402) prolyl hydroxylation sites.
Whereas interaction through the C-terminal site could be promoted
by all enzymes, VHL E3 capture was less efficient when only the
N-terminal site (P402) was intact, and was only promoted by EGLN2
(PHD1) and EGLN1(PHD2). No activity at all was observed with a
double HIF-1.alpha. mutant, P402A P564G, that ablates both
hydroxylation sites.
[0731] In keeping with these results, all enzymes strongly promoted
interaction of pVHL with isolated HIF-1.alpha. sequences {residues
549-582) from the C-terminal site. Further analysis demonstrated
that this activity was strongly inhibited by iron chelation,
cobaltous ions, and the 2-oxogluarate analogue N-oxalylglycine.
[0732] To confirm direct action on HIF-.alpha. sequences, we
prepared purified EGLN2 as an MBP fusion protein in E.coli and
assayed activity using either purified His-tagged HIF-1.alpha.
polypeptides containing the N-terminal (344-503) or C-terminal
(530-698) hydroxylation sites, or a synthetic peptide consisting of
the minimal HIF-1.alpha. C-terminal substrate (B19Pro, residues
556-574). These experiments demonstrated activity by pVHL capture
assays, HPLC/MS detection of the hydroxylated peptide product or
derivatised 4-hydroxyproline, and by 2-oxoglutarate decarboxylation
assays.
[0733] To verify expression of all three isoforms, we performed
RNase protection analysis using riboprobes specific for each
transcript. Since recently published work has indicated that the
rate of HIF degradation in normoxia is enhanced by prior exposure
of cells to a period of hypoxia (Berra et al.(2001) FEBS Letters
491-85-90) it has been predicted that HIF-PH would itself be
induced by the transcriptional response to hypoxia.
[0734] RNase protection demonstrated that all three HIF-PH mRNAs
are expressed in HeLa cells and that, in this cell line,
transcripts for EGLN1 (PHD2) and EGLN3 (PHD3) but not EGLN2 (PHD1)
are induced by hypoxia. In keeping with this, semi-quantitative
analysis of lysates prepared from HeLa cells that had been grown in
normoxla or exposed to hypoxia for 16 hours then assayed for HIF-PH
activity in vitro using the pVHL capture assay, demonstrated
induction of total HIF-PH activity that was blocked by actinomycin
D.
[0735] Finally, we used pVHL capture assays to measure the activity
in vitro of recombinant EGLN2 on a HIF-1.alpha. 549-582 substrate
at graded levels of hypoxia in a controlled hypoxia work-station.
We first measured the effect of graded hypoxia on the HIF modifying
activity of extracts of vhl-defective RCC4 cells that contain a
relatively high level of total HIF-PH activity. A progressive
reduction in activity was observed with graded hypoxia. Similar
assays were then performed using EGLN2 produced in reticulocyte
lysate by IVTT, or purified MBP/PHD-1 obtained by expression in E.
coli. Closely similar progressive reductions in the activity of
each preparation were observed with graded hypoxia. Thus, the
oxygen dependent activity of recombinant PHD-1 from either source
parallels that observed in crude cell extracts, and mirrors the
progressive increases in HIF-1.alpha. protein and DNA binding that
are observed when cells are exposed to graded hypoxia in culture
(Jiang et al., (1996) Am. J. Physiol. 271 C1172-C] 180).
[0736] 4.6 HIF Prolyl Hydroxylase Activity
[0737] Full length rat SM20, a truncated form of rat SM 20 lacking
the amino terminal 59 amino acids and the human homologue EGLN-2
were shown to modify HIF amino acids 549-582 in a manner which
facilitates interaction with the VHL protein. This is known to
depend on hydroxylation of proline 564.
[0738] Wheat germ lysate was programmed with pcDNA3 based plasmids
containing no insert, an insert encoding the full open reading
frame of rat SM20, a truncated form of rat SM20 lacking the amino
terminal 59 amino acids or the human homologue known as EGLN-2 in
the absence of exogenous iron or with the addition of 100 .mu.M
ferrous chloride. The nucleotide sequence corresponding to these
putative proteins were generated by PCR and their identity was
confirmed by sequencing. The protein products generated conformed
with their predicted molecular weights.
[0739] Protein containing HIF-1 sequence (amino acids 549-582) was
generated in a reticulocyte in vitro transcription translation
reaction in the presence of 100 micromolar desferrioxamine,
retrieved and then exposed to wheat germ translates containing the
putative enzymes as described above. These proteins contain the
critical proline (564) which can be modified by hydroxylation and
which enables recognition by von Hippel Lindau tumour suppressor
protein (pVHL).
[0740] The modification of the HIF-1 sequence was assayed by the
binding of the HIF-1 to radiolabelled pVHL, generated by in vitro
transcription and translation of a pcDNA3 vector containing the
human wild type pVHL open reading frame in a rabbit reticulocyte
lysate in the presence of .sup.35S-methionine.
[0741] SDS PAGE analysis indicated that the plasmids encoding the
genes EGLN-2, full length and truncated rat SM20 produced, in the
presence of Fe.sup.2+, a clear modification of HIF-1 which allowed
capture of labeled pVHL.
[0742] No pVHL binding was observed in the absence of Fe.sup.2+ for
SM2O, or EGLN-2 and only a low level of binding was observed for
truncated SM20.
[0743] 4.7 Mutation of EGLN2
[0744] Modification and pVHL binding assays were performed as
described above. Rabbit reticulocyte lysate was programmed with
pcDNA3 based plasmids containing no insert, an insert encoding the
full open reading frame of the human homologue known as EGLN-2, a
mutant form of EGLN-2 with a Histidine to Alanine substitution at
amino acid residue 358 or a naturally occurring splice variant
licking amino acids 369-389.
[0745] pVHL binding was observed only with the full length wild
type PHD-3 polypeptide. The full length wild type enzyme was able
to modify HIF-1 sequence whilst neither the mutant form nor the
deleted splice variant was able to do so. This demonstrates that
His358 and the region between residues 369 and 389 are necessary
for HIF hydroxylase activity.
[0746] 4.8 Effects of HIF and pVHL Mutations
[0747] Modification and pVHL binding assays were performed as
described above. Wheat germ lysate was programmed with pcDNA3 based
plasmids containing no insert, an insert encoding the full open
reading frame of the human homologue known as EGLN-2, or an insert
encoding the full open reading frame of the human homologue known
as PHD-3 or EGLN-3.
[0748] HIF substrates for modification were wild type or contained
mutation of proline 564 to glycine. The pVHL target for capture was
wild type or contained the mutation of tyrosine 98 to
histidine.
[0749] Binding of labeled pVHL was observed in assays using wild
type HIF and pVHL. No binding was observed in assays using mutant
HIF or pVHL.
[0750] Both EGLN2 and EGLN3 were therefore able to modify wild type
but not mutant HIF in a manner allowing capture of wild type but
not mutant pVHL.
[0751] 4.9 Oxygen Dependence of Modification of HIF by Recombinant
Enzymes
[0752] Modification and pVHL binding assays were performed as
described above except that enzymes were generated by expression in
COS cells or rabbit reticulocyte lysate.
[0753] Plasmids used to generate enzymes were as follows; pcDNA3
(without insert); pcDNA3 containing sequence encoding rat SM20
lacking the first 59 amino acids; pcDNA3 containing, sequence
encoding EGLN2 (PHD 1). Modification of HIF substrate by enzymes
was performed in either normoxia (21% O2) or anoxia (2% O.sub.2)
conditions using a hypoxia workstation.
[0754] Given the regulation of HIF-1 by oxygen and the known
substrate requirement of 2-oxoglutarate dependent dioxygenases for
oxygen, the oxygen dependence of the HIF modifying activity was
examined.
[0755] In anoxia, EGLN2(PHD1) or rat SM20 (lacking the amino
terminal 59 amino acids) were unable to modify HIF for pVHL
binding, in contrast to the clear modification at 21% O.sub.2. This
demonstrates oxygen dependence and a means of oxygen sensing in the
regulation of HIF-1.
[0756] 4.10 Effects of Oxalylglycine and 2-Oxoglutarate on EGLN2
and EGLN3 in vitro
[0757] Modification and pVHL binding assays were performed as
described above. Rabbit reticulocyte lysate was programmed with
pcDNA3 based plasmids containing no insert, an insert encoding the
full open reading frame of the human homologue of C. elegans Egl-9
known as EGLN-2, or an insert encoding the full open reading frame
of the human homologue known as EGLN-3. Modification was performed
in the absence of additives, in the presence of oxalylglycine,
oxalylglycine plus 2-oxoglutarate, 200 .mu.M desferrioxamine or 200
.mu.M cobaltous chloride.
[0758] Enzyme activity was observed to be diminished by
oxalylglycine, desferrioxamine and cobaltous ions. The inhibitory
effect of oxalylglycine was partially competed by addition of
excess 2-oxoglutarate. The family of 2-oxoglutarate dependent
dioxygenases demonstrate a requirement for oxygen, iron and
2-oxoglutarate. The ability of these gene products to modify HIF-1
to a VHL binding form was examined in differing conditions of iron
availability, 2-oxoglutarate availability and in the presence of a
2-oxoglutarate inhibitor. These results demonstrated an iron and
2-oxoglutarate dependence of activity in reticulocyte lysate.
[0759] 4.11 Effects of Dimethyl Oxalyl Glycine on HIF Activity in
vivo.
[0760] Hep3b and U2OS cells were co-transfected with a mixture of
three plasmids; pUAS.tk.luc, encoding a GAL 4 responsive luciferase
gene, pgal-hif775-826, a mammalian expression plasmid leading to
expression of a fusion between a 147 amino acid DNA binding domain
of GAL 4 and the carboxy terminal transactivator of human HIF-1
alpha, and pCMV.beta-gal, encoding a constitutively expressed
beta-galactosidase gene as a transfection control.
[0761] 48 hours following transfection, cells were incubated in
normoxia or 2% hypoxia overnight in the presence or absence of
dimethyl oxalyl glycine as indicated. Cell lysates were assayed for
luciferase and beta galactosidase activity and the relative
luciferase activity in each sample determined.
[0762] In both cell lines, the presence of the HIF prolyl
hydroxylase inhibitor resulted in enhanced activity of the carboxy
terminal transactivator in both normoxia and hypoxia compared to
the untreated samples (FIG. 6). This result shows a potentiating
action of this inhibitor on a domain of HIF which is not normally
considered to be dependent on proteolytic destruction for its
activity.
[0763] Potentiation of the action of the carboxy terminal
transactivator coupled with inhibition of destruction via the
oxygen dependent degradation domains enhances the overall inhibitor
mediated increase in HIF activity.
[0764] Addition of dimethyloxalylglycine to Hep3B and U2OS cells in
tissue culture (0.1 mM, 1 mM) was also observed to increase
intracellular levels of HIF-1 in Western Blot experiments.
[0765] Effects of forced expression of EGLN2 (PHD 1) or a naturally
occurring splice variant lacking amino acids 369-389 (PHD4) on HIF
Activity
[0766] Hep3b cells were co-transfected with a mixture of three
plasmids; pHRE.luc, encoding a HIF responsive luciferase gene,
pcDNA3 or pcDNA3.HIF, mammalian expression plasmids leading to
expression of no product or full length human HIF-1 alpha, and
pCMV-beta-gal, encoding a constitutively expressed
betagalactosidase gene as a transfection control.
[0767] 48 hours following transfection, cells were incubated in
normoxia or 2% hypoxia overnight. Cell lysates were assayed for
luciferase and beta galactosidase activity and the relative
luciferase activity in each sample determined.
[0768] In all circumstances, relative luciferase activity was lower
when co-expression included the fill length EGLN2 (PHD 1) rather
than the deleted, non-functional version (PHD4)(FIG. 7), providing
indication that expression of full length EGLN2 reduces functional
HIF by enhancing the generation of the rapidly destroyed
hydroxylated HIF protein.
[0769] The effect was even more prominent in circumstances where
the level of HIF-1.alpha. would be expected to be higher (e.g. when
co-expressed from a plasmid or partially stabilised by modest
hypoxia). This demonstrates that expression of these gene products
is able to enhance HIF-1 degradation in vivo.
[0770] 4.12 Effect of Inhibitors of HIF Prolyl Hydroxylase
Activity
[0771] Modification and pVHL binding assays were performed as
described above (and by reverse phase HPLC) to determine the effect
of inhibitors on the ability of cell extract to modify
Gal-Hif49-582-VPI6, thereby allowing capture of radiolabelled
recombinant pVHL.
[0772] Binding of pVHL was determined using SDS-PAGE and
autoradiography.
[0773] In the absence of treatment with cell extract, no binding of
pVHL was observed, showing the Gal-Hif549-582-VPI6 was
unmodified.
[0774] Treatment with cell extract in the absence of inhibitor
showed strong binding of pVHL. Treatment with cell extract in the
presence of oxalyl glycine (NK 87) showed reduced binding of pVHL,
showing that oxalyl glycine inhibits the HIP prolyl hydroxylase.
Treatment with cell extract supplemented with additional
2-oxoglutarate produced strong pVHL binding. Treatment with cell
extract in the presence of NK87 and additional 2-oxoglutarate also
produced strong binding, indicating that HK87 competes with the
oxoglutarate co-substrate.
[0775] Treatment with cell extract in the presence of 1 mM NMPG
produces strong binding of pVHL. However, treatment in the presence
of 5 mM NMPG reduced the amount of pVHL binding.
[0776] As a positive control, pVHL was captured when Gal-HIF-VP16
substrate was synthesised in rabbit reticulocyte lysate in the
presence of additional ferrous chloride. Other potential inhibitors
as shown in Table 3 were screened for the ability to inhibit HIF
hydroxylase activity as described above.
[0777] Of the compounds screened in this assay, reduced pVHL
binding indicative of inhibition of HIF hydroxylation was observed
for Is1, Is3, Is8, benzohydroxamic acid, ethyl dihydroxybenzoate,
and NK45.
[0778] The present application relates to the characterization of a
HIF-1/VHL prolyl hydroxylase system and the identification of a new
functional group of 2-oxoglutarate dependent oxygenase that
function as HIF prolyl hydroxylases (HIF-PHs). The critical role of
these enzymes in the regulation of HIF is emphasised by analysis of
vhl-1 and egl-9 mutant worms, which show essentially complete loss
of regulation of HIF-1 by oxygen. The availability of recombinant
HIF-PHs permits further investigation of the HIF/VHL system and an
important challenge will be to determine the extent to which the
complex demands of physiological oxygen homeostasis are met by the
biochemical properties of these enzymes.
[0779] Identification of the HIF system in nematode worms that
obtain oxygen directly by diffusion reveals that this system of
gene regulation must have evolved before the development of complex
systemic oxygen delivery systems, presumably to regulate; responses
to oxygen availability at the cellular level.
[0780] In mammals, the HIF system regulates not only cellular
responses to oxygen, but also a range of systemic functions such as
the control of oxygen delivery through effects on angiogenesis,
vasomotor control, and erythropoiesis. These complex requirements
have argued against the concept of a single oxygen sensor. However,
the existence in mammalian cells of (at least) three isoforms of
HIF.PH, and (at least) two isoforms of HIF-.alpha., each with more
than one site of prolyl hydroxylation (Masson et al., 2001 supra),
may provide the potential for different physiological responses to
oxygen availability to be generated through combinatorial
interactions amongst these molecules.
[0781] The characterisation of the HIF PH enzymes described herein
has various therapeutic applications, in particular as targets in
the development of pharmacological agents which modulate
HIF-.alpha. levels in a cell.
Example 5
[0782] In this Example it is shown that HIF-1.alpha. protein and
the endogenous HIF target gene encoding carbonic anhydrase 9 (CA-9)
are induced by exposure of cells to the PHD inhibitor, dimethyl
oxalylglycine. In previous studies we have demonstrated that
N-oxalylglycine is an inhibitor of PHD activity in vitro, but seems
to be incapable of entering intact cells. The esterified form,
dimethyloxalylglycine, has therefore been used to deliver the
compound to tissue culture cells.
[0783] Hep3B and U2OS cells were exposed to either 0.1 mM or 1 mM
dimethyloxalylglycine for 6 hours, harvested and assayed by
immunoblotting (Western blotting) for changes in the level of
HIF-1.alpha. and CA-9 expression. Controls, where no inhibitor was
added, were also performed. Clear upregulation of both HIF-1.alpha.
and the HIF target gene product CA-9 are observed in the presence
of dimethyloxalylglycine. Upregulation of HIF-1' increased with
increasing concentration of dimethyloxalylglycine, whilst the level
of CA-9 expression was similar after-exposure to both 0.1 and 1 mM
of dimethyloxalylglycine.
Example 6
[0784] In this example it is shown that enhanced new vessel growth
can be stimulated in a murine subcutaneous sponge angiogenesis
assay by injection of the HIF prolyl hydroxylase inhibitor,
dimethyl oxalylglycine.
[0785] In previous studies we have demonstrated that
N-oxalylglycine is an inhibitor of PHD activity in vitro but seems
to be incapable of entering intact cells. Application of an
esterified form, dimethyloxalylglycine, to tissue culture cells
results in stabilisation of HIF alpha chains (Example 1) and
activation of transcription of endogenous HIF target genes (Example
5).
[0786] Implantation of a polyurethane sponge subcutaneously in a
mouse provides an inflammatory stimulus to angiogenesis and is a
well established model for assessing the pro- and anti-angiogenic
effects of compounds. To test the effects of dimethyl oxalylglycine
in vivo, sterile 8 mm sponge discs were inserted under the dorsal
skin of C57 Black mice on Day, 0. Test solutions were injected
through the skin into the sporiges of mice once per day on days 1,
2, 4 and. 5. Individual mice received 100 microlitres aliquots of
either sterile dimethyloxalylglycine (0.1 mM, 1 mM or 10 mM) or
carrier solution. Animals were sacrificed on day 7 and the sponges
removed. The sponges were fixed in 3.7% formaldehyde, paraffin
embedded and stained immunohistochemically for von Willebrand
factor to identify blood vessels.
[0787] Considerably more blood vessels were observed in
sponges-injected with dimethyloxalylglycine (1 mM) than those
receiving solvent alone.
Example 7
The Effect of PK-Tagged PHD1 Expression on HIF-1-.alpha. Induction
by Hypoxia
[0788] In this Example it is shown that a recombinant PHD (PHD1)
may be overexpressed in a tissue culture cell line in such a manner
as to affect the metabolism of a HIF polypeptide.
[0789] U2OS cells were stably transfected with a binary system
encoding a tetracycline operator fused to an activator, and a
plasmid encoding C-terminal PK epitope tagged PHD1 under control of
a tetracycline response element. The transfected cells were
incubated with 21%, 3% or 0%/oxygen in either the presence or
absence of doxycycline for 16 hours. Immunoblots were then
performed on cell lysates to quantify levels of HIF-1.alpha. and
also to check for expression of PHD via the PK tag.
[0790] Exposure of cells to doxycycline for 16 hours induced
expression of PK tagged PHD1. The induced expression of PHD1
substantially reduced expression of HIF-1.alpha. in modest hypoxia
(3% oxygen) and also reduced expression to a lesser extent under
total hypoxia (0% oxygen). Thus in this Example, the expression of
endogenous HIF-1 is shown to be strikingly dependent on the
activity of the specifically induced PHD1 isoform under the
conditions of assay.
Example 8
(S)-2-(Methoxyoxalyl-amino)-pentanedioic acid diethylester (IS12)
or: Diethyl N-methoxyoxalyl-(L)-glutamate (IS12)
[0791] 22
[0792] To a stirred solution of 10 mmol (2.40 g) of diethyl
(L)-glutamate hydrochloride in 10 ml of toluene, 10 mmol (1.23 g,
0.93 ml) of methyl oxalyl chloride was added and heated until no
further HCl gas evolved (4-6 hr). The solvent was evaporated
yielding 2.86 g (9.9 mmol, 99%) of IS12 as a yellowish oil,
[.alpha.].sub.D.sup.25-28.3.degree.(c 1 in methanol); .nu..sub.max
(NaCl)/cm.sup.-1 1738, 1705 (C.dbd.O); .delta..sub.H(200 MHz;
CDCl.sub.3) 1.22, 1.26 (6 H, 2 t, .sup.3J.sub.HH 7.3,
OCH.sub.2CH.sub.3), 1.95-2.47 (3 H, m, CHCH.sub.2CH.sub.2), 3.88 (3
H, s, OCH.sub.3), 4.10 4.20 (4 H, 2 quart, .sup.3J.sub.HH 7.3,
OCH.sub.2CH.sub.3), 4.60 (1 H, ddd, .sup.3J.sub.HH 8.1,
.sup.3J.sub.HH 8.1, .sup.3J.sub.HH 4.8, CH), 7.76 (1 H, d,
.sup.3J.sub.HH 8.1, NH); .delta..sub.C(50 MHz; CDCl.sub.3) 14.1
(OCH.sub.2CH.sub.3), 27.0, 30.1 (CH.sub.2CH.sub.2), 52.1, 53.6 (CH,
OCH.sub.3), 60.8, 62.0 (OCH.sub.2CH.sub.3), 156.1, 160.4, 170.5,
172.4 (C.dbd.O); m/z(AP+) 290 (MH.sup.+, 68%).
Example 9
(S)-2-7(Oxalyl-amino)pentanedioic acid (IS13) or:
N-Oxalyl-(L)-glutamate (IS13)
[0793] 23
[0794] 3 mmol (0.87 g) of IS12 was heated with 5.0 ml of 2 N
aqueous sodium hydroxide solution ensuring 1.1 equivalents of
sodium hydroxide for the sum of the ester functions to be cleaved
in the compound. The reaction was percolated through a column of
`Amberlite IR 120 H` ion exchange resin (previously washed with
water to about pH 4) and cluated with water until pH raised to 4
again. The water evaporated in vacuo and the residue dried in
vacuum. This yielded 0.65 g (2.9 mmol, 97%) of IS13 as ayellowish
hygroscopic solid, mp ca. 60.degree. C.; [.alpha.].sub.D.sup.25
-2.2 (c 1 in methanol); .nu..sub.max (NaCl, MeOH)/cm.sup.-1 1697
(C.dbd.O); .delta..sub.H(200 MHz; D.sub.2O) 1.77-2.35 (3 H, m,
CHCH.sub.2CH.sub.2), 4.30 (1 H, dd, .sup.3J.sub.HH 9.1,
.sup.3J.sub.HH 5.0, CH); .delta..sub.C(50 MHz; D.sub.2O) 25.8, 30.3
(CH.sub.2CH.sub.2), 52.6, (CH), 161.4, 162.9, 174.5, 177.3
(C.dbd.O); m/z(AP-) 218 (M-H.sup.+, 5%), 168 (M-H.sup.+-oxalyl,
85%).
Example 10
(S)-2-(Methoxyoxalyl-amino)-propionic acid (IS68) or:
Methyloxalyl-L-alanine (IS68)
[0795] 24
[0796] This compound was prepared as in Example 8 using 10 mmol
(0.89 g) of (L)-alanine and 10 mmol (1.23 g, 0.93 ml) of methyl
oxalyl chloride yielding 1.96 g crude yellow oil. The crude product
was chromatographed over silica gel (ethyl acetate eluent)
resulting in 1.42 g of IS68 as a yellowish oil, which still
contained traces of impuritics. A pure sample was obtained from
recrysallization from a mixture of ethyl acetate and diethyl ether
(0.39 g, 2.2 mmol, 22%), mp 129-130.degree. C.; 1.1.degree. (c 1 in
MeOH); .nu..sub.max (NaCl, MeOH)/cm.sup.-1 1744, 1693 (C.dbd.O);
.delta..sub.H(200 MHz; DMSO-d.sub.6) 1.35 (3 H, d, .sup.3J.sub.HH
7.3, CHCH.sub.3), 3.43 (1 H, br, COOH), 3.81 (3 H, s, OCH.sub.3),
4.28 (1 H, pseudo-quint, .sup.3J.sub.HH 7.4, CH), 9.16 (1 H, d,
.sup.3J.sub.HH 7.5, NH); .delta..sub.C(50 MHz; DMSO-d.sub.6) 17.3
(CHCH.sub.3), 48.8 (CH), 53.7 (OCH.sub.3), 157.6, 161.8, 173.8
(C.dbd.O).
Example 11
(R)-2-(Methoxyoxalyl-amino)-propionic acid (IS69) or:
Methyloxalyl-D-alanine (IS69)
[0797] 25
[0798] This compound was prepared as for IS68 but with (L)-alanine
substituted by (D)-alanine yielding 0.36 g (2.1 mmol, 21%) of IS69
as a colourless solid, mp 131-132.degree. C.;
[.alpha.].sub.D.sup.25+1.9.degre- e. (c 1 in MeOH). Analytical data
except optical rotation corresponded to those of IS68.
Example 12
(S)-2-(3-Mercapto-propionylamino)-propionic acid (IS37) or:
N-(3-Mercaptopropanoyl)-(L)-alanine (IS37)
[0799] 26
[0800] Prepare according to literature procedure: M. A. Ondetti, D.
W. Cushman, U.S. Pat. No. 4,053,651, 1977, E. R. Squibb & Sons
(Chem. Abstr., Volume 88, 136977). No analytical details but
melting point were given in the literature work. A solution of 4
mmol (1.13 g) of IS20 in 2 ml of water was treated with 1.6 ml of
conc. aqueous ammonia solution for one hour at room temperature,
while a colourless precipitate formed. The mixture was diluted with
water and the solids filtered off. The filtrate was washed with
ethyl acetate, the aqueous phase was acidified with conc.
hydrochloric acid and extracted with ethyl acetate. The combined
organics were washed with water, dried over magnesium sulfate and
evaporated in vacuo resulting in 0.49 g of crude IS37.
Recrystallization from a mixture of ethyl acetate and n-hexane
yielded 0.32 g (1.8 mmol, 45%) of IS37 as a colourless solid, mp
78-79.degree. C.; [.alpha.].sub.D.sup.25 -39.4 (c 1 in methanol);
V.sub.max (NaCl, MeOH)/cm.sup.-1 1728, 1638 (C.dbd.O);
.delta..sub.H(200 MHz; DMSO-d.sub.6) 1.28 (3 H, d, .sup.3J.sub.HH
7.3, CH.sub.3), 2.31 (1 H, t, .sup.3J.sub.HH 7.9, SH), 2.40-2.48,
2.60-2.73 (4 H, 2 m, CH.sub.2CH.sub.2), 4.22 (1 H, quint,
.sup.3J.sub.HH 7.3, CH), 8.26 (1 H, d, .sup.3J.sub.HH 7.3, NH),
12.56 (1 H, br s, COOH); .delta..sub.C(50 MHz; DMSO-d.sub.6) 18.0
(CH.sub.3), 20.8 (CH.sub.2CH.sub.2, second signal covered by DMSO,
recording in CDCl.sub.3 revealed it at 40.0), 48.3, (CH), 171.0,
175.1 (C.dbd.O); m/z(AP-) 176 (M-H.sup.+, 100%).
Example 13
(R) -2-(3-Mercapto-propionylamino)-propionic acid (IS38) or;
N-(3-Mercaptopropanoyl)-(D)-alanine (IS38)
[0801] 27
[0802] The title compound was prepared as for IS37 but with IS20
substituted by IS21 yielding 0.18 g (1.0 mmol, 25%) of IS38 as a
colourless solid, mp 64.degree. C.; [.alpha.].sub.D.sup.25 +39.5 (c
1 in methanol). Analytical data except optical rotation correspond
to those of IS37.
Example 14
(S)-2-(3-Benzoylsulfanyl-propionylamino)-propioptic acid (IS20) or:
N-(3-Benzoylthiopropanoyl)-(L)-alanine (IS20)
[0803] 28
[0804] Prepared according to literature procedure: M. A. Ondetti,
D. W. Cushman, U.S. Pat. No. 4,053,651, 1977, E. R. Squibb &
Sons (Chem. Abstr., Volume 88, 136977). No analytical details but
melting point were given in the literature work.
[0805] In 16.7 ml of 1 N aqueous sodium hydroxide solution, 16.7
mmol (1.48 g) of (L)-alanine were dissolved. After adding another 9
ml of 2 N sodium hydroxide solution at ice temperature, 16.7 mmol
(2.85 g) of 3-bromopropionic acid were added and the reaction was
stirred for 3.5 h at room temperature. A mixture of 18.1 mmol (2.50
g) of thiobenzoic acid and 11.6 mmol (1.6 g) of potassium carbonate
in 16.7 ml of water and 5 ml of THF was than added to the reaction,
which was then stirred overnight. The resultant mixture was
acidified with conc. hydrochloric acid, stirred for 30 min. and
extracted with ethyl acetate. The combined organics were dried and
the solvents evaporated in vacuo. The remaining thick (5.15 g)
yellow oil was crystallized from ether yielding 1.83 g.(6.5 mmol,
39%) of IS20 as a colourless powder, mp 98-99.degree. C.;
[.alpha.].sub.D.sup.25 -19.1 (c 1 in methanol); .nu..sub.max (NaCl,
MeOH)/cm.sup.-1 1730, 1660 (C.dbd.O); .delta..sub.H(200 MHz;
CDCl.sub.3) 1.44 (3 H, d, .sup.3J.sub.HH 7.1, CH.sub.3), 2.66; 3.32
(4 H, 2 d, .sup.3J.sub.HH 7.1, CH.sub.2CH.sub.2), 4.61 (1 H, quint,
.sup.3J.sub.HH 7.1, CH), 6.77 (1 H, d, .sup.3J.sub.HH 7.1, NH),
7.37-7.61, 7.89-7.96 (5 H, 2 m, ar), 10.08 (1 H, br s, COOH);
.delta..sub.C(50 MHz; CDCl.sub.3) 18.0 (CH.sub.3), 24.6, 36.1
(CH.sub.2CH.sub.2), 48.3, (CH), 127.2, 128.7,133.6,136.7 (ar),
171.5, 176.0, 192.4 (C.dbd.O); m/z(AP-) 280 (M-H.sup.+, 10%).
Example 15
(R)-2-(3-Benzoylsulfanyl-propionylamino)-propionic acid (IS21) or:
N-(3-Benzoylthiopropanoyl)-(D)-alanine (IS21)
[0806] 29
[0807] This compound was prepared as for IS20 but with (L)-alanine
substituting for (D)-alanine yielding 1.78 g (6.3 mmol, 38%) of
IS20 as a colourless powder, mp 98-99.degree. C.;
[.alpha.].sub.D.sup.25 +19.1 (c 1 in methanol). Analytical data
except optical rotation corresponded to those of IS20.
Example 16
Peptide Blockade of HIF-.alpha. Degradation Modulates Cellular
Metabolism and Angiogenesis
[0808] 16.1 Introduction
[0809] Ischaemia is a major cause of morbidity and mortality and
effective molecular therapies are being intensively sought.sup.1 2.
The transcription factor hypoxia-inducible factor-1 (HIF) is a
master regulator of the hypoxic response, controlling genes
involved in diverse processes that balance metabolic supply and
demand within tissues.sup.3 4 5. Modulation of HIF activity
therefore provides an attractive approach for the treatment of
ischaemic disease. Furthermore, HIF driven angiogcnesis produces
more mature and less leaky vessels than those generated by
individual growth factors.sup.6 7 8.
[0810] Regulation of HIF is mediated at multiple levels via its a
chain.sup.9 10 11 12 13. It has been reported that PR39, a
macrophage derived peptide, results in HIF accumulation and
angiogenesis.sup.14. Analysis of the HIF.alpha. oxygen-dependent
degradation domains (ODD) by transient transfection studies.sup.11
12 15 16 17 18 suggested a possible specific, alternative approach
to HIF stabilisation. We have used peptides containing the sites of
regulated prolyl hydroxylation identified as necessary in the
previous Examples for proteasomal destruction in the presence of
oxygen mediated by the von Hippel-Lindau E3 (VHL E3) ubiquitin
ligase complex.sup.19 20 21 22. Despite the multiple steps involved
in HIF activation we demonstrate unequivocally that peptides from
two regions of the ODD not only stabilise HIF.alpha. but produce a
transcriptional response that modulates normoxic angiogenesis and
metabolism in vivo, suggesting that the peptides affect mechanisms
that are common to all activation steps. These results indicate
that these polypeptides, or molecules based on them, provide a
possible therapeutic approach for ischaemic tissues.
[0811] 16.2 Overexpression of CODD and NODD Polypeptides can Induce
HRE-Dependent Reporter Gene Expression
[0812] Since normoxic HIF.alpha. degradation is saturable.sup.17
and depends on sub-regions within the ODD we tested whether
peptides encoding the HIF-1.alpha. amino terminal ODD (NODD) and
carboxy terminal ODD (CODD).sup.21 could affect HIF activity as
measured by hypoxia response element (HRE)-dependent reporter gene
expression. The proposed model by which the NODD and CODD peptides
inhibit HIF activity is shown in FIG. 13. Briefly, Degradation is
prevented by the NODD and CODD polypeptides competing for prolyl
hydroxylation and/or VHL binding, thereby blocking subsequent
ubiquitination. We constructed plasmids encoding NODD
(HIF-1-.alpha. aa 343-417) or CODD (HIF-1-.alpha. aa 549-582)
linked to nuclear localisation sequences and a c-myc epitope tag.
These plasmids were transiently co-transfected into U2OS and Hep3B
cells with an HRE-dependent luciferase reporter plasmid. Under
normoxic conditions expression of either ODD derived polypeptide
increased relative luciferase activity after 24 hours to levels
comparable to those induced in the absence of peptide by hypoxia
(results shown in FIG. 13). Transfection of the NODD and CODD
expression plasmids had no effect on luciferase activity from
reporter plasmids lacking functional HRE's, demonstrating that the
effect was mediated via the HRE (results not shown). To confirm
that polypeptide action was being mediated via the endogenous HIF
pathway we repeated this experiment in a mutant Chinese hamster
ovary cell line lacking HIF.alpha. chains (Ka13), and a
HIF-1.alpha. complemented transfectant (KH-1).sup.23. Transfection
of the NODD and CODD plasmids led to enhanced luciferase activity
in KH-1 but not in Ka13 cells (results shown in FIG. 13).
[0813] We tested shorter fragments of NODD and CODD peptides,
defining amino acids 390-417 and amino acids 556-74 as minimal
domains capable of HRE-dependent luciferase activation. (results
shown in FIG. 13). A sequence alignment of these minimal domains
with the equivalent regions from mouse HIF-1-.alpha. is shown in
FIG. 13.
[0814] HIF.alpha. chain degradation depends on recognition by the
VHL E3 ubiquitin ligase following oxygen-dependent enzymatic
hydroxylation of prolyl residues at positions 402 and 564.sup.19 20
21. Using mutated expression plasmids (P402A or P564G) we
demonstrated complete ablation of HRE-dependent induction of
luciferase activity (results shown in FIG. 13), showing the
critical role of these residues for polypeptide function. This
suggested that expression of the NODD and CODD fusion proteins
interfered with degradation of endogenous HIF.alpha. chains, most
likely by interfering with VHL recognition or prolyl hydroxylation
in normoxic cells, and hence provides a potential route to the
therapeutic manipulation of the HIF system.
[0815] 16.3 Stable NODD and CODD Polypeptide Expression Results in
Endogenous HIF-1.alpha. Accumulation
[0816] To explore further the therapeutic potential of NODD and
CODD polypeptides to activate endogenous HIF we stably transfected
U2OS cells with doxycycline-inducible NODD (doxNODD) and CODD
constructs (doxCODD) encoding identical sequences to those used for
transient transfections.
[0817] Cells stably transfected with NODD or CODD controlled by the
tetracycline-inducible system were exposed to doxycycline. Cell
extracts were prepared 0, 16, 24 and 48 hours after exposure to
doxycycline. The extracts of doxNODD (HIF-1.alpha. aa343-417),
doxCODD (HIF-1.alpha. aa549-82) and control cells (empty vector)
were then immunoblotted for HIF-1.alpha. protein. Increased
HIF-1.alpha. signals were detected from 16-48 hours following
doxycycline administration in doxNODD and doxCODD cells but not in
empty vector cells. Levels of HIF-1.alpha. induced by hypoxia were
also measured for comparison. The endogenous HIF-1.alpha. induction
was doxycycline dose (0.2-3.2 .mu.g/ml) and time dependent, peaking
after 48 hours. Maximal levels were about 20% and about 70% of
HIF-1.alpha. levels seen following hypoxic or DFO treatment of the
doxNODD and doxCODD cells respectively. Doxycycline did not induce
HIF-1.alpha. protein in cells transfected with empty vector despite
its weak ability to chelate iron.
[0818] Immunofluorescence microscopy allowed visualisation of both
the c-myc tag of the expressed fusion proteins and endogenous
HIF-1.alpha.. In doxycycline activated doxCODD cells both were
located in nuclei. HIF-1.alpha. expression varied considerably from
cell to cell. In cells which had not been exposed to doxycycline
strong staining was only seen from the endogenous
HIF-1-.alpha..
[0819] Combined treatment of doxCODD cells with doxycycline and
optimal DFO (75 .mu.m) or hypoxic stimuli (1% 0.sub.2) did not lead
to further increases in HIF-1.alpha. signals on immunoblots
confirming that the peptides had no additional action when
endogenous HIF.alpha. chains were fully induced by physiological
stimuli.
[0820] To test directly whether the polypeptides prevented cellular
HIF-1.alpha. targeting by the VHL-ubiquitin-proteasome system we
showed that ubiquitination of exogenous .sup.35S-methionine
labelled HIF-1.alpha. was markedly reduced in the presence of
doxCODD extracts compared with control cell extracts lacking the
peptide transfected with empty vector.
[0821] HIF and HIF-dependent target gene expression has been
suggested to be subject to a number of negative feedback controls.
To investigate the consequences of continuous activation of the
system we exposed doxCODD cells to doxycycline for two, four, six
or eight days. HIF-1.alpha. protein levels were significantly
elevated on days 2 and 4 but decreased thereafter. Switching off
the system by removing doxycycline from the medium for 48 hours
prior to re-exposure resulted in re-induction of elevated
HIF-1.alpha. protein levels, indicating that the suppressive
effects were reversible. This phenomenon will need to be considered
in using the HIF system to modulate complex physiological
downstream effects for example through the administration of
modulators of the invention at spaced intervals or alternatively by
inducing the constructs of the invention at spaced intervals.
[0822] 16.4 NODD and CODD Fusion Proteins Induce Target Gene mRNA
and Protein Levels
[0823] Results thus far presented indicate that under normoxic
conditions NODD and CODD polypeptide expression results in
stabilisation of endogenous HIF-1.alpha. chains and consequent
activation of transiently transfected artificial HRE-dependent
promoters. Expression of natural HIF target genes in chromosomal
DNA may be constrained by other factors. We therefore investigated
peptide modulation of endogenous genes known to be HIF targets.
[0824] Carbonic anhydrase IX (CAIX) is transcriptionally
up-regulated under hypoxic conditions.sup.24. We used a
ribonuclease protection assay to measure CAIX mRNA at intervals
following doxycycline treatment in doxCODD and empty vector
transfected cells. Levels of mRNA were measured at 0, 24 and 48
hours following treatment. SnRNA (small nuclear RNA) was also
probed to ensure equivalent loading. Doxycycline markedly induced
mRNA levels in doxCODD cells after 24 and 48 hours to levels
similar to those obtained by hypoxic incubation. Cells transfected
with empty vector showed no induction of CAIX. Immunoblots
demonstrated an associated increase of CAIX protein, paralleling
detection of the CODD peptide, visualised by immunoblotting using
the c-myc tag. To test the generality of this effect we performed
comparable experiments on glucose transporter-1 (Glut-1) mRNA
expression, obtaining similar results. When doxycycline was
repeatedly added to cellculture medium Glut-1 mRNA, detected by
ribonuclease protection, measured after 0, 2, 4, 6 and 8 days
Glut-1 mRNA levels increased for the first 4 days and then declined
in parallel with the HIF-1.alpha. protein levels as observed above.
Maximal Glut-1 mRNA levels were comparable to those induced
following exposure to 75 .mu.M desferrioxamine.
[0825] To test for the physiological relevance of increased Glut-1
expression we conducted glucose uptake experiments. .sup.3H-glucose
uptake was measured. An enhanced uptake of .sup.3H-glucose was
measured in doxCODD cells compared with empty vector transfected
cells after 24 hours induction with doxycycline. In contrast, basal
levels in cells untreated with doxycycline and hypoxically induced
levels (hypoxia) of .sup.3H-glucose uptake were comparable between
cell lines. (*:P<0.01; Error bars represent the SEM of 3
replicates.)
[0826] Thus, in contrast to control cells, expression of the CODD
polypeptides mimicked the effect of hypoxia by inducing glucose
uptake in stably transfected cells.
[0827] 16.5 Tat-NODD and Tat-CODD Fusion Proteins Enter Cultured
Cells and Induce HIF-1.alpha. under Normoxic Conditions
[0828] Experiments presented above show that-oxygen-dependent gene
expression can be modulated in normoxia by plasmid based expression
of NODD and CODD polypeptides. To extend this approach we chose to
study the effects of transducing comparable peptides into cells.
The transduction domain of HIV tat-protein delivers fused proteins
across cell membranes in a transporter independent mechanism.sup.25
26. We fused the NODD and CODD peptides to the tat-sequence in
combination with HA and HIS tags to facilitate detection and nickel
affinity purification. We did not include exogenous nuclear
localisation sequences because the tat sequence itself is
sufficient for nuclear entry.sup.27.
[0829] We performed VHL E3 interaction assays.sup.21 with tat-NODD
and tat-CODD, demonstrating their ability to undergo the necessary
modifications for interaction with VHL. .sup.31S-Methionine
labelled IVTT products of tat-ODD expression vectors were tested
for their ability to bind to VHL E3 ligase. Concordant with the
ubiquitination assays discussed above NODD (HIF-1.alpha. 343-417)
and CODD (HIF-1.alpha. 549-582) polypeptides, but not their
corresponding proline mutants (HIF-1.alpha. 343-417/P402A and
HIF-1.alpha. 549-582/P564G), bound to VHL E3 ubiquitin ligase after
modification by cell extracts. The .sup.35S-methionine labelled
recombinant polypeptides therefore interacted with VHL, supporting
the results of the ubiquitination experiments discussed above. The
interaction was enhanced by the presence of cell extracts which
promote hydroxylation of the prolines at positions 402 and
564.sup.19. In contrast, no binding occurred using peptides in
which prolines were mutated.
[0830] We next tested, by immunoblotting, if these tat-fusion
proteins could traverse cell membranes and induce HIF-1.alpha.. Two
hours following addition of tat-NODD or tat-CODD fusion proteins to
cell cultures intact peptide was detectable in whole cell protein
extracts by immunoblotting for the HA tag. The HA tag of tat-NODD
(tat-343-417) and tat-CODD (tat-549-582) polypeptides were detected
in cell extracts, following repetitive polypeptide administration
indicating their uptake by the cells. HIF-1.alpha. protein was
induced by the tat-NODD and the tat-CODD polypeptides (0.5 .mu.M),
but not by the corresponding proline mutants. Maximal levels were
comparable to those induced following exposure to 75 .mu.M
desferrioxamine (DFO). In experiments with repetitive polypeptide
administration, endogenous HIF-1.alpha. was detectable in normoxia
20 hours after initial exposure of the cells to fusion proteins. In
controls, using the corresponding mutant peptides lacking the
prolines, we detected no HIF-1.alpha. signals. It has been reported
that denaturation enhances uptake of tat-fused proteins.sup.25.
Denatured tat-NODD and tat-CODD peptides were still able to enter
cells, but were inactive in mediating HIF-1.alpha. upregulation,
perhaps because they were no longer capable of being
hydroxylated.
[0831] 16.6 Endothelial Activation and in vivo Angiogenesis
Assays
[0832] Artificial activation of the HIF signalling pathway using
the methods of the invention should induce angiogenesis and will
therefore be of potential therapeutic use in ischaemic disease. We
tested the effect of polypeptide induced HIF stabilisation in an in
vitro angiogenesis assay, co-culturing human microvascular
endothelial cells (HMEC-1) with empty vector transfected or doxCODD
cells. In view of the possibility of sustained activation inducing
a negative feedback loop we opted to test the effects of
intermittent induction. In doxCODD, but not in empty vector
transfected cells, intermittent exposure to doxycycline over a
period of 5 days led to assembly of co-cultured endothelial cells
into complex tubular structures visualised by immunostaining for
von Willebrand factor but not in control cells. As a positive
control epidermal Growth Factor (EGF; 5 ng/ml), which is known to
induce growth of HMEC-1, was used. To extend these observations
into an in viva model we assayed the effects of injecting
tat-fusion proteins into polyurethane sponges implanted
subcutaneously in mice intermittent injections on days 1, 2, 4 and
5 led to a markedly accelerated angiogenic response assayed on day
7 when compared with sponges injected with proline mutant fusion
proteins, excluding a contribution from the tat component.sup.28.
Immunohistochemistry for von Willebrand factor revealed increased
vessel density in sponges explanted after 7 days following
treatment with tat-CODD, but not with mutant peptide
(tat-CODD/P564G). Staining for VEGF and Glut-1 was enhanced in
tat-NODD or tat-CODD treated animals compared to controls. Cells
surrounding the sponge showed particularly intense staining. The
vessel endothelium within sponges was surrounded by cells
expressing smooth muscle actin.
[0833] 16.7 Summary
[0834] Hypoxia-inducible factor-1 (HIF) is a transcription factor
known to regulate pro-angiogenic genes and modulate metabolism in
response to hypoxic stress. Modulation of HIF activity therefore
provides an attractive theoretical route to ameliorating ischaemic
disease. Under normoxic conditions HIF.alpha. chains are
ubiquitylated and destroyed by the proteasome following enzymatic
hydroxylation of critical prolyl residues. Here we demonstrate use
of polypeptides bearing these prolyl residues to stabilise
endogenous HIF, thereby up-regulating HIF target genes. Peptide
expression in cell cultures affects physiologically important
functions such as glucose transport and leads to tubule formation
by co-cultured endothelial cells. Subcutaneous injection of
polypeptides-results in a markedly accelerated local angiogenic
response and induction of glucose transporter-1 gene expression.
These results demonstrate the feasibility of utilising these
polypeptides to enhance normoxic HIF activity, opening new
therapeutic avenues for ischaemic diseases.
[0835] In this Example we have described the use of polypeptides
which stabilise the hypoxia-regulated transcription factor
HIF-1.alpha.. We provide evidence that complex physiological
systems like glucose uptake and angiogenesis can be induced
strongly, even under normoxic conditions.
[0836] Related molecular approaches to treating ischaemic disease
include use of single growth factors.sup.2 or gene therapy with HIF
based sequences lacking the degradation domains.sup.6 29. The
approach used here has advantages over the former in that it
co-opts the entire physiological response resulting in metabolic
adaptation as well as angiogenesis and provides an alternative to
gene therapy that should be easier to apply.
[0837] Influences of HIF on cancer growth and apoptosis.sup.30 31
lead to concerns that long-term HIF activation might have
deleterious effects, including pro-neoplastic actions. However,
these processes probably require additional events beyond HIF
activation and are likely to have a much longer time course than
that required for therapeutic angiogenesis. Furthermore, the
peptides used here are inherently unstable and act locally,
allowing circumscribed dosing schedules that avoid continued and
general exposure.
[0838] The NODD and CODD polypeptides were effective alone and in
combination. Mechanisms of polypeptide action within cells include
competition for HIF prolyl hydroxylase activity or VHL binding
capacity. Three lines of evidence suggest the latter is more
probable. Firstly, we have demonstrated that these NODD and CODD
fusion proteins bind to VHL, presumably following their own
hydroxylation. Secondly, the action of either peptide is sufficient
to stabilise HIF.alpha. even though it contains both prolyl
residues, which can be targetted by different hydroxylase
isoforms.sup.22. Thirdly, concentrations of synthetic peptide
necessary to quench HIF prolyl hydroxylase activity in cell
extracts are unlikely to be produced in cells.
[0839] Comparison of the NODD and CODD sequences coupled with
structural studies clarifying the nature of their interactions with
VHL and different prolyl hydroxylase isoforms will allow further
refinements to these agents. Use of other protein transduction
domains and/or tissue specific targetting sequences, including
tripeptides such as GFE for lung or RDV for retina, will lead to
new formulations with lower risks of side effects.sup.32 33. The
polypeptides reported here are exciting reagents, allowing
controlled activation of the HIF pathway in normoxia. Animal models
of ischaemia may be used to demonstrate the net therapeutic
benefits of the peptides followed by clinical trials.
[0840] 16.8 Methods
[0841] Plasmids, Transient and Stable Transfections
[0842] Plasmid constructs: For reporter gene assays DNA fragments
encoding HIF-1.alpha. amino acids 343-417, 380-417, 390-417,
390-410, 343-400, 530-95, 530-82, 549-82, 556-74 and 530-62 were
generated by PCR using oligonucleotides containing 5' SacII or 3'
AscI sites and inserted into a pCMV/myc/nuc (Invitrogen) derivative
bearing these sites in frame with the NLS and epitope tag. Site
directed mutagenesis (QuikChange, Stratagene) was used to mutate
the constructs containing HIF-1.alpha. aa343-417 or aa549-82 at
aa402 [cca to gca] or aa564 [cca to ggc] converting prolines to
alanine or glycine respectively. To generate tet-operator dependent
plasmids the open reading frames from the aa343-417 and aa549-82
constructs were subcloned into pUHD 10.sup.34. Fragments coding for
HIF-1.alpha. aa343-417 and aa530-82 (with and without P402A and
P564G mutants) were subcloned into ptat-HA.sup.25. All constructs
were confirmed by DNA sequencing.
[0843] Reporter gene assays. Cells were co-transfected with an HRE
containing reporter gene, pCMV/myc/nuc constructs and a
constitutively expressed beta-galactosidase gene using Fugene6
(RocheMolecular).sup.35. Transfectants were maintained in normoxia
for 24 hours or in hypoxia for the final 16 hours. Luciferase
activities in cell extracts were determined using a commercial kit
(Promega) and a TD-20e luminometer (Turner Designs).
Beta-galactosidase activity was measured spectrophotometrically
using o-nitrophenyl-beta-D-galactopyranoside as substrate.
[0844] Stably transfected cell lines were generated by transfecting
U2OS cells bearing the reverse tetracycline responsive
transactivator.sup.34 and the tetKRAB silencer construct.sup.36
with pUHD/HIF plasmids. Following selection in G418 (1 mg/ml)
individual colonies were picked. DoxNODD (F21) and doxCODD (myc19)
clones expressed pUHD/HIF-1 aa343-417/3 NLS/c-myc and pUHD/HIF-1
aa549-82/3NLS/c-myc respectively.
[0845] mRNA and Protein Detection
[0846] RNA analysis: Total RNA extracted using RNAzol B (Biotec
Laboratories) was analysed by ribonuclease protection using
.sup.32P-GTP labelled Glut-1,CAIX and snRNA (as internal control)
riboprobes using templates previously described.sup.10 24
Immunoblotting: Cell extracts were prepared in buffer (8M urea, 10%
glycerol, 1% SDS, 5 mM DTT, 10 rnM Tris/pH 6.8), separated by
SDS-PAGE and transferred to Immobilon-P membrane (Millipore).
Primary antibodies against HIF-1.alpha., c-myc tag and HA tag were
from Transduction Laboratories, Innogenex and Roche Molecular
respectively.
[0847] Ubiquitination and Interaction Assays
[0848] Empty vector and doxCODD cells grown to confluence were
induced with doxycycline (0.8 .mu.g/ml) for 48 hours and
ubiquitination assays performed using cytoplasmic extracts as
described previously.sup.37.
[0849] For VHL E3 interaction assays .sup.35S-methionine labelled
HIF-1.alpha. substrates were prepared by transcription/translation
using TnT7 rabbit reticulocyte lysate (Promega). 100 .mu.M DFO was
added to the reaction to suppress prolyl hydroxylase activity of
the reticulocyte lysate. Substrate modification was achieved by
incubation of HIF-1.alpha. translate with RCC4 cell lysate in the
presence of ferrous chloride (100 .mu.M). Interaction with VHL E3
was analysed as described previously.sup.21.
[0850] Glucose Uptake
[0851] Empty vector and doxCODD cells were grown to confluence,
exposed to 1%, 21% oxygen or 0.8 .mu.g/ml doxycycline for 16 h,
washed with glucose-ftee DMEM and incubated for 10 min with 1
.mu.Ci/ml 2-deoxy-D .sup.3H-glucose (Amersham, UK), before lysis in
0.5% NT-40, 0.25 M NaCl, 10 mM HEPES/pH 7.6. Glucose uptake was
determined by liquid scintillation counting.sup.35.
[0852] tat-Protein Synthesis and Purification
[0853] HIF-1.alpha.-tat-fusion proteins were purified by sonication
of transformed BL21 pLysS (Novagen) in lysis buffer (0.5% Tween-20,
50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 5 mM imidazole) after 4 hours
induction with 1 mM IPTG. Lysates were spun down at 10.sup.3 g
before loading on a Ni-NTA column (Qiagen). Proteins were eluted
with 100 mM imidazole and desalted on a PD10 column
(Amersham).sup.25 in 10 mM Tris/pH 7.0 or in 10 mM Tris/pH 8.0, 30
mM KCl. Aliquots were snap frozen in liquid nitrogen. Tat-proteins
(0.5 .mu.M final concentration) were given to cultures in DMEM/1%
FCS at the beginning and 8 hours after starting the experiments
before harvesting the cells after 24 hours.
[0854] Angiogenesis Assays
[0855] Tubule Formation Assay. doxCODD or empty vector cells were
co-cultivated with human microvascular endothelial cells (HMEC-1)
in a ratio of 2:1 in DMEM 10% doxycyline free FCS (Clontech), 2 mM
Glutamrine and 100 U/ml Penicillin/100 .mu.g/ml Streptomycin.
Stimulation with doxycycline (0.8 .mu.g/ml) or epidermal growth
factor (5 ng/ml) (Sigma) or control medium was renewed every second
day. On day 5 cells were. fixed with 70% ethanol, pre-blocked with
1% BSA/PBS. Endothelial cells were detected using antibodies to von
Willebrand factor (vWP) (Dako).
[0856] Murine sponge model: Sterile polyurethane sponges (8 mm
diameter) were inserted subcutaneously under the dorsal skin of
anaesthetised black C57 female mice on day 0. On the 1.sup.st,
2.sup.nd, 4.sup.th and 5.sup.th days 100 .mu.l of tat-fusion
proteins (1 .mu.M) in Tris buffer (10 mM, pH 7.0) were injected
into the sponges. On day 7 mice were sacrificed and sponges were
excised with surrounding tissue and fixed in 3.5%
paraformaldehyde.
[0857] Immunohistochemistry. Paraffin embedded sponges were cut
into 6 .mu.m sections, dewaxed with xylene, rehydrated and stained
with vWF (Dako), Glut-1 (Alpha Labs), EGF (Santa Cruz) and smooth
muscle cell actin (DAKO) antibodies. Antigen retrieval, blocking of
sections, secondary, HRP labelled antibodies and chromogenic
reactions were performed according to the manufacturers'
recommendations (DAKO Envision System and Vector Labs ABC
Vectastain).
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Example 17
[0896] Effect of Iron chelation on N-Oxalylglycine Inhibitory
Activity and Direct Comparison of the Inhibitory Activity of a Pair
of Enantiomers.
[0897] To determine whether N-oxalylglycine inhibits HIF-1.alpha.
modification via iron chelation we performed capture assays using a
Gal/HIF-1.alpha./VP 16 fusion protein expressing HIF-1.alpha.
residues 549-582 in the presence of varying concentrations of
inhibitor and iron.
[0898] The unlabelled HIF-1.alpha. substrate was immunopurified on
beads, washed, and aliquots incubated in the presence of RCC4 cell
extract With 0, 20, 50, 200, 500 or 2000 .mu.M N-oxalylglycine and
either 5 or 100 .mu.M FeCl.sub.2. After washing, the beads were
assayed for their ability to interact with 35-S labelled pVHL IVTT,
which was then visualised by fluorography. The results obtained are
shown in FIG. 14A. The amount of pVHL captured is expressed as
relative counts per band. Inhibition of pVHL capture by
N-oxalylglycine increased with the concentration of inhibitor and
was the same regardless of iron concentration. As iron
supplementation has little or no effect on inhibition this shows
that the inhibition of HIF-1.alpha. modification by N-oxalylglycine
is not mediated by iron chelation but by an alternative
mechanism.
[0899] The inhibitory effect of the pair of enantiomers,
N-oxalyl-2S-alanine and N-oxalyl-2R-alanine, on HIF-1.alpha.
modification was also studied. This was done using the same pVHL
capture assay described above again using the Gal/549-582/VP16
fusion protein as a substrate. The effect of 0, 20, 50, 200, 500
and 2000 .mu.M concentrations of each enantiomer on pVHL capture
was then assessed. The results obtained are shown in FIG. 14B.
Again, the-amount of labelled pVHL captured is expressed as
relative counts per band. The results show that there is
approximately one log difference in the ability of the enantiomers
to inhibit modification of the HIF substrate and hence pVHL
capture. N-oxalyl-2S-alanine enantiomer had the higher inhibitory
activity of the two enantiomers.
Example 18
[0900] In vitro screening of potential inhibitors of HIF
modification was performed using a capture assay. A
Gal/HIF-1.alpha./VP16 fusion protein expressing HIF-1.alpha.
residues 549-582 was prepared by IVTT and used as a substrate in
the assay. The unlabelled substrate was immunopurified on beads,
washed, and aliquots incubated in the presence of RCC4 cell
extract, with 100 .mu.M FeCl.sub.2 and 2 mM of the potential
inhibitor. The inhibitors were either dissolved in DMSO or Tris as
indicated. Controls, where no inhibitor but the equivalent amount
of DMSO or Tris was added, were also performed. After washing, the
beads were assayed for their ability to interact with 35-S labelled
pVHL WTT. Hydroxylation of the fusion protein by HIF hydroxylase
present in the cell extract leads to the ability to capture the
labelled pVHL and the amount of labelled protein bound to the
fusion protein can then be measured to determine relative HIF
hydroxylase activity. FIGS. 15 -20 show HIF hydroxylase activity in
the presence of a particular inhibitor relative to that seen in the
absence of the inhibitor (the DMSO/Tris control).
7TABLE 1 Protein Name Species Nucleotide Accession Accession(s)
Egl-9 C. elegans AF178536 GI5923812 GI5923811 CG1114 D.
melanogaster AE003603 AAF52050 Clorf12 H. sapiens AF229245 NP071334
PHD2 AJ310543 gi14547145 Gi14547146 M. musculus AJ310546 PHD1 H.
sapiens BC01723 NP071334 AJ310543 gi14547147 Gi14547148 FALKOR M.
musculus AF340231 Gi13649965 gi13649964 FLJ21620 H. sapiens
AK025273 BAB15101 Colo7838 PHD3 H. sapiens AJ310545 Gi14547150
gi14547149 M. musculus AJ310548 Gi14547243 gi14547242
[0901]
8TABLE 2 Disruption of HIF-VHL In-vivo esterified Induction of HIF
In-vitro inhibitor interaction equivalent in tissue culture NK80 No
NK81 Yes Methylmethoxalyl- No D/L-alanine NK82 No Methylmethoxalyl-
No L/D-alanine NK87 Yes Methylmethoxalyl Yes glycine 2,4 pyridine
Yes 2,4 diethylpyridine No dicarboxylic acid dicarboxylate 2,5
pyridine Not tested 2,5 diethylpyridine No dicarboxylic acid
dicarboxylate 2,6 pyridine Not tested 2,6 diethylpyridine No
dicarboxylic acid dicarboxylate
[0902]
9 TABLE 3 NK80 is 30 NK81 is 31 NK82 is 32 NK87 is 33 IS 1: 34 IS 3
35 IS 4 36 IS 5 37 IS 6 38 IS 7 39 IS 8 40 IS 9 41 NK5 42 NK36 43
NK45 44 NK46 45 NK47 46 NK84 47 EDB 48 Benzohydoxamic acid (BHAA)
49 N-oxaloylamino-L-alanine 50 Dimethyloxaloylglycine (MMOG) 51
Protocatechuic acid 52 IS68 53 IS69 54 IS70 55 IS71 56 C8 57 C9 58
C10 59 C11 60 C14 61 C15 62 IS12 63 IS13 64 IS20 65 IS21 66 IS37 67
IS38 68
[0903] 6970717273747576
10 TABLE 4 Designation Accession No. Protected Fragment EGLN1/PHD2
BC 001723 1136-1481 EGLN2/PHD1 AF 229245 4050-4213 EGLN3/PHD3 AK
025273 817-1046 F22B5.4(C. elegans) 210-359 HIF-1 (C. elegans)
1366-1496
[0904]
11 TABLE 5 Gene Strain Allele daf-18 CB1375 e1375 daf-2 CB1370
e1370 age-1 TJ1052 hx546 mev-1 TK22 knl clk-1 CB4876 e2519 gas-1
CW152 fc21 ctl-1 TU2463 u800 mev-2 TK93 kn2 2mev-3 TK66 kn10 dpy-18
CB364 e364 phy-2 JK2757 ok177 egl-9 MT1201 n571 egl-9 MT1216 n586
egl-9 JT307 sa307 egl-9 JT330 sa330 vhl-1 CB5603 ok161
[0905]
Sequence CWU 1
1
48 1 2110 DNA Homo sapiens CDS (297)...(1517) 1 gctttcccct
gcctgcctgt ctctagtttc tctcacatcc cttttttttt tcctttctct 60
agccaccctg aagggtccct tcccaagccc ttagggaccg cagaggactt ggggaccagc
120 aagcaacccc cagggcacga gaagagctct tgctgtctgc cctgcctcac
cctgccccac 180 accaggcccg gtggccccca gctgcatcaa gtggaggcgg
aggaggaggc ggaggagggt 240 ggcaccatgg gcccgggcgg tgccctccat
gcccggggga tgaagacact gctgcc atg 299 Met 1 gac agc ccg tgc cag ccg
cag ccc cta agt cag gct ctc cct cag tta 347 Asp Ser Pro Cys Gln Pro
Gln Pro Leu Ser Gln Ala Leu Pro Gln Leu 5 10 15 cca ggg tct tcg tca
gag ccc ttg gag cct gag cct ggc cgg gcc agg 395 Pro Gly Ser Ser Ser
Glu Pro Leu Glu Pro Glu Pro Gly Arg Ala Arg 20 25 30 atg gga gtg
gag agt tac ctg ccc tgt ccc ctg ctc ccc tcc tac cac 443 Met Gly Val
Glu Ser Tyr Leu Pro Cys Pro Leu Leu Pro Ser Tyr His 35 40 45 tgt
cca gga gtg cct agt gag gcc tcg gca ggg agt ggg acc ccc aga 491 Cys
Pro Gly Val Pro Ser Glu Ala Ser Ala Gly Ser Gly Thr Pro Arg 50 55
60 65 gcc aca gcc acc tct acc act gcc agc cct ctt cgg gac ggt ttt
ggc 539 Ala Thr Ala Thr Ser Thr Thr Ala Ser Pro Leu Arg Asp Gly Phe
Gly 70 75 80 ggg cag gat ggt ggt gag ctg cgg ccg ctg cag agt gaa
ggc gct gca 587 Gly Gln Asp Gly Gly Glu Leu Arg Pro Leu Gln Ser Glu
Gly Ala Ala 85 90 95 gcg ctg gtc acc aag ggg tgc cag cga ttg gca
gcc cag ggc gca cgg 635 Ala Leu Val Thr Lys Gly Cys Gln Arg Leu Ala
Ala Gln Gly Ala Arg 100 105 110 cct gag gcc ccc aaa cgg aaa tgg gcc
gag gat ggt ggg gat gcc cct 683 Pro Glu Ala Pro Lys Arg Lys Trp Ala
Glu Asp Gly Gly Asp Ala Pro 115 120 125 tca ccc agc aaa cgg ccc tgg
gcc agg caa gag aac cag gag gca gag 731 Ser Pro Ser Lys Arg Pro Trp
Ala Arg Gln Glu Asn Gln Glu Ala Glu 130 135 140 145 cgg gag ggt ggc
atg agc tgc agc tgc agc agt ggc agt ggt gag gcc 779 Arg Glu Gly Gly
Met Ser Cys Ser Cys Ser Ser Gly Ser Gly Glu Ala 150 155 160 agt gct
ggg ctg atg gag gag gcg ctg ccc tct gcg ccc gag cgc ctg 827 Ser Ala
Gly Leu Met Glu Glu Ala Leu Pro Ser Ala Pro Glu Arg Leu 165 170 175
gcc ctg gac tat atc gtg ccc tgc atg cgg tac tac ggc atc tgc gtc 875
Ala Leu Asp Tyr Ile Val Pro Cys Met Arg Tyr Tyr Gly Ile Cys Val 180
185 190 aag gac agc ttc ctg ggg gca gca ctg ggc ggt cgc gtg ctg gcc
gag 923 Lys Asp Ser Phe Leu Gly Ala Ala Leu Gly Gly Arg Val Leu Ala
Glu 195 200 205 gtg gag gcc ctc aaa cgg ggt ggg cgc ctg cga gac ggg
cag cta gtg 971 Val Glu Ala Leu Lys Arg Gly Gly Arg Leu Arg Asp Gly
Gln Leu Val 210 215 220 225 agc cag agg gcg atc ccg ccg cgc agc atc
cgt ggg gac cag att gcc 1019 Ser Gln Arg Ala Ile Pro Pro Arg Ser
Ile Arg Gly Asp Gln Ile Ala 230 235 240 tgg gtg gaa ggc cat gaa cca
ggc tgt cga agc att ggt gcc ctc atg 1067 Trp Val Glu Gly His Glu
Pro Gly Cys Arg Ser Ile Gly Ala Leu Met 245 250 255 gcc cat gtg gac
gcc gtc atc cgc cac tgc gca ggg cgg ctg ggc agc 1115 Ala His Val
Asp Ala Val Ile Arg His Cys Ala Gly Arg Leu Gly Ser 260 265 270 tat
gtc atc aac ggg cgc acc aag gcc atg gtg gcg tgt tac cca ggc 1163
Tyr Val Ile Asn Gly Arg Thr Lys Ala Met Val Ala Cys Tyr Pro Gly 275
280 285 aac ggg ctc ggg tac gta agg cac gtt gac aat ccc cac ggc gat
ggg 1211 Asn Gly Leu Gly Tyr Val Arg His Val Asp Asn Pro His Gly
Asp Gly 290 295 300 305 cgc tgc atc acc tgt atc tat tac ctg aat cag
aac tgg gac gtt aag 1259 Arg Cys Ile Thr Cys Ile Tyr Tyr Leu Asn
Gln Asn Trp Asp Val Lys 310 315 320 gtg cat ggc ggc ctg ctg cag atc
ttc cct gag ggc cgg ccc gtg gta 1307 Val His Gly Gly Leu Leu Gln
Ile Phe Pro Glu Gly Arg Pro Val Val 325 330 335 gcc aac atc gag cca
ctc ttt gac cgg ttg ctc att ttc tgg tct gac 1355 Ala Asn Ile Glu
Pro Leu Phe Asp Arg Leu Leu Ile Phe Trp Ser Asp 340 345 350 cgg cgg
aac ccc cac gag gtg aag cca gcc tat gcc acc agg tac gcc 1403 Arg
Arg Asn Pro His Glu Val Lys Pro Ala Tyr Ala Thr Arg Tyr Ala 355 360
365 atc act gtc tgg tat ttt gat gcc aag gag cgg gca gca gcc aaa gac
1451 Ile Thr Val Trp Tyr Phe Asp Ala Lys Glu Arg Ala Ala Ala Lys
Asp 370 375 380 385 aag tat cag cta gca tca gga cag aaa ggt gtc caa
gta cct gta tca 1499 Lys Tyr Gln Leu Ala Ser Gly Gln Lys Gly Val
Gln Val Pro Val Ser 390 395 400 cag ccg cct acg ccc acc tagtggccag
tcccagagcc gcatggcaga 1547 Gln Pro Pro Thr Pro Thr 405 cagcttaaat
gacttcagga gagccctggg cctgtgctgg ctgctccttc cctgccaccg 1607
ctgctgcttc tgactttgcc tctgtcctgc ctggtgtgga gggctctgtc tgttgctgag
1667 gaccaaggag gagaagagac ctttgctgcc ccatcatggg ggctggggtt
gtcacctgga 1727 cagggggcag ccgtggaggc caccgttacc aactgaagct
gggggcctgg gtcctaccct 1787 gtctggtcat gaccccatta ggtatggaga
gctgggagga ggcattgtca cttcccacca 1847 ggatgcagga cttggggttg
aggtgagtca tggcctcttg ctggcaatgg ggtgggagga 1907 gtacccccaa
gtcctctcac tcctccagcc tggaatgtga agtgactccc caaccccttt 1967
ggccatggca ggcacctttt ggactgggct gccactgctt gggcagagta aaaggtgcca
2027 ggaggagcat gggtgtggaa gtcctgtcag ccaagaaata aaagtttacc
tcagagctgc 2087 aaaaaaaaaa aaaaaaaaaa aaa 2110 2 407 PRT Homo
sapiens 2 Met Asp Ser Pro Cys Gln Pro Gln Pro Leu Ser Gln Ala Leu
Pro Gln 1 5 10 15 Leu Pro Gly Ser Ser Ser Glu Pro Leu Glu Pro Glu
Pro Gly Arg Ala 20 25 30 Arg Met Gly Val Glu Ser Tyr Leu Pro Cys
Pro Leu Leu Pro Ser Tyr 35 40 45 His Cys Pro Gly Val Pro Ser Glu
Ala Ser Ala Gly Ser Gly Thr Pro 50 55 60 Arg Ala Thr Ala Thr Ser
Thr Thr Ala Ser Pro Leu Arg Asp Gly Phe 65 70 75 80 Gly Gly Gln Asp
Gly Gly Glu Leu Arg Pro Leu Gln Ser Glu Gly Ala 85 90 95 Ala Ala
Leu Val Thr Lys Gly Cys Gln Arg Leu Ala Ala Gln Gly Ala 100 105 110
Arg Pro Glu Ala Pro Lys Arg Lys Trp Ala Glu Asp Gly Gly Asp Ala 115
120 125 Pro Ser Pro Ser Lys Arg Pro Trp Ala Arg Gln Glu Asn Gln Glu
Ala 130 135 140 Glu Arg Glu Gly Gly Met Ser Cys Ser Cys Ser Ser Gly
Ser Gly Glu 145 150 155 160 Ala Ser Ala Gly Leu Met Glu Glu Ala Leu
Pro Ser Ala Pro Glu Arg 165 170 175 Leu Ala Leu Asp Tyr Ile Val Pro
Cys Met Arg Tyr Tyr Gly Ile Cys 180 185 190 Val Lys Asp Ser Phe Leu
Gly Ala Ala Leu Gly Gly Arg Val Leu Ala 195 200 205 Glu Val Glu Ala
Leu Lys Arg Gly Gly Arg Leu Arg Asp Gly Gln Leu 210 215 220 Val Ser
Gln Arg Ala Ile Pro Pro Arg Ser Ile Arg Gly Asp Gln Ile 225 230 235
240 Ala Trp Val Glu Gly His Glu Pro Gly Cys Arg Ser Ile Gly Ala Leu
245 250 255 Met Ala His Val Asp Ala Val Ile Arg His Cys Ala Gly Arg
Leu Gly 260 265 270 Ser Tyr Val Ile Asn Gly Arg Thr Lys Ala Met Val
Ala Cys Tyr Pro 275 280 285 Gly Asn Gly Leu Gly Tyr Val Arg His Val
Asp Asn Pro His Gly Asp 290 295 300 Gly Arg Cys Ile Thr Cys Ile Tyr
Tyr Leu Asn Gln Asn Trp Asp Val 305 310 315 320 Lys Val His Gly Gly
Leu Leu Gln Ile Phe Pro Glu Gly Arg Pro Val 325 330 335 Val Ala Asn
Ile Glu Pro Leu Phe Asp Arg Leu Leu Ile Phe Trp Ser 340 345 350 Asp
Arg Arg Asn Pro His Glu Val Lys Pro Ala Tyr Ala Thr Arg Tyr 355 360
365 Ala Ile Thr Val Trp Tyr Phe Asp Ala Lys Glu Arg Ala Ala Ala Lys
370 375 380 Asp Lys Tyr Gln Leu Ala Ser Gly Gln Lys Gly Val Gln Val
Pro Val 385 390 395 400 Ser Gln Pro Pro Thr Pro Thr 405 3 5163 DNA
Homo sapiens CDS (3157)...(4434) 3 ttaggggcag aaaaacattt gtaataatta
atggctttga gagacacaag gctttgtttg 60 ccccagagta ttagttaacc
cacctagtgc tcctaatcat acaatattaa ggattgggag 120 ggacattcat
tgcctcactc tctatttgtt tcaccttctg taaaattggt agaataatag 180
tacccacttc atagcattgt atgatgatta aattggttaa tatttttaaa atgcttagaa
240 cacagattgg gcacataaca gcaagcacca catgtgttta taagataaat
tcctttgtgt 300 tgccttccgt taaagtttaa ataagtaaat aaataaataa
atacttgcat gacattttga 360 agtctctcta taacatctga gtaagtggcg
gctgcgacaa tgctactgga gttccagaat 420 cgtgttggtg acaagattgt
tcaccagcat atggtgtggt gaaaactcac taatttggaa 480 ttagttcaga
ttattaagcc tgaataggtg aaaatcctga aatcaaggat ctttggaact 540
atttgaaatc agtattttat attttcctgt tgtattcatt aaagtgttgc aagtgttcta
600 tttgatggat taagtatatt taggatatac atgttcaatt tgtgattttg
tatacttaat 660 tggaacaaga aagctaataa aggttttgat atggacatct
attcttttaa gtaaacttca 720 atgaaaatat atgagtagag catatagaga
tgtaaataat ttgtggacac accacagact 780 gaaatagcaa atttaaaaga
aattgttgga agaatcaagt gtttgtggaa tgagtcctcc 840 tagtaaagtt
cctgctcttg tgaataatta agcctcatgt ataattacta tagcaaaagg 900
aagcctaaga agtattagac tctacttgta tttaaattac attttacata atttatgtgt
960 atgaaaaatg ttttaaatgc ttattttcgt aagccatgag atagctcctt
tatattttaa 1020 gaatttctga attaatttgc ttggatttta ttagtgcaaa
tggcagagct agcaattcct 1080 ttttctgtgt tcccattcca tcctattcat
ccctctttta ggaaactctg aactctggat 1140 tgtccttgtt tacatacctg
cctcctgcat tggactatgt gtctctgagt gtagtatgac 1200 taattcattt
gtttgtcaag gactctcaat gcatttgttg aacagcctaa ttagtaatgt 1260
ctgcaacaat gacattttac tgtatttaat aaagctctgg gaaagtagga tacacataag
1320 acaggtctag gtctaaattc tttacagaaa cttggatttt tagttcggtt
tgaaatttga 1380 agatgtgagt atatttatct cagtttccca aaggacaagc
taattggaat tatcatcctc 1440 tttcacttga ttggatcccc agaatgccat
ttacgcatgc agcaggattt tataacagtt 1500 ttaaattctg tatatttgat
gaagaggttt tatatttttg gattcaagcc tctttttaaa 1560 cttctacaat
atggtttaca ataattcctt atatcctgct tttgaaatac atattacaac 1620
tttttaagtt tggaaggcta tatttcaagg actgaagtta cagtatactc aagtgataca
1680 caagcctagc accccacttt ccacatagtg ttcgataaag attgataaac
tcgaaatcac 1740 agacctttta attcttaaga caaatagcag cagaaagaaa
catctttggc ttatttctgg 1800 taaggttttt atgctctgta aaacaaagaa
ttgtattcat ccgcgcagca cagattctat 1860 taaaaataaa tgtgagagtc
gttaatgtag tactgctcat ttaccatcaa aattcacttt 1920 tcaggaataa
tcccatcagt ttaaattgga tattggaatg agcattgatt acatttaact 1980
tggtagccca aaatttcttc atggggtttt gaactcggcg ggatttcaaa ggttttaaaa
2040 atgagttttt gatttttttt aaaaccctca aatttcatta cctttaaact
aggtcgaaac 2100 ggggcgcaag agattggatt aacaccatag taatacttat
tttgttctta accatttcag 2160 ggcttcttga aatagaggct gtatggtgta
atggaaaaaa cagccttgga atctgggagc 2220 ctgattcctg gattcagtcc
cagttttgcg tgaccttggg caagttactt tacttctctg 2280 aatttccgtt
tcctcctctg caaaatgagg atcgcaatag ccaccttgca accttgactg 2340
gagcgagcct cgcacacccc gcgccggcct ggaggaagag cagccatgat tacgccgcct
2400 tcgctccgct acccgcttgc ggctggcgcc ctcctccagc aggtgtaggc
gctgccgcgc 2460 tgccccacgc ctttccgccg ctcgcgggcc tgcgcctcgg
cgtccccgag gaggccgctg 2520 cgggctgagg tagcgcaccg gcctctcggc
gtcccagtcc ggtcccgggc ggagggaaag 2580 cgggcgaccc acctccgagg
cagaagccga ggcccggccc cgccgagtgc ggaggagcgc 2640 aggcagcccc
cgcccctcgg ccctcccccc ggccctcccg gccctccctc cgccccctcc 2700
gccctcgcgc gccgcccgcc cgggtcgccg cggggccgtg gtgtacgtgc agagcgcgca
2760 gagcgagtgg cgcccgtatg ccctgcgctc ctccacagcc tgggccgggc
cgcccgggac 2820 gctgaggcgg cggcggcggc cgagggggcc ggtcttgcgc
tccccaggcc cgcgcgcctg 2880 agcccaggtt gccattcgcc gcacaggccc
tattctctca gccctcggcg gcgatgaggc 2940 gctgaggcgg ctgccggcgc
tgcgccggag cttaggactc ggaagcggcc gggccgaggg 3000 cgtggggtgc
cggcctccct gaggcgaggg tagcgggtgc atggcgcagt aacggcccct 3060
atctctctcc ccgctcccca gcctcgggcg aggccgtccg gccgctaccc ctcctgctcg
3120 gccgccgcag tcgccgtcgc cgccgccgcc gccgcc atg gcc aat gac agc
ggc 3174 Met Ala Asn Asp Ser Gly 1 5 ggg ccc ggc ggg ccg agc ccg
agc gag cga gac cgg cag tac tgc gag 3222 Gly Pro Gly Gly Pro Ser
Pro Ser Glu Arg Asp Arg Gln Tyr Cys Glu 10 15 20 ctg tgc ggg aag
atg gag aac ctg ctg cgc tgc agc cgc tgc cgc agc 3270 Leu Cys Gly
Lys Met Glu Asn Leu Leu Arg Cys Ser Arg Cys Arg Ser 25 30 35 tcc
ttc tac tgc tgc aag gag cac cag cgt cag gac tgg aag aag cac 3318
Ser Phe Tyr Cys Cys Lys Glu His Gln Arg Gln Asp Trp Lys Lys His 40
45 50 aag ctc gtg tgc cag ggc agc gag ggc gcc ctc ggc cac gga gtg
ggc 3366 Lys Leu Val Cys Gln Gly Ser Glu Gly Ala Leu Gly His Gly
Val Gly 55 60 65 70 cca cac cag cat tcc ggc ccc gcg ccg ccg gct gca
gtg ccg ccg ccc 3414 Pro His Gln His Ser Gly Pro Ala Pro Pro Ala
Ala Val Pro Pro Pro 75 80 85 agg gcc ggg gcc cgg gag ccc agg aag
gca gcg gcg cgc cgg gac aac 3462 Arg Ala Gly Ala Arg Glu Pro Arg
Lys Ala Ala Ala Arg Arg Asp Asn 90 95 100 gcc tcc ggg gac gcg gcc
aag gga aaa gta aag gcc aag ccc ccg gcc 3510 Ala Ser Gly Asp Ala
Ala Lys Gly Lys Val Lys Ala Lys Pro Pro Ala 105 110 115 gac cca gcg
gcg gcc gcg tcg ccg tgt cgt gcg gcc gcc ggc ggc cag 3558 Asp Pro
Ala Ala Ala Ala Ser Pro Cys Arg Ala Ala Ala Gly Gly Gln 120 125 130
ggc tcg gcg gtg gct gcc gaa gcc gag ccc ggc aag gag gag ccg ccg
3606 Gly Ser Ala Val Ala Ala Glu Ala Glu Pro Gly Lys Glu Glu Pro
Pro 135 140 145 150 gcc cgc tca tcg ctg ttc cag gag aag gcg aac ctg
tac ccc cca agc 3654 Ala Arg Ser Ser Leu Phe Gln Glu Lys Ala Asn
Leu Tyr Pro Pro Ser 155 160 165 aac acg ccc ggg gat gcg ctg agc ccc
ggc ggc ggc ctg cgg ccc aac 3702 Asn Thr Pro Gly Asp Ala Leu Ser
Pro Gly Gly Gly Leu Arg Pro Asn 170 175 180 ggg cag acg aag ccc ctg
ccg gcg ctg aag ctg gcg ctc gag tac atc 3750 Gly Gln Thr Lys Pro
Leu Pro Ala Leu Lys Leu Ala Leu Glu Tyr Ile 185 190 195 gtg ccg tgc
atg aac aag cac ggc atc tgt gtg gtg gac gac ttc ctc 3798 Val Pro
Cys Met Asn Lys His Gly Ile Cys Val Val Asp Asp Phe Leu 200 205 210
ggc aag gag acc gga cag cag atc ggc gac gag gtg cgc gcc ctg cac
3846 Gly Lys Glu Thr Gly Gln Gln Ile Gly Asp Glu Val Arg Ala Leu
His 215 220 225 230 gac acc ggg aag ttc acg gac ggg cag ctg gtc agc
cag aag agt gac 3894 Asp Thr Gly Lys Phe Thr Asp Gly Gln Leu Val
Ser Gln Lys Ser Asp 235 240 245 tcg tcc aag gac atc cga ggc gat aag
atc acc tgg atc gag ggc aag 3942 Ser Ser Lys Asp Ile Arg Gly Asp
Lys Ile Thr Trp Ile Glu Gly Lys 250 255 260 gag ccc ggc tgc gaa acc
att ggg ctg ctc atg agc agc atg gac gac 3990 Glu Pro Gly Cys Glu
Thr Ile Gly Leu Leu Met Ser Ser Met Asp Asp 265 270 275 ctg ata cgc
cac tgt aac ggg aag ctg ggc agc tac aaa atc aat ggc 4038 Leu Ile
Arg His Cys Asn Gly Lys Leu Gly Ser Tyr Lys Ile Asn Gly 280 285 290
cgg acg aaa gcc atg gtt gct tgt tat ccg ggc aat gga acg ggt tat
4086 Arg Thr Lys Ala Met Val Ala Cys Tyr Pro Gly Asn Gly Thr Gly
Tyr 295 300 305 310 gta cgt cat gtt gat aat cca aat gga gat gga aga
tgt gtg aca tgt 4134 Val Arg His Val Asp Asn Pro Asn Gly Asp Gly
Arg Cys Val Thr Cys 315 320 325 ata tat tat ctt aat aaa gac tgg gat
gcc aag gta agt gga ggt ata 4182 Ile Tyr Tyr Leu Asn Lys Asp Trp
Asp Ala Lys Val Ser Gly Gly Ile 330 335 340 ctt cga att ttt cca gaa
ggc aaa gcc cag ttt gct gac att gaa ccc 4230 Leu Arg Ile Phe Pro
Glu Gly Lys Ala Gln Phe Ala Asp Ile Glu Pro 345 350 355 aaa ttt gat
aga ctg ctg ttt ttc tgg tct gac cgt cgc aac cct cat 4278 Lys Phe
Asp Arg Leu Leu Phe Phe Trp Ser Asp Arg Arg Asn Pro His 360 365 370
gaa gta caa cca gca tat gct aca agg tac gca ata act gtt tgg tat
4326 Glu Val Gln Pro Ala Tyr Ala Thr Arg Tyr Ala Ile Thr Val Trp
Tyr 375 380 385 390 ttt gat gca gat gag aga gca cga gct aaa gta aaa
tat cta aca ggt 4374 Phe Asp Ala Asp Glu Arg Ala Arg Ala Lys Val
Lys Tyr Leu Thr Gly 395 400 405 gaa aaa ggt gtg agg gtt gaa ctc aat
aaa cct tca gat tcg gtc ggt 4422 Glu Lys Gly Val Arg Val Glu Leu
Asn Lys Pro Ser Asp Ser Val Gly 410 415 420 aaa gac gtc ttc
tagagccttt gatccagcaa taccccactt cacctacaat 4474 Lys Asp Val Phe
425 attgttaact atttgttaac ttgtgaatac gaataaatgg gataaagaaa
aatagacaac 4534 cagttcgcat tttaataagg aaacagaaac aactttttgt
gttgcatcaa acagaagatt 4594 ttgactgctg tgactttgta ctgcatgatc
aacttcaaat ctgtgattgc ttacaggagg 4654 aagataagct actaattgaa
aatggttttt acatctggat atgaaataag tgccctgtgt 4714 agaatttttt
tcattcttat attttgccag atctgttatc tagctgagtt catttcatct 4774
ctcccttttt tatatcaagt ttgaatttgg gataattttt ctatattagg tacaatttat
4834 ctaaactgaa ttgagaaaaa attacagtat tattcctcaa aataacatca
atctattttt 4894 gtaaacctgt tcatactatt aaattttgcc ctaaaagacc
tcttaataat gattgttgcc 4954 agtgactgat gattaatttt attttactta
aaataagaaa aggagcactt taattacaac 5014 tgaaaaatca gattgttttg
cagtccttcc ttacactaat ttgaactctt aaagattgct 5074 gctttttttt
tgacattgtc aataacgaaa cctaattgta aaacagtcac catttactac 5134
caataacttt tagttaatgt tttacaagg 5163 4 426 PRT Homo sapiens 4 Met
Ala Asn Asp Ser Gly Gly Pro Gly Gly Pro Ser Pro Ser Glu Arg 1 5 10
15 Asp Arg Gln Tyr Cys Glu Leu Cys Gly Lys Met Glu Asn Leu Leu Arg
20 25 30 Cys Ser Arg Cys Arg Ser Ser Phe Tyr Cys Cys Lys Glu His
Gln Arg 35 40 45 Gln Asp Trp Lys Lys His Lys Leu Val Cys Gln Gly
Ser Glu Gly Ala 50 55 60 Leu Gly His Gly Val Gly Pro His Gln His
Ser Gly Pro Ala Pro Pro 65 70 75 80 Ala Ala Val Pro Pro Pro Arg Ala
Gly Ala Arg Glu Pro Arg Lys Ala 85 90 95 Ala Ala Arg Arg Asp Asn
Ala Ser Gly Asp Ala Ala Lys Gly Lys Val 100 105 110 Lys Ala Lys Pro
Pro Ala Asp Pro Ala Ala Ala Ala Ser Pro Cys Arg 115 120 125 Ala Ala
Ala Gly Gly Gln Gly Ser Ala Val Ala Ala Glu Ala Glu Pro 130 135 140
Gly Lys Glu Glu Pro Pro Ala Arg Ser Ser Leu Phe Gln Glu Lys Ala 145
150 155 160 Asn Leu Tyr Pro Pro Ser Asn Thr Pro Gly Asp Ala Leu Ser
Pro Gly 165 170 175 Gly Gly Leu Arg Pro Asn Gly Gln Thr Lys Pro Leu
Pro Ala Leu Lys 180 185 190 Leu Ala Leu Glu Tyr Ile Val Pro Cys Met
Asn Lys His Gly Ile Cys 195 200 205 Val Val Asp Asp Phe Leu Gly Lys
Glu Thr Gly Gln Gln Ile Gly Asp 210 215 220 Glu Val Arg Ala Leu His
Asp Thr Gly Lys Phe Thr Asp Gly Gln Leu 225 230 235 240 Val Ser Gln
Lys Ser Asp Ser Ser Lys Asp Ile Arg Gly Asp Lys Ile 245 250 255 Thr
Trp Ile Glu Gly Lys Glu Pro Gly Cys Glu Thr Ile Gly Leu Leu 260 265
270 Met Ser Ser Met Asp Asp Leu Ile Arg His Cys Asn Gly Lys Leu Gly
275 280 285 Ser Tyr Lys Ile Asn Gly Arg Thr Lys Ala Met Val Ala Cys
Tyr Pro 290 295 300 Gly Asn Gly Thr Gly Tyr Val Arg His Val Asp Asn
Pro Asn Gly Asp 305 310 315 320 Gly Arg Cys Val Thr Cys Ile Tyr Tyr
Leu Asn Lys Asp Trp Asp Ala 325 330 335 Lys Val Ser Gly Gly Ile Leu
Arg Ile Phe Pro Glu Gly Lys Ala Gln 340 345 350 Phe Ala Asp Ile Glu
Pro Lys Phe Asp Arg Leu Leu Phe Phe Trp Ser 355 360 365 Asp Arg Arg
Asn Pro His Glu Val Gln Pro Ala Tyr Ala Thr Arg Tyr 370 375 380 Ala
Ile Thr Val Trp Tyr Phe Asp Ala Asp Glu Arg Ala Arg Ala Lys 385 390
395 400 Val Lys Tyr Leu Thr Gly Glu Lys Gly Val Arg Val Glu Leu Asn
Lys 405 410 415 Pro Ser Asp Ser Val Gly Lys Asp Val Phe 420 425 5
2770 DNA Homo sapiens CDS (327)...(1043) 5 gagtctggcc gcagtcgcgg
cagtggtggc ttcccatccc caaaaggcgc cctccgactc 60 cttgcgccgc
actgctcgcc gggccagtcc ggaaacgggt cgtggagctc cgcaccactc 120
ccgctggttc ccgaaggcag atcccttctc ccgagagttg cgagaaactt tcccttgtcc
180 ccgacgctgc agcggctcgg gtaccgtggc agccgcaggt ttctgaaccc
cgggccacgc 240 tccccgcgcc tcggcttcgc gctcgtgtag atcgttccct
ctctggttgc acgctgggga 300 tcccggacct cgattctgcg ggcgag atg ccc ctg
gga cac atc atg agg ctg 353 Met Pro Leu Gly His Ile Met Arg Leu 1 5
gac ctg gag aaa att gcc ctg gag tac atc gtg ccc tgt ctg cac gag 401
Asp Leu Glu Lys Ile Ala Leu Glu Tyr Ile Val Pro Cys Leu His Glu 10
15 20 25 gtg ggc ttc tgc tac ctg gac aac ttc ctg ggc gag gtg gtg
ggc gac 449 Val Gly Phe Cys Tyr Leu Asp Asn Phe Leu Gly Glu Val Val
Gly Asp 30 35 40 tgc gtc ctg gag cgc gtc aag cag ctg cac tgc acc
ggg gcc ctg cgg 497 Cys Val Leu Glu Arg Val Lys Gln Leu His Cys Thr
Gly Ala Leu Arg 45 50 55 gac ggc cag ctg gcg ggg ccg cgc gcc ggc
gtc tcc aag cga cac ctg 545 Asp Gly Gln Leu Ala Gly Pro Arg Ala Gly
Val Ser Lys Arg His Leu 60 65 70 cgg ggc gac cag atc acg tgg atc
ggg ggc aac gag gag ggc tgc gag 593 Arg Gly Asp Gln Ile Thr Trp Ile
Gly Gly Asn Glu Glu Gly Cys Glu 75 80 85 gcc atc agc ttc ctc ctg
tcc ctc atc gac agg ctg gtc ctc tac tgc 641 Ala Ile Ser Phe Leu Leu
Ser Leu Ile Asp Arg Leu Val Leu Tyr Cys 90 95 100 105 ggg agc cgg
ctg ggc aaa tac tac gtc aag gag agg tct aag gca atg 689 Gly Ser Arg
Leu Gly Lys Tyr Tyr Val Lys Glu Arg Ser Lys Ala Met 110 115 120 gtg
gct tgc tat ccg gga aat gga aca ggt tat gtt cgc cac gtg gac 737 Val
Ala Cys Tyr Pro Gly Asn Gly Thr Gly Tyr Val Arg His Val Asp 125 130
135 aac ccc aac ggt gat ggt cgc tgc atc acc tgc atc tac tat ctg aac
785 Asn Pro Asn Gly Asp Gly Arg Cys Ile Thr Cys Ile Tyr Tyr Leu Asn
140 145 150 aag aat tgg gat gcc aag cta cat ggt ggg atc ctg cgg ata
ttt cca 833 Lys Asn Trp Asp Ala Lys Leu His Gly Gly Ile Leu Arg Ile
Phe Pro 155 160 165 gag ggg aaa tca ttc ata gca gat gtg gag ccc att
ttt gac aga ctc 881 Glu Gly Lys Ser Phe Ile Ala Asp Val Glu Pro Ile
Phe Asp Arg Leu 170 175 180 185 ctg ttc ttc tgg tca gat cgt agg aac
cca cac gaa gtg cag ccc tct 929 Leu Phe Phe Trp Ser Asp Arg Arg Asn
Pro His Glu Val Gln Pro Ser 190 195 200 tac gca acc aga tat gct atg
act gtc tgg tac ttt gat gct gaa gaa 977 Tyr Ala Thr Arg Tyr Ala Met
Thr Val Trp Tyr Phe Asp Ala Glu Glu 205 210 215 agg gca gaa gcc aaa
aag aaa ttc agg aat tta act agg aaa act gaa 1025 Arg Ala Glu Ala
Lys Lys Lys Phe Arg Asn Leu Thr Arg Lys Thr Glu 220 225 230 tct gcc
ctc act gaa gac tgaccgtgct ctgaaatctg ctggccttgt 1073 Ser Ala Leu
Thr Glu Asp 235 tcattttagt aacggttcct gaattctctt aaattctttg
agatccaaag atggcctctt 1133 cagtgacaac aatctccctg ctacttcttg
catccttcac atccctgtct tgtgtgtggt 1193 acttcatgtt ttcttgccaa
gactgtgttg atcttcagat actctctttg ccagatgaag 1253 ttatttgcta
actccagaaa ttcctgcaga catcctactc ggccagcggt ttacctgata 1313
gattcggtaa tactatcaag agaagagcct aggagcacag cgagggaatg aaccttactt
1373 gcactttatg tatacttcct gatttgaaag gaggaggttt gaaaagaaaa
aaatggaggt 1433 ggtagatgcc acagagaggc atcacggaag ccttaacagc
aggaaacaga gaaatttgtg 1493 tcatctgaac aatttccaga tgttcttaat
ccagggctgt tggggtttct ggagaattat 1553 cacaacctaa tgacattaat
acctctagaa agggctgctg tcatagtgaa caatttataa 1613 gtgtcccatg
gggcagacac tccttttttc ccagtcctgc aacctggatt ttctgcctca 1673
gctccatttt gctgaaaata atgactttct gaataaagat ggcaacacaa ttttttctcc
1733 attttcagtt cttacctggg aacctaattc cccagaagct aaaaaactag
acattagttg 1793 ttttggttgc tttgttggaa tggaatttaa atttaaatga
aaggaaaaat atatccctgg 1853 tagttttgtg ttaaccactg ataactgtgg
aaagagctag gtctactgat atacaataaa 1913 catgtgtgca tcttgaacaa
tttgagaggg gaggtggagt tggaaatgtg ggtgttcctg 1973 tttttttttt
tttttttttt tttttttagt tttccttttt aatgagctca ccctttaaca 2033
caaaaaaagc agggtgatgt attttaaaaa aggaagtgga aataaaaaaa tctcaaagct
2093 atttgagttc tcgtctgtcc ctagcagtct ttcttcagct cacttggctc
tctagatcca 2153 ctgtggttgg cagtatgacc agaatcatgg aacttgctag
aactgtggaa gcttctactc 2213 ctgcagtaag cacagatcgc actgcctcaa
taacttggta ttgagcacgt attttgcaaa 2273 agctactttt cctagttttc
agtattactt tcatgtttta aaaatccctt taatttcttg 2333 cttgaaaatc
ccatgaacat taaagagcca gaaatatttt cctttgttat gtacggatat 2393
atatatatat atagtcttcc aagatagaag tttacttttt cctcttctgg ttttggaaaa
2453 tttccagata agacatgtca ccattaattc tcaacgactg ctctattttg
ttgtacggta 2513 atagttatca ccttctaaat tactatgtaa tttactcact
tattatgttt attgtcttgt 2573 atcctttctc tggagtgtaa gcacaatgaa
gacaggaatt ttgtatattt ttaaccaatg 2633 caacatactc tcagcaccta
aaatagtgcc gggaacatag taagggctca gtaaatactt 2693 gttgaataaa
ctcagtctcc tacattagca ttctaaaaaa aaaaaaaaaa aaaaaaaaaa 2753
aaaaaaaaaa aaaaaag 2770 6 239 PRT Homo sapiens 6 Met Pro Leu Gly
His Ile Met Arg Leu Asp Leu Glu Lys Ile Ala Leu 1 5 10 15 Glu Tyr
Ile Val Pro Cys Leu His Glu Val Gly Phe Cys Tyr Leu Asp 20 25 30
Asn Phe Leu Gly Glu Val Val Gly Asp Cys Val Leu Glu Arg Val Lys 35
40 45 Gln Leu His Cys Thr Gly Ala Leu Arg Asp Gly Gln Leu Ala Gly
Pro 50 55 60 Arg Ala Gly Val Ser Lys Arg His Leu Arg Gly Asp Gln
Ile Thr Trp 65 70 75 80 Ile Gly Gly Asn Glu Glu Gly Cys Glu Ala Ile
Ser Phe Leu Leu Ser 85 90 95 Leu Ile Asp Arg Leu Val Leu Tyr Cys
Gly Ser Arg Leu Gly Lys Tyr 100 105 110 Tyr Val Lys Glu Arg Ser Lys
Ala Met Val Ala Cys Tyr Pro Gly Asn 115 120 125 Gly Thr Gly Tyr Val
Arg His Val Asp Asn Pro Asn Gly Asp Gly Arg 130 135 140 Cys Ile Thr
Cys Ile Tyr Tyr Leu Asn Lys Asn Trp Asp Ala Lys Leu 145 150 155 160
His Gly Gly Ile Leu Arg Ile Phe Pro Glu Gly Lys Ser Phe Ile Ala 165
170 175 Asp Val Glu Pro Ile Phe Asp Arg Leu Leu Phe Phe Trp Ser Asp
Arg 180 185 190 Arg Asn Pro His Glu Val Gln Pro Ser Tyr Ala Thr Arg
Tyr Ala Met 195 200 205 Thr Val Trp Tyr Phe Asp Ala Glu Glu Arg Ala
Glu Ala Lys Lys Lys 210 215 220 Phe Arg Asn Leu Thr Arg Lys Thr Glu
Ser Ala Leu Thr Glu Asp 225 230 235 7 2369 DNA Caenorhabditis
elegans CDS (11)...(2182) 7 agcacatgac atg agc agt gcc cca aat gat
gac tgt gag atc gac aag 49 Met Ser Ser Ala Pro Asn Asp Asp Cys Glu
Ile Asp Lys 1 5 10 gga aca cct tct acc gct tca ctt ttt aca acg ctg
atg ctc agt caa 97 Gly Thr Pro Ser Thr Ala Ser Leu Phe Thr Thr Leu
Met Leu Ser Gln 15 20 25 cca tct tct tct aca gct gtt tta cag tgt
aca tat tgt gga agc tcg 145 Pro Ser Ser Ser Thr Ala Val Leu Gln Cys
Thr Tyr Cys Gly Ser Ser 30 35 40 45 tgc aca tct tcc caa ttg caa aca
tgt tta ttc tgt gga aca gtg gct 193 Cys Thr Ser Ser Gln Leu Gln Thr
Cys Leu Phe Cys Gly Thr Val Ala 50 55 60 tat tgt tcc aag gag cac
cag caa ctc gat tgg cta aca cat aaa atg 241 Tyr Cys Ser Lys Glu His
Gln Gln Leu Asp Trp Leu Thr His Lys Met 65 70 75 ata tgc aag tca
ctt caa aca agt ggc atg gtg cca agt aat ttg atg 289 Ile Cys Lys Ser
Leu Gln Thr Ser Gly Met Val Pro Ser Asn Leu Met 80 85 90 cct cag
gca gca cct gct gtt atg gct cca att cca cct act gtt tcg 337 Pro Gln
Ala Ala Pro Ala Val Met Ala Pro Ile Pro Pro Thr Val Ser 95 100 105
ttt gat gat cct gca ctt acc acg tca ctt ctt cta tct ctt caa aat 385
Phe Asp Asp Pro Ala Leu Thr Thr Ser Leu Leu Leu Ser Leu Gln Asn 110
115 120 125 aat cca att ctg aat caa act att tca aat ttt ccg cca aca
ttt tcg 433 Asn Pro Ile Leu Asn Gln Thr Ile Ser Asn Phe Pro Pro Thr
Phe Ser 130 135 140 atc aca tcg aag acc gaa cca gag cca tcg att cca
atc caa att cca 481 Ile Thr Ser Lys Thr Glu Pro Glu Pro Ser Ile Pro
Ile Gln Ile Pro 145 150 155 caa agg ata tca tca aca agt aca gta ccg
ttc agt agt gaa gga agt 529 Gln Arg Ile Ser Ser Thr Ser Thr Val Pro
Phe Ser Ser Glu Gly Ser 160 165 170 gca ttc aaa cca tac aga aat acg
cat gtg ttt aat tca att tct tct 577 Ala Phe Lys Pro Tyr Arg Asn Thr
His Val Phe Asn Ser Ile Ser Ser 175 180 185 gaa tca atg tct tcc atg
tgc aca tca cat gaa gca tca ctt gaa cac 625 Glu Ser Met Ser Ser Met
Cys Thr Ser His Glu Ala Ser Leu Glu His 190 195 200 205 atg tca tca
gct tcc ctt gca atg ttc cca aca agt agt act gct caa 673 Met Ser Ser
Ala Ser Leu Ala Met Phe Pro Thr Ser Ser Thr Ala Gln 210 215 220 agt
gat atc agt aga ctc gct caa gtt ttg agt ctt gct gga gat tca 721 Ser
Asp Ile Ser Arg Leu Ala Gln Val Leu Ser Leu Ala Gly Asp Ser 225 230
235 cca gct tcg ttg gct ctt gtc aca act tcg gta ccg tca act gct tcc
769 Pro Ala Ser Leu Ala Leu Val Thr Thr Ser Val Pro Ser Thr Ala Ser
240 245 250 aca gca act att cca cct cca gcg aca acg aca agt tca gct
aca agt 817 Thr Ala Thr Ile Pro Pro Pro Ala Thr Thr Thr Ser Ser Ala
Thr Ser 255 260 265 tca ggc aaa agc gag aca ata act gtt gga aaa gaa
aag ata att caa 865 Ser Gly Lys Ser Glu Thr Ile Thr Val Gly Lys Glu
Lys Ile Ile Gln 270 275 280 285 act gat gat ccg gat att cag atc atc
gaa aca gaa ggt gga tca aaa 913 Thr Asp Asp Pro Asp Ile Gln Ile Ile
Glu Thr Glu Gly Gly Ser Lys 290 295 300 cca acg gta tcc aga aca cgg
aaa cga cca act cct tct aac tcc gct 961 Pro Thr Val Ser Arg Thr Arg
Lys Arg Pro Thr Pro Ser Asn Ser Ala 305 310 315 gac cca aaa att aat
tac aag gat cac aat aag aat gtc gtt tac tcg 1009 Asp Pro Lys Ile
Asn Tyr Lys Asp His Asn Lys Asn Val Val Tyr Ser 320 325 330 aca acc
ctc caa gaa cat cag aag cat ctt cag aat cgt ggt ctc gca 1057 Thr
Thr Leu Gln Glu His Gln Lys His Leu Gln Asn Arg Gly Leu Ala 335 340
345 cta agc att cac caa gca atg gtt cta cgg tta aga tac att gcc gag
1105 Leu Ser Ile His Gln Ala Met Val Leu Arg Leu Arg Tyr Ile Ala
Glu 350 355 360 365 cat gtg atc aga agc ctg aat gag ttt gga tgg gcc
gtt gtt gac aat 1153 His Val Ile Arg Ser Leu Asn Glu Phe Gly Trp
Ala Val Val Asp Asn 370 375 380 ttt ctg ggc tcg gat cac tac aaa ttt
acc gcg aaa gaa att gag cga 1201 Phe Leu Gly Ser Asp His Tyr Lys
Phe Thr Ala Lys Glu Ile Glu Arg 385 390 395 ctc tat gaa cgg gga ctc
ttc agc cct ggt cag ttg atg gaa gca aaa 1249 Leu Tyr Glu Arg Gly
Leu Phe Ser Pro Gly Gln Leu Met Glu Ala Lys 400 405 410 cac aaa gac
gaa ttt cac atc aaa gat att cga tct gac cac att tac 1297 His Lys
Asp Glu Phe His Ile Lys Asp Ile Arg Ser Asp His Ile Tyr 415 420 425
tgg tat gat ggt tat gat gga cgt gcc aag gat gct gca act gtt cgt
1345 Trp Tyr Asp Gly Tyr Asp Gly Arg Ala Lys Asp Ala Ala Thr Val
Arg 430 435 440 445 cta ttg att tca atg att gat tct gta att caa cat
ttc aaa aaa cga 1393 Leu Leu Ile Ser Met Ile Asp Ser Val Ile Gln
His Phe Lys Lys Arg 450 455 460 att gat cat gat att gga gga cgt tct
cgt gca atg ctt gcc atc tat 1441 Ile Asp His Asp Ile Gly Gly Arg
Ser Arg Ala Met Leu Ala Ile Tyr 465 470 475 cct gga aat gga act cgt
tat gtg aag cat gta gat aat ccg gta aaa 1489 Pro Gly Asn Gly Thr
Arg Tyr Val Lys His Val Asp Asn Pro Val Lys 480 485 490 gat gga aga
tgt ata acc act att tat tac tgt aat gaa aat tgg gat 1537 Asp Gly
Arg Cys Ile Thr Thr Ile Tyr Tyr Cys Asn Glu Asn Trp Asp 495 500 505
atg gca act gat ggt ggt act ctc aga tta tat cca gag act tca atg
1585 Met Ala Thr Asp Gly Gly Thr Leu Arg Leu Tyr Pro Glu Thr Ser
Met 510 515 520 525 act cca atg gat att gat cca agg gct gat cgt ctg
gta ttc ttc tgg 1633 Thr Pro Met Asp Ile Asp Pro Arg Ala Asp Arg
Leu Val Phe Phe Trp 530 535 540 tcc gat cgt cgc aat cct cat gaa gtc
atg cca gtc ttc cgt cat cgt 1681 Ser Asp Arg Arg Asn Pro His Glu
Val Met Pro Val Phe Arg His Arg 545 550 555
ttc gca att act att tgg tat atg gat aaa tcc gaa aga gat aag gct
1729 Phe Ala Ile Thr Ile Trp Tyr Met Asp Lys Ser Glu Arg Asp Lys
Ala 560 565 570 ttg gca aaa gga aaa gag tca gat gcg gca tgt gct tca
aag aaa gag 1777 Leu Ala Lys Gly Lys Glu Ser Asp Ala Ala Cys Ala
Ser Lys Lys Glu 575 580 585 aat gat cca aca agc tct tca cta aat tcc
ctt att gga tca ctt ttg 1825 Asn Asp Pro Thr Ser Ser Ser Leu Asn
Ser Leu Ile Gly Ser Leu Leu 590 595 600 605 aga cca cgg aaa aat cca
agt act cac gat tta tca aaa ctt gac ctt 1873 Arg Pro Arg Lys Asn
Pro Ser Thr His Asp Leu Ser Lys Leu Asp Leu 610 615 620 cga ctc ttc
ccg tcc aca tca tcc gat cca gct ctg gta tct gca gca 1921 Arg Leu
Phe Pro Ser Thr Ser Ser Asp Pro Ala Leu Val Ser Ala Ala 625 630 635
gat gaa gat aga gtt gac atc tct gcc gac ttt caa tcc act tca agt
1969 Asp Glu Asp Arg Val Asp Ile Ser Ala Asp Phe Gln Ser Thr Ser
Ser 640 645 650 ctg gct cat ccg gaa tct act gac tcg gga gta tct ctc
tcc acc ttc 2017 Leu Ala His Pro Glu Ser Thr Asp Ser Gly Val Ser
Leu Ser Thr Phe 655 660 665 aat gtc gct cat aat cac atg gaa cgt act
acc agt ctc cag tcg atc 2065 Asn Val Ala His Asn His Met Glu Arg
Thr Thr Ser Leu Gln Ser Ile 670 675 680 685 tcc gat cat ttc cgt tcc
gaa aga tca cac gaa cgt cgc agc tca aca 2113 Ser Asp His Phe Arg
Ser Glu Arg Ser His Glu Arg Arg Ser Ser Thr 690 695 700 agc agc gat
caa gat cta gac gaa ggg ctc cca cca cct cct tcc aca 2161 Ser Ser
Asp Gln Asp Leu Asp Glu Gly Leu Pro Pro Pro Pro Ser Thr 705 710 715
aac cca gag tat tac atc tga agtttttctg gtttttgtta ctttctatat 2212
Asn Pro Glu Tyr Tyr Ile * 720 atatatatgt caccttcatt caatacgggt
aaagtcaatc ttgaaattcc gattcccgag 2272 aaaatcattg tcattcgagt
ttttttatgt atggactcta caaattatta tctcgacttt 2332 tccatgtgag
atagagtacc aattcaacat ggttttt 2369 8 723 PRT Caenorhabditis elegans
8 Met Ser Ser Ala Pro Asn Asp Asp Cys Glu Ile Asp Lys Gly Thr Pro 1
5 10 15 Ser Thr Ala Ser Leu Phe Thr Thr Leu Met Leu Ser Gln Pro Ser
Ser 20 25 30 Ser Thr Ala Val Leu Gln Cys Thr Tyr Cys Gly Ser Ser
Cys Thr Ser 35 40 45 Ser Gln Leu Gln Thr Cys Leu Phe Cys Gly Thr
Val Ala Tyr Cys Ser 50 55 60 Lys Glu His Gln Gln Leu Asp Trp Leu
Thr His Lys Met Ile Cys Lys 65 70 75 80 Ser Leu Gln Thr Ser Gly Met
Val Pro Ser Asn Leu Met Pro Gln Ala 85 90 95 Ala Pro Ala Val Met
Ala Pro Ile Pro Pro Thr Val Ser Phe Asp Asp 100 105 110 Pro Ala Leu
Thr Thr Ser Leu Leu Leu Ser Leu Gln Asn Asn Pro Ile 115 120 125 Leu
Asn Gln Thr Ile Ser Asn Phe Pro Pro Thr Phe Ser Ile Thr Ser 130 135
140 Lys Thr Glu Pro Glu Pro Ser Ile Pro Ile Gln Ile Pro Gln Arg Ile
145 150 155 160 Ser Ser Thr Ser Thr Val Pro Phe Ser Ser Glu Gly Ser
Ala Phe Lys 165 170 175 Pro Tyr Arg Asn Thr His Val Phe Asn Ser Ile
Ser Ser Glu Ser Met 180 185 190 Ser Ser Met Cys Thr Ser His Glu Ala
Ser Leu Glu His Met Ser Ser 195 200 205 Ala Ser Leu Ala Met Phe Pro
Thr Ser Ser Thr Ala Gln Ser Asp Ile 210 215 220 Ser Arg Leu Ala Gln
Val Leu Ser Leu Ala Gly Asp Ser Pro Ala Ser 225 230 235 240 Leu Ala
Leu Val Thr Thr Ser Val Pro Ser Thr Ala Ser Thr Ala Thr 245 250 255
Ile Pro Pro Pro Ala Thr Thr Thr Ser Ser Ala Thr Ser Ser Gly Lys 260
265 270 Ser Glu Thr Ile Thr Val Gly Lys Glu Lys Ile Ile Gln Thr Asp
Asp 275 280 285 Pro Asp Ile Gln Ile Ile Glu Thr Glu Gly Gly Ser Lys
Pro Thr Val 290 295 300 Ser Arg Thr Arg Lys Arg Pro Thr Pro Ser Asn
Ser Ala Asp Pro Lys 305 310 315 320 Ile Asn Tyr Lys Asp His Asn Lys
Asn Val Val Tyr Ser Thr Thr Leu 325 330 335 Gln Glu His Gln Lys His
Leu Gln Asn Arg Gly Leu Ala Leu Ser Ile 340 345 350 His Gln Ala Met
Val Leu Arg Leu Arg Tyr Ile Ala Glu His Val Ile 355 360 365 Arg Ser
Leu Asn Glu Phe Gly Trp Ala Val Val Asp Asn Phe Leu Gly 370 375 380
Ser Asp His Tyr Lys Phe Thr Ala Lys Glu Ile Glu Arg Leu Tyr Glu 385
390 395 400 Arg Gly Leu Phe Ser Pro Gly Gln Leu Met Glu Ala Lys His
Lys Asp 405 410 415 Glu Phe His Ile Lys Asp Ile Arg Ser Asp His Ile
Tyr Trp Tyr Asp 420 425 430 Gly Tyr Asp Gly Arg Ala Lys Asp Ala Ala
Thr Val Arg Leu Leu Ile 435 440 445 Ser Met Ile Asp Ser Val Ile Gln
His Phe Lys Lys Arg Ile Asp His 450 455 460 Asp Ile Gly Gly Arg Ser
Arg Ala Met Leu Ala Ile Tyr Pro Gly Asn 465 470 475 480 Gly Thr Arg
Tyr Val Lys His Val Asp Asn Pro Val Lys Asp Gly Arg 485 490 495 Cys
Ile Thr Thr Ile Tyr Tyr Cys Asn Glu Asn Trp Asp Met Ala Thr 500 505
510 Asp Gly Gly Thr Leu Arg Leu Tyr Pro Glu Thr Ser Met Thr Pro Met
515 520 525 Asp Ile Asp Pro Arg Ala Asp Arg Leu Val Phe Phe Trp Ser
Asp Arg 530 535 540 Arg Asn Pro His Glu Val Met Pro Val Phe Arg His
Arg Phe Ala Ile 545 550 555 560 Thr Ile Trp Tyr Met Asp Lys Ser Glu
Arg Asp Lys Ala Leu Ala Lys 565 570 575 Gly Lys Glu Ser Asp Ala Ala
Cys Ala Ser Lys Lys Glu Asn Asp Pro 580 585 590 Thr Ser Ser Ser Leu
Asn Ser Leu Ile Gly Ser Leu Leu Arg Pro Arg 595 600 605 Lys Asn Pro
Ser Thr His Asp Leu Ser Lys Leu Asp Leu Arg Leu Phe 610 615 620 Pro
Ser Thr Ser Ser Asp Pro Ala Leu Val Ser Ala Ala Asp Glu Asp 625 630
635 640 Arg Val Asp Ile Ser Ala Asp Phe Gln Ser Thr Ser Ser Leu Ala
His 645 650 655 Pro Glu Ser Thr Asp Ser Gly Val Ser Leu Ser Thr Phe
Asn Val Ala 660 665 670 His Asn His Met Glu Arg Thr Thr Ser Leu Gln
Ser Ile Ser Asp His 675 680 685 Phe Arg Ser Glu Arg Ser His Glu Arg
Arg Ser Ser Thr Ser Ser Asp 690 695 700 Gln Asp Leu Asp Glu Gly Leu
Pro Pro Pro Pro Ser Thr Asn Pro Glu 705 710 715 720 Tyr Tyr Ile 9
19 PRT Artificial Sequence 4Hyp 9 Asp Leu Asp Leu Glu Met Leu Ala
Xaa Tyr Ile Pro Met Asp Asp Asp 1 5 10 15 Phe Gln Leu 10 19 PRT
Artificial Sequence 4Hyp 10 Asp Leu Asp Leu Glu Met Leu Ala Xaa Tyr
Ile Ser Met Asp Asp Asp 1 5 10 15 Phe Gln Leu 11 19 PRT Artificial
Sequence 4Hyp 11 Asp Leu Asp Leu Glu Met Leu Leu Xaa Tyr Ile Pro
Met Asp Asp Asp 1 5 10 15 Phe Gln Leu 12 19 PRT Artificial Sequence
4Hyp 12 Asp Leu Asp Leu Glu Met Leu Val Xaa Tyr Ile Pro Met Asp Asp
Asp 1 5 10 15 Phe Gln Leu 13 19 PRT Artificial Sequence 4Hyp 13 Asp
Leu Asp Leu Glu Met Ile Ala Xaa Tyr Ile Pro Met Asp Asp Asp 1 5 10
15 Phe Gln Leu 14 19 PRT Artificial Sequence 4Hyp 14 Asp Leu Asp
Leu Glu Met Ile Ala Xaa Tyr Ile Pro Met Glu Asp Asp 1 5 10 15 Phe
Gln Leu 15 19 PRT Artificial Sequence 4Hyp 15 Asp Leu Asp Leu Glu
Met Leu Val Xaa Tyr Ile Ser Met Asp Asp Asp 1 5 10 15 Phe Gln Leu
16 34 PRT Artificial Sequence 4Hyp 16 Pro Phe Ser Thr Gln Asp Thr
Asp Leu Asp Leu Glu Met Leu Ala Pro 1 5 10 15 Tyr Ile Pro Met Asp
Asp Asp Phe Gln Leu Arg Ser Phe Asp Gln Leu 20 25 30 Ser Pro 17 6
PRT Artificial Sequence X=any amino acid 17 Leu Xaa Xaa Leu Ala Pro
1 5 18 5 PRT Artificial Sequence X=any amino acid 18 His Xaa Asp
Xaa His 1 5 19 62 PRT Artificial Sequence X=any amino acid 19 His
Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa His 50 55 60 20 85 PRT Artificial Sequence X=any amino acid 20
Met Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa His Xaa 1 5
10 15 Asp Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa
Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu
Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Pro Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa
Xaa Xaa His Xaa Val Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Arg 85 21
110 PRT Artificial Sequence X=any amino acid 21 Arg Xaa Xaa Xaa Met
Xaa Xaa Xaa Tyr Pro Gly Asn Gly Xaa Xaa Tyr 1 5 10 15 Val Xaa His
Val Asp Asn Pro Xaa Xaa Asp Gly Arg Cys Xaa Thr Xaa 20 25 30 Ile
Tyr Tyr Xaa Asn Xaa Xaa Trp Asp Xaa Xaa Xaa Xaa Gly Gly Xaa 35 40
45 Leu Xaa Xaa Phe Pro Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro
50 55 60 Xaa Xaa Asp Arg Leu Xaa Phe Xaa Trp Ser Asp Arg Arg Asn
Pro His 65 70 75 80 Glu Val Xaa Pro Xaa Xaa Xaa Xaa Arg Xaa Ala Xaa
Thr Val Trp Tyr 85 90 95 Xaa Asp Xaa Xaa Glu Arg Xaa Xaa Ala Xaa
Ala Lys Xaa Lys 100 105 110 22 6 PRT Artificial Sequence 4Hyp 22
Leu Xaa Xaa Leu Xaa Xaa 1 5 23 19 PRT Homo sapiens 23 Asp Leu Asp
Leu Glu Met Leu Ala Pro Tyr Ile Pro Met Asp Asp Asp 1 5 10 15 Phe
Gln Leu 24 20 PRT Homo sapiens 24 Glu Leu Asp Leu Glu Thr Leu Ala
Pro Tyr Ile Pro Met Asp Gly Glu 1 5 10 15 Asp Phe Gln Leu 20 25 19
PRT Xenopus laevis 25 Asp Leu Asp Leu Glu Met Leu Ala Pro Tyr Ile
Pro Met Asp Asp Asp 1 5 10 15 Phe Gln Leu 26 19 PRT Drosophila
melanogaster 26 Phe Glu Ala Phe Ala Met Arg Ala Pro Tyr Ile Pro Ile
Asp Asp Asp 1 5 10 15 Met Pro Leu 27 19 PRT Caenorhabditis elegans
27 Glu Pro Asp Leu Ser Cys Leu Ala Pro Phe Val Asp Thr Tyr Asp Met
1 5 10 15 Met Gln Met 28 34 PRT Artificial Sequence Synthetic
polypeptide 28 Pro Phe Ser Thr Gln Asp Thr Asp Leu Asp Leu Glu Met
Leu Ala Pro 1 5 10 15 Tyr Ile Pro Met Asp Asp Asp Phe Gln Leu Arg
Ser Phe Asp Gln Leu 20 25 30 Ser Pro 29 19 PRT Artificial Sequence
Synthetic polypeptide 29 Asp Leu Asp Leu Glu Met Leu Ala Pro Tyr
Ile Pro Met Asp Asp Asp 1 5 10 15 Phe Gln Leu 30 16 PRT Artificial
Sequence Synthetic polypeptide 30 Leu Glu Met Leu Ala Pro Tyr Ile
Pro Met Asp Asp Asp Phe Gln Leu 1 5 10 15 31 13 PRT Artificial
Sequence Synthetic polypeptide 31 Leu Ala Pro Tyr Ile Pro Met Asp
Asp Asp Phe Gln Leu 1 5 10 32 14 PRT Artificial Sequence Synthetic
polypeptide 32 Asp Leu Asp Leu Glu Met Leu Ala Pro Tyr Ile Pro Met
Asp 1 5 10 33 19 PRT Artificial Sequence Synthetic polypeptide 33
Asp Leu Asp Leu Glu Met Leu Ala Gly Tyr Ile Pro Met Asp Asp Asp 1 5
10 15 Phe Gln Leu 34 19 PRT Artificial Sequence Synthetic
polypeptide 34 Asp Leu Asp Leu Glu Met Leu Ala Pro Tyr Ile Pro Met
Asp Asp Asp 1 5 10 15 Phe Gln Leu 35 826 PRT Homo sapiens 35 Met
Glu Gly Ala Gly Gly Ala Asn Asp Lys Lys Lys Ile Ser Ser Glu 1 5 10
15 Arg Arg Lys Glu Lys Ser Arg Asp Ala Ala Arg Ser Arg Arg Ser Lys
20 25 30 Glu Ser Glu Val Phe Tyr Glu Leu Ala His Gln Leu Pro Leu
Pro His 35 40 45 Asn Val Ser Ser His Leu Asp Lys Ala Ser Val Met
Arg Leu Thr Ile 50 55 60 Ser Tyr Leu Arg Val Arg Lys Leu Leu Asp
Ala Gly Asp Leu Asp Ile 65 70 75 80 Glu Asp Asp Met Lys Ala Gln Met
Asn Cys Phe Tyr Leu Lys Ala Leu 85 90 95 Asp Gly Phe Val Met Val
Leu Thr Asp Asp Gly Asp Met Ile Tyr Ile 100 105 110 Ser Asp Asn Val
Asn Lys Tyr Met Gly Leu Thr Gln Phe Glu Leu Thr 115 120 125 Gly His
Ser Val Phe Asp Phe Thr His Pro Cys Asp His Glu Glu Met 130 135 140
Arg Glu Met Leu Thr His Arg Asn Gly Leu Val Lys Lys Gly Lys Glu 145
150 155 160 Gln Asn Thr Gln Arg Ser Phe Phe Leu Arg Met Lys Cys Thr
Leu Thr 165 170 175 Ser Arg Gly Arg Thr Met Asn Ile Lys Ser Ala Thr
Trp Lys Val Leu 180 185 190 His Cys Thr Gly His Ile His Val Tyr Asp
Thr Asn Ser Asn Gln Pro 195 200 205 Gln Cys Gly Tyr Lys Lys Pro Pro
Met Thr Cys Leu Val Leu Ile Cys 210 215 220 Glu Pro Ile Pro His Pro
Ser Asn Ile Glu Ile Pro Leu Asp Ser Lys 225 230 235 240 Thr Phe Leu
Ser Arg His Ser Leu Asp Met Lys Phe Ser Tyr Cys Asp 245 250 255 Glu
Arg Ile Thr Glu Leu Met Gly Tyr Glu Pro Glu Glu Leu Leu Gly 260 265
270 Arg Ser Ile Tyr Glu Tyr Tyr His Ala Leu Asp Ser Asp His Leu Thr
275 280 285 Lys Thr His His Asp Met Phe Thr Lys Gly Gln Val Thr Thr
Gly Gln 290 295 300 Tyr Arg Met Leu Ala Lys Arg Gly Gly Tyr Val Trp
Val Glu Thr Gln 305 310 315 320 Ala Thr Val Ile Tyr Asn Thr Lys Asn
Ser Gln Pro Gln Cys Ile Val 325 330 335 Cys Val Asn Tyr Val Val Ser
Gly Ile Ile Gln His Asp Leu Ile Phe 340 345 350 Ser Leu Gln Gln Thr
Glu Cys Val Leu Lys Pro Val Glu Ser Ser Asp 355 360 365 Met Lys Met
Thr Gln Leu Phe Thr Lys Val Glu Ser Glu Asp Thr Ser 370 375 380 Ser
Leu Phe Asp Lys Leu Lys Lys Glu Pro Asp Ala Leu Thr Leu Leu 385 390
395 400 Ala Pro Ala Ala Gly Asp Thr Ile Ile Ser Leu Asp Phe Gly Ser
Asn 405 410 415 Asp Thr Glu Thr Asp Asp Gln Gln Leu Glu Glu Val Pro
Leu Tyr Asn 420 425 430 Asp Val Met Leu Pro Ser Pro Asn Glu Lys Leu
Gln Asn Ile Asn Leu 435 440 445 Ala Met Ser Pro Leu Pro Thr Ala Glu
Thr Pro Lys Pro Leu Arg Ser 450 455 460 Ser Ala Asp Pro Ala Leu Asn
Gln Glu Val Ala Leu Lys Leu Glu Pro 465 470 475 480 Asn Pro Glu Ser
Leu Glu Leu Ser Phe Thr Met Pro Gln Ile Gln Asp 485 490 495 Gln Thr
Pro Ser Pro Ser Asp Gly Ser Thr Arg Gln Ser Ser Pro Glu 500 505 510
Pro Asn Ser Pro Ser Glu Tyr Cys Phe Tyr Val Asp Ser Asp Met Val 515
520 525 Asn Glu Phe Lys Leu Glu Leu Val Glu Lys Leu Phe Ala Glu Asp
Thr 530 535 540 Glu Ala Lys Asn Pro Phe Ser Thr Gln Asp Thr Asp Leu
Asp Leu Glu 545 550 555 560 Met Leu Ala Pro Tyr Ile Pro Met Asp Asp
Asp Phe Gln Leu Arg Ser 565 570 575 Phe Asp Gln Leu Ser Pro Leu Glu
Ser Ser Ser Ala Ser Pro Glu Ser 580 585 590 Ala Ser Pro Gln Ser Thr
Val Thr Val Phe Gln Gln Thr Gln Ile Gln
595 600 605 Glu Pro Thr Ala Asn Ala Thr Thr Thr Thr Ala Thr Thr Asp
Glu Leu 610 615 620 Lys Thr Val Thr Lys Asp Arg Met Glu Asp Ile Lys
Ile Leu Ile Ala 625 630 635 640 Ser Pro Ser Pro Thr His Ile His Lys
Glu Thr Thr Ser Ala Thr Ser 645 650 655 Ser Pro Tyr Arg Asp Thr Gln
Ser Arg Thr Ala Ser Pro Asn Arg Ala 660 665 670 Gly Lys Gly Val Ile
Glu Gln Thr Glu Lys Ser His Pro Arg Ser Pro 675 680 685 Asn Val Leu
Ser Val Ala Leu Ser Gln Arg Thr Thr Val Pro Glu Glu 690 695 700 Glu
Leu Asn Pro Lys Ile Leu Ala Leu Gln Asn Ala Gln Arg Lys Arg 705 710
715 720 Lys Met Glu His Asp Gly Ser Leu Phe Gln Ala Val Gly Ile Gly
Thr 725 730 735 Leu Leu Gln Gln Pro Asp Asp His Ala Ala Thr Thr Ser
Leu Ser Trp 740 745 750 Lys Arg Val Lys Gly Cys Lys Ser Ser Glu Gln
Asn Gly Met Glu Gln 755 760 765 Lys Thr Ile Ile Leu Ile Pro Ser Asp
Leu Ala Cys Arg Leu Leu Gly 770 775 780 Gln Ser Met Asp Glu Ser Gly
Leu Pro Gln Leu Thr Ser Tyr Asp Cys 785 790 795 800 Glu Val Asn Ala
Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Glu 805 810 815 Glu Leu
Leu Arg Ala Leu Asp Gln Val Asn 820 825 36 719 PRT Caenorhabditis
elegans 36 Met Glu Asp Asn Arg Lys Arg Asn Met Glu Arg Arg Arg Glu
Thr Ser 1 5 10 15 Arg His Ala Ala Arg Asp Arg Arg Ser Lys Glu Ser
Asp Ile Phe Asp 20 25 30 Asp Leu Lys Met Cys Val Pro Ile Val Glu
Glu Gly Thr Val Thr His 35 40 45 Leu Asp Arg Ile Ala Leu Leu Arg
Val Ala Ala Thr Ile Cys Arg Leu 50 55 60 Arg Lys Thr Ala Gly Asn
Val Leu Glu Asn Asn Leu Asp Asn Glu Ile 65 70 75 80 Thr Asn Glu Val
Trp Thr Glu Asp Thr Ile Ala Glu Cys Leu Asp Gly 85 90 95 Phe Val
Met Ile Val Asp Ser Asp Ser Ser Ile Leu Tyr Val Thr Glu 100 105 110
Ser Val Ala Met Tyr Leu Gly Leu Thr Gln Thr Asp Leu Thr Gly Arg 115
120 125 Ala Leu Arg Asp Phe Leu His Pro Ser Asp Tyr Asp Glu Phe Asp
Lys 130 135 140 Gln Ser Lys Met Leu His Lys Pro Arg Gly Glu Asp Thr
Asp Thr Thr 145 150 155 160 Gly Ile Asn Met Val Leu Arg Met Lys Thr
Val Ile Ser Pro Arg Gly 165 170 175 Arg Cys Leu Asn Leu Lys Ser Ala
Leu Tyr Lys Ser Val Ser Phe Leu 180 185 190 Val His Ser Lys Val Ser
Thr Gly Gly His Val Ser Phe Met Gln Gly 195 200 205 Ile Thr Ile Pro
Ala Gly Gln Gly Thr Thr Asn Ala Asn Ala Ser Ala 210 215 220 Met Thr
Lys Tyr Thr Glu Ser Pro Met Gly Ala Phe Thr Thr Arg His 225 230 235
240 Thr Cys Asp Met Arg Ile Thr Phe Val Ser Asp Lys Phe Asn Tyr Ile
245 250 255 Leu Lys Ser Glu Leu Lys Thr Leu Met Gly Thr Ser Phe Tyr
Glu Leu 260 265 270 Val His Pro Ala Asp Met Met Ile Val Ser Lys Ser
Met Lys Glu Leu 275 280 285 Phe Ala Lys Gly His Ile Arg Thr Pro Tyr
Tyr Arg Leu Ile Ala Ala 290 295 300 Asn Asp Thr Leu Ala Trp Ile Gln
Thr Glu Ala Thr Thr Ile Thr His 305 310 315 320 Thr Thr Lys Gly Gln
Lys Gly Gln Tyr Val Ile Cys Val His Tyr Val 325 330 335 Leu Gly Ile
Gln Gly Ala Glu Glu Ser Leu Val Val Cys Thr Asp Ser 340 345 350 Met
Pro Ala Gly Met Gln Val Asp Ile Lys Lys Glu Val Asp Asp Thr 355 360
365 Arg Asp Tyr Ile Gly Arg Gln Pro Glu Ile Val Glu Cys Val Asp Phe
370 375 380 Thr Pro Leu Ile Glu Pro Glu Asp Pro Phe Asp Thr Val Ile
Glu Pro 385 390 395 400 Val Val Gly Gly Glu Glu Pro Val Lys Gln Ala
Asp Met Gly Ala Arg 405 410 415 Lys Asn Ser Tyr Asp Asp Val Leu Gln
Trp Leu Phe Arg Asp Gln Pro 420 425 430 Ser Ser Pro Pro Pro Ala Arg
Tyr Arg Ser Ala Asp Arg Phe Arg Thr 435 440 445 Thr Glu Pro Ser Asn
Phe Gly Ser Ala Leu Ala Ser Pro Asp Phe Met 450 455 460 Asp Ser Ser
Ser Arg Thr Ser Arg Pro Lys Thr Ser Tyr Gly Arg Arg 465 470 475 480
Ala Gln Ser Gln Gly Ser Arg Thr Thr Gly Ser Ser Ser Thr Ser Ala 485
490 495 Ser Ala Thr Leu Pro His Ser Ala Asn Tyr Ser Pro Leu Ala Glu
Gly 500 505 510 Ile Ser Gln Cys Gly Leu Asn Ser Pro Pro Ser Ile Lys
Ser Gly Gln 515 520 525 Val Val Tyr Gly Asp Ala Arg Ser Met Gly Arg
Ser Cys Asp Pro Ser 530 535 540 Asp Ser Ser Arg Arg Phe Ser Ala Leu
Ser Pro Ser Asp Thr Leu Asn 545 550 555 560 Val Ser Ser Thr Arg Gly
Ile Asn Pro Val Ile Gly Ser Asn Asp Val 565 570 575 Phe Ser Thr Met
Pro Phe Ala Asp Ser Ile Ala Ile Ala Glu Arg Ile 580 585 590 Asp Ser
Ser Pro Thr Leu Thr Ser Gly Glu Pro Ile Leu Cys Asp Asp 595 600 605
Leu Gln Trp Glu Glu Pro Asp Leu Ser Cys Leu Ala Pro Phe Val Asp 610
615 620 Thr Tyr Asp Met Met Gln Met Asp Glu Gly Leu Pro Pro Glu Leu
Gln 625 630 635 640 Ala Leu Tyr Asp Leu Pro Asp Phe Thr Pro Ala Val
Pro Gln Ala Pro 645 650 655 Ala Ala Arg Pro Val His Ile Asp Arg Ser
Pro Pro Ala Lys Arg Met 660 665 670 His Gln Ser Gly Pro Ser Asp Leu
Asp Phe Met Tyr Thr Gln His Tyr 675 680 685 Gln Pro Phe Gln Gln Asp
Glu Thr Tyr Trp Gln Gly Gln Gln Gln Gln 690 695 700 Asn Glu Gln Gln
Pro Ser Ser Tyr Ser Pro Phe Pro Met Leu Ser 705 710 715 37 38 PRT
Homo sapiens 37 Ser Glu Asp Thr Ser Ser Leu Phe Asp Lys Leu Lys Lys
Glu Pro Asp 1 5 10 15 Ala Leu Thr Leu Leu Ala Pro Ala Ala Gly Asp
Thr Ile Ile Ser Leu 20 25 30 Asp Phe Gly Ser Asn Asp 35 38 38 PRT
Homo sapiens 38 Ser Glu Lys Ser Asn Phe Leu Phe Thr Lys Leu Lys Glu
Glu Pro Glu 1 5 10 15 Glu Leu Ala Gln Leu Ala Pro Thr Pro Gly Asp
Ala Ile Ile Ser Leu 20 25 30 Asp Phe Gly Asn Gln Asn 35 39 19 PRT
Homo sapiens 39 Asp Leu Asp Leu Glu Met Leu Ala Pro Tyr Ile Pro Met
Asp Asp Asp 1 5 10 15 Phe Gln Leu 40 119 PRT Caenorhabditis elegans
40 Ile Asp His Asp Ile Gly Gly Arg Ser Arg Ala Met Leu Ala Ile Tyr
1 5 10 15 Pro Gly Asn Gly Thr Arg Tyr Val Lys His Val Asp Asn Pro
Val Lys 20 25 30 Asp Gly Arg Cys Ile Thr Thr Ile Tyr Tyr Cys Asn
Glu Asn Trp Asp 35 40 45 Met Ala Thr Asp Gly Gly Thr Leu Arg Leu
Tyr Pro Glu Thr Ser Met 50 55 60 Thr Pro Met Asp Ile Asp Pro Arg
Ala Asp Arg Leu Val Phe Phe Trp 65 70 75 80 Ser Asp Arg Arg Asn Pro
His Glu Val Met Pro Val Phe Arg His Arg 85 90 95 Phe Ala Ile Thr
Ile Trp Tyr Met Asp Lys Ser Glu Arg Asp Lys Ala 100 105 110 Leu Ala
Lys Gly Lys Glu Ser 115 41 116 PRT Homo sapiens 41 Gly Ser Tyr Lys
Ile Asn Gly Arg Thr Lys Ala Met Val Ala Cys Tyr 1 5 10 15 Pro Gly
Asn Gly Thr Gly Tyr Val Arg His Val Asp Asn Pro Asn Gly 20 25 30
Asp Gly Arg Cys Val Thr Cys Ile Tyr Tyr Leu Asn Lys Asp Trp Asp 35
40 45 Ala Lys Val Ser Gly Gly Ile Leu Arg Ile Phe Pro Glu Gly Lys
Ala 50 55 60 Gln Phe Ala Asp Ile Glu Pro Lys Phe Asp Arg Leu Leu
Phe Phe Trp 65 70 75 80 Ser Asp Arg Arg Asn Pro His Glu Val Gln Pro
Ala Tyr Ala Thr Arg 85 90 95 Tyr Ala Ile Thr Val Trp Tyr Phe Asp
Ala Asp Glu Arg Ala Arg Ala 100 105 110 Lys Val Lys Tyr 115 42 116
PRT Homo sapiens 42 Gly Ser Tyr Val Ile Asn Gly Arg Thr Lys Ala Met
Val Ala Cys Tyr 1 5 10 15 Pro Gly Asn Gly Leu Gly Tyr Val Arg His
Val Asp Asn Pro His Gly 20 25 30 Asp Gly Arg Cys Ile Thr Cys Ile
Tyr Tyr Leu Asn Gln Asn Trp Asp 35 40 45 Val Lys Val His Gly Gly
Leu Leu Gln Ile Phe Pro Glu Gly Arg Pro 50 55 60 Val Val Ala Asn
Ile Glu Pro Leu Phe Asp Arg Leu Leu Ile Phe Trp 65 70 75 80 Ser Asp
Arg Arg Asn Pro His Glu Val Lys Pro Ala Tyr Ala Thr Arg 85 90 95
Tyr Ala Ile Thr Val Trp Tyr Phe Asp Ala Lys Glu Arg Ala Ala Ala 100
105 110 Lys Asp Lys Tyr 115 43 115 PRT Homo sapiens 43 Lys Tyr Tyr
Val Lys Glu Arg Ser Lys Ala Met Val Ala Cys Tyr Pro 1 5 10 15 Gly
Asn Gly Thr Gly Tyr Val Arg His Val Asp Asn Pro Asn Gly Asp 20 25
30 Gly Arg Cys Ile Thr Cys Ile Tyr Tyr Leu Asn Lys Asn Trp Asp Ala
35 40 45 Lys Leu His Gly Gly Ile Leu Arg Ile Phe Pro Glu Gly Lys
Ser Phe 50 55 60 Ile Ala Asp Val Glu Pro Ile Phe Asp Arg Leu Leu
Phe Phe Trp Ser 65 70 75 80 Asp Arg Arg Asn Pro His Glu Val Gln Pro
Ser Tyr Ala Thr Arg Tyr 85 90 95 Ala Met Thr Val Trp Tyr Phe Asp
Ala Glu Glu Arg Ala Glu Ala Lys 100 105 110 Lys Lys Phe 115 44 116
PRT Rattus sp. 44 Gly Lys Tyr Tyr Val Lys Glu Arg Ser Lys Ala Met
Val Ala Cys Tyr 1 5 10 15 Pro Gly Asn Gly Thr Gly Tyr Val Arg His
Val Asp Asn Pro Asn Gly 20 25 30 Asp Gly Arg Cys Ile Thr Cys Ile
Tyr Tyr Leu Asn Lys Asn Trp Asp 35 40 45 Ala Lys Leu His Gly Gly
Val Leu Arg Ile Phe Pro Glu Gly Lys Ser 50 55 60 Phe Val Ala Asp
Val Glu Pro Ile Phe Asp Arg Leu Leu Phe Ser Trp 65 70 75 80 Ser Asp
Arg Arg Asn Pro His Glu Val Gln Pro Ser Tyr Ala Thr Arg 85 90 95
Tyr Ala Met Thr Val Trp Tyr Phe Asp Ala Glu Glu Arg Ala Glu Ala 100
105 110 Lys Lys Lys Phe 115 45 92 PRT Streptomyces sp. 45 Phe Asp
Gly Thr His Leu Gln Met Ala Arg Ser Arg Asn Leu Lys Asn 1 5 10 15
Ala Ile Val Ile Pro His Arg Asp Phe Val Glu Leu Asp Arg Glu Val 20
25 30 Asp Arg Tyr Phe Arg Thr Phe Met Val Leu Glu Asp Ser Pro Leu
Ala 35 40 45 Phe His Ser Asn Glu Asp Thr Val Ile His Met Arg Pro
Gly Glu Ile 50 55 60 Trp Phe Leu Asp Ala Ala Thr Val His Ser Ala
Val Asn Phe Ser Glu 65 70 75 80 Ile Ser Arg Gln Ser Leu Cys Val Asp
Phe Ala Phe 85 90 46 37 DNA Artificial Sequence Forward primer 46
gatttatcgt gcttggcagg attcgttgac acttatg 37 47 36 DNA Artificial
Sequence Reverse primer 47 gtgtcaacga atcctgccaa gcacgataaa tcaggc
36 48 6 PRT Artificial Sequence X=any amino acid 48 Leu Xaa Xaa Leu
Ala Xaa 1 5
* * * * *
References