U.S. patent application number 10/211133 was filed with the patent office on 2003-02-06 for hprp4s as modifiers of the p53 pathway and methods of use.
Invention is credited to Costa, Michael, Francis-Lang, Helen, Friedman, Lori, Funke, Roel P., Hung, Tak, Li, Danxi, Plowman, Gregory D..
Application Number | 20030027230 10/211133 |
Document ID | / |
Family ID | 26977360 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030027230 |
Kind Code |
A1 |
Friedman, Lori ; et
al. |
February 6, 2003 |
HPRP4s as modifiers of the p53 pathway and methods of use
Abstract
Human hPRP4 genes are identified as modulators of the p53
pathway, and thus are therapeutic targets for disorders associated
with defective p53 function. Methods for identifying modulators of
p53, comprising screening for agents that modulate the activity of
hPRP4 are provided.
Inventors: |
Friedman, Lori; (San
Francisco, CA) ; Plowman, Gregory D.; (San Carlos,
CA) ; Hung, Tak; (Foster City, CA) ;
Francis-Lang, Helen; (San Francisco, CA) ; Li,
Danxi; (San Francisco, CA) ; Funke, Roel P.;
(South San Francisco, CA) ; Costa, Michael; (San
Francisco, CA) |
Correspondence
Address: |
JAN P. BRUNELLE
EXELIXIS, INC.
170 HARBOR WAY
P.O. BOX 511
SOUTH SAN FRANCISCO
CA
94083-0511
US
|
Family ID: |
26977360 |
Appl. No.: |
10/211133 |
Filed: |
August 2, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60310362 |
Aug 6, 2001 |
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60357501 |
Feb 15, 2002 |
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Current U.S.
Class: |
435/7.23 ;
435/15 |
Current CPC
Class: |
G01N 33/5008 20130101;
G01N 33/57419 20130101; G01N 33/6842 20130101; G01N 33/5041
20130101; A61P 9/00 20180101; G01N 2510/00 20130101; G01N 33/68
20130101; G01N 33/57415 20130101; A61P 35/00 20180101; G01N 33/5091
20130101; C12Q 1/485 20130101; G01N 2500/00 20130101; G01N 33/57449
20130101; G01N 33/6875 20130101; A61P 43/00 20180101; G01N 33/5011
20130101; G01N 33/57423 20130101 |
Class at
Publication: |
435/7.23 ;
435/15 |
International
Class: |
C12Q 001/68; G01N
033/574; C12Q 001/48 |
Claims
What is claimed is:
1. A method of identifying a candidate p53 pathway modulating
agent, said method comprising the steps of: (a) providing an assay
system comprising a purified hPRP4 polypeptide or nucleic acid or a
functionally active fragment or derivative thereof; (b) contacting
the assay system with a test agent under conditions whereby, but
for the presence of the test agent, the system provides a reference
activity; and (c) detecting a test agent-biased activity of the
assay system, wherein a difference between the test agent-biased
activity and the reference activity identifies the test agent as a
candidate p53 pathway modulating agent.
2. The method of claim 1 wherein the assay system comprises
cultured cells that express the hPRP4 polypeptide.
3. The method of claim 2 wherein the cultured cells additionally
have defective p53 function.
4. The method of claim 1 wherein the assay system includes a
screening assay comprising a hPRP4 polypeptide, and the candidate
test agent is a small molecule modulator.
5. The method of claim 4 wherein the assay is a kinase assay.
6. The method of claim 1 wherein the assay system is selected from
the group consisting of an apoptosis assay system, a cell
proliferation assay system, an angiogenesis assay system, and a
hypoxic induction assay system.
7. The method of claim 1 wherein the assay system includes a
binding assay comprising a hPRP4 polypeptide and the candidate test
agent is an antibody.
8. The method of claim 1 wherein the assay system includes an
expression assay comprising a hPRP4 nucleic acid and the candidate
test agent is a nucleic acid modulator.
9. The method of claim 8 wherein the nucleic acid modulator is an
antisense oligomer.
10. The method of claim 8 wherein the nucleic acid modulator is a
PMO.
11. The method of claim 1 additionally comprising: (d)
administering the candidate p53 pathway modulating agent identified
in (c) to a model system comprising cells defective in p53 function
and, detecting a phenotypic change in the model system that
indicates that the p53 function is restored.
12. The method of claim 11 wherein the model system is a mouse
model with defective p53 function.
13. A method for modulating a p53 pathway of a cell comprising
contacting a cell defective in p53 function with a candidate
modulator that specifically binds to a hPRP4 polypeptide comprising
an amino acid as set forth in SEQ ID NO:7, whereby p53 function is
restored.
14. The method of claim 13 wherein the candidate modulator is
administered to a vertebrate animal predetermined to have a disease
or disorder resulting from a defect in p53 function.
15. The method of claim 13 wherein the candidate modulator is
selected from the group consisting of an antibody and a small
molecule.
16. The method of claim 1, comprising the additional steps of: (d)
providing a secondary assay system comprising cultured cells or a
non-human animal expressing hPRP4, (e) contacting the secondary
assay system with the test agent of (b) or an agent derived
therefrom under conditions whereby, but for the presence of the
test agent or agent derived therefrom, the system provides a
reference activity; and (f) detecting an agent-biased activity of
the second assay system, wherein a difference between the
agent-biased activity and the reference activity of the second
assay system confirms the test agent or agent derived therefrom as
a candidate p53 pathway modulating agent, and wherein the second
assay detects an agent-biased change in the p53 pathway.
17. The method of claim 16 wherein the secondary assay system
comprises cultured cells.
18. The method of claim 16 wherein the secondary assay system
comprises a non-human animal.
19. The method of claim 18 wherein the non-human animal
mis-expresses a p53 pathway gene.
20. A method of modulating p53 pathway in a mammalian cell
comprising contacting the cell with an agent that specifically
binds a hPRP4 polypeptide or nucleic acid.
21. The method of claim 20 wherein the agent is administered to a
mammalian animal predetermined to have a pathology associated with
the p53 pathway.
22. The method of claim 20 wherein the agent is a small molecule
modulator, a nucleic acid modulator, or an antibody.
23. A method for diagnosing a disease in a patient comprising: (a)
obtaining a biological sample from the patient; (b) contacting the
sample with a probe for hPRP4 expression; (c) comparing results
from step (b) with a control; (d) determining whether step (c)
indicates a likelihood of disease.
24. The method of claim 23 wherein said disease is cancer.
25. The method according to claim 24, wherein said cancer ovarian
cancer.
Description
[0001] REFERENCE TO RELATED APPLICATIONS
[0002] This application claims priority to U.S. provisional patent
applications Nos. 60/310,362 filed Aug. 6, 2001, and 60/357,501
filed Feb. 2, 2002. The contents of the prior applications are
hereby incorporated in their entirety.
BACKGROUND OF THE INVENTION
[0003] The p53 gene is mutated in over 50 different types of human
cancers, including familial and spontaneous cancers, and is
believed to be the most commonly mutated gene in human cancer
(Zambetti and Levine, FASEB (1993) 7:855-865; Hollstein, et al.,
Nucleic Acids Res. (1994) 22:3551-3555). Greater than 90% of
mutations in the p53 gene are missense mutations that alter a
single amino acid that inactivates p53 function. Aberrant forms of
human p53 are associated with poor prognosis, more aggressive
tumors, metastasis, and short survival rates (Mitsudomi et al.,
Clin Cancer Res Oct. 6, 2000 (10):4055-63; Koshland, Science (1993)
262:1953).
[0004] The human p53 protein normally functions as a central
integrator of signals including DNA damage, hypoxia, nucleotide
deprivation, and oncogene activation (Prives, Cell (1998) 95:5-8).
In response to these signals, p53 protein levels are greatly
increased with the result that the accumulated p53 activates cell
cycle arrest or apoptosis depending on the nature and strength of
these signals. Indeed, multiple lines of experimental evidence have
pointed to a key role for p53 as a tumor suppressor (Levine, Cell
(1997) 88:323-331). For example, homozygous p53 "knockout" mice are
developmentally normal but exhibit nearly 100% incidence of
neoplasia in the first year of life (Donehower et al., Nature
(1992) 356:215-221).
[0005] The biochemical mechanisms and pathways through which p53
functions in normal and cancerous cells are not fully understood,
but one clearly important aspect of p53 function is its activity as
a gene-specific transcriptional activator. Among the genes with
known p53-response elements are several with well-characterized
roles in either regulation of the cell cycle or apoptosis,
including GADD45, p21/Waf1/Cip1, cyclin G, Bax, IGF-BP3, and MDM2
(Levine, Cell (1997) 88:323-331).
[0006] Mitogen-activated protein kinases (MAPKs) and
cyclin-dependent kinases (CDKs) are important proline-directed
Ser/Thr kinases that play critical roles in cell differentiation
and proliferation. PRP (pre-mRNA processing gene) is a CDK-like
kinase with homology to MAPKs (Huang, Y. et al. (2000) Biochem
Biophys Res Commun; 271(2): 456-63).
[0007] Pre-mRNA processing 4 (PRP4) is a nuclear serine-threonine
kinase activated by EGF stimulation, plays a role in
transcriptional regulation, and may be involved in pre-mRNA
splicing and intracellular signaling (Kojima, T.et al. (2001) J
Biol Chem 276, 32247-56; Gross, T. et al. (1997) Nucleic Acids Res.
25: 1028-1035). The Prp4 gene of Schizosaccharomyces pombe encodes
a protein kinase that appears to be involved in pre-mRNA splicing
(Gross et al., supra). The sequence of PRP4 kinase and its function
in pre-mRNA splicing are highly conserved in yeast and humans
(Wang, et al., Hum Mol Genet. (1997) Nov;6(12):2117-26; Schwelnus
et al., EMBO Rep. (2001) Jan;2(1):35-41). Based on kinase domain
sequence, Prp4 belongs to the Clk (CDC-like kinase) family and
interacts with CLK1 (Kojima et al., supra).
[0008] The ability to manipulate the genomes of model organisms
such as C. elegans provides a powerful means to analyze biochemical
processes that, due to significant evolutionary conservation, have
direct relevance to more complex vertebrate organisms. Due to a
high level of gene and pathway conservation, the strong similarity
of cellular processes, and the functional conservation of genes
between these model organisms and mammals, identification of the
involvement of novel genes in particular pathways and their
functions in such model organisms can directly contribute to the
understanding of the correlative pathways and methods of modulating
them in mammals (see, for example, Dulubova I, et al, J Neurochem
2001 Apr;77(1):229-38; Cai T, et al., Diabetologia 2001
Jan;44(1):81-8; Pasquinelli AE, et al., Nature. 2000 Nov
2;408(6808):37-8; Ivanov IP, et al., EMBO J 2000 Apr
17;19(8):1907-17; Vajo Z et al., Mamm Genome Oct. 10, 1999 ;(10):
1000-4). For example, a genetic screen can be carried out in an
invertebrate model organism having underexpression (e.g. knockout)
or overexpression of a gene (referred to as a "genetic entry
point") that yields a visible phenotype. Additional genes are
mutated in a random or targeted manner. When a gene mutation
changes the original phenotype caused by the mutation in the
genetic entry point, the gene is identified as a "modifier"
involved in the same or overlapping pathway as the genetic entry
point. When the genetic entry point is an ortholog of a human gene
implicated in a disease pathway, such as p53, modifier genes can be
identified that may be attractive candidate targets for novel
therapeutics.
[0009] All references cited herein, including sequence information
in referenced Genbank identifier numbers and website references,
are incorporated herein in their entireties.
SUMMARY OF THE INVENTION
[0010] We have discovered genes that modify the p53 pathway in C.
elegans, and identified their human orthologs, hereinafter referred
to as hPRP4. The invention provides methods for utilizing these p53
modifier genes and polypeptides to identify hPRP4-modulating agents
that are candidate therapeutic agents that can be used in the
treatment of disorders associated with defective or impaired p53
function and/or hPRP4 function. Preferred hPRP4-modulating agents
specifically bind to hPRP4 polypeptides and restore p53 function.
Other preferred hPRP4-modulating agents are nucleic acid modulators
such as antisense oligomers and RNAi that repress hPRP4 gene
expression or product activity by, for example, binding to and
inhibiting the respective nucleic acid (i.e. DNA or mRNA).
[0011] hPRP4 modulating agents may be evaluated by any convenient
in vitro or in vivo assay for molecular interaction with an hPRP4
polypeptide or nucleic acid. In one embodiment, candidate hPRP4
modulating agents are tested with an assay system comprising a
hPRP4 polypeptide or nucleic acid.
[0012] Agents that produce a change in the activity of the assay
system relative to controls are identified as candidate p53
modulating agents. The assay system may be cell-based or cell-free.
hPRP4-modulating agents include hPRP4 related proteins (e.g.
dominant negative mutants, and biotherapeutics); hPRP4-specific
antibodies; hPRP4-specific antisense oligomers and other nucleic
acid modulators; and chemical agents that specifically bind to or
interact with hPRP4 or compete with hPRP4 binding partner (e.g. by
binding to an hPRP4 binding partner). In one specific embodiment, a
small molecule modulator is identified using a kinase assay. In
specific embodiments, the screening assay system is selected from a
binding assay, an apoptosis assay, a cell proliferation assay, an
angiogenesis assay, and a hypoxic induction assay.
[0013] In another embodiment, candidate p53 pathway modulating
agents are further tested using a second assay system that detects
changes in the p53 pathway, such as angiogenic, apoptotic, or cell
proliferation changes produced by the originally identified
candidate agent or an agent derived from the original agent. The
second assay system may use cultured cells or non-human animals. In
specific embodiments, the secondary assay system uses non-human
animals, including animals predetermined to have a disease or
disorder implicating the p53 pathway, such as an angiogenic,
apoptotic, or cell proliferation disorder (e.g. cancer).
[0014] The invention further provides methods for modulating the
hPRP4 function and/or the p53 pathway in a mammalian cell by
contacting the mammalian cell with an agent that specifically binds
a hPRP4 polypeptide or nucleic acid. The agent may be a small
molecule modulator, a nucleic acid modulator, or an antibody and
may be administered to a mammalian animal predetermiined to have a
pathology associated the p53 pathway.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Genetic screens were designed to identify modifiers of the
p53 pathway in C. elegans, where a homozygous p53 deletion mutant
was used. Various specific genes were silenced by RNA inhibition
(RNAi). Methods for using RNAi to silence genes in C. elegans are
known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A.
Trends Genet. 15, 358-363 (1999); WO9932619). Genes causing altered
phenotypes in the worms were identified as modifiers of the p53
pathway. Modifiers of particular interest, F22D6.5, were identified
followed by identification of their human orthologs.
[0016] In vitro and in vivo methods of assessing hPRP4 function are
provided herein. Modulation of the hPRP4 or their respective
binding partners is useful for understanding the association of the
p53 pathway and its members in normal and disease conditions and
for developing diagnostics and therapeutic modalities for p53
related pathologies. hPRP4-modulating agents that act by inhibiting
or enhancing hPRP4 expression, directly or indirectly, for example,
by affecting an hPRP4 function such as enzymatic (e.g., catalytic)
or binding activity, can be identified using methods provided
herein. hPRP4 modulating agents are useful in diagnosis, therapy
and pharmaceutical development.
[0017] Nucleic Acids and Polypeptides of the Invention
[0018] Sequences related to hPRP4 nucleic acids and polypeptides
that can be used in the invention are disclosed in Genbank
(referenced by Genbank identifier (GI) number) as GI#s 14571505
(SEQ ID NO:1), 17999534 (SEQ ID NO:2), 14746978 (SEQ ID NO:3),
14714402 (SEQ ID NO:4), and 1399461 (SEQ ID NO:5) for nucleic acid,
and GI# 14571506 (SEQ ID NO:7)for polypeptides. Additionally,
sequences of clone N5A08 (SEQ ID NO:6) can be used in the methods
of the invention.
[0019] hPRP4s are nuclear serine/threonine kinase proteins with
kinase domains. The term "hPRP4 polypeptide" refers to a
full-length hPRP4 protein or a functionally active fragment or
derivative thereof. A "functionally active" hPRP4 fragment or
derivative exhibits one or more functional activities associated
with a full-length, wild-type hPRP4 protein, such as antigenic or
immunogenic activity, enzymatic activity, ability to bind natural
cellular substrates, etc. The functional activity of hPRP4
proteins, derivatives and fragments can be assayed by various
methods known to one skilled in the art (Current Protocols in
Protein Science (1998) Coligan et al., eds., John Wiley & Sons,
Inc., Somerset, N.J.) and as further discussed below. For purposes
herein, functionally active fragments also include those fragments
that comprise one or more structural domains of an hPRP4, such as a
kinase domain or a binding domain. Protein domains can be
identified using the PFAM program (Bateman A., et al., Nucleic
Acids Res, 1999, 27:260-2). For example, the kinase domain of hPRP4
from GI# 14571506 (SEQ ID NO:7) is located at approximately amino
acid residues 687-1003 (PFAM 00069). Methods for obtaining hPRP4
polypeptides are also further described below. In some embodiments,
preferred fragments are functionally active, domain-containing
fragments comprising at least 25 contiguous amino acids, preferably
at least 50, more preferably 75, and most preferably at least 100
contiguous amino acids of SEQ ID NO:7 (an hPRP4). In further
preferred embodiments, the fragment comprises the entire kinase
(functionally active) domain.
[0020] The term "hPRP4 nucleic acid" refers to a DNA or RNA
molecule that encodes a hPRP4 polypeptide. Preferably, the hPRP4
polypeptide or nucleic acid or fragment thereof is from a human,
but can also be an ortholog, or derivative thereof with at least
70% sequence identity, preferably at least 80%, more preferably
85%, still more preferably 90%, and most preferably at least 95%
sequence identity with hPRP4. Normally, orthologs in different
species retain the same function, due to presence of one or more
protein motifs and/or 3-dimensional structures. Orthologs are
generally identified by sequence homology analysis, such as BLAST
analysis, usually using protein bait sequences. Sequences are
assigned as a potential ortholog if the best hit sequence from the
forward BLAST result retrieves the original query sequence in the
reverse BLAST (Huynen MA and Bork P, Proc Natl Acad Sci (1998)
95:5849-5856; Huynen MA et al., Genome Research (2000)
10:1204-1210). Programs for multiple sequence alignment, such as
CLUSTAL (Thompson JD et al, 1994, Nucleic Acids Res 22:4673-4680)
may be used to highlight conserved regions and/or residues of
orthologous proteins and to generate phylogenetic trees. In a
phylogenetic tree representing multiple homologous sequences from
diverse species (e.g., retrieved through BLAST analysis),
orthologous sequences from two species generally appear closest on
the tree with respect to all other sequences from these two
species. Structural threading or other analysis of protein folding
(e.g., using software by ProCeryon, Biosciences, Salzburg, Austria)
may also identify potential orthologs. In evolution, when a gene
duplication event follows speciation, a single gene in one species,
such as C. elegans, may correspond to multiple genes (paralogs) in
another, such as human. As used herein, the term "orthologs"
encompasses paralogs. As used herein, "percent (%) sequence
identity" with respect to a subject sequence, or a specified
portion of a subject sequence, is defined as the percentage of
nucleotides or amino acids in the candidate derivative sequence
identical with the nucleotides or amino acids in the subject
sequence (or specified portion thereof), after aligning the
sequences and introducing gaps, if necessary to achieve the maximum
percent sequence identity, as generated by the program
WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1997) 215:403-410)
with all the search parameters set to default values. The HSP S and
HSP S2 parameters are dynamic values and are established by the
program itself depending upon the composition of the particular
sequence and composition of the particular database against which
the sequence of interest is being searched. A % identity value is
determined by the number of matching identical nucleotides or amino
acids divided by the sequence length for which the percent identity
is being reported. "Percent (%) amino acid sequence similarity" is
determined by doing the same calculation as for determining % amino
acid sequence identity, but including conservative amino acid
substitutions in addition to identical amino acids in the
computation.
[0021] A conservative amino acid substitution is one in which an
amino acid is substituted for another amino acid having similar
properties such that the folding or activity of the protein is not
significantly affected. Aromatic amino acids that can be
substituted for each other are phenylalanine, tryptophan, and
tyrosine; interchangeable hydrophobic amino acids are leucine,
isoleucine, methionine, and valine; interchangeable polar amino
acids are glutamine and asparagine; interchangeable basic amino
acids are arginine, lysine and histidine; interchangeable acidic
amino acids are aspartic acid and glutamic acid; and
interchangeable small amino acids are alanine, serine, threonine,
cysteine and glycine.
[0022] Alternatively, an alignment for nucleic acid sequences is
provided by the local homology algorithm of Smith and Waterman
(Smith and Waterman, 1981, Advances in Applied Mathematics
2:482-489; database: European Bioinformatics Institute; Smith and
Waterman, 1981, J. of Molec.Biol., 147:195-197; Nicholas et al.,
1998, "A Tutorial on Searching Sequence Databases and Sequence
Scoring Methods" (www.psc.edu) and references cited therein.; W. R.
Pearson, 1991, Genomics 11:635-650). This algorithm can be applied
to amino acid sequences by using the scoring matrix developed by
Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O.
Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research
Foundation, Washington, D.C., USA), and normalized by Gribskov
(Gribskov 1986 Nucl. Acids Res. 14(6):6745-6763). The
Smith-Waterman algorithm may be employed where default parameters
are used for scoring (for example, gap open penalty of 12, gap
extension penalty of two). From the data generated, the "Match"
value reflects "sequence identity."
[0023] Derivative nucleic acid molecules of the subject nucleic
acid molecules include sequences that hybridize to the nucleic acid
sequence of any of SEQ ID NOs:1, 2, 3, 4, 5, or 6. The stringency
of hybridization can be controlled by temperature, ionic strength,
pH, and the presence of denaturing agents such as formamide during
hybridization and washing. Conditions routinely used are set out in
readily available procedure texts (e.g., Current Protocol in
Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons,
Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring
Harbor (1989)). In some embodiments, a nucleic acid molecule of the
invention is capable of hybridizing to a nucleic acid molecule
containing the nucleotide sequence of any one of SEQ ID NOs:1, 2,
3, 4, 5, or 6 under stringent hybridization conditions that
comprise: prehybridization of filters containing nucleic acid for 8
hours to overnight at 65.degree. C. in a solution comprising
6.times. single strength citrate (SSC) (1.times.SSC is 0.15 M NaCl,
0.015 M Na citrate; pH 7.0), 5.times.Denhardt's solution, 0.05%
sodium pyrophosphate and 100 .mu.g/ml herring sperm DNA;
hybridization for 18-20 hours at 65.degree. C. in a solution
containing 6.times.SSC, 1.times.Denhardt's solution, 100 .mu.g/ml
yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters
at 65.degree. C. for 1h in a solution containing 0.2.times.SSC and
0.1% SDS (sodium dodecyl sulfate).
[0024] In other embodiments, moderately stringent hybridization
conditions are used that comprise: pretreatment of filters
containing nucleic acid for 6 h at 40.degree. C. in a solution
containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl (pH7.5), 5mM
EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/ml denatured
salmon sperm DNA; hybridization for 18-20h at 40.degree. C. in a
solution containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl
(pH7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml
salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by
washing twice for 1 hour at 55.degree. C. in a solution containing
2.times.SSC and 0.1% SDS.
[0025] Alternatively, low stringency conditions can be used that
comprise: incubation for 8 hours to overnight at 37.degree. C. in a
solution comprising 20% formamide, 5.times.SSC, 50 mM sodium
phosphate (pH 7.6), 5.times.Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured sheared salmon sperm DNA;
hybridization in the same buffer for 18 to 20 hours; and washing of
filters in 1.times.SSC at about 37.degree. C. for 1 hour.
[0026] Isolation, Production, Expression, and Mis-expression of
hPRP4 Nucleic Acids and Polypeptides
[0027] hPRP4 nucleic acids and polypeptides, useful for identifying
and testing agents that modulate hPRP4 function and for other
applications related to the involvement of hPRP4 in the p53
pathway. hPRP4 nucleic acids and derivatives and orthologs thereof
may be obtained using any available method. For instance,
techniques for isolating cDNA or genomic DNA sequences of interest
by screening DNA libraries or by using polymerase chain reaction
(PCR) are well known in the art. In general, the particular use for
the protein will dictate the particulars of expression, production,
and purification methods. For instance, production of proteins for
use in screening for modulating agents may require methods that
preserve specific biological activities of these proteins, whereas
production of proteins for antibody generation may require
structural integrity of particular epitopes. Expression of proteins
to be purified for screening or antibody production may require the
addition of specific tags (e.g., generation of fusion proteins).
Overexpression of an hPRP4 protein for assays used to assess hPRP4
function, such as involvement in cell cycle regulation or hypoxic
response, may require expression in eukaryotic cell lines capable
of these cellular activities. Techniques for the expression,
production, and purification of proteins are well known in the art;
any suitable means therefore may be used (e.g., Higgins S J and
Hames B D (eds.) Protein Expression: A Practical Approach, Oxford
University Press Inc., New York 1999; Stanbury PF et al.,
Principles of Fermentation Technology, .sub.2nd edition, Elsevier
Science, New York, 1995; Doonan S (ed.) Protein Purification
Protocols, Humana Press, New Jersey, 1996; Coligan JE et al,
Current Protocols in Protein Science (eds.), 1999, John Wiley &
Sons, New York). In particular embodiments, recombinant hPRP4 is
expressed in a cell line known to have defective p53 function (e.g.
SAOS-2 osteoblasts, H1299 lung cancer cells, C33A and HT3 cervical
cancer cells, HT-29 and DLD-1 colon cancer cells, among others,
available from American Type Culture Collection (ATCC), Manassas,
Va.). The recombinant cells are used in cell-based screening assay
systems of the invention, as described further below.
[0028] The nucleotide sequence encoding an hPRP4 polypeptide can be
inserted into any appropriate expression vector. The necessary
transcriptional and translational signals, including
promoter/enhancer element, can derive from the native hPRP4 gene
and/or its flanking regions or can be heterologous. A variety of
host-vector expression systems may be utilized, such as mammalian
cell systems infected with virus (e.g. vaccinia virus, adenovirus,
etc.); insect cell systems infected with virus (e.g. baculovirus);
microorganisms such as yeast containing yeast vectors, or bacteria
transformed with bacteriophage, plasmid, or cosmid DNA. A host cell
strain that modulates the expression of, modifies, and/or
specifically processes the gene product may be used.
[0029] To detect expression of the hPRP4 gene product, the
expression vector can comprise a promoter operably linked to an
hPRP4 gene nucleic acid, one or more origins of replication, and,
one or more selectable markers (e.g. thymidine kinase activity,
resistance to antibiotics, etc.). Alternatively, recombinant
expression vectors can be identified by assaying for the expression
of the hPRP4 gene product based on the physical or functional
properties of the hPRP4 protein in in vitro assay systems (e.g.
immunoassays).
[0030] The hPRP4 protein, fragment, or derivative may be optionally
expressed as a fusion, or chimeric protein product (i.e. it is
joined via a peptide bond to a heterologous protein sequence of a
different protein), for example to facilitate purification or
detection. A chimeric product can be made by ligating the
appropriate nucleic acid sequences encoding the desired amino acid
sequences to each other using standard methods and expressing the
chimeric product. A chimeric product may also be made by protein
synthetic techniques, e.g. by use of a peptide synthesizer
(Hunkapiller et al., Nature (1984) 310:105-111).
[0031] Once a recombinant cell that expresses the hPRP4 gene
sequence is identified, the gene product can be isolated and
purified using standard methods (e.g. ion exchange, affinity, and
gel exclusion chromatography; centrifugation; differential
solubility; electrophoresis). Alternatively, native hPRP4 proteins
can be purified from natural sources, by standard methods (e.g.
immunoaffinity purification). Once a protein is obtained, it may be
quantified and its activity measured by appropriate methods, such
as immunoassay, bioassay, or other measurements of physical
properties, such as crystallography.
[0032] The methods of this invention may also use cells that have
been engineered for altered expression (mis-expression) of hPRP4 or
other genes associated with the p53 pathway. As used herein,
mis-expression encompasses ectopic expression, over-expression,
under-expression, and non-expression (e.g. by gene knock-out or
blocking expression that would otherwise normally occur).
[0033] Genetically Modified Animals
[0034] Animal models that have been genetically modified to alter
hPRP4 expression may be used in in vivo assays to test for activity
of a candidate p53 modulating agent, or to further assess the role
of hPRP4 in a p53 pathway process such as apoptosis or cell
proliferation. Preferably, the altered hPRP4 expression results in
a detectable phenotype, such as decreased or increased levels of
cell proliferation, angiogenesis, or apoptosis compared to control
animals having normal hPRP4 expression. The genetically modified
animal may additionally have altered p53 expression (e.g. p53
knockout). Preferred genetically modified animals are mammals such
as primates, rodents (preferably mice), cows, horses, goats, sheep,
pigs, dogs and cats. Preferred non-mammalian species include
zebrafish, C. elegans , and Drosophila. Preferred genetically
modified animals are transgenic animals having a heterologous
nucleic acid sequence present as an extrachromosomal element in a
portion of its cells, i.e. mosaic animals (see, for example,
techniques described by Jakobovits, 1994, Curr. Biol. 4:761-763.)
or stably integrated into its germ line DNA (i.e., in the genomic
sequence of most or all of its cells). Heterologous nucleic acid is
introduced into the germ line of such transgenic animals by genetic
manipulation of, for example, embryos or embryonic stem cells of
the host animal.
[0035] Methods of making transgenic animals are well-known in the
art (for transgenic mice see Brinster et al., Proc. Nat. Acad. Sci.
USA 82: 4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009,
both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al., and
Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1986); for particle
bombardment see U.S. Pat. No., 4,945,050, by Sandford et al.; for
transgenic Drosophila see Rubin and Spradling, Science (1982)
218:348-53 and U.S. Pat. No. 4,670,388; for transgenic insects see
Berghammer A. J. et al., A Universal Marker for Transgenic Insects
(1999) Nature 402:370-371; for transgenic Zebrafish see Lin S.,
Transgenic Zebrafish, Methods Mol Biol. (2000);136:375-3830); for
microinjection procedures for fish, amphibian eggs and birds see
Houdebine and Chourrout, Experientia (1991) 47:897-905; for
transgenic rats see Hammer et al., Cell (1990) 63:1099-1112; and
for culturing of embryonic stem (ES) cells and the subsequent
production of transgenic animals by the introduction of DNA into ES
cells using methods such as electroporation, calcium phosphate/DNA
precipitation and direct injection see, e.g., Teratocarcinomas and
Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed.,
IRL Press (1987)). Clones of the nonhuman transgenic animals can be
produced according to available methods (see Wilmut, I. et al.
(1997) Nature 385:810-813; and PCT International Publication Nos.
WO 97/07668 and WO 97/07669).
[0036] In one embodiment, the transgenic animal is a "knock-out"
animal having a heterozygous or homozygous alteration in the
sequence of an endogenous hPRP4 gene that results in a decrease of
hPRP4 function, preferably such that hPRP4 expression is
undetectable or insignificant. Knock-out animals are typically
generated by homologous recombination with a vector comprising a
transgene having at least a portion of the gene to be knocked out.
Typically a deletion, addition or substitution has been introduced
into the transgene to functionally disrupt it. The transgene can be
a human gene (e.g., from a human genomic clone) but more preferably
is an ortholog of the human gene derived from the transgenic host
species. For example, a mouse hPRP4 gene is used to construct a
homologous recombination vector suitable for altering an endogenous
hPRP4 gene in the mouse genome. Detailed methodologies for
homologous recombination in mice are available (see Capecchi,
Science (1989) 244:1288-1292; Joyner et al., Nature (1989)
338:153-156). Procedures for the production of non-rodent
transgenic mammals and other animals are also available (Houdebine
and Chourrout, supra; Pursel et al., Science (1989) 244:1281-1288;
Simms et al., Bio/Technology (1988) 6:179-183). In a preferred
embodiment, knock-out animals, such as mice harboring a knockout of
a specific gene, may be used to produce antibodies against the
human counterpart of the gene that has been knocked out (Claesson
MH et al., (1994) Scan J Immunol 40:257-264; Declerck PJ et al.,
(1995) J Biol Chem. 270:8397-400).
[0037] In another embodiment, the transgenic animal is a "knock-in"
animal having an alteration in its genome that results in altered
expression (e.g., increased (including ectopic) or decreased
expression) of the hPRP4 gene, e.g., by introduction of additional
copies of hPRP4, or by operatively inserting a regulatory sequence
that provides for altered expression of an endogenous copy of the
hPRP4 gene. Such regulatory sequences include inducible,
tissue-specific, and constitutive promoters and enhancer elements.
The knock-in can be homozygous or heterozygous.
[0038] Transgenic nonhuman animals can also be produced that
contain selected systems allowing for regulated expression of the
transgene. One example of such a system that may be produced is the
cre/loxP recombinase system of bacteriophage P1 (Lakso et al., PNAS
(1992) 89:6232-6236; U.S. Pat. No. 4,959,317). If a cre/loxP
recombinase system is used to regulate expression of the transgene,
animals containing transgenes encoding both the Cre recombinase and
a selected protein are required. Such animals can be provided
through the construction of "double" transgenic animals, e.g., by
mating two transgenic animals, one containing a transgene encoding
a selected protein and the other containing a transgene encoding a
recombinase. Another example of a recombinase system is the FLP
recombinase system of Saccharomyces cerevisiae (O'Gorman et al.
(1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182). In a
preferred embodiment, both Cre-LoxP and Flp-Frt are used in the
same system to regulate expression of the transgene, and for
sequential deletion of vector sequences in the same cell (Sun X et
al (2000) Nat Genet 25:83-6).
[0039] The genetically modified animals can be used in genetic
studies to further elucidate the p53 pathway, as animal models of
disease and disorders implicating defective p53 function, and for
in vivo testing of candidate therapeutic agents, such as those
identified in screens described below. The candidate therapeutic
agents are administered to a genetically modified animal having
altered hPRP4 function and phenotypic changes are compared with
appropriate control animals such as genetically modified animals
that receive placebo treatment, and/or animals with unaltered hPRP4
expression that receive candidate therapeutic agent.
[0040] In addition to the above-described genetically modified
animals having altered hPRP4 function, animal models having
defective p53 function (and otherwise normal hPRP4 function), can
be used in the methods of the present invention. For example, a p53
knockout mouse can be used to assess, in vivo, the activity of a
candidate p53 modulating agent identified in one of the in vitro
assays described below. p53 knockout mice are described in the
literature (Jacks et al., Nature 2001 ;410:1111-1116, 1043-1044;
Donehower et al., supra). Preferably, the candidate p53 modulating
agent when administered to a model system with cells defective in
p53 function, produces a detectable phenotypic change in the model
system indicating that the p53 function is restored, i.e., the
cells exhibit normal cell cycle progression.
[0041] Modulating Agents
[0042] The invention provides methods to identify agents that
interact with and/or modulate the function of hPRP4 and/or the p53
pathway. Modulating agents identified by the methods are also part
of the invention. Such agents are useful in a variety of diagnostic
and therapeutic applications associated with the p53 pathway, as
well as in further analysis of the hPRP4 protein and its
contribution to the p53 pathway. Accordingly, the invention also
provides methods for modulating the p53 pathway comprising the step
of specifically modulating hPRP4 activity by administering a
hPRP4-interacting or -modulating agent.
[0043] As used herein, an "hPRP4-modulating agent" is any agent
that modulated hPRP4 function, for example, an agent that interacts
with hPRP4 to inhibit or enhance hPRP4 activity or otherwise affect
normal hPRP4 function. hPRP4 function can be affected at any level,
including transcription, protein expression, protein localization,
and cellular or extra-cellular activity. In a preferred embodiment,
the hPRP4 - modulating agent specifically modulates the function of
the hPRP4. The phrases "specific modulating agent", "specifically
modulates", etc., are used herein to refer to modulating agents
that directly bind to the hPRP4 polypeptide or nucleic acid, and
preferably inhibit, enhance, or otherwise alter, the function of
the hPRP4. These phrases also encompasses modulating agents that
alter the interaction of the hPRP4 with a binding partner,
substrate, or cofactor (e.g. by binding to a binding partner of an
hPRP4, or to a protein/binding partner complex, and altering hPRP4
function). In a further preferred embodiment, the hPRP4-modulating
agent is a modulator of the p53 pathway (e.g. it restores and/or
upregulates p53 function) and thus is also a p53-modulating
agent.
[0044] Preferred hPRP4-modulating agents include small molecule
compounds; hPRP4-interacting proteins, including antibodies and
other biotherapeutics; and nucleic acid modulators such as
antisense and RNA inhibitors. The modulating agents may be
formulated in pharmaceutical compositions, for example, as
compositions that may comprise other active ingredients, as in
combination therapy, and/or suitable carriers or excipients.
Techniques for formulation and administration of the compounds may
be found in "Remington's Pharmaceutical Sciences" Mack Publishing
Co., Easton, Pa., 19.sup.th edition.
[0045] Small Molecule Modulators
[0046] Small molecules, are often preferred to modulate function of
proteins with enzymatic function, and/or containing protein
interaction domains. Chemical agents, referred to in the art as
"small molecule" compounds are typically organic, non-peptide
molecules, having a molecular weight less than 10,000, preferably
less than 5,000, more preferably less than 1,000, and most
preferably less than 500. This class of modulators includes
chemically synthesized molecules, for instance, compounds from
combinatorial chemical libraries. Synthetic compounds may be
rationally designed or identified based on known or inferred
properties of the hPRP4 protein or may be identified by screening
compound libraries. Alternative appropriate modulators of this
class are natural products, particularly secondary metabolites from
organisms such as plants or fungi, which can also be identified by
screening compound libraries for hPRP4-modulating activity. Methods
for generating and obtaining compounds are well known in the art
(Schreiber SL, Science (2000) 151: 1964-1969; Radmann J and Gunther
J, Science (2000) 151:1947-1948).
[0047] Small molecule modulators identified from screening assays,
as described below, can be used as lead compounds from which
candidate clinical compounds may be designed, optimized, and
synthesized. Such clinical compounds may have utility in treating
pathologies associated with the p53 pathway. The activity of
candidate small molecule modulating agents may be improved
several-fold through iterative secondary functional validation, as
further described below, structure determination, and candidate
modulator modification and testing. Additionally, candidate
clinical compounds are generated with specific regard to clinical
and pharmacological properties. For example, the reagents may be
derivatized and re-screened using in vitro and in vivo assays to
optimize activity and minimize toxicity for pharmaceutical
development.
[0048] Protein Modulators
[0049] Specific hPRP4-interacting proteins are useful in a variety
of diagnostic and therapeutic applications related to the p53
pathway and related disorders, as well as in validation assays for
other hPRP4-modulating agents. In a preferred embodiment,
hPRP4-interacting proteins affect normal hPRP4 function, including
transcription, protein expression, protein localization, and
cellular or extra-cellular activity. In another embodiment,
hPRP4-interacting proteins are useful in detecting and providing
information about the function of hPRP4 proteins, as is relevant to
p53 related disorders, such as cancer (e.g., for diagnostic
means).
[0050] An hPRP4-interacting protein may be endogenous, i.e. one
that naturally interacts genetically or biochemically with an
hPRP4, such as a member of the hPRP4 pathway that modulates hPRP4
expression, localization, and/or activity. hPRP4-modulators include
dominant negative forms of hPRP4-interacting proteins and of hPRP4
proteins themselves. Yeast two-hybrid and variant screens offer
preferred methods for identifying endogenous hPRP4-interacting
proteins (Finley, R. L. et al. (1996) in DNA Cloning-Expression
Systems: A Practical Approach, eds. Glover D. & Hames B. D
(Oxford University Press, Oxford, England), pp. 169-203; Fashema SF
et al., Gene (2000) 250:1-14; Drees BL Curr Opin Chem Biol (1999)
3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29;
and U.S. Pat. No. 5,928,868). Mass spectrometry is an alternative
preferred method for the elucidation of protein complexes (reviewed
in, e.g., Pandley A and Mann M, Nature (2000) 405:837-846; Yates JR
3.sup.rd, Trends Genet (2000) 16:5-8).
[0051] An hPRP4-interacting protein may be an exogenous protein,
such as an hPRP4-specific antibody or a T-cell antigen receptor
(see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual,
Cold Spring Harbor Laboratory; Harlow and Lane (1999) Using
antibodies: a laboratory manual. Cold Spring Harbor, N.Y.: Cold
Spring Harbor Laboratory Press). hPRP4 antibodies are further
discussed below.
[0052] In preferred embodiments, an hPRP4-interacting protein
specifically binds an hPRP4 protein. In alternative preferred
embodiments, an hPRP4-modulating agent binds an hPRP4 substrate,
binding partner, or cofactor.
[0053] Antibodies
[0054] In another embodiment, the protein modulator is an hPRP4
specific antibody agonist or antagonist. The antibodies have
therapeutic and diagnostic utilities, and can be used in screening
assays to identify hPRP4 modulators. The antibodies can also be
used in dissecting the portions of the hPRP4 pathway responsible
for various cellular responses and in the general processing and
maturation of the hPRP4.
[0055] Antibodies that specifically bind hPRP4 polypeptides can be
generated using known methods. Preferably the antibody is specific
to a mammalian ortholog of hPRP4 polypeptide, and more preferably,
to human hPRP4. Antibodies may be polyclonal, monoclonal (mAbs),
humanized or chimeric antibodies, single chain antibodies, Fab
fragments, F(ab').sub.2 fragments, fragments produced by a FAb
expression library, anti-idiotypic (anti-Id) antibodies, and
epitope-binding fragments of any of the above. Epitopes of hPRP4
which are particularly antigenic can be selected, for example, by
routine screening of hPRP4 polypeptides for antigenicity or by
applying a theoretical method for selecting antigenic regions of a
protein (Hopp and Wood (1981), Proc. Nati. Acad. Sci. U.S.A.
78:3824-28; Hopp and Wood, (1983) Mol. Immunol. 20:483-89;
Sutcliffe et al., (1983) Science 219:660-66) to the amino acid
sequence shown in SEQ ID NO:7. Monoclonal antibodies with
affinities of 10.sup.8 M.sup.-1 preferably 10.sup.9 M.sup.-1 to
10.sup.10 M.sup.-1, or stronger can be made by standard procedures
as described (Harlow and Lane, supra; Goding (1986) Monoclonal
Antibodies: Principles and Practice (2d ed) Academic Press, New
York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and 4,618,577).
Antibodies may be generated against crude cell extracts of hPRP4 or
substantially purified fragments thereof. If hPRP4 fragments are
used, they preferably comprise at least 10, and more preferably, at
least 20 contiguous amino acids of an hPRP4 protein. In a
particular embodiment, hPRP4-specific antigens and/or immunogens
are coupled to carrier proteins that stimulate the immune response.
For example, the subject polypeptides are covalently coupled to the
keyhole limpet hemocyanin (KLH) carrier, and the conjugate is
emulsified in Freund's complete adjuvant, which enhances the immune
response. An appropriate immune system such as a laboratory rabbit
or mouse is immunized according to conventional protocols.
[0056] The presence of hPRP4-specific antibodies is assayed by an
appropriate assay such as a solid phase enzyme-linked immunosorbant
assay (ELISA) using immobilized corresponding hPRP4 polypeptides.
Other assays, such as radioimmunoassays or fluorescent assays might
also be used.
[0057] Chimeric antibodies specific to hPRP4 polypeptides can be
made that contain different portions from different animal species.
For instance, a human immunoglobulin constant region may be linked
to a variable region of a murine mAb, such that the antibody
derives its biological activity from the human antibody, and its
binding specificity from the murine fragment. Chimeric antibodies
are produced by splicing together genes that encode the appropriate
regions from each species (Morrison et al., Proc. Natl. Acad. Sci.
(1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604-608;
Takeda et al., Nature (1985) 31:452-454). Humanized antibodies,
which are a form of chimeric antibodies, can be generated by
grafting complementary-determining regions (CDRs) (Carlos, T. M.,
J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a
background of human framework regions and constant regions by
recombinant DNA technology (Riechmann L M, et al., 1988 Nature 323:
323-327). Humanized antibodies contain .about.10% murine sequences
and .about.90% human sequences, and thus further reduce or
eliminate immunogenicity, while retaining the antibody
specificities (Co MS, and Queen C. 1991 Nature 351: 501-501;
Morrison SL. 1992 Ann. Rev.
[0058] Immun. 10:239-265). Humanized antibodies and methods of
their production are well-known in the art (U.S. Pat. Nos.
5,530,101, 5,585,089, 5,693,762, and 6,180,370).
[0059] hPRP4-specific single chain antibodies which are
recombinant, single chain polypeptides formed by linking the heavy
and light chain fragments of the Fv regions via an amino acid
bridge, can be produced by methods known in the art (U.S. Pat. No.
4,946,778; Bird, Science (1988) 242:423-426; Huston et al., Proc.
Natl. Acad. Sci. USA (1988) 85:5879-5883; and Ward et al., Nature
(1989) 334:544-546).
[0060] Other suitable techniques for antibody production involve in
vitro exposure of lymphocytes to the antigenic polypeptides or
alternatively to selection of libraries of antibodies in phage or
similar vectors (Huse et al., Science (1989) 246:1275-1281). As
used herein, T-cell antigen receptors are included within the scope
of antibody modulators (Harlow and Lane, 1988, supra).
[0061] The polypeptides and antibodies of the present invention may
be used with or without modification. Frequently, antibodies will
be labeled by joining, either covalently or non-covalently, a
substance that provides for a detectable signal, or that is toxic
to cells that express the targeted protein (Menard S, et al., Int
J. Biol Markers (1989) 4:131-134). A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, fluorescent emitting lanthanide metals,
chemiluminescent moieties, bioluminescent moieties, magnetic
particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also,
recombinant immunoglobulins may be produced (U.S. Pat. No.
4,816,567). Antibodies to cytoplasmic polypeptides may be delivered
and reach their targets by conjugation with membrane-penetrating
toxin proteins (U.S. Pat. No. 6,086,900).
[0062] When used therapeutically in a patient, the antibodies of
the subject invention are typically administered parenterally, when
possible at the target site, or intravenously. The therapeutically
effective dose and dosage regimen is determined by clinical
studies. Typically, the amount of antibody administered is in the
range of about 0.1 mg/kg--to about 10 mg/kg of patient weight. For
parenteral administration, the antibodies are formulated in a unit
dosage injectable form (e.g., solution, suspension, emulsion) in
association with a pharmaceutically acceptable vehicle. Such
vehicles are inherently nontoxic and non-therapeutic. Examples are
water, saline, Ringer's solution, dextrose solution, and 5% human
serum albumin. Nonaqueous vehicles such as fixed oils, ethyl
oleate, or liposome carriers may also be used. The vehicle may
contain minor amounts of additives, such as buffers and
preservatives, which enhance isotonicity and chemical stability or
otherwise enhance therapeutic potential. The antibodies'
concentrations in such vehicles are typically in the range of about
1 mg/ml to about10 mg/ml. Immunotherapeutic methods are further
described in the literature (US Pat. No. 5,859,206; WO0073469).
[0063] Nucleic Acid Modulators
[0064] Other preferred hPRP4-modulating agents comprise nucleic
acid molecules, such as antisense oligomers or double stranded RNA
(dsRNA), which generally inhibit hPRP4 activity. Preferred nucleic
acid modulators interfere with the function of the hPRP4 nucleic
acid such as DNA replication, transcription, translocation of the
hPRP4 RNA to the site of protein translation, translation of
protein from the hPRP4 RNA, splicing of the hPRP4 RNA to yield one
or more mRNA species, or catalytic activity which may be engaged in
or facilitated by the hPRP4 RNA.
[0065] In one embodiment, the antisense oligomer is an
oligonucleotide that is sufficiently complementary to an hPRP4 mRNA
to bind to and prevent translation, preferably by binding to the 5'
untranslated region. hPRP4-specific antisense oligonucleotides,
preferably range from at least 6 to about 200 nucleotides. In some
embodiments the oligonucleotide is preferably at least 10, 15, or
20 nucleotides in length. In other embodiments, the oligonucleotide
is preferably less than 50, 40, or 30 nucleotides in length. The
oligonucleotide can be DNA or RNA or a chimeric mixture or
derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone. The oligonucleotide
may include other appending groups such as peptides, agents that
facilitate transport across the cell membrane,
hybridization-triggered cleavage agents, and intercalating
agents.
[0066] In another embodiment, the antisense oligomer is a
phosphothioate morpholino oligomer (PMO). PMOs are assembled from
four different morpholino subunits, each of which contain one of
four genetic bases (A, C, G, or T) linked to a six-membered
morpholine ring. Polymers of these subunits are joined by non-ionic
phosphodiamidate intersubunit linkages. Details of how to make and
use PMOs and other antisense oligomers are well known in the art
(e.g. see WO99/18193; Probst J C, Antisense Oligodeoxynucleotide
and Ribozyme Design, Methods. (2000) 22(3):271-281; Summerton J,
and Weller D. 1997 Antisense Nucleic Acid Drug Dev. :7:187-95; U.S.
Pat. Nos. 5,235,033; and 5,378,841).
[0067] Alternative preferred hPRP4 nucleic acid modulators are
double-stranded RNA species mediating RNA interference (RNAi). RNAi
is the process of sequence-specific, post-transcriptional gene
silencing in animals and plants, initiated by double-stranded RNA
(dsRNA) that is homologous in sequence to the silenced gene.
Methods relating to the use of RNAi to silence genes in C. elegans
, Drosophila, plants, and humans are known in the art (Fire A, et
al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363
(1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490
(2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119
(2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A.
et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature
404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000);
Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M.,
et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619;
Elbashir S M, et al., 2001 Nature 411:494-498).
[0068] Nucleic acid modulators are commonly used as research
reagents, diagnostics, and therapeutics. For example, antisense
oligonucleotides, which are able to inhibit gene expression with
exquisite specificity, are often used to elucidate the function of
particular genes (see, for example, U.S. Pat. No. 6,165,790).
Nucleic acid modulators are also used, for example, to distinguish
between functions of various members of a biological pathway. For
example, antisense oligomers have been employed as therapeutic
moieties in the treatment of disease states in animals and man and
have been demonstrated in numerous clinical trials to be safe and
effective (Milligan J F, et al, Current Concepts in Antisense Drug
Design, J Med Chem. (1993) 36:1923-1937; Tonkinson J L et al.,
Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents,
Cancer Invest. (1996) 14:54-65). Accordingly, in one aspect of the
invention, an hPRP4-specific nucleic acid modulator is used in an
assay to further elucidate the role of the hPRP4 in the p53
pathway, and/or its relationship to other members of the pathway.
In another aspect of the invention, an hPRP4-specific antisense
oligomer is used as a therapeutic agent for treatment of
p53-related disease states.
[0069] Assay Systems
[0070] The invention provides assay systems and screening methods
for identifying specific modulators of hPRP4 activity. As used
herein, an "assay system" encompasses all the components required
for performing and analyzing results of an assay that detects
and/or measures a particular event. In general, primary assays are
used to identify or confirm a modulator's specific biochemical or
molecular effect with respect to the hPRP4 nucleic acid or protein.
In general, secondary assays further assess the activity of a hPRP4
modulating agent identified by a primary assay and may confirm that
the modulating agent affects hPRP4 in a manner relevant to the p53
pathway. In some cases, hPRP4 modulators will be directly tested in
a secondary assay.
[0071] In a preferred embodiment, the screening method comprises
contacting a suitable assay system comprising an hPRP4 polypeptide
or nucleic acid with a candidate agent under conditions whereby,
but for the presence of the agent, the system provides a reference
activity (e.g. kinase activity), which is based on the particular
molecular event the screening method detects. A statistically
significant difference between the agent-biased activity and the
reference activity indicates that the candidate agent modulates
hPRP4 activity, and hence the p53 pathway. The hPRP4 polypeptide or
nucleic acid used in the assay may comprise any of the nucleic
acids or polypeptides described above.
[0072] Primary Assays
[0073] The type of modulator tested generally determines the type
of primary assay.
[0074] Primary Assays for Small Molecule Modulators
[0075] For small molecule modulators, screening assays are used to
identify candidate modulators. Screening assays may be cell-based
or may use a cell-free system that recreates or retains the
relevant biochemical reaction of the target protein (reviewed in
Sittampalam G S et al., Curr Opin Chem Biol (1997) 1:384-91 and
accompanying references). As used herein the term "cell-based"
refers to assays using live cells, dead cells, or a particular
cellular fraction, such as a membrane, endoplasmic reticulum, or
mitochondrial fraction. The term "cell free" encompasses assays
using substantially purified protein (either endogenous or
recombinantly produced), partially purified or crude cellular
extracts. Screening assays may detect a variety of molecular
events, including protein-DNA interactions, protein-protein
interactions (e.g., receptor-ligand binding), transcriptional
activity (e.g., using a reporter gene), enzymatic activity (e.g.,
via a property of the substrate), activity of second messengers,
immunogenicty and changes in cellular morphology or other cellular
characteristics. Appropriate screening assays may use a wide range
of detection methods including fluorescent, radioactive,
colorimetric, spectrophotometric, and amperometric methods, to
provide a read-out for the particular molecular event detected.
[0076] Cell-based screening assays usually require systems for
recombinant expression of hPRP4 and any auxiliary proteins demanded
by the particular assay. Appropriate methods for generating
recombinant proteins produce sufficient quantities of proteins that
retain their relevant biological activities and are of sufficient
purity to optimize activity and assure assay reproducibility. Yeast
two-hybrid and variant screens, and mass spectrometry provide
preferred methods for determining protein-protein interactions and
elucidation of protein complexes. In certain applications, when
hPRP4-interacting proteins are used in screens to identify small
molecule modulators, the binding specificity of the interacting
protein to the hPRP4 protein may be assayed by various known
methods such as substrate processing (e.g. ability of the candidate
hPRP4-specific binding agents to function as negative effectors in
hPRP4-expressing cells), binding equilibrium constants (usually at
least about 10.sup.7 M.sup.-1, preferably at least about 10.sup.8
M.sup.-1, more preferably at least about 10.sup.9 M.sup.-1), and
immunogenicity (e.g. ability to elicit hPRP4 specific antibody in a
heterologous host such as a mouse, rat, goat or rabbit). For
enzymes and receptors, binding may be assayed by, respectively,
substrate and ligand processing.
[0077] The screening assay may measure a candidate agent's ability
to specifically bind to or modulate activity of a hPRP4
polypeptide, a fusion protein thereof, or to cells or membranes
bearing the polypeptide or fusion protein. The hPRP4 polypeptide
can be full length or a fragment thereof that retains functional
hPRP4 activity. The hPRP4 polypeptide may be fused to another
polypeptide, such as a peptide tag for detection or anchoring, or
to another tag. The hPRP4 polypeptide is preferably human hPRP4, or
is an ortholog or derivative thereof as described above. In a
preferred embodiment, the screening assay detects candidate
agent-based modulation of hPRP4 interaction with a binding target,
such as an endogenous or exogenous protein or other substrate that
has hPRP4-specific binding activity, and can be used to assess
normal hPRP4 gene function.
[0078] Suitable assay formats that may be adapted to screen for
hPRP4 modulators are known in the art. Preferred screening assays
are high throughput or ultra high throughput and thus provide
automated, cost-effective means of screening compound libraries for
lead compounds (Fernandes P B, Curr Opin Chem Biol (1998)
2:597-603; Sundberg S A, Curr Opin Biotechnol 2000, 11:47-53). In
one preferred embodiment, screening assays uses fluorescence
technologies, including fluorescence polarization, time-resolved
fluorescence, and fluorescence resonance energy transfer. These
systems offer means to monitor protein-protein or DNA-protein
interactions in which the intensity of the signal emitted from
dye-labeled molecules depends upon their interactions with partner
molecules (e.g., Selvin P R, Nat Struct Biol (2000) 7:730-4;
Fernandes P B, supra; Hertzberg R P and Pope A J, Curr Opin Chem
Biol (2000) 4:445-451).
[0079] A variety of suitable assay systems may be used to identify
candidate hPRP4 and p53 pathway modulators (e.g. U.S. Pat. No.
6,165,992 (kinase assays); U.S. Pat. Nos. 5,550,019 and 6,133,437
(apoptosis assays); U.S. Pat. No. 6,020,135 (p53 modulation), among
others). Specific preferred assays are described in more detail
below.
[0080] Kinase assays. In some preferred embodiments the screening
assay detects the ability of the test agent to modulate the kinase
activity of an hPRP4 polypeptide. In further embodiments, a
cell-free kinase assay system is used to identify a candidate p53
modulating agent, and a secondary, cell-based assay, such as an
apoptosis or hypoxic induction assay (described below), may be used
to further characterize the candidate p53 modulating agent. Many
different assays for kinases have been reported in the literature
and are well known to those skilled in the art (e.g. U.S. Pat. No.
6,165,992; Zhu et al., Nature Genetics (2000) 26:283-289; and
WO0073469). Radioassays, which monitor the transfer of a gamma
phosphate are frequently used. For instance, a scintillation assay
for p56 (lck) kinase activity monitors the transfer of the gamma
phosphate from gamma--.sup.33P ATP to a biotinylated peptide
substrate; the substrate is captured on a streptavidin coated bead
that transmits the signal (Beveridge M et al., J Biomol Screen
(2000) 5:205-212). This assay uses the scintillation proximity
assay (SPA), in which only radio-ligand bound to receptors tethered
to the surface of an SPA bead are detected by the scintillant
immobilized within it, allowing binding to be measured without
separation of bound from free ligand.
[0081] Other assays for protein kinase activity may use antibodies
that specifically recognize phosphorylated substrates. For
instance, the kinase receptor activation (KIRA) assay measures
receptor tyrosine kinase activity by ligand stimulating the intact
receptor in cultured cells, then capturing solubilized receptor
with specific antibodies and quantifying phosphorylation via
phosphotyrosine ELISA (Sadick Md., Dev Biol Stand (1999)
97:121-133).
[0082] Another example of antibody based assays for protein kinase
activity is TRF (time-resolved fluorometry). This method utilizes
europium chelate-labeled anti-phosphotyrosine antibodies to detect
phosphate transfer to a polymeric substrate coated onto microtiter
plate wells. The amount of phosphorylation is then detected using
time-resolved, dissociation-enhanced fluorescence (Braunwalder A F,
et al., Anal Biochem Jul. 1, 1996 ;238(2):159-64).
[0083] Apoptosis assays. Assays for apoptosis may be performed by
terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP
nick end labeling (TUNEL) assay. The TUNEL assay is used to measure
nuclear DNA fragmentation characteristic of apoptosis (Lazebnik et
al., 1994, Nature 371, 346), by following the incorporation of
fluorescein-dUTP (Yonehara et al., 1989, J. Exp. Med. 169, 1747).
Apoptosis may further be assayed by acridine orange staining of
tissue culture cells (Lucas, R., et al., 1998, Blood 15:4730-41).
An apoptosis assay system may comprise a cell that expresses an
hPRP4, and that optionally has defective p53 function (e.g. p53 is
over-expressed or under-expressed relative to wild-type cells). A
test agent can be added to the apoptosis assay system and changes
in induction of apoptosis relative to controls where no test agent
is added, identify candidate p53 modulating agents. In some
embodiments of the invention, an apoptosis assay may be used as a
secondary assay to test a candidate p53 modulating agents that is
initially identified using a cell-free assay system. An apoptosis
assay may also be used to test whether hPRP4 function plays a
direct role in apoptosis. For example, an apoptosis assay may be
performed on cells that over- or under-express hPRP4 relative to
wild type cells. Differences in apoptotic response compared to wild
type cells suggests that the hPRP4 plays a direct role in the
apoptotic response. Apoptosis assays are described further in US
Pat. No. 6,133,437.
[0084] Cell proliferation and cell cycle assays. Cell proliferation
may be assayed via bromodeoxyuridine (BRDU) incorporation. This
assay identifies a cell population undergoing DNA synthesis by
incorporation of BRDU into newly-synthesized DNA. Newly-synthesized
DNA may then be detected using an anti-BRDU antibody (Hoshino et
al., 1986, Int. J. Cancer 38, 369; Campana et al., 1988, J.
Immunol. Meth. 107, 79), or by other means.
[0085] Cell Proliferation may also be examined using
[.sup.3H]-thymidine incorporation (Chen, J., 1996, Oncogene
13:1395-403; Jeoung, J., 1995, J. Biol. Chem. 270:18367-73). This
assay allows for quantitative characterization of S-phase DNA
syntheses. In this assay, cells synthesizing DNA will incorporate
[.sup.3H]-thymidine into newly synthesized DNA. Incorporation can
then be measured by standard techniques such as by counting of
radioisotope in a scintillation counter (e.g., Beckman LS 3800
Liquid Scintillation Counter).
[0086] Cell proliferation may also be assayed by colony formation
in soft agar (Sambrook et al., Molecular Cloning, Cold Spring
Harbor (1989)). For example, cells transformed with hPRP4 are
seeded in soft agar plates, and colonies are measured and counted
after two weeks incubation.
[0087] Involvement of a gene in the cell cycle may be assayed by
flow cytometry (Gray JW et al. (1986) Int J Radiat Biol Relat Stud
Phys Chem Med 49:237-55). Cells transfected with an hPRP4 may be
stained with propidium iodide and evaluated in a flow cytometer
(available from Becton Dickinson).
[0088] Accordingly, a cell proliferation or cell cycle assay system
may comprise a cell that expresses an hPRP4, and that optionally
has defective p53 function (e.g. p53 is over-expressed or
under-expressed relative to wild-type cells). A test agent can be
added to the assay system and changes in cell proliferation or cell
cycle relative to controls where no test agent is added, identify
candidate p53 modulating agents. In some embodiments of the
invention, the cell proliferation or cell cycle assay may be used
as a secondary assay to test a candidate p53 modulating agents that
is initially identified using another assay system such as a
cell-free kinase assay system. A cell proliferation assay may also
be used to test whether hPRP4 function plays a direct role in cell
proliferation or cell cycle. For example, a cell proliferation or
cell cycle assay may be performed on cells that over- or
under-express hPRP4 relative to wild type cells. Differences in
proliferation or cell cycle compared to wild type cells suggests
that the hPRP4 plays a direct role in cell proliferation or cell
cycle.
[0089] Angiogenesis. Angiogenesis may be assayed using various
human endothelial cell systems, such as umbilical vein, coronary
artery, or dermal cells. Suitable assays include Alamar Blue based
assays (available from Biosource International) to measure
proliferation; migration assays using fluorescent molecules, such
as the use of Becton Dickinson Falcon HTS FluoroBlock cell culture
inserts to measure migration of cells through membranes in presence
or absence of angiogenesis enhancer or suppressors; and tubule
formation assays based on the formation of tubular structures by
endothelial cells on Matrigel.RTM. (Becton Dickinson). Accordingly,
an angiogenesis assay system may comprise a cell that expresses an
hPRP4, and that optionally has defective p53 function (e.g. p53 is
over-expressed or under-expressed relative to wild-type cells). A
test agent can be added to the angiogenesis assay system and
changes in angiogenesis relative to controls where no test agent is
added, identify candidate p53 modulating agents. In some
embodiments of the invention, the angiogenesis assay may be used as
a secondary assay to test a candidate p53 modulating agents that is
initially identified using another assay system. An angiogenesis
assay may also be used to test whether hPRP4 function plays a
direct role in cell proliferation. For example, an angiogenesis
assay may be performed on cells that over- or under-express hPRP4
relative to wild type cells. Differences in angiogenesis compared
to wild type cells suggests that the hPRP4 plays a direct role in
angiogenesis.
[0090] Hypoxic induction. The alpha subunit of the transcription
factor, hypoxia inducible factor-1 (HF-1), is upregulated in tumor
cells following exposure to hypoxia in vitro. Under hypoxic
conditions, HIF-1 stimulates the expression of genes known to be
important in tumour cell survival, such as those encoding glyolytic
enzymes and VEGF. Induction of such genes by hypoxic conditions may
be assayed by growing cells transfected with hPRP4 in hypoxic
conditions (such as with 0.1% O2, 5% CO2, and balance N2, generated
in a Napco 7001 incubator (Precision Scientific)) and normoxic
conditions, followed by assessment of gene activity or expression
by Taqman.RTM.. For example, a hypoxic induction assay system may
comprise a cell that expresses an hPRP4, and that optionally has a
mutated p53 (e.g. p53 is over-expressed or under-expressed relative
to wild-type cells). A test agent can be added to the hypoxic
induction assay system and changes in hypoxic response relative to
controls where no test agent is added, identify candidate p53
modulating agents. In some embodiments of the invention, the
hypoxic induction assay may be used as a secondary assay to test a
candidate p53 modulating agents that is initially identified using
another assay system. A hypoxic induction assay may also be used to
test whether hPRP4 function plays a direct role in the hypoxic
response. For example, a hypoxic induction assay may be performed
on cells that over- or under-express hPRP4 relative to wild type
cells. Differences in hypoxic response compared to wild type cells
suggests that the hPRP4 plays a direct role in hypoxic
induction.
[0091] Cell adhesion. Cell adhesion assays measure adhesion of
cells to purified adhesion proteins, or adhesion of cells to each
other, in presence or absence of candidate modulating agents.
Cell-protein adhesion assays measure the ability of agents to
modulate the adhesion of cells to purified proteins. For example,
recombinant proteins are produced, diluted to 2.5 g/mL in PBS, and
used to coat the wells of a microtiter plate. The wells used for
negative control are not coated. Coated wells are then washed,
blocked with 1% BSA, and washed again. Compounds are diluted to
2.times.final test concentration and added to the blocked, coated
wells. Cells are then added to the wells, and the unbound cells are
washed off. Retained cells are labeled directly on the plate by
adding a membrane-permeable fluorescent dye, such as calcein-AM,
and the signal is quantified in a fluorescent microplate
reader.
[0092] Cell-cell adhesion assays measure the ability of agents to
modulate binding of cell adhesion proteins with their native
ligands. These assays use cells that naturally or recombinantly
express the adhesion protein of choice. In an exemplary assay,
cells expressing the cell adhesion protein are plated in wells of a
multiwell plate. Cells expressing the ligand are labeled with a
membrane-permeable fluorescent dye, such as BCECF , and allowed to
adhere to the monolayers in the presence of candidate agents.
Unbound cells are washed off, and bound cells are detected using a
fluorescence plate reader.
[0093] High-throughput cell adhesion assays have also been
described. In one such assay, small molecule ligands and peptides
are bound to the surface of microscope slides using a microarray
spotter, intact cells are then contacted with the slides, and
unbound cells are washed off. In this assay, not only the binding
specificity of the peptides and modulators against cell lines are
determined, but also the functional cell signaling of attached
cells using immunofluorescence techniques in situ on the microchip
is measured (Falsey J R et al., Bioconjug Chem. 2001
May-Jun;12(3):346-53).
[0094] Cell Migration. An invasion/migration assay (also called a
migration assay) tests the ability of cells to overcome a physical
barrier and to migrate towards pro-angiogenic signals. Migration
assays are known in the art (e.g., Paik J H et al., 2001, J Biol
Chem 276:11830-11837). In a typical experimental set-up, cultured
endothelial cells are seeded onto a matrix-coated porous lamina,
with pore sizes generally smaller than typical cell size. The
matrix generally simulates the environment of the extracellular
matrix, as described above. The lamina is typically a membrane,
such as the transwell polycarbonate membrane (Coming Costar
Corporation, Cambridge, Ma.), and is generally part of an upper
chamber that is in fluid contact with a lower chamber containing
pro-angiogenic stimuli. Migration is generally assayed after an
overnight incubation with stimuli, but longer or shorter time
frames may also be used. Migration is assessed as the number of
cells that crossed the lamina, and may be detected by staining
cells with hemotoxylin solution (VWR Scientific, South San
Francisco, Calif.), or by any other method for determining cell
number. In another exemplary set up, cells are fluorescently
labeled and migration is detected using fluorescent readings, for
instance using the Falcon HTS FluoroBlok (Becton Dickinson). While
some migration is observed in the absence of stimulus, migration is
greatly increased in response to pro-angiogenic factors. As
described above, a preferred assay system for migration/invasion
assays comprises testing an hPRP4's response to a variety of
pro-angiogenic factors, including tumor angiogenic and inflammatory
angiogenic agents, and culturing the cells in serum free
medium.
[0095] Sprouting assay. A sprouting assay is a three-dimensional in
vitro angiogenesis assay that uses a cell-number defined spheroid
aggregation of endothelial cells ("spheroid"), embedded in a
collagen gel-based matrix. The spheroid can serve as a starting
point for the sprouting of capillary-like structures by invasion
into the extracellular matrix (termed "cell sprouting") and the
subsequent formation of complex anastomosing networks (Korff and
Augustin, 1999, J Cell Sci 112:3249-58). In an exemplary
experimental set-up, spheroids are prepared by pipetting 400 human
umbilical vein endothelial cells into individual wells of a
nonadhesive 96-well plates to allow overnight spheroidal
aggregation (Korff and Augustin: J Cell Biol 143: 1341-52, 1998).
Spheroids are harvested and seeded in 900 .mu.l of
methocel-collagen solution and pipetted into individual wells of a
24 well plate to allow collagen gel polymerization. Test agents are
added after 30 min by pipetting 100 .mu.l of 10-fold concentrated
working dilution of the test substances on top of the gel. Plates
are incubated at 37.degree. C. for 24 h. Dishes are fixed at the
end of the experimental incubation period by addition of
paraformaldehyde. Sprouting intensity of endothelial cells can be
quantitated by an automated image analysis system to determine the
cumulative sprout length per spheroid.
[0096] Primary Assays for Antibody Modulators
[0097] For antibody modulators, appropriate primary assays test is
a binding assay that tests the antibody's affinity to and
specificity for the hPRP4 protein. Methods for testing antibody
affinity and specificity are well known in the art (Harlow and
Lane, 1988, 1999, supra). The enzyme-linked immunosorbant assay
(ELISA) is a preferred method for detecting hPRP4-specific
antibodies; others include FACS assays, radioimmunoassays, and
fluorescent assays.
[0098] Primary Assays for Nucleic Acid Modulators
[0099] For nucleic acid modulators, primary assays may test the
ability of the nucleic acid modulator to inhibit or enhance hPRP4
gene expression, preferably mRNA expression. In general, expression
analysis comprises comparing hPRP4 expression in like populations
of cells (e.g., two pools of cells that endogenously or
recombinantly express hPRP4) in the presence and absence of the
nucleic acid modulator. Methods for analyzing mRNA and protein
expression are well known in the art. For instance, Northern
blotting, slot blotting, ribonuclease protection, quantitative
RT-PCR (e.g., using the TaqMan.RTM., PE Applied Biosystems), or
microarray analysis may be used to confirm that hPRP4 mRNA
expression is reduced in cells treated with the nucleic acid
modulator (e.g., Current Protocols in Molecular Biology (1994)
Ausubel FM et al., eds., John Wiley & Sons, Inc., chapter 4;
Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O
P, Ann Med 2001, 33:142-147; Blohm D H and Guiseppi-Elie, A Curr
Opin Biotechnol 2001, 12:41-47). Protein expression may also be
monitored. Proteins are most commonly detected with specific
antibodies or antisera directed against either the hPRP4 protein or
specific peptides. A variety of means including Western blotting,
ELISA, or in situ detection, are available (Harlow E and Lane D,
1988 and 1999, supra).
[0100] Secondary Assays
[0101] Secondary assays may be used to further assess the activity
of hPRP4-modulating agent identified by any of the above methods to
confirm that the modulating agent affects hPRP4 in a manner
relevant to the p53 pathway. As used herein, hPRP4-modulating
agents encompass candidate clinical compounds or other agents
derived from previously identified modulating agent. Secondary
assays can also be used to test the activity of a modulating agent
on a particular genetic or biochemical pathway or to test the
specificity of the modulating agent's interaction with hPRP4.
[0102] Secondary assays generally compare like populations of cells
or animals (e.g., two pools of cells or animals that endogenously
or recombinantly express hPRP4) in the presence and absence of the
candidate modulator. In general, such assays test whether treatment
of cells or animals with a candidate hPRP4-modulating agent results
in changes in the p53 pathway in comparison to untreated (or mock-
or placebo-treated) cells or animals. Certain assays use
"sensitized genetic backgrounds", which, as used herein, describe
cells or animals engineered for altered expression of genes in the
p53 or interacting pathways.
[0103] Cell-based Assays
[0104] Cell based assays may use a variety of mammalian cell lines
known to have defective p53 function (e.g. SAOS-2 osteoblasts,
H1299 lung cancer cells, C33A and HT3 cervical cancer cells, HT-29
and DLD-1 colon cancer cells, among others, available from American
Type Culture Collection (ATCC), Manassas, Va.). Cell based assays
may detect endogenous p53 pathway activity or may rely on
recombinant expression of p53 pathway components. Any of the
aforementioned assays may be used in this cell-based format.
Candidate modulators are typically added to the cell media but may
also be injected into cells or delivered by any other efficacious
means.
[0105] Animal Assays
[0106] A variety of non-human animal models of normal or defective
p53 pathway may be used to test candidate hPRP4 modulators. Models
for defective p53 pathway typically use genetically modified
animals that have been engineered to mis-express (e.g.,
over-express or lack expression in) genes involved in the p53
pathway. Assays generally require systemic delivery of the
candidate modulators, such as by oral administration, injection,
etc.
[0107] In a preferred embodiment, p53 pathway activity is assessed
by monitoring neovascularization and angiogenesis. Animal models
with defective and normal p53 are used to test the candidate
modulator's affect on hPRP4 in Matrigel.RTM. assays. Matrigel.RTM.
is an extract of basement membrane proteins, and is composed
primarily of laminin, collagen IV, and heparin sulfate
proteoglycan. It is provided as a sterile liquid at 4.degree. C.,
but rapidly forms a solid gel at 37.degree. C. Liquid Matrigel.RTM.
is mixed with various angiogenic agents, such as bFGF and VEGF, or
with human tumor cells which over-express the hPRP4. The mixture is
then injected subcutaneously(SC) into female athymic nude mice
(Taconic, Germantown, N.Y.) to support an intense vascular
response. Mice with Matrigel.RTM. pellets may be dosed via oral
(PO), intraperitoneal (IP), or intravenous (IV) routes with the
candidate modulator. Mice are euthanized 5-12 days post-injection,
and the Matrigel.RTM. pellet is harvested for hemoglobin analysis
(Sigma plasma hemoglobin kit). Hemoglobin content of the gel is
found to correlate the degree of neovascularization in the gel.
[0108] In another preferred embodiment, the effect of the candidate
modulator on hPRP4 is assessed via tumorigenicity assays. In one
example, xenograft human tumors are implanted SC into female
athymic mice, 6-7 week old, as single cell suspensions either from
a pre-existing tumor or from in vitro culture. The tumors which
express the hPRP4 endogenously are injected in the flank,
1.times.10.sup.5 to 1.times.10.sup.7 cells per mouse in a volume of
100 .mu.L using a 27gauge needle. Mice are then ear tagged and
tumors are measured twice weekly. Candidate modulator treatment is
initiated on the day the mean tumor weight reaches 100 mg.
Candidate modulator is delivered IV, SC, IP, or PO by bolus
administration. Depending upon the pharmacokinetics of each unique
candidate modulator, dosing can be performed multiple times per
day. The tumor weight is assessed by measuring perpendicular
diameters with a caliper and calculated by multiplying the
measurements of diameters in two dimensions. At the end of the
experiment, the excised tumors maybe utilized for biomarker
identification or further analyses. For immunohistochemistry
staining, xenograft tumors are fixed in 4% paraformaldehyde, 0.1M
phosphate, pH 7.2, for 6 hours at 4.degree. C., immersed in 30%
sucrose in PBS, and rapidly frozen in isopentane cooled with liquid
nitrogen.
[0109] Diagnostic and Therapeutic Uses
[0110] Specific hPRP4-modulating agents are useful in a variety of
diagnostic and therapeutic applications where disease or disease
prognosis is related to defects in the p53 pathway, such as
angiogenic, apoptotic, or cell proliferation disorders.
Accordingly, the invention also provides methods for modulating the
p53 pathway in a cell, preferably a cell pre-determined to have
defective or impaired p53 function (e.g. due to overexpression,
underexpression, or misexpression of p53, or due to gene
mutations), comprising the step of administering an agent to the
cell that specifically modulates hPRP4 activity. Preferably, the
modulating agent produces a detectable phenotypic change in the
cell indicating that the p53 function is restored. The phrase
"function is restored", and equivalents, as used herein, means that
the desired phenotype is achieved, or is brought closer to normal
compared to untreated cells. For example, with restored p53
function, cell proliferation and/or progression through cell cycle
may normalize, or be brought closer to normal relative to untreated
cells. The invention also provides methods for treating disorders
or disease associated with impaired p53 function by administering a
therapeutically effective amount of an hPRP4 -modulating agent that
modulates the p53 pathway. The invention further provides methods
for modulating hPRP4 function in a cell, preferably a cell
pre-determined to have defective or impaired hPRP4 function, by
administering an hPRP4 -modulating agent. Additionally, the
invention provides a method for treating disorders or disease
associated with impaired hPRP4 function by administering a
therapeutically effective amount of an hPRP4-modulating agent.
[0111] The discovery that hPRP4 is implicated in p53 pathway
provides for a variety of methods that can be employed for the
diagnostic and prognostic evaluation of diseases and disorders
involving defects in the p53 pathway and for the identification of
subjects having a predisposition to such diseases and
disorders.
[0112] Various expression analysis methods can be used to diagnose
whether hPRP4 expression occurs in a particular sample, including
Northern blotting, slot blotting, ribonuclease protection,
quantitative RT-PCR, and microarray analysis. (e.g., Current
Protocols in Molecular Biology (1994) Ausubel F M et al., eds.,
John Wiley & Sons, Inc., chapter 4; Freeman W M et al.,
Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001,
33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001,
12:41-47). Tissues having a disease or disorder implicating
defective p53 signaling that express an hPRP4, are identified as
amenable to treatment with an hPRP4 modulating agent. In a
preferred application, the p53 defective tissue overexpresses an
hPRP4 relative to normal tissue. For example, a Northern blot
analysis of mRNA from tumor and normal cell lines, or from tumor
and matching normal tissue samples from the same patient, using
full or partial hPRP4 cDNA sequences as probes, can determine
whether particular tumors express or overexpress hPRP4.
Alternatively, the TaqMan.RTM. is used for quantitative RT-PCR
analysis of hPRP4 expression in cell lines, normal tissues and
tumor samples (PE Applied Biosystems).
[0113] Various other diagnostic methods may be performed, for
example, utilizing reagents such as the hPRP4 oligonucleotides, and
antibodies directed against an hPRP4, as described above for: (1)
the detection of the presence of hPRP4 gene mutations, or the
detection of either over- or under-expression of hPRP4 mRNA
relative to the non-disorder state; (2) the detection of either an
over- or an under-abundance of hPRP4 gene product relative to the
non-disorder state; and (3) the detection of perturbations or
abnormalities in the signal transduction pathway mediated by
hPRP4.
[0114] Thus, in a specific embodiment, the invention is drawn to a
method for diagnosing a disease or disorder in a patient that is
associated with alterations in hPRP4 expression, the method
comprising: a) obtaining a biological sample from the patient; b)
contacting the sample with a probe for hPRP4 expression; c)
comparing results from step (b) with a control; and d) determining
whether step (c) indicates a likelihood of the disease or disorder.
Preferably, the disease is cancer, most preferably ovarian
cancer.
[0115] The probe may be either DNA or protein, including an
antibody.
EXAMPLES
[0116] The following experimental section and examples are offered
by way of illustration and not by way of limitation.
[0117] I. C. elegans p53 screen
[0118] A systematic RNAi of various genes was carried out in worms
homozygous for p53 deletion. p53 (-/-) worms have a normal
phenotype, but are defective in germline apoptotic response to
ionizing radiation as p53 is involved in the DNA damage response.
After silencing of each gene by RNAi, worms were subject to
gamma-irradiation, and phenotypes were scored. The worm F22D6.5
suppressed the p53 block in DNA-damage induced apoptosis in
germline.
[0119] BLAST analysis (Altschul et al., supra) was employed to
identify Targets from C. elegans modifiers. For example,
representative sequence from hPRP4, GI# 14571506 (SEQ ID NO:7),
shares 40% amino acid identity with the C. elegans F22D6.5.
[0120] Various domains, signals, and functional subunits in
proteins were analyzed using the PSORT (Nakai K., and Horton P.,
Trends Biochem Sci, 1999, 24:34-6; Kenta Nakai, Protein sorting
signals and prediction of subcellular localization, Adv. Protein
Chem. 54, 277-344 (2000)), PFAM (Bateman A., et al., Nucleic Acids
Res, 1999, 27:260-2), SMART (Ponting CP, et al., SMART:
identification and annotation of domains from signaling and
extracellular protein sequences. Nucleic Acids Res. 1999 Jan
1;27(1):229-32), TM-HMM (Erik L. L. Sonnhammer, Gunnar von Heijne,
and Anders Krogh: A hidden Markov model for predicting
transmembrane helices in protein sequences. In Proc. of Sixth Int.
Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J.
Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C.
Sensen Menlo Park, CA: AAAI Press, 1998), and clust (Remm M, and
Sonnhammer E. Classification of transmembrane protein families in
the Caenorhabditis elegans genome and identification of human
orthologs. Genome Res. 2000 Nov; 10(11): 1679-89) programs. For
example, the kinase domain of hPRP4 from GI# 14571506 (SEQ ID NO:7)
is located at approximately amino acid residues 687-1003 (PFAM
00069).
[0121] II. High-Throughput In Vitro Fluorescence Polarization
Assay
[0122] Fluorescently-labeled hPRP4 peptide/substrate are added to
each well of a 96-well microtiter plate, along with a test agent in
a test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH
7.6). Changes in fluorescence polarization, determined by using a
Fluorolite FPM-2 Fluorescence Polarization Microtiter System
(Dynatech Laboratories, Inc), relative to control values indicates
the test compound is a candidate modifier of hPRP4 activity.
[0123] III. High-Throughput In Vitro Binding Assay
[0124] .sup.33P-labeled hPRP4 peptide is added in an assay buffer
(100 mM KCI, 20 mM HEPES pH 7.6, 1 mM MgCl.sub.2, 1% glycerol, 0.5%
NP-40, 50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of
protease inhibitors) along with a test agent to the wells of a
Neutralite-avidin coated assay plate and incubated at 25.degree. C.
for 1 hour. Biotinylated substrate is then added to each well and
incubated for 1 hour. Reactions are stopped by washing with PBS,
and counted in a scintillation counter. Test agents that cause a
difference in activity relative to control without test agent are
identified as candidate p53 modulating agents.
[0125] IV. Immunoprecipitations and Immunoblotting
[0126] For coprecipitation of transfected proteins,
3.times.10.sup.6 appropriate recombinant cells containing the hPRP4
proteins are plated on 10-cm dishes and transfected on the
following day with expression constructs. The total amount of DNA
is kept constant in each transfection by adding empty vector. After
24 h, cells are collected, washed once with phosphate-buffered
saline and lysed for 20 min on ice in 1 ml of lysis buffer
containing 50 mM Hepes, pH 7.9, 250 mM NaCl, 20 mM
-glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenyl
phosphate, 2 mM dithiothreitol, protease inhibitors (complete,
Roche Molecular Biochemicals), and 1% Nonidet P-40. Cellular debris
is removed by centrifugation twice at 15,000.times.g for 15 min.
The cell lysate is incubated with 25 .mu.l of M2 beads (Sigma) for
2 h at 4.degree. C. with gentle rocking.
[0127] After extensive washing with lysis buffer, proteins bound to
the beads are solubilized by boiling in SDS sample buffer,
fractionated by SDS-polyacrylamide gel electrophoresis, transferred
to polyvinylidene difluoride membrane and blotted with the
indicated antibodies. The reactive bands are visualized with
horseradish peroxidase coupled to the appropriate secondary
antibodies and the enhanced chemiluminescence (ECL) Western
blotting detection system (Amersham Pharmacia Biotech).
[0128] V. Kinase Assay
[0129] A purified or partially purified hPRP4 is diluted in a
suitable reaction buffer, e.g., 50 mM Hepes, pH 7.5, containing
magnesium chloride or manganese chloride (1-20 mM) and a peptide or
polypeptide substrate, such as myelin basic protein or casein (1-10
.mu.g/ml). The final concentration of the kinase is 1-20 nM. The
enzyme reaction is conducted in microtiter plates to facilitate
optimization of reaction conditions by increasing assay throughput.
A 96-well microtiter plate is employed using a final volume 30-100
.mu.l. The reaction is initiated by the addition of
.sup.33P-gamma-ATP (0.5 .mu.Ci/ml) and incubated for 0.5 to 3 hours
at room temperature. Negative controls are provided by the addition
of EDTA, which chelates the divalent cation (Mg2.sup.+ or
Mn.sup.2+) required for enzymatic activity. Following the
incubation, the enzyme reaction is quenched using EDTA. Samples of
the reaction are transferred to a 96-well glass fiber filter plate
(MultiScreen, Millipore). The filters are subsequently washed with
phosphate-buffered saline, dilute phosphoric acid (0.5%) or other
suitable medium to remove excess radiolabeled ATP. Scintillation
cocktail is added to the filter plate and the incorporated
radioactivity is quantitated by scintillation counting
(Wallac/Perkin Elmer). Activity is defined by the amount of
radioactivity detected following subtraction of the negative
control reaction value (EDTA quench).
[0130] VI. Expression Analysis
[0131] All cell lines used in the following experiments are NCI
(National Cancer Institute) lines, and are available from ATCC
(American Type Culture Collection, Manassas, Va. 20110-2209).
Normal and tumor tissues were obtained from Impath, UC Davis,
Clontech, Stratagene, and Ambion.
[0132] TaqMan analysis was used to assess expression levels of the
disclosed genes in various samples.
[0133] RNA was extracted from each tissue sample using Qiagen
(Valencia, Calif.) RNeasy kits, following manufacturer's protocols,
to a final concentration of 50 ng/.mu.l. Single stranded cDNA was
then synthesized by reverse transcribing the RNA samples using
random hexamers and 500 ng of total RNA per reaction, following
protocol 4304965 of Applied Biosystems (Foster City, Calif.).
[0134] Primers for expression analysis using TaqMan assay (Applied
Biosystems, Foster City, Calif.) were prepared according to the
TaqMan protocols, and the following criteria: a) primer pairs were
designed to span introns to eliminate genomic contamination, and b)
each primer pair produced only one product.
[0135] Taqman reactions were carried out following manufacturer's
protocols, in 25 .mu.l total volume for 96-well plates and 10 .mu.l
total volume for 384-well plates, using 300 nM primer and 250 nM
probe, and approximately 25 ng of cDNA. The standard curve for
result analysis was prepared using a universal pool of human cDNA
samples, which is a mixture of cDNAs from a wide variety of tissues
so that the chance that a target will be present in appreciable
amounts is good. The raw data were normalized using 18S rRNA
(universally expressed in all tissues and cells).
[0136] For each expression analysis, tumor tissue samples were
compared with matched normal tissues from the same patient. A gene
was considered overexpressed in a tumor when the level of
expression of the gene was 2 fold or higher in the tumor compared
with its matched normal sample. In cases where normal tissue was
not available, a universal pool of cDNA samples was used instead.
In these cases, a gene was considered overexpressed in a tumor
sample when the difference of expression levels between a tumor
sample and the average of all normal samples from the same tissue
type was greater than 2 times the standard deviation of all normal
samples (i.e., Tumor--average(all normal samples)>2.times.STDEV-
(all normal samples) ).
[0137] hPRP4 (GI#14571505, SEQ ID NO:1) was overexpressed in 2 of 7
ovarian tumors. A modulator identified by an assay described herein
can be further validated for therapeutic effect by administration
to a tumor in which the gene is overexpressed. A decrease in tumor
growth confirms therapeutic utility of the modulator. Prior to
treating a patient with the modulator, the likelihood that the
patient will respond to treatment can be diagnosed by obtaining a
tumor sample from the patient, and assaying for expression of the
gene targeted by the modulator. The expression data for the gene(s)
can also be used as a diagnostic marker for disease progression.
The assay can be performed by expression analysis as described
above, by antibody directed to the gene target, or by any other
available detection method.
Sequence CWU 1
1
7 1 3024 DNA Homo sapiens 1 atggccgccg cggagaccca gtcgctacgg
gagcagccag agatggaaga tgctaattct 60 gaaaagagta taaatgaaga
aaatggagaa gtatcagaag accagtctca aaataagcac 120 agtcgtcaca
aaaaaaagaa gcataaacac agaagtaaac ataagaaaca taaacattcc 180
tcagaagaag acaaggataa aaaacataaa cataagcata aacataagaa acacaaaaga
240 aaagaggtta ttgatgcttc tgataaagag ggtatgtctc cagcaaaaag
aactaaactt 300 gatgatttag ctttgctaga agacttggaa aaacagagag
ccttgattaa ggccgaactt 360 gataatgagt taatggaagg aaaggtccag
tctggtatgg ggctcatttt gcaaggttat 420 gagtctggct ctgaagaaga
gggggaaatt catgaaaagg caagaaatgg aaataggtct 480 agtactagat
cttcaagtac aaaggggaaa cttgaacttg tggacaataa aattactaca 540
aagaaacgaa gtaaaagcag atccaaagaa cggactagac ataggtctga taaaaagaaa
600 agtaaggggg gtattgaaat cgttaaagag aaaacaacta ggagcaagtc
aaaggagagg 660 aaaaaatcta aaagcccatc caaaagaagt aagtctcaag
atcaagcaag gaaatcaaaa 720 tcccctaccc ttagaaggcg atctcaagag
aaaattggta aggccagatc tcctactgat 780 gataaggtta aaattgaaga
taaaagtaaa tcaaaagata ggaaaaaatc cccaattata 840 aatgaaagta
gaagtcgcga tcgaggtaaa aaatccagat ccccagttga tttaagaggt 900
aaatccaaag acagaaggtc acggtccaaa gagagaaaat caaaacggtc tgaaactgat
960 aaagaaaaga agccaattaa atctccctct aaagatgctt catctgggaa
agaaaatagg 1020 tcacccagca gaagacctgg tcgtagtcct aaaagaagaa
gtttgtctcc aaaaccacgt 1080 gataaatcaa gaagaagcag gtctccactt
ttgaatgata gaagatctaa gcagagcaaa 1140 tccccctcgc ggacactgtc
tcctgggaga agagccaaga gccgatcctt agaaagaaaa 1200 cgacgagaac
cagagaggag acgactttct tctccaagaa cacgacctcg agatgatatc 1260
ctcagtagac gtgaaagatc aaaagatgcc agccccatca atagatggtc tccaacccga
1320 agaagaagta gatctcccat tagaaggagg tctcgttccc cactcagacg
tagcaggtct 1380 ccaagaagaa gaagcagatc tcctcggaga agggacagag
gtcggaggag cagatcacgc 1440 ttgcgaaggc ggtctcgatc acgcggtggt
cgtagacgaa ggagcagaag caaagtaaag 1500 gaagataaat ttaaaggaag
tctttctgaa ggaatgaaag ttgagcagga atcttcgtct 1560 gatgataacc
ttgaagactt tgatgtagag gaagaagatg aagaagccct aatagaacag 1620
agaagaatcc aaaggcaggc aattgttcag aaatataaat accttgctga agatagcaac
1680 atgtctgtgc catctgaacc aagcagcccc cagagcagta cgagaacacg
atcaccatct 1740 ccagatgaca ttctggagcg agtagctgct gatgttaaag
agtatgaacg ggaaaatgtt 1800 gatacatttg aggcctcagt gaaagccaag
cataatctaa tgacagttga acagaataat 1860 ggttcatctc agaagaagtt
gttggcacct gatatgttta cagaatctga tgatatgttt 1920 gctgcgtatt
ttgatagtgc tcgtcttcgg gccgctggca ttggaaaaga tttcaaagag 1980
aatcccaacc tcagagataa ctggaccgat gcagaaggct attatcgtgt gaacataggt
2040 gaagtcctag ataaacgtta caatgtgtat ggctacactg ggcaaggtgt
attcagtaat 2100 gttgtacgag ccagagataa tgcaagagcc aaccaagaag
tggctgtaaa gatcatcaga 2160 aacaatgagc tcatgcaaaa gactggttta
aaagaattag agttcttgaa aaaacttaat 2220 gatgctgatc ctgatgacaa
atttcattgt ctgagactct tcaggcactt ctatcacaag 2280 cagcatcttt
gtctggtatt cgagcctctc agcatgaact tacgagaggt gttaaaaaaa 2340
tatggtaaag atgttggtct tcatattaaa gctgtaagat cctatagtca gcagttgttc
2400 ctggcattga aactccttaa aagatgcaat atcctacatg cagatatcaa
gccagacaat 2460 atcctggtta atgaatccaa aactatttta aagctttgcg
attttgggtc ggcttcacat 2520 gttgcggata atgacataac accttatctt
gtcagtagat tttatcgtgc tcctgaaatc 2580 attataggta aaagctatga
ctatggtata gatatgtggt ctgtaggttg caccttatac 2640 gaactctata
ctggaaaaat tttattccct ggcaaaacca ataaccatat gctgaagctt 2700
gcaatggatc tcaaaggaaa gatgccaaat aagatgattc gaaaaggtgt gttcaaagat
2760 cagcattttg atcaaaatct caacttcatg tacatagaag ttgataaagt
aacagagagg 2820 gagaaagtta ctgttatgag caccattaat ccaactaagg
acctgttggc tgacttgatt 2880 gggtgccaga gacttcctga agaccaacgt
aagaaagtac accagctaaa ggacttgttg 2940 gaccagattc tgatgttgga
cccagctaaa cgaattagca tcaaccaggc cctacagcac 3000 gccttcatcc
aggaaaaaat ttaa 3024 2 3202 DNA Homo sapiens 2 cttcccctac
cctccaccgt ccgggagccg ccgccaccgc cgccgaggag tcaggaagtt 60
caagatggcc gccgcggaga cccagtcgct acgggagcag ccagagatgg aagatgctaa
120 ttctgaaaag agtataaatg aagaaaatgg agaagtatca gaagaccagt
ctcaaaataa 180 gcacagtcgt cacaaaaaaa agaagcataa acacagaagt
aaacataaga aacataaaca 240 ttcctcagaa gaagacaagg ataaaaaaca
taaacataag cataaacata agaaacacaa 300 aagaaaagag gttattgatg
cttctgataa agagggtatg tctccagcaa aaagaactaa 360 acttgatgat
ttagctttgc tagaagactt ggaaaaacag agagccttga ttaaggccga 420
acttgataat gagttaatgg aaggaaaggt ccagtctggt atggggctca ttttgcaagg
480 ttatgagtct ggctctgaag aagaggggga aattcatgaa aaggcaagaa
atggaaatag 540 gtctagtact agatcttcaa gtacaaaggg gaaacttgaa
cttgtggaca ataaaattac 600 tacaaagaaa cgaagtaaaa gcagatccaa
agaacggact agacataggt ctgataaaaa 660 gaaaagtaag gggggtattg
aaatcgttaa agagaaaaca actaggagca agtcaaagga 720 gaggaaaaaa
tctaaaagcc catccaaaag aagtaagtct caagatcaag caaggaaatc 780
aaaatcccct acccttagaa ggcgatctca agagaaaatt ggtaaggcca gatctcctac
840 tgatgataag gttaaaattg aagataaaag taaatcaaaa gataggaaaa
aatccccaat 900 tataaatgaa agtagaagtc gcgatcgagg taaaaaatcc
agatccccag ttgatttaag 960 aggtaaatcc aaagacagaa ggtcacggtc
caaagagaga aaatcaaaac ggtctgaaac 1020 tgataaagaa aagaagccaa
ttaaatctcc ctctaaagat gcttcatctg ggaaagaaaa 1080 taggtcaccc
agcagaagac ctggtcgtag tcctaaaaga agaagtttgt ctccaaaacc 1140
acgtgataaa tcaagaagaa gcaggtctcc acttttgaat gatagaagat ctaagcagag
1200 caaatccccc tcgcggacac tgtctcctgg gagaagagcc aagagccgat
ccttagaaag 1260 aaaacgacga gaaccagaga ggagacgact ttcttctcca
agaacacgac ctcgagatga 1320 tatcctcagt agacgtgaaa gatcaaaaga
tgccagcccc atcaatagat ggtctccaac 1380 ccgaagaaga agtagatctc
ccattagaag gaggtctcgt tccccactca gacgtagcag 1440 gtctccaaga
agaagaagca gatctcctcg gagaagggac agaggtcgga ggagcagatc 1500
acgcttgcga aggcggtctc gatcacgcgg tggtcgtaga cgaaggagca gaagcaaagt
1560 aaaggaagat aaatttaaag gaagtctttc tgaaggaatg aaagttgagc
aggaatcttc 1620 gtctgatgat aaccttgaag actttgatgt agaggaagaa
gatgaagaag ccctaataga 1680 acagagaaga atccaaaggc aggcaattgt
tcagaaatat aaataccttg ctgaagatag 1740 caacatgtct gtgccatctg
aaccaagcag cccccagagc agtacgagaa cacgatcacc 1800 atctccagat
gacattctgg agcgagtagc tgctgatgtt aaagagtatg aacgggaaaa 1860
tgttgataca tttgaggcct cagtgaaagc caagcataat ctaatgacag ttgaacagaa
1920 taatggttca tctcagaaga agttgttggc acctgatatg tttacagaat
ctgatgatat 1980 gtttgctgcg tattttgata gtgctcgtct tcgggccgct
ggcattggaa aagatttcaa 2040 agagaatccc aacctcagag ataactggac
cgatgcagaa ggctattatc gtgtgaacat 2100 aggtgaagtc ctagataaac
gttacaatgt gtatggctac actgggcaag gtgtattcag 2160 taatgttgta
cgagccagag ataatgcaag agccaaccaa gaagtggctg taaagatcat 2220
cagaaacaat gagctcatgc aaaagactgg tttaaaagaa ttagagttct tgaaaaaact
2280 taatgatgct gatcctgatg acaaatttca ttgtctgaga ctcttcaggc
acttctatca 2340 caagcagcat ctttgtctgg tattcgagcc tctcagcatg
aacttacgag aggtgttaaa 2400 aaaatatggt aaagatgttg gtcttcatat
taaagctgta agatcctata gtcagcagtt 2460 gttcctggca ttgaaactcc
ttaaaagatg caatatccta catgcagata tcaagccaga 2520 caatatcctg
gttaatgaat ccaaaactat tttaaagctt tgcgattttg ggtcggcttc 2580
acatgttgcg gataatgaca taacacctta tcttgtcagt agattttatc gtgctcctga
2640 aatcattata ggtaaaagct atgactatgg tatagatatg tggtctgtag
gttgcacctt 2700 atacgaactc tatactggaa aaattttatt ccctggcaaa
accaataacc atatgctgaa 2760 gcttgcaatg gatctcaaag gaaagatgcc
aaataagatg attcgaaaag gtgtgttcaa 2820 agatcagcat tttgatcaaa
atctcaactt catgtacata gaagttgata aagtaacaga 2880 gagggagaaa
gttactgtta tgagcaccat taatccaact aaggacctgt tggctgactt 2940
gattgggtgc cagagacttc ctgaagacca acgtaagaaa gtacaccagc taaaggactt
3000 gttggaccag attctgatgt tggacccagc taaacgaatt agcatcaacc
aggccctaca 3060 gcacgccttc atccaggaaa aaatttaaac aagatgaaga
aactccaagg gtttgagtaa 3120 atacaaagac tgaagaaatt tcacagcagt
ttattaatgt atataaactt ataaatattt 3180 ctccagcaaa tttgaggaag ca 3202
3 1719 DNA Homo sapiens 3 ggtcggagga gcagatcacg cttgcgaagg
cggtctcgat cacgcggtgg tcgtagacga 60 aggagcagaa gcaaagtaaa
ggaagataaa tttaaaggaa gtctttctga aggaatgaaa 120 gttgagcagg
aatcttcgtc tgatgataac cttgaagact ttgatgtaga ggaagaagat 180
gaagaagccc taatagaaca gagaagaatc caaaggcagg caattgttca gaaatataaa
240 taccttgctg aagatagcaa catgtctgtg ccatctgaac caagcagccc
ccagagcagt 300 acgagaacac gatcaccatc tccagatgac attctggagc
gagtagctgc tgatgttaaa 360 gagtatgaac gggaaaatgt tgatacattt
gaggcctcag tgaaagccaa gcataatcta 420 atgacagttg aacagaataa
tggttcatct cagaagaagt tgttggcacc tgatatgttt 480 acagaatctg
atgatatgtt tgctgcgtat tttgatagtg ctcgtcttcg ggccgctggc 540
attggaaaag atttcaaaga gaatcccaac ctcagagata actggaccga tgcagaaggc
600 tattatcgtg tgaacatagg tgaagtccta gataaacgtt acaatgtgta
tggctacact 660 gggcaaggtg tattcagtaa tgttgtacga gccagagata
atgcaagagc caaccaagaa 720 gtggctgtaa agatcatcag aaacaatgag
ctcatgcaaa agactggttt aaaagaatta 780 gagttcttga aaaaacttaa
tgatgctgat cctgatgaca aatttcattg tctgagactc 840 ttcaggcact
tctatcacaa gcagcatctt tgtctggtat tcgagcctct cagcatgaac 900
ttacgagagg tgttaaaaaa atatggtaaa gatgttggtc ttcatattaa agctgtaaga
960 tcctatagtc agcagttgtt cctggcattg aaactcctta aaagatgcaa
tatcctacat 1020 gcagatatca agccagacaa tatcctggtt aatgaatcca
aaactatttt aaagctttgc 1080 gattttgggt cggcttcaca tgttgcggat
aatgacataa caccttatct tgtcagtaga 1140 ttttatcgtg ctcctgaaat
cattataggt aaaagctatg actatggtat agatatgtgg 1200 tctgtaggtt
gcaccttata cgaactctat actggaaaaa ttttattccc tggcaaaacc 1260
aataaccata tgctgaagct tgcaatggat ctcaaaggaa agatgccaaa taagatgatt
1320 cgaaaaggtg tgttcaaaga tcagcatttt gatcaaaatc tcaacttcat
gtacatagaa 1380 gttgataaag taacagagag ggagaaagtt actgttatga
gcaccattaa tccaactaag 1440 gacctgttgg ctgacttgat tgggtgccag
agacttcctg aagaccaacg taagaaagta 1500 caccagctaa aggacttgtt
ggaccagatt ctgatgttgg acccagctaa acgaattagc 1560 atcaaccagg
ccctacagca cgccttcatc caggaaaaaa tttaaacaag atgaagaaac 1620
tccaagggtt tgagtaaata caaagactga agaaatttca cagcagttta ttaatgtata
1680 taaacttata aatatttctc cagcaaattt gaggaagca 1719 4 2393 DNA
Homo sapiens 4 ggcacgaggc tcagtagacg tgaaagatca aaagatgcca
gcccatcaat agatggtctc 60 caacccgaag aagaagtaga tctcccatta
gaaggaggtc tcgttcccca ctcagacgta 120 gcaggtctcc aagaagaaga
agcagatctc ctcggagaag ggacagaggt cggaggagca 180 gatcacgctt
gcgaaggcgg tctcgatcac gcggtggtcg tagacgaagg agcagaagca 240
aagtaaagga agataaattt aaaggaagtc tttctgaagg aatgaaagtt gagcaggaat
300 cttcgtctga tgataacctt gaagactttg atgtagagga agaagatgaa
gaagccctaa 360 tagaacagag aagaatccaa aggcaggcaa ttgttcagaa
atataaatac cttgctgaag 420 atagcaacat gtctgtgcca tctgaaccaa
gcagccccca gagcagtacg agaacacgat 480 caccatctcc agatgacatt
ctggagcgag tagctgctga tgttaaagag tatgaacggg 540 aaaatgttga
tacatttgag gcctcagtga aagccaagca taatctaatg acagttgaac 600
agaataatgg ttcatctcag aagaagttgt tggcacctga tatgtttaca gaatctgatg
660 atatgtttgc tgcgtatttt gatagtgctc gtcttcgggc cgctggcatt
ggaaaagatt 720 tcaaagagaa tcccaacctc agagataact ggaccgatgc
agaaggctat tatcgtgtga 780 acataggtga agtcctagat aaacgttaca
atgtgtatgg ctacactggg caaggtgtat 840 tcagtaatgt tgtacgagcc
agagataatg caagagccaa ccaagaagtg gctgtaaaga 900 tcatcagaaa
caatgagctc atgcaaaaga ctggtttaaa agaattagag ttcttgaaaa 960
aacttaatga tgctgatcct gatgacaaat ttcattgtct gagactcttc aggcacttct
1020 atcacaagca gcatctttgt ctggtattcg agcctctcag catgaactta
cgagaggtgt 1080 taaaaaaata tggtaaagat gttggtcttc atattaaagc
tgtaagatcc tatagtcagc 1140 agttgttcct ggcattgaaa ctccttaaaa
gatgcaatat cctacatgca gatatcaagc 1200 cagacaatat cctggttaat
gaatccaaaa ctattttaaa gctttgcgat tttgggtcgg 1260 cttcacatgt
tgcggataat gacataacac cttatcttgt cagtagattt tatcgtgctc 1320
ctgaaatcat tataggtaaa agctatgact atggtataga tatgtggtct gtaggttgca
1380 ccttatacga actctatact ggaaaaattt tattccctgg caaaaccaat
aaccatatgc 1440 tgaagcttgc aatggatctc aaaggaaaga tgccaaataa
gatgattcga aaaggtgtgt 1500 tcaaagatca gcattttgat caaaatctca
acttcatgta catagaagtt gataaagtaa 1560 cagagaggga gaaagttact
gttatgagca ccattaatcc aactaaggac ctgttggctg 1620 acttgattgg
gtgccagaga cttcctgaag accaacgtaa gaaagtacac cagctaaagg 1680
acttgttgga ccagattctg atgttggacc cagctaaacg aattagcatc aaccaggccc
1740 tacagcacgc cttcatccag gaaaaaattt aaacaagatg aagaaactcc
aagggtttga 1800 gtgtgtgtgt gcaggccaca gcagcatgcc cttggtgtag
tcagtgccga aaggggtctg 1860 ttccttcttg agcctgcctg cagggatggt
ctccttttaa agcaggttgt gtgcagcatt 1920 cagtacactg aaggcataaa
ccttccactc ttgaacaaag cagctgcttt ttaaaagcga 1980 gaaaaaggaa
aacggggcac aggccattcg acgccttctc caaggggtct gatttgctga 2040
gacaccagct tcaccttctt aacaaggcac ctaattacaa caagcatgca cattttggtg
2100 cattcaagaa tggaaaatca gaatagcagc attgattctt ctggtgcagc
tcagtggaag 2160 atgatgacaa ccagaagaca tgagctaagg gtaagggact
gttctgaaga acctttccat 2220 ttagtgatca agatatggaa gctgatttct
gaaaatgctc agtgtgtact ctaattattt 2280 atggtaccat ttgaattgta
acttgcattt tagcagtgca tgtttctaat tgacttactg 2340 ggaaactgaa
taaaatatgc ctcttattat caaaaaaaaa aaaaaaaaaa aaa 2393 5 1718 DNA
Homo sapiens 5 ggtcggagga gcagatcacg cttgcgaagg cggtctcgat
cacgcggtgg tcgtagacga 60 aggagcagaa gcaaagtaag gaagataaat
ttaaaggaag tctttctgaa ggaatgaaag 120 ttgagcagga atcttcgtct
gatgataacc ttgaagactt tgatgtagag gaagaagatg 180 aagaagccct
aatagaacag agaagaatcc aaaggcaggc aattgttcag aaatataaat 240
accttgctga agatagcaac atgtctgtgc catctgaacc aagcagcccc cagagcagta
300 cgagaacacg atcaccatct ccagatgaca ttctggagcg agtagctgct
gatgttaaag 360 agtatgaacg ggaaaatgtt gatacatttg aggcctcagt
gaaagccaag cataatctaa 420 tgacagttga acagaataat ggttcatctc
agaagaagtt gttggcacct gatatgttta 480 cagaatctga tgatatgttt
gctgcgtatt ttgatagtgc tcgtcttcgg gccgctggca 540 ttggaaaaga
tttcaaagag aatcccaacc tcagagataa ctggaccgat gcagaaggct 600
attatcgtgt gaacataggt gaagtcctag ataaacgtta caatgtgtat ggctacactg
660 ggcaaggtgt attcagtaat gttgtacgag ccagagataa tgcaagagcc
aaccaagaag 720 tggctgtaaa gatcatcaga aacaatgagc tcatgcaaaa
gactggttta aaagaattag 780 agttcttgaa aaagcttaat gatgctgatc
ctgatgacaa atttcattgt ctgagactct 840 tcaggcactt ctatcacaag
cagcatcttt gtctggtatt cgagcctctc agcatgaact 900 tacgagaggt
gttaaaaaaa tatggtaaag atgttggtct tcatattaaa gctgtaagat 960
cctatagtca gcagttgttc ctggcattga aactccttaa aagatgcaat atcctacatg
1020 cagatatcaa gccagacaat atcctggtta atgaatccaa aactatttta
aagctttgcg 1080 attttgggtc ggcttcacat gttgcggata atgacataac
accttatctt gtcagtagat 1140 tttatcgtgc tcctgaaatc attataggta
aaagctatga ctatggtata gatatgtggt 1200 ctgtaggttg caccttatac
gaactctata ctggaaaaat tttattccct ggcaaaacca 1260 ataaccatat
gctgaagctt gcaatggatc tcaaaggaaa gatgccaaat aagatgattc 1320
gaaaaggtgt gttcaaagat cagcattttg atcaaaatct caacttcatg tacatagaag
1380 ttgataaagt aacagagagg gagaaagtta ctgttatgag caccattaat
ccaactaagg 1440 acctgttggc tgacttgatt gggtgccaga gacttcctga
agaccaacgt aagaaagtac 1500 accagctaaa ggacttgttg gaccagattc
tgatgttgga cccagctaaa cgaattagca 1560 tcaaccaggc cctacagcac
gccttcatcc aggaaaaaat ttaaacaaga tgaagaaact 1620 ccaagggttt
gagtaaatac aaagatgaag aaatttcaca gcagtttcat taatgtatat 1680
aaacttataa atatttctcc agcaaatttg aggaagca 1718 6 3024 DNA Homo
sapiens 6 atggccgccg cggagaccca gtcgctacgg gagcagccag agatggaaga
tgctaattct 60 gaaaagagta taaatgaaga aaatggagaa gtatcagaag
accagtctca aaataagcac 120 agtcgtcaca aaaaaaagaa gcataaacac
agaagtaaac ataagaaaca taaacattcc 180 tcagaagaag acaaggataa
aaaacataaa cataagcata aacataagaa acacaaaaga 240 aaagaggtta
ttgatgcttc tgataaagag ggtatgtctc cagcaaaaag aactaaactt 300
gatgatttag ctttgctaga agacttggaa aaacagagag ccttgattaa ggccgaactt
360 gataatgagt taatggaagg aaaggtccag tctggtatgg ggctcatttt
gcaaggttat 420 gagtctggct ctgaagaaga gggggaaatt catgaaaagg
caagaaatgg aaataggtct 480 agtactagat cttcaagtac aaaggggaaa
cttgaacttg tggacaataa aattactaca 540 aagaaacgaa gtaaaagcag
atccaaagaa cggactagac ataggtctga taaaaagaaa 600 agtaaggggg
gtattgaaat cgttaaagag aaaacaacta ggagcaagtc aaaggagagg 660
aaaaaatcta aaagcccatc caaaagaagt aagtctcaag atcaagcaag gaaatcaaaa
720 tcccctaccc ttagaaggcg atctcaagag aaaattggta aggccagatc
tcctactgat 780 gataaggtta aaattgaaga taaaagtaaa tcaaaagata
ggaaaaaatc cccaattata 840 aatgaaagta gaagtcgcga tcgaggtaaa
aaatccagat ccccagttga tttaagaggt 900 aaatccaaag acagaaggtc
acggtccaaa gagagaaaat caaaacggtc tgaaactgat 960 aaagaaaaga
agccaattaa atctccctct aaagatgctt catctgggaa agaaaatagg 1020
tcacccagca gaagacctgg tcgtagtcct aaaagaagaa gtttgtctcc aaaaccacgt
1080 gataaatcaa gaagaagcag gtctccactt ttgaatgata gaagatctaa
gcagagcaaa 1140 tccccctcgc ggacactgtc tcctgggaga agagccaaga
gccgatcctt agaaagaaaa 1200 cgacgagaac cagagaggag acgactttct
tctccaagaa cacgacctcg agatgatatc 1260 ctcagtagac gtgaaagatc
aaaagatgcc agccccatca atagatggtc tccaacccga 1320 agaagaagta
gatctcccat tagaaggagg tctcgttccc cactcagacg tagcaggtct 1380
ccaagaagaa gaagcagatc tcctcggaga agggacagag gtcggaggag cagatcacgc
1440 ttgcgaaggc ggtctcgatc acgcggtggt cgtagacgaa ggagcagaag
caaagtaaag 1500 gaagataaat ttaaaggaag tctttctgaa ggaatgaaag
ttgagcagga atcttcgtct 1560 gatgataacc ttgaagactt tgatgtagag
gaagaagatg aagaagccct aatagaacag 1620 agaagaatcc aaaggcaggc
aattgttcag aaatataaat accttgctga agatagcaac 1680 atgtctgtgc
catctgaacc aagcagcccc cagagcagta cgagaacacg atcaccatct 1740
ccagatgaca ttctggagcg agtagctgct gatgttaaag agtatgaacg ggaaaatgtt
1800 gatacatttg aggcctcagt gaaagccaag cataatctaa tgacagttga
acagaataat 1860 ggttcatctc agaagaagtt gttggcacct gatatgttta
cagaatctga tgatatgttt 1920 gctgcgtatt ttgatagtgc tcgtcttcgg
gccgctggca ttggaaaaga tttcaaagag 1980 aatcccaacc tcagagataa
ctggaccgat gcagaaggct attatcgtgt gaacataggt 2040 gaagtcctag
ataaacgtta caatgtgtat ggctacactg ggcaaggtgt attcagtaat 2100
gttgtacgag ccagagataa tgcaagagcc aaccaagaag tggctgtaaa gatcatcaga
2160 aacaatgagc tcatgcaaaa gactggttta aaagaattag agttcttgaa
aaaacttaat 2220 gatgctgatc ctgatgacaa atttcattgt ctgagactct
tcaggcactt ctatcacaag 2280 cagcatcttt gtctggtatt cgagcctctc
agcatgaact tacgagaggt gttaaaaaaa 2340 tatggtaaag atgttggtct
tcatattaaa gctgtaagat cctatagtca gcagttgttc 2400 ctggcattga
aactccttaa aagatgcaat atcctacatg cagatatcaa gccagacaat 2460
atcctggtta atgaatccaa aactatttta aagctttgcg attttgggtc ggcttcacat
2520 gttgcggata atgacataac accttatctt gtcagtagat tttatcgtgc
tcctgaaatc 2580 attataggta aaagctatga ctatggtata gatatgtggt
ctgtaggttg caccttatac 2640 gaactctata ctggaaaaat tttattccct
ggcaaaacca ataaccatat gctgaagctt 2700 gcaatggatc tcaaaggaaa
gatgccaaat aagatgattc gaaaaggtgt gttcaaagat 2760 cagcattttg
atcaaaatct caacttcatg tacatagaag ttgataaagt aacagagagg 2820
gagaaagtta ctgttatgag caccattaat ccaactaagg acctgttggc tgacttgatt
2880 gggtgccaga gacttcctga agaccaacgt aagaaagtac accagctaaa
ggacttgttg 2940 gaccagattc tgatgttgga cccagctaaa cgaattagca
tcaaccaggc cctacagcac 3000 gccttcatcc aggaaaaaat ttaa 3024 7 1007
PRT Homo sapiens 7 Met Ala Ala Ala Glu Thr Gln Ser Leu Arg Glu Gln
Pro Glu Met Glu 1 5 10 15 Asp Ala Asn Ser Glu Lys Ser Ile Asn Glu
Glu Asn Gly Glu Val Ser 20 25 30 Glu Asp Gln Ser Gln Asn Lys His
Ser Arg His Lys Lys Lys Lys His 35 40 45 Lys His Arg Ser Lys His
Lys Lys His Lys His Ser Ser Glu Glu Asp 50 55 60 Lys Asp Lys Lys
His Lys His Lys His Lys His Lys Lys His Lys Arg 65 70 75 80 Lys Glu
Val Ile Asp Ala Ser Asp Lys Glu Gly Met Ser Pro Ala Lys 85 90 95
Arg Thr Lys Leu Asp Asp Leu Ala Leu Leu Glu Asp Leu Glu Lys Gln 100
105 110 Arg Ala Leu Ile Lys Ala Glu Leu Asp Asn Glu Leu Met Glu Gly
Lys 115 120 125 Val Gln Ser Gly Met Gly Leu Ile Leu Gln Gly Tyr Glu
Ser Gly Ser 130 135 140 Glu Glu Glu Gly Glu Ile His Glu Lys Ala Arg
Asn Gly Asn Arg Ser 145 150 155 160 Ser Thr Arg Ser Ser Ser Thr Lys
Gly Lys Leu Glu Leu Val Asp Asn 165 170 175 Lys Ile Thr Thr Lys Lys
Arg Ser Lys Ser Arg Ser Lys Glu Arg Thr 180 185 190 Arg His Arg Ser
Asp Lys Lys Lys Ser Lys Gly Gly Ile Glu Ile Val 195 200 205 Lys Glu
Lys Thr Thr Arg Ser Lys Ser Lys Glu Arg Lys Lys Ser Lys 210 215 220
Ser Pro Ser Lys Arg Ser Lys Ser Gln Asp Gln Ala Arg Lys Ser Lys 225
230 235 240 Ser Pro Thr Leu Arg Arg Arg Ser Gln Glu Lys Ile Gly Lys
Ala Arg 245 250 255 Ser Pro Thr Asp Asp Lys Val Lys Ile Glu Asp Lys
Ser Lys Ser Lys 260 265 270 Asp Arg Lys Lys Ser Pro Ile Ile Asn Glu
Ser Arg Ser Arg Asp Arg 275 280 285 Gly Lys Lys Ser Arg Ser Pro Val
Asp Leu Arg Gly Lys Ser Lys Asp 290 295 300 Arg Arg Ser Arg Ser Lys
Glu Arg Lys Ser Lys Arg Ser Glu Thr Asp 305 310 315 320 Lys Glu Lys
Lys Pro Ile Lys Ser Pro Ser Lys Asp Ala Ser Ser Gly 325 330 335 Lys
Glu Asn Arg Ser Pro Ser Arg Arg Pro Gly Arg Ser Pro Lys Arg 340 345
350 Arg Ser Leu Ser Pro Lys Pro Arg Asp Lys Ser Arg Arg Ser Arg Ser
355 360 365 Pro Leu Leu Asn Asp Arg Arg Ser Lys Gln Ser Lys Ser Pro
Ser Arg 370 375 380 Thr Leu Ser Pro Gly Arg Arg Ala Lys Ser Arg Ser
Leu Glu Arg Lys 385 390 395 400 Arg Arg Glu Pro Glu Arg Arg Arg Leu
Ser Ser Pro Arg Thr Arg Pro 405 410 415 Arg Asp Asp Ile Leu Ser Arg
Arg Glu Arg Ser Lys Asp Ala Ser Pro 420 425 430 Ile Asn Arg Trp Ser
Pro Thr Arg Arg Arg Ser Arg Ser Pro Ile Arg 435 440 445 Arg Arg Ser
Arg Ser Pro Leu Arg Arg Ser Arg Ser Pro Arg Arg Arg 450 455 460 Ser
Arg Ser Pro Arg Arg Arg Asp Arg Gly Arg Arg Ser Arg Ser Arg 465 470
475 480 Leu Arg Arg Arg Ser Arg Ser Arg Gly Gly Arg Arg Arg Arg Ser
Arg 485 490 495 Ser Lys Val Lys Glu Asp Lys Phe Lys Gly Ser Leu Ser
Glu Gly Met 500 505 510 Lys Val Glu Gln Glu Ser Ser Ser Asp Asp Asn
Leu Glu Asp Phe Asp 515 520 525 Val Glu Glu Glu Asp Glu Glu Ala Leu
Ile Glu Gln Arg Arg Ile Gln 530 535 540 Arg Gln Ala Ile Val Gln Lys
Tyr Lys Tyr Leu Ala Glu Asp Ser Asn 545 550 555 560 Met Ser Val Pro
Ser Glu Pro Ser Ser Pro Gln Ser Ser Thr Arg Thr 565 570 575 Arg Ser
Pro Ser Pro Asp Asp Ile Leu Glu Arg Val Ala Ala Asp Val 580 585 590
Lys Glu Tyr Glu Arg Glu Asn Val Asp Thr Phe Glu Ala Ser Val Lys 595
600 605 Ala Lys His Asn Leu Met Thr Val Glu Gln Asn Asn Gly Ser Ser
Gln 610 615 620 Lys Lys Leu Leu Ala Pro Asp Met Phe Thr Glu Ser Asp
Asp Met Phe 625 630 635 640 Ala Ala Tyr Phe Asp Ser Ala Arg Leu Arg
Ala Ala Gly Ile Gly Lys 645 650 655 Asp Phe Lys Glu Asn Pro Asn Leu
Arg Asp Asn Trp Thr Asp Ala Glu 660 665 670 Gly Tyr Tyr Arg Val Asn
Ile Gly Glu Val Leu Asp Lys Arg Tyr Asn 675 680 685 Val Tyr Gly Tyr
Thr Gly Gln Gly Val Phe Ser Asn Val Val Arg Ala 690 695 700 Arg Asp
Asn Ala Arg Ala Asn Gln Glu Val Ala Val Lys Ile Ile Arg 705 710 715
720 Asn Asn Glu Leu Met Gln Lys Thr Gly Leu Lys Glu Leu Glu Phe Leu
725 730 735 Lys Lys Leu Asn Asp Ala Asp Pro Asp Asp Lys Phe His Cys
Leu Arg 740 745 750 Leu Phe Arg His Phe Tyr His Lys Gln His Leu Cys
Leu Val Phe Glu 755 760 765 Pro Leu Ser Met Asn Leu Arg Glu Val Leu
Lys Lys Tyr Gly Lys Asp 770 775 780 Val Gly Leu His Ile Lys Ala Val
Arg Ser Tyr Ser Gln Gln Leu Phe 785 790 795 800 Leu Ala Leu Lys Leu
Leu Lys Arg Cys Asn Ile Leu His Ala Asp Ile 805 810 815 Lys Pro Asp
Asn Ile Leu Val Asn Glu Ser Lys Thr Ile Leu Lys Leu 820 825 830 Cys
Asp Phe Gly Ser Ala Ser His Val Ala Asp Asn Asp Ile Thr Pro 835 840
845 Tyr Leu Val Ser Arg Phe Tyr Arg Ala Pro Glu Ile Ile Ile Gly Lys
850 855 860 Ser Tyr Asp Tyr Gly Ile Asp Met Trp Ser Val Gly Cys Thr
Leu Tyr 865 870 875 880 Glu Leu Tyr Thr Gly Lys Ile Leu Phe Pro Gly
Lys Thr Asn Asn His 885 890 895 Met Leu Lys Leu Ala Met Asp Leu Lys
Gly Lys Met Pro Asn Lys Met 900 905 910 Ile Arg Lys Gly Val Phe Lys
Asp Gln His Phe Asp Gln Asn Leu Asn 915 920 925 Phe Met Tyr Ile Glu
Val Asp Lys Val Thr Glu Arg Glu Lys Val Thr 930 935 940 Val Met Ser
Thr Ile Asn Pro Thr Lys Asp Leu Leu Ala Asp Leu Ile 945 950 955 960
Gly Cys Gln Arg Leu Pro Glu Asp Gln Arg Lys Lys Val His Gln Leu 965
970 975 Lys Asp Leu Leu Asp Gln Ile Leu Met Leu Asp Pro Ala Lys Arg
Ile 980 985 990 Ser Ile Asn Gln Ala Leu Gln His Ala Phe Ile Gln Glu
Lys Ile 995 1000 1005
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