U.S. patent application number 10/395048 was filed with the patent office on 2004-02-05 for modulation of angiogenesis through targeting of cysteine oxygenase activity.
Invention is credited to Davydov, Ilia V., Du, Fangyong, Hu, Rong-gui, Kwon, Yong Tae, Varshavsky, Alexander.
Application Number | 20040023311 10/395048 |
Document ID | / |
Family ID | 28457129 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040023311 |
Kind Code |
A1 |
Kwon, Yong Tae ; et
al. |
February 5, 2004 |
Modulation of angiogenesis through targeting of cysteine oxygenase
activity
Abstract
The invention provides methods and compositions for modulating
angiogenesis in a subject. The methods of modulating angiogenesis
in a subject include administering to the subject a modulator of
N-terminal cysteine oxygenase activity. The invention also provides
a method of identifying such a modulator and a method of in vitro
screening for modulators of N-terminal cysteine oxygenase activity.
Additionally, the invention provides a method of treating an
angiogenesis-related disorder.
Inventors: |
Kwon, Yong Tae; (Wexford,
PA) ; Davydov, Ilia V.; (North Potomac, MD) ;
Hu, Rong-gui; (Pasadena, CA) ; Du, Fangyong;
(New Haven, CT) ; Varshavsky, Alexander; (La
Canada, CA) |
Correspondence
Address: |
GRAY CARY WARE & FREIDENRICH LLP
4365 EXECUTIVE DRIVE
SUITE 1100
SAN DIEGO
CA
92121-2133
US
|
Family ID: |
28457129 |
Appl. No.: |
10/395048 |
Filed: |
March 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60366218 |
Mar 21, 2002 |
|
|
|
60366207 |
Mar 21, 2002 |
|
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Current U.S.
Class: |
435/7.2 ;
514/1 |
Current CPC
Class: |
G01N 2333/515 20130101;
C12Q 1/48 20130101; G01N 2500/10 20130101; G01N 2500/00 20130101;
G01N 2500/04 20130101 |
Class at
Publication: |
435/7.2 ;
514/1 |
International
Class: |
G01N 033/53; G01N
033/567; A61K 031/00 |
Goverment Interests
[0002] This invention was made in part with government support
under Grant No. GM31530 awarded by the National Institutes of
Health. The United States government may have certain rights in
this invention.
Claims
What is claimed is:
1. A method of modulating angiogenesis in a subject, comprising
administering to a subject a modulator of cysteine oxygenase,
thereby modulating angiogenesis in the subject.
2. The method of claim 1, wherein the subject is a mammal.
3. A method of identifying a modulator of cysteine oxygenase,
comprising: a) providing a cell that expresses a reporter protein
with an N-terminal cysteine residue; b) contacting at least one
potential modulator with the cell; and c) measuring the level of
the reporter protein expressed in the presence and absence of the
modulator, wherein a change in the level of reporter protein
expressed is indicative of modulation of cysteine oxygenase
activity.
4. The method of claim 3, wherein the cell is a cultured mammalian
cell.
5. The method of claim 3, wherein the cell is a yeast cell.
6. The method of claim 3, wherein the cell is a bacterial cell.
7. The method of claim 3, wherein the reporter protein is expressed
as a cleavable fusion protein comprising a reporter portion and a
ubiquitin domain functionally linked to the reporter protein,
wherein the reporter portion comprises an N-terminal cysteine.
8. The method of claim 3, wherein the reporter protein comprises a
selectable marker protein, and the measuring comprises a selective
genetic screen.
9. The method of claim 8, wherein the selectable marker protein is
an antibiotic resistance protein.
10. The method of claim 3, wherein the measuring comprises
fluorescence detection of the reporter protein.
11. The method of claim 3, wherein the reporter portion comprises a
fluorescent protein.
12. The method of claim 11, wherein the fluorescent protein is
green fluorescent protein.
13. The method of claim 3, wherein the reporter portion comprises a
light-generating protein.
14. The method of claim 12, wherein the light-generating protein is
luciferase.
15. The method of claim 3, wherein the reporter portion comprises
an enzyme.
16. The method of claim 15, wherein the enzyme is
beta-galactosidase.
17. The method of claim 3, wherein the expression of the reporter
protein is inducible.
18. The method of claim 3, wherein the reporter protein has a
half-life in the first cell of less than about an hour in the
absence of a modulator.
19. The method of claim 3, wherein the reporter protein has a
half-life in the first cell of less than about 10 minutes in the
absence of a modulator.
20. The method of claim 3, wherein the change in the level of
reporter protein expressed is increased expression.
21. The method of claim 3, further comprising: d) providing a
second cell that expresses a second reporter protein, wherein the
second reporter portion comprises an N-terminal argenine, aspartic
acid or glutamic acid; e) contacting at least one potential
modulator of cysteine oxygenase activity with the second cell; and
f) measuring the level of second reporter protein expressed within
the second cell relative to the level of reporter protein expressed
within the first cell.
22. The method of claim 21, wherein the cell in step a) and the
second cell are the same cell.
23. The method of claim 22, wherein the reporter protein of step a)
and the second reporter protein are fluorescent proteins having
distinct spectral properties.
24. A modulator of cysteine oxygenase identified by the method of
claim 3.
25. The modulator of claim 23, wherein the modulator of cysteine
oxygenase also modulates angiogenesis.
26. A method of modulating angiogenesis in a subject, comprising
administration of a modulator identified by the method of claim
3.
27. An in vitro method of screening for modulators of cysteine
oxygenase, comprising: a) contacting one or more potential
modulators of cysteine oxygenase with a cell extract; b) providing
a peptide substrate having an N-terminal cysteine residue; and c)
determining the level of N-terminal cysteine oxidation of the
peptide substrate, wherein a change in the level of cysteine
oxidation of the peptide substrate is indicative of modulation of
cysteine oxygenase activity.
28. The method of claim 27, wherein the determining of the level of
cysteine oxidation comprises electrophoresis, chromatography, mass
spectrometry or imunoassay.
29. The method of claim 27, wherein the determining of the level of
cysteine oxidation comprises reaction with an antibody that
distinguishes between the substrate peptide and an oxidized
substrate peptide.
30. The method of claim 27, wherein the modulator of cysteine
oxygenase also modulates angiogenesis.
31. The method of claim 27, wherein the change in the level of
oxidation of the peptide substrate is increased oxidation.
32. A modulator of cysteine oxygenase identified by the method of
claim 27.
33. A method of modulating angiogenesis in a subject, comprising
administration of a modulator identified by the method of claim
27.
34. A method of treating an angiogenesis-related disorder in a
subject comprising administering to a subject a modulator of
angiogenesis, thereby treating the angiogenesis-related disorder in
the subject.
35. The method of claim 34, wherein the modulator inhibits cysteine
oxygenase.
36. The method of claim 34, wherein the subject is a mammal.
37. The method of claim 34, wherein the modulator of angiogenesis
inhibits angiogenesis in the subject.
38. The method of claim 34, wherein the angiogenesis-related
disorder is cancer, tumors, rheumatoid arthritis, psoriasis or
metastasis of cancerous cells in the subject.
39. The method of claim 34, wherein the modulator of angiogenesis
stimulates angiogenesis in the subject.
40. The method of claim 34, wherein the angiogenesis-related
disorder is coronary artery disease, stroke or delayed wound
healing.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) of U.S. Ser. No. 60/366,218, filed Mar. 21,
2002 and U.S. Ser. No. 60/366,207, filed Mar. 21, 2002, the entire
contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The invention relates generally to methods of identification
of modulators of angiogenesis and more specifically to modulators
of N-terminal cysteine oxygenase activity.
BACKGROUND INFORMATION
[0004] Substrates of the ubiquitin (Ub)-dependent N-end rule
pathway include proteins with destabilizing N-terminal residues. A
set of amino acids that are destabilizing in a given cell yields a
rule, called the N-end rule, which relates the in vivo half-life of
a protein to the identity of its N-terminal residue. The essential
component of a degradation signal called the N-degron is a
destabilizing N-terminal residue of a protein. Variants of the
N-end rule pathway are present in all organisms examined, from
animals and plants to fungi and prokaryotes (FIG. 1A). In
eukaryotes, an N-degron of a protein consists of a destabilizing
N-terminal residue and an internal lysine, the latter being the
site of formation of a substrate-linked multi-Ub chain. The N-end
rule has a hierarchic structure (FIG. 1 A). N-terminal Asn and Gln
are tertiary destabilizing residues in that they function through
their deamidation, by N-terminal amidohydrolases, to yield the
secondary destabilizing residues Asp and Glu. The activity of
N-terminal Asp and Glu requires their conjugation, by ATE I-encoded
Arg-tRNA-protein transferases (R-transferases), to Arg, one of the
primary destabilizing residues. The latter are recognized by the E3
(Ub ligase) components of the N-end rule pathway (FIG. 1A). In
mammals, the set of destabilizing residues that function through
their arginylation contains not only Asp and Glu but also Cys,
which is a stabilizing (non-arginylated) residue in the yeast
Saccharomyces cerevisiae. The known species of mammalian
R-transferase, ATE1-1 and ATE1-2, are produced through alternative
splicing of ATE1 pre-mRNA, and have the same substrate specificity
as the yeast R-transferase, they arginylate N-terminal Asp or Glu,
but cannot arginylate N-terminal Cys.
[0005] Angiogenesis is the growth of new blood vessels. Generally
in the body, a balance of angiogenesis growth factors and
angiogenesis inhibitors keeps this process of blood vessel growth
under control. However, a lack or excess of either angiogenesis
growth factors or angiogenesis inhibitors can cause undesired
growth of blood vessels or a failure to produce blood vessels.
SUMMARY OF THE INVENTION
[0006] The present invention is based on the seminal discovery that
the N-terminal cysteine of a polypeptide is specifically oxidized
in vivo, and that it is the oxidized form of N-terminal cysteine,
rather than the initial (unoxidized) cysteine moiety, that is
actually arginylated by R-transferase.
[0007] In one embodiment, the invention is directed to a method of
modulating angiogenesis in a subject. The method of the invention
includes administering a modulator of cysteine oxygenase to a
subject.
[0008] In another embodiment, the invention provides a method of
identifying a modulator of cysteine oxygenase. The method includes
providing a cell that expresses a reporter protein with an
N-terminal cysteine residue, contacting that cell with at least one
potential modulator and measuring the level of the reporter protein
expressed in the presence and absence of the modulator. A change of
the level of reporter protein expressed in the presence of the
modulator as compared to in the absence of the modulator is
indicative of modulation. Such a change may be an increase in
expression or a decrease in expression. The level of reporter
protein may be indicative of the level of protein expressed or may
be indicative of the half life of the reporter.
[0009] In another embodiment, the method further includes providing
a second cell that expresses a second reporter protein with an
N-terminal argenine, aspartic acid or glutamic acid residue. The
second cell is contacted with at least one potential modulator of
cysteine oxygenase and the level of second reporter protein
expressed within the second cell relative to the level of reporter
protein expressed within the first cell is measured.
[0010] In still another embodiment, the invention provides an in
vitro method of screening for modulators of cysteine oxygenase
including contacting one or more potential modulators of cysteine
oxygenase with a cell extract, providing a peptide substrate having
an N-terminal cysteine residue and determining the level of
N-terminal cysteine oxidation of the peptide substrate. A change of
the level of N-terminal cysteine oxidation in the presence of the
modulator as compared to in the absence of the modulator is
indicative of modulation. Such a change may be an increase in
expression or a decrease in level of oxidation.
[0011] In yet another embodiment the invention is directed to a
method of treating an angiogenesis-related disorder in a subject.
The method includes administering a modulator of angiogenesis to a
subject.
[0012] The invention also includes a modulator identified by a
method of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. N-terminal residues are indicated by single-letter
abbreviations for amino acids. Ovals denote the rest of a protein
substrate. Type 1 and type 2 primary destabilizing N-terminal
residues (Arg, Lys, His, Phe, Leu, Trp, Tyr, and Ile) are
recognized by functionally overlapping Ub ligases (E3s) that
include UBR1 (E3.alpha.) and UBR2. N-terminal Ala, Ser and Thr are
recognized by a distinct, unidentified E3.
[0014] FIG. 2A shows quantitation of results of the stabilization
of N-end rule substrates in mouse ATE1.sup.-/- cells, and
oxidation-arginylation of N-terminal cysteine. ATE1.sup.+/+ and
ATE1.sup.-/- EF cells were transfected with plasmids expressing
.sup.fDHFR.sup.h-Ub.sup.R48-X-nsP4.s- up.f, whose cleavage yielded
the .sup.fDFHR.sup.h-Ub.sup.R48 reference protein, denoted as DHFR,
and X-nsP4.sup.f test proteins (X=Met, Arg, Asp, Glu, Cys), denoted
as X-nsP4. Cells were pulse-labeled for 10 min with
.sup.35S-methionine, and chased for 1 and 2 hr. For each time
point, the ratio of .sup.35S in X-nsP4.sup.f (X=Arg, Asp, Glu, Cys)
to .sup.35S in the .sup.fDHFR-Ub.sup.R48 reference protein at the
same time point, was plotted as the percentage of this ratio
relative to that for Met-nsP4.sup.f(which bore a stabilizing
N-terminal residue) at time 0. Open and closed symbols: +/+ and
ATE1.sup.-/- cells, respectively. .DELTA., .tangle-solidup.,
Met-nsP4.sup.f; .smallcircle., .circle-solid., Arg-nsP4.sup.f;
.quadrature., .box-solid., Asp-nsP4.sup.f; .gradient.,
.tangle-soliddn., Glu-nsP4.sup.f; .diamond., .diamond-solid.,
Cys-nsP4.sup.f.
[0015] FIG. 2B shows a cell-free assay for mouse R-transferase
using .sup.3H-Arg, S105 extracts from +/+and ATE1.sup.-/- EF cells,
and unlabeled X-.beta.gals (X=Cys, Asp, Glu, Met, Arg) and
.alpha.-lactalbumin as test substrates. Arginylated test substrates
are indicated on the right. Asterisks indicate arginylated
endogenous proteins in the extracts.
[0016] FIGS. 2C and 2D show Mouse ATE1.sup.-/- EFs were
co-transfected with a plasmid expressing X-nsP4.sup.f and a plasmid
expressing either wild-type S. cerevisiae ATE1 (pCDNA3yATE1) or its
enzymatically impaired mutant (pCDNA3yATE1C23A). Curve
designations: Met-nsP4.sup.f coexpressed with either a vector alone
(open gray square), or yeast ATE1 (solid gray square), or mutant
yeast ATE1 (solid black square). Cys-nsP4.sup.f the same except for
diamonds instead of squares. Asp-nsP4.sup.f: the same except for
circles instead of squares.
[0017] FIG. 2E shows the N-terminal sequences, as determined
through Edman degradation, of mouse RGS4-flag-His.sub.6 isolated
from mouse L cells. Calk denotes alkylated Cys residue.
[0018] FIG. 2F shows the N-terminal sequence of mouse RGS4 deduced
from mass spectrometric data in shown in FIG. 2G. The M.sub.r of
residue 2 (Cys-2 in the arginylated RGS4) identified it as cysteic
acid (M.sub.r of 151.1 Da, 48.1 Da higher than M.sub.r of Cys). M*
denotes homoserine lactone.
[0019] FIG. 2G shows a mass spectrometric analysis of the
CNBr-produced 19-residue N-terminal peptide of RGS4. Both the
observed molecular masses of peaks and the differences between
observed and expected values are shown.
[0020] FIG. 2H shows the chemical formulas of N-terminal Cys
residue, Cys sulfinic acid, cysteic acid, and Asp residues.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Methods are provided herein, based on the discovery that the
N-terminal cysteine of a polypeptide is specifically oxidized in
vivo, and that it is the oxidized form of N-terminal cysteine that
is arginylated by R-transferase. In particular, the invention
provides methods for modulating angiogenisis in a subject by
providing a therapeutically effective amount of a modulator of
enzymatic oxidation of N-terminal cysteine in polypeptides. The
invention described herein provides screening methods for
modulators of cysteine oxygenase, and methods for treating an
animal by providing a modulator of N-terminal cysteine
oxidation.
[0022] As used herein, the term "modulate" or "modulation" is used
with regard to modulation of activity. In particular, the present
invention provides modulators of N-terminal cysteine oxidation. As
used herein, modulate refers to changing the normal activity.
Modulation may be inhibition of an activity or stimulation of the
same.
[0023] As used herein, the term "angiogenesis" is used to refer to
the growth of new blood vessels. Accordingly, an
"angiogenesis-related disorder" is a disorder in which the activity
of angiogenesis is altered. Such diseases may be completely caused
by angiogenesis or may be exacerbated by angiogenesis activity.
Angiogenesis-related disorders due to excessive angiogenesis may
include, but are not limited to, cancer, tumors, rheumatoid
arthritis, psoriasis, rosacea and metastasis of cancerous cells.
Angiogenesis-related disorders due to insufficient angiogenesis may
include, but are not limited to, coronary artery disease, stroke,
ulcers and delayed wound healing.
[0024] In one embodiment, the invention provides methods for
screening for modulators of cysteine oxygenase, in which one or
more potential modulators of cysteine oxygenase are contacted with
a first cell. The cell expresses a first reporter protein having an
N-terminal cysteine residue. The level of said first reporter
protein present is then determined in the cell either directly or
through an indirect means. A change of the level of reporter
protein expressed in the presence of the modulator as compared to
in the absence of the modulator is indicative of modulation. Such a
change may be an increase in expression or a decrease in
expression. The level of reporter protein may be indicative of the
level of protein expressed or may be indicative of the half life of
the reporter. In another embodiment of the invention, the
expression of the reporter protein is inducible.
[0025] The reporter protein is selected such that it has a
relatively short half-life in the cell. Generally, the reporter
protein has a half-life in the first cell of less than about an
hour in the absence of a modulator. The reporter protein can have a
half-life in the first cell of less than about 10 minutes in the
absence of the modulator.
[0026] In one embodiment of the invention, the reporter protein is
expressed as a cleavable fusion protein. The cleavable fusion
protein can have an ubiquitin domain functionally linked to the
reporter protein. In an appropriate cell, the ubiquitin domain is
rapidly cleaved by one of several deubiquitylating enzymes.
[0027] In yet another embodiment of the invention, the screening
methods described can be accomplished in a variety of cells,
including in a cultured mammalian cell, a yeast cell or a bacterial
cell.
[0028] The first type of screen for inhibitors of ATE1-mediated
N-terminal arginylation can be carried in the setting of either
mammalian cells in culture or yeast (S. cerevisiae) cells in
culture. In mammalian cells and yeast, the ATE1-encoded
R-transferases are solely responsible for N-terminal arginylation.
The logic of either yeast- or mammalian cells-based screens is
essentially the same. The advantage of a yeast-based screen is the
ease of handling and analyzing yeast cell cultures; the
disadvantage is that potential inhibitors of R-transferase that
would cross the plasma membrane of mammalian cells may be incapable
of gaining entry into yeast cells, given substantial differences in
the permeability (and transport) properties of the plasma membrane
between yeast and mammals. A mammalian cells-based screen is
preferred, since the sought inhibitors would be intended for use in
mammals. The screen: a mammalian (mouse or human) cell line is set
up that expresses a short-lived reporter whose ubiquitin-dependent
degradation by the N-end rule pathway involves N-terminal
arginylation. Such a reporter would be a protein with either a
genetically selectable or "screenable" (visually detectable)
influence on the test cell culture.
[0029] A number of classes of reporter proteins are suitable for
use with the methods of the invention. For example, the reporter
protein can be a genetically selectable marker protein, and the
relative level of the reporter protein in the presence and absence
of a modulator can accomplished indirectly through a selective
genetic screen which requires the presence of said reporter protein
for survival of said first cell. In such an example, a modulator
which inhibits the cysteine oxygenase activity would therefore
lengthen the half-life of the reporter protein. For example, the
selectable marker protein can be an antibiotic resistance protein.
In one such example, the reporter protein is designed to be
short-lived in a cell, the cell will be relatively sensitive to the
relevant antibiotic. By contrast, if the protein is made long-lived
in vivo, for example through the inhibition of a proteolytic
pathway, and more specifically through inhibition of cysteine
oxygenase activity, that normally destroys this protein, its
steady-state level would, by definition, increase, and the cell
would become resistant to the same dose of antibiotic. This readout
would enable a selection-based screen. A number of reporter
proteins that confer antibiotic resistance are known to those
skilled in the art.
[0030] Detection of the relative abundance of the reporter protein
also can be accomplished by more direct means. For example, the
reporter protein can be a screenable reporter protein. Such
proteins may include, but are not limited to Green Fluorescent
Protein (GFP) and E. coli .beta.-galactosidase (.beta.gal). A
number of fluorescent proteins are known to those of skill in the
art. These proteins, if expressed in a cell, can be detected either
through their fluorescence (GFP) or through their enzymatic
activity (.beta.gal). The suitable reporter protein can be
expressed in the cell type used for the assay and has sufficient
fluorescence intensity to be detected within the cell at the
appropriate concentrations. Making a reporter of this class
short-lived in vivo would strongly diminish its steady-state level
in a cell. If degradation of such a short-lived reporter is
inhibited, its steady-state level will rise, enabling the detection
of reporter. The level of protein present can be determined using
fluorescence detection, for example, using a fluorometer or
fluorescence microscope. The relative amount of fluorescence can be
compared between cells treated with a potential modulator versus no
treatment. Similarly, the reporter protein can be a
light-generating protein, for example, luciferase. The amount of
light produced can be measured and compared in cells treated with a
modulator and in untreated cells.
[0031] The level of reporter protein present can also be determined
enzymatically. For example, enzymes capable of making a
colorimetric change in a substrate, can be detected indirectly. One
such protein, E. coli .beta.-galactosidase (.beta.gal), can be
detected through a colorimetric change it causes in the substrate
.beta.-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactosidase).
Making a reporter of this class short-lived in vivo would strongly
diminish its steady-state level in a cell. If degradation of such a
short-lived reporter is inhibited, its steady-state level will
rise, enabling the detection of the reporter protein (through an
enzymatic reaction).
[0032] Other reporter protein systems are known to one of skill in
the art and can be applied to the methods of the invention. As will
be apparent to one of skill in the art, the invention is not
limited to those currently known, but can also be extended to newly
discovered reporter protein systems.
[0033] The invention also provides modulators of N-terminal
cysteine oxygenase activity. In summary, the invention provides
methods for identifying modulators of cysteine oxygenase that
modifies the N-terminal cysteine of a polypeptide. This modulation
is also essential for a cysteine-specific subset of N-terminal
arginylation reactions, which, in turn, are important for
cardiovascular functions, including angiogenesis.
[0034] According to another embodiment of the invention, the
screening methods can include additional features, including
contacting the potential modulators of cysteine oxygenase with a
second cell, wherein the second cell expresses a second reporter
protein having an N-terminal arginine, aspartic acid or glutamic
acid residue and determining the level of expression of said second
reporter protein relative to said first reporter protein. These
settings serve as negative-control settings for those in which a
reporter protein contains N-terminal cysteine. According to the
invention, the second cell can be the same cell as the first cell
(i.e., a cell expressing both the first reporter protein and the
second reporter protein) or can be a different cell (i.e., one not
expressing the first reporter protein). Such a screening method
allows for the selection of modulators specific for the cysteine
oxygenase activity that do not inhibit the rest of the N-end rule
pathway, including the rest of its arginylation branch. According
to this embodiment of the invention, a desired modulator of
cysteine oxygenase activity would alter the relative abundance of
the N-terminal cysteine-containing first reporter protein, but
would not significantly alter the relative abundance of the second
reporter protein having an arginine, aspartic acid or glutamic acid
residue at the N-terminus. In one embodiment of such an assay
system, the first reporter protein and the second reporter proteins
are fluorescent proteins having distinct spectral properties.
[0035] In one embodiment of the invention, the screen is carried
out with a GFP-based reporter. Mammalian cells growing in
microtiter plates and expressing Cys-GFP that is designed to be
N-terminally arginylated and rapidly destroyed by the N-end rule
pathway, are exposed to a library of potential inhibitors of
cysteine oxidase (one tested compound per well, with different
concentrations of a compound in adjacent wells). After incubation
with the library of compounds, the microtiter plates are scanned by
plate fluorimeter to detect wells where the levels of Cys-GFP
increased. Compounds of the library that caused the increase of GFP
level are then re-tested in control assays, to identify those among
them that act by inhibiting oxidation of the N-terminal cysteine or
its subsequent arginylation. One way to do that is to add these
compounds, one at a time, to an otherwise identical mammalian cell
culture that expresses Asp-GFP, a reporter identical to Cys-GFP
except having Asp instead of Cys at its N-terminus. Such a reporter
would be degraded by the N-end rule pathway in
arginylation-dependent manner, but would not require the action of
the cysteine oxygenase activity. Likewise, the screen can
differentiate between compounds that act on the cysteine oxygenase
versus the rest of the N-end rule pathway, including the rest of
its arginylation branch. For example, these compounds can be added,
one at a time, to an otherwise identical mammalian cell culture
that expresses Arg-GFP, a reporter identical to Cys-GFP and Asp-GFP
except having Arg residue at its N-terminus. Such a reporter would
be degraded by the N-end rule pathway in arginylation-independent
manner; and therefore it would not require the action of the
cysteine oxygenase activity or the arginylation activity. Any
compound that inhibits the enzymatic oxidation of N-terminal
cysteine would be, immediately, a strong candidate for an inhibitor
of N-terminal cysteine oxidation. Any compound that inhibits
degradation of both Cys-GFP and Asp-GFP but does not significantly
inhibit degradation of Arg-GFP would be, immediately, a strong
candidate for a specific inhibitor of R-transferase.
[0036] Confirmation of inhibition of R-transferase may then be
obtained in at least two ways: Whether this is, in fact, the case
could then be determined straightforwardly in at least two ways: 1)
in vivo, by isolating Asp-GFP from cells treated with inhibitor
versus control (untreated) cells, and N-terminally sequencing
Asp-GFP from these two sources, to determine whether its N-terminal
arginylation was, in fact, inhibited in vivo by the compound in
question; or 2) a direct enzymatic assay with purified
R-transferase and test the identified compound or compounds
(identified through initial screen) for inhibition of N-terminal
arginylation of a model substrate in vitro.
[0037] To carry out a screen for inhibitors of N-terminal cysteine
oxidation through linkage to arginylation and degradation, a
reporter of either class will have to be extended N-terminally with
a sequence of amino acid residues known as the N-degron, a class of
degradation signals (degrons) recognized by the universally
present, ubiquitin-dependent N-end rule pathway of protein
degradation. A particular class of N-degrons that would require
N-terminal arginylation for its activity would have cysteine (Cys)
as the N-terminal residue of the reporter, since oxidized Cys is
arginylated by the N-end rule pathway, by R-transferase, prior to
reporter's degradation. Previous extensive work by this and other
laboratories made the construction of N-degron-equipped reporters a
well-developed art known to researchers in the field.
[0038] A number of techniques are available to express a reporter
protein having a desired N-terminal residue. Universally,
translated proteins are initiated with an amino-terminal methionine
residue. Processing of the protein by various peptidases can expose
an internal residue as an amino-terminal residue. One such
technique involves expressing a fusion protein having a ubiquitin
domain and a reporter protein domain. The ubiquitin domain is
rapidly cleaved from the fusion protein by a class of enzymes known
as the deubiquitinylating enzymes (DUBs). This strategy is well
known to one of skill in the art and is taught, for example, in
references 3, 5 and 16, which are incorporated herein by reference
in their entireties.
[0039] The invention also provides in vitro methods for screening
for modulators of cysteine oxygenase. The method involves
contacting one or more potential modulators of cysteine oxygenase
with a cell extract, providing a peptide substrate having an
N-terminal cysteine residue; and determining the level of cysteine
oxidation of said peptide substrate. A change of the level of
cysteine oxidation in the presence of the modulator as compared to
in the absence of the modulator is indicative of modulation. Such a
change may be an increase in oxidation or a decrease in
oxidation.
[0040] The level of cysteine oxidation in the peptide substrate can
be determined by any suitable method including, for example, gel
electrophoresis, capillary electrophoresis, thin layer
chromatography (TLC); high pressure liquid chromatography (HPLC),
mass spectrometry and immunoassay. The type of detection used in
such a scheme can be determined by one of skill in the art and will
be chosen as to be compatible with the type of separation used. For
example, when the technique to be used is HPLC, detection can be UV
detection. The degree or rate of N-terminal cysteine oxidation can
be compared using such techniques, or combinations of techniques to
determine the impact of a modulator of cysteine oxygenase
activity.
[0041] The cysteine oxidation level also can be determined using an
antibody based system. An antibody is generated that selectively
recognizes the peptide substrate having an N-terminal cysteine
residue versus a peptide having an oxidized N-terminal cysteine
residue. In such an arrangement, a peptide substrate having an
N-terminal cysteine residue is covalently or otherwise affixed to
the surface of a vessel, for example the well of a microtiter
plate. A cell extract having N-terminal cysteine oxygenase activity
is added to the wells of such a plate in the presence or absence of
a potential modulator of the cysteine oxidase activity. The degree
of cysteine oxidation is determined using the antibody. A number of
suitable detection schemes are known to one of skill in the art of
immunological assays, including directly linking the antibody to a
colorimetric or fluorescent moiety, or using a sandwich type assay,
in which the detection means is provided by a second protein,
typically an antibody which binds to the first antibody.
[0042] In still another embodiment, the invention provides
modulators of cysteine oxygenase identified by any of the methods
of the invention. In yet another embodiment, the modulators of
cysteine oxygenase are also modulators of angiogenesis.
[0043] In another embodiment the invention provides methods for
modulating angiogenesis in a subject, including administering to a
mammal a modulator of cysteine oxygenase. In one embodiment, the
subject is a mammal, such as a human or a mouse. Inhibitors of
cysteine oxygenase activity can be used to treat patients, for
example, who are in need of inhibited angiogenic activity, for
example, those with tumors.
[0044] In still another embodiment of the invention, a method of
treating an angiogenesis-related disorder is provided. The method
involves administering a modulator of angiogenesis to a subject,
thereby treating the angiogenesis-related disorder in the subject.
Such a modulator may inhibit cysteine oxygenase. The subject may be
a mammal. The angiogenesis modulator may inhibit or stimulate
angiogenesis in the subject, thereby treating the
angiogenesis-related disorder. Angiogenesis-related disorders
treated by this method of the invention may include, but are not
limited to: cancer, tumors, rheumatoid arthritis, psoriasis,
rosacea, metastasis of cancerous cells, coronary artery disease,
stroke, ulcers and delayed wound healing.
[0045] In one embodiment, the potential modulators are small
organic compounds, compounds having a molecular weight of less than
about 1,000. Such compounds are available from natural sources
including plant, bacterial, and fungal extracts, from techniques
such as combinatorial chemistry. One of skill in the art will
recognize that libraries of compounds are commercially
available.
[0046] It is noted that Cys dioxygenase (CDO) converts free Cys, in
the presence of oxygen, to Cys sulfinic acid (FIG. 1). A rigorous
identification of the physiologically relevant enzyme(s) that
oxidizes N-terminal Cys will require genetic analysis of a
candidate enzyme(s).
[0047] The following examples are intended to illustrate but not
limit the invention.
EXAMPLE 1
Assays of Arginyl Transferase Activity
[0048] Mouse ATE1 was isolated using screening, with an ATE1 cDNA
fragment (nt 638-1,491), a BAC library (Genome Systems) from
129/SvJ ES mouse cells. The exon/intron organization of the
first.about.20 kb of ATE1 was determined using exon-specific PCR
primers to produce genomic DNA fragments flanked by exons. The
targeting vector was linearized with HindIII and electroporated
into CJ7 embryonic stem (ES) cells, followed by selection and
identification of the correctly targeted ATE1.sup.+/- ES cell
clones with normal karyotype. Standard techniques were then used to
produce chimeric and ATE1.sup.-/- mice. Phenotypes of ATE1.sup.-/-
embryos were observed mainly with mice of the C57BL/6J-129SvEv
(mixed) background, and confirmed in the 129SvEv (inbred)
background. RT-PCR, Southern and Northern analyses, and
PCR-mediated genotyping of embryos and pups were performed.
Standard procedures were used for thin sectioning and staining with
hematoxylin/eosin, X-Gal, or anti-PECAM-1 antibody (clone MEC 13.3,
Pharmingen). The TUNEL assay was performed using a kit (Roche) and
fluorescein-dUTP. For R-transferase assays (FIG. 2B), Ub-X-bgal
proteins were purified from E. coli carrying pKKUbXbgal plasmids.
The R-transferase reaction (50 ml) contained S105 supernatant (0.5
mg of protein per ml) from either EF cells or whole embryos,
Ub-X-.beta.gal or .alpha.-lactalbumin (0.2 mg/ml), E. coli tRNA (1
mg/ml), E. coli aminoacyl-tRNA synthetases (50 mg/ml), puromycin
(0.2 mM), bestatin (0.15 mM), 5 mM MG132 (proteasome inhibitor),
0.4 mM Lys-Ala dipeptide (inhibitor of post-arginylation steps in
the N-end rule pathway), 1 mM ATP, 10 mM creatine phosphate, 0.1 M
KC1, 5 mM MgCl.sub.2, 50 mM b-mercaptoethanol, 50 mM Tris-HC1 (pH
8.0) and 0.3 mM .sup.3H-arginine (New England Nuclear). The
reaction mixture was incubated for 3 hr (2 hr with embryo extracts)
at 37.degree. C. A 20 ml sample was precipitated with 10% TCA, and
analyzed by SDS-12% PAGE and fluorography.
[0049] Primary mouse EFs were established from E13.5 ATE1.sup.-/-
and littermate +/+ embryos and immortalized to increase
transfection efficiency. Cells were transiently transfected with
pcDNA3flagDHFRhaUbXnsP4flag, which expressed
.sup.fDFHR.sup.h-UbR48-X-nsP- 4.sup.f (and the main text) from the
PCMV promoter. Cells were labeled with 35S-EXPRESS (New England
Nuclear) for 10 min at 37.degree. C., followed by a chase for 0, 1,
and 2 hr in the presence of cycloheximide, preparation of extracts,
precipitation with anti-flag antibody, SDS-10% PAGE,
autoradiography, and quantitation using PhosphorImager. In other
pulse-chases, ATE1.sup.-/- EFs were co-transfected with a plasmid
expressing X-nsP4.sup.f(.sup.fDFHR.sup.h-UbR48-X-nsP4f) and either
pCDNA3yATE1, expressing S. cerevisiae ATE1, or pCDNA3yATE1C23A
(derived from a plasmid supplied by Dr. C. Pickart, Johns Hopkins
University), which expressed ATE1C23SA, bearing Cys.fwdarw.Ala
mutation at position 23.
EXAMPLE 2
Arginylation of N-terminal Cysteine-Containing Polypeptides
[0050] To measure the N-terminal arginylation directly, either
purified Ub-X-.beta.gal proteins (X=Met, Arg, Glu, Cys) or purified
human .alpha.-lactalbumin (bearing N-terminal Glu) were added to
+/+ and ATE1.sup.-/- immortalized embryonic fibroblast (EF) cell
extracts supplemented with ATP, total E. coli tRNA and a mixture of
E. coli aminoacyl-tRNA synthetases. SDS-PAGE and fluorography were
used to detect covalent conjugation of .sup.3H-Arg to test proteins
in these extracts (FIG. 2B). Ub-X-.beta.gals are rapidly
deubiquitylated in vivo and in cell-free extracts, yielding
X-.beta.gal test proteins. As expected, Asp-.beta.gal,
Glu-.beta.gal and .alpha.-lactalbumin were arginylated in the
extracts from +/+ EF cells, whereas Arg-.beta.gal and
Met-.beta.gal, bearing a primary destabilizing and a stabilizing
N-terminal residue, respectively, were not arginylated (FIG. 2B).
Crucially, no arginylation of Asp-.beta.gal, Glu-.beta.gal and
.alpha.-lactalbumin could be detected in ATE1.sup.-/- EF extracts
(FIG. 2B), even after prolonged fluorographic exposures. Identical
results were obtained with extracts from +/+ and ATE1.sup.-/-
embryos. In addition to being consistent with the conclusions from
pulse-chase analyses in EF cells (FIG. 2A), these findings
confirmed the absence of R-transferase activity from ATE1.sup.-/-
embryos.
[0051] The N-terminal Cys, of Cys-.beta.gal, was not arginylated in
either +/+ or ATE1.sup.-/- extracts, in contrast to N-terminal Asp
and Glu (FIG. 2B), suggesting that the previously observed
arginylation of N-terminal Cys, and the demonstrated ATE1
dependence of the in vivo degradation of Cys-bearing N-end rule
substrates in mouse cells (FIG. 2A) involved a modification of
N-terminal Cys prior to its arginylation. In this interpretation,
the absence of arginylation of Cys-.beta.gal in an extract from +/+
mouse cells could be caused, for example, by inactivation of a
Cys-modifying enzyme in the extract. A comparison of Asp and Cys
structures suggested that either the Cys sulfinic acid residue
(CysO.sub.2, an oxidized derivative of Cys) or the cysteic acid
residue (CysO.sub.3, a further oxidized Cys derivative), may be
sufficiently close in structure and charge distribution to Asp
(FIG. 2H) to serve as a substrate of R-transferases. Consistent
with this possibility, a protease called Asp-N has been shown to
cleave peptide bonds N-terminal to either the Asp or CysO.sub.3
residues. Another class of enzymes, aspartate aminotransferases,
can utilize either Asp or oxidized Cys as substrates.
[0052] This example illustrates that degradation of a test protein
bearing N-terminal Cys is tRNA-dependent (implying the involvement
of R-transferase), similarly to the degradation of otherwise
identical proteins bearing N-terminal Asp, Glu, Asn or Gln, and in
contrast to degradation of otherwise identical proteins bearing
primary destabilizing N-terminal residues (FIG. 1). The normally
short-lived Cys-bearing N-end rule substrates are stabilized in
mouse ATE1.sup.-/- cells (FIG. 2A), although the ATE1-encoded
R-transferases that are absent from these cells cannot arginylate
N-terminal Cys upon their expression in atel.DELTA.S.
cerevisiae.
EXAMPLE 3
Generation of RGS4
[0053] To produce RGS4, mouse L cells were transiently transfected
with pCDNA3RGS4flagHis.sub.6, which expressed mouse
RGS4-flag-His.sub.6 from the PCMV promoter and was constructed from
the pcDNA3RGS4 plasmid. Cell extracts were prepared 30 hr later;
RGS4-flag-His.sub.6 was purified using Ni-NTA Magnetic Agarose
Beads (Qiagen), then treated with 25 mM iodoacetamide in 7 M urea,
followed by SDS-PAGE, the transfer onto Immobilon-P membrane, and
sequencing by Edman-degradation, using the 476A sequencer
(Perkin-Elmer). For mass spectrometry, RGS4-flag-His.sub.6 was
treated with 90 mM iodoacetic acid in 8 M urea for 50 min at
.about.20.degree. C., then cleaved with CNBr in 55% HCOOH under
argon atmosphere for 12 hr in the dark, followed by reverse phase
HPLC and on-line, fragmentation-based mass spectrometric sequencing
of peptides.
EXAMPLE 4
Arginylation of N-terminal Cysteine-Containing RGS4
[0054] The existence of amino-terminal arginylated Cys residues as
CysO.sub.2 or CysO.sub.3 was verified and confirmed with mouse
RGS4, a GTPase-activating (GAP) protein that bears N-terminal Cys
and was previously shown to be arginylated and degraded by the
N-end rule pathway in rabbit reticulocyte extracts. RGS4-His.sub.6
was transiently expressed in mouse L cells, purified, treated with
iodoacetamide to alkylate Cys residues (thereby making them
identifiable by the sequencing procedure used), and was
N-terminally sequenced by Edman degradation. The results indicated
the presence of two RGS4 proteins, an arginylated and unarginylated
one, the former being a major species (FIG. 2E). Remarkably,
whereas the expected Cys residue at position 12 of arginylated RGS4
could be identified as alkylated Cys, the expected (alkylated) Cys
residue at position 2 (position 1 in the unarginylated RGS4) could
not be identified by the Edman procedure (FIG. 2E), indicating that
a residue at this position existed as a derivative of Cys prior to
alkylation, and thereby precluded it. To determine the identity of
a residue at position 2, the purified, alkylated RGS4 was cleaved
with cyanogen bromide (CNBr), followed by HPLC fractionation and
on-line mass spectrometric sequencing of CNBr-produced peptides.
Mass spectra derived from the arginylated N-terminal peptide of
RGS4 demonstrated that the mass of a residue at position 2 was
increased by 48 (+0.1) Da in comparison to the expected mass of
Cys-2 (FIGS. 2F, G). These results (FIGS. 2E-G) identified residue
2 as a cysteic acid (CysO.sub.3) residue (FIG. 2H).
EXAMPLE 5
Rescue of the Destabilizing Activity of CYS in Mouse ATE1.sup.-/-
CELLS
[0055] Verification and confirmation of the fact that the yeast
R-transferase should be able to rescue the destabilizing activity
of Cys in mouse ATE1.sup.-/- cells, owing to the presence of
Cys-oxidation activity in these cells was obtained by the present
example. Pulse-chase assays were carried out with mouse
ATE1.sup.-/- EF cells that expressed X-nsP4.sup.f proteins (X=Met,
Asp, Cys) and either the wild-type S. cerevisiae ATE1
(R-transferase) or ATE1.sup.C23SA, an enzymatically impaired
missense mutant. The metabolic stability of long-lived
Met-nsP4.sup.f (bearing a stabilizing N-terminal residue) was
unchanged in the presence of yeast ATE1. In contrast, both
Asp-nsP4.sup.f and Cys-nsP4.sup.f, which were long-lived in mouse
ATE1.sup.-/- EF cells, became short-lived in the presence of yeast
ATE1 (FIGS. 2C, D). The complementation by yeast R-transferase
required its enzymatic activity, since ATE1.sup.C23SA, a
catalytically impaired missense mutant, had a significantly weaker
effect (FIGS. 2C, D). In addition to supporting the
Cys-oxidation/arginylation hypothesis, these results suggested that
the oxidation of N-terminal Cys is an enzymatic (rather than
uncatalyzed) reaction, since the intracellular solvent conditions,
including redox potential, are likely to be similar in mammalian
and yeast cells. The fact of stoichiometric oxidation of N-terminal
Cys in mouse cells (FIGS. 2E-G) indicated the same conclusion.
[0056] Thus, S. cerevisiae R-transferase, which cannot arginylate
N-terminal Cys, can rescue the in vivo arginylation and degradation
of Cys-bearing N-end rule substrates in mouse ATE1.sup.-/- cells
(FIGS. 2C, D). Such a cell-based system can be used with the
methods of the invention described above to identify modulators of
cysteine oxygenase activity, which is necessary for degradation of
proteins having an N-terminal cysteine residue.
[0057] In contrast to most of the destabilizing residues, including
Asp and Glu (FIG. 1), Cys can be exposed at the N-terminus of a
protein substrate by Met-aminopeptidases, which cleave off the
N-terminal Met of a newly formed protein if the side chain of a
second residue is small enough; only Cys, Ala, Thr, and Ser of the
mammalian N-end rule satisfy this condition. Two Cys-bearing mouse
proteins, RGS4 and RGS16, were recently identified as N-end rule
substrates. A mammalian genome encodes a few hundred proteins
containing the N-terminal Met-Cys sequence. However, given the
constraints of N-degron's organization, the presence of Cys at the
N-terminus of a protein is not, by itself, sufficient to render
this protein an N-end rule substrate. In addition, the oxidation
(and subsequent arginylation) of N-terminal Cys may compete with
its other known modifications, including acetylation and
palmitoylation. N-end rule substrates that bear the
arginylation-dependent destabilizing N-terminal residues (Asn, Gln,
Asp, Glu, and Cys) (FIG. 1) can also be produced through cleavages
anywhere in a protein's polypeptide chain. For example, the
conditional cleavage of a subunit of the mammalian cohesin complex
at the metaphase-anaphase transition is predicted to produce a
putative N-end rule substrate whose degradation would require
N-terminal arginylation. Since the failure to degrade, through the
N-end rule pathway, a cohesin fragment has been shown to impair the
fidelity of chromosome segregation in S. cerevisiae, mouse
ATE1.sup.-/- cells may exhibit an increased chromosome
instability.
[0058] The N-end rule is implicated in a variety of physiological
processes, including angiogenesis. Mice lacking the gene ATE1 were
shown to have extensive defects in angiogenic development. Because
of the importance of the arginylation pathway to angiogenesis,
inhibitors of the cysteine oxygenase activity can be used as
anti-angiogenic compounds for the treatment of tumors, and
particularly of solid tumors.
[0059] Recent studies have identified the mammalian
Met-aminopeptidase MetAP2 as the target of fumagillin and related
inhibitors of angiogenesis. Upon inhibition of MetAP2, some
intracellular proteins partially retain their N-terminal Met
residues. The N-terminal Met-Cys bond (but neither Met-Asp nor
Met-Glu) can be cleaved by MetAP2. The results of the invention
suggest that metabolic stabilization of an arginylation-dependent
N-end rule substrate(s) in mouse ATE1.sup.-/- cells causes
angiogenic and cardiogenic defects. Thus, one possibility is that
fumagillin and related drugs may act by partially inhibiting the
N-terminal Met-Cys cleavage and thereby partially stabilizing an
otherwise short-lived repressor(s) of angiogenesis that bears a
Cys-containing N-degron. If the repressor's half-life is normally
short enough, even a small fraction of repressor molecules that
retain Met-Cys and are, therefore, long-lived, would yield a strong
increase in the repressor's steady-state level, thereby possibly
accounting for the finding that even a partial inhibition of MetAP2
is sufficient to block proliferation of endothelial cells.
Likewise, inhibition of the cysteine oxidase pathway also is likely
to block such proliferation.
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[0092] Although the invention has been described with reference to
the above examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
* * * * *