U.S. patent application number 09/956425 was filed with the patent office on 2002-04-18 for arf and hdm2 interaction domains and methods of use thereof.
This patent application is currently assigned to St. Jude Children's Research Hospital. Invention is credited to Bothner, Brian, Kriwacki, Richard, Lewis, William.
Application Number | 20020045192 09/956425 |
Document ID | / |
Family ID | 25498230 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020045192 |
Kind Code |
A1 |
Kriwacki, Richard ; et
al. |
April 18, 2002 |
Arf and HDM2 interaction domains and methods of use thereof
Abstract
The present invention discloses that the binding of Arf with Dm2
results in specific domains of both proteins undergoing a dramatic
transition from disordered conformations to extended structures
comprised of .beta.-strands. The presence of these specific domains
is necessary and sufficient for the formation of the highly stable
extended .beta. structures formed between these two proteins. The
present invention further exploits this discovery by providing
unique methods for identifying and/or designing compounds that
mimic, inhibit and/or enhance the effect of Arf on Dm2. The present
invention also provides specific protein fragments derived from Arf
and Dm2 that play a critical role in the binding of these two
important regulatory proteins.
Inventors: |
Kriwacki, Richard; (Memphis,
TN) ; Bothner, Brian; (Memphis, TN) ; Lewis,
William; (Memphis, TN) |
Correspondence
Address: |
KLAUBER AND JACKSON
CONTINENTAL PLAZA
411 HACKENSACK AVENUE
HACKENSACK
NJ
07601
US
|
Assignee: |
St. Jude Children's Research
Hospital
332 North Lauderdale Street
Memphis
TN
38105-2794
|
Family ID: |
25498230 |
Appl. No.: |
09/956425 |
Filed: |
September 19, 2001 |
Current U.S.
Class: |
435/7.1 ; 514/1;
702/19 |
Current CPC
Class: |
G01N 2500/04 20130101;
G16B 15/00 20190201; G16B 15/20 20190201; G01N 33/57496 20130101;
G01N 33/574 20130101; C07K 5/1019 20130101; G01N 2500/02 20130101;
C07K 14/4703 20130101; G16B 20/00 20190201 |
Class at
Publication: |
435/7.1 ; 702/19;
514/1 |
International
Class: |
G01N 033/53; A61K
031/00; G06F 019/00; G01N 033/48; G01N 033/50 |
Goverment Interests
[0001] The research leading to the present invention was supported
in part by the American Cancer Society and a Cancer Center (CORE)
Support Grant CA21765. The government may have certain rights in
the present invention. Support for this invention was also provided
by the AMERICAN LEBANESE SYRIAN ASSOCIATED CHARITIES and the ASSISI
FOUNDATION OF MEMPHIS INC.
Claims
What is claimed is:
1. A method of identifying a compound that can induce the formation
of .beta.-strand assembly of Dm2 comprising: (a) contacting the
compound with Dm2 or an inducible fragment of Dm2; and (b)
determining whether Dm2 or the inducible fragment of Dm2 is induced
to form a .beta.-strand assembly; wherein a compound is identified
when Dm2 or the inducible fragment of Dm2 is induced to form a
.beta.-strand assembly.
2. The method of claim 1 wherein said determining is performed by
circular dichroism measurements.
3. The method of claim 1 wherein said determining is performed by
nuclear magnetic resonance.
4. The method of claim 1 wherein said determining is performed by
Fourier Transform Infra-red spectroscopy.
5. The method of claim 1 wherein said determining is performed by
fluorescence spectroscopy.
6. The method of claim 5, wherein said determining is performed by
monitoring the fluorescence of a native tryptophan of Dm2 or of the
inducible fragment of Dm2.
7. The method of claim 5, wherein Dm2 or the inducible fragment of
Dm2 is labeled with a fluorescent probe, and wherein said
determining is performed by monitoring the fluorescence of the
fluorescent probe.
8. The method of claim 1 wherein the Dm2 is Hdm2 having the amino
acid sequence of SEQ ID NO:8.
9. The method of claim 1 wherein the inducible fragment of Dm2
comprises amino acid residues 235-259 of SEQ ID NO:8, the H1
segment.
10. The method of claim 9 wherein the inducible fragment of Dm2
further comprises amino acid residues 275-289 of SEQ ID NO:8, the
H2 segment.
11. The method of claim 1 wherein the inducible fragment of Dm2
comprises amino acid residues 275-289 of SEQ ID NO:8, the H2
segment.
12. A compound identified by the method of claim 1; wherein said
compound is not a peptide comprising five or more consecutive amino
acids comprised by a naturally occurring protein.
13. A method of identifying a compound that can enhance the rate of
.beta.-strand assembly of Dm2 induced by Arf comprising: (a)
contacting the compound with Dm2 or an inducible fragment of Dm2,
and Arf or an inducing fragment of Arf; and (b) determining the
rate of the .beta.-strand assembly of Dm2 or of the inducible
fragment of Dm2; wherein a compound is identified when the rate of
the .beta.-strand assembly of Dm2 or of the inducible fragment of
Dm2 increases in the presence of the compound relative to in the
absence of the compound.
14. A method of identifying a compound that can inhibit the
formation of .beta.-strand assembly of Dm2 comprising: (a)
contacting the compound with Dm2 or an inducible fragment of Dm2,
and Arf or an inducing fragment of Arf; and (b) determining the
rate of formation of a .beta.-strand assembly of Dm2 or the
inducible fragment of Dm2; wherein when the rate of formation of
the .beta.-strand assembly of Dm2 or the inducible fragment of Dm2
decreases in the presence of the compound relative to in its
absence, the compound is identified as a compound that can inhibit
the formation of .beta.-strand assembly of Dm2.
15. A method of identifying a compound that can inhibit the
formation of .beta.-strand assembly of Dm2 comprising: (a)
contacting the compound with Dm2 or an inducible fragment of Dm2,
and Arf or an inducing fragment of Arf; and (b) determining the
amount of formation of a .beta.-strand assembly of Dm2 or the
inducible fragment of Dm2; wherein when the amount of formation of
the .beta.-strand assembly of Dm2 or the inducible fragment of Dm2
decreases in the presence of the compound relative to in its
absence, the compound is identified as a compound that can inhibit
the formation of .beta.-strand assembly of Dm2.
16. A method of identifying a compound that can induce the
formation of supramolecular assemblies comprised of .beta.-strands
of Dm2 comprising: (a) contacting the compound with Dm2 or an
inducible fragment of Dm2; and (b) determining whether Dm2 or the
inducible fragment of Dm2 is induced to form supramolecular
assemblies comprised of .beta.-strands of Dm2; wherein when Dm2 or
the inducible fragment of Dm2 is induced to form supramolecular
assemblies the compound is identified as a compound that can induce
the formation of supramolecular assemblies comprised of
.beta.-strands of Dm2
17. The method of claim 16 wherein said determining is performed by
size exclusion determinations.
18. The method of claim 16 wherein the Dm2 is Hdm2 having the amino
acid sequence of SEQ ID NO:8.
19. The method of claim 16 wherein the fragment of Dm2 comprises
amino acid residues 235-259 of SEQ ID NO:8, the H1 segment.
20. The method of claim 19 wherein the fragment of Dm2 further
comprises amino acid residues 275-289 of SEQ ID NO:8, the H2
segment.
21. The method of claim 16 wherein the fragment of Dm2 comprises
amino acid residues 275-289 of SEQ ID NO:8, the H2 segment.
22. A compound identified by the method of claim 16; wherein said
compound is not a peptide comprising five or more consecutive amino
acids comprised by a naturally occurring protein.
23. A method of treating a patient with cancer or a predisposition
for getting cancer comprising administering to the patient a
compound that can induce .beta.-strand assembly of Hdm2 in a
cell.
24. The method of claim 23 wherein the patient has a tumor with
cells that are characterized by a lack of sufficient Arf activity
to induce cell cycle arrest and/or apoptosis; but wherein the cells
retain functional p53.
25. A kit for identifying a compound that can induce .beta.-strand
assembly of Dm2 comprising: (a) a peptide comprising an amino acid
sequence selected from the group consisting of amino acid residues
235-259 of SEQ ID NO:8; amino acid residues 275-289 of SEQ ID NO:8,
and both amino acid residues 235-259 and amino acid residues
275-289 of SEQ ID NO:8; and (b) a peptide that comprises two copies
of the Arf motif comprising the amino acid sequence of SEQ ID
NO:13.
26. The kit of claim 25 further comprising instructions for
identifying a compound that can induce .beta.-strand assembly of
Dm2.
27. An antibody raised against a peptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:13, amino
acid residues 235-259 of SEQ ID NO:8, and amino acid residues
275-289 of SEQ ID NO:8.
28. The antibody of claim 26 that is a humanized antibody.
29. A method of inducing apoptosis in a cell by administering the
antibody of claim 28 to the cell.
30. A method of treating a patient having a tumor comprising
administering the antibody of claim 29 to the patient; wherein the
tumor contains cells characterized by having functional Arf,
functional Hdm2 and functional p53.
31. A method of designing a compound that is predicted to mimic the
ability of Arf to induce the formation of the .beta.-strand
assembly of Dm2, said method comprising: (a) generating a computer
model of a structure of an Arf-Dm2 complex based on: (i) the amino
acid sequence of the portions of Arf and Dm2 involved in the
Arf-Dm2 complex; and (ii) the circular dichroism and Fourier
Transform Infra-red spectra obtained for the Arf-Dm2 complex; and
(b) designing a compound to bind to Dm2 as Arf does using the
computer model of the structure of the Arf-Dm2 binding complex
generated in step (a); wherein said compound is predicted to mimic
the ability of Arf to induce the formation of the .beta.-strand
assembly of Dm2.
32. The method of claim 31, further comprising: (c) organically
synthesizing said compound; (d) contacting the synthesized compound
with a Dm2 or an inducible fragment of Dm2; and (e) determining
whether the Dm2 or the inducible fragment of Dm2 has formed of a
.beta.-strand assembly; wherein when the Dm2 or the inducible
fragment of Dm2 is induced to form a .beta.-strand assembly in step
(d), the synthesized compound is identified as a compound that
mimics the ability of Arf to induce the formation of the
.beta.-strand assembly of Dm2.
33. A peptide consisting of the amino acid sequence of SEQ ID
NO:13.
34. The peptide of claim 33 consisting of the amino acid sequence
selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11 and SEQ ID NO:12.
35. A fusion protein comprising a peptide consisting of the amino
acid sequence of SEQ ID NO:13.
36. A peptide consisting of two segments of an Arf protein, wherein
each segment consists of the amino acid sequence of SEQ ID
NO:13.
37. The peptide of claim 36 wherein at least one segment consists
of the amino acid sequence selected from the group consisting of
SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.
38. The peptide of claim 37 wherein the other segment consists of
the amino acid sequence selected from the group consisting of SEQ
ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.
39. A fusion protein comprising a peptide consisting of two
segments of an Arf protein, wherein each segment consists of the
amino acid sequence of SEQ ID NO:13.
40. A peptide consisting of amino acid residues 235-259 of SEQ ID
NO:8, the H1 segment.
41. A fusion protein comprising a peptide consisting of amino acid
residues 235-259 of SEQ ID NO:8.
42. A peptide consisting of amino acid residues 275-289 of SEQ ID
NO:8.
43. A fusion protein comprising a peptide consisting of amino acid
residues 275-289 of SEQ ID NO:8.
44. A peptide consisting of amino acid residues 235-259 and amino
acid residues 275-289 of SEQ ID NO:8.
45. A fusion protein comprising a peptide consisting of amino acid
residues 235-259 and amino acid residues 275-289 of SEQ ID
NO:8.
46. A composition comprising two segments of an Arf protein
chemically joined via a non-peptide linkage, wherein each segment
comprises the amino acid sequence of SEQ ID NO:13.
Description
FIELD OF THE INVENTION
[0002] The present invention relates to the interaction of Arf and
Hdm2, as well as to specific protein fragments derived from Arf and
Hdm2 that play a critical role in the binding of these two
regulatory proteins. The present invention also relates to the use
of Arf and Hdm2 and specific fragments thereof in unique assays for
identifying compounds that can be used in the treatment of cancer.
The present invention further relates to identifying and using
specific domains of a given protein that interact with one or more
specific domains of a second protein.
BACKGROUND OF THE INVENTION
[0003] Disruption of cell cycle control mechanisms contributes
significantly to the development of cancer in humans [Sherr, Cancer
Res., 60:3689-95 (2000)]. Consistently, the two most frequently
inactivated tumor suppressor genes in human cancer irrespective of
tumor type, site, and patient age, are the p53 gene and the
INK4a-Arf gene locus, both of which encode proteins involved in the
regulation of cellular replication [Hall and Peters, Adv. Cancer
Res., 68:67-108 (1996); Hainaut et al., Nucleic Acid Res.,
25:151-157 (1997)]. The p53 gene encodes the transcription factor
p53. Activation of the p53 gene in response to oncogenic stress
signals results in cell cycle arrest or apoptosis, thereby enabling
cells to repair genotoxic damage or alternatively, to be eliminated
from the organism [reviewed in Ko et al., Genes & Devel.
10:1054-1072 (1996); Levine, Cell 88:323-331 (1997)]. Loss of p53
function cancels these surveillance functions thereby allowing
defective cells to replicate and predisposing the cell to cancer
development.
[0004] The INK4a/Arf gene locus has been shown to encode two
unrelated proteins from alternative but partially overlapping
reading frames: (i) p16.sup.Ink4a and (ii) Arf (p14.sup.Arf in
humans and p19.sup.ARF in the mouse) [Quelle et al., Cell,
83:993-1000 (1995)]. These proteins independently target two cell
cycle control pathways. The N-terminal 62 amino acid residues of
the 132 amino acid p14.sup.ARF protein and the N-terminal 63 amino
acid residues of the 169 amino acid p19.sup.ARF protein are encoded
by a unique first exon (1.beta.), whereas the remaining amino acid
residues are encoded by exon 2. An alternative reading frame of
exon 2 also encodes the bulk of p16.sup.INK4a.
[0005] p16.sup.INK4a is an antagonist of cell replication. More
specifically, p16.sup.INK4a inhibits the cyclin D-dependent kinases
CDK4 and CDK6 [Serrano et al., Nature, 366:704-707 (1993)]. CDK4
and CDK6 play an important role in the cell replication cycle
through their phosphorylation of the retinoblastoma protein (Rb).
Hyperphosphorylation of Rb stimulates the cell to exit from the G1
phase and begin DNA synthesis, a required step prior to cell
division. Thus, the inhibition of CDK4 and CDK6 by p16.sup.INK4a
prevents hyperphosphorylated Rb-dependent DNA synthesis, thereby
maintaining the cell in its non-replicating mode.
[0006] Disruption in mice of either the entire INVK4a/Arf locus
[Serrano et al., Cell, 85: 27-37 (1996)] or exon 1.beta. [Kamijo et
al., Cell, 91:649-59 (1997)] leads to multi-type tumor growth and
early death, identifying Arf as a bona fide tumor suppressor.
Interestingly, it has been suggested that disruption of INK4a does
not contribute to spontaneous tumor formation in mice and that Arf
disruption accounts for the high rate of spontaneous tumor
formation in INK4a/Arf-null mice [Sherr, Cancer Res., 60: 3689-95
(2000)]. Since the INK4a/Arf locus is frequently disrupted in human
cancers [Raus and Peters, Biochim. Biophys. Acta Rev. Cancer,
1378:F115-F177 (1998)], the loss of Arf function appears to be a
major contributor to human cancers.
[0007] Indeed, Arf, in concert with other cell cycle regulators and
tumor suppressors such as p53 and Rb, plays a central role in
cellular responses to oncogenic stress, such as inappropriate
mitogenic signaling. For example, Arf expression is activated by
overexpression of proteins involved in mitogenic signaling, such as
Myc [Zindy et al., Genes & Dev., 12:2424-2434 (1998)], E1A [de
Stanchina et al., Genes Dev., 12:2434-42 (1998)], E2F [Bates et
al., Nature, 395:124-5 (1998)], Ras [Palmero et al., Nature,
395:125-6 (1998)], and v-Ab1 [Radfar et al., Proc. Natl. Acad. Sci.
U.S.A., 95:13194-13199 (1998)]. Activation of Arf leads to
stabilization of p53 [Pomerantz et al., Cell, 92:713-23 (1998);
Kamijo et al., Proc. Natl. Acad. Sci., 95:8292-8297 (1998); Stott
et al., Embo J, 17: 5001-14 (1998); and Zhang et al., Cell,
92:725-734 (1998)] followed by cell cycle arrest. Arf therefore
connects the Rb and p53 pathways [Sherr, Cancer Res., 60: 3689-95
(2000)] so that excessive proliferative signaling via the Rb
pathway activates arrest mechanisms controlled by p53.
[0008] Arf stabilizes p53 by interfering with an auto-regulatory
loop involving p53 and Double Minute 2 (Hdm2 in humans, Mdm2 in
mice) [Wu et al., Genes Dev., 7:1126-1132 (1993)] that maintains
p53 at low levels under normal cellular conditions (i.e. in the
absence of oncogenic stress, DNA damage, etc.). The positive
component of this auto-regulatory loop involves activation of Mdm2
transcription by p53 [Barak et al., EMBO J, 12:461-468 (1993)]. The
negative component has several facets. First, Mdm2 binds p53
[Kussie et al., Science, 274:948-953 (1996)] and inhibits the
transactivation function of p53 [Oliner et al., Nature, 362:857-860
(1993); Momand et al., Cell, 69:1237-1245 (1992)]. Second, Mdm2
shuttles p53 from the nucleus to the cytoplasm and facilitates p53
degradation [Roth et al., Embo J, 17:554-64 (1998)]; Freedman et
al., Mol Cell Biol, 18:7288-93 (1998)]. Third, Mdm2 acts as an E3
ubiquitin ligase toward p53 within the ubiquitin-dependent 26S
proteosome pathway [Honda et al., FEBSLett, 420:25-7 (1997)].
Therefore, Mdm2 inhibits p53 activity in the nucleus through
multiple and diverse mechanisms. Balance between the positive and
negative components of this auto-regulatory system is essential for
cell survival. When p53 is inactivated, mice develop tumors at an
unusually high rate [Donehower et al., Nature, 356:215-221 (1992)],
indicating that p53-dependent tumor suppression is compromised.
Additionally, when Mdm2 is inactivated, mice are not viable [Jones
et al., Nature, 378:206-8 (1995); Montes de Oca Luna et al.,
Nature, 378:203-206 (1995)], suggesting that unregulated p53
expression is lethal. Mdm2.sup.-/- mice are rescued, however, by
the additional inactivation of p53 [Jones et al., Nature, 378:206-8
(1995); Montes de Oca Luna et al., Nature, 378:203-206 (1995)].
Thus, proper regulation of p53 activity relies on appropriate
balance between the positive and negative components of the
p53-Mdm2 auto-regulatory system.
[0009] The first direct biochemical connection between p19.sup.ARF
and p53 was established when it was found that p19.sup.ARF could
bind to Mdm2, [Pomerantz et al., Cell, 92:713-723 (1998); Zhang et
al., Cell, 92:725-734 (1998)]. Arf was subsequently found to
inhibit the negative components of the p53-Mdm2 auto-regulatory
loop by interfering with several of Mdm2's activities toward p53.
First, by binding Mdm2, Arf inhibits Mdm2-dependent
nucleo-cytoplasmic shuttling of p53 which leads to stabilization
and activation of p53 [Tao et al., Proc Natl Acad Sci USA,
96:6937-41(1999)]. Second, Arf inhibits the E3 ubiquitin ligase
activity of Mdm2 toward p53 in vitro [Honda et al., Embo J, 18:22-7
(1999); Midgley et al., Oncogene, 19:2312-23 (2000)] and is thought
to be an important aspect of Arf-dependent activation of p53 in
vivo [Midgley et al., Oncogene, 19:2312-23 (2000); Llanos et al.,
Nat. Cell Bio., 3:445-452 (2001)]. Finally, Arf binds and
sequesters Mdm2 in the nucleolus, physically separating Mdm2 and
p53 in different sub-cellular compartments [Weber et al., Nat. Cell
Biol., 1:20-26 (1999); Lohrum et al., Nat. Cell Biol., 2:179-81
(2000); Weber et al., Mol. Cell Biol., 20:2517-2528 (2000)]. The
relative importance of these three mechanisms to Arf-dependent
stabilization and activation of p53 is a matter of debate. For
example, a recent report shows that Mdm2 binding but not nucleolar
localization is the functional property of Arf that is required for
p53 activation [Llanos et al., Nat. Cell Biol., 3:445-452 (2001)].
This report, however, does not rule out earlier reports that
nucleolar co-localization of Arf and Mdm2 contribute to p53
stabilization through sequestration [Weber et al., Nat. Cell Biol.,
1:20-26 (1999)]. It is likely that Arf acts via several mechanisms
to stabilize p53 and these have evolved in concert with the
multiplicity of Mdm2's effects on p53. Importantly, direct
interaction between Arf and Hdm2 is required for the multiple
mechanisms of p53 stabilization.
[0010] Therefore, there is a need to further characterize the
Arf-Hdm2 complex. In addition, there is a need to determine the
specific domains of Arf and Hdm2 that are involved in this complex.
Furthermore, there is a need to identify compounds that can mimic
the effect of Arf on Hdm2, since the absence of functional Arf is
commonplace in tumor cells. Alternatively, there is a need to
identify compounds that inhibit the binding of Arf to Hdm2 to
prevent undesired activation of p53-dependent pathways by, for
example, DNA damaging agents, in normal cells.
[0011] The citation of any reference herein should not be deemed as
an admission that such reference is available as prior art to the
instant invention.
SUMMARY OF THE INVENTION
[0012] Through disclosing that the binding of Arf with Hdm2 results
in specific domains of both proteins undergoing a dramatic
transition from disordered conformations to extended structures
comprised of .beta.-strands, the present invention provides new
insight towards the identification/design of novel anti-cancer
therapeutics. Thus, in a particular aspect of the present invention
unique assays are provided for identifying compounds that mimic
and/or enhance, or alternatively inhibit the effect of Arf on Hdm2.
In a related aspect of the present invention specific protein
fragments derived from Arf and Hdm2 that play a direct role in the
binding of these two important regulatory proteins are
provided.
[0013] Therefore, the present invention provides methods of
identifying a compound that can induce .beta.-strand assembly of
Dm2 (e.g., Hdm2 or Mdm2). One such method comprises contacting the
compound with Dm2 or an inducible fragment of Dm2 (e.g., a fragment
of Dm2 that is capable of being induced to .beta.-strand assembly
by Arf) and then determining whether Dm2 or the inducible fragment
of Dm2 is induced to form a .beta.-strand assembly by the compound.
A compound is identified when Dm2 or the inducible fragment of Dm2
is induced to form a .beta.-strand assembly. In a particular
embodiment, a peptide or protein comprising the Arf motif (i.e.,
the amino acid sequence of SEQ ID NO:13) responsible for inducing
.beta. strand assembly in Hdm2, can be used as a positive
control.
[0014] The present invention also provides methods of identifying a
compound that can enhance the rate of .beta.-strand assembly of Dm2
induced by Arf. One such embodiment comprises contacting the
compound with Dm2 or an inducible fragment of Dm2, and Arf or an
inducing fragment of Arf and then determining the rate of the
.beta.-strand assembly of Dm2 or of the inducible fragment of Dm2.
A compound is identified that can enhance the rate of .beta.-strand
assembly of Dm2 induced by Arf when the rate of the .beta.-strand
assembly of Dm2 or of the inducible fragment of Dm2 increases in
the presence of the compound relative to in the absence of the
compound.
[0015] The present invention further provides methods of
identifying a compound that can inhibit the formation of
.beta.-strand assembly of Dm2. In a particular embodiment of this
type the compound is contacted with Dm2 or an inducible fragment of
Dm2, and Arf or an inducing fragment of Arf, and the rate of
formation of a .beta.-strand assembly of Dm2 or the inducible
fragment of Dm2 is determined. A compound is identified that can
inhibit the formation of .beta.-strand assembly of Dm2 when the
rate of formation of the .beta.-strand assembly of Dm2 and/or the
rate of formation of the .beta.-strand assembly of the inducible
fragment of Dm2 decreases in the presence of the compound relative
to in its absence.
[0016] In a related embodiment, the compound is contacted with Dm2
or an inducible fragment of Dm2, and Arf or an inducing fragment of
Arf, and the amount of formation of a .beta.-strand assembly of Dm2
or the inducible fragment of Dm2 is determined. A compound is
identified that can inhibit the formation of .beta.-strand assembly
of Dm2 when the amount of formation of the .beta.-strand assembly
of Dm2 or the inducible fragment of Dm2 decreases in the presence
of the compound relative to in its absence.
[0017] In a particular embodiment, the inducing and/or inhibiting
of .beta.-strand assembly of Dm2 or the inducible fragment of Dm2
is determined by circular dichroism (CD) measurements. In another
embodiment, the inducing and/or inhibiting of .beta.-strand
assembly of Dm2 or the inducible fragment of Dm2 is determined by
nuclear magnetic resonance (NMR) measurements. In yet another
embodiment, the inducing and/or inhibiting of .beta.-strand
assembly of Dm2 or the inducible fragment of Dm2 is determined by
Fourier Transform Infra-red (FTIR) spectroscopy. In still another
embodiment, the inducing and/or inhibiting of .beta.-strand
assembly of Dm2 or the inducible fragment of Dm2 is determined by
fluorescence spectroscopy. In another embodiment the natural
fluorescence of one or more tryptophan residues in Dm2 are used to
monitor the induction and/or inhibition of .beta.-strand assembly.
In one such embodiment changes in the intensity, wavelength, and/or
anisotropy of tryptophan emission are used to monitor the binding
of Arf to Dm2 and the formation of .beta.-strand assemblies. In an
alternative embodiment, a fluorescent probe, such as Texas Red.TM.,
is covalently bound to either Dm2 or Arf. Changes in the
fluorescence intensity, excitation and/or emission wavelength,
and/or anisotropty of the probe can be monitored when the unlabeled
and labeled species are mixed together.
[0018] The present invention also provides methods of identifying a
compound that can induce supramolecular assemblies comprised of
.beta.-strands of Dm2 or a inducible fragment of Dm2 (e.g., Hdm2 or
Mdm2). One such method comprises contacting the compound with Dm2
or an inducible fragment of Dm2 that is capable of being induced to
form supramolecular assemblies by Arf, and then determining whether
the compound induces Dm2 or the inducible fragment of Dm2 to form
supramolecular assemblies. A compound is identified when Dm2 or the
inducible fragment of Dm2 is induced to form supramolecular
assemblies. In a particular embodiment, a peptide or protein
comprising the Arf motif, (i.e., the amino acid sequence of SEQ ID
NO:13) can be used as a positive control. In one embodiment, the
inducing of supramolecular assemblies of Dm2 or the inducible
fragment of Dm2 is determined by size exclusion measurements. In a
preferred embodiment of this type, the inducing of supramolecular
assemblies of Dm2 or the inducible fragment of Dm2 is determined by
gel filtration chromatography.
[0019] In one embodiment, the Dm2 used in the methods of the
invention is Hdm2. In a preferred embodiment of this type, the Hdm2
comprises the amino acid sequence of SEQ ID NO:8. In still another
embodiment, the inducible fragment of Hdm2 comprises amino acid
residues 235-259 of SEQ ID NO:8, which is the H1 segment. In a
related embodiment, the inducible fragment of Hdm2 comprises amino
acid residues 275-289 of SEQ ID NO:8, which is the H2 segment. In a
preferred embodiment, the inducible fragment of Hdm2 comprises both
amino acid residues 235-259 and amino acid residues 275-289 of SEQ
ID NO:8.
[0020] The present invention also provides a compound that is
identified by a method of the present invention. Preferably the
compound does not comprise five or more consecutive amino acids of
a naturally occurring protein. More preferably the compound is
neither an amino acid nor made up of animal acids (i.e., a compound
which is not a peptide). Even more preferably, the compound is a
small molecule that that has a molecular weight of less than 3
Kilodaltons.
[0021] In related embodiments, compounds can be tested for their
ability to either enhance the effect of Arf or alternatively
interfere with the formation of the Arf-Dm2 complex using similar
protocols as outlined above except both Arf or an inducing fragment
of Arf, and Dm2 or an inducible fragment of Dm2 are included in the
assay.
[0022] The formation of supramolecular assemblies and/or
.beta.-strand assembly can be readily monitored, e.g., by NMR, CD,
FTIR, fluorescence and/or size exclusion. Therefore, a compound can
be contacted with the Arf and Dm2 (and/or fragments thereof) and
the amount of formation of the supramolecular assemblies and/or
.beta.-strand assembly of the Arf-Dm2 can be determined. When the
compound decreases or eliminates the supramolecular assemblies
and/or .beta.-strand assembly of the Arf-Dm2 complex, the compound
is identified as an inhibitor of the Arf-Dm2 interaction. Similarly
the kinetics of the rate of formation of the supramolecular
assemblies and/or .beta.-strand assembly can be measured, and
compounds can be assayed to select inhibitors or enhancers of the
rate of formation of the supramolecular assemblies and/or
.beta.-strand assembly, as exemplified herein.
[0023] All of the methods for identifying compounds of the present
invention can be performed by adding a compound to the assay
solution at any time during the assay, including making additions
at multiple times. Thus the compound can be added: (i) prior to the
addition of Arf and/or an inducing fragment of Arf; and/or (ii)
prior to the addition of Dm2 and/or an inducible fragment of Dm2;
and/or (iii) together with Arf and/or an inducing fragment of Arf;
and/or (iv) together with Dm2 and/or an inducible fragment of Dm2;
and/or (v) after the addition of Arf and/or an inducing fragment of
Arf; and/or (vi) after the addition of Dm2 and/or an inducible
fragment of Dm2.
[0024] In addition the present invention provides methods of
designing compounds that are predicted to mimic, enhance or
alternatively inhibit the Arf-induced formation of .beta.-strand
assembly of Dm2. One such method comprises defining the structure
of the Arf-Dm2 complex by using computer-based molecular modeling
and docking techniques. In this approach, ensembles of molecular
models for segments of Arf (for example, the Arf motif, or the
segments of human or mouse Arf embodied by this motif) and Dm2 (for
example, the H1 and/or H2 segments) are generated that are
consistent with CD and FT-IR spectra for the Arf-Dm2 complex,
namely that the polypeptide backbone torsion angles adopt values
allowed in .beta.-strands. Then, each member of the Arf ensemble is
systematically docked with each member of the Dm2 ensemble using
programs such as DOCK, or AUTODOCK. During the docking stage of the
procedure, the binding energy for each of a large number of
alternative Arf-Dm2 binding configurations is calculated and the
docked configurations ranked according to binding energy. The
Arf-Dm2 binding models with the lowest overall binding energy will
then be used to design and/or identify a compound that is predicted
to mimic, enhance, or alternatively inhibit the Arf-induced
formation of .beta.-strand assembly of Dm2.
[0025] As the skilled artisan would readily recognize, compounds
designed and/or identified by this method can then be synthesized
(if necessary) and tested in any of a number of assays, including
those described above. For example, in one such embodiment the
method further comprises contacting the compound with Dm2 or an
inducible fragment of Dm2 and then determining whether Dm2 or the
inducible fragment of Dm2 is induced to form a .beta.-strand
assembly. The compound is identified as a mimic of Arf if Dm2 or
the inducible fragment of Dm2 is induced to form a .beta.-strand
assembly.
[0026] The present invention further provides methods of treating
patients with cancer and/or patients having a predisposition for
developing cancer. In a particular embodiment of this type, the
patient has a tumor with cells that are characterized by a lack of
sufficient Arf activity (e.g., lacking of a functional Arf), but
still retain functional p53. One specific embodiment comprises
administering to a patient a compound that mimics and/or enhances
Arf activity that was identified by a method of the present
invention. In a related embodiment, a compound is administered that
can induce .beta.-strand assembly of Dm2 in a cell that is lacking
a functional Arf protein and/or sufficient Arf activity to
de-repress the repression of p53 mediated apoptosis by Dm2 and
thereby arrest cell growth.
[0027] The present invention also provides specific fragments of
the Arf and Dm2 proteins and peptides comprising the amino acid
sequences of such fragments that can induce .beta.-strand assembly
of Dm2. Fusion proteins (including chimeric proteins) comprising
these fragments/peptides are also provided, as are nucleic acids
encoding such fragments/peptides, and corresponding fusion
proteins. Preferably the fragments/peptides are between 8 and 50
amino acids in length. In one such embodiment the fragment/peptide
comprises and/or consists of the amino acid sequence of SEQ ID
NO:13. In a particular embodiment of this type, the
fragment/peptide comprises and/or consists of the amino acid
sequence of SEQ ID NO:9. In a another embodiment of this type, the
fragment/peptide comprises and/or consists of the amino acid
sequence of SEQ ID NO:10. In yet another embodiment, the
fragment/peptide comprises and/or consists of the amino acid
sequence of SEQ ID NO:11. In still another embodiment of this type,
the fragment/peptide comprises and/or consists of the amino acid
sequence of SEQ ID NO:12.
[0028] In a preferred embodiment the fragment/peptide comprises or
alternatively consists of two or more segments of the Arf protein
each segment comprising and/or consisting of the amino acid
sequence of SEQ ID NO:13. In one embodiment of this type, the
fragment/peptide comprises and/or consists of both the amino acid
sequence of SEQ ID NO:9 and SEQ ID NO:10. In another embodiment,
the fragment/peptide comprises and/or consists of both the amino
acid sequence of SEQ ID NO:11 and SEQ ID NO:12. In still another
embodiment, the fragment/peptide comprises and/or consists of both
the amino acid sequence of SEQ ID NO:9 and SEQ ID NO:12. In yet
another embodiment, the fragment/peptide comprises and/or consists
of both the amino acid sequence of SEQ ID NO:10 and SEQ ID
NO:11.
[0029] In addition to peptides comprising the Arf segments
described above, the invention also provides compositions comprised
of at least one pair of the peptide segments described above linked
together by a non-peptide linkage, e.g., a non-peptide chemical
linkage.
[0030] In a related embodiment, the present invention provides a
fragment/peptide that comprises and/or consists of amino acid
residues 235-259 of SEQ ID NO:8. In still another embodiment of
this type the fragment/peptide comprises and/or consists of amino
acid residues 275-289 of SEQ ID NO:8. In a preferred embodiment,
the fragment/peptide comprises and/or consists of amino acid
residues 235-259 and amino acid residues 275-289 of SEQ ID
NO:8.
[0031] In another aspect of the present invention, antibodies
raised against specific fragments/peptides of Arf and/or Dm2 are
provided. In one such embodiment the antibody is raised against a
fragment/peptide comprising an amino acid sequence of SEQ ID NO:13.
In another embodiment, the antibody is raised against a
fragment/peptide comprising amino acid residues 235-259 of SEQ ID
NO:8. In yet another embodiment, the antibody is raised against a
fragment/peptide comprising amino acid residues 275-289 of SEQ ID
NO:8. In a preferred embodiment, the antibody is raised against a
fragment/peptide comprising amino acid residues 235-259 and amino
acid residues 275-289 of SEQ ID NO:8. In one embodiment, the
antibody is a polyclonal antibody. In another embodiment, the
antibody is a monoclonal antibody. In still another embodiment, the
antibody is a chimeric and/or humanized antibody.
[0032] The present invention further provides methods of inducing
apoptosis in a cell. In one such embodiment, apoptosis is induced
by administering an antibody of the present invention to a cell. In
a preferred embodiment of this type the antibody is a humanized
antibody. In a related embodiment, apoptosis is induced by
administering a compound identified by a method of the present
invention to the cell.
[0033] The present invention further provides methods of treating a
patient for which induced apoptosis in targeted cells is a
desirable treatment, such as various forms of cancer in which p53
is functional and Dm2 is overexpressed and/or p53 is functional and
Arf is not functional. One or both of these conditions is thought
to be a contributing factor in the development of a wide variety of
cancers, including acute myeloid leukemia [Faderl, et. al. Cancer,
89:1976-82 (2000) ], breast cancer [Takami etal. Breast Cancer, 30:
95-102 (1994)], Burkitt lymphoma [Lindstrom et. al. Oncogene, 20:
2117-7 (2001)], clear cell renal cell carcinoma [Haitel et. al.
Clin. Cancer Res., 6: 1840-4 (2000)], colon carcinomas [Burri et.
al. Lab. Invest., 81: 217-29 (2001)], ependymomas [Suzuki and Iwaki
Mod. Pathol. 13:548-53 (2000)], gastric cancer [Villaseca et. al.
Rev. Med. Chil., 128:127-36 (2000)], glioblastoma [Fulci et. al.
Oncogene, 19: 3816-22 (2000)], Hodgkin's disease [Kupper et. al.
Br. J. Haematol., 112: 768-75 (2001)], intrahepatic
cholangiocarcinoma [Horie. Archet. al. Virchows., 437: 25-30
(2000)], intimal sarcomas arising in the pulmonary artery
[Bode-Lesniewska et. al. Virchows. Arch., 438: 57-65 (2001)],
malignant pleural mesothelioma [Yang et. al. Cancer Res., 61:
5959-63 (2001)], melanoma-neural system tumour syndrome
[Randerson-Moor et. al. Hum. Molec. Genet., 10: 55-62 (2001)],
non-Hodgkin's lymphomas [Pagnano et. al. Am. J. Hematol., 67: 84-92
(2001)], non-small cell lung carcinomas [Gorgoulis et. al. Mol.
Med., 6: 208-37 (2000)], ovarian tumors [Palazzo et. al. Hum.
Pathol., 31: 698-704 (2000)], oral cancer [Ralhan et. al. Am. J.
Pathol., 157: 587-96 (2000)], oral squamous cell carcinoma [Sano
et. al. Pathol. Int., 50: 709-16 (2000)], paragangliomas [Lam et.
al. J. Clin. Pathol., 54: 443-8 (2001)], phaeochromocytomas [Lam
et. al. J. Clin. Pathol., 54: 443-8 (2001)], primary central
nervous system lymphomas [Nakamura et. al. Cancer Res., 61 6335-39
(2001)], prostate carcinoma [Leite et. al. Mod. Pathol. 14: 428-36
(2001)], soft tissue sarcoma [Bartel et. al. Int. J. Cancer,
95:168-75 (2001)], and urinary bladder carcinoma [Ioachim et. al.
Histol. Histopathol., 15: 721-7 (2000)].
[0034] One such method comprises administering an antibody of the
present invention to a patient. Preferably, the treatment is
administered to a patient that has a tumor containing cells
characterized by the presence of functional Hdm2, and functional
p53.
[0035] The present invention also provides kits for identifying a
compound that can induce .beta.-strand assembly of Dm2 in the
absence and/or presence of Arf. One such kit comprises a fragment
of Hdm2 that comprises amino acid residues 235-259 of SEQ ID NO:8.
In another embodiment, the kit comprises a fragment of Hdm2 that
comprises amino acid residues 275-289 of SEQ ID NO:8. In a
particular embodiment the kit comprises a fragment of Hdm2 that
comprises amino acid residues 235-259 and amino acid residues
275-289 of SEQ ID NO:8. In another embodiment a peptide that
comprises the amino acid sequence of SEQ ID NO:13 is also included.
Preferably this peptide comprises two copies of the amino acid
sequence of SEQ ID NO:13. More preferably the kit further comprises
instructions for identifying a compound that can induce
.beta.-strand assembly of Dm2.
[0036] Accordingly, it is a principal object of the present
invention to provide an assay for selecting drugs that can be used
to treat cancer.
[0037] It is a further object of the present invention to provide
agents that can mimic and/or enhance the ability of Arf to
stimulate .beta.-strand assembly of Dm2.
[0038] It is a further object of the present invention to provide
peptides consisting of defined minimal domains of Arf and Dm2 that
are necessary and sufficient for Arf-Dm2 binding.
[0039] It is a further object of the present invention to provide
methods of treating diseases that are adversely affected by the
existence of cells that do not contain sufficient Arf activity.
[0040] It is a further object of the present invention to provide
methods of identifying agents that can interfere with the ability
of Arf to bind Dm2, including antibodies to Arf or Dm2.
[0041] It is a further object of the present invention to provide
agents that can interfere with the ability of Arf to bind Dm2.
[0042] It is a further object of the present invention to provide
antibodies that can interfere with the ability of Arf to bind
Dm2.
[0043] These and other aspects of the present invention will be
better appreciated by reference to the following drawings and
Detailed Description.
BRIEF DESCRIPTION OF THE SEQUENCES
[0044]
1 SEQ ID NO: TYPE ORGANISM PROTEIN 1 Nucleic Acid Mouse Arf 2 Amino
Acid Mouse Arf 3 Nucleic Acid Human Arf 4 Amino Acid Human Arf 5
Nucleic Acid Mouse Mdm2 6 Amino Acid Mouse Mdm2 7 Nucleic Acid
Human Hdm2 8 Amino Acid Human Hdm2 9 Amino Acid Mouse mA1 10 Amino
Acid Mouse mA2 11 Amino Acid Human hA1 12 Amino Acid Human hA2 13
Amino Acid Consensus Arf Motif 14 Amino Acid Consensus RRPR 15
Amino Acid Human N-terminal 1-37 of Arf 16 Amino Acid Mouse
N-terminal 1-37 of Arf 17 Amino Acid Opossum N-terminal 1-37 of Arf
18 Amino Acid Human .about.210-304 of Dm2 19 Amino Acid Mouse
.about.210-304 of Dm2 20 Amino Acid Hamster .about.210-304 of Dm2
21 Amino Acid Horse .about.210-304 of Dm2 22 Amino Acid Dog
.about.210-304 of Dm2 23 Amino Acid Chicken .about.210-304 of Dm2
24 Amino Acid Zebrafish .about.210-304 of Dm2 25 Amino Acid Tree
Frog .about.210-304 of Dm2
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIGS. 1a-1b show the surface plasmon resonance sensograms of
Hdm2 constructs binding to mArfN37. His-tagged mArN37 was
immobilized on the SPR biosensor surface using a covalently linked
His antibody. Binding of Hdm2 210-304 of SEQ ID NO:8 (FIG. 1a) and
Hdm2 210-275 of SEQ ID NO:8 (FIG., 1b).
[0046] FIGS. 2a-2c show the structure prediction for Arf and Hdm2
binding domains. Alignment of sequences for residues 1-37 of human
(SEQ ID NO:15), mouse (SEQ ID NO:16) and opossum (SEQ ID NO:17) Arf
(FIG. 2a) and .about. residues 210-304 of Mdm2 (FIG. 2b) from
several species listed in THE BRIEF DESCRIPTION OF THE SEQUENCES
above, corresponding to SEQ ID NOs:18-25. Residues that are
underlined are conserved in all sequences and those in bold type
are conserved in several sequences. The program Jnet was used to
predict secondary structure and solvent exposure within the aligned
regions. The secondary structure predictions are labeled "Jnet
pred."; .beta.-strand secondary structure is abbreviated "E",
.alpha.-helix "H", and random coil "-". The prediction confidence
score is labeled "Jnet conf.", with 0 the lowest and 9 the highest
confidence values. The prediction of solvent exposure is labeled
"Solv. Exp.", with "B" indicating that a residue is predicted to be
less than 25% solvent exposed and "-" indicating greater than 25%
solvent exposure. The schematic illustration of peptides derived
from Hdm2 210-304 that bind mArfN37 is shown in FIG. 2c.
[0047] FIG. 3 shows the characterization of Arf:Hdm2 assemblies. CD
spectra of mArfN37 complexed with Hdm2 210-304 (solid line) and
Hdm2 210-275 (dotted line) are consistent with .beta.-strand
secondary structure. The molar ratio of the two species in each
sample is given.
[0048] FIG. 4 shows the "Arf motif" (consensus sequence, SEQ ID
NO:13). A short sequence of 8 or 9 amino acids is repeated twice in
mouse and human Arf. An alternating hydrophobic/charge pattern
mediates binding.
[0049] FIGS. 5a-5c show the surface plasmon resonance (SPR) binding
experiments with Arf and Hdm2 peptides which reveal sites of
interaction. His-tagged Hdm2 210-304 (FIGS. 5a-5b) and His-tagged
mArfN37 (FIG. 5c) were captured on the SPR surface with an anti-His
antibody. The binding of peptides derived from the N-terminus of
mouse Arf (FIG. 5a), human Arf (FIG. 5b), or the central, acidic
domain of Hdm2 (FIG. 5c) was monitored. *, this peptide is
anomalous because it binds extensively to the reference cell.
DETAILED DESCRIPTION OF THE INVENTION
[0050] Isolated Arf and Hdm2 domains are dynamically disordered in
solution, yet they retain the ability to interact in vitro and in
cellular assays. As shown below, upon binding, domains of both Arf
and Hdm2 undergo a dramatic transition from disordered
conformations to extended structures comprised of .beta.-strands.
The presence of domains from both proteins is necessary and
sufficient for the formation of the highly stable extended .alpha.
structures. Sites within Arf and Hdm2 that interact at a resolution
of 5 amino acids have been mapped using surface plasmon resonance
(SPR). SPR and circular dichroism (CD) spectropolarimetry confirm
the presence of multiple interaction domains within each protein
(see Example below).
[0051] As disclosed herein, small peptide segments are identified
within Arf and Hdm2 that are responsible for the interactions of
these two proteins and mediate their nucleolar localization.
Furthermore, pure Arf and Hdm2 are both shown to be dynamically
disordered in solution but that, when mixed in vitro, they adopt
highly stable .beta.-sheet structures. The .beta.-structures
prepared in vitro, however, are extended networks and are relevant
to the structures that form when Arf and Hdm2 interact in cells
within the nucleoplasm and/or nucleoli.
[0052] Both p14.sup.Arf (human) and p19.sup.Arf (mouse) interact
with Hdm2 through two short motifs present in their N-termini. The
Arf interacting region of Hdm2 is also composed of two short
sequences located in the central acidic domain, between residues
235-264 and 270-289 of SEQ ID NO:8. The binding-induced structural
transition is also induced by short peptides, 15 amino acids in
length, which contain the binding motifs. Micro-injection and live
cell imaging of proteins tagged with fluorescent labels was used to
confirm the in vivo function of the interaction domains. Arf and
Hdm2 thus appear to interact through a novel mechanism that exerts
control over the cell division cycle. A detailed analysis of
Arf/Hdm2 interactions is disclosed herein. The present invention
therefore provides unique opportunities for the development of
anticancer therapeutics due to the novel interaction between Dm2
and Hdm2 and the limited size of the protein domains involved.
[0053] Furthermore, small segments of Arf and Hdm2 have been
identified that mediate binding and that can play a role in
regulating Hdm2's repressor function toward p53. Using this
information, it is now possible to inhibit p53 destruction by
disrupting inter-domain interactions within Hdm2 with molecules
that mimic and/or enhance Arf function. The Arf motif is relatively
small, and may be mimicked by yet smaller molecules. Similarly, the
molecular targets of this motif, the H1 and H2 segments of Hdm2,
are small. These findings provide a method for searching for small
molecules that bind Hdm2 in a manner that mimics Arf, and that may
produce biological effects similar to those produced by Arf. Since
many human cancers are characterized by Arf loss while p53 is
maintained in wild-type form [Sherr, Cancer Res., 60: 3689-95
(2000)], this methodology should have wide-ranging implications in
the treatment of cancer in humans.
2 Abbreviations: BrdU 5-bromodeoxyuridine CD circular dichroism
CHAPSO 3-[(3-Cholamidopropyl) dimethylammonio]-2-
hydroxypropanesulfonic acid DAPI 4'-6-Diamidino-2-phenylindole-2HCl
DMEM Dulbecco's modified Eagle's medium EDC
N-Ethyl-N'-(3-Dimethylamino- propyl)Carbodiimide FBS fetal bovine
serum FMOC- .alpha.-(9-fluorenylmethyloxycarbonyl)-amino acid GFP
green fluorescent protein HBS-N 0.01 M HEPES, pH 7.4, 0.15 M NaCl
buffer HBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium-
hexafluorophosphate HEPES 4-(2-Hydroxyethyl)-1-Piperazinee-
thanesulfonic Acid HMP hydroxymethylphenyl-polystyrene resin HOBt
N-hydroxybenzotriazole NHS N-Hydroxysuccinimide NLS nuclear
localization signal NMR nuclear magnetic resonance NoLS nucleolar
localization signal PBS phosphate-buffered saline SDS-PAGE sodium
dodecyl sulfate polyacrylamide gel electrophoresis SPR surface
plasmon resonance TFA trifluoroacetic acid
[0054] Therefore, if appearing herein, the following terms shall
have the definitions set out below.
[0055] As used herein the terms "Arf", "ARF", "p19.sup.ARF
protein," "p19.sup.ARF", "ARF-p19", "ARF-p19/ARF-p14", "p14.sup.ARF
protein," "p14.sup.ARF", or "ARF-p14" are all used interchangeably
except that "p14.sup.ARF protein," "p14.sup.ARF", "ARF-p14" in
general refer specifically to the human protein. Arf is involved in
regulation of the eukaryotic cell cycle. The Arf protein is encoded
by a nucleic acid derived from the gene locus, INK4A-Arf, which
also encodes an inhibitor of D-type cyclin-dependent kinases termed
"p16.sup.InK4a protein," "p16.sup.InK4" or simply "InK4a-p16." [see
also Quelle et al., Cell, 83:993-1000 (1995)]
[0056] An "active fragment" of an Arf protein is a peptide or
polypeptide that comprises a fragment of Arf and retains at least
one physiological activity of the Arf e.g. by acting as a tumor
suppressor and/or having the ability to bind to Dm2. Examples of
active fragments of Arf are the peptides encoded by exon 1.beta.,
e.g. amino acid residues 1-62 of SEQ ID NO:4 and the peptide
encoded by amino acid residues 1-37 of SEQ ID NO:2. A fusion
protein comprising an active fragment of an Arf protein can often
be used interchangeably with an active fragment of an Arf protein,
and such fusion proteins are meant to be included when the term
"active fragment" of an Arf protein is used.
[0057] As used herein a peptide "consisting of a minimal domain of
Arf" is a peptide comprising the minimum portion of the full-length
Arf that still retains the ability of the full-length Arf protein
to bind Dm2 and act as a tumor repressor.
[0058] As used herein, an "inducing fragment" of an Arf protein is
an active fragment of an Arf protein that can induce supramolecular
assemblies and/or .beta.-strand assembly of (i) DM2; and/or (ii) an
inducible fragment of Dm2; and/or (iii) an Arf-Dm2 complex.
Preferably an inducing fragment of Arf comprises two copies of the
Arf motif each comprising the amino acid sequence of SEQ ID NO:13.
A fusion protein comprising an inducing fragment of an Arf protein
can often be used interchangeably with an inducing fragment of an
Arf protein, and such fusion proteins are meant to be included when
the term inducing fragment of an Arf protein is used.
[0059] As used herein the term "sufficient Arf activity" means
activity sufficient to arrest the entry of cells into the cell
division cycle, which is a hallmark of Arf activity. This activity
has been characterized [Weber, et al., Nat. Cell Biol. 1:20-26
(2000); Weber, et al., Mol. Cell Biol. 20:2517-2528 (2000) and U.S.
application Ser. No. 09/480,718, filed Jan. 7, 2000, the contents
of which are hereby incorporated by reference in their entireties].
Further, this activity has been characterized for a fragment of
mouse p19.sup.Arf containing residues 1-37 [DiGiammarino, et al.,
Biochemistry 40:2379-2386 (2001), the contents of which are hereby
incorporated by reference in their entireties].
[0060] The abbreviation "DM2" or "Dm2" as used herein refers to the
generic form of the protein "Mdm2" and its human ortholog "Hdm2"
which are Murine Double Minute 2 and Human Double Minute 2
respectively. Hdm2 has the GenBank accession number of M92424, an
amino acid sequence of SEQ ID NO:8 and a nucleic acid sequence of
SEQ ID NO:7. Mdm2 has the GenBank accession number of X58876, an
amino acid sequence of SEQ ID NO:6 and a nucleic acid sequence of
SEQ ID NO:5. Mdm2, for example, can bind to the N-terminal
transcriptional activation domain of p53 to block expression of
p53-responsive genes [Momand et al., Cell 69:1237-1245 (1992);
Oliner et al., Nature 362:857-860 (1993)], it has an intrinsic E3
ligase activity that conjugates ubiquitin to p53 [Honda and Yasuda,
Oncogene 19:1473-1476 (2000)] and it also appears to play a role in
shuttling p53 from the nucleus to the cytoplasm, where p53 is
degraded in cytoplasmic proteasomes [Freedman and Levine, Mol.
Cell. Biol. 18:7288-7293 (1998); Roth et al., EMBO J. 17:554-564
(1998); Tao and Levine, Proc. Natl. Acad. Sci. 96:3077-3080
(1999)].
[0061] As used herein a peptide "consisting of a minimal domain of
Dm2" is a peptide comprising the minimum portion of the full-length
Dm2 that still retains the ability to bind Arf and thereby
competitively inhibit the binding of Arf with the full-length
DM2.
[0062] As used herein, an "inducible fragment" of a Dm2 protein is
a fragment of a Dm2 protein that can be induced to form
supramolecular assemblies and/or .beta.-strand assemblies by Arf,
and/or an inducing fragment of Arf. An inducible fragment of Dm2
may be part of an Arf-Dm2 complex. Preferably an inducible fragment
of Dm2 comprises the amino acid residues 235-259 of SEQ ID NO:8
and/or the amino acid residues 275-289 of SEQ ID NO:8. A fusion
protein comprising an inducible fragment of a Dm2 protein can often
be used interchangeably with an inducible fragment of a Dm2
protein, and such fusion proteins are meant to be included when the
term an inducible fragment of a Dm2 protein is used.
[0063] As used herein the terms "fusion protein" and "fusion
peptide" are used interchangeably and encompass "chimeric proteins
and/or chimeric peptides". A fusion protein comprises at least a
portion of one protein such as ARF-p19 joined via a peptide bond to
at least another portion of a protein or peptide that it is not
naturally contiguously connected to. For example, a fusion peptide
of the present invention includes a peptide that consists of two
consecutive nonamers and/or octamers each having the amino acid
sequence of SEQ ID NO:13. In another embodiment, the fusion peptide
can comprise amino acid residues of SEQ ID NO:11 that is covalently
joined to a linker peptide which in turn is bound to amino acid
residues of SEQ ID NO:12. Fusion proteins and peptides can also,
and/or alternatively comprise a marker protein or peptide as
exemplified below, or a protein or peptide that aids in the
isolation and/or purification of the fusion protein.
[0064] A "heterologous nucleotide sequence" as used herein is a
nucleotide sequence that is added to a nucleotide sequence of the
present invention by recombinant methods to form a nucleic acid
which is not naturally formed in nature. Such nucleic acids can
encode fusion (e.g. chimeric) proteins. Thus the heterologous
nucleotide sequence can encode peptides and/or proteins which
contain regulatory and/or structural properties. In another such
embodiment the heterologous nucleotide sequence can encode a
protein or peptide that functions as a means of detecting the
protein or peptide encoded by the nucleotide sequence of the
present invention after the recombinant nucleic acid is expressed.
In still another embodiment the heterologous nucleotide sequence
can function as a means of detecting a nucleotide sequence of the
present invention. A heterologous nucleotide sequence can comprise
non-coding sequences including restriction sites, regulatory sites,
promoters and the like.
[0065] As used herein a "polypeptide" is used interchangeably with
the term "protein" and denotes a polymer comprising two or more
amino acids connected by peptide bonds. Preferably, a polypeptide
is further distinguished from a "peptide" with a peptide comprising
about twenty or less amino acids, and a polypeptide or protein
comprising more than about twenty amino acids. Preferably a protein
fragment is defined as a peptide or a polypeptide employing the
same size criteria.
[0066] As used herein "supramolecular assemblies comprised of
.beta.-strands" describes peptides or polypeptides that bind
together to form high molecular weight assemblies. In the present
case, Arf and Dm2 bind together to form assemblies comprised of
.beta.-strands. These assemblies are comprised of many molecules of
Arf and Dm2. The molecular size of these assemblies is
characterized using, for example, gel filtration chromatography,
wherein the assemblies elute at early times in the excluded volume
and appear to have a molecular weight of 200 Kilodaltons, or
greater.
[0067] As used herein a "small organic molecule" is an organic
compound [or organic compound complexed with an inorganic compound
(e.g., metal)] that has a molecular weight of less than 3
Kilodaltons, and preferably less than 1.5 Kilodaltons. A "compound"
of the present invention is preferably a small organic molecule.
Preferably, the small organic molecules identified by the methods
of the present invention are not peptides.
[0068] As used herein the terms "solid substrate" and "solid
support" are used interchangeably and represent a solid material
that provides an inert surface that allows a biological reaction to
be performed. Solid supports include biological chip plates as
exemplified by Rava et al., U.S. Pat. No. 5,874,219, the contents
of which are hereby incorporated by reference in their entireties
and multi-well (multi-titer) quartz and polystyrene plates.
Examples of material that can be used as solid substrates include
glass, peptide polymers (e.g., collagen), peptoid polymers,
polysaccharides (including commercial beads, e.g., SEPHADEX and the
like), carbohydrates, hydrophobic polymers, polymers, tissue
culture polystyrene, metals, derivatized plastic films, glass
beads, plastic beads, alumina gels, magnetic beads, nitrocellulose,
cellulose, and nylon membranes.
[0069] A molecule is "antigenic" when it is capable of specifically
interacting with an antigen recognition molecule of the immune
system, such as an immunoglobulin (antibody) or T cell antigen
receptor. An antigenic polypeptide contains at least about 5, and
preferably at least about 10, amino acids. An antigenic portion of
a molecule can be that portion that is immunodominant for antibody
or T cell receptor recognition, or it can be a portion used to
generate an antibody to the molecule by conjugating the antigenic
portion to a carrier molecule for immunization. A molecule that is
antigenic need not be itself immunogenic, i.e., capable of
eliciting an immune response without a carrier.
[0070] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are physiologically tolerable and do
not typically produce an allergic or similar untoward reaction,
such as gastric upset, dizziness and the like, when administered to
a human. Preferably, as used herein, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the compound is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water or aqueous solution
saline solutions and aqueous dextrose and glycerol solutions are
preferably employed as carriers, particularly for injectable
solutions. Suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin.
[0071] The phrase "therapeutically effective amount" is used herein
to mean an amount sufficient to reduce by at least about 15
percent, preferably by at least 50 percent, more preferably by at
least 90 percent, and most preferably prevent, a clinically
significant deficit in the activity, function and response of the
host. Alternatively, a therapeutically effective amount is
sufficient to cause an improvement in a clinically significant
condition/symptom in the host, i.e., a shrinkage of a tumor.
[0072] As used herein, the term "ortholog" refers to the
relationship between proteins that have a common evolutionary
origin and differ because they originate from different species or
strains. For example, mouse ARF-p19 is an ortholog of human
ARF-p14.
[0073] As used herein an amino acid sequence is 100% "homologous"
to a second amino acid sequence if the two amino acid sequences are
identical, and/or differ only by neutral or conservative
substitutions as defined below. Accordingly, an amino acid sequence
is 50% "homologous" to a second amino acid sequence if 50% of the
two amino acid sequences are identical, and/or differ only by
neutral or conservative substitutions.
[0074] As used herein, DNA and protein sequence percent identity
can be determined using MacVector 6.0.1, Oxford Molecular Group PLC
(1996) and the Clustal W algorithm with the alignment default
parameters, and default parameters for identity. These commercially
available programs can also be used to determine sequence
similarity using the same or analogous default parameters.
[0075] Polypeptides, peptides, or protein fragments of the present
invention include, but are not limited to, those containing part of
the amino acid sequences of an Arf protein and/or an Dm2 protein,
including altered sequences in which functionally equivalent amino
acid residues are substituted for residues within the sequence
resulting in a conservative amino acid substitution. Such
alterations define the term "a conservative substitution" as used
herein. For example, one or more amino acid residues within the
sequence can be substituted by another amino acid of a similar
polarity, which acts as a functional equivalent, resulting in a
silent alteration. Substitutes for an amino acid within the
sequence may be selected from other members of the class to which
the amino acid belongs. For example, the nonpolar (hydrophobic)
amino acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. Amino acids containing
aromatic ring structures are phenylalanine, tryptophan, and
tyrosine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine. The
positively charged (basic) amino acids include arginine, lysine and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid. Such alterations will not be
expected to affect apparent molecular weight as determined by
polyacrylamide gel electrophoresis, or isoelectric point.
[0076] Particularly preferred conservative substitutions are:
[0077] Lys for Arg and vice versa such that a positive charge may
be maintained;
[0078] Glu for Asp and vice versa such that a negative charge may
be maintained;
[0079] Ser for Thr such that a free --OH can be maintained; and
[0080] Gln for Asn such that a free NH2 can be maintained.
[0081] As used herein the term "approximately" is used
interchangeably with the term "about" and signifies that a value is
within twenty percent of the indicated value i.e., a protein
containing "approximately" 50 amino acid residues can contain
between 40 and 60 amino acid residues.
Candidate Compounds
[0082] A candidate compound can be obtained by a number of means,
including from a commercially available chemical library or an "in
house" pharmaceutical library.
[0083] Examples of libraries of compounds that are commercially
available include the Available Chemicals Directory (ACD,) the
Specs and BioSpecs database, the Maybridge database, and the
Chembridge database. Examples of pharmaceutical companies with "in
house" chemical libraries include Merck, GlaxoSmithKline, Bristol
Myers Squibb, Eli Lilly, Novartis, and Pharmacia.
[0084] Alternatively, candidate compounds can also be synthesized
de novo either individually or as combinatorial libraries [Gordon
et al., J. Med. Chem. 37:1385-1401(1994)]. They may also be
obtained from phage libraries. Phage libraries have been
constructed which when infected into host E. coli produce random
peptide sequences of approximately 10 to 15 amino acids [Parmley
and Smith, Gene 73:305-318 (1988); Scott and Smith, Science
249:386-390 (1990)]. Once a phage encoding a peptide that can act
as a potential drug has been purified, the sequence of the peptide
contained within the phage can be determined by standard DNA
sequencing techniques. Once the DNA sequence is known, synthetic
peptides can be generated which are encoded by these sequences.
[0085] Since the present invention discloses the critical portions
of the mutual binding domains of Arf and Dm2, the bound Arf-Dm2
peptide binding complex can be readily analyzed to determine their
three-dimensional structure. Using this structural information,
potential mimics for the Arf peptides or inhibitors of the Arf-Dm2
binding can be examined through the use of computer modeling using
a docking program such as DOCK, GRAM, or AUTODOCK [Dunbrack et al.,
Folding & Design, 2;27-42 (1997)]. This procedure can include
computer fitting of candidate compounds to Dm2 for example, to
determine how well the shape and the chemical structure of the
candidate compound can bind to the Dm2 fragment [Bugg et al.,
Scientific American, December:92-98 (1993); West et al., TIPS
16:67-74 (1995)]. Computer programs can also be employed to
estimate the attraction, repulsion, and steric hindrance of the Arf
or Dm2 peptides with a candidate compound.
[0086] Generally, the greater the steric complementarity and the
greater the attractive forces, the more potent the candidate
compound since these properties are consistent with a tighter
binding constant. Furthermore, the more specificity in the design
of a candidate compound, the more likely that the resulting drug
will not interact as well with other proteins. This will minimize
potential side-effects due to unwanted interactions with other
proteins.
[0087] Systematic modification of selected compounds by computer
modeling programs can then be performed until one or more compounds
are identified. Such analysis has been shown to be effective in the
development of HIV protease inhibitors [Lam et al, Science 263:
380-384 (1994); Wlodawer et al, Ann. Rev. Biochem. 62:543-585
(1993); Appelt, Perspectives in Drug Discovery and Design 1:23-48
(1993); Erickson, Perspectives in Drug Discovery and Design
1:109-128 (1993)].
[0088] In another such example, Selzer et al. [Exp. Parasitol.
87(3):212-221 (1997)] screened the Available Chemicals Directory (a
database of about 150,000 commercially available compounds) for
potential cysteine protease inhibitors, using DOCK3.5. Based on
both steric and force field considerations, they selected 69
compounds. Of these, three had IC50's below 50 .mu.M (i.e., the
concentration of the compound required to inhibit the reaction rate
by 50%)
[0089] In addition, amino acid analogs, or peptidomimetics can be
used that employ one or more unnatural or synthetic amino acids,
such as using a D amino acid. The subunits may be linked by peptide
bonds or by other the bonds, e.g., an ester, ether, etc. A good
starting point for designing such a peptidomimetic is of course a
peptide of the present invention, e.g., one comprising the amino
acid sequence of SEQ ID NO:13.
[0090] Synthetic peptides prepared using the well known techniques
of solid phase, liquid phase, or peptide condensation techniques,
or any combination thereof, can thus include natural and unnatural
amino acids. Amino acids used for peptide synthesis may be standard
Boc (N-amino protected N-t-butyloxycarbonyl) amino acid resin with
the standard deprotecting, neutralization, coupling and wash
protocols of the original solid phase procedure of Merrifield [J.
Am. Chem. Soc. 85:2149-2154 (1963)], or the base-labile N-amino
protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first
described by Carpino and Han [J. Org. Chem. 37:3403-3409 (1972)].
Both Fmoc and Boc N.sup..alpha.-amino protected amino acids can be
obtained from Fluka, Bachem, Advanced Chemtech, Sigma, Cambridge
Research Biochemical, Bachem, or Peninsula Labs or other chemical
companies familiar to those who practice this art. In addition, the
method of the invention can be used with other N-protecting groups
that are familiar to those skilled in this art. Solid phase peptide
synthesis may be accomplished by techniques familiar to those in
the art and provided, [for example, in Stewart and Young Solid
Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, IL
(1984); and Fields and Noble, Int. J. Pept. Protein Res. 35:161-214
(1990)], or using automated synthesizers, such as sold by ABS.
Thus, polypeptides of the invention may comprise D-amino acids, a
combination of D- and L-amino acids, and various "designer" amino
acids (e.g., .beta.-methyl amino acids, C .alpha.-methyl amino
acids, and N-.alpha.-methyl amino acids, etc.) to convey special
properties. Synthetic amino acids include ornithine for lysine,
fluorophenylalanine for phenylalanine, and norleucine for leucine
or isoleucine. Additionally, by assigning specific amino acids at
specific coupling steps, .alpha.-helices, .beta. turns, .beta.
sheets, .gamma.-turns, and cyclic peptides can be generated.
[0091] In one aspect of the invention, the peptides may comprise a
special amino acid at the C-terminus which incorporates either a
CO.sub.2H or CONH.sub.2 side chain to simulate a free glycine or a
glycine-amide group. Another way to consider this special residue
would be as a D or L amino acid analog with a side chain consisting
of the linker or bond to the bead. In one embodiment, the
pseudo-free C-terminal residue may be of the D or the L optical
configuration; in another embodiment, a racemic mixture of D and
L-isomers may be used.
[0092] In any case, compounds can be selected, for example, for
their ability to induce .beta.-strand formation of the Hdm2
fragments disclosed herein. A "lead" compound can then be
identified for use as a focus of a drug development project.
Methods of Identifying Compounds that Affect the Arf-Dm2
Interaction
[0093] As disclosed by the present invention, new .beta.-strand
secondary structure forms when Arf and Hdm2 interact. This
.beta.-strand secondary structure is due to the interaction of
small, specific domains present in Arf (e.g., the Arf motif having
the amino acid sequence of SEQ ID NO:13) and Dm2 (e.g., H1 and H2
as defined in the example below). In addition, as disclosed herein,
the binding of Arf and Dm2 does not lead to a bimolecular complex
as expected, but rather involves large, extended structures with
predominantly .beta.-strand secondary structure, i.e.,
supramolecular assemblies comprised of .beta.-strands. Therefore,
measuring the formation of this .beta.-strand secondary structure
directly or indirectly via measurement of Arf-Dm2 binding provides
the unique ability to select specific compounds based on their
capacity to either mimic the effect of Arf on Dm2 , or
alternatively to interfere with the Arf-Hdm2 binding complex and
associated .beta.-strand formation. As detailed below, such
measurements can be performed with NMR spectroscopy, circular
dichroism spectropolarimetry, Fourier Transform Infra-Red
spectroscopy (FT-IR), fluorescence spectroscopy, or surface plasmon
resonance. In addition, determining the size of the resulting
protein-protein, or peptide-peptide complex, can also be used to
select such specific compounds. In this case, any size
distinguishing methodology such as gel filtration chromatography,
gel electrophoresis, ultra centrifugation, dynamic light scattering
etc. may be used.
[0094] Thus, as detailed below, gel filtration chromatography
demonstrated that mArfN37 and Hdm2 210-304 elute together in the
void volume, whereas the uncomplexed peptides elute at times
consistent with monodisperse, conformationally extended
polypeptides. Similarly, NMR resonances for .sup.15N-mArfN37 or
.sup.15N-Hdm2 210-304 (and .sup.15N-Hdm2 210-275) are broadened
beyond detection when an unlabeled form of the appropriate binding
partner is added to the solution. Furthermore, the NMR spectra are
consistent with slow exchange between the free and bound states. In
addition, at mArfN37:Hdm2 210-304 molar ratios that produce maximal
.beta.-strand secondary structure based on ellipticity at 200 nm
using CD resonances cannot be observed for the isotope-labeled
component of Arf/Hdm2 mixtures.
[0095] Dm2 or Dm2 peptides comprising one or more specific
interacting domains, such as Hdm2 210-275 and 210-304, exemplified
below, can be labeled with .sup.15N. NMR spectra can be performed
and .sup.1H-.sup.15N steady-state {.sup.1H}-.sup.15N nuclear
Overhauser effect (NOE) values can be determined as the ratio of
peak intensities in 2D .sup.1H-.sup.15N correlation spectra with
and without .sup.1H saturation. Alternatively, or in addition, a
circular dichroism spectropolarimeter, as exemplified below, can be
used to monitor the structural changes. Such measurements can be
performed in the presence and absence of test compounds to
determine the effect of the compound on the formation of
.beta.-strand secondary structure in the H1 and H2 domains, for
example. In one embodiment, the assay is performed in the absence
of Arf and Arf fragments, and a compound is selected which can
stimulate the formation of .beta.-strand secondary structure of Dm2
(fragment thereof) and/or the formation of supramolecular
assemblies. In another embodiment, the assay is performed in the
presence of Arf and/or an Arf fragment, and a compound is selected
that can enhance the rate of formation of .beta.-strand secondary
structure of Dm2 (or fragment thereof) and/or the formation of
supramolecular assemblies. In still another embodiment, a compound
is selected that interferes with and/or inhibits the rate of
formation of .beta.-strand secondary structure of Dm2 (fragment
thereof) and/or the formation of supramolecular assemblies in the
presence of Arf and/or an Arf fragment.
[0096] The measurements outlined above can be preceded or
alternatively followed by other determinations. Thus, compounds can
be initially selected for their ability to bind specific Dm2
peptides (such as those comprising H1 and H2 as defined in the
Example below) or Arf peptides (such as those comprising the amino
acid sequence of SEQ ID NO:13). Alternatively, compounds can be
selected for their ability to interfere with the binding of Arf
with Dm2 (using either the full-length proteins or fragments,
including peptides that comprise the Arf and Dm2 binding domains as
defined in the Example below). As exemplified below, such binding
studies can be performed using Surface Plasmon Resonance (SPR).
[0097] Thus, initial screens can be performed in a high throughput
format using any of a large number of methodologies. One such
method employs a solid support that comprises multiple compartments
(e.g., wells). Currently a solid support comprising between 96 and
1516 compartments is relatively standard in drug assays. Each
compartment can include a separate reaction mixture. In a
particular embodiment, a selected target molecule (e.g., an Arf
peptide) can be introduced into the compartments either in solution
or on a solid support such as a chip [see U.S. Pat. No. 5,874,219,
Issued Feb. 23, 1999, the contents of which are hereby incorporated
by reference in their entireties]. The remaining components of the
reaction mixture can be added to the compartment and a compound can
then be added to determine if the compound binds to the peptide,
using radioactive compounds for example and a wash step.
[0098] If a chip is employed, a chip reader is generally used to
measure the reaction. Accordingly, the compartments in which the
detectable signal appears can be readily identified. The
interaction between reactants can be characterized in a number of
ways including in terms of kinetics and/or thermodynamics.
[0099] Assays on biological arrays generally include carrying out
the particular binding reaction under selected conditions,
optionally washing the compartment to remove unreacted molecules,
and analyzing the biological array for evidence of binding between
the reactants. Since the process can involve multiple steps, it is
preferred that such steps be automated so as to allow multiple
assays to be performed concurrently. Accordingly high throughput
analysis can employ automated fluid handling systems for
concurrently performing the reaction steps in each of the
compartments. Fluid handling allows uniform treatment of samples in
the compartments. Microtiter robotic and fluid-handling devices are
commercially available, including from Tecan AG.
[0100] A fluid handling device can be used to manipulate the
reaction conditions in any given compartment by, for example, (i)
adding or removing fluid from the compartments, including for
manipulating the concentration of the reactants; (ii) maintaining
and/or manipulating the temperature of the liquid in the
compartment; (iii) altering the ionic strength of the reaction
mixture; and (iv) agitating the compartments to ensure proper
mixing. A reader can then be used to measure the reaction and a
computer with an appropriate program can further analyze the
results from the reaction [see U.S. Pat. No. 5,874,219, Issued Feb.
23, 1999, the contents of which are hereby incorporated by
reference in their entireties]. Data analysis can include removing
"outliers" (data deviating from a predetermined statistical
distribution), and calculating the relative reaction activity of
each compartment. In a particular embodiment, the resulting data
are displayed as an image with color in each region varying
according to the amount of detectable binding measured. A solid
support can be introduced into a holder in the fluid-handling
device.
[0101] Preferably the fluid-handling device is a robotic device
that is programmed to: (i) set appropriate reaction conditions,
such as temperature, and volumes; (ii) to add specific reactants to
the compartments; (iii) incubate the binding partners for an
appropriate time; (iv) remove unreacted reactants; (v) wash the
compartments; (vi) add reactants/test compounds as appropriate to
the compartments; and (viii) allow the detection of the
reaction.
[0102] As part of the binding studies performed herein, it is often
desiresable to label one or more of the reagents. Suitable labels
include enzymes as discussed above, fluorophores (e.g., fluorescein
isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR)
rhodamine, free or chelated lanthanide series salts, especially
Eu.sup.3+, to name a few fluorophores), chromophores,
radioisotopes, chelating agents, dyes, colloidal gold, latex
particles, ligands (e.g., biotin), and chemiluminescent agents. In
the instance where a radioactive label, such as the isotopes
.sup.3H, .sup.14C, .sup.32P, .sup.35S, .sup.36Cl, .sup.51Cr,
.sup.57Co, .sup.58Co, .sup.59Fe, 90Y, .sup.125I, .sup.131I, and
.sup.186Re are used, known currently available counting procedures
may be utilized. In the instance where the label is an enzyme,
detection may be accomplished by any of the presently utilized
techniques known in the art including ultraviolet, visible, and
infra-red spectroscopy, circular dichroism, magnetic circular
dichroism, fluorescence (including measuring changes in fluorescent
lifetimes and fluorescent anisotropy), bioluminescence,
luminescence, phosphorescence, mass spectrometry, NMR, ESR,
amperometric or gasometric techniques.
[0103] Direct labels are one example of labels that can be used
according to the present invention. A direct label has been defined
as an entity which in its natural state is readily visible, either
to the naked eye or with the aid of an optical filter and/or
applied stimulation, e.g. ultraviolet light to promote
fluorescence. Examples of colored labels that can be used according
to the present invention include metallic sol particles, for
example, gold sol particles such as those described by Leuvering
(U.S. Pat. No. 4,313,734); dye sol particles such as described by
Gribnau et al. (U.S. Pat. No. 4,373,932) and May et al. (WO
88/08534); dyed latex such as described by May, Supra, Snyder (EP-A
0 280 559 and 0 281 327); or dyes encapsulated in liposomes as
described by Campbell et al. (U.S. Pat. No. 4,703,017). Other
direct labels include a radionucleotide, a fluorescent moiety or a
luminescent moiety. In addition to these direct labeling devices,
indirect labels comprising enzymes can also be used according to
the present invention. Various types of enzyme linked immunoassays
are well known in the art, for example, alkaline phosphatase and
horseradish peroxidase, lysozyme, glucose-6-phosphate
dehydrogenase, lactate dehydrogenase, and urease. These and others
have been discussed in detail by Eva Engvall in Enzyme Immunoassay
ELISA and EMIT in Methods in Enzymology, 70:419-439 (1980) and in
U.S. Pat. No. 4,857,453. The protein/peptides of the present
invention can be modified to contain a marker protein such as
luciferase or green fluorescent protein as described in U.S. Pat.
No. 5,625,048 filed Apr. 29, 1997, WO 97/26333, published Jul. 24,
1997 and WO 99/64592, published Dec. 16, 1999, all of which are
hereby incorporated by reference in their entireties. Suitable
marker enzymes include, but are not limited to, alkaline
phosphatase and horseradish peroxidase. Other labels for use in the
invention include magnetic beads or magnetic resonance imaging
labels.
[0104] In another embodiment, a phosphorylation site can be created
on an antibody of the invention for labeling with .sup.32P, e.g.,
as described in European Patent No. 0372707 (application No.
89311108.8) by Sidney Pestka, or U.S. Pat. No. 5,459,240, issued
Oct. 17, 1995 to Foxwell et al.
[0105] Polypeptides, and peptides, including antibodies, can be
labeled by metabolic labeling. Metabolic labeling occurs during in
vitro incubation of the cells that express the protein in the
presence of culture medium supplemented with a metabolic label,
such as [.sup.35S]-methionine or [.sup.32P]-orthophosphate. In
addition to metabolic (or biosynthetic) labeling with
[.sup.35S]-methionine, the invention further contemplates labeling
with [.sup.15N]-amino acids, [.sup.14C]-amino acids and
[.sup.3H]-amino acids (with the tritium substituted at non-labile
positions).
[0106] Once a lead compound is identified it can be tested for its
ability to affect one or more of the activities attributed to Arf,
including to induce .beta.-strand assembly of Dm2 (e.g., Hdm2 or
Mdm2) or a fragment thereof. For example, its ability to bind Mdm2,
to inhibit Mdm2-dependent nucleo-cytoplasmic shuttling of p53, to
inhibit the E3 ubiquitin ligase activity of Mdm2 toward p53 in
vitro and/or its ability to sequester Mdm2 in the nucleolus can be
determined. Such effects can be measured in Arf.sup.-/- cells such
as NIH 3T3 cells in which Mdm2 is present, for example.
Alternatively, microinjection and live cell imaging, as exemplified
below can be used to determine whether Hdm2, for example, is
sequestered within nucleoli by a particular compound.
[0107] As detailed below, Hdm2 deletion constructs tagged with a
fluorescent label (Texas Red.TM.) were microinjected into the
nucleus of NIH 3T3 cells that lack the gene for Arf. Nuclear
microinjection was used because the Hdm2 constructs containing the
central Arf-binding domain lack the nuclear localization signal
found between residues 181-186.
[0108] Thus labeled Hdm2 constructs can be individually injected
into cells in the absence or presence of the compound and the
localization of the labeled Hdm2 in the nucleoplasm and nucleoli
can be determined. If the labeled Hdm2 is sequestered within
nucleoli in the presence of the compound relative to in its
absence, the compound is identified as an Arf mimic. Analogously,
if the compound interferes with Hdm2 being sequestered in the
nucleoli, when Arf is present, the compound is identified as an
inhibitor of the Arf-Hdm2 interaction. Next, a lead compound can be
tested in animal models to determine its effect on tumors for
example, and then finally, tested in humans.
[0109] Furthermore, the effect of a lead compound on cell division
can be determined by monitoring the incorporation of BrdU into
chromosomal DNA in NIH 3T3 cells, as previously described
[DiGiammarino, et al., Biochemistry 40:2379-2386 (2001), the
contents of which are hereby incorporated by reference in their
entireties]. NIH 3T3 cells, for example, can be cultured in the
presence or absence of a lead compound and, after a set time
interval, such as 8 hours, the amount of BrdU incorporated into
chromosomal DNA can be determined using immunofluorescence
microscopy. A lead compound is then selected (identified) when the
amount of BrdU incorporated in the presence of the compound
decreases relative to the amount incorporated in the absence of the
compound.
Antibodies to the Arf and DM2 peptides of the Present Invention
[0110] The Arf and Dm2 polypeptides and peptides of the present
invention, as produced by a recombinant source or through chemical
synthesis, or isolated from natural sources, or from a digestion of
a recombinant/natural polypeptide, and derivatives or analogs
thereof, including fusion proteins, may be used as an immunogen to
generate antibodies that recognize the corresponding peptide. Such
antibodies include but are not limited to polyclonal, monoclonal,
chimeric including humanized chimeric, single chain, Fab fragments,
and a Fab expression library. Polyclonal antibodies have greater
likelihood of cross reactivity.
[0111] Thus the present invention provides compositions and uses of
antibodies that are immunoreactive with the Arf and Dm2 peptides of
the present invention. Such antibodies "bind specifically" to such
peptides, meaning that they bind via antigen-binding sites of the
antibody as compared to non-specific binding interactions. The
terms "antibody" and "antibodies" are used herein in their broadest
sense, including but not limited to intact monoclonal and
polyclonal antibodies as well as fragments such as Fv, Fab, and
F(ab')2 fragments, single-chain antibodies such as scFv, and
various chain combinations. In some embodiments, the antibodies of
the present invention are humanized antibodies or human antibodies.
The antibodies may be prepared using a variety of well-known
methods including but not limited to immunization of animals having
native or transgenic immune repertoires, phage display, hybridoma
and recombinant cell culture, and transgenic plant and animal
bioreactors.
[0112] Both polyclonal and monoclonal antibodies may be prepared by
conventional techniques. See, for example, Monoclonal Antibodies,
Hybridomas: A New Dimension in Biological Analyses, Kennet et al.
(eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory
Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., (1988).
[0113] Various procedures known in the art may be used for the
production of polyclonal antibodies to the Arf and Dm2 peptides of
the present invention or derivatives or analogs thereof. For the
production of antibody, various host animals can be immunized by
injection with such a peptide, or a derivative (e.g., or fusion
protein) thereof, including but not limited to rabbits, mice, rats,
sheep, goats, etc. In one embodiment, the peptide can be conjugated
to an immunogenic carrier, e.g., bovine serum albumin (BSA) or
keyhole limpet hemocyanin (KLH). Various adjuvants may be used to
increase the immunological response, depending on the host species,
including but not limited to Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and Corynebacterium parvum.
[0114] For preparation of monoclonal antibodies directed toward the
Arf and Dm2 peptides of the present invention, or analogs, or
derivatives thereof, any technique that provides for the production
of antibody molecules by continuous cell lines in culture may be
used. These include but are not limited to the hybridoma technique
originally developed by Kohler and Milstein [Nature, 256:495-497
(1975)], as well as the trioma technique, the human B-cell
hybridoma technique [Kozbor et al., Immunology Today, 4:72 (1983);
Cote et al., Proc. Natl. Acad. Sci. U.S.A., 80:2026-2030 (1983)],
and the EBV-hybridoma technique to produce human monoclonal
antibodies [Cole et al., in Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)]. In an additional
embodiment of the invention, monoclonal antibodies can be produced
in germ-free animals utilizing recent technology
[PCT/US90/02545].
[0115] The monoclonal antibodies of the present invention include
chimeric antibodies, e.g., "humanized" versions of antibodies
originally produced in mice or other non-human species. Such
humanized antibodies may be prepared by known techniques and offer
the advantage of reduced immunogenicity when the antibodies are
administered to humans. In fact, according to the invention,
techniques developed for the production of "chimeric antibodies"
[Morrison et al., J. Bacteriol., 159:870 (1984); Neuberger et al.,
Nature, 312:604-608 (1984); Takeda et al., Nature, 314:452-454
(1985)] by splicing the genes from a mouse antibody molecule
specific for an Arf and/or Dm2 peptide of the present invention
together with genes from a human antibody molecule of appropriate
biological activity can be used. Antibodies such as these are
within the scope of this invention.
[0116] Thus, a humanized antibody is an engineered antibody that
typically comprises the variable region of a non-human (e.g.,
murine) antibody, or at least complementarity determining regions
(CDRs) thereof, and the remaining immunoglobulin portions derived
from a human antibody. Procedures for the production of chimeric
and further engineered monoclonal antibodies include those
described Riechmann et al., [Nature 332:323, (1988)]; Liu et al.,
[Proc.Nat.Acad.Sci. 84:3439 (1987)]; Larrick et al.,
[Bio/Technology 7:934, (1989)]; and Winter and Harris, [TIBS
14:139, (May, 1993)]. Such human or humanized chimeric antibodies
are preferred for use in therapy of human diseases or disorders
(described infra), since the human or humanized antibodies are much
less likely than xenogenic antibodies to induce an immune response,
in particular an allergic response, themselves.
[0117] Therefore, procedures that have been developed for
generating human antibodies in non-human animals may be employed in
producing antibodies of the present invention. The antibodies may
be partially human or preferably completely human. For example,
transgenic mice into which genetic material encoding one or more
human immunoglobulin chains has been introduced may be employed.
Such mice may be genetically altered in a variety of ways. The
genetic manipulation may result in human immunoglobulin polypeptide
chains replacing endogenous immunoglobulin chains in at least some,
and preferably virtually all, antibodies produced by the animal
upon immunization. Mice in which one or more endogenous
immunoglobulin genes have been inactivated by various means have
been prepared. Human immunoglobulin genes have been introduced into
the mice to replace the inactivated mouse genes. Antibodies
produced in the animals incorporate human immunoglobulin
polypeptide chains encoded by the human genetic material introduced
into the animal. Examples of techniques for the production and use
of such transgenic animals to make antibodies (which are sometimes
called "transgenic antibodies") are described in U.S. Pat. Nos.
5,814,318, 5,569,825, and 5,545,806, which are incorporated by
reference herein.
[0118] Hybridoma cell lines that produce monoclonal antibodies
specific for the polypeptides of the invention are also provided by
the present invention. Such hybridomas may be produced and
identified by conventional techniques. One method for producing
such a hybridoma cell line comprises immunizing an animal with a
polypeptide, harvesting spleen cells from the immunized animal,
fusing said spleen cells to a myeloma cell line, thereby generating
hybridoma cells, and identifying a hybridoma cell line that
produces a monoclonal antibody that binds the polypeptide. The
monoclonal antibodies produced by hybridomas may be recovered by
conventional techniques.
[0119] According to the invention, techniques described for the
production of single chain antibodies [U.S. Pat. Nos. 5,476,786 and
5,132,405 to Huston; U.S. Pat. No. 4,946,778] can be adapted to
produce e.g., Arf peptide-specific single chain antibodies. An
additional embodiment of the invention utilizes the techniques
described for the construction of Fab expression libraries [Huse et
al., Science, 246:1275-1281 (1989)] to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity for an Arf peptide, or its derivatives, or analogs.
[0120] Antibody fragments which contain the idiotype of the
antibody molecule can be generated by known techniques. For
example, such fragments include but are not limited to: the
F(ab').sub.2 fragment which can be produced by pepsin digestion of
the antibody molecule; the Fab' fragments which can be generated by
reducing the disulfide bridges of the F(ab').sub.2 fragment, and
the Fab fragments which can be generated by treating the antibody
molecule with papain and a reducing agent.
[0121] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.,
radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitin reactions, immunodiffusion assays, in situ immunoassays
(using colloidal gold, enzyme or radioisotope labels, for example),
Western blots, precipitation reactions, agglutination assays (e.g.,
gel agglutination assays, hemagglutination assays), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. Antibody binding can be detected
by detecting a label on the primary antibody, or by detecting
binding of a secondary antibody or reagent to the primary antibody.
In an alternative embodiment, the secondary antibody is labeled.
Many means are known in the art for detecting binding in an
immunoassay and are within the scope of the present invention. For
example, to select antibodies which recognize a specific epitope of
Arf, one may assay generated hybridomas for a product which binds
to the Arf fragment containing such epitope and choose those which
do not cross-react with the full-length Arf protein.
[0122] In a specific embodiment, antibodies that agonize or
antagonize the binding of Arf to Hdm2 can be generated. Such
antibodies can be tested using the assays described infra. In
addition an antibody that mimics the effect of Arf on Hdm2 can also
be assayed for using the assays disclosed herein.
Administration of the Therapeutic Compositions of the Present
Invention
[0123] According to the present invention, the component or
components of a therapeutic composition of the invention may be
introduced topically, parenterally, transmucosally, e.g., orally,
nasally, or rectally, or transdermally. When the administration is
parenteral, it may be via intravenous injection, and also
including, but is not limited to, intra-arteriole, intramuscular,
intradermal, subcutaneous, intraperitoneal, intraventricular, and
intracranial administration.
[0124] In a particular embodiment, the therapeutic compound can be
delivered in a vesicle, in particular a liposome [see Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss: New York, pp. 353-365 (1989); Lopez-Berestein,
ibid., pp. 317-327; see generally ibid.].
[0125] In yet another embodiment, the therapeutic compound can be
delivered in a controlled release system. For example, a small
organic molecule as described above, may be administered using
intravenous infusion, an implantable osmotic pump, a transdermal
patch, liposomes, or other modes of administration. In one
embodiment, a pump may be used [see Langer, supra; Sefton, CRC
Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery
88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)]. In
another embodiment, polymeric materials can be used [see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Press: Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley:
New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev.
Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190
(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al.,
J. Neurosurg. 71:105 (1989)]. In yet another embodiment, a
controlled release system can be placed in proximity of a
therapeutic target, thus requiring only a fraction of the systemic
dose [see, e.g., Goodson, in Medical Applications of Controlled
Release, supra, vol. 2, pp. 115-138 (1984)].
[0126] Other controlled release systems are discussed in the review
by Langer [Science 249:1527-1533 (1990)].
[0127] Thus, a therapeutic composition of the present invention can
be delivered by intravenous, intraarterial, intraperitoneal,
intramuscular, or subcutaneous routes of administration.
Alternatively, the therapeutic composition, properly formulated,
can be administered by nasal or oral administration. A constant
supply of the therapeutic composition can be ensured by providing a
therapeutically effective dose (i.e., a dose effective to induce
metabolic changes in a subject) at the necessary intervals, e.g.,
daily, every 12 hours, etc. These parameters will depend on the
severity of the condition being treated, other actions, such as
diet modification, that are implemented, the weight, age, and sex
of the subject, and other criteria, which can be readily determined
according to standard good medical practice by those of skill in
the art.
[0128] A subject in whom administration of the therapeutic
composition is an effective therapeutic regiment for cancer
treatment for example, is preferably a human, but can be any
primate, other mammals or even avians suffering from cancer,
including domestic animals such as dogs and cats, laboratory
animals such as rats, rabbits and mice, livestock, such as cattle
(including cows), pigs, horses, and goats, and animals maintained
in a zoo such as elephants, lions, zebras, and gorillas. Thus, as
can be readily appreciated by one of ordinary skill in the art, the
methods and pharmaceutical compositions of the present invention
are particularly suited to administration to a number of animal
subjects, but particularly humans.
Kits
[0129] The materials for use in this aspect of the invention are
ideally suited for the preparation of a kit. Specifically, the
invention provides a compartmentalized kit to receive in close
confinement, one or more containers which comprise one or more of
the peptides of the present invention; and optionally one or more
other containers comprising reagents, such as additional buffers
etc.
[0130] In detail, a compartmentalized kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers or strips of
plastic or paper. Such containers allow one to efficiently transfer
reagents from one compartment to another compartment such that the
samples and reagents are not cross-contaminated and the agents or
solutions of each container can be added in a quantitative fashion
from one compartment to another. Such containers will include a
container which will accept the test sample, a container which
contains the peptides used in the assay, and containers which
contain additional reagents such as specific buffers with defined
ionic strengths.
[0131] The present invention may be better understood by reference
to the following non-limiting Example, which is provided as
exemplary of the invention. This example is presented in order to
more fully illustrate the preferred embodiments of the
invention.
[0132] It should in no way be construed, however, as limiting the
broad scope of the invention.
EXAMPLE
Defining the Molecular Basis of Arf And Hdm2 Interactions
Introduction
[0133] Understanding the interaction of Arf and Hdm2 has recently
become a central issue in cancer biology. In response to
hyperproliferative signals, p14.sup.Arf stabilizes p53 by binding
to Hdm2 and inhibits the ubiquitination and subsequent
proteosome-dependent degradation of p53. The medical importance of
the Arf-Hdm2-p53 regulatory system is highlighted by the finding
that either p53 or p14.sup.Arf are lost or modified in virtually
all human cancers.
[0134] Human and mouse Arf are highly basic proteins (.about.20%
Arg residues) of 132 and 169 residues, respectively, that localize
to nucleoli. The extreme N-terminal segments of the two proteins
are very similar (17/29 identity; 21/29 similarity) and contain a
repeated motif of 8 or 9 residues that comprises hydrophobic
residues flanked by Arg residues [DiGiammarino et al., Biochem.,
40:2379-2386 (2001)]. Exon 1.beta. of the human and mouse
p16.sup.Ink4a/Arf locus uniquely encodes the first 62 and 63 amino
acids of human and mouse Arf, respectively, while exon 2 encodes
the C-terminal domains. An alternative reading frame within exon 2
also encodes the central segment of p16.sup.Ink4a [Quelle et al.,
Cell, 83:993-1000 (1995)]. Importantly, peptides containing highly
conserved N-terminal residues of human or mouse Arf have been shown
to possess biological activity [Midgley et al., Oncogene,
19:2312-23 (2000); Weber et al., Mol. Cell Biol., 20:2517-2528
(2000); DiGiammarino et al., Biochem., 40:2379-2386 (2001)]. For
example, a peptide containing the N-terminal 37 amino acids of
mouse Arf (termed mArfN37) localizes to nucleoli, binds and
sequesters Hdm2 within nucleoli, and activates p53 leading to cell
cycle arrest [Weber et al., Mol. Cell Biol., 20:2517-2528 (2000);
DiGiammarino et al., Biochem., 40:2379-2386 (2001)]. Additionally,
a 20 amino acid peptide from the human Arf N-terminus inhibits
Mdm2-dependent ubiquitination of p53 and activates p53 in vivo
[Midgley et al., Oncogene, 19:2312-23 (2000)]. Further study of the
Arf N-terminus has shown that the two repeated motifs within
mArfN37 bind individually to Hdm2 and are both required for normal
Arf function [Weber et al., Mol. Cell Biol., 20:2517-2528 (2000)].
Two segments of p14.sup.Arf also are reported to mediate
interactions with Hdm2 but one of these is found in a different
region of the polypeptide; these are residues 1-14 and 82-101
[Weber et al., Mol. Cell Biol., 20:2517-2528 (2000); Zhang et al.,
Mol. Cell, 3:579-91 (1999)]. Nucleolar localization of mouse and
human Arf is specified by the amino acid sequence RRPR (SEQ ID
NO:14) (the nucleolar localization signal, NoLS); the NoLS in
p19.sup.Arf is found between residues 31-34 of SEQ ID NO:2 and in
p14.sup.Arf between residues 87-90 of SEQ ID NO:4. Interestingly,
when Arf binds Mdm2 (or Hdm2), the Arf NoLS is masked and nucleolar
colocalization of the Arf/Mdm2 complex relies on the exposure of a
cryptic NoLS within the RING domain of Mdm2 [Lohrum et al., Nat.
Cell Biol., 2:179-81 (2000); Weber et al., Mol. Cell Biol.,
20:2517-2528 (2000)]. Despite the wealth of information on how Arf
functions in cells, detailed information on the Hdm2-bound state of
mArfN37, or the mechanism of Hdm2 binding or nucleolar localization
is completely lacking.
[0135] Mdm2 is a multifunctional protein that is reported to
interact with p53 [Wu et al., Genes Dev., 7:1126-1132 (1993)],
CBP/p300 [Grossman et al., Mol Cell, 2:405-15 (1998)], E2F1 [Martin
et al., Nature, 375:691-4 (1995); O.degree. C.onnor et al., Embo J,
14:6184-92 (1995)], Rb [Xiao et al, Nature, 375:694-8 (1995)], L5
[Marechal et al., Mol Cell Biol, 14:7414-20 (1994)], TBP
[Leveillard et al., Mech Dev, 74:189-93 (1998)] and Arf [Weber et
al., Nat. Cell Biol., 1:20-26 (1999); Weber et al., Mol. Cell
Biol., 20:2517-2528 (2000); DiGiammarino et al., Biochem.,
40:2379-2386 (2001)]. The human (Hdm2) and mouse (Mdm2) orthologs
are 72% identical and can functionally substitute for one another.
The domains responsible for some of the above interactions are well
characterized. For example, the N-terminal domain of .about.100
amino acids adopts a globular, helical structure and binds a small
peptide within the p53 N-terminus; this interaction inhibits the
transcriptional activation function of p53 [Kussie et al., Science,
274:948-953 (1996); Oliner et al., Nature, 362:857-860 (1993);
Momand et al., Cell, 69:1237-1245 (1992)]. Two zinc-binding motifs
have been identified in Mdm2, a C.sub.4 zinc finger motif (amino
acid residues 305-325 of SEQ ID NO:6) and a C.sub.3HC.sub.4 RING
domain (amino aid residues 438-478 of SEQ ID NO:6) [Boddy et al.,
Trends Biochem Sci, 19:198-9 (1994)]. The latter domain has been
shown to mediate ubiquitin ligase activity toward p53 in vitro
[Honda et al., FEBS Lett, 420:25-7 (1997); Honda et al., Embo J,
18:22-7 (1999)] and to bind RNA [Elenbaas et al., Mol. Med.,
2:439-51 (1996); Lai et al., Biochem., 37:17005-15 (1998)].
Further, the RING domain has been shown to bind zinc ions and to
exhibit globular but disordered structure [Lai et al., Biochem.,
37:17005-15 (1998)]. The C.sub.4 zinc finger motif has not been
functionally characterized. The Arf-binding domain of Hdm2 has been
mapped to amino acids 210-304 of SEQ ID NO:8 (termed Hdm2 210-304)
[Weber et al., Mol. Cell Biol., 20:2517-2528 (2000)]. The L5
binding domain also maps to this region [Marechal et al., Mol Cell
Biol, 14:7414-20 (1994)]. Between humans and mice, this segment is
92% similar and, in striking contrast to Arg-rich Arf, is highly
acidic (for Hdm2, 32% Asp/Glu, predicted pI .about.3.2; for Mdm2,
33% Asp/Glu; predicted pI .about.3.5). This central segment has
been shown to bind N-terminal fragments of mouse Arf, including
1-37, 1-14 and 26-37 [Weber et al., Mol. Cell Biol., 20:2517-2528
(2000)]. Furthermore, a peptide composed of the first 20 amino
acids of human p14.sup.Arf has been shown to bind the central,
acidic segment of Hdm2 and to inhibit Hdm2-dependent ubiquitination
of p53 in vitro [Midgley et al., Oncogene, 19:2312-23 (2000)]. The
interaction motif within Hdm2 has been mapped to residues 212-244
[Midgley et al., Oncogene, 19:2312-23 (2000)]. The studies
summarized above contribute significantly to the understanding of
the cellular functions of Arf and Hdm2 but provide little insight
into the physical and structural basis for these functions, such as
the binding of Arf to Hdm2, nucleolar colocalization, inhibition of
Hdm2-dependent nucleo-cytoplasmic shuttling of p53, and E3
ubiquitin ligase activity toward p53.
Materials and Methods
[0136] Hdm2 and p19.sup.Arf Protein Purification:
[0137] Fragments of Hdm2 corresponding to residues 210-275 and
210-304 of SEQ ID NO:8 (termed Hdm2 210-275 and Hdm2 210-304) were
subcloned into the expression plasmid pET28a (Novagen) using
standard methods; pET28a allows expression of polypeptides with a
thrombin cleavable poly-His affinity purification tag. Following
protein expression in E. coli BL21 (DE3) (Novagen, Inc.), bacterial
cells were harvested by centrifugation followed by resuspension in
20 mM Tris-HCl (pH 8.0) 500 mM NaCl at 4.degree. C., and lysed by
sonication. Urea was added to a final concentration of 6 M to the
soluble fraction after centrifugation (20,000 g, 20 min.). Soluble,
His-tagged proteins were purified using Ni.sup.2+-affinity
chromatography (Chelating-Sepharose, Amersham Pharmacia Biotech,
Inc.) in the presence of 6 M urea using otherwise standard
procedures (Novagen, Inc.). An N-terminal fragment of mouse
p19.sup.Arf corresponding to residues 1-37 (mArfN37) was purified
in a similar manner, as previously reported [DiGiammarino et al.,
Biochem., 40:2379-2386 (2001)]. His tag-cleaved mArfN37 was further
purified using C.sub.4 reverse-phase high-performance liquid
chromatography (HPLC) (C.sub.4 column; Vydac, Inc.) using a 0.1%
trifluoroacetic acid (TFA)/acetonitrile buffer system. Lyophilized
proteins were directly dissolved in the appropriate buffer for each
experiment, as described below. Fractions containing Hdm2 proteins
were dialyzed into 20 mM Tris-HCl (pH 8.0), 500 mM NaCl and treated
with thrombin (Novagen) 1 U/mg protein at room temperature for 16
hours to cleave the His tag. Constructs were further purified using
anion exchange chromatography (Q SEPHAROSE, Amersham Pharmacia
Biotech, Inc.) using 20 mM Tris-HCl buffer, pH 7.0 with elution
using a 50 mM--1.0 M NaCl gradient over 0.05 liters.
[0138] Peptide Synthesis.
[0139] Peptides were synthesized using standard methods by an
Advanced Chemtech 396 synthesizer. The peptide amides were
synthesized on HMP-amide resin (Applied Biosystems, Inc.) and the
FMOC-amino acids were coupled using HOBt/HBTU chemistries.
N-terminal acetylation was performed using acetic anhydride and
HOBt. Peptides were cleaved from the resin in 91% TFA containing 2%
phenol, 2% ethanedithiol and 5% thioanisole. The peptides were
precipitated and washed twice with diethyl ether. Peptide
concentrations for circular dichroism (CD) and surface plasmon
resonance (SPR) were determined using quantitative amino acid
analysis.
[0140] Surface Plasmon Resonance:
[0141] Binding studies were performed using a Biacore 3000 surface
plasmon resonance (SPR) instrument (Biacore, Inc.). A Tetra-His.TM.
Antibody (Qiagen, Inc.; Cat. #34670) was covalently attached to a
carboxymethylated gold surface (C-1 chip; Biacore, Inc.). The
carboxymethyl groups on the surface were activated with
N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and
N-hydroxysuccinimide (NHS) and the antibody was attached at pH 7.4
in 20 mM sodium phosphate buffer, 150 mM NaCl (PBS). Reactive sites
remaining on the surface were blocked by reaction with
ethanolamine. The His-tagged ligand was attached to the antibody by
injecting a 5-10 .mu.g/ml solution of the ligand in 10 mM HEPES,
150 mM NaCl, pH 7.4 (HBS-N buffer, Biacore) at a flow rate of 10
.mu.L/min through the flow cell. A reference cell was prepared
similarly except that no His-tagged ligand was added. Binding was
measured by flowing the non-His-tagged analyte through the
reference and ligand-containing flow cells in sequence. Prior to
injection of ligands, the chip surfaces were equilibrated in HBS-N
buffer. Changes in the SPR response due to solvent differences and
the injection process were monitored by injection of HBS-N buffer
alone. Regeneration of the chip surface to remove bound analyte and
ligand was accomplished by two 100 .mu.L injections of 10 mM
glycine, pH 2.0 through both flow cells. Data reported is the
difference in SPR signal between the flow cell containing analyte
and the reference cell. Duplicate injections were made and the SPR
response values reported are the average of these two
injections.
[0142] NMR Spectroscopy:
[0143] Uniformly .sup.15N-labeled Hdm2 210-275 and 210-304 were
prepared using standard procedures [Kriwacki et al., Proc. Natl.
Acad. Sci. USA, 93:11504-11509 (1996)]. Samples were concentrated
by ultra-filtration using a 3000 Da cutoff filter (Millipore,
Corp., Centricon 3) to 1-2 MM in 10 mM potassium phosphate buffer,
pH 6.0, 5% D.sub.2O (vol:vol) and placed in 5 mm micro-cell NMR
tubes (Shegimi, Inc.). All NMR spectra were acquired using a 600
MHz Varian Inova NMR spectrometer (Varian Associates, Inc.) fitted
with a 5 mm triple resonance probe equipped with x, y, z axis
pulsed magnetic field gradients. 2D .sup.1H-.sup.15N HSQC spectra
[Muller, J. Amer. Chem. Soc., 101:4481-4484 (1979); Bodenhausen and
Ruben, D. J. Chem. Phys. Lett., 69:185-189 (1980)] were acquired
using standard procedures provided in the Varian Protein Pack pulse
sequence library. .sup.1H-.sup.15N steady-state {.sup.1H}-.sup.15N
nuclear Overhauser effect (NOE) values were determined as the ratio
of peak intensities in 2D .sup.1H-.sup.15N correlation spectra with
and without .sup.1H saturation [Farrow et al., Biochem.,
33:5984-6003 (1994)].
[0144] Circular Dichroism:
[0145] Spectra were recorded at 25.degree. C. using an Aviv Model
62A DS circular dichroism spectropolarimeter (Aviv Instruments,
Inc.) equipped with a thermoelectric temperature control unit. All
samples were prepared in 10 mM Tris-HCl, pH 7.0 at 0.02-0.2 mM as
determined by amino acid analysis. Spectra were recorded using 1 mm
quartz cuveftes and reported spectra are the average of 10 scans
over the range 195-260 nM in 1 nM steps. Melting experiments were
recorded in 10 mm quartz cuvettes with active stirring. The signal
at 216 nm (.beta. strand minimum) was used to monitor structural
changes and the signal was averaged over 15 sec. The sample
temperature was increased in 1.degree. C. steps.
[0146] Fluorescent Labeling:
[0147] Proteins for fluorescence microimaging studies were
covalently modified using an amine-reactive, sulfosuccinimidyl
ester of Texas Red.TM. (Molecular Probes, Inc., Cat. # C-1171). In
brief, purified Hdm2-derived proteins were dialyzed into 100 mM
sodium bicarbonate buffer, pH 8.3 at 2-4 mg/ml. The Texas Red.TM.
dye was dissolved in dimethylsulfoxide at 10 mg/ml. Protein and dye
3:1 (vol:vol) were reacted for 1 hour at room temperature followed
by purification using size-exclusion chromatography (NAP 5 G-25
columns, Amersham Pharmacia Biotech, Inc.). Labeled protein bands
migrated more slowly than unlabeled protein in SDS-polyacrylamide
gels consistent with the covalent attachment of dye.
[0148] Cell Culture and Microinjection:
[0149] NIH 3T3 Arf.sup.-/- cells passage 10-15 were cultured in
Dulbecco's modified Eagle's medium (Dulbecco's modified Eagle's
medium (DMEM); Gibco-Invitrogen, Inc.), 10% fetal bovine serum
(FBS) at 37.degree. C., 7% CO.sub.2. 24 hours prior to
transfection, cells were plated on 35 mm dishes coated with
poly-D-Lysine (Mattek, Corp.) at a density of 3.times.10.sup.4
cells per dish. Cells were transfected with a pcDNA plasmid
(Invitrogen) containing the GFP-p19.sup.ARF fusion protein using
Fugene-6 (Roche Molecular Biochemicals, Inc.) in media according to
the protocol supplied by the manufacturer. Sixteen hours
post-transfection, the transfection media was refreshed with DMEM,
10% FBS and the cells were allowed to recover for four hours.
GFP-Arf positive cells were then located and co-injected with Texas
Red-labeled Hdm2 polypeptides (2 mg/ml in 20 mM Tris-HCl, 100 mM
NaCl, pH 7.0) using a Micromanipulator 5171/Transjector 5246
(Eppendorf, AG) in media supplemented with 10 mM HEPES, pH 7.2 to
buffer cell media during injection and imaging. Representative
cells were imaged over time. An Axiovert 135 TV inverted
fluorescence microscope with an automated stage controller (Carl
Zeiss, Inc.) and circulating water bath heater (Fisher) was used
for both microinjection and imaging. Images were acquired with a
Zeiss 40.times. NA=1.30 oil objective and MicroMax CCD camera
(Princeton Instruments Inc.) operated with MetaMorph Version 4.01
imaging software.
Results
[0150] Small Segments of Arf and Hdm2 Participate in Arf/Hdm2
Interactions.
[0151] It has been previously shown that a fragment of mouse
p19.sup.Arf containing the N-terminal 37 amino acids (mArfN37) can
(i) bind Hdm2, (ii) cause the relocalization of Hdm2 to nucleoli,
and (iii) induce cell cycle arrest in MEFs [see, U.S. patent
application Ser. No. 09/480,718, Filed Jan. 7, 2000, the contents
of which are hereby incorporated by reference in their entireties;
Weber et al., Nat. Cell Biol., 1:20-26 (1999)]. Further, a fragment
of Hdm2 containing amino acids 140-350 has been show to be capable
of binding mArfN37 and to be relocalized to nucleoli in a
mArfN37-dependent manner. A smaller fragment, Hdm2 210-304, has
also been shown to be capable of binding mArfN37 on the basis of
Arf affinity chromatography [Weber et al., Mol. Cell Biol.,
20:2517-2528 (2000)]. However, because mArfN37 is a relatively
small polypeptide, it was determined whether it could bind a
correspondingly small segment of Hdm2. To test this hypothesis and
to monitor Arf/Hdm2 binding reactions quantitatively, Hdm2
constructs were prepared spanning amino acids 210-304 of SEQ ID
NO:8 and 210-275 of SEQ ID NO:8 and then it was determined whether
they could bind mArfN37 using surface plasmon resonance (SPR). Both
Hdrn2 210-304 and 210-275 bind tightly to His-tagged mArf37 that
was immobilized on the SPR biosensor surface using a covalently
linked His antibody (FIG. 1). The two Hdm2 fragments did not bind a
control surface that lacked His-mArfN37.
[0152] The binding results discussed above were verified (based on
an in vitro assay using SPR) in a biological setting by monitoring
the interaction of p19.sup.Arf and Hdm2 fragments in NIH 3T3 cells
that lack the gene for Arf using fluorescence microscopy.
p19.sup.Arf was tagged with GFP (green fluorescent protein) and
expressed in cells after transfection with an expression plasmid,
as previously described [Weber et al., Nat. Cell Biol., 1:20-26
(1999); Weber et al., Mol. Cell Biol., 20:2517-2528 (2000)], while
Hdm2 was chemically tagged with the fluorescent dye Texas Red.TM.
and introduced into cells by microinjection. The results show that
GFP-p19.sup.Arf is localized in nucleoli after transfection,
whereas, in direct contrast, Hdm2 210-304 is evenly dispersed in
the nucleoplasm after microinjection in the absence of p19.sup.Arf.
When GFP-p19.sup.Arf and Hdm2 210-304 are introduced into cells
together, the two proteins become co-localized within nucleoli. The
smaller fragment of Hdm2, Hdm2 210-275, exhibits similar
localization properties in the absence and presence of
GFP-p19.sup.Arf. Together, the SPR and cell localization results
show that a .about.100 amino acid central segment of Hdm2 interacts
with Arf and that fragments containing this segment can be
sequestered within nucleoli in the same manner as shown previously
for full-length Hdm2 [Weber et al., Nat. Cell Biol., 1:20-26
(1999); Weber et al., Mol. Cell Biol., 20:2517-2528 (2000)].
[0153] Structural Properties of mArfN37 and Arf-binding Hdm2
Fragments.
[0154] Knowing that mArfN37 and Hdm2 210-304 (and Hdm2 210-275)
interact and that the interactions appear to be biologically
relevant, the structural properties of these domains were
investigated. Previous work showed that mArfN37 is unstructured in
aqueous solution and that the peptide adopts a bi-helical
conformation in 30% trifluoroethanol [DiGiammarino et al.,
Biochem., 40:2379-2386 (2001)]. CD spectra for Hdm2 210-304 and
210-275 show that these two Arf-binding polypeptides are also
unstructured. The CD spectrum for mArfN37 is also shown, for
reference. A similar conclusion can be reached on the basis of the
.sup.1H-.sup.15N 2D correlation spectra for Hdm2 210-304 and
210-275. The observed chemical shift values in both the .sup.1H and
.sup.15N dimensions cluster near random coil values [Schwarzinger
et al., J. Amer. Chem. Soc., 123:2970-2978 (2001)] and are
consistent with a general lack of secondary and tertiary structure
[Kriwacki et al., Proc. Natl. Acad. Sci. USA, 93:11504-11509
(1996)]. Furthermore, heteronuclear {.sup.1H}-.sup.15N NOE values
for Hdm2 210-275 are all negative and are consistent with the
conclusion that the Arf-binding segment of Hdm2, prior to binding
Arf, is conformationally disordered and highly flexible.
[0155] Secondary structure prediction methods were used to gain
further insight into the structural properties of the N-terminal
segment of Arf, and the Arf-binding segment of Hdm2. Prior analysis
of mArfN37 alone using a variety of secondary structure prediction
algorithms did not yield consistent results [DiGiammarino et al.,
Biochem., 40:2379-2386 (2001)]. However, when the neural net-based
Jnet algorithm [Cuff et al., Proteins, 40:502-11 (2000)] (available
through the Jpred site at jura.ebi.ac.uk:8888) was used to analyze
the human, mouse, and opossum Arf sequences simultaneously, two
short .beta.-strands are predicted within the N-terminal 37 amino
acids (FIG. 2a), between residues 4-12 and 20-27, respectively. The
Jnet approach, which is based on the principle that secondary
structure is conserved within evolutionarily related proteins, has
been shown to predict secondary structure with 73% or higher
accuracy. For Arf, both .beta.-strands are predicted with high
confidence (FIG. 2a). Prior structure predictions were probably
hampered by the unusual nature of the mArfN37 sequence, which
contains 27% Arg residues (10/37) and has a correspondingly high
predicted pI value (12.6).
[0156] In contrast to mArfN37, Hdm2 210-304 is highly acidic (17/95
Asp and 14/95 Glu, 31/95 total, or 33%; the predicted pI is 3.5)
and very hydrophilic due additionally to its high Ser content
(17/95). Interestingly, as for the Arf N-terminus, two short
segments of .beta.-strand are predicted by Jnet within the
Arf-binding domain of Hdm2, between residues 245-253 and 275-282
(FIG. 2b). Many residues within the .beta.-strands are also
predicted to be less than 25% solvent exposed. The remainder of the
polypeptide is predicted to be unstructured and solvent exposed.
The opposed charge characteristics of mArfN37 and Hdm2 210-304
suggest that electrostatic forces play a role in the interactions
between the two polypeptides. The prediction of two .beta.-strands
in both mArfN37 and Hdm2 210-304 suggests that .beta.-strand
secondary structure is involved in Arf/Hdm2 interactions.
[0157] .beta.-strand Secondary Structure Forms when Arf and Hdm2
Interact.
[0158] Interestingly and in accord with the secondary structure
predictions discussed above, a striking transition from random
conformations to .beta.-strand secondary structure is observed when
mArfN37 and Hdm2 210-304 (and Hdm2 210-275) are mixed (FIG. 3).
This structural transition is induced when mArfN37 is added to an
Hdm2 fragment and when an Hdm2 fragment is added to mArfN37. Data
from gel filtration chromatography and NMR spectroscopy shows that
the mArfN37 and Hdm2 210-304 do not form a bimolecular complex
involving small numbers of molecules but rather form large,
extended structures with predominantly .beta.-strand secondary
structure. First, gel filtration chromatography shows that, when
mixed, mArfN37 and Hdm2 210-304 elute together in the void volume.
In contrast, the uncomplexed species elute at times consistent with
monodisperse, conformationally extended polypeptides. Second, NMR
resonances for .sup.15N-mArfN37 or .sup.15N-Hdm2 210-304 (and
.sup.15N-Hdm2 210-275) are broadened beyond detection when an
unlabeled form of the appropriate binding partner is added to the
solution. At mArfN37:Hdm2 210-304 (or mArfN37:Hdm2 210-275) molar
ratios that produce maximal .beta.-strand secondary structure based
on ellipticity at 200 nm using CD, resonances cannot be observed
for the isotope-labeled component of Arf/Hdm2 mixtures. Further,
the NMR spectra are consistent with slow exchange between the free
and bound states. These results indicate that .beta.-strand
secondary structure forms when mArfN37 and Hdm2 210-304 (and Hdm2
210-275) interact and that this secondary structure exists in the
context of supramolecular assemblies that can be described as
.beta. networks.
[0159] Characterization of Arf:Hdm2 Assemblies.
[0160] As discussed above, the addition of mArfN37 to Hdm2 210-304
results in the formation of supramolecular assemblies comprised of
.beta.-strands. Stepwise addition of sub-stoichiometric amounts of
mArfN37 to Hdm2 210-304 increased .beta.-strand secondary structure
content as judged by the change in ellipticity at 200 nm using CD.
Ellipticity at 200 nm was monitored because the largest difference
in ellipticity between random and .beta.-strand conformations was
observed at this wavelength. The binding of mArfN37 to Hdm2
210-304, as monitored in this way, is saturable. Similar results
were obtained when mArfN37 was mixed with the shorter fragment of
Hdm2, Hdm2 210-275. Further, saturable binding is also observed
when the Hdm2 fragments were added to an excess of mArfN37. These
findings indicate that there are a limited number of binding sites
for mArfN37 within Hdm2 210-304 and that discreet structures
(containing .beta.-strands) form when mArfN37 and Hdm2 210-304 (or
210-275) are mixed. For mArfN37 added to Hdm2 210-304, the plot of
ellipticity versus amount of protein added is linear, indicating
that each molecule of mArfN37 added binds completely to the Hdm2
fragment. Similar behavior is observed when mArfN37 binds Hdm2
210-275. These findings suggest that the equilibrium dissociation
constant (K.sub.D) for the interactions is less than the
concentrations used (.about.5.times.10.sup.6 M).
[0161] Formation of mArfN37:Hdm2 210-304 (and mArfN37:Hdm2 210-275)
assemblies was observed under a variety of conditions, including pH
values between 4 and 10, and salt concentrations from 0 to 2 M
NaCl. Furthermore, the assemblies were not disrupted by chemical
denaturation (4 M urea), treatment with an organic solvent (50%
acetonitrile (vol:vol)), or treatment with a mild detergent (10 mM
CHAPSO) These results indicate that the mArfN37:Hdm2 210-304
assemblies are thermodynamically stable. This conclusion is further
supported by the results of thermal denaturation experiments using
CD. The assembly formed by adding mArfN37 to an excess of Hdm2
210-304 to a final molar ratio of 2:1 (mArfN37:Hdm2 210-304) is
stable to heating up to .about.75.degree. C. For example, the CD
spectrum for the mArfN37:Hdm2 210-304 assembly prepared in this way
does not change when the sample temperature is increased from
25.degree. C. to 75.degree. C. The CD spectrum does change above
75.degree. C., and the changes are consistent with unfolding of the
supramolecular assemblies. In particular, the intensity of spectral
features indicative of .beta.-strands is reduced and finally
completely eliminated at 95.degree. C. When CD ellipticity at 216
nm is plotted versus temperature, the shape of the curve above
75.degree. C. resembles other protein denaturation curves that are
known to involve a cooperative unfolding process. The melting data
was fit with a sigmoidal function to determine the melting
temperature (T.sub.m), defined as the midpoint of the melting
curve. Denaturation experiments were performed at three different
ionic strengths, 0, 150 and 300 mM NaCl, and yield T.sub.m values
of 77, 65 and 52.degree. C., respectively. Similar T.sub.m values
were obtained with the assembly formed by adding mArfN37 to Hdm2
210-275.
[0162] Dual Binding Motifs in Arf and Hdm2 Mediate Intermolecular
Interactions.
[0163] The N-terminal domains of human and mouse Arf have similar
primary structure (FIG. 2a) and contain an arginine-rich, repeated
motif (FIG. 4) [DiGiammarino et al., Biochem., 40:2379-2386
(2001)]; this is termed the "Arf motif" the first and second
repeats are termed "A1" and "A2", respectively. [In the mouse Arf
protein (p19.sup.Arf) A1 and A2 have the amino acid sequences of
SEQ ID NO:s 9 and 10 respectively, whereas in the human Arf
(p14.sup.Arf), A1 and A2 have the amino acid sequences of SEQ ID
NOs:11 and 12 respectively.
[0164] Importantly, mArfN37, which contains both of the Arf motifs
in the mouse Arf sequence, has been shown to possess biological
properties comparable to full-length mouse Arf. These properties
include nucleolar localization, the ability to sequester Mdm2 and
Hdm2 in nucleoli and the ability to cause cell cycle arrest [Weber
et al., Mol. Cell Biol., 20:2517-2528 (2000); DiGiammarino et al.,
Biochem., 40:2379-2386 (2001)]. Furthermore, a construct containing
residues 1-20 of human Arf has been shown to activate p53 in
cellular assays [Midgley et al., Oncogene, 19:2312-23 (2000)]. The
existence of two structural motifs within mArfN37 was suggested on
the basis of the solution structure of mArfN37 determined in the
presence of trifluoroethanol (TFE) using NMR spectroscopy
[DiGiammarino et al., Biochem., 40:2379-2386 (2001)]. In TFE,
mArfN37 is bi-helical, with the two Arf motifs contained by helices
that are 12 amino acids in length. Based on the observation that
.beta.-strands form when mArfN37 and Hdm2 210-304 interact, and the
prediction of .beta.-strands within the interacting segments, the
bi-helical conformation of mArfN37 in TFE is probably not relevant
to the Hdm2-bound conformation. However, the observation of similar
structure for the mouse A1 and A2 segments in mArfN37 did suggest
that the two Arf motifs may function in Hdm2 binding in a
structurally similar and mechanistically coordinated manner. With
these ideas in mind, the role the two Arf motifs play in Hdm2
binding was investigated by monitoring the binding of short
peptides derived from human and mouse Arf to Hdm2 fragments using
surface plasmon resonance and CD.
[0165] Two libraries of peptides 15 amino acids in length were
synthesized based on the sequence of exon 1.beta. of mouse
p19.sup.Arf and the entire sequence of human p14.sup.Arf. The
sequence for the first peptide in each library corresponded to the
first 15 amino acids of the protein sequences. The N-terminus for
the second peptide was shifted forward 5 residues to position 6 and
spanned residues 6-20, and each subsequent peptide had an
additional 5-residue forward shift of the N-terminus. This approach
yielded two libraries of overlapping peptides spanning mouse
p19.sup.Arf residues 1-64 and human p14.sup.Arf residues 1-132. The
ability of these peptides to bind Hdm2 fragments was determined
using the surface plasmon resonance (SPR) technique with a Biacore
3000 instrument. His affinity tagged Hdm2 210-304 was immobilized
on the surface of a C-1 chip through capture by a covalently
cross-linked anti-His antibody. Library peptides were allowed to
flow over the Hdm2 surface, or over a control surface lacking an
Hdm2 fragment. Relative binding affinity was judged on the basis of
the maximal SPR signal detected after binding for 10 minutes. This
unusually long inject time was used because some peptides exhibited
slow association kinetics; the long association time allowed weak
binding peptides to be identified. The peptides that exhibited the
largest relative binding affinity map to the N-termini of mouse and
human Arf (FIGS. 5a and 5b); however, the length of the
Hdm2-binding segment for the two is slightly different. For
example, the first four peptides from the human library bind Hdm2
210-304; these span the segment of human p14.sup.Arf that encloses
the two Arf motifs. In contrast, 6 of the first 7 peptides of the
mouse p19.sup.Arf library bind Hdm2 210-304. The first five of
these enclose the two Arf motifs while the last, spanning residues
31-45, lacks elements of an Arf motif but contains the RRPR motif
that is the nucleolar localization signal (NoLS) for mouse Arf. One
additional human Arf peptide binds Hdm2 210-304; this spans
residues 86-100 and contains the NoLS (with the sequence RRPR) for
human Arf. Through an unknown mechanism, the RRPR motif within
these nucleolar localization signals causes the polypeptides to
become localized in nucleoli of eukaryotic cells.
[0166] A similar approach using short synthetic peptides and SPR
was used to map the segments of Hdm2 that bind mArfN37 (FIG. 5c).
Peptides within two segments of the central region of Hdm2 bind
with relatively high affinity to mArfN37, including those spanning
residues 235-259 and 275-289 of SEQ ID NO:8. The first of these as
Arf-binding segment is termed "H1" and the second is termed segment
"H2". The peptide containing residues 290-304 of SEQ ID NO:8
exhibits modest affinity for mArfN37; however, based on results
discussed below this appears to be due to non-specific
electrostatic forces.
[0167] To investigate the roles of individual Arf motifs (A1 and
A2) and Arf-binding segments of Hdm2 (H1 and H2) in the
interactions between Arf and Hdm2 the binding of peptides
containing these small segments (A1, A2, H1, or H2) to larger
protein fragments of the corresponding binding partner (mArfN37 or
Hdm2 210-304) were monitored using the CD-based binding assay.
Peptides corresponding to the mouse Arf motifs, A1 (1-14) and A2
(16-30), bind independently to Hdm2 210-304 and induce the
transition from random conformations to .beta.-strand secondary
structure. Similarly, peptides containing the human Arf motifs, A1
(1-14) and A2 (16-30), bind Hdm2 210-304 and produce the
random-to-.beta.-strand structure transition. Peptides from the
Hdm2 library that were positive for mArfN37 binding on the basis of
SPR (i.e. those within H1 and H2) were mixed with mArfN37 and
binding was monitored in the same manner. Three peptides from the
H1 segment and one from the H2 segment of Hdm2 induce .beta.-strand
secondary structure when mixed with mArfN37 (FIG. 2c). These
experiments indicate that peptides containing A1, A2, H1 or H2 can
interact with larger fragments of the target protein and that the
interactions also occur through the formation of .beta.-sheet
secondary structure. Peptides lacking A1, A2, H1 or H2 failed to
induce the structural transition. Importantly, however, further
reduction in the size of the interacting species led to a loss of
Arf/Hdm2 binding as judged by a failure to induce structure in CD
titration experiments. For example, while peptides derived from the
A1 or A2 segments of Arf bind Hdm2 210-304, they fail to bind short
peptides (15 amino acids in length) derived from the H1 or H2
segments of Hdm2. Similarly, while peptides derived from the H1 or
H2 segments of Hdm2 bind mArfN37, they fail to bind peptides
derived from the A1 or A2 segments of mouse or human Arf.
Furthermore, mixtures of two, three, or four different peptides
that contain the important binding segments (A1, A2, H1, and H2)
fail to interact, in contrast to the results obtained when A1 and
A2, or H1 and H2, are covalent linked in larger protein fragments.
Apparently, cooperative interactions between covalently linked
binding segments from one protein (A1 and A2 of Arf, or H1 and H2
of Hdm2) are minimally required for Arf/Hdm2 interactions.
[0168] Arf/Hdm2 Interactions Mediate Nucleolar Colocalization.
[0169] Microinjection and live cell imaging were used to determine
whether the Hdm2 domains that were shown to bind Arf in the SPR and
CD binding studies bound to Arf in cells and whether these domains
are sequestered within nucleoli by Arf. Hdm2 deletion constructs
tagged with a fluorescent label (Texas Red.TM.) were microinjected
into the nucleus of NIH 3T3 cells. importantly, the NIH 3T3 cell
line used lacks the gene for Arf. Nuclear microinjection was used
because the Hdm2 constructs containing the central Arf-binding
domain lack the nuclear localization signal found between residues
181-186. A covalently bound fluorescent label was used to detect
Hdm2 fragments instead of immuno fluorescence to avoid nonspecific
background staining and staining variability due to differential
antibody reactivity. In addition, preliminary results using the
antibody of choice to detect Hdm2 210-326 (2A10) [Weber et al.,
Nat. Cell Biol., 1:20-26 (1999)] gave poor staining results,
possibly because the antibody epitope is very near the Arf binding
site and Arf binding inhibits antibody binding [Midgley et al.,
Oncogene, 19:2312-23 (2000)]. Arf was delivered to cells prior to
microinjection by transfection with a plasmid expressing
GFP-p19.sup.Arf.
[0170] Texas Red.TM. labeled Hdm2 deletion constructs containing
residues 210-275, 210-304, 277-350 and 277-491 were individually
injected into cells in the absence or presence of the
GFP-p19.sup.Arf fusion protein. In cells that did not express Arf,
all constructs were localized in the nucleoplasm and appeared to be
excluded from nucleoli. In contrast, when GFP-Arf was expressed,
three of these constructs that contain all or portions of the
central, Arf-binding domain exhibited a distinct nucleolar
localization pattern. For example, Hdm2 210-275, 210-304 and
277-491 all displayed nucleolar localization when Arf was
expressed. Hdm2 277-350 also binds GFP-Arf, but causes the complex
to be localized in the nucleoplasm rather than in nucleoli. These
results are consistent with a report [Weber et al., Mol. Cell
Biol., 20:2517-2528 (2000)] showing that Hdm2 constructs that
contain a segment spanning residues 210-304 bind Arf and are
sequestered within nucleoli. The use of live cell imaging also
allowed the kinetics of localization to be monitored. Within 5
minutes after injection of Hdm2 constructs, complex relocalization
was complete.
Discussion
[0171] The studies disclosed above focused on understanding the
molecular basis of Arf and Hdm2 interactions and their relationship
to biological function. Previously, the domains mediating Arf and
Hdm2 interactions to the N-terminal 37 amino acids of p19.sup.Arf
[Weber et al., Nat. Cell Biol., 1:20-26 (1999); Weber et al., Mol.
Cell Biol., 20:2517-2528 (2000)] and the central acidic domain of
Hdm2 (210-304) [Weber et al., Mol Cell Biol., 20:2517-2528 (2000)]
had been localized. Structural analysis of mArfN37 in solution
showed that this domain is dynamically disordered in the unbound
state [DiGiammarino et al., Biochem., 40:2379-2386 (2001)].
Interestingly, the Arf interacting domain of Hdm2 is shown herein
to also be dynamically disordered in solution. For example, CD
spectra for Hdm2 acidic domain-containing fragments (210-275 and
210-304) show no signs of secondary structure and NMR spectra
reveal poorly dispersed resonances consistent with random coil
chemical shift values. Further, steady-state heteronuclear
{.sup.1H}-.sup.15N NOE values for Hdm2 210-275 indicated that
amides throughout the entire polypeptide are highly dynamic.
Importantly, as shown herein, these disordered Arf and Hdm2 domains
bind to each other in both in vitro and cellular assays
demonstrating the relevance of the disordered states to biological
function.
[0172] The importance of dynamically disordered proteins or domains
in biological systems, and in the regulation of cell division, is
well established [Kriwacki et al., Proc. Natl. Acad. Sci. USA,
93:11504-11509 (1996); Uversky et al., Protein Sci., 8:161-73
(1999); Sosnick et al., Proteins, 24:427-32 (1996); Dyson et al.,
Biol., 5:499-503 (1998); Plaxco et al., Nature, 386:657-658 (1997);
Wright et al., J. Mol Biol, 293:321-31 (1999)]. For example, the
N-terminal domains of the cyclin dependent kinase inhibitors p21
[Kriwacki et al., Proc. Natl. Acad. Sci. USA, 93:11504-11509
(1996); Kriwacki et al., J. Amer. Chem. Soc., 118:5320-5321 (1996)]
and p27 are largely unstructured prior to binding of their cellular
targets. Currently, the functional advantage(s) of the
`folding-on-binding` mechanism is not well understood. Intuitively,
the loss of conformational entropy associated with folding will
reduce the Gibbs free energy of binding for dynamically disordered
proteins binding their targets [Kriwacki et al., Proc. Natl. Acad.
Sci. USA, 93:11504-11509 (1996); Spolar et al., Science, 263:777-84
(1994)]. It has been suggested that the advantage of this mechanism
is to enhance specificity [Spolar et al., Science, 263:777-84
(1994)] and/or to allow multiple, structurally distinct substrates
to be bound [Kriwacki et al., Proc. Natl. Acad. Sci. USA,
93:11504-11509 (1996); Kim et al., Nature, 404:151-8 (2000)]. A
recent computational study focused on understanding the impact of
conformational entropy in protein folding [Pappu et al., Proc Natl
Acad Sci USA, 97:12565-70 (2000)] suggests that the entropy penalty
may not be as great as commonly envisioned due to steric
restrictions by amino acid side chains on the vastness of
polypeptide conformational space. While dynamic and highly
disordered, flexible polypeptides are probably conformationally
restrained in solution by steric and other interaction forces; the
challenges for the future are to develop approaches to
quantitatively describe these biased conformations and to relate
them to biological function. The need for such studies continues to
grow as more examples of biologically active, dynamically
disordered proteins appear in the literature. The significance of
the observations disclosed herein with the Arf:Hdm2 system is that,
in contrast to previous observations of the folding-on-binding
phenomenon involving a single disordered protein, both components
of the Arf:Hdm2 system undergo folding-on-binding.
[0173] .beta.-strands form when dynamically disordered segments of
Arf and Hdm2 interact. The formation of .beta.-stand secondary
structure is accompanied by the cooperative assembly of large
supramolecular structures. The large size of these assemblies has
been confirmed by gel filtration chromatography and NMR
spectroscopy. For example, resonances for .sup.15N-Hdm2 210-304 are
broadened beyond detection when an excess of unlabeled mArfN37 is
added to the solution. Arf:Hdm2 complexes appear to be formed by
the cooperative assembly of like structural units into extended
.beta.-networks. This is supported by the appearance of the thermal
denaturation curves for the mArfN37:Hdm2 210-304 assembly, which
show the characteristic sigmoidal shape of a cooperative, two-state
protein unfolding transition [Creighton et al., Proteins:
Structures and Molecular Properties, W. H. Freeman & Co., New
York, N.Y. (1993)]. Thermal unfolding is not reversible, however.
Electrostatic forces stabilize the .beta.-assemblies as shown by
the salt-dependence of the T.sub.m values.
[0174] Through in vitro binding assays two segments of similar
sequence in mouse and human Arf-the consensus for which is termed
herein the Arf motif-that mediate binding to the central acidic
domain of Hdm2 and that, individually, induce the formation of
.beta.-strands when mixed with the central, acidic domain of Hdm2
have been identified herein. Further, peptides derived from the
Arf-binding segments of Hdm2-termed H1 and H2-induce .beta.-strands
when mixed with mArfN37. Importantly, however, the mixing of a
short peptide containing either A1 or A2 with another containing
either H1 or H2 does not lead to the formation of .beta.-strands.
CD was used to monitor the structural effects of peptide mixing and
the concentrations used (1-10 .mu.M) may have been below the
threshold for binding of individual domains. The results disclosed
herein with larger protein fragments containing either contiguous
A1-A2 or H1-H2 mixed with short peptides containing a single
binding segment (i.e. H1 or H2, or A1 or A2, respectively) indicate
that cooperative assembly of .beta.-strands requires two binding
elements of one protein (Arf or Hdm2) and one element of the other.
An exception to this is the interaction of Hdm2 210-275, which
contains only the H1 binding segment, with short peptides
containing either the human or mouse A1 segment. Hdm2 210-275 may
contain a portion of the H2 segment that participates in
cooperative interactions with its own H1 segment and the A1
segments of the peptides.
[0175] Peptides from the human and mouse A1 and A2 segments of Arf
induce .beta.-strand assembly when mixed with Hdm2 210-304. In
contrast, peptides from the human and mouse A2 segment of Arf fail
to induce .beta.-strand assembly with Hdm2 210-275 but do induce
.beta.-strand assembly with Hdm2 210-304. These findings indicate
that the H2 binding segment interacts only with the A2 segment of
Arf. Similarly, H1 may selectively interact with A1 but, based on
the data disclosed herein, A1 may also interact with H2.
[0176] It is difficult to rank the relative importance of the
various modes of Arf:Hdm2 interaction (i.e. A1-H1, A2-H2, and
A1-H2) with regard to biological function. However, several
findings indicate that the A1 segment of human and mouse Arf plays
a dominant biological role. First, Weber, et al. have examined the
effects of deleting the A1 or A2 segments of full-length mouse Arf,
either individually or in combination, on the ability of mouse Arf
to cause cell cycle arrest [Weber et al., Mol. Cell Biol.,
20:2517-2528 (2000)]. Deletion of A1, or A1 and A2 together, from
mouse Arf almost completely eliminated the ability to arrest cell
division while deletion of residues 26-37 that partially contain
the A2 segment produced arrest in some cells but not in others.
While it is difficult to quantify the differences in the biological
effects of the different binding site deletion constructs, deletion
of the A1 segment uniformly eliminated the ability to arrest cell
division and can be ranked as the most essential Hdm2-binding
element. Second, Midgley, et al., have shown that a GFP fusion
protein linked to the N-terminal 20 amino acids of human Arf, which
contains the human A1 segment that is almost identical to that from
mouse Arf (FIG. 2a), produces Arf-like biological effects in
cellular assays [Midgley et al., Oncogene, 19:2312-23 (2000)]. This
result has been confirmed by Llanos, et al., using both N- and
C-terminal fusions of residues 2-29 of human Arf to GFP. For Hdm2,
biological data for constructs with deletions of the H1 and/or H2
segments is not available. However, the absolute conservation of
amino acids within H1 from humans and mice to zebra fish and tree
frogs (FIGS. 2b-2c) suggests that this segment is important for
biological function. Amino acids within the H2 segment are also
evolutionarily conserved but not to the same degree as the H1
segment. The A1 segment of Arf and H1 of Hdm2 may play dominant
biological roles; these interactions, however, may not be strong
enough to support high affinity interactions and are assisted by
interactions between the A2 segment of Arf and H2 of Hdm2 in the
formation of Arf:Hdm2 assemblies in cells.
[0177] As further evidence that isolated domains from Arf
(containing the A1 and A2 segments) and Hdm2 (containing the H1
and/or H2 segments) are functionally competent, microinjection and
live cell imaging experiments demonstrate that the domains of Hdm2
that have been characterized in vitro herein, retain the ability to
interact with Arf in vivo. The Hdm2 constructs 210-304 and 210-275
contain two (H1 and H2) and one (H1 only) of the Hdm2 binding
segments, respectively. After nuclear microinjection, each of these
constructs was re-localized to the nucleolus in the presence of
GFP-p19.sup.Arf. Since the Hdm2 NoLS has been deleted from both of
these constructs, the p19.sup.Arf NoLS signal must be driving the
nucleolar localization for both proteins. In contrast, the Hdm2
construct 277-350 relocalizes Arf to the nucleoplasm. This result
confirms the presence of an Arf binding motif in Hdm2 beyond amino
acid 277 and shows that, upon binding of Hdm2 277-350 to GFP-p19A,
the Arf NoLS becomes inaccessible to the localization machinery.
This result is consistent with localization experiments performed
using full length Hdm2 [Lohrum et al., Nat. Cell Biol., 2:179-81
(2000); Weber et al., Mol. Cell Biol., 20:2517-28 (2000)].
Localization of Hdm2 277-491 to nucleoli only in the presence of
p19.sup.Arf demonstrates that the mechanism for exposure of the
cryptic Hdm2 NoLS is operative even in the absence of residues
1-176 of Hdm2. These biological results are consistent with the
existence of two Arf-binding segments within the 210-350 segment of
Hdm2, as identified through in vitro assays using short peptides
and protein fragments.
[0178] A novel mechanism of protein-protein interaction mediates
Arf:Hdm2 binding. The strict requirement for the presence of both
proteins--Arf and Hdm2--for the formation of
.beta.-strand-containing assemblies differentiates this system from
others that utilize the folding-on-binding mechanism. Further,
while the Arf:Hdm2 system is similar to amyloid proteins in that
they form extended networks comprised of .beta.-strands, the
individual components of the Arf:Hdm2 system fail to form
.beta.-assemblies alone under a wide range of solution conditions.
While many proteins will form amyloid-like aggregates with P-fibril
structure [Booth et al., Nature, 385:787-93 (1997); Ohnishi et al.,
J. Mol. Biol., 301:477-89 (2000); Alexandrescu and Rathgeb-Szabo,
J. Mol Biol, 291:1191-206 (1999); [Esposito et al., Protein Sci.,
9:831-845 (2000); Chiti et al., EMBO J, 19:1441-1449 (2000);
Wilkins et al., Eur. J. Biochem., 267:2609-2616 (2000)], Arf and
Hdm2 form .beta.-assemblies only when both components are present.
Insight into the two-component, folding-on-binding phenomenon can
be gained by considering the unusual amino acid composition of the
interacting segments of Arf and Hdm2 (i.e. A1, and A2, and H1 and
H2). As previously reported, the N-termini of both human and mouse
Arf are unusually rich in Arg residues [DiGiammarino et al.,
Biochem., 40:2379-2386 (2001)]. Electrostatic repulsion between
these Arg residues may cause the polypeptide to be dynamically
disordered. Interestingly, the Arf-binding segments of Hdm2
identified here (H1 and H2) are rich in acidic residues. The
carboxylate groups of these acidic residues probably interact with
the guanidinium groups of Arg residues within the A1 and A2
segments of Arf via electrostatic interactions. The ability of salt
to reduce the T.sub.m value for the mArfN37:Hdm2 210-304 assembly
strongly supports the idea that electrostatic interactions
stabilize Arf:Hdm2 assemblies. In the absence of Arf, the acidic
residues within the Arf-binding segments of Hdm2 (H1 and H2) repel
each other, producing the dynamically disordered conformations
observed here. Interestingly, the Jpred secondary structure
prediction algorithm predicts .beta.-strand conformations exactly
within the segments of Arf and Hdm2 which have been shown to be
important for molecular interactions (FIGS. 2a-2c). Thus, the
sequences within these segments (A1 and A2, and H1 and H2) are
consistent with extended, .beta.-strand conformations. However, the
high frequency of like-charged residues within them may cause the
individual polypeptides to be dynamically disordered. It is also
possible that the .beta.-strand regions of .phi., .psi.
conformational space are populated within the H1 and H2 segments
and that the methods of analysis (CD and NMR) used have failed to
detect them.
[0179] In addition to electrostatic interactions, Arf:Hdm2
interactions are likely to be mediated by hydrophobic interactions.
The Arg residues of the Arf motif (FIG. 4) are separated by a
string of hydrophobic residues, including a highly conserved
segment with the sequence FLV. In an extended conformation, the
arrangement of Arg and hydrophobic residues within the Arf motif
would give rise to both types of residues (hydrophobic and Arg) on
both faces of a .beta.-pleated sheet. In the H1 segment of Hdm2,
acidic residues and hydrophobic residues alternate within the
sequence (FIG. 2b). This situation would allow acidic residues of
Hdm2 to interact with Arg residues of Arf in the context of
.beta.-strands composed of intermingled A1 (or A2) and H1 segments.
Hydrophobic residues of H1 would align, on the opposite face, with
hydrophobic residues of the Arf motif. The conclusion that both
electrostatic and hydrophobic forces stabilize Arf:Hdm2
.beta.-assemblies is consistent with their high degree of
stability, as exemplified by resistance to denaturation by urea,
detergent, salt and extremes of pH. It would appear reasonable that
these highly stable structures would be favored within the cellular
environment as well as in vitro. It is of interest that the
nucleolus, where Arf and Hdm2 are localized, was originally
characterized by its granular and fibrillar nature [Olson et al.,
Trends Cell Biol., 10:189-96 (2000)] that is, in principle,
consistent with the extended structures reported here for Arf and
Hdm2.
[0180] The biological function of Hdm2 is, in part, to maintain p53
at low levels by actively controlling its ubiquitination, nuclear
export and proteosome-dependent degradation. The early observation
that Arf leads to sequestration of Hdm2 within nucleoli suggested
that Arf inhibited Hdm2-dependent degradation of p53 by physically
separating the destroyer, Hdm2, from it target, p53. However,
whether Arf inhibits the destroyer function of nucleoplasmic Hdm2,
which has access to p53, has been an open question. Llanos, et al.,
have recently reported that truncated forms of human Arf that fail
to localize within nucleoli maintain the ability to stabilize and
activate p53 [Llanos et al., Nat. Cell Biol., 3:445-452 (2001)].
This suggests that the binding of Arf to Hdm2 within the
nucleoplasm, which may occur prior to nucleolar colocalization,
directly inhibits some or all of the destroyer functions of Hdm2
toward p53. Hdm2 is comprised of several domains that mediate p53
binding (N-terminal helical domain) [Kussie et al., Science,
274:948-953 (1996)], Arf binding (acidic domain studied here), and
p53 ubiquitination (C-terminal RING domain) [Geyer et al., Nat.
Cell Biol., 2:569-73 (2000); Fang et al., J. Biol. Chem.,
275:8945-8951 (2000)]. Lohrum, et al., have shown that when Arf
binds Hdm2, the NoLS of Arf is hidden and a cryptic NoLS within the
RING domain of Hdm2 is revealed, leading to nucleolar
colocalization [Lohrum et al., Nat. Cell Biol., 2:179-81 (2000)].
Consistent with the RING domain of Hdm2 (and Mdm2) playing an
important role in p53 ubiquitination, nuclear export and
degradation [Geyer et al., Nat. Cell Biol., 2:569-73 (2000); Fang
et al., J. Biol. Chem., 275:8945-8951 (2000); Honda and Yasuda,
Oncogene, 19:1473-1476 (2000); Argentini et al., Oncogene,
19:3849-3857 (2000); Boyd et al., Nat. Cell Biol., 2:563-568
(2000)], when Arf binds the central, acidic domain of Hdm2 it not
only exposes a cryptic NoLS within the Hdm2 RING domain but also
alters the structure and function of this domain in the context of
E3 ubiquitin ligase activity. The structure of the UbcH7/Cbl E2/E3
complex [Zheng et al., Cell, 102:533-539 (2000)] shows that the
RING domain within the Cbl E3 subunit interacts with the UbcH7 E2
and serves to orient the E2 with respect to a small peptide derived
from Zap-70, a target of this E2/E3 complex. The RING domain of
Hdm2 can play a similar `orienting` role within the E2/E3 complex
that targets p53. The binding of Arf to Hdm2 can alter the
conformation of the Hdm2 RING domain to expose the cryptic NoLS and
inhibit the orienting function of the RING domain. It is likely
that intramolecular interaction between the domains of Hdm2 hide
the cryptic NoLS and maintain the RING domain in an active E3
ubiquitin ligase conformation. Arf binding, through the
interactions described herein, can disrupt inter-domain
interactions, revealing the cryptic NoLS and inhibiting E3
ubiquitin ligase activity toward p53.
[0181] While the invention has been described and illustrated
herein by references to the specific embodiments, various specific
material, procedures and examples, it is understood that the
invention is not restricted to the particular material combinations
of material, and procedures selected for that purpose. Indeed,
various modifications of the invention in addition to those
described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are intended to
fall within the scope of the appended claims.
[0182] It is further to be understood that all base sizes or amino
acid sizes, and all molecular weight or molecular mass values,
given for nucleic acids or polypeptides are approximate, and are
provided for description.
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NMR relaxation. Biochem., 33, 5984-6003.
[0257] Various publications, patent applications and patents are
cited herein, the disclosures of which are incorporated by
reference in their entireties.
Sequence CWU 1
1
25 1 713 DNA Mus musculus CDS (43)..(549) 1 gtcacagtga ggccgccgct
gagggagtac agcagcggga gc atg ggt cgc agg 54 Met Gly Arg Arg 1 ttc
ttg gtc act gtg agg att cag cgc gcg ggc cgc cca ctc caa gag 102 Phe
Leu Val Thr Val Arg Ile Gln Arg Ala Gly Arg Pro Leu Gln Glu 5 10 15
20 agg gtt ttc ttg gtg aag ttc gtg cga tcc cgg aga ccc agg aca gcg
150 Arg Val Phe Leu Val Lys Phe Val Arg Ser Arg Arg Pro Arg Thr Ala
25 30 35 agc tgc gct ctg gct ttc gtg aac atg ttg ttg agg cta gag
agg atc 198 Ser Cys Ala Leu Ala Phe Val Asn Met Leu Leu Arg Leu Glu
Arg Ile 40 45 50 ttg aga aga ggg ccg cac cgg aat cct gga cca ggt
gat gat gat ggg 246 Leu Arg Arg Gly Pro His Arg Asn Pro Gly Pro Gly
Asp Asp Asp Gly 55 60 65 caa cgt tca cgt agc agc tct tct gct caa
cta cgg tgc aga ttc gaa 294 Gln Arg Ser Arg Ser Ser Ser Ser Ala Gln
Leu Arg Cys Arg Phe Glu 70 75 80 ctg cga gga ccc cac tac ctt ctc
ccg ccc ggt gca cga cgc agc gcg 342 Leu Arg Gly Pro His Tyr Leu Leu
Pro Pro Gly Ala Arg Arg Ser Ala 85 90 95 100 gga agg ctt cct gga
cac gct ggt ggt gct gca cgg gtc agg ggc tcg 390 Gly Arg Leu Pro Gly
His Ala Gly Gly Ala Ala Arg Val Arg Gly Ser 105 110 115 gct gga tgt
gcg cga tgc ctg ggg tcg cct gcc gct cga ctt ggc cca 438 Ala Gly Cys
Ala Arg Cys Leu Gly Ser Pro Ala Ala Arg Leu Gly Pro 120 125 130 aga
gcg ggg aca tca aga cat cgt gcg ata ttt gcg ttc cgc tgg gtg 486 Arg
Ala Gly Thr Ser Arg His Arg Ala Ile Phe Ala Phe Arg Trp Val 135 140
145 ctc ttt gtg ttc cgc tgg gtg gtc ttt gtg tac cgc tgg gaa cgt cgc
534 Leu Phe Val Phe Arg Trp Val Val Phe Val Tyr Arg Trp Glu Arg Arg
150 155 160 cca gac cga cgg gca tagcttcagc tcaagcacgc ccagggccct
ggaacttcgc 589 Pro Asp Arg Arg Ala 165 ggccaatccc aagagcagag
ctaaatccgg cctcagcccg cctttttctt cttagcttca 649 cttctagcga
tgctagcgtg tctagcatgt ggctttaaaa aatacataat aatgcttttt 709 tttt 713
2 169 PRT Mus musculus 2 Met Gly Arg Arg Phe Leu Val Thr Val Arg
Ile Gln Arg Ala Gly Arg 1 5 10 15 Pro Leu Gln Glu Arg Val Phe Leu
Val Lys Phe Val Arg Ser Arg Arg 20 25 30 Pro Arg Thr Ala Ser Cys
Ala Leu Ala Phe Val Asn Met Leu Leu Arg 35 40 45 Leu Glu Arg Ile
Leu Arg Arg Gly Pro His Arg Asn Pro Gly Pro Gly 50 55 60 Asp Asp
Asp Gly Gln Arg Ser Arg Ser Ser Ser Ser Ala Gln Leu Arg 65 70 75 80
Cys Arg Phe Glu Leu Arg Gly Pro His Tyr Leu Leu Pro Pro Gly Ala 85
90 95 Arg Arg Ser Ala Gly Arg Leu Pro Gly His Ala Gly Gly Ala Ala
Arg 100 105 110 Val Arg Gly Ser Ala Gly Cys Ala Arg Cys Leu Gly Ser
Pro Ala Ala 115 120 125 Arg Leu Gly Pro Arg Ala Gly Thr Ser Arg His
Arg Ala Ile Phe Ala 130 135 140 Phe Arg Trp Val Leu Phe Val Phe Arg
Trp Val Val Phe Val Tyr Arg 145 150 155 160 Trp Glu Arg Arg Pro Asp
Arg Arg Ala 165 3 540 DNA Homo sapiens CDS (142)..(537) 3
cgcgcctgcg gggcggagat gggcaggggg cggtgcgtgg gtcccagtct gcagttaagg
60 gggcaggagt ggcgctgctc acctctggtg ccaaagggcg gcgcagcggc
tgccgagctc 120 ggccctggag gcggcgagaa c atg gtg cgc agg ttc ttg gtg
acc ctc cgg 171 Met Val Arg Arg Phe Leu Val Thr Leu Arg 1 5 10 att
cgg cgc gcg tgc ggc ccg ccg cga gtg agg gtt ttc gtg gtt cac 219 Ile
Arg Arg Ala Cys Gly Pro Pro Arg Val Arg Val Phe Val Val His 15 20
25 atc ccg cgg ctc acg ggg gag tgg gca gcg cca ggg gcg ccc gcc gct
267 Ile Pro Arg Leu Thr Gly Glu Trp Ala Ala Pro Gly Ala Pro Ala Ala
30 35 40 gtg gcc ctc gtg ctg atg cta ctg agg agc cag cgt cta ggg
cag cag 315 Val Ala Leu Val Leu Met Leu Leu Arg Ser Gln Arg Leu Gly
Gln Gln 45 50 55 ccg ctt cct aga aga cca ggt cat gat gat ggg cag
cgc ccg agt ggc 363 Pro Leu Pro Arg Arg Pro Gly His Asp Asp Gly Gln
Arg Pro Ser Gly 60 65 70 gga gct gct gct gct cca cgg cgc gga gcc
caa ctg cgc cga ccc cgc 411 Gly Ala Ala Ala Ala Pro Arg Arg Gly Ala
Gln Leu Arg Arg Pro Arg 75 80 85 90 cac tct cac ccg acc cgt gca cga
cgc tgc ccg gga ggg ctt cct gga 459 His Ser His Pro Thr Arg Ala Arg
Arg Cys Pro Gly Gly Leu Pro Gly 95 100 105 cac gct ggt ggt gct gca
ccg ggc cgg ggc gcg gct gga cgt gcg cga 507 His Ala Gly Gly Ala Ala
Pro Gly Arg Gly Ala Ala Gly Arg Ala Arg 110 115 120 tgc ctg ggg ccg
tct gcc cgt gga cct ggc tga 540 Cys Leu Gly Pro Ser Ala Arg Gly Pro
Gly 125 130 4 132 PRT Homo sapiens 4 Met Val Arg Arg Phe Leu Val
Thr Leu Arg Ile Arg Arg Ala Cys Gly 1 5 10 15 Pro Pro Arg Val Arg
Val Phe Val Val His Ile Pro Arg Leu Thr Gly 20 25 30 Glu Trp Ala
Ala Pro Gly Ala Pro Ala Ala Val Ala Leu Val Leu Met 35 40 45 Leu
Leu Arg Ser Gln Arg Leu Gly Gln Gln Pro Leu Pro Arg Arg Pro 50 55
60 Gly His Asp Asp Gly Gln Arg Pro Ser Gly Gly Ala Ala Ala Ala Pro
65 70 75 80 Arg Arg Gly Ala Gln Leu Arg Arg Pro Arg His Ser His Pro
Thr Arg 85 90 95 Ala Arg Arg Cys Pro Gly Gly Leu Pro Gly His Ala
Gly Gly Ala Ala 100 105 110 Pro Gly Arg Gly Ala Ala Gly Arg Ala Arg
Cys Leu Gly Pro Ser Ala 115 120 125 Arg Gly Pro Gly 130 5 1710 DNA
Mus musculus 5 gaggagccgc cgccttctcg tcgctcgagc tctggacgac
catggtcgct caggccccgt 60 ccgcggggcc tccgcgctcc ccgtgaaggg
tcggaagatg cgcgggaagt agcagccgtc 120 tgctgggcga gcgggagacc
gaccggacac ccctggggga ccctctcgga tcaccgcgct 180 tctcctgcgg
cctccaggcc aatgtgcaat accaacatgt ctgtgtctac cgagggtgct 240
gcaagcacct cacagattcc agcttcggaa caagagactc tggttagacc aaaaccattg
300 cttttgaagt tgttaaagtc cgttggagcg caaaacgaca cttacactat
gaaagagatt 360 atattttata ttggccagta tattatgact aagaggttat
atgacgagaa gcagcagcac 420 attgtgtatt gttcaaatga tctcctagga
gatgtgtttg gagtcccgag tttctctgtg 480 aaggagcaca ggaaaatata
tgcaatgatc tacagaaatt tagtggctgt aagtcagcaa 540 gactctggca
catcgctgag tgagagcaga cgtcagcctg aaggtgggag tgatctgaag 600
gatcctttgc aagcgccacc agaagagaaa ccttcatctt ctgatttaat ttctagactg
660 tctacctcat ctagaaggag atccattagt gagacagaag agaacacaga
tgagctacct 720 ggggagcggc accggaagcg ccgcaggtcc ctgtcctttg
atccgagcct gggtctgtgt 780 gagctgaggg agatgtgcag cggcggcacg
agcagcagta gcagcagcag cagcgagtcc 840 acagagacgc cctcgcatca
ggatcttgac gatggcgtaa gtgagcattc tggtgattgc 900 ctggatcagg
attcagtttc tgatcagttt agcgtggaat ttgaagttga gtctctggac 960
tcggaagatt acagcctgag tgacgaaggg cacgagctct cagatgagga tgatgaggtc
1020 tatcgggtca cagtctatca gacaggagaa agcgatacag actcttttga
aggagatcct 1080 gagatttcct tagctgacta ttggaagtgt acctcatgca
atgaaatgaa tcctcccctt 1140 ccatcacact gcaaaagatg ctggaccctt
cgtgagaact ggcttccaga cgataagggg 1200 aaagataaag tggaaatctc
tgaaaaagcc aaactggaaa actcagctca ggcagaagaa 1260 ggcttggatg
tgcctgatgg caaaaagctg acagagaatg atgctaaaga gccatgtgct 1320
gaggaggaca gcgaggagaa ggccgaacag acgcccctgt cccaggagag tgacgactat
1380 tcccaaccat cgacttccag cagcattgtt tatagcagcc aagaaagcgt
gaaagagttg 1440 aaggaggaaa cgcagcacaa agacgagagt gtggaatcta
gcttctccct gaatgccatc 1500 gaaccatgtg tgatctgcca ggggcggcct
aaaaatggct gcattgttca cggcaagact 1560 ggacacctca tgtcatgttt
cacgtgtgca aagaagctaa aaaaaagaaa caagccctgc 1620 ccagtgtgca
gacagccaat ccaaatgatt gtgctaagtt acttcaacta gctgacctgc 1680
tcacaaaaat agaattttat atttctaact 1710 6 489 PRT Mus musculus 6 Met
Cys Asn Thr Asn Met Ser Val Ser Thr Glu Gly Ala Ala Ser Thr 1 5 10
15 Ser Gln Ile Pro Ala Ser Glu Gln Glu Thr Leu Val Arg Pro Lys Pro
20 25 30 Leu Leu Leu Lys Leu Leu Lys Ser Val Gly Ala Gln Asn Asp
Thr Tyr 35 40 45 Thr Met Lys Glu Ile Ile Phe Tyr Ile Gly Gln Tyr
Ile Met Thr Lys 50 55 60 Arg Leu Tyr Asp Glu Lys Gln Gln His Ile
Val Tyr Cys Ser Asn Asp 65 70 75 80 Leu Leu Gly Asp Val Phe Gly Val
Pro Ser Phe Ser Val Lys Glu His 85 90 95 Arg Lys Ile Tyr Ala Met
Ile Tyr Arg Asn Leu Val Ala Val Ser Gln 100 105 110 Gln Asp Ser Gly
Thr Ser Leu Ser Glu Ser Arg Arg Gln Pro Glu Gly 115 120 125 Gly Ser
Asp Leu Lys Asp Pro Leu Gln Ala Pro Pro Glu Glu Lys Pro 130 135 140
Ser Ser Ser Asp Leu Ile Ser Arg Leu Ser Thr Ser Ser Arg Arg Arg 145
150 155 160 Ser Ile Ser Glu Thr Glu Glu Asn Thr Asp Glu Leu Pro Gly
Glu Arg 165 170 175 His Arg Lys Arg Arg Arg Ser Leu Ser Phe Asp Pro
Ser Leu Gly Leu 180 185 190 Cys Glu Leu Arg Glu Met Cys Ser Gly Gly
Thr Ser Ser Ser Ser Ser 195 200 205 Ser Ser Ser Glu Ser Thr Glu Thr
Pro Ser His Gln Asp Leu Asp Asp 210 215 220 Gly Val Ser Glu His Ser
Gly Asp Cys Leu Asp Gln Asp Ser Val Ser 225 230 235 240 Asp Gln Phe
Ser Val Glu Phe Glu Val Glu Ser Leu Asp Ser Glu Asp 245 250 255 Tyr
Ser Leu Ser Asp Glu Gly His Glu Leu Ser Asp Glu Asp Asp Glu 260 265
270 Val Tyr Arg Val Thr Val Tyr Gln Thr Gly Glu Ser Asp Thr Asp Ser
275 280 285 Phe Glu Gly Asp Pro Glu Ile Ser Leu Ala Asp Tyr Trp Lys
Cys Thr 290 295 300 Ser Cys Asn Glu Met Asn Pro Pro Leu Pro Ser His
Cys Lys Arg Cys 305 310 315 320 Trp Thr Leu Arg Glu Asn Trp Leu Pro
Asp Asp Lys Gly Lys Asp Lys 325 330 335 Val Glu Ile Ser Glu Lys Ala
Lys Leu Glu Asn Ser Ala Gln Ala Glu 340 345 350 Glu Gly Leu Asp Val
Pro Asp Gly Lys Lys Leu Thr Glu Asn Asp Ala 355 360 365 Lys Glu Pro
Cys Ala Glu Glu Asp Ser Glu Glu Lys Ala Glu Gln Thr 370 375 380 Pro
Leu Ser Gln Glu Ser Asp Asp Tyr Ser Gln Pro Ser Thr Ser Ser 385 390
395 400 Ser Ile Val Tyr Ser Ser Gln Glu Ser Val Lys Glu Leu Lys Glu
Glu 405 410 415 Thr Gln His Lys Asp Glu Ser Val Glu Ser Ser Phe Ser
Leu Asn Ala 420 425 430 Ile Glu Pro Cys Val Ile Cys Gln Gly Arg Pro
Lys Asn Gly Cys Ile 435 440 445 Val His Gly Lys Thr Gly His Leu Met
Ser Cys Phe Thr Cys Ala Lys 450 455 460 Lys Leu Lys Lys Arg Asn Lys
Pro Cys Pro Val Cys Arg Gln Pro Ile 465 470 475 480 Gln Met Ile Val
Leu Ser Tyr Phe Asn 485 7 2372 DNA Homo sapiens 7 gcaccgcgcg
agcttggctg cttctggggc ctgtgtggcc ctgtgtgtcg gaaagatgga 60
gcaagaagcc gagcccgagg ggcggccgcg acccctctga ccgagatcct gctgctttcg
120 cagccaggag caccgtccct ccccggatta gtgcgtacga gcgcccagtg
ccctggcccg 180 gagagtggaa tgatccccga ggcccagggc gtcgtgcttc
cgcagtagtc agtccccgtg 240 aaggaaactg gggagtcttg agggaccccc
gactccaagc gcgaaaaccc cggatggtga 300 ggagcaggca aatgtgcaat
accaacatgt ctgtacctac tgatggtgct gtaaccacct 360 cacagattcc
agcttcggaa caagagaccc tggttagacc aaagccattg cttttgaagt 420
tattaaagtc tgttggtgca caaaaagaca cttatactat gaaagaggtt cttttttatc
480 ttggccagta tattatgact aaacgattat atgatgagaa gcaacaacat
attgtatatt 540 gttcaaatga tcttctagga gatttgtttg gcgtgccaag
cttctctgtg aaagagcaca 600 ggaaaatata taccatgatc tacaggaact
tggtagtagt caatcagcag gaatcatcgg 660 actcaggtac atctgtgagt
gagaacaggt gtcaccttga aggtgggagt gatcaaaagg 720 accttgtaca
agagcttcag gaagagaaac cttcatcttc acatttggtt tctagaccat 780
ctacctcatc tagaaggaga gcaattagtg agacagaaga aaattcagat gaattatctg
840 gtgaacgaca aagaaaacgc cacaaatctg atagtatttc cctttccttt
gatgaaagcc 900 tggctctgtg tgtaataagg gagatatgtt gtgaaagaag
cagtagcagt gaatctacag 960 ggacgccatc gaatccggat cttgatgctg
gtgtaagtga acattcaggt gattggttgg 1020 atcaggattc agtttcagat
cagtttagtg tagaatttga agttgaatct ctcgactcag 1080 aagattatag
ccttagtgaa gaaggacaag aactctcaga tgaagatgat gaggtatatc 1140
aagttactgt gtatcaggca ggggagagtg atacagattc atttgaagaa gatcctgaaa
1200 tttccttagc tgactattgg aaatgcactt catgcaatga aatgaatccc
ccccttccat 1260 cacattgcaa cagatgttgg gcccttcgtg agaattggct
tcctgaagat aaagggaaag 1320 ataaagggga aatctctgag aaagccaaac
tggaaaactc aacacaagct gaagagggct 1380 ttgatgttcc tgattgtaaa
aaaactatag tgaatgattc cagagagtca tgtgttgagg 1440 aaaatgatga
taaaattaca caagcttcac aatcacaaga aagtgaagac tattctcagc 1500
catcaacttc tagtagcatt atttatagca gccaagaaga tgtgaaagag tttgaaaggg
1560 aagaaaccca agacaaagaa gagagtgtgg aatctagttt gccccttaat
gccattgaac 1620 cttgtgtgat ttgtcaaggt cgacctaaaa atggttgcat
tgtccatggc aaaacaggac 1680 atcttatggc ctgctttaca tgtgcaaaga
agctaaagaa aaggaataag ccctgcccag 1740 tatgtagaca accaattcaa
atgattgtgc taacttattt cccctagttg acctgtctat 1800 aagagaatta
tatatttcta actatataac cctaggaatt tagacaacct gaaatttatt 1860
cacatatatc aaagtgagaa aatgcctcaa ttcacataga tttcttctct ttagtataat
1920 tgacctactt tggtagtgga atagtgaata cttactataa tttgacttga
atatgtagct 1980 catcctttac accaactcct aattttaaat aatttctact
ctgtcttaaa tgagaagtac 2040 ttggtttttt ttttcttaaa tatgtatatg
acatttaaat gtaacttatt attttttttg 2100 agaccgagtc ttgctctgtt
acccaggctg gagtgcagtg ggtgatcttg gctcactgca 2160 agctctgccc
tccccgggtt cgcaccattc tcctgcctca gcctcccaat tagcttggcc 2220
tacagtcatc tgccaccaca cctggctaat tttttgtact tttagtagag acagggtttc
2280 accgtgttag ccaggatggt ctcgatctcc tgacctcgtg atccgcccac
ctcggcctcc 2340 caaagtgctg ggattacagg catgagccac cg 2372 8 491 PRT
Homo sapiens 8 Met Cys Asn Thr Asn Met Ser Val Pro Thr Asp Gly Ala
Val Thr Thr 1 5 10 15 Ser Gln Ile Pro Ala Ser Glu Gln Glu Thr Leu
Val Arg Pro Lys Pro 20 25 30 Leu Leu Leu Lys Leu Leu Lys Ser Val
Gly Ala Gln Lys Asp Thr Tyr 35 40 45 Thr Met Lys Glu Val Leu Phe
Tyr Leu Gly Gln Tyr Ile Met Thr Lys 50 55 60 Arg Leu Tyr Asp Glu
Lys Gln Gln His Ile Val Tyr Cys Ser Asn Asp 65 70 75 80 Leu Leu Gly
Asp Leu Phe Gly Val Pro Ser Phe Ser Val Lys Glu His 85 90 95 Arg
Lys Ile Tyr Thr Met Ile Tyr Arg Asn Leu Val Val Val Asn Gln 100 105
110 Gln Glu Ser Ser Asp Ser Gly Thr Ser Val Ser Glu Asn Arg Cys His
115 120 125 Leu Glu Gly Gly Ser Asp Gln Lys Asp Leu Val Gln Glu Leu
Gln Glu 130 135 140 Glu Lys Pro Ser Ser Ser His Leu Val Ser Arg Pro
Ser Thr Ser Ser 145 150 155 160 Arg Arg Arg Ala Ile Ser Glu Thr Glu
Glu Asn Ser Asp Glu Leu Ser 165 170 175 Gly Glu Arg Gln Arg Lys Arg
His Lys Ser Asp Ser Ile Ser Leu Ser 180 185 190 Phe Asp Glu Ser Leu
Ala Leu Cys Val Ile Arg Glu Ile Cys Cys Glu 195 200 205 Arg Ser Ser
Ser Ser Glu Ser Thr Gly Thr Pro Ser Asn Pro Asp Leu 210 215 220 Asp
Ala Gly Val Ser Glu His Ser Gly Asp Trp Leu Asp Gln Asp Ser 225 230
235 240 Val Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu Asp
Ser 245 250 255 Glu Asp Tyr Ser Leu Ser Glu Glu Gly Gln Glu Leu Ser
Asp Glu Asp 260 265 270 Asp Glu Val Tyr Gln Val Thr Val Tyr Gln Ala
Gly Glu Ser Asp Thr 275 280 285 Asp Ser Phe Glu Glu Asp Pro Glu Ile
Ser Leu Ala Asp Tyr Trp Lys 290 295 300 Cys Thr Ser Cys Asn Glu Met
Asn Pro Pro Leu Pro Ser His Cys Asn 305 310 315 320 Arg Cys Trp Ala
Leu Arg Glu Asn Trp Leu Pro Glu Asp Lys Gly Lys 325 330 335 Asp Lys
Gly Glu Ile Ser Glu Lys Ala Lys Leu Glu Asn Ser Thr Gln 340 345 350
Ala Glu Glu Gly Phe Asp Val Pro Asp Cys Lys Lys Thr Ile Val Asn 355
360 365 Asp Ser Arg Glu Ser Cys Val Glu Glu Asn Asp Asp Lys Ile Thr
Gln 370 375 380 Ala Ser Gln Ser Gln
Glu Ser Glu Asp Tyr Ser Gln Pro Ser Thr Ser 385 390 395 400 Ser Ser
Ile Ile Tyr Ser Ser Gln Glu Asp Val Lys Glu Phe Glu Arg 405 410 415
Glu Glu Thr Gln Asp Lys Glu Glu Ser Val Glu Ser Ser Leu Pro Leu 420
425 430 Asn Ala Ile Glu Pro Cys Val Ile Cys Gln Gly Arg Pro Lys Asn
Gly 435 440 445 Cys Ile Val His Gly Lys Thr Gly His Leu Met Ala Cys
Phe Thr Cys 450 455 460 Ala Lys Lys Leu Lys Lys Arg Asn Lys Pro Cys
Pro Val Cys Arg Gln 465 470 475 480 Pro Ile Gln Met Ile Val Leu Thr
Tyr Phe Pro 485 490 9 8 PRT Mus musculus 9 Arg Arg Phe Leu Val Thr
Val Arg 1 5 10 9 PRT Mus musculus 10 Arg Val Phe Leu Val Lys Phe
Val Arg 1 5 11 8 PRT Homo sapiens 11 Arg Arg Phe Leu Val Thr Leu
Arg 1 5 12 9 PRT Homo sapiens 12 Arg Val Phe Val Val His Ile Pro
Arg 1 5 13 9 PRT CONSENSUS SEQUENCE MISC_FEATURE (2)..(2) X can be
R or V 13 Arg Xaa Phe Xaa Val Xaa Xaa Xaa Arg 1 5 14 4 PRT
Nucleolar Localization Sequence 14 Arg Arg Pro Arg 1 15 37 PRT Homo
sapiens 15 Met Val Arg Arg Phe Leu Val Thr Leu Arg Ile Arg Arg Ala
Cys Gly 1 5 10 15 Pro Pro Arg Val Arg Val Phe Val Val His Ile Pro
Arg Leu Thr Gly 20 25 30 Glu Trp Ala Ala Pro 35 16 37 PRT Mus
musculus 16 Met Gly Arg Arg Phe Leu Val Thr Val Arg Ile Gln Arg Ala
Gly Arg 1 5 10 15 Pro Leu Gln Glu Arg Val Phe Leu Val Lys Phe Val
Arg Ser Arg Arg 20 25 30 Pro Arg Thr Ala Ser 35 17 37 PRT opossum
17 Met Ile Arg Arg Val Arg Val Thr Val Arg Val Ser Arg Ala Cys Arg
1 5 10 15 Pro His His Val Arg Ile Phe Val Ala Lys Ile Val Gln Ala
Leu Cys 20 25 30 Arg Ala Ser Ala Ser 35 18 95 PRT Homo sapiens 18
Ser Ser Ser Ser Glu Ser Thr Gly Thr Pro Ser Asn Pro Asp Leu Asp 1 5
10 15 Ala Gly Val Ser Glu His Ser Gly Asp Trp Leu Asp Gln Asp Ser
Val 20 25 30 Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu
Asp Ser Glu 35 40 45 Asp Tyr Ser Leu Ser Glu Glu Gly Gln Glu Leu
Ser Asp Glu Asp Asp 50 55 60 Glu Val Tyr Gln Val Thr Val Tyr Gln
Ala Gly Glu Ser Asp Thr Asp 65 70 75 80 Ser Phe Glu Glu Asp Pro Glu
Ile Ser Leu Ala Asp Tyr Trp Lys 85 90 95 19 95 PRT Mus musculus 19
Ser Ser Ser Ser Glu Ser Thr Glu Thr Pro Ser His Gln Asp Leu Asp 1 5
10 15 Asp Gly Val Ser Glu His Ser Gly Asp Cys Leu Asp Gln Asp Ser
Val 20 25 30 Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu
Asp Ser Glu 35 40 45 Asp Tyr Ser Leu Ser Asp Glu Gly His Glu Leu
Ser Asp Glu Asp Asp 50 55 60 Glu Val Tyr Arg Val Thr Val Tyr Gln
Thr Gly Glu Ser Asp Thr Asp 65 70 75 80 Ser Phe Glu Gly Asp Pro Glu
Ile Ser Leu Ala Asp Tyr Trp Lys 85 90 95 20 95 PRT hamster 20 Ser
Ser Ser Ser Glu Ser Thr Asp Thr Pro Ser Asn Gln Asp Leu Asp 1 5 10
15 Asp Gly Val Ser Glu His Ser Gly Asp Trp Leu Asp Gln Asp Ser Val
20 25 30 Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu Asp
Ser Glu 35 40 45 Asp Tyr Ser Leu Ser Glu Gly Gly Gln Glu Leu Ser
Asp Glu Asp Asp 50 55 60 Glu Val Tyr Arg Val Thr Val Tyr Gln Ser
Gly Glu Ser Asp Val Asp 65 70 75 80 Ser Phe Glu Gly Asp Pro Glu Ile
Ser Leu Ala Asp Tyr Trp Lys 85 90 95 21 95 PRT horse 21 Ser Ser Ser
Ser Glu Ser Thr Gly Thr Pro Ser Asn Pro Asp Leu Asp 1 5 10 15 Ala
Gly Val Ser Glu His Ser Gly Asp Trp Leu Asp Gln Asp Ser Val 20 25
30 Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu Asp Ser Glu
35 40 45 Asp Tyr Ser Leu Ser Glu Glu Gly Gln Glu Leu Ser Asp Glu
Asp Asp 50 55 60 Glu Val Tyr Arg Val Thr Val Tyr Gln Ala Gly Glu
Ser Asp Thr Asp 65 70 75 80 Ser Phe Glu Glu Asp Pro Glu Ile Ser Leu
Ala Asp Tyr Trp Lys 85 90 95 22 95 PRT dog 22 Ser Ser Ser Ser Glu
Ser Thr Gly Thr Pro Ser Asn Pro Asp Leu Asp 1 5 10 15 Ala Gly Val
Ser Glu His Ser Gly Asp Trp Leu Asp Gln Asp Ser Val 20 25 30 Ser
Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu Asp Ser Glu 35 40
45 Asp Tyr Ser Leu Ser Glu Glu Gly Gln Glu Leu Ser Asp Glu Asp Asp
50 55 60 Glu Val Tyr Arg Val Thr Val Tyr Gln Ala Gly Glu Ser Asp
Thr Asp 65 70 75 80 Ser Phe Glu Glu Asp Pro Glu Ile Ser Leu Ala Asp
Tyr Trp Lys 85 90 95 23 95 PRT chicken 23 Ser Asn Ser Ser Asp Ser
Thr Asp Ser Val Ser Ile Pro Asp Leu Asp 1 5 10 15 Ala Ser Ser Leu
Ser Glu Asn Ser Asp Trp Phe Asp His Gly Ser Val 20 25 30 Ser Asp
Gln Phe Ser Val Glu Phe Glu Val Glu Ser Ile Tyr Ser Glu 35 40 45
Asp Tyr Ser His Asn Glu Glu Gly Gln Glu Leu Thr Asp Glu Asp Asp 50
55 60 Glu Val Tyr Gln Leu Thr Ile Tyr Gln Asp Glu Asp Ser Asp Ser
Asp 65 70 75 80 Ser Phe Asn Glu Asp Pro Glu Ile Ser Leu Ala Asp Tyr
Trp Lys 85 90 95 24 90 PRT zebrafish 24 Arg Gly Asn Ser Glu Ser Ser
Asp Ala Asn Ser Asn Ser Asp Val Gly 1 5 10 15 Ile Ser Arg Ser Glu
Gly Ser Glu Glu Ser Glu Asp Ser Asp Ser Asp 20 25 30 Ser Asp Asn
Phe Ser Val Glu Phe Glu Val Glu Ser Ile Asn Ser Asp 35 40 45 Ala
Tyr Ser Glu Asn Asp Val Asp Ser Val Pro Gly Glu Asn Glu Ile 50 55
60 Tyr Glu Val Thr Ile Phe Ala Glu Asp Glu Asp Ser Phe Asp Glu Asp
65 70 75 80 Thr Glu Ile Thr Glu Ala Asp Tyr Lys Trp 85 90 25 98 PRT
treefrog 25 Gly Leu Arg Cys Asp Arg Asn Ser Ser Glu Ser Thr Asp Ser
Ser Ser 1 5 10 15 Asn Ser Asp Pro Glu Arg His Ser Thr Asn Asp Asn
Ser Glu His Asp 20 25 30 Ser Asp Gln Phe Ser Val Glu Phe Glu Val
Glu Ser Val Cys Ser Asp 35 40 45 Asp Tyr Ser Pro Ser Gly Asp Glu
His Gly Val Ser Glu Glu Glu Glu 50 55 60 Ile Asn Asp Glu Val Tyr
Gln Val Thr Ile Tyr Glu Thr Glu Glu Ser 65 70 75 80 Glu Thr Asp Ser
Phe Asp Val Asp Thr Glu Ile Ser Glu Ala Asp Tyr 85 90 95 Trp
Lys
* * * * *