U.S. patent number 7,867,500 [Application Number 11/510,859] was granted by the patent office on 2011-01-11 for cofactor that modulates steroid receptor activities.
This patent grant is currently assigned to N/A, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Yuanzheng He, S. Stoney Simons, Jr..
United States Patent |
7,867,500 |
Simons, Jr. , et
al. |
January 11, 2011 |
Cofactor that modulates steroid receptor activities
Abstract
The invention provides a new glucocorticoid receptor coactivator
named STAMP (Steroid receptor coactivator-1 and Transcription
intermediary factor-2 Associated Modulatory Protein) that can
modulate transcription of glucocorticoid-, progesterone-,
mineralocorticoid- and androgen-responsive genes. The invention
also provides antibodies that can bind STAMP and modulate its
activity. In addition, the invention provides antisense, ribozyme
and siRNA STAMP nucleic acids that can modulate the expression of
STAMP. Also provided are compositions and methods for modulating
glucocorticoid-responsive gene expression and for treating a
variety of diseases and conditions.
Inventors: |
Simons, Jr.; S. Stoney
(Bethesda, MD), He; Yuanzheng (Bethesda, MD) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
N/A (N/A)
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Family
ID: |
34910977 |
Appl.
No.: |
11/510,859 |
Filed: |
August 25, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20070128627 A1 |
Jun 7, 2007 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/US2005/006393 |
Feb 25, 2005 |
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60548039 |
Feb 26, 2004 |
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Current U.S.
Class: |
424/198.1;
424/185.1; 514/20.6 |
Current CPC
Class: |
C07K
14/4702 (20130101); A61K 38/00 (20130101) |
Current International
Class: |
A61K
39/00 (20060101); A61K 38/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
He et al. 2007. Mol and Cell Biology, 27:1467-1485. cited by
examiner .
Glass et al. 1997. Curr Opin. Cell Biol. 9:222-232. cited by
examiner .
Wang et al. 2004. J. Steroid Biochem and Mol Biol. 91:197-210.
cited by examiner .
Database EMBL `Online! Apr. 9, 1999, "Homo Sapiens mRNA for
KIAA0998 protein, partial cds." XP002332405, retrieved from EBI
accession No. EM.sub.--PRO:ABO23215. Database accession No.
AB023215 abstract. cited by other .
Database Geneseq `Online! Oct. 22, 2001, "Human polynucleotide SEQ
ID No. 464." XP002332406 retrieved from EBI accession No.
GSN:AAI58261. Database accession No. AAI58261 abstract. cited by
other .
Szapary Daniele et al. "Opposing effects of corepressor and
coactivators in determining the dose-response curve of agonists,
and residual agonist activity of antagonists, for glucocorticoid
receptor-regulated gene expression," Molecular Endocrinology,
13(12):2108-2121, Dec. 1999. cited by other .
Chen Shiyou et al. "A second pathway for modulating glucocorticoid
receptor transactivation properties," Molecular and Cellular
Endocrinology 199(1-2):129-142, Jan. 2003. cited by other .
He Yuanzheng et al., "Modulation of induction properties of
glucocorticoid receptor-agonist and --antagonist complexes by
coactivators involves binding to receptors but is independent of
ability of coactivators to augment transactivation." J. Biological
Chemistry 277(51):49256-49266, Dec. 2002. cited by other.
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Primary Examiner: Shafer; Shulamith H.
Attorney, Agent or Firm: Swanson & Bratschun, L.L.C.
Parent Case Text
This application is a continuation-in-part under 35 U.S.C. 111(a)
of International Application No. PCT/US2005/006393 filed Feb. 25,
2005 and published in English as WO 2005/082935 on Sep. 9, 2005,
which claimed the benefit of U.S. Provisional Application Ser. No.
60/548,039, filed Feb. 26, 2004, the contents of which applications
and publication are incorporated herein in their entireties.
The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
Claims
What is claimed is:
1. A method of modulating the activity of a steroid or nuclear
receptor selected from the group consisting of a glucocorticoid
receptor, a progesterone receptor, an androgen receptor and a
mineralocorticoid receptor, in a mammal comprising administering to
the mammal a composition comprising a carrier and an effective
amount of a Steroid receptor coactivator-1 and Transcription
intermediary factor 2 Associated Modulatory Protein (STAMP)
polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:70 and a combination thereof.
2. The method of claim 1, wherein the receptor is a glucocorticoid
receptor.
3. The method of claim 1, wherein the composition also comprises a
ligand, coactivator, agonist, partial agonist or antagonist for the
receptor.
Description
FIELD OF THE INVENTION
The invention relates to a new factor, termed STAMP (Steroid
receptor coactivator-1 and Transcription intermediary factor-2
(TIF2) Associated Modulatory Protein), a coactivator that can
modulate steroid receptors and glucocorticoid-sensitive gene
expression.
BACKGROUND OF THE INVENTION
Nuclear receptors are classically defined as a family of ligand
dependent transcription factors that are activated in response to
ligand binding. Members of this family include the following
receptors: glucocorticoid, mineralocorticoid, androgen,
progesterone and estrogen. Naturally occurring ligands to these
receptors are low molecular weight molecules that play an important
role in health and in many diseases. Excesses or deficiencies of
these ligands can have profound physiological consequences. By way
of example, glucocorticoid excess results in Cushing's Syndrome,
while glucocorticoid insufficiency results in Addison's
Disease.
The glucocorticoid receptor (GR) is present in glucocorticoid
responsive cells where it resides in the cytosol in an inactive
state until it is stimulated by the binding of a ligand. Upon
stimulation, the glucocorticoid receptor translocates to the cell
nucleus where it specifically interacts with DNA and/or protein(s)
and either activates or represses transcription in a glucocorticoid
responsive manner. Activation involves glucocorticoid receptor
binding to specific DNA sequences (glucocorticoid response elements
or GREs) and the association with other factors, including
coactivators that were discovered on the basis of their ability to
increase the total levels of induced gene product. Repression
usually involves glucocorticoid receptor becoming tethered to
proteins bound to non-GRE DNA sequences as opposed to the direct
binding of glucocorticoid receptors to DNA sequences. Two examples
of DNA-bound proteins that interact with glucocorticoid receptors,
and participate in glucocorticoid receptor-mediated gene
repression, are AP-1 and NF.kappa.-B. These interactions of
glucocorticoid receptor are believed to be responsible for some of
the anti-inflammatory activity of exogenously administered
glucocorticoids. In addition, glucocorticoids may also exert
physiologic effects independent of nuclear transcription.
Three distinguishing properties of ligand-regulated gene induction
by glucocorticoid receptors are 1) the level of activated gene
expression with agonist steroids, 2) the concentration of agonist
required for half-maximal induction (EC.sub.50), and 3) the amount
of partial agonist activity with antagonists or antisteroids. The
last two properties are independent of the total amount of gene
expression, and basal level activity, due to the mathematical
method by which they are defined. Physiological concentrations of
steroid are often similar to the EC.sub.50 of inducible genes.
Therefore, those regulated genes that have lower EC.sub.50s, and
dose-response curves that are more left-shifted, will be
preferentially induced by the circulating hormone, thereby
affording differential gene induction during development,
differentiation, and homeostasis. Endocrine therapies using
antisteroids will be much more specific if one can increase the
partial agonist activity for most genes and limit the repression to
one or a few regulated genes.
Biologically relevant glucocorticoid receptor agonists include
cortisol and corticosterone. Many synthetic glucocorticoid receptor
agonists exist including dexamethasone, prednisone and
prednisilone. By definition, glucocorticoid receptor antagonists
bind to the receptor and prevent glucocorticoid receptor agonists
from eliciting GR mediated events, including transcription. RU486
is an example of a non-selective glucocorticoid receptor
antagonist.
However, while glucocorticoid receptor agonists can be beneficial
agents for the treatment or prevention of various diseases and
conditions, such treatment is often accompanied by undesirable side
effects. These side effects include, for example, metabolic
effects, weight gain, muscle wasting, decalcification of the
skeleton, osteoporosis, thinning of the skin and thinning of the
skeleton. Hence, agents are needed that modulate glucocorticoid
receptor activity without causing these types of side effects.
Moreover, glucocorticoid antagonists and agonists that act
selectively may be desirable in therapies for the selective
blockage/enhancement of specific genes without simultaneously
influencing other glucocorticoid-responsive genes.
SUMMARY OF THE INVENTION
The invention provides a new factor, termed STAMP (SRC-1 and TIF2
Associated Modulatory Protein) that can modulate transcription of
glucocorticoid-responsive genes. In other embodiments, STAMP can
influence the activity of a variety of receptors, including
glucocorticoid receptors, androgen receptors, estrogen receptors,
mineralocorticoid receptors, progesterone receptors, thyroid
receptors, retinoid receptors (RAR and RXR), and peroxisome
proliferator-activated receptors (PPARs). Hence, STAMP can be
included in compositions and methods for modulating such
receptors.
Also provided are STAMP nucleic acids, antibodies that can bind
STAMP polypeptides from various species, and agents that can
modulate the transcription of a STAMP RNA, or the translation or
activity of a STAMP polypeptide. Use of STAMP polypeptides,
anti-STAMP antibodies, STAMP nucleic acids and agents that can
modulate STAMP may reduce the need for steroids and avoid some of
the negative side effects observed upon administration of
steroids.
Thus, one aspect of the invention is an isolated STAMP polypeptide
comprising any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,
SEQ ID NO:70, or a combination thereof.
Another aspect of the invention is an isolated antibody that can
bind a STAMP polypeptide comprising any one of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:62, SEQ
ID NO:63, SEQ ID NO:64, SEQ ID NO:70, or a combination thereof.
Another aspect of the invention is an isolated nucleic acid
encoding a STAMP polypeptide comprising any one of SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:62, SEQ
ID NO:63, SEQ ID NO:64, SEQ ID NO:70, or a combination thereof.
Such a nucleic acid can, for example, comprise SEQ ID NO:2 or SEQ
ID NO:61.
Another aspect of the invention is a nucleic acid that can modulate
the expression of a STAMP polypeptide comprising any one of SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:70, or a
combination thereof. Such a nucleic acid can, for example, be an
antisense nucleic acid, a ribozyme or a siRNA. These nucleic acids
can hybridize to a nucleic acid comprising SEQ ID NO:2 OR SEQ ID
NO:61 under physiological conditions. In other embodiments, these
nucleic acids can hybridize to a nucleic acid comprising SEQ ID
NO:2 or SEQ ID NO:61 under stringent hybridization conditions.
Examples of nucleic acids that can modulate the expression of a
STAMP polypeptide include a siRNA that consists essentially of a
double-stranded RNA with any one of SEQ ID NO:17-36.
According to the present invention, STAMP may be formulated into a
composition. Such a composition can contain a therapeutically
effective amount of a STAMP polypeptide comprising any one of SEQ
ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:70, or a
combination thereof. The composition can also contain a
glucocorticoid receptor coactivator, a glucocorticoid receptor
agonist, a glucocorticoid receptor partial agonist or a
glucocorticoid receptor antagonist. Examples of coactivators that
can be included in the STAMP compositions include, for example,
transcription intermediary factor-2 (TIF2) and steroid receptor
coactivator-1 (SRC-1). Glucocorticoid receptor and glucocorticoids
can also be included in the compositions of the invention.
Another aspect of the invention is a composition comprising a
carrier and an agent that can modulate the expression or activity
of a STAMP polypeptide comprising any one of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:62, SEQ
ID NO:63, SEQ ID NO:64, SEQ ID NO:70, or a combination thereof. One
type of agent that can modulate the expression or activity of a
STAMP polypeptide is a nucleic acid that can hybridize to a nucleic
acid comprising SEQ ID NO:2 or SEQ ID NO:61 under physiological
conditions. In some embodiments, the nucleic acid that can modulate
the expression or activity of a STAMP polypeptide is a nucleic acid
can hybridize to a nucleic acid comprising SEQ ID NO:2 or SEQ ID
NO:61 under stringent hybridization conditions.
In another embodiment, the agent that can modulate the expression
or activity of a STAMP polypeptide is an antisense nucleic acid, a
ribozyme or a siRNA. Examples of siRNA that may modulate the
expression of a STAMP polypeptide include double-stranded RNA
molecules with SEQ ID NO:17-36. In another embodiment, the agent
that can modulate the expression or activity of a STAMP polypeptide
is a sense nucleic acid, for example, a STAMP nucleic acid having
SEQ ID NO:2 or SEQ ID NO:61.
In another embodiment, the agent that can modulate the expression
or activity of a STAMP polypeptide is an antibody that can bind to
a STAMP polypeptide comprising any one of SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:62, SEQ
ID NO:63, SEQ ID NO:64, SEQ ID NO:70, or a combination thereof.
Another aspect of the invention is a method of modulating
glucocorticoid-responsive gene expression in a mammalian cell
comprising contacting the cell with a composition comprising a
carrier and an effective amount of a STAMP polypeptide comprising
any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ
ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ
ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,
SEQ ID NO:16, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID
NO:70, or a combination thereof. Compositions containing a STAMP
polypeptide can contain other active ingredients, as described
herein.
Another aspect of the invention is a method of modulating
glucocorticoid-responsive gene expression in a mammalian cell
comprising contacting the cell with a composition comprising a
carrier and an agent that can modulate the expression or activity
of a STAMP polypeptide comprising any one of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:62, SEQ
ID NO:63, SEQ ID NO:64, SEQ ID NO:70, or a combination thereof.
Agents that can modulate the expression or activity of a STAMP
polypeptide include nucleic acids that can hybridize to a nucleic
acid comprising SEQ ID NO:2 or SEQ ID NO:61, an antisense nucleic
acid, a ribozyme, a siRNA or an antibody that can bind to STAMP.
Compositions containing an agent that can modulate the expression
or activity of a STAMP polypeptide can contain other active
ingredients, as described herein.
Another aspect of the invention is a method of modulating
glucocorticoid-responsive gene expression in a mammal comprising
administering to the mammal a composition comprising a carrier and
an effective amount of a STAMP polypeptide comprising any one of
SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:70, or a
combination thereof. Compositions containing a STAMP polypeptide
can contain other active ingredients, as described herein. In
another embodiment, the method for modulating
glucocorticoid-responsive gene expression in a mammal comprises
administering to the mammal a composition comprising a carrier and
an agent that can modulate the expression or activity of a STAMP
polypeptide. Agents that can modulate the expression or activity of
a STAMP polypeptide include nucleic acids that can hybridize to a
nucleic acid comprising SEQ ID NO:2 or SEQ ID NO:61, an antisense
nucleic acid, a ribozyme, a siRNA or an antibody that can bind to
STAMP. Compositions containing an agent that can modulate the
expression or activity of a STAMP polypeptide can contain other
active ingredients, as described herein.
Another aspect of the invention is a method of treating or
preventing a disease or a condition in a mammal comprising
administering to the mammal a composition comprising a carrier and
an effective amount of a STAMP polypeptide comprising any one of
SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:70, or a
combination thereof. Compositions containing a STAMP polypeptide
can contain other active ingredients, as described herein.
Another aspect of the invention is a method of treating or
preventing a disease or a condition in a mammal comprising
administering to the mammal a composition comprising a carrier and
an effective amount of an agent that can modulate the expression or
activity of a STAMP polypeptide comprising any one of SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:70, or a combination
thereof. Agents that can modulate the expression or activity of a
STAMP polypeptide include nucleic acids that can hybridize to a
nucleic acid comprising SEQ ID NO:2 or SEQ ID NO:61, an antisense
nucleic acid, a ribozyme, a siRNA or an antibody that can bind to
STAMP. Compositions containing an agent that can modulate the
expression or activity of a STAMP polypeptide can contain other
active ingredients, as described herein.
Examples of the diseases and conditions that can be treated by the
methods and compositions of the invention include conception
(contraception), infertility, diabetes, menopause, cancer (e.g.,
hormone- or steroid-sensitive cancers, such as prostate and breast
cancer), hypertension, osteoporosis, and the like. Other examples
of diseases and conditions that can be treated by the compositions
and methods of the invention are described below.
DESCRIPTION OF THE DRAWINGS
FIG. 1A provides a schematic diagram of the various constructs of
the coactivator TIF2 employed in several of the experiments
described herein. The amino acid positions of the various domains
for TIF2 are given above the schematic diagram of each polypeptide.
See Voegel et al., 1998, EMBO J. 17, 507-519; Ma et al., 1999, Mol.
Cell. Biol. 19, 6164-6173. The abbreviations for the domains listed
above each diagram are as follows: RID, receptor interaction
domain; AD1 and AD2, activation domain 1 and 2; CBP, CBP
interaction domain; Q-rich, glutamine rich; and Neg, negative
suppressor domain. See Onate et al., 1995, Science 270,
1354-1357.
FIG. 1B provides a schematic diagram of the various constructs of
the coactivator SRC-1 employed in several of the experiments
described herein. The amino acid positions of the various domains
for the SRC-1 polypeptides are shown. See Onate et al., 1998 J.
Biol. Chem. 273, 12101-12108; Kalkhoven et al., 1998, EMBO J. 17,
232-243. The abbreviations for the domains listed above each
diagram are as follows: RID, receptor interaction domain; AD1 and
AD2, activation domain 1 and 2; CBP, CBP interaction domain;
Q-rich, glutamine rich; and Neg, negative suppressor domain. See
Onate et al., 1995, Science 270, 1354-1357.
FIG. 1C provides a schematic diagram of a Sos-Ras two-hybrid screen
to isolate STAMP. The bait, present as a chimera with Sos, will
activate the membrane-bound Ras protein in a GDP-dependent coupled
reaction to cause colony growth only if it binds to a target
protein that is fused to a membrane localization sequence.
FIG. 2A-2C illustrate the isolation and characterization of the
partial clone of the protein that binds to TIF2.4. FIG. 2A shows
the growth of yeast clones from the Sos-Ras two-hybrid screen for
TIF2.4 interacting proteins. Five clones were obtained but only two
(clones I and V) contained a protein that was needed for colony
growth in the presence of the pSos/TIF2.4 fusion protein. After
initial screening, only clone I (also named 6-6CL1) was selected
for further investigation. FIG. 2B and FIG. 2C illustrate the
selectivity of 6-6CL1 interactions with TIF2.4 in mammalian
two-hybrid assays. Triplicate samples of CV-1 cells in 24-well
plates were transiently transfected with the indicated GAL-DBD and
VP16-AD plasmids along with the GAL4-regulated reporter, pFRLuc.
The relative luciferase activity, normalized for the activity of
the internal Renilla control, was determined and plotted as the
average.+-.S.D. The much lower activity of GAL/TIF2.4m123 was
previously shown not to be due to unequal levels of protein
expression (He et al., 2002). Similar results were obtained in
three additional experiments. FIG. 2D and FIG. 2E provide a
comparison of binding to 6-6CL1 by similarly positioned domains of
SRC-1 and TIF2. Triplicate samples of CV-1 cells in 24-well plates
were analyzed as in (B&C) after being transiently transfected
with GAL.+-.6-6CL1 constructs and pFRLuc reporter along with VP16
chimeras of SRC-1 (FIG. 2D) or TIF2 (FIG. 2E) segments. Similar
results for SRC-1 were obtained in two additional experiments
(ND=not determined). Western blots (FIG. 2F) indicate that the low
activity of TIF2(1140C) (see FIG. 1) is not due to inadequate
protein expression.
FIG. 3A-F provide STAMP sequences and expression patterns. FIG.
3A1-2 provides the nucleotide sequence for human STAMP (nucleotide
sequence: SEQ ID NO:74; amino acid sequence: SEQ ID NO:72). Stop
codons that are 5' and 3' to the coding sequence are given in bold
type. The double dagger above the DNA sequence indicate the
positions of two sets of consensus Kozak sequence nucleotides
(positions 211-229) for the start of translation at the first two
codons for methionine, which would afford proteins containing
either 1281 or 1277 amino acids. The first methionine is thought
not to be the major start site as only the nucleotide at -3 to the
ATG fits the consensus Kozak sequence (Kozak, 1989). The asterisk
marks the end of the predicted, more abundant protein sequence with
1277 amino acids. A poly-A signal sequence is marked by a double
underline. FIG. 3B shows a gel with endogenous STAMP mRNA in a
human testis library as identified by RT-PCR. The human STAMP cDNA
sequence was use to select primers (single underlined sequences in
FIG. 3A) that were used to amplify STAMP mRNA sequences present in
a human testis mRNA library. The position of the predicted 4.1 kb
band is indicated by the arrow. FIG. 3C provides Northern blots of
mRNAs from different tissues probed with a [.sup.32P]labeled STAMP
oligonucleotide (bp 2093-2426). The membrane contained mRNAs of the
indicated human tissues (BD Clonetech). The presence of a common
species at about 4.6 kb was detected in all samples by
radioautography. The level of the internal control protein
.beta.-actin was determined by probing with a [.sup.32P]labeled
actin fragment (BD Clonetech). FIG. 3D1-4 provide a comparison of
human (top; SEQ ID NO:71) vs. monkey (bottom, SEQ ID NO:61) STAMP
cDNAs. FIG. 3E1-2 provide a comparison of the predicted human (top;
SEQ ID NO:72) vs. monkey (bottom, SEQ ID NO:62) STAMP amino acid
sequences.
FIG. 3F1-6 illustrate the immunocytochemical localization of
transiently transfected STAMP. U2OS.rGR cells were treated as
described in Example 1. Illumination at 570-590 or 330-360 nm was
used to locate glucocorticoid receptors (FIG. 3F2 and FIG. 3F5,
red) or HA/STAMP (FIG. 3F1 and FIG. 3F4, green), respectively. FIG.
3F1-3 show cells that were not treated with dexamethasone, while
FIG. 3F4-6 show cells that were treated with 100 nM dexamethasone.
FIG. 3F3 and FIG. 3F6 shows the signal for both glucocorticoid and
STAMP together when illuminating at both wavelengths.
Colocalization is revealed by the yellow fluorescence of the merged
pictures.
FIGS. 4A1, 4A2 and 4B illustrate STAMP modulation of GR-mediated
gene induction. FIG. 4A1-2 show that exogenous STAMP can modulate
the dose-response curve and partial agonist activity for
GR-regulated induction. Triplicate samples of CV-1 cells were
transiently transfected with GR (6 ng).+-.TIF2 (20 ng).+-.STAMP
(160 ng) plasmids, GREtkLUC reporter, and Renilla control plasmid.
Gene expression was induced with EtOH.+-. the indicated
concentrations of dexamethasone (Dex) or 1 .mu.M
dexamethasone-21-mesylate (Dex-Mes). Luciferase activities,
normalized to the internal Renilla control values, were then
expressed as a percent of the maximal response with 1 .mu.M Dex as
described in Example 1. Similar results were obtained in three
additional experiments. FIG. 4B illustrates the fold changes in GR
induction parameters by STAMP and TIF2. The absolute value of the
EC.sub.50, the partial agonist activity with 1 .mu.M Dex-Mes, and
the fold induction with 1 .mu.M Dex, were determined in four
independent experiments as described for FIG. 4A. The fold-increase
in each parameter was determined as follows: for EC.sub.50s, fold
increase=[EC.sub.50].sub.GR/[EC.sub.50].sub.GR+factor; for partial
agonist activity and fold Dex induction, fold
increase=activity.sub.GR+factor/activity.sub.GR). The average fold
increases were then plotted.+-.S.E.M (n=4).
FIG. 5A-F illustrate STAMP modulation of GR-mediated repression.
FIG. 5A shows that ectopic STAMP augments GR-repression of an AP-1
induced gene. U2OS.rGR cells were transiently transfected with
.+-.TIF2 (20 ng).+-.STAMP (100 ng) plasmids, AP-1/Luc reporter (20
ng), and Renilla control (10 ng) plasmid. The cells were induced
with ethanol (EtOH) or 25 .mu.M PMA.+-.0.1 .mu.M Dex. The
luciferase activities, normalized to the internal Renilla control
values, were then expressed as the fold increase over the basal
level response with EtOH as described in Example 1. The average
values.+-.S.E.M. of four experiments are plotted. The numbers in
parentheses above the bars labeled "PMA+Dex" represent the average
fold repression caused by Dex.+-. the various factors.
FIG. 5B illustrates that functional TIF2 is needed for optimal
STAMP activity. U2OS.rGR cells were transiently transfected and
processed as described for FIG. 5A but less STAMP (50 ng) was used
and two concentrations of TIF2 or TIF2m123 (10 and 20 ng) were
employed. The average values.+-.S.E.M. of 4-5 experiments are
plotted. An asterisk indicates a significant difference
(P.ltoreq.0.031) between samples containing equal amounts of TIF2
and TIF2m123.
FIG. 5C illustrates that STAMP and GR are co-localized on the
promoter of an endogenous regulated gene as detected by chromatin
immunoprecipitation (ChIP) assays. U2OS.rGR cells were treated with
1 .mu.M Dex and then processed after 0 to 80 min as described in
Example 1. The binding of GR and STAMP to the coll3 promoter was
determined by the ability of PCR to amplify coll3 promoter
sequences in the DNA that had been co-immunoprecipitated by anti-GR
and anti-HA (to immobilize HA/STAMP) antibodies, respectively
(upper panel). As a control for specificity, the same treatment
does not co-precipitate hsp70DNA sequences (lower panel). Similar
results were obtained in two additional experiments.
FIGS. 5D1 and 5D2 illustrate selective repression of STAMP protein
levels by STAMP siRNAs. Cos-7 cells were transiently transfected
with HA/STAMP (2 .mu.g) and GR (1 .mu.g) plasmids and .+-.5 .mu.g
of one out of the four STAMP siRNAs, or a .beta.-actin siRNA, per
60 mm dish. Cytoplasmic extracts were separated on SDS-PAGE gels
and Western blotted with anti-HA (for STAMP) or anti-GR antibodies
to detect STAMP (downward pointing open arrow) and GR (thin sideway
pointing arrows). The specificity of the effects of the siRNAs is
exemplified by the lack of effect on the other, non-specifically
detected proteins (*). Major decreases in protein level were seen
only for STAMP (open downward pointing arrow) in the presence of
STAMP siRNA and not in the presence of .beta.-actin siRNA (FIGS.
5D1 and 5D2).
FIGS. 5E and 5F show that STAMP siRNAs significantly inhibit
increased repression by exogenous (E) and endogenous (F) STAMP in
U2OS.rGR cells relative to .beta.-actin siRNA. U2OS.rGR cells were
transiently transfected with (FIG. 5E) or without (FIG. 5F)
HA/STAMP plasmid (100 ng) and .+-.500 ng of STAMP siRNA, or
.beta.-actin siRNA, plus AP-1/Luc reporter (20 ng) and the Renilla
control (10 ng) plasmid. The cells were processed and the fold
repression with each treatment was calculated as described in FIG.
5A. The average values were then plotted (.+-.S.E.M., n=4-5 (FIG.
5D) and 7 (FIG. 5E); *, P.ltoreq.0.032; **, P=0.0022).
FIGS. 6A and 6B illustrate the domains of STAMP that interact with
TIF2.4 (FIG. 6A) and GR (FIG. 6B) in the mammalian two-hybrid
assay. Triplicate samples of CV-1 cells were transiently
transfected with VP16 or VP16/TIF2.4 in FIG. 6A experiments. In
FIG. 6B experiments, triplicate samples of CV-1 cells were
transiently transfected with VP16 or VP16/GR.+-.1 .mu.M Dex. These
CV-1 cells were also transfected with GAL fused to the indicated
segments of STAMP along with the FRLuc reporter and Renilla control
plasmids. After determining the relative luciferase activities as
described in FIG. 2B and 2C, the average values.+-.S.E.M. from 2-5
experiments were plotted. The average fold increase for VP16/TIF2.4
with GAL/STAMP(623-834) in FIG. 6A was 11.0.+-.3.0 (SEM, n=5) above
the VP16 control. The values in parentheses above the data bars in
FIG. 6B indicate the fold increased interaction .+-.1 .mu.M
Dex.
FIG. 6C1-3 illustrate the association of STAMP with assorted
steroid/nuclear receptors, including glucocorticoid receptors
(GRs), progesterone receptors (PRs), androgen receptors (ARs),
estrogen receptors (ER .alpha. or .beta.), PPAR.gamma.2 receptors,
thyroid receptor beta (TR), and retinoid (.alpha.) receptors (RAR
.alpha. or RXR .alpha.). Triplicate samples of CV-1 cells were
transfected (as described for FIG. 6B) with GAL, or GAL fused to
TIF2.4 or STAMP834C, plus VP16 fused to the indicated full length
receptors and incubated with the appropriate steroid (1 .mu.M Dex,
20 nM R5020, 1 nM R1881, 0.1 .mu.M triiodothyronine, or 1 .mu.M
estradiol, roziglitazone, or 9-cis-retinoic acid). The average
relative luciferase activities of 2-3 experiments.+-.S.E.M. were
then plotted. FIG. 6C1 illustrates the effects of STAMP association
upon GR, PR and AR induced expression. FIG. 6C2 illustrates the
effects of STAMP association upon ER.alpha., ER.beta. and
PPAR.gamma.2 induced expression. FIG. 6C3 illustrates the effects
of STAMP association upon TR, RAR.alpha. and RXR.alpha. induced
expression.
FIG. 6D1-2 illustrate the effects of STAMP and TIF2 on androgen
receptor (AR, FIG. 6D1) and thyroid receptor .beta. (TR.beta., FIG.
6D2) expression. CV-1 cells were transiently transfected with AR (2
ng) or TR.beta. (10 ng).+-.TIF2 (20 ng).+-.STAMP (160 ng) plasmids,
GREtkLUC reporter, and Renilla control plasmid, and induced with
ethanol.+-.various concentrations of R1881 or triiodothyronine.
Luciferase activities, normalized to the internal Renilla control
values, and the fold changes in EC.sub.50 and fold induction were
determined and plotted as in FIG. 4B. The average fold increases
were then plotted.+-.S.E.M (n=3-4).
FIGS. 7A and 7B illustrate the domains of STAMP that bind to TIF2.4
(FIG. 7A) and GR (FIG. 7B) in cell-free pulldown assays.
Bacterially expressed GST-chimeras (GST/TIF2.4 in FIG. 7A and
GST/STAMP segments in FIG. 7B) were immobilized on
glutathione-Sepharose beads. The [.sup.35S]methionine labeled STAMP
fragments (FIG. 7A) or wild type GR (FIG. 7B) that were retained on
the matrix by binding to the GST chimeras were separated on SDS
PAGE gels and visualized by autoradiography. Specific binding was
seen as when the GST/TIF2.4 or GST/STAMP bands were darker than the
GST control bands. "10% input" shows 10% of the
[.sup.35S]methionine label that was initially loaded onto the
matrix beads. Similar results were obtained in a second
experiment.
FIG. 7C1-3 illustrate whole cell co-immunoprecipitation of STAMP,
GR, and TIF2/GRIP1. Cos-7 cells that had been co-transfected with
Flag/STAMP (or Flag itself), HA/GRIP1, and GR plasmids (10 .mu.g of
each/150 mm dish) for 48 hr were treated for 2 hr with EtOH.+-.1
.mu.M Dex before being lysed as described in Example 1. Lysate was
either analyzed on SDS-PAGE gels or treated with anti-Flag antibody
to immunoprecipitate Flag/STAMP. The pellets were separated on
SDS-PAGE gels and Western blotted with anti-Flag (FIG. 7C1),
anti-HA (FIG. 7C2), or anti-GR antibodies (FIG. 7C3). In each
panel, lane 1=5.7% of input lysate, lane 2=IP pellet of Flag/STAMP,
HA/GRIP1, and GR+EtOH, lane 3=IP pellet of Flag/STAMP, HA/GRIP1,
and GR+Dex, and lane 4=control IP pellet of Flag, HA/GRIP1, and
GR+EtOH. Similar results were obtained in a second experiment.
FIG. 8A-D illustrate the modulatory activity of STAMP versus
STAMP623C on glucocorticoid receptor (GR) mediated gene induction.
FIG. 8A schematically illustrates the structural domains of STAMP
compared to the truncated STAMP polypeptide, STAMP623C. FIG. 8B
graphically illustrates the degree of gene induction at various
dexamethasone concentration by various factors, including TIF2
(open triangles), TIF2+STAMP (closed squares) and TIF2+STAMP623C
(open squares), compared to a control where no such factor was
present (open circles). FIG. 8C-D illustrate the percent of maximal
response and fold induction, respectively, of GR-induced gene
expression by various factors, as shown. CV-1 cells were
transiently transfected with GR (6 ng).+-.TIF2 (20 ng).+-.STAMP
(160 ng) plasmids, GREtkLUC reporter, and Renilla control plasmid,
and induced with ethanol.+-. the indicated concentrations of Dex.
Luciferase activities, normalized to the internal Renilla control
values, were then expressed as percent of the maximal response with
1 .mu.M Dex as described in Example 1 and plotted as in FIG. 4.
Similar results were obtained in two additional experiments.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a new factor that can modulate
glucocorticoids-responsive gene expression. This new factor is
termed STAMP ((SRC-1 and TIF2 Associated Modulatory Protein). STAMP
polypeptides can bind to glucocorticoid receptors as well as to a
variety of glucocorticoid receptor coactivators, for example, the
TIF2 and SRC-1 glucocorticoid receptor co-activators. One target of
STAMP (i.e., TIF2) can modulate the dose-response curve and partial
agonist activity of several steroid receptors and can affect the
properties of steroid and nuclear receptors. Examples of receptors
that can be influenced by STAMP-TIF2 include androgen receptors,
estrogen receptors, mineralocorticoid receptors, progesterone
receptors, thyroid receptors, retinoid receptors (RAR and RXR), and
peroxisome proliferator-activated receptors (PPARs). Hence, STAMP
can be included in compositions and methods for modulating such
receptors. STAMP polypeptides, anti-STAMP antibodies, and STAMP
nucleic acids (including, for example, antisense nucleic acids,
ribozymes and siRNAs) can be used to modulate the activity of such
receptors and gene expression that is controlled by such receptors.
The compositions and methods of the invention can also be used to
treat or prevent a variety of conditions and diseases.
Stamp
The full-length STAMP polypeptide is about 1277 amino acid long
with a predicted molecular weight of about 143 kDa. Note that an
alternate start codon would give rise to a 1281 amino acid STAMP
polypeptide. However, the 1277 amino acid STAMP polypeptide is
predicted to be more abundant. The full-length amino acid sequence
for the 1277 human STAMP polypeptide is provided below (SEQ ID
NO:1).
TABLE-US-00001 1 MARDLEETAS SSEDEEVISQ EDHPCIMWTG GCRRIPVLVF 41
HADAILTKDN NIRVIGERYH LSYKIVRTDS RLVRSILTAH 81 GFHEVHPSST
DYNLMWTGSH LKPFLLRTLS EAQKVNHFPR 121 SYELTRKDRL YKNIIRMQHT
HGEKVFHILP QTFLLPAEYA 161 EFCNSYSKDR GPWIVKPVAS SRGRGVYLIN
NPNQISLEEN 201 ILVSRYINNP LLIDDFKFDV RLYVLVTSYD PLVIYLYEEG 241
LARFATVRYD QGAKNIRNQF MHLTNYSVNK KSGDYVSCDD 281 PEVEDYGNKW
SMSAMLRYLK QEGRDTTALM AHVEDLIIKT 321 ITSAELAIAT ACKTFVPHRS
SCFELYGFDV LIDSTLKPWL 361 LEVNLSPSLA CDAPLDLKIK ASMISDMFTV
VGFVCQDPAQ 401 RASTRPIYPT FESSRRNPFQ KPQRCRPLSA SDAEMKNLVG 441
SAREKGPGKL GGSVLGLSME EIKVLRRVKE ENDRRGGFIR 481 IFPTSETWEI
YGSYLEHKTS MNYMLATRLF QDRMTADGAP 521 ELKIESLNSK AKLHAALYER
KLLSLEVRKR RRRSSRLRAM 561 RPKYPVITQP AEMNVKTETE SEEEEEVALD
NEDEEQEASQ 601 EESAGFLREN QAKYTPSLTA LVENTPKENS MKVREWNNKG 641
GHCCKLETQE LEPKFNLMQI LQDNGNLSKM QARIAFSAYL 681 QHVQIRLMKD
SGGQTFSASW AAKEDEQMEL VVRFLKRASN 721 NLQHSLRMVL PSRRLALLER
RRILAHQLGD FIIVYNKETE 761 QMAEKKSKKK VEEEEEDGVN MENFQEFIRQ
ASEAELEEVL 801 TFYTQKNKSA SVFLGTHSKI SKNNNNYSDS GAKGDHPETI 841
MEEVKIKPPK QQQTTEIHSD KLSRFTTSAE KEAKLVYSNS 881 SSGPTATLQK
IPNTHLSSVT TSDLSPGPCH HSSLSQIPSA 921 IPSMPHQPTI LLNTVSASAS
PCLHPGAQNI PSPTGLPRCR 961 SGSHTIGPES SFQSAAHIYS QKLSRPSSAK
AGSCYLNKHH 1001 SGIAKTQKEG EDASLYSKRY NQSMVTAELQ RLAEKQAARQ 1041
YSPSSHINLL TQQVTNLNLA TGIINRSSAS APPTLRPIIS 1081 PSGPTWSTQS
DPQAPENHSS SPGSRSLQTG GFAWEGEVEN 1121 NVYSQATGVV PQHKYHPTAG
SYQLQFALQQ LEQQKLQSRQ 1161 LLDQSRARHQ AIFGSQTLPN SNLWTMNNGA
GCRISSATAS 1201 GQKPTTLPQK VVPPPSSCAS LVPKPPPNHE QVLRRATSQK 1241
ASKGSSAEGQ LNGLQSSLNP AASVPITSST DPAHTKI
This human STAMP polypeptide is encoded within a 4.6 kb cDNA (newly
deposited as Genbank Accession No. AY237126). As indicated above,
two start codons are found in naturally occurring human STAMP
nucleic acids. The first codon starts at about nucleotide 227 of
the human STAMP cDNA and has the sequence ATGCCAATCGTG (SEQ ID
NO:66), which encodes Met-Pro-Ile-Val (SEQ ID NO:67), while the
second start codon starts immediately thereafter at ATGGCCCGGGAC
(SEQ ID NO:68), which encodes Met-Ala-Arg-Asp (SEQ ID NO:69).
The sequence for this STAMP cDNA is as follows (SEQ ID NO:2).
TABLE-US-00002 1 GGCACGAGGG GGAAGCAGCC GTCGGCGGCT GCCCTGAGCC 41
TTCCTGGGGA AGGAGGAGGG AGGTAGGCGC AGAGCGCGGT 81 CCACGCCTGC
TCGCCCCGAA CCATGGGAAG ATGAGACAGG 121 AATCTGTGCC ATCCAAATTG
CTTGATCCAG TGAATCTGCT 161 AGGAAAGGTC TCTGAGGCCC CCGTCTGCTG
ACTGCATGAC 201 AAACCCTAAA GGAAATGCCA ATCGTGATGG CCCGGGACCT 241
GGAGGAAACA GCATCATCCT CAGAGGATGA GGAGGTCATA 281 AGTCAAGAGG
ATCATCCATG CATCATGTGG ACTGGAGGCT 321 GCAGGAGAAT TCCAGTTTTG
GTATTCCATG CCGACGCTAT 361 TCTTACAAAG GACAACAATA TTAGAGTAAT
TGGAGAACGT 401 TATCATTTGT CTTATAAGAT TGTACGAACG GACAGTCGCC 441
TAGTACGCAG CATTCTGACA GCCCATGGAT TTCATGAAGT 481 TCACCCAAGC
AGCACTGACT ATAACCTAAT GTGGACAGGA 521 TCCCACCTGA AGCCCTTCTT
ACTGCGCACC CTCTCTGAAG 561 CACAAAAAGT TAATCACTTT CCCAGGTCTT
ATGAACTTAC 601 CCGGAAGGAC CGACTGTACA AAAACATTAT TCGAATGCAG 641
CATACACATG GATTCAAGGT TTTTCACATC CTCCCCCAGA 681 CCTTCCTCCT
GCCAGCTGAG TACGCGGAAT TTTGTAATTC 721 ATATTCGAAG GACCGGGGAC
CTTGGATAGT AAAACCAGTG 761 GCATCTTCAA GGGGGCGGGG CGTCTACCTG
ATCAACAATC 801 CAAACCAGAT CTCCCTGGAA GAGAACATTT TGGTCTCCCG 841
TTACATTAAC AACCCCCTGC TCATAGATGA TTTCAAGTTT 881 GACGTGCGCC
TCTATGTGCT CGTGACTTCC TATGATCCTC 921 TTGTCATCTA TCTCTATGAA
GAAGGATTGG CTAGGTTTGC 961 AACTGTGCGA TATGATCAAG GAGCCAAGAA
CATTCGGAAC 1001 CAGTTCATGC ATCTGACAAA CTACAGTGTC AACAAGAAAA 1041
GTGGAGATTA CGTCAGTTGT GACGATCCAG AAGTGGAGGA 1081 TTATGGAAAC
AAATGGAGCA TGAGTGCTAT GCTTAGGTAC 1121 CTGAAACAAG AAGGCAGAGA
TACAACCGCA TTGATGGCCC 1161 ATGTAGAAGA CCTGATCATT AAGACTATAA
TCTCTGCTGA 1201 ACTAGCTATT GCTACTGCCT GTAAAACCTT TGTTCCTCAT 1241
CGCAGCAGTT GTTTTGAACT CTATGGCTTT GACGTGCTCA 1281 TAGATTCTAC
TCTGAAGCCA TGGTTGTTGG AAGTGAATCT 1321 CTCTCCTTCT TTGGCCTGTG
ATGCGCCTCT GGACCTAAAG 1361 ATTAAAGCCA GTATGATTTC AGATATGTTC
ACTGTTGTAG 1401 GATTTGTGTG CCAAGATCCT GCCCAGCGGG CATCAACTCG 1441
GCCAATTTAT CCCACCTTTG AGTCTTCCAG GCGAAACCCT 1481 TTCCAGAAAC
CTCAGCGTTG CCGTCCACTC TCTGCCAGTG 1521 ATGCGGAAAT GAAAAACCTC
GTGGGCTCAG CCCGGGAGAA 1561 AGGGCCAGGG AAGTTGGGTG GTTCTGTGCT
TGGTCTGTCA 1601 ATGGAGGAGA TCAAAGTTTT ACGAAGGGTG AAGGAGGAGA 1641
ATGATCGGCG AGGTGGATTT ATTCGCATAT TTCCTACATC 1681 TGAGACATGG
GAAATATATG GGTCCTACCT CGAGCATAAG 1721 ACCTCAATGA ACTATATGCT
GGCAACACGC CTCTTCCAGG 1761 ACAGAATGAC TGCTGATGGA GCGCCAGAAT
TGAAGATAGA 1801 GAGTCTGAAT TCAAAGGCCA AGCTGCATGC TGCACTTTAC 1841
GAGAGGAAGC TCCTGTCTCT GGAGGTGCGA AAACGTAGAC 1881 GACGGAGTAG
CAGATTGAGG GCAATGAGGC CAAAATACCC 1921 AGTGATTACC CAACCAGCTG
AAATGAATGT TAAAACTGAG 1961 ACAGAGAGTG AAGAGGAGGA AGAAGTCGCA
TTAGATAATG 2001 AAGATGAAGA ACAGGAGGCT TCCCAGGAGG AGTCTGCAGG 2041
ATTTCTTAGA GAAAATCAAG CCAAATATAC ACCCTCATTG 2081 ACAGCTTTGG
TAGAAAATAC ACCCAAAGAA AATTCCATGA 2121 AAGTTCGTGA ATGGAATAAT
AAAGGTGGAC ACTGCTGCAA 2161 ACTTGAGACT CAGGAGCTAG AGCCTAAATT
TAACCTGATG 2201 CAGATTCTTC AAGATAATGG CAATCTTAGC AAAATGCAGG 2241
CCCGAATAGC ATTCTCTGCC TATCTCCAGC ATGTTCAAAT 2281 TCGCCTGATG
AAAGACAGTG GCGGTCAGAC GTTCAGTGCC 2321 AGTTGGGCTG CCAAAGAGGA
TGAACAGATG GAGCTGGTTG 2361 TTCGTTTCCT CAAGCGAGCA TCAAATAACC
TCCAGCATTC 2401 ACTGAGGATG GTATTACCCA GTCGACGATT GGCACTTCTG 2441
GAACGCAGAA GAATCCTGGC CCACCAGCTG GGTGACTTTA 2481 TCATTGTATA
CAACAAGGAA ACAGAACAAA TGGCTGAAAA 2521 GAAATCAAAG AAGAAAGTTG
AGGAAGAAGA GGAAGATGGG 2561 GTGAATATGG AAAACTTTCA GGAGTTCATC
AGACAAGCAA 2601 GTGAGGCTGA ACTGGAGGAG GTGTTGACTT TTTATACCCA 2641
AAAGAACAAG TCTGCTAGTG TCTTCCTGGG GACTCACTCT 2681 AAAATTTCTA
AGAACAACAA CAATTATTCT GATAGTGGGG 2721 CAAAAGGTGA TCACCCTGAG
ACTATAATGG AAGAAGTGAA 2761 AATAAAGCCA CCTAAACAGC AACAGACGAC
AGAAATTCAT 2801 TCTGATAAAT TATCTCGATT TACCACTTCA GCAGAAAAAG 2841
AGGCAAAATT AGTTTATAGC AATTCCTCCT CTGGTCCTAC 2881 TGCTACTCTG
CAGAAAATTC CCAACACCCA TTTGTCATCT 2921 GTTACAACCT CTGACCTCTC
TCCAGGGCCT TGCCACCATT 2961 CTTCTTTATC TCAAATTCCT TCAGCTATCC
CCAGCATGCC 3001 TCACCAGCCA ACAATTTTAC TGAACACAGT CTCTGCCAGT 3041
GCTTCTCCCT GCCTACATCC CGGGGCACAG AACATCCCAA 3081 GCCCTACTGG
CCTGCCACGC TGTCGATCAG GAAGTCACAC 3121 CATTGGTCCC TTTTCTTCCT
TCCAAAGTGC TGCACACATC 3161 TATAGCCAGA AACTGTCTCG TCCCTCTTCA
GCAAAGGCAG 3201 GATCGTGCTA TCTAAACAAG CATCATTCAG GAATAGCCAA 3241
AACACAAAAA GAGGGAGAAG ATGCTTCTTT ATATAGCAAA 3281 CGGTACAACC
AAAGTATGGT TACAGCTGAA CTTCAGCGGC 3321 TAGCTGAGAA GCAGGCAGCG
AGACAGTATT CTCCATCCAG 3361 CCACATCAAC CTCCTCACCC AACAGGTAAC
AAACCTGAAT 3401 TTGGCAACTG GCATCATAAA CAGAAGCAGT GCTTCAGCTC 3441
CCCCAACCCT CCGACCCATC ATCAGTCCTA GTGGCCCGAC 3481 ATGGTCTACA
CAGTCAGACC CCCAAGCTCC CGAGAATCAC 3521 TCCAGCTCTC CTGGAAGCAG
GAGCCTGCAG ACAGGGGGAT 3561 TTGCCTGGGA AGGAGAAGTA GAAAACAACG
TGTACAGCCA 3601 GGCTACAGGG GTGGTCCCCC AGCACAAGTA TCACCCCACA 3641
GCAGGCAGCT ATCAGCTTCA ATTTGCCCTG CAGCAACTTG 3681 AACAACAAAA
ACTTCAGTCC CGGCAGCTCC TGGACCAGAG 3721 TCGAGCCCGG CACCAGGCAA
TCTTTGGCAG CCAGACACTA 3761 CCTAACTCCA ATTTATGGAC AATGAATAAT
GGTGCAGGTT 3801 GTAGAATTTC CAGTGCCACA GCTAGTGGCC AGAAGCCAAC 3841
CACTCTGCCA CAAAAAGTGG TACCACCTCC AAGTTCTTGC 3881 GCCTCCCTGG
TTCCCAAACC CCCACCCAAC CACGAACAAG 3921 TGCTCAGAAG GGCAACATCC
CAGAAAGCTT CCAAAGGGTC 3961 CTCCGCGGAA GGGCAGCTGA ATGGACTCCA
GAGCAGCCTT 4001 AACCCTGCAG CCTCTGTGCC CATCACCAGC TCTACAGATC 4041
CTGCTCACAC TAAAATATGA ACCACAAACA CACAGAGAAA 4081 CAACCTGTTC
ACCACTCCTG GGTGCATGAT TGAGGGTGAA 4121 GCATCCACCA GCACTTCAAG
GGGTCCATAG TATTTTTTTT 4161 TTTGCTGCCT CAAAGTCCCC AAAGCCTTCG
AGCAGAAGTG 4201 GCAGTAGATG GTTGCCAATC AGCCAATGCA GACTTTCACT 4241
GGGACAACAA GAAAGCAGAT CTTCTGGGTT TTGATGGAAC 4281 TTGGCACTGG
GGACATTCAG CTGATGCATT ATATACCCCG 4321 TCAGAGCACA CTTGTATCTT
TTACCTTCCC TTTGCCCCAT 4361 GCCCCCAAAC TGCTTAGGTC TTCTCTGTCC
CTTTACTGCT 4401 GCTGCACAGA GATGATATAA AAGAGGCTCT TTGGCTATTT 4441
GCATTTTGCT TCCTCTTCTT TTCCAGATTA CAGTATGAAG 4481 CTTTATTTTC
TTTGTACAAG CTTAAAATTT CAACATCATC 4521 ATCCGCCAAA GTTGTTCCTC
CCTTTTCGGA GGATCTAGGG 4561 GGAAAGAGGA GCATTCATCA CAAGTTTCCT
AGAGAGAGGA 4601 GACAAATCGG TGTGCCATTG ACAACATGAG CCAGGGTAAA 4641
GGCACCCTTT GGAATTACTG ATTTCAAAGA TTAATAAAGT 4681 AATTCTATTT TT
The human STAMP gene is located on chromosome 14q24.3 and has 32
introns (GenBank Accession No. NM.sub.--015072).
According to the invention, STAMP and related STAMP polypeptides
(like those STAMP-related polypeptides provided herein) can
modulate transcription of glucocorticoid-responsive genes. As used
herein, modulating transcription of glucocorticoid-responsive genes
means that STAMP can inhibit or enhance transcription from genes
that are glucocorticoid-responsive. In some embodiments, when
modulation of transcription from glucocorticoid-responsive genes is
by an agonist, modulating transcription of
glucocorticoid-responsive genes means that the concentration at
which the glucocorticoid agonist exhibits 50% of maximal activity
is shifted, meaning that more or less glucocorticoid is needed to
observe 50% activity. In other embodiments, when modulation of
transcription from glucocorticoid-responsive genes is by an
antisteroid, modulation of transcription from
glucocorticoid-responsive genes means that the amount of partial
agonist activity of an antisteroid changes, meaning that the
ability of the antisteroid to repress the induction of that gene by
agonist steroids is increased or decreased.
STAMP and STAMP-related polypeptides have a number of domains,
including a coactivator interaction domain (CID), a receptor
interaction domain (RID) and a tubulin-tyrosine ligase (TTL)
domain. The coactivator interaction domain in SEQ ID NO:1 is found
at about amino acid 623 to about amino acid 834. The receptor
interaction domain is found in SEQ ID NO:1 at about amino acid 834
to about amino acid 1277. The tubulin-tyrosine ligase domain in SEQ
ID NO:1 is found at about amino acid 113 to about amino acid 391.
These domains can readily be identified in STAMP-related
polypeptides by aligning the STAMP-related polypeptide sequences
with SEQ ID NO:1 and identifying regions of sequence identity or
homology.
STAMP nucleic acids were cloned using a Sos-Ras based yeast
two-hybrid assay, generally as described by Aronheim, 1997, Nucleic
Acids Res 25, 3373-3374. This assay was used to detect polypeptides
that bind to a TIF2 fragment (TIF2.4). The first STAMP clone
isolated using this assay encoded only amino acids 623-834 of the
STAMP polypeptide, and therefore had the following sequence (SEQ ID
NO:3).
TABLE-US-00003 623 ENTPKENS MKVREWNNKG 641 GHCCKLETQE LEPKFNLMQI
LQDNGNLSKM QARIAFSAYL 681 QHVQIRLMKD SGGQTFSASW AAKEDEQMEL
VVRFLKRASN 721 NLQHSLRMVL PSRRLALLER RRILAHQLGD FIIVYNKETE 761
QMAEKKSKKK VEEEEEDGVN MENFQEFIRQ ASEAELEEVL 801 TFYTQKNKSA
SVFLGTHSKI SKNNNNYSDS GAKG
This STAMP fragment can bind to TIF2 and acts as a coactivator
interaction domain (CID). This STAMP polypeptide fragment can also
be incorporated into compositions for modulating
glucocorticoid-responsive gene expression.
A region of STAMP that is C-terminal to the CID region interacts
with GR in a steroid-responsive manner. This region of STAMP
includes amino acids 834-1277 and has the following sequence (SEQ
ID NO:4).
TABLE-US-00004 834 GDHPETI 841 MEEVKIKPPK QQQTTEIHSD KLSRFTTSAE
KEAKLVYSNS 881 SSGPTATLQK IPNTHLSSVT TSDLSPGPCH HSSLSQIPSA 921
IPSMPHQPTI LLNTVSASAS PCLHPGAQNI PSPTGLPRCR 961 SGSHTIGPFS
SFQSAAHIYS QKLSRPSSAK AGSCYLNKHH 1001 SGIAKTQKEG EDASLYSKRY
NQSMVTAELQ RLAEKQAARQ 1041 YSPSSHINLL TQQVTNLNLA TGIINRSSAS
APPTLRPIIS 1081 PSGPTWSTQS DPQAPENHSS SPGSRSLQTG GFAWEGEVEN 1121
NVYSQATGVV PQHKYHPTAG SYQLQFALQQ LEQQKLQSRQ 1161 LLDQSRARHQ
AIFGSQTLPN SNLWTMNNGA GCRISSATAS 1201 GQKPTTLPQK VVPPPSSCAS
LVPKPPPNHE QVLRRATSQK 1241 ASKGSSAEGQ LNGLQSSLNP AASVPITSST
DPAHTKI
This C-terminal STAMP fragment can bind to glucocorticoid receptor
and provides a receptor interaction domain (RID). This STAMP
polypeptide fragment can be incorporated into compositions for
modulating glucocorticoid-responsive gene expression.
Other STAMP peptidyl fragments also have utility. For example, the
following STAMP polypeptide fragment, with amino acids 623 to 1277
has the CID and the RID regions of STAMP (SEQ ID NO:5), but not the
tubulin-tyrosine ligase (TTL) domain
TABLE-US-00005 601 ENTPKENS MKVREWNNKG 641 GHCCKLETQE LEPKFNLMQI
LQDNGNLSKM QARIAFSAYL 681 QHVQIRLMKD SGGQTFSASW AAKEDEQMEL
VVRFLKRASN 721 NLQHSLRMVL PSRRLALLER RRILAHQLGD FIIVYNKETE 761
QMAEKKSKKK VEEEEEDGVN MENFQEFIRQ ASEAELEEVL 801 TFYTQKNKSA
SVFLGTHSKI SKNNNNYSDS GAKGDHPETI 841 MEEVKIKPPK QQQTTEIHSD
KLSRFTTSAE KEAKLVYSNS 881 SSGPTATLQK IPNTHLSSVT TSDLSPGPCH
HSSLSQIPSA 921 IPSMPHQPTI LLNTVSASAS PCLHPGAQNI PSPTGLPRCR 961
SGSHTIGPFS SFQSAAHIYS QKLSRPSSAK AGSCYLNKHH 1001 SGIAKTQKEG
EDASLYSKRY NQSMVTAELQ RLAEKQAARQ 1041 YSPSSHINLL TQQVTNLNLA
TGIINRSSAS APPTLRPIIS 1081 PSGPTWSTQS DPQAPENHSS SPGSRSLQTG
GFAWEGEVEN 1121 NVYSQATGVV PQHKYHPTAG SYQLQEALQQ LEQQKLQSRQ 1161
LLDQSRARHQ AIFGSQTLPN SNLWTMNNGA GCRISSATAS 1201 GQKPTTLPQK
VVPPPSSCAS LVPKPPPNHE QVLRRATSQK 1241 ASKGSSAEGQ LNGLQSSLNP
AASVPITSST DPAHTKT
This STAMP polypeptide fragment (SEQ ID NO:5) can also be
incorporated into compositions for modulating
glucocorticoid-responsive gene expression.
The following STAMP polypeptide fragment, with amino acids 518 to
1277 has the CID and the RID regions of STAMP, as well as part of
the region between the tubulin-tyrosine ligase (TTL) domain and the
CID region(SEQ ID NO:6).
TABLE-US-00006 518 GAP 521 ELKIESLNSK AKLHAALYER KLLSLEVRKR
RRRSSRLRAM 561 RPKYPVITQP AEMNVKTETE SEEEEEVALD NEDEEQEASQ 601
EESAGFLREN QAKYTPSLTA LVENTPKENS MKVREWNNKG 641 GHCCKLETQE
LEPKFNLMQI LQDNGNLSKM QARIAFSAYL 681 QHVQIRLMKD SGGQTFSASW
AAKEDEQMEL VVRFLKRASN 721 NLQHSLRMVL PSRRLALLER RRILAHQLGD
FIIVYNKETE 761 QMAEKKSKKK VEEEEEDGVN MENFQEFIRQ ASEAELEEVL 801
TFYTQKNKSA SVFLGTHSKI SKNNNNYSDS GAKGDHPETI 841 MEEVKIKPPK
QQQTTEIHSD KLSRFTTSAE KEAKLVYSNS 881 SSGPTATLQK IPNTHLSSVT
TSDLSPGPCH HSSLSQIPSA 921 IPSMPHQPTI LLNTVSASAS PCLHPGAQNI
PSPTGLPRCR 961 SGSHTIGPES SFQSAAHIYS QKLSRPSSAK AGSCYLNKHH 1001
SGIAKTQKEG EDASLYSKRY NQSMVTAELQ RLAEKQAARQ 1041 YSPSSHINLL
TQQVTNLNLA TGIINRSSAS APPTLRPIIS 1081 PSGPTWSTQS DPQAPENHSS
SPGSRSLQTG GFAWEGEVEN 1121 NVYSQATGVV PQHKYHPTAG SYQLQFALQQ
LEQQKLQSRQ 1161 LLDQSRARHQ AIFGSQTLPN SNLWTMNNGA GCRISSATAS 1201
GQKPTTLPQK VVPPPSSCAS LVPKPPPNHE QVLRRATSQK 1241 ASKGSSAEGQ
LNGLQSSLNP AASVPITSST DPAHTKT
This STAMP polypeptide fragment (SEQ ID NO:6) can also be
incorporated into compositions for modulating
glucocorticoid-responsive gene expression.
The following STAMP polypeptide fragment, with amino acids 435 to
1242 has the CID and most of the RID regions of STAMP (SEQ ID
NO:7).
TABLE-US-00007 435 MKNLVG 441 SAREKGPGKL GGSVLGLSME EIKVLRRVKE
ENDRRGGFIR 481 IFPTSETWEI YGSYLEHKTS MNYMLATRLF QDRMTADGAP 521
ELKIESLNSK AKLHAALYER KLLSLEVRKR RRRSSRLRAM 561 RPKYPVITQP
AEMNVKTETE SEEEEEVALD NEDEEQEASQ 601 EESAGFLREN QAKYTPSLTA
LVENTPKENS MKVREWNNKG 641 GHCCKLETQE LEPKFNLMQI LQDNGNLSKM
QARIAFSAYL 681 QHVQIRLMKD SGGQTFSASW AAKEDEQMEL VVRFLKRASN 721
NLQHSLRMVL PSRRLALLER RRILAHQLGD FIIVYNKETE 761 QMAEKKSKKK
VEEEEEDGVN MENFQEFIRQ ASEAELEEVL 801 TFYTQKNKSA SVFLGTHSKI
SKNNNNYSDS GAKGDHPETI 841 MEEVKIKPPK QQQTTEIHSD KLSRFTTSAE
KEAKLVYSNS 881 SSGPTATLQK IPNTHLSSVT TSDLSPGPCH HSSLSQIPSA 921
IPSMPHQPTI LLNTVSASAS PCLHPGAQNI PSPTGLPRCR 961 SGSHTIGPFS
SFQSAAHIYS QKLSRPSSAK AGSCYLNKHH 1001 SGIAKTQKEG EDASLYSKRY
NQSMVTAELQ RLAEKQAARQ 1041 YSPSSHINLL TQQVTNLNLA TGIINRSSAS
APPTLRPIIS 1081 PSGPTWSTQS DPQAPENHSS SPGSRSLQTG GFAWEGEVEN 1121
NVYSQATGVV PQHKYHPTAG SYQLQEALQQ LEQQKLQSRQ 1161 LLDQSRARHQ
AIFGSQTLPN SNLWTMNNGA GCRISSATAS 1201 GQKPTTLPQK VVPPPSSCAS
LVPKPPPNHE QVLRRATSQK 1242 AS
This STAMP polypeptide fragment (SEQ ID NO:7) can also be
incorporated into compositions for modulating
glucocorticoid-responsive gene expression.
The following STAMP polypeptide fragment, with amino acids 378 to
1046, also has the CID region and somewhat less of the RID regions
of STAMP (SEQ ID NO:8).
TABLE-US-00008 378 KIK ASMISDMFTV VCFVCQDPAQ 401 RASTRPIYPT
FESSRRNPFQ KPQRCRPLSA SDAEMKNLVG 441 SAREKGPGKL GGSVLGLSME
EIKVLRRVKE ENDRRGGFIR 481 IFPTSETWEI YGSYLEHKTS MNYMLATRLF
QDRMTADGAP 521 ELKIESLNSK AKLHAALYER KLLSLEVRKR RRRSSRLRAM 561
RPKYPVITQP AEMNVKTETE SEEEEEVALD NEDEEQEASQ 601 EESAGFLREN
QAKYTPSLTA LVENTPKENS MKVREWNNKG 641 GHCCKLETQE LEPKFNLMQI
LQDNGNLSKM QARIAFSAYL 681 QHVQIRLMKD SGGQTFSASW AAKEDEQMEL
VVRFLKRASN 721 NLQHSLRMVL PSRRLALLER RRILAHQLGD FIIVYNKETE 761
QMAEKKSKKK VEEEEEDGVN MENFQEFIRQ ASEAELEEVL 801 TFYTQKNKSA
SVFLGTHSKI SKNNNNYSDS GAKGDHPETI 841 MEEVKIKPPK QQQTTEIHSD
KLSRFTTSAE KEAKLVYSNS 881 SSGPTATLQK IPNTHLSSVT TSDLSPGPCH
HSSLSQIPSA 921 IPSMPHQPTI LLNTVSASAS PCLHPGAQNI PSPTGLPRCR 961
SGSHTIGPFS SFQSAAHIYS QKLSRPSSAK AGSCYLNKHH 1001 SGIAKTQKEG
EDASLYSKRY NQSMVTAELQ RLAEKQAARQ 1041 YSPSSH
This STAMP polypeptide fragment (SEQ ID NO:8) can also be
incorporated into compositions for modulating
glucocorticoid-responsive gene expression.
The following STAMP polypeptide fragment, with amino acids 429 to
834, has the CID region but not the RID regions of STAMP (SEQ ID
NO:9).
TABLE-US-00009 429 SA SDAEMKNLVG 441 SAREKGPGKL GGSVLGLSME
EIKVLRRVKE ENDRRGGFIR 481 IFPTSETWEI YGSYLEHKTS MNYMLATRLF
QDRMTADGAP 521 ELKIESLNSK AKLHAALYER KLLSLEVRKR RRRSSRLRAM 561
RPKYPVITQP AEMNVKTETE SEEEEEVALD NEDEEQEASQ 601 EESAGFLREN
QAKYTPSLTA LVENTPKENS MKVREWNNKG 641 GHCCKLETQE LEPKFNLMQI
LQDNGNLSKM QARIAFSAYL 681 QHVQIRLMKD SGGQTFSASW AAKEDEQMEL
VVRFLKRASN 721 NLQHSLRMVL PSRRLALLER RRILAHQLGD FIIVYNKETE 761
QMAEKKSKKK VEEEEEDGVN MENFQEFIRQ ASEAELEEVL 801 TFYTQKNKSA
SVFLGTHSKI SKNNNNYSDS GAKG
This STAMP polypeptide fragment (SEQ ID NO:9) can also be
incorporated into compositions for modulating
glucocorticoid-responsive gene expression.
The following STAMP polypeptide fragment, with amino acids 518 to
834, has the CID region but not the RID regions of STAMP (SEQ ID
NO:10).
TABLE-US-00010 518 GAP 521 ELKIESLNSK AKLHAALYER KLLSLEVRKR
RRRSSRLRAM 561 RPKYPVITQP AEMNVKTETE SEEEEEVALD NEDEEQEASQ 601
EESAGFLREN QAKYTPSLTA LVENTPKENS MKVREWNNKG 641 GHCCKLETQE
LEPKFNLMQI LQDNGNLSKM QARIAFSAYL 681 QHVQIRLMKD SGGQTFSASW
AAKEDEQMEL VVRFLKRASN 721 NLQHSLRMVL PSRRLALLER RRILAHQLGD
FIIVYNKETE 761 QMAEKKSKKK VEEEEEDGVN MENFQEFIRQ ASEAELEEVL 801
TPYTQKNKSA SVFLGTHSKI SKNNNNYSDS GAKG
This STAMP polypeptide fragment (SEQ ID NO:10) can also be
incorporated into compositions for modulating
glucocorticoid-responsive gene expression.
The following STAMP polypeptide fragment, with amino acids 956 to
1277 has most of the RID regions of STAMP but no CID region (SEQ ID
NO:11).
TABLE-US-00011 956 LPRCR 961 SGSHTIGPFS SFQSAAHIYS QKLSRPSSAK
AGSCYLNKHH 1001 SGIAKTQKEG EDASLYSKRY NQSMVTAELQ RLAEKQAARQ 1041
YSPSSHINLL TQQVTNLNLA TGIINRSSAS APPTLRPIIS 1081 PSGPTWSTQS
DPQAPENHSS SPGSRSLQTG GFAWEGEVEN 1121 NVYSQATGVV PQHKYHPTAG
SYQLQFALQQ LEQQKLQSRQ 1161 LLDQSRARHQ AIFGSQTLPN SNLWTMNNGA
GCRISSATAS 1201 CQKPTTLPQK VVPPPSSCAS LVPKPPPNHE QVLRRATSQK 1241
ASKGSSAEGQ LNGLQSSLNP AASVPITSST DPAHTKI
This STAMP polypeptide fragment (SEQ ID NO:11) can also be
incorporated into compositions for modulating
glucocorticoid-responsive gene expression.
The following STAMP polypeptide fragment, with amino acids 956 to
1127 has a portion of the RID region of STAMP (SEQ ID NO:12).
TABLE-US-00012 956 LPRCR 961 SGSHTIGPFS SFQSAAHIYS QKLSRPSSAK
AGSCYLNKHH 1001 SGIAKTQKEG EDASLYSKRY NQSMVTAELQ RLAEKQAARQ 1041
YSPSSHINLL TQQVTNLNLA TCIINRSSAS APPTLRPIIS 1081 PSGPTWSTQS
DPQAPENHSS SPGSRSLQTG GFAWEGEVEN 1121 NVYSQAT
This STAMP polypeptide fragment (SEQ ID NO:12) can also be
incorporated into compositions for modulating
glucocorticoid-responsive gene expression.
The following STAMP polypeptide fragment, with amino acids 1127 to
1277 has the C-terminal part of the RID regions of STAMP (SEQ ID
NO:13).
TABLE-US-00013 1127 TGVV PQHKYHPTAG SYQLQFALQQ LEQQKLQSRQ 1161
LLDQSRARHQ AIFGSQTLPN SNLWTMNNGA GCRISSATAS 1201 GQKPTTLPQK
VVPPPSSCAS LVPKPPPNHE QVLRRATSQK 1241 ASKGSSAEGQ LNGLQSSLNP
AASVPITSST DPAHTKI
This STAMP polypeptide fragment (SEQ ID NO:13) can also be
incorporated into compositions for modulating
glucocorticoid-responsive gene expression.
The following STAMP polypeptide fragment, with amino acids 1-834
has the tubulin-tyrosine ligase (TTL) domain (at amino acids
113-391) and the CID region (SEQ ID NO:14).
TABLE-US-00014 1 MARDLEETAS SSEDEEVISQ EDHPCIMWTG GCRRIPVLVF 41
HADAILTKDN NIRVIGERYH LSYKIVRTDS RLVRSILTAH 81 GFHEVHPSST
DYNLMWTGSH LKPFLLRTLS EAQKVNHFPR 121 SYELTRKDRL YKNIIRMQHT
HGFKVFHILP QTFLLPAEYA 161 EFCNSYSKDR GPWIVKPVAS SRGRGVYLIN
NPNQISLEEN 201 ILVSRYINNP LLIDDFKFDV RLYVLVTSYD PLVIYLYEEG 241
LARFATVRYD QGAKNIRNQF MHLTNYSVNK KSGDYVSCDD 281 PEVEDYGNKW
SMSAMLRYLK QEGRDTTALM AHVEDLIIKT 321 IISAELAIAT ACKTFVPHRS
SCFELYGFDV LIDSTLKPWL 361 LEVNLSPSLA CDAPLDLKIK ASMISDMFTV
VGFVCQDPAQ 401 RASTRPIYPT FESSRRNPFQ KPQRCRPLSA SDAEMKNLVG 441
SAREKGPGKL GGSVLGLSME EIKVLRRVKE ENDRRGGFIR 481 IFPTSETWEI
YGSYLEHKTS MNYMLATRLF QDRMTADGAP 521 ELKIESLNSK AKLHAALYER
KLLSLEVRKR RRRSSRLRAM 561 RPKYPVITQP AEMNVKTETE SEEEEEVALD
NEDEEQEASQ 601 EESAGFLREN QAKYTPSLTA LVENTPKENS MKVREWNNKG 641
GHCCKLETQE LEPKFNLMQI LQDNGNLSKM QARIAFSAYL 681 QHVQIRLMKD
SGGQTFSASW AAKEDEQMEL VVRFLKRASN 721 NLQHSLRMVL PSRRLALLER
RRILAHQLGD FIIVYNKETE 761 QMAEKKSKKK VEEEEEDGVN MENFQEFIRQ
ASEAELEEVL 801 TFYTQKNKSA SVFLGTHSKI SKNNNNYSDS GAKG
This STAMP polypeptide fragment (SEQ ID NO:14) can also be
incorporated into compositions for modulating
glucocorticoid-responsive gene expression.
The following STAMP polypeptide fragment, with amino acids 1-623
has the tubulin-tyrosine ligase (TTL) domain (at amino acids
113-391) but not the CID region (SEQ ID NO:15).
TABLE-US-00015 1 MARDLEETAS SSEDEEVISQ EDHPCIMWTG GCRRIPVLVF 41
HADAILTKDN NIRVIGERYH LSYKIVRTDS RLVRSILTAH 81 GFHEVHPSST
DYNLMWTGSH LKPFLLRTLS EAQKVNHFPR 121 SYELTRKDRL YKNIIRMQHT
HGFKVFHILP QTFLLPAEYA 161 EFCNSYSKDR GPWIVKPVAS SRGRGVYLIN
NPNQISLEEN 201 ILVSRYINNP LLIDDFKFDV RLYVLVTSYD PLVIYLYEEG 241
LARFATVRYD QGAKNIRNQF MHLTNYSVNK KSGDYVSCDD 281 PEVEDYGNKW
SMSAMLRYLK QEGRDTTALM AHVEDLIIKT 321 IISAELAIAT ACKTFVPHRS
SCFELYGFDV LIDSTLKPWL 361 LEVNLSPSLA CDAPLDLKIK ASMISDMFTV
VGFVCQDPAQ 401 RASTRPTYPT FESSRRNPFQ KPQRCRPLSA SDAEMKNLVG 441
SAREKGPGKL GGSVLGLSME EIKVLRRVKE ENDRRGGFIR 481 IFPTSETWEI
YGSYLEHKTS MNYMLATRLF QDRMTADGAP 521 ELKIESLNSK AKLHAALYER
KLLSLEVRKR RRRSSRLRAM 561 RPKYPVITQP AEMNVKTETE SEEEEEVALD
NEDEEQEASQ 601 EESAGFLREN QAKYTPSLTA LVE
The following STAMP polypeptide fragment, with amino acids 834-956
has the N-terminal portion the RID regions of STAMP (SEQ ID
NO:16).
TABLE-US-00016 834 GDHPETI 841 MEEVKIKPPK QQQTTEIHSD KLSRFTTSAE
KEAKLVYSNS 881 SSGPTATLQK IPNTHLSSVT TSDLSPGPCH HSSLSQIPSA 921
IPSMPHQPTI LLNTVSASAS PCLHPGAQNI PSPTGL
Moreover, according to the invention, STAMP-related polypeptides
with sequences similar but not necessarily identical to SEQ ID NO:1
can be used in the compositions of the invention. Thus, for
example, a sequence related to human STAMP has been newly deposited
as Genbank Accession No. NP 055887 (gi: 50658079). According to the
invention, this STAMP-related polypeptide, and fragments thereof,
can be used in the compositions and methods of the invention. This
STAMP-related polypeptide sequence is provided below for easy
reference (SEQ ID NO:63).
TABLE-US-00017 1 MPIVMARDLE ETASSSEDEE VISQEDHPCI MWTGGCRRIP 41
VLVFHADAIL TKDNNIRVIG ERYHLSYKIV RTDSRLVRSI 81 LTAHGFHEVH
PSSTDYNLMW TGSHLKPFLL RTLSEAQKVN 121 HFPRSYELTR KDRLYKNIIR
MQHTHGFKAF HILPQTFLLP 161 AEYAEFCNSY SKDRGPWIVK PVASSRGRGV
YLINNPNQIS 201 LEENILVSRY INNPLLIDDF KFDVRLYVLV TSYDPLVIYL 241
YEEGLARFAT VRYDQGAKNI RNQFMHLTNY SVNKKSGDYV 281 SCDDPEVEDY
GNKWSMSAML RYLKQEGRDT TALMAHVEDL 321 IIKTIISAEL AIATACKTFV
PHRSSCFELY GFDVLIDSTL 361 KPWLLEVNLS PSLACDAPLD LKIKASMISD
MFTVVGFVCQ 401 DPAQRASTRP IYPTFESSRR NPFQKPQRCR PLSASDAEMK 441
NLVGSAREKG PGKLGGSVLG LSMEEIKVLR RVKEENDRRG 481 GFIRIFPTSE
TWEIYGSYLE HKTSMNYMLA TRLFQDRMTA 521 DGAPELKIES LNSKAKLHAA
LYERKLLSLE VRKRRRRSSR 561 LRAMRPKYPV ITQPAEMNVK TETESEEEEE
VALDNEDEEQ 601 EASQEESAGF LRENQAKYTP SLTALVENTP KENSMKVREW 641
NNKGGHCCKL ETQELEPKFN LMQILQDNGN LSKMQARIAF 681 SAYLQHVQIR
LMKDSGGQTF SASWAAKEDE QMELVVRFLK 721 RASNNLQHSL RMVLPSRRLA
LLERRRILAH QLGDFIIVYN 761 KETEQMAEKK SKKKVEEEEE DGVNMENFQE
FIRQASEAEL 801 EEVLTFYTQK NKSASVFLGT HSKISKNNNN YSDSGAKGDH 841
PETIMEEVKI KPPKQQQTTE IHSDKLSRFT TSAEKEAKLV 881 YSNSSSGPTA
TLQKIPNTHL SSVTTSDLSP GPCHHSSLSQ 921 IPSAIPSMPH QPTILLNTVS
ASASPCLHPG AQNIPSPTGL 961 PRCRSGSHTI GPFSSFQSAA HIYSQKLSRP
SSAKAGSCYL 1001 NKHHSGIAKT QKEGEDASLY SKRYNQSMVT AELQRLAEKQ 1041
AARQYSPSSH INLLTQQVTN LNLATGIINR SSASAPPTLR 1081 PIISPSGPTW
STQSDPQAPE NHSSSPGSRS LQTGGFAWEG 1121 EVENNVYSQA TGVVPQHKYH
PTAGSYQLQF ALQQLEQQKL 1161 QSRQLLDQSR ARHQAIFGSQ TLPNSNLWTM
NNGAGCRISS 1201 ATASGQKPTT LPQKVVPPPS SCASLVPKPP PNHEQVLRRA 1241
TSQKASKGSS AEGQLNGLQS SLNPAAFVPI TSSTDPAHTK 1281 I
An orangutan (Pongo pygmaeus) sequence related to human STAMP has
been newly deposited as Genbank Accession No. NP CAH91681 (gi:
55729911). According to the invention, this STAMP-related
polypeptide, and fragments thereof, can be used in the compositions
and methods of the invention. This STAMP-related orangutan
polypeptide sequence is provided below for easy reference (SEQ ID
NO:64).
TABLE-US-00018 1 MPIVMARDLE ETASSSEDEE VISQEDHPCI MWTGGCRRIP 41
VLVFHADAIL TKDNNIRVIG ERYHLSYKIV RTDSRLVRSI 81 LTAHGFHEVH
PSSTDYNLMW TGSHLKPFLL RTLSEAQKVN 121 HFPRSYELTR KDRLYKNIIR
MQHTHGFKAF HILPQTELLP 161 AEYAEFCNSY SKDRGPWIVK PVASSRGRGV
YLINNPNQIS 201 LEENILVSRY INNPLLIDDF KFDVRLYVLV TSYDPLVIYL 241
YEEGLARFAT VRYDQGAKNI RNQFMHLTNY SVNKKSGDYV 281 SCDDPEVEDY
GNKWSMSAML RYLKQEGRDT TALMAHVEDL 321 IIKTIISAEL AIATACKTFV
PHRSSCFELY GFDVLIDSTL 361 KPWLLEVNLS PSLACDAPLD LKIKASMISD
MFTVVGFVCQ 401 DPAQRASTRP IYPTFESSRR NPFQKPQRCR PLSASDAEMK 441
NLVGSAREKG PGKLGGSVLG LSMEEIKVLR RVKEENDRRG 481 GFIRIFPTSE
TWEIYGSYLE HKTSMNYMLA TRLFQDRGNP 521 RRSLLTGRTR MTADGAPELK
IESLNSKAKL HAALYERKLL 561 SLEVRKRRRR SSRLRAMRPK YPVITQPAEM
NVKTETESEE 601 EEEVALDNEE EEQEASQEES AGFLRENQAK YTPSLTALVE 641
NTPKEHSMKV REWNNKGGHC CKLETQELEP KFNLVQILQD 681 NGNLSKVQAR
IAFSAYLQHV QIRLMKDSGG QTFSASWAAK 721 EDEQMELVVR FLKRASNNLQ
HSLRMVLPSR RLALLERRRI 761 LAHQLGDFII VYNKETEQMA EKKSKKKVEE
EEEDGVNMEN 801 FQEFIRQASE AELEEVLTFY TQKNKSASVF LGTHSKSSKN 841
NNSYSDSGAK GDHPETIMEE VKIKPPKQQQ TTEIHSDKLS 881 RFTTSAEKEA
KLVYSNSSST PFSGPTATLQ KIPNTHLSSV 921 TTSDLSPGPG HHSSLSQIPS
AIPSMPHQPT VLLNTVSASA 961 SPCLHTGTQN IPNPAGLPRC RSGSHTIGPF
SSFQSAAHIY 1001 SQKLSRPSSA KAAGSCYLNK HHSGIAKTQK EGEDASSYSK 1041
RYNQSMVTAE LQRLAEKQAA RQYSPSSHIN LLTQQVTNLN 1081 LATGIINRSS
ASTPPTLRPI ISPSGPTWST QSDPQAPENH 1121 SSPPGSRSLQ TGVFAWEGEV
ENNVYSKATG VVPQHKYHPT 1161 AGSYQLHFAL QQLEQQKLQS RQLLDQSRAR
HQAIFGSQTL 1201 PNSNLWTMNN GAGCRISSAT ASGQKPTTLP QKVVPPPSSC 1241
ASLVPKPPPN HKQVLRRATS QRASKGSSAE GQLNGLQSSL 1281 NPAAFVPITS
STDPAHTKI
A human STAMP polypeptide with a 1281 amino acid sequence, where
translation began at the first start codon has SEQ ID NO:70,
provided below. The amino acid numbering provided below is used
simply to illustrate the N-terminal extension that constitutes the
difference between the predominant form of STAMP (SEQ ID NO:1) and
the 1281 amino acid form of STAMP with SEQ ID NO:70.
TABLE-US-00019 -4 MPIV 1 MARDLEETAS SSEDEEVISQ EDHPCIMWTG
GCRRIPVLVF 41 HADAILTKDN NIRVIGERYH LSYKIVRTDS RLVRSILTAH 81
GFHEVHPSST DYNLMWTGSH LKPFLLRTLS EAQKVNHFPR 121 SYELTRKDRL
YKNIIRMQHT HGFKVFHILP QTFLLPAEYA 161 EFCNSYSKDR GPWIVKPVAS
SRGRGVYLIN NPNQISLEEN 201 ILVSRYINNP LLIDDFKFDV RLYVLVTSYD
PLVIYLYEEG 241 LARFATVRYD QGAKNIRNQF MHLTNYSVNK KSGDYVSCDD 281
PEVEDYGNKW SMSAMLRYLK QEGRDTTALM AHVEDLIIKT 321 IISAELAIAT
ACKTFVPHRS SCFELYGFDV LIDSTLKPWL 361 LEVNLSPSLA CDAPLDLKIK
ASMISDMFTV VGFVCQDPAQ 401 RASTRPIYPT FESSRRNPFQ KPQRCRPLSA
SDAEMKNLVG 441 SAREKGPGKL GGSVLGLSME EIKVLRRVKE ENDRRGGFIR 481
IFPTSETWEI YGSYLEHKTS MNYMLATRLF QDRMTADGAP 521 ELKIESLNSK
AKLHAALYER KLLSLEVRKR RRRSSRLRAM 561 RPKYPVITQP AEMNVKTETE
SEEEEEVALD NEDEEQEASQ 601 EESAGFLREN QAKYTPSLTA LVENTPKENS
MKVREWNNKG 641 GHCCKLETQE LEPKFNLMQI LQDNGNLSKM QARIAFSAYL 681
QHVQIRLMKD SGGQTFSASW AAKEDEQMEL VVRFLKRASN 721 NLQHSLRMVL
PSRRLALLER RRILAHQLGD FIIVYNKETE 761 QMAEKKSKKK VEEEEEDGVN
MENFQEFIRQ ASEAELEEVL 801 TFYTQKNKSA SVFLGTHSKI SKNNNNYSDS
GAKGDHPETI 841 MEEVKIKPPK QQQTTEIHSD KLSRFTTSAE KEAKLVYSNS 881
SSGPTATLQK IPNTHLSSVT TSDLSPGPCH HSSLSQIPSA 921 IPSMPHQPTI
LLNTVSASAS PCLHPGAQNI PSPTGLPRCR 961 SGSHTTGPFS SFQSAAHIYS
QKLSRPSSAK AGSCYLNKHH 1001 SGIAKTQKEG EDASLYSKRY NQSMVTAELQ
RLAEKQAARQ 1041 YSPSSHINLL TQQVTNLNLA TGIINRSSAS APPTLRPIIS 1081
PSGPTWSTQS DPQAPENHSS SPGSRSLQTG GFAWEGEVEN 1121 NVYSQATGVV
PQHKYHPTAG SYQLQFALQQ LEQQKLQSRQ 1161 LLDQSRARHQ AIFGSQTLPN
SNLWTMNNGA GCRISSATAS 1201 GQKPTTLPQK VVPPPSSCAS LVPKPPPNHE
QVLRRATSQK 1241 ASKGSSAEGQ LNGLQSSLNP AASVPITSST DPAHTKI
The invention is directed to all these STAMP polypeptides and to
functionally active fragments thereof. STAMP Nucleic Acids
As described herein STAMP polypeptides are encoded by STAMP nucleic
acids, for example, a nucleic acid comprising SEQ ID NO:2 or SEQ ID
NO:61. In one embodiment, the invention provides STAMP sense and/or
anti-sense nucleic acids, as well as related RNA or DNA molecules.
Such STAMP sense and/or anti-sense nucleic acids, as well as
related RNA or DNA molecules can be used to modulate STAMP
expression, translation and/or the degradation of STAMP
transcripts. For example, an anti-sense RNA or DNA that can
hybridize to a nucleic acid having SEQ ID NO:2 or SEQ ID NO:61 can
be used for diminishing the expression of STAMP. The degradation of
STAMP mRNA may also be increased upon exposure to small duplexes of
synthetic double-stranded RNA through the use of RNA interference
(RNAi) technology (Scherr, M. et al. 2003, Curr. Med. Chem. 10:245;
Martinez, L. A. et al. 2002 PNAS 99: 14849).
In one embodiment, a disease where glucocorticoid responsive gene
expression is undesirably active, or inactive, can be treated by
administering to a mammal a nucleic acid that can inhibit, or
augment, the functioning of a STAMP RNA. Nucleic acids that can
inhibit the function of a STAMP RNA can be generated from coding
and non-coding regions of the STAMP gene. However, nucleic acids
that can inhibit the function of a STAMP RNA are often selected to
be complementary to sequences near the 5' end of the coding region.
Hence, in some embodiments, the nucleic acid that can inhibit the
functioning of a STAMP RNA can be complementary to sequences near
the 5' end of SEQ ID NO:2. In other embodiments, nucleic acids that
can inhibit the function of a STAMP RNA can be complementary to SEQ
ID NO:2 or to STAMP RNAs from other species (e.g., mouse or a
monkey STAMP RNA).
A nucleic acid that can inhibit the functioning of a STAMP RNA need
not be 100% complementary to a selected region of SEQ ID NO:2.
Instead, some variability the sequence of the nucleic acid that can
inhibit the functioning of a STAMP RNA is permitted. It has
recently been shown that a string of eleven nucleotides in a 21-23
RNA duplex is sufficient to complex with an mRNA species and cause
silencing of expression from the gene encoding this mRNA. See
Jackson et al. (2003) Nat. Biotechnol. 21: 635-37. Thus, perfect
complementarity of a 21-23 bp siRNA is not needed for gene
silencing. A nucleic acid that can inhibit the functioning of a
mouse STAMP RNA, for example, can therefore be complementary to a
nucleic acid encoding a mouse or monkey STAMP gene product. A
nucleic acid encoding African green monkey (Cercopithecus aethiops)
STAMP gene product, for example, can be found in the NCBI database
at GenBank Accession No. AY383558). This monkey STAMP cDNA is 98%
identical to the human STAMP cDNA (FIG. 3D; alignment from BLAST on
GCG). The sequence for this monkey STAMP cDNA is as follows (SEQ ID
NO:61).
TABLE-US-00020 1 TGAATCTGCt aGGAAAGGtc tcTGAGGCCC CCGTCTGCCG 41
ACTGCATGAC AAACCCTAAA GGAAATGCCA GTCGTGATGG 81 CCCGGGACCT
GGAGGAAACA GCATCATCCT CAGAGGATGA 121 GGAGGTCATA AGTCAAGAGG
ATCATCCATG CATCATGTGG 161 ACTGGAGGCT GTAGGAGAAT TCCAGTTTTG
GTATTCCATG 201 CCGACGCTAT TCTTACAAAG GACAACAATA TTAGAGTAAT 241
TGGAGAACGT TATCATTTGT CTTATAAGAT TGTACGAACG 281 GACAGTCGCC
TAGTACGCAG CATTCTGACA GCCCATGGAT 321 TTCATGAAGT TCACCCAAGC
AGCACTGACT ATAACCTAAT 361 GTGGACAGGA TCCCACCTGA AGCCCTTCTT
ACTGCGCACC 401 CTCTCTGAAG CACAAAAAGT TAATCACTTT CCCAGGTCTT 441
ATGAACTTAC CCGGAAGGAC CGACTGTACA AAAACATTAT 481 TCGAATGCAg
CATACACATG GATTCAAGGC TTTtCACATC 521 CTCCCCCaGA CCTTCCTCCT
GCCAGCTGAG TACGCGGAAT 561 TTTGTAATTC ATATTCGAAG GACCGGGGAC
CTTGGATAGT 601 AAAACCAGTG GCATCTTCTA GGGGGCGGGG CGTCTACCTG 641
ATCAACAATC CAAACCAGAT TTCCCTGGAA GAAAACATTC 681 TGGTCTCCCG
TTATATTAAC AACCCCCTGC TCATAGATGA 721 TTTCAAGTTT GATGTGCGCC
TCTATGTGCT GGTGACTTCC 761 TATGATCCTC TTGTCATCTA TCTCTATGAA
GAAGGATTGG 801 CTAGGTTTGC AACTGTGCGA TATGATCAAG GAGCCAAGAA 841
CATTCGGAAC CAGTTCATGC ATCTGACAAA CTACAGTGTG 881 AACAAGAAGA
GTGGAGACTA CGTCAGTTGT GATGATCCAG 921 AAgtGGAGGA CTATGGAAAC
AAATGGAGCA TGAgTGCTAT 961 GCTTAGGTAC CTGAAACAAG AAGGCAGAGA
TACAACTGCA 1001 TTGATGGCCC ATGTAGAAGA CCTGATCATT AAGACTATAA 1041
TCTCTGCTGA ACTAGCTATT GCTACTGCCT GTAAAACCTT 1081 TGTTCCTCAT
CGCAGCAGTT GTTTTGAACT CTATGGCTTT 1121 GACGTGCTCA TAGATGCTAC
TCTGAAGCCA TGGTTGTTGG 1161 AAGTGAATCT CTCTCCTTCT TTGGCCTGTG
ATGCACCTCT 1201 GGACCTAAAG ATTAAAGCCA GTATGAtTTC AGATATGTTC 1241
ACTGTTGTTG GATTTGTGTG CCAAGATCCT GCCCAGCGGG 1281 CATCAACCCG
GCCAATTTAT CCCACCTTTG AGTCTTCCAG 1321 GCGAAACCCT TTCCAGAAAC
CTcagcgtcc acttccagca 1361 cagtttcatt catcagagcc aaagCAGCgT
TCCCGTCCAC 1401 TCTCTGCcAg TgatGCGGAA ATGAAAAACC TCGTGGGCTC 1441
AGCCCGGGAG AAAGGGCCAG GGAAGTTGGG TGGTTCTGTG 1481 CTTGGTCTGT
CAATGGAGGA GATCAAAGTT TTACGGAGGG 1521 TGAAGGAGGA GAATGATCGG
AGAGGTGGAT TTATTCGCAT 1561 ATTTCCTACA TCTGAGACAT GGGAAATATA
TGGGTCCTAC 1601 CTCGAGCATA AGACCTCAAT GAACTATATG CTGGCAACAC 1641
GCCTCTTCCA GGACAGaatg ACtGCtGAtG GAGCACCAGA 1681 ATTGAAGATA
GAGGGCCTGA ATTCAAAGGC CAAGCTGCAT 1721 GCTGCACTTT ACGAGAGGAA
GCTCCTGTCT CTGGAGGTGC 1761 GAAAACGTAG ACGACGGAGT AGCAGATTGA
GGGCAATGAG 1801 GCCAAAATAC CCAGTGATTA CCCAACCAGC TGAAATGAAT 1841
GTTAAAACTG AGACAGAGAG TGAAGAGGAG GAAGAAGTCG 1881 CATTAGACAA
TGAAGATGAA GAGCAGGAAG CTTCCCAGGA 1921 GGAGTCTGCA GGATTTCTTA
GAgAAAATCA AGCCAAAGAT 1961 ACACCCTCAT TGACAACTTT GGTAGAAAAT
ACACCCAAAG 2001 AAAATTCCGT GAAAGTTCGT GAATGGAGTA aAAAAGGTGA 2041
ACGGTGCTGC AAACTTGAGA CTCAGGAGCT GGAGCCTAAA 2081 TTTAACCTGA
TGCAGGTTCT TCAAGATAAC GGCAATCTTA 2121 GCAAAGTGCA GGCCCGAATA
GCATTCTCTA CCTATCTCCA 2161 GCATGTTCAA ATTCGCCTGA TGAAAGACAG
TGGAGGTCAG 2201 ACGTTCAGTG CCAGTTGGGC TGCCAAAGAG GATGAACAGA 2241
TGGAGCTGGT CGTTCGTTTC CTCAAGCGAG CATCAAATAA 2281 CCTTCAGCAG
TCACTGAGGA TGGTATTACC CAGCCGACGA 2321 TTGGCACTTC TGGAACGCAG
AAGAATCCTG GCCCACCAGC 2361 TGGGTGACTT TATCATTGTA TACAACAAGG
AAACAGAACA 2401 AATGGCTGAA AAGAAaTCAA AGAAGAAAGT TGAAGAAGAA 2441
GAGGAgGATG GaGTGAATAT GGAAAACTTT CAGGAGTTCA 2481 TCAGACAAGC
AAGTGAGGCT GAACTGGAGG AGGTGTTGAC 2521 TTTTTATACC CAAAAGAACA
AGTCTGCTAG TGTCTTCCTG 2561 GGGACTCACT CTAAAAGTTC TAAGAACAAC
AACAGTTATT 2601 CTGATAGTGG GGCAAAAGGT GATCACCCTG AGACTGTAAT 2641
GGAAGAAGCG AAAATGAAGC CGCCTAAACA GCAACAGACA 2681 ACAGAAATTC
ACTCTGATAA ATTATCTCGA TTTACCACTT 2721 CAGCAgAAAA AGAGGCAAAA
TTAGTTTATA CCAGTTCTTC 2761 GTCGaCTCCT TTCTCTGGTC CTACTGCTAC
TCTGCAGAAA 2801 ATTCCCAACA CCCATTTGTC ATCTGTTACA ACCTCAGACC 2841
TCTCTCCAGG GCCTGGCCAC CATTCTTCTT TATCTCAAAT 2881 TCCTTCAGCT
ATCCCCAGCA TGCCTCACCA GCCAACAATT 2921 TTACTGAACA CAGTCTCTGC
CAGTGCTTCT CCCTCCCTAC 2961 ATCCTGGGAC ACAGAACATC CCAAGCCCTG
CTGGCCTGCC 3001 TCGCTGTCGA TCAGGAAGTC ACACCATTGG CTCCTTTTCT 3041
TCCTTCCAAA GTGCTGCACA CATCTATAGC CAGAAACTGT 3081 CTCGTCCCTC
TTCAGCAAAG GCAGGATcgT GCTaTCTAaa 3121 cAAgCATCAT TCAgGAAtAG
CCAAaACACA ACAAGAGGGA 3161 GAAGATGCTT CTTTATATAG CAAACGGTAC
AACCAAAGTA 3201 TGGTTACAGC TgAACTTCAG CGgCTAGCtG AGAAGCAGGC 3241
AGCGAGACAG TATTCTCCAt CCAGCCACAT CAACCTCCTC 3281 ACCCAACAGG
TGACAAACTT GAATTTGGCC ACTGGCATCA 3321 TAAACAGAAG CAGTGCTTCA
ACTCCCCCCA CCCTCCAACC 3361 CATCATCAGC CCTAGTGGCC CCACATGGTT
GGTGCAGTCG 3401 GACCCTCAAG CTCCTGAGAA TCACTCCAGC CCTCCCAGAA 3441
GCAGGAGCCT CCAGACAGGT GGGTTTGCCT GGGAAGGAGA 3481 GGTAGAAAAC
AACGTGTACA GCAAGGCTAC CGGGGTGGTC 3521 CCCCAGCACA AGTATCACCC
CACAGCAGGC AGCTATCAGC 3561 TCCATTTTGC CCTGCAGCAA CTTGAACAAC
AAAAACTTCA 3601 GTCCCGGCAG CTCCTGGACC AGAGTCGAGc CCGGCACCAG 3641
GCAATCTTTG GCAGCCAGAC ACTACCTAAC TCCAATTTAT 3681 GGACAATGAA
TAATGGTGCA ggTTGTAGAA TTTCCAGTGC 3721 CACAGCTAGT GGCCAGAAGC
CAACCACTCT GCCACAAAAA 3761 GCAGTACCAC CTCCAAGCTC TTGCGCCTCC
CTGGTCCCCA 3801 AACCCCCTCC CAACCACAAA CAAGTGCTCA GAAGGGCAAC 3841
ATCCCAgAGC GCTTCCAAAG GGTCCTCgGC AtATGCGCAG 3881 CTGAaTGGAC
TCCAgAGCAg CCTTaAcccT GCAgCCTcTG 3921 TGCCCATCAC CAGCTCCACA
GATCCTGCTC ACACTAAAAG 3961 ATGAACCACA AACACACAGA GAAACGAcCT
GTTCACCACT 4001 CCTGGGGTGC ATCTAGAGCA T
This green monkey cDNA clone is predicted to encode a protein that
is 97% identical to the human STAMP protein (FIG. 3E). The green
monkey STAMP polypeptide has the following sequence (SEQ ID
NO:62).
TABLE-US-00021 1 MPVVMARDLE ETASSSEDEE VISQEDHPCI MWTGGCRRIP 41
VLVFHADAIL TKDNNIRVIG ERYHLSYKIV RTDSRLVRSI 81 LTAHGFHEVH
PSSTDYNLMW TGSHLKPFLL RTLSEAQKVN 121 HFPRSYELTR KDRLYKNIIR
MQHTHGFKAF HILPQTFLLP 161 AEYAEFCNSY SKDRGPWIVK PVASSRGRGV
YLINNPNQIS 201 LEENILVSRY INNPLLIDDF KFDVRLYVLV TSYDPLVIYL 241
YEEGLARFAT VRYDQGAKNI RNQFMHLTNY SVNKKSGDYV 281 SCDDPEVEDY
GNKWSNSAML RYLKQEGRDT TALMAHVEDL 321 IIKTIISAEL AIATACKTFV
PHRSSCFELY GFDVLIDATL 361 KPWLLEVNLS PSLACDAPLD LKIKASMISD
MFTVVGFVCQ 401 DPAQRASTRP IYPTFESSRR NPFQKPQRPL PAQFHSSEPK 441
QRSRPLSASD AEMKNLVGSA REKGPGKLGG SVLGLSMEEI 481 KVLRRVKEEN
DRRGGEIRIF PTSETWEIYG SYLEHKTSMN 521 YMLATRLFQD RMTADGAPEL
KIEGLNSKAK LHAALYERKL 561 LSLEVRKRRR RSSRLRAMRP KYPVITQPAE
MNVKTETESE 601 EEEEVALDNE DEEQEASQEE SAGFLRENQA KDTPSLTTLV 641
ENTPKENSVK VREWSKKGER CCKLETQELE PKFNLMQVLQ 681 DNGNLSKVQA
RIAFSTYLQH VQIRLMKDSG GQTFSASWAA 721 KEDEQMELVV RFLKRASNNL
QQSLRMVLPS RRLALLERRR 761 ILAHQLGDFI IVYNKETEQM AEKKSKKKVE
EEEEDGVNME 801 NFQEFIRQAS EAELEEVLTF YTQKNKSASV FLGTHSKSSK 841
NNNSYSDSGA KGDHPETVME EAKMKPPKQQ QTTEIHSDKL 881 SRFTTSAEKE
AKLVYTSSSS TPFSGPTATL QKIPNTHLSS 921 VTTSDLSPGP GHHSSLSQIP
SAIPSMPHQP TILLNTVSAS 961 ASPSLHPGTQ NIPSPAGLPR CRSGSHTIGS
FSSEQSAAHI 1001 YSQKLSRPSS AKAGSCYLNK HHSGIAKTQQ EGEDASLYSK 1041
RYNQSMVTAE LQRLAEKQAA RQYSPSSHIN LLTQQVTNLN 1081 LATGIINRSS
ASTPPTLQPI ISPSGPTWLV QSDPQAPENH 1121 SSPPRSRSLQ TGGFAWEGEV
ENNVYSKATG VVPQHKYHPT 1161 AGSYQLHFAL QQLEQQKLQS RQLLDQSRAR
HQAIFGSQTL 1241 PNSNLWTMNN GAGCRISSAT ASGQKPTTLP QKAVPPPSSC 1281
ASLVPKPPPN HKQVLRRATS QRASKGSSAY AQLNGLQSSL 1321 NPAASVPITS
STDPAHTKR
Mice contain a cDNA encoding a protein of unknown function that is
89% identical to the human STAMP cDNA (GenBank Accession No.
XM.sub.--126935). Similarly, nucleic acids encoding STAMP-related
polypeptides such as those provided herein as SEQ ID NO:63-65 are
sufficiently closely related to the human STAMP sequence to be used
for generating STAMP-related polypeptides or for making nucleic
acids that can inhibit the functioning of a STAMP RNA. Thus, a cDNA
sequence for STAMP-related polypeptide having SEQ ID NO:63 is
available in the NCBI database at accession number NM 015072 (gi:
50658078). A cDNA sequence for STAMP-related orangutan polypeptide
having SEQ ID NO:64 is available in the NCBI database at accession
number CR859514 (gi: 55729910). Therefore, variant STAMP sequences
exist and the STAMP gene and protein are conserved among different
species.
Moreover, nucleic acids that can hybridize under moderately or
highly stringent hybridization conditions are sufficiently
complementary to inhibit the functioning of a STAMP RNA and can be
utilized in the compositions of the invention. Generally, stringent
hybridization conditions are selected to be about 5.degree. C.
lower than the thermal melting point (T.sub.m) for the specific
sequence at a defined ionic strength and pH. However, stringent
conditions encompass temperatures in the range of about 1.degree.
C. to about 20.degree. C. lower than the thermal pointing point of
the selected sequence, depending upon the desired degree of
stringency as otherwise qualified herein. In some embodiments, the
nucleic acids that can inhibit the functioning of STAMP RNA can
hybridize to a STAMP RNA under physiological conditions, for
example, physiological temperatures and salt concentrations.
Precise complementarity is therefore not required for successful
duplex formation between a nucleic acid that can inhibit a STAMP
RNA and the complementary coding sequence of a STAMP RNA.
Inhibitory nucleic acid molecules that comprise, for example, 2, 3,
4, or 5 or more stretches of contiguous nucleotides that are
precisely complementary to a STAMP coding sequence, each separated
by a stretch of contiguous nucleotides that are not complementary
to adjacent STAMP coding sequences, can inhibit the function of
STAMP mRNA. In general, each stretch of contiguous nucleotides is
at least 4, 5, 6, 7, or 8 or more nucleotides in length.
Non-complementary intervening sequences are preferably 1, 2, 3, or
4 nucleotides in length. One skilled in the art can easily use the
calculated melting point of a nucleic acid hybridized to a sense
nucleic acid to estimate the degree of mismatching that will be
tolerated between a particular nucleic acid for inhibiting
expression of a particular STAMP RNA.
In some embodiments a nucleic acid that can inhibit the function of
an endogenous STAMP RNA is an anti-sense oligonucleotide. The
anti-sense oligonucleotide is complementary to at least a portion
of the coding sequence of a gene comprising SEQ ID NO:2 or SEQ ID
NO:61. Such anti-sense oligonucleotides are generally at least six
nucleotides in length, but can be about 8, 12, 15, 20, 25, 30, 35,
40, 45, or 50 nucleotides long. Longer oligonucleotides can also be
used. STAMP anti-sense oligonucleotides can be provided in a DNA
construct and introduced into cells whose division is to be
decreased, for example, into cells expressing a glucocorticoid
receptor such as immune cells or lymphocyte precursor cells.
In one embodiment of the invention, expression of a STAMP gene is
decreased using a ribozyme. A ribozyme is an RNA molecule with
catalytic activity. See, e.g., Cech, 1987, Science 236: 1532-1539;
Cech, 1990, Ann. Rev. Biochem. 59:543-568; Cech, 1992, Curr. Opin.
Struct. Biol. 2: 605-609; Couture and Stinchcomb, 1996, Trends
Genet. 12: 510-515. Ribozymes can be used to inhibit gene function
by cleaving an RNA sequence, as is known in the art (see, e.g.,
Haseloffet al., U.S. Pat. No. 5,641,673).
STAMP nucleic acids complementary to SEQ ID NO:2 or SEQ ID NO:61
can be used to generate ribozymes that will specifically bind to
mRNA transcribed from a STAMP gene. Methods of designing and
constructing ribozymes that can cleave other RNA molecules in trans
in a highly sequence specific manner have been developed and
described in the art (see Haseloff et al. (1988), Nature
334:585-591). For example, the cleavage activity of ribozymes can
be targeted to specific RNAs by engineering a discrete
"hybridization" region into the ribozyme. The hybridization region
contains a sequence complementary to the target RNA and thus
specifically hybridizes with the target (see, for example, Gerlach
et al., EP 321,201). The target sequence can be a segment of about
10, 12, 15, 20, or 50 contiguous nucleotides selected from a
nucleotide sequence having SEQ ID NO:2 or SEQ ID NO:61. Longer
complementary sequences can be used to increase the affinity of the
hybridization sequence for the target. The hybridizing and cleavage
regions of the ribozyme can be integrally related; thus, upon
hybridizing to the target RNA through the complementary regions,
the catalytic region of the ribozyme can cleave the target.
RNA interference (RNAi) involves post-transcriptional gene
silencing (PTGS) induced by the direct introduction of dsRNA into a
cell or organism. Small interfering RNAs (siRNAs) are generally
21-23 nucleotide dsRNAs and can mediate post-transcriptional gene
silencing in mammalian cells. siRNAs can also be produced in vivo
by cleavage of dsRNA introduced directly or via a transgene or
virus. Amplification by an RNA-dependent RNA polymerase may occur
in some organisms. siRNAs are incorporated into the RNA-induced
silencing complex, guiding the complex to the homologous endogenous
mRNA where the complex cleaves the transcript.
Rules for designing siRNAs are available. See, e.g., Elbashir S M,
Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001).
Duplexes of 21-nucleotide RNAs mediate RNA interference in
mammalian cell culture. Nature 411: 494-498; J. Harborth, S. M.
Elbashir, K. Vandenburgh, H. Manninga, S. A. Scaringe, K. Weber and
T. Tuschl (2003). Sequence, chemical, and structural variation of
small interfering RNAs and short hairpin RNAs and the effect on
mammalian gene silencing, Antisense Nucleic Acid Drug Dev. 13:
83-106.
Thus, an effective siRNA can be made by selecting target sites
within SEQ ID NO:2 or SEQ ID NO:61 that begin with AA, that have 3'
UU overhangs for both the sense and antisense siRNA strands, and
that have an approximate 50% G/C content. For example, a siRNA of
the invention can be a double-stranded RNA having one strand with
the following sequence:
TABLE-US-00022 (SEQ ID NO: 17) AAGCAGCCGUCGGCGGCUGUU (SEQ ID NO:
18) AACCAUGGGAAGAUGAGACUU (SEQ ID NO: 19) AAUCUGUGCCAUCCAAAUUUU
(SEQ ID NO: 20) AAAGGUCUCUGAGGCCCCCUU (SEQ ID NO: 21)
AAACCCUAAAGGAAAUGCCUU (SEQ ID NO: 22) AAACAGCAUCAUCCUCAGAUU (SEQ ID
NO: 23) AAAAAGUUAAUCACUUUCCUU (SEQ ID NO: 24) AAAACAUUAUUCGAAUGCAUU
(SEQ ID NO: 25) AAGGUUUUUCACAUCCUCCUU (SEQ ID NO: 26)
AAUUUUGUAAUUCAUAUUCUU (SEQ ID NO: 27) AAGGACCGGGGACCUUGGAUU (SEQ ID
NO: 28) AAACCAGUGGCAUCUUCAAUU (SEQ ID NO: 29) AACAAUCCAAACCAGAUCUUU
(SEQ ID NO: 30) AAGAGAACAUUUUGGUCUCUU (SEQ ID NO: 31)
AACCCCCUGCUCAUAGAUGAUU (SEQ ID NO: 32) AAGAAGGAUUGGCUAGGUUUUU (SEQ
ID NO: 33) CAGCACUGACUAUAACCUAAUUU (SEQ ID NO: 34)
CACCCUCUCUGAAGCACAAAAUU (SEQ ID NO: 35) GCCAGCUGAGUACGCGGAAUUUU
(SEQ ID NO: 36) GAGGGCAAUGAGGCCAAAAUAUU
Examples of siRNAs that seem to work in preliminary experiments are
the double stranded RNAs corresponding to the following DNA
sequences of human STAMP:
TABLE-US-00023 (SEQ ID NO: 37) 490-CAGCACTGACTATAACCTAAT-510 (SEQ
ID NO: 38) 547-CACCCTCTCTGAAGCACAAAA-567 (SEQ ID NO: 39)
691-GCCAGCTGAGTACGCGGAATT-711 (SEQ ID NO: 40)
1897-GAGGGCAATGAGGCCAAAATA-1917
Hence, the invention contemplated RNAi and siRNAs that can be used
to control STAMP expression. In some embodiments, the mechanism
used to decrease expression of the STAMP, whether by ribozyme,
anti-sense, siRNA, or antibody, decreases expression of the gene by
50%, 55%, 60%, 65%, 70%, 75% or 80%. In other embodiments,
expression of the STAMP gene is decreased by 85%, 90%, 95%, 99%, or
100%. The effectiveness of the mechanism chosen to alter expression
of the gene can be assessed using methods well known in the art,
such as hybridization of nucleotide probes to mRNA from the human
or monkey STAMP cDNA, quantitative RT-PCR, or detection of a STAMP
protein using antibodies of the invention.
Anti-sense oligonucleotides, ribozymes and siRNAs can be composed
of deoxyribonucleotides, ribonucleotides, or a combination of both.
Oligonucleotides can be synthesized endogenously from transgenic
expression cassettes or vectors as described herein. Alternatively,
such oligonucleotides can be synthesized manually or by an
automated synthesizer, by covalently linking the 5' end of one
nucleotide with the 3' end of another nucleotide with
non-phosphodiester internucleotide linkages such alkylphosphonates,
phosphorothioates, phosphorodithioates, alkylphosphonothioates,
alkylphosphonates, phosphoramidates, phosphate esters, carbamates,
acetamidate, carboxymethyl esters, carbonates, and phosphate
triesters. See Brown, 1994, Meth. Mol. Biol. 20:1-8; Sonveaux,
1994, Meth. Mol. Biol. 26:1-72; Uhlmann et al., 1990, Chem. Rev.
90:543-583.
STAMP anti-sense oligonucleotides, ribozymes and siRNAs can be
modified without affecting their ability to hybridize to a STAMP
coding sequence. These modifications can be internal or at one or
both ends of the selected nucleic acid. For example,
internucleoside phosphate linkages can be modified by adding
peptidyl, cholesteryl or diamine moieties with varying numbers of
carbon residues between these moieties and the terminal ribose.
Modified bases and/or sugars, such as arabinose instead of ribose,
or a 3', 5'-substituted oligonucleotide in which the 3' hydroxyl
group or the 5' phosphate group are substituted, can also be
employed in a modified nucleic acid or oligonucleotide. These
modified nucleic acids and oligonucleotides can be prepared by
methods available in the art. Agrawal et al., 1992, Trends
Biotechnol. 10:152-158; Uhlmann et al., 1990, Chem. Rev.
90:543-584; Uhlmann et al., 1987, Tetrahedron. Lett.
215:3539-3542.
Receptors, Coactivators and Other Factors
Compositions and methods employing STAMP polypeptides and nucleic
acids can also include other factors, for example, hormones,
receptor agonists, other coactivators, repressors, receptors,
receptor ligands and factors that modulate gene expression. As
described herein STAMP can bind to glucocorticoid receptors, and to
the coactivators such as transcription intermediary factor-2 (TIF2)
and steroid receptor coactivator-1 (SRC-1). These and other factors
can be included in the compositions of the invention.
Glucocorticoid receptors (GRs) mediate both gene induction and gene
repression by increasing or decreasing the rates of transcription
from glucocorticoids-responsive genes. For gene induction, GR binds
directly to a glucocorticoid response element that consists of a
consensus palindromic sequence of six nucleotides (TGTTCT, SEQ ID
NO:41), each of which is separated by three additional nucleotides.
No such common DNA sequence exists among those genes that are
down-regulated by GRs due to the diversity of mechanisms involved
in GR-mediated repression.
Glucocorticoids, a class of corticosteroids, are endogenous
hormones with profound effects on the immune system and multiple
organ systems. They suppress a variety of immune and inflammatory
functions by inhibition of inflammatory cytokines such as IL-1,
IL-2, IL-6, and TNF, inhibition of arachidonic acid metabolites
including prostaglandins and leukotrienes, depletion of
T-lymphocytes, and reduction of the expression of adhesion
molecules on endothelial cells (P. J. Barnes, Clin. Sci., 1998, 94,
pp. 557-572; P. J. Barnes et al., Trends Pharmacol. Sci., 1993, 14,
pp. 436-441). In addition to these effects, glucocorticoids
stimulate glucose production in the liver and catabolism of
proteins, play a role in electrolyte and water balance, reduce
calcium absorption, and inhibit osteoblast function.
The anti-inflammatory and immune suppressive activities of
endogenous glucocorticoids have stimulated the development of
synthetic glucocorticoid derivatives including dexamethasone,
prednisone, and prednisolone (L. Parente, Glucocorticoids, N. J.
Goulding and R. J. Flowers (eds.), Boston: Birkhauser, 2001, pp.
35-54). These have found wide use in the treatment of inflammatory,
immune, and allergic disorders including rheumatic diseases such as
rheumatoid arthritis, juvenile arthritis, and ankylosing
spondylitis, dermatological diseases including psoriasis and
pemphigus, allergic disorders including allergic rhinitis, atopic
dermatitis, and contact dermatitis, pulmonary conditions including
asthma and chronic obstructive pulmonary disease (COPD), and other
immune and inflammatory diseases including Crohn's disease,
ulcerative colitis, systemic lupus erythematosus, autoimmune
chronic active hepatitis, osteoarthritis, tendonitis, and bursitis
(J. Toogood, Glucocorticoids, N. J. Goulding and R. J. Flowers
(eds.), Boston: Birkhauser, 2001, pp. 161-174). They have also been
used to help prevent rejection in organ transplantation.
The responses in GR-regulated induction, and often in GR-directed
repression, are augmented by cofactors including coactivators such
as transcription intermediary factor-2 (TIF2) and hBRM. Voegel et
al., 1996, EMBO J. 15, 3667-3675; Hong et al., 1996, Proc. Natl.
Acad. Sci. USA 93, 4948-4952; He et al., 2002, J. Biol. Chem. 277,
49256-49266; Rogatsky et al., 2001, EMBO. J. 20, 6071-6083;
Rogatsky et al., 2002, Proc. Natl. Acad. Sci. U. S. A. 99,
16701-16706; Singh et al., 2001, J. Biol. Chem. 276, 13762-13770.
These coactivators are recruited to DNA-associated receptors. Two
activation domains called AD1 and AD2 have been found to be
required for the ability of the p160 coactivators, such as TIF2, to
increase the total levels of transactivation.
As described herein, the coactivators TIF2 and SRC-1 bind to STAMP.
The regions of the coactivators TIF2 and SRC-1 that participate in
the modulation of the EC.sub.50 and partial agonist activity of
glucocorticoid receptor complexes are separable from the AD1 and
AD2 domains that are critical for increasing the total levels of
gene activation. Moreover, TIF2 can modulate the activities of
several receptors, including androgen receptors, estrogen
receptors, mineralocorticoid receptors, progesterone receptors,
thyroid receptors, retinoid receptors (RAR and RXR), and peroxisome
proliferator-activated receptors (PPARs).
Hence, the invention contemplates compositions containing STAMP
polypeptides or STAMP nucleic acids along with hormones, receptor
agonists, antisteroids, other coactivators, repressors, steroid
receptors, nuclear receptors, and factors that modulate
glucocorticoid-responsive gene expression. Examples of receptors
that STAMP can influence include steroid and nuclear receptors such
as glucocorticoid receptors, androgen receptors, estrogen
receptors, mineralocorticoid receptors, progesterone receptors,
thyroid receptors, retinoid receptors (RAR and RXR), and peroxisome
proliferator-activated receptors (PPARs).
Nucleic acid and amino acid sequences for coactivators, repressors,
glucocorticoid receptors, and factors that modulate
glucocorticoid-responsive gene expression can be found in the art,
for example, in the NCBI database. See website at ncbi.nlm.nih.gov.
For example, one amino acid sequence for human transcription
intermediary factor-2 (TIF2) can be found in the NCBI database as
accession number Q15596 (gi: 13626594). See website at
ncbi.nlm.nih.gov. This sequence is provided below as follows (SEQ
ID NO:42).
TABLE-US-00024 1 MSGMGENTSD PSRAETRKRK ECPDQLGPSP KRNTEKRNRE 41
QENKYIEELA ELIFANFNDI DNFNEKPDKC AILKETVKQI 81 RQIKEQEKAA
AANIDEVQKS DVSSTGQGVI DKDALGPMML 121 EALDGFFFVV NLEGNVVFVS
ENVTQYLRYN QEELMNKSVY 161 SILHVGDHTE FVKNLLPKSI VNGGSWSGEP
PRRNSHTFNC 201 RMLVKPLPDS EEEGHDNQEA HQKYETMQCF AVSQPKSIKE 241
EGEDLQSCLI CVARRVPMKE RPVLPSSESF TTRQDLQGKI 281 TSLDTSTMRA
AMKPGWEDLV RRCIQKFHAQ HEGESVSYAK 321 RHHHEVLRQG LAFSQIYRFS
LSDGTLVAAQ TKSKLIRSQT 361 TNEPQLVISL HMLHREQNVC VMNPDLTGQT
MGKPLNPISS 401 NSPAHQALCS GNPGQDMTLS SNINFPINGP KEQMGMPMGR 441
FGGSGGMNHV SGMQATTPQG SNYALKMNSP SQSSPGMNPG 481 QPTSMLSPRH
RMSPGVAGSP RIPPSQFSPA GSLHSPVGVC 521 SSTGNSHSYT NSSLNALQAL
SEGHGVSLGS SLASPDLKMG 561 NLQNSPVNMN PPPLSKMGSL DSKDCFGLYG
EPSEGTTGQA 601 ESSCHPGEQK ETNDPNLPPA VSSERADGQS RLHDSKGQTK 641
LLQLLTTKSD QMEPSPLASS LSDTNKDSTG SLPGSGSTHG 681 TSLKEKHKIL
HRLLQDSSSP VDLAKLTAEA TGKDLSQESS 721 STAPGSEVTI KQEPVSPKKK
ENALLRYLLD KDDTKDIGLP 761 EITPKLERLD SKTDPASNTK LIAMKTEKEE
MSFEPGDQPG 801 SELDNLEEIL DDLQNSQLPQ LFPDTRPGAP AGSVDKQAII 841
NDLMQLTAEN SPVTPVGAQK TALRISQSTF NNPRPGQLGR 881 LLPNQNLPLD
ITLQSPTGAG PFPPIRNSSP YSVIPQPGMM 921 GNQGMIGNQG NLGNSSTGMI
GNSASRPTMP SGEWAPQSSA 961 VRVTCAATTS AMNRPVQGGM IRNPAASIPM
RPSSQPGQRQ 1001 TLQSQVMNIG PSELEMNMGG PQYSQQQAPP NQTAPWPESI 1041
LPIDQASFAS QNRQPFGSSP DDLLCPHPAA ESPSDEGALL 1081 DQLYLALRNF
DGLEEIDRAL GIPELVSQSQ AVDPEQFSSQ 1121 DSNIMLEQKA PVFPQQYASQ
AQMAQGSYSP MQDPNFHTMG 1161 QRPSYATLRM QPRPGLRPTG LVQNQPNQLR
LQLQHRLQAQ 1201 QNRQPLMNQI SNVSNVNLTL RPGVPTQAPI NAQMLAQRQR 1241
EILNQHLRQR QMHQQQQVQQ RTLMMRGQGL NMTPSMVAPS 1281 GMPATMSNPR
IPQANAQQFP FPPNYGISQQ PDPGFTGATT 1321 PQSPLMSPRM AHTQSPMMQQ
SQANPAYQAP SDINGWAQGN 1361 MGGNSMFSQQ SPPHFGQQAN TSMYSNNMNI
NVSMATNTGG 1401 MSSMNQMTGQ ISMTSVTSVP TSGLSSMGPE QVNDPALRGG 1441
NLFPNQLPGM DMIKQEGDTT RKYC
See also, Voegel et al., 1998, EMBO J. 17, 507-519.
As described herein, STAMP interacts with a region of the
coactivator TIF2 defined by amino acids 623-834. The TIF2
coactivator modulates the properties of not only glucocorticoid
receptors but also of progesterone (Giannoukos et al., 2001, Mol.
Endocrinol., 15, 255-270) and mineralocorticoid (data not shown)
receptors. Thus, a target of STAMP (i.e., TIF2) does modulate the
dose-response curve and partial agonist activity of several steroid
receptors and can affect the properties of steroid and nuclear
receptors such as androgen receptors, estrogen receptors,
mineralocorticoid receptors, progesterone receptor, thyroid
receptors, retinoid receptors (RAR and RXR), and peroxisome
proliferator-activated receptors (PPARs). Hence, STAMP can be
included in compositions and methods for modulating such
receptors.
One example of a sequence for a human steroid receptor
coactivator-1 (SRC-1) has accession number AAC50631 (gi: 1480646).
See website at ncbi.nlm.nih.gov. This sequence for this human
glucocorticoid receptor is provided below (SEQ ID NO:43).
TABLE-US-00025 1 MSGLGDSSSD PANPDSHKRK GSPCDTLASS TEKRRREQEN 41
KYLEELAELL SANISDIDSL SVKPDKCKIL KKTVDQIQLM 81 KRMEQEKSTT
DDDVQKSDIS SSSQGVIEKE SLGPLLLEAL 121 DGFFFVVNCE GRIVFVSENV
TSYLGYNQEE LMNTSVYSIL 161 HVGDHAEFVK NLLPKSLVNG VPWPQEATRR
NSHTFNCRML 201 IHPPDEPGTE NQEACQRYEV MQCFTVSQPK SIQEDGEDFQ 241
SCLICIARRL PRPPAITGVE SFMTKQDTTG KIISIDTSSL 281 RAAGRTGWED
LVRKCIYAFF QPQGREPSYA RQLFQEVMTR 321 GTASSPSYRF ILNDGTMLSA
HTKCKLCYPQ SPDMQPFIMG 361 IHIIDREHSG LSPQDDTNSG MSIPRVNPSV
NPSISPAHGV 401 ARSSTLPPSN SNMVSTRINR QQSSDLHSSS HSNSSNSQGS 441
FGCSPGSQIV ANVALNQGQA SSQSSNPSLN LNNSPMEGTG 481 ISLAQFMSPR
RQVTSGLATR PRMPNNSFPP NISTLSSPVG 521 MTSSACNNNN RSYSNIPVTS
LQGMNEGPNN SVGFSASSPV 561 LRQMSSQNSP SRLNIQPAKA ESKDNKEIAS
ILNEMIQSDN 601 SSSDGKPLDS GLLHNNDRLS DGDSKYSQTS HKLVQLLTTT 641
AEQQLRHADI DTSCKDVLSC TGTSNSASAN SSGGSCPSSH 681 SSLTERHKIL
HRLLQEGSPS DITTLSVEPD KKDSASTSVS 721 VTGQVQGNSS IKLELDASKK
KESKDHQLLR YLLDKDEKDL 761 RSTPNLSLDD VKVKVEKKEQ MDPCNTNPTP
MTKPTPEEIK 801 LEAQSQFTAD LDQFDQLLPT LEKAAQLPGL CETDRMDGAV 841
TSVTIKSEIL PASLQSATAR PTSRLNRLPE LELEAIDNQF 881 GQPGTGDQIP
WTNNTVTAIN QSKSEDQCIS SQLDELLCPP 921 TTVEGRNDEK ALLEQLVSFL
SGKDETELAE LDRALGIDKL 961 VQGGGLDVLS ERFPPQQATP PLIMEERPNL
YSQPYSSPSP 1001 TANLPSPFQG MVRQKPSLGT MPVQVTPPRG AFSPGMGMQP 1041
RQTLNRPPAA PNQLRLQLQQ RLQGQQQLIH QNRQAILNQF 1081 AATAPVGINM
RSGMQQQITP QPPLNAQMLA QRQRELYSQQ 1121 HRQRQLIQQQ RAMLMRQQSF
GNNLPPSSGL PVQMGNPRLP 1161 QGAPQQFPYP PNYGTNPGTP PASTSPFSQL
AANPEASLAN 1201 RNSMVSRGMT GNIGGQFGTG INPQMQQNVF QYPGAGMVPQ 1241
GEANFAPSLS PGSSMVPMPI PPPQSSLLQQ TPPASGYQSP 1281 DMKAWQQGAI
GNNNVFSQAV QNQPTPAQPG VYNNMSITVS 1321 MAGGNTNVQN MNPMMAQMQM
SSLQMPGMNT VCPEQINDPA 1361 LRHTGLYCNQ LSSTDLLKTE ADGTQVQQVQ
VEADVQCTVN 1401 LVGGDPYLNQ PGPLGTQKPT SGPQTPQAQQ KSLLQQLLTE
See also, Takeshita et al., Molecular cloning and properties of a
full-length putative thyroid hormone receptor coactivator,
Endocrinology 137 (8), 3594-3597 (1996).
Numerous sequences for human glucocorticoid receptor isoforms and
variants are available, for example, in the NCBI database. One
example of a sequence for a human glucocorticoid receptor has
accession number P04150 (gi: 121069). See website at
ncbi.nlm.nih.gov. This sequence for this human glucocorticoid
receptor is provided below (SEQ ID NO:44).
TABLE-US-00026 1 MDSKESLTPG REENPSSVLA QERGDVMDFY KTLRGGATVK 41
VSASSPSLAV ASQSDSKQRR LLVDFPKGSV SNAQQPDLSK 81 AVSLSMGLYM
GETETKVMGN DLGFPQQGQI SLSSGETDLK 121 LLEESIANLN RSTSVPENPK
SSASTAVSAA PTEKEFPKTH 161 SDVSSEQQHL KGQTGTNGGN VKLYTTDQST
FDILQDLEFS 201 SGSPGKETNE SPWRSDLLID ENCLLSPLAG EDDSFLLEGN 241
SNEDCKPLIL PDTKPKIKDN GDLVLSSPSN VTLPQVKTEK 281 EDFIELCTPG
VIKQEKLGTV YCQASFPGAN IIGNKMSAIS 321 VHGVSTSGGQ MYHYDMNTAS
LSQQQDQKPI FNVIPPIPVG 361 SENWNRCQGS GDDNLTSLGT LNFPGRTVFS
NGYSSPSMRP 401 DVSSPPSSSS TATTGPPPKL CLVCSDEASG CHYGVLTCGS 441
CKVFFKRAVE GQHNYLCAGR NDCIIDKIRR KNCPACRYRK 481 CLQAGMNLEA
RKTKKKIKGI QQATTGVSQE TSENPGNKTI 521 VPATLPQLTP TLVSLLEVIE
PEVLYAGYDS SVPDSTWRIM 561 TTLNMLGGRQ VIAAVKWAKA IPGFRNLHLD
DQMTLLQYSW 601 MFLMAFALGW RSYRQSSANL LCFAPDLIIN EQRMTLPCMY 641
DQCKHMLYVS SELHRLQVSY EEYLCMKTLL LLSSVPKDGL 681 KSQELFDEIR
MTYIKELGKA IVKREGNSSQ NWQREYQLTK 721 LLDSMHEVVE NLLNYCFQTF
LDKTMSIEFP EMLAEIITNQ 761 IPKYSNGNIK KLLFHQK
One of skill in the art can readily obtain the sequence for other
human glucocorticoid receptor, human transcription intermediary
factor-2 and steroid receptor coactivator-1 in the art, for
example, in the NCBI database. Compositions containing a STAMP
polypeptide of the invention and such other human glucocorticoid
receptors, human transcription intermediary factor-2 or steroid
receptor coactivator-1 can readily be made by one of skill in the
art.
The invention also contemplates a method of identifying a factor
that can interact with a STAMP polypeptide that involves contacting
the STAMP polypeptide with a test factor and observing whether a
complex forms between the STAMP polypeptide and the test factor.
Formation of a complex can be identified by immunoprecipitation,
western analysis, yeast two-hybrid assays and other procedures
available to one of skill in the art. Factors that can be tested
include, for example, receptors, coactivators, agonists,
antagonists and cDNA libraries encoding segments of known and
unknown proteins.
Modulating Glucocorticoid-Responsive Gene Expression
Compositions containing STAMP that may also contain any available
GR coactivators, agonists, partial agonists, steroids, antisteroids
and antagonists can be used to modulate glucocorticoid-responsive
gene expression and glucocorticoid receptor-mediated diseases. As
such, these compounds can be used to influence several systems in
the body, including carbohydrate, protein and lipid metabolism,
electrolyte and water balance, and the functions of the
cardiovascular, kidney, central nervous, immune, skeletal muscle
and other organ and tissue systems. In this regard, GR modulators
are used for the treatment of diseases or conditions associated
with an excess or a deficiency of steroids and/or glucocorticoids
in the body. As such, they may be used to treat the following:
obesity, diabetes, cardiovascular disease, hypertension, cancer,
Syndrome X, depression, anxiety, glaucoma, human immunodeficiency
virus (HIV) or acquired immunodeficiency syndrome (AIDS),
neurodegeneration (for example, Alzheimer's disease and Parkinson's
disease), cognition enhancement, Cushing's Syndrome, Addison's
Disease, osteoporosis, frailty, inflammatory diseases (such as
osteoarthritis, rheumatoid arthritis, asthma and rhinitis), tests
of adrenal function, viral infection, immunodeficiency,
immunomodulation, autoimmune diseases, allergies, wound healing,
compulsive behavior, multi-drug resistance, addiction, psychosis,
anorexia, cachexia, post-traumatic stress syndrome, post-surgical
bone fracture, medical catabolism and prevention of muscle frailty.
In some embodiments, the disease or condition that can be treated
by the compositions of the invention include conception (prevention
or enhancement of the probability of conception), contraception,
infertility, diabetes, menopause, cancer (e.g., hormone- or
steroid-sensitive cancers, such as prostate and breast cancer),
hypertension, and osteoporosis.
The present invention relates to methods of treating conditions and
disorders such as the following: endocrine disorders (such as
primary or secondary adrenocortical insufficiency; congenital
adrenal hyperplasia, nonsuppurative thyroiditis and hypercalcemia
associated with cancer); hormone-sensitive cancer,
steroid-sensitive cancer, conception (prevention of conception or
enhancement of the probability of conception), contraception,
infertility, arthritis (such as osteoarthritis; psoriatic
arthritis; rheumatoid arthritis; juvenile rheumatoid arthritis;
ankylosing spondylitis; acute and subacute bursitis; acute
nonspecific tenosynovitis; acute gouty arthritis; post-traumatic
osteoarthritis; synovitis of osteoarthritis and epicondylitis);
collagen diseases (such as exacerbation or as maintenance therapy
in systemic lupus erythematosus, acute rheumatic carditis and
systemic dermatomyositis (polymyositis)); dermatologic diseases
(such as pemphigus, bullous dermatitis herpetiformis, erythema
multiforme, Stevens-Johnson syndrome, exfoliative dermatitis,
mycosis fungoides; psoriasis and seborrheic dermatitis); allergic
states (such as control of severe or incapacitating allergic
conditions intractable to adequate trials of conventional
treatment, rhinitis including seasonal or perennial allergic
rhinitis, asthma including bronchial asthma, contact dermatitis,
atopic dermatitis, serum sickness, food allergies and drug
hypersensitivity reactions); ophthalmic diseases (such as the
treatment of severe acute and chronic allergic and inflammatory
processes involving the eye and its adnexa, such as allergic
conjunctivitis, keratitis, allergic corneal marginal ulcers, herpes
zoster ophthalmicus, iritis and iridocyclitis, chorioretinitis,
anterior segment inflammation, diffuse posterior uveitis and
choroiditis, optic neuritis and sympathetic ophthalmia);
ameliorating or treating postmenopausal symptoms; respiratory
diseases (such as chronic obstructive pulmonary disease, acute
respiratory distress syndrome, symptomatic sarcoidosis, Loeffler's
syndrome; berylliosis, fulminating or disseminated pulmonary
tuberculosis and aspiration pneumonitis); hematologic disorders
(such as idiopathic thrombocytopenic purpura, secondary
thrombocytopenia, acquired (autoimmune) hemolytic anemia,
erythroblastopenia (RBC anemia) and congenital (erythroid)
hypoplastic anemia); neoplastic diseases (such as leukemias and
lymphomas); edematous states (such as to induce a diuresis or
remission of proteinuria in the nephrotic syndrome, without uremia,
of the idiopathic type or that due to lupus erythematosus);
gastrointestinal diseases (such as ulcerative colitis, inflammatory
bowel diseases, Crohns disease and regional enteritis);
tuberculosis, tuberculosis meningitis, trichinosis with
neurological or myocardial involvement, immunomodulation such as
graft vs. host transplant rejection, multiple sclerosis,
glucocorticoid insufficiency and systemic fungal infections, in a
mammal comprising administering to said mammal a therapeutically
effective amount of a composition of the invention.
Cancers that can be treated by the present compositions and methods
include autoimmune deficiency syndrome-associated Kaposi's sarcoma,
cancer of the adrenal cortex, cancer of the cervix, cancer of the
breast, cancer of the endometrium, cancer of the esophagus, cancer
of the head and neck, cancer of the liver, cancer of the pancreas,
cancer of the prostate, cancer of the thymus, carcinoid tumors,
chronic lymphocytic leukemia, Ewing's sarcoma, gestational
trophoblastic tumors, hepatoblastoma, multiple myeloma, non-small
cell lung cancer, retinoblastoma, or tumors in the ovaries. A
cancer at any stage of progression can be treated or detected, such
as primary, metastatic, and recurrent cancers. Information
regarding numerous types of cancer can be found e.g., from the
American Cancer Society, or from, e.g., Wilson et al. (1991)
Harrison's Principles of Internal Medicine, 12.sup.th Edition,
McGraw-Hill, Inc. Both human and veterinary uses are
contemplated.
The compositions of the present invention, and pharmaceutically
acceptable salts thereof, are useful to induce weight loss in
mammals needing or desiring to lose weight. While not intending to
limit the present invention to a specific mechanism of action, the
compositions of the present invention, and salts thereof, are able
to induce weight loss by a variety of mechanisms, such as appetite
suppression, decreasing food intake and stimulation of the
metabolic rate in peripheral tissue, thereby increasing energy
expenditure. In addition, the compositions of the present
invention, and salts thereof are useful to induce a more favorable
partitioning of nutrients from fat to muscle tissue in mammals.
Thus, while not necessarily resulting in weight loss, this increase
in muscle mass may be useful in preventing or treating diseases,
such as obesity and frailty. In addition, the compounds of the
present invention, prodrugs and pharmaceutically acceptable salts
thereof, may also be useful to increase lean meat deposition,
improve lean meat to fat ratio, and trim unwanted fat from
non-human animals.
Furthermore, it will be understood by those skilled in the art that
the compositions and pharmaceutically acceptable salts thereof can
be used in a wide variety of combination therapies to treat the
conditions and diseases described above. Thus, the compositions of
the present invention can be used in conjunction with (e.g.,
simultaneously or sequentially) other pharmaceutical agents for the
treatment of the disease/conditions described herein. For example,
they may be used in combination with pharmaceutical agents that
treat obesity, diabetes, inflammatory disease, immunodefficiency,
hypertension, cardiovascular disease, viral infection, HIV,
Alzheimer's disease, Parkinson's disease, anxiety, depression, or
psychosis. In combination therapy treatment, both the compositions
of this invention and the other drug therapies are administered to
mammals (e.g., humans, male or female) by conventional methods.
Thus, the STAMP polypeptides of the invention can be formulated
into compositions containing a variety of other active and inactive
agents. For example, the compositions can also contain
glucocorticoid receptor agonists that are efficacious for the
treatment of various inflammatory diseases. Such treatment is often
accompanied by undesirable side effects such as metabolic effects,
weight gain, muscle wasting, decalcification of the skeleton,
osteoporosis, thinning of the skin and thinning of the skeleton.
However, according to the present invention, glucocorticoid
receptor modulators may be used in combination with glucocorticoid
receptor agonists to minimize or reduce the incidence of these side
effects, without inhibiting the efficacy of the treatment. As
demonstrated by the inventors, STAMP can shift the dose response
curve of steroids and glucocorticoid receptor agonists to lower
concentrations of ligand, thereby reducing the dosage required for
activity of such steroids and glucocorticoid receptor agonists and
minimizing or eliminating their side effects.
Thus, any glucocorticoid receptor agonist may be used as an
additional active agent in the STAMP compositions of the present
invention. This composition can be used in the treatment of various
inflammatory diseases, such as arthritis (osteo and rheumatoid),
asthma, rhinitis, or immunomodulation.
Examples of additional glucocorticoid receptor modulators that can
be used in the compositions of the invention include those known in
the art as well as the polypeptides, compounds and other agents
described herein. Examples of glucocorticoid receptor modulators
known in the art include, but are not limited to, certain
nonsteroidal compounds, such as 5H-chromeno[3,4-f]quinolines, which
are selective modulators of steroid receptors, as disclosed in U.S.
Pat. No. 5,696,127; and certain steroid compounds which possess
antiglucocorticoid activity, and some of which have glucocorticoid
activity, as disclosed in Published European Patent Application 0
188 396, published Jul. 23, 1986. Examples of glucocorticoid
receptor agonists include prednisone
(17,21-dihydroxypregnane-1,4-diene-3,11,20-trione), progesterone,
prednylidene
((11.beta.)-11,17,21-trihydroxy-16-methylenepregna-1,4-diene-3,20-dione),
prednisolone
((11.beta.)-11,17,21-trihydroxypregna-1,4-diene-3,20-dione),
cortisone (17.alpha.,21-dihydroxy-4-pregnene-3,11,20-trione),
dexamethasone
((11.beta.,16.alpha.)-9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-di-
ene-3,20-dione), hydrocortisone
(11.beta.,17.alpha.,21-trihydroxypregn-4-ene-3,20-dione), androgen
(e.g., 5.alpha.-dihydrotestosterone, testosterone and/or
methyltrienolone), and/or 5H-chromeno[3,4-f]quinoline. Another
highly potent glucocorticoid agonist that may be used in the
compositions of the invention is deacylcortivazol. See Simons Jr.
et al. (1979) Biochem. Biophys. Res. Comm. 86: 793-800.
These glucocorticoid receptor modulators and agonists will
generally be administered in the form of a dosage unit at a
therapeutically effective amount of such compound. For example,
prednisone or an equivalent compound may be administered from about
5 to about 80 mg, depending on the condition; hydrocortisone may be
administered from about 100 to about 400 mg, depending on the
condition; and dexamethasone may be administered from about 4 to
about 16 mg, depending on the condition. These doses are typically
administered once to twice daily, and for maintenance purposes,
sometimes on alternate days.
The compositions of the invention can also include antisteroids.
Antisteroids are defined as compounds that block the action of
agonist steroids. For example, certain dexamethasone derivatives
can act as antiglucocorticoids or antiprogestins, that block or
partially block the activities of glucocorticoids and progestin.
See. U.S. Patent Application 20030207854.
For the treatment of Alzheimer's disease, any cholinomimetic drug,
such as donepezil hydrochloride (ARICEPT.TM.), may be used in the
compositions of this invention.
For the treatment of Parkinson's disease, any anti-Parkinson's
drug, such as L-dopa, bromocriptine, or selegiline, may be used in
the compositions of this invention.
For the treatment of anxiety, any antianxiolytic drug, such as
benzodiazepine, valium, or librium, may be used in the compositions
of this invention.
For the treatment of depression, any tricyclic antidepressant such
as, desipramine, or any selective serotonin reuptake inhibitor
(SSRI's), such as sertraline hydrochloride or fluoxetine
hydrochloride, may be used in the compositions of this
invention.
For the treatment of psychosis, any typical or atypical
antipsychotic drug, such as haloperidol or clozapine may be used in
the compositions of this invention.
For the treatment of diabetes, any aldose reductase inhibitor may
be used in the compositions of this invention. The term aldose
reductase inhibitor refers to a compound, which inhibits the
bioconversion of glucose to sorbitol catalyzed by the enzyme aldose
reductase. Such inhibition is readily determined by those skilled
in the art according to standard assays (J. Malone, Diabetes,
29:861-864, 1980, "Red Cell Sorbitol, an Indicator of Diabetic
Control"). A variety of aldose reductase inhibitors are available
in the art. Examples of aldose reductase inhibitors include
compounds such as those disclosed and described in WO 99/43663,
published Sep. 2, 1999.
Any glycogen phosphorylase inhibitor may be used in the
compositions of this invention. The term glycogen phosphorylase
inhibitor refers to any substance or agent or any combination of
substances and/or agents which reduces, retards or eliminates the
enzymatic action of glycogen phosphorylase. The currently known
enzymatic action of glycogen phosphorylase is the degradation of
glycogen by catalysis of the reversible reaction of a glycogen
macromolecule and inorganic phosphate to glucose-1-phosphate and a
glycogen macromolecule which is one glucosyl residue shorter than
the original glycogen macromolecule (forward direction of
glycogenolysis). Such actions are readily determined by those
skilled in the art according to standard assays (e.g., as described
in WO 99/43664, published Sep. 2, 1999). A variety of these
compounds are described in the following published international
patent applications: WO 96139384, published Dec. 12, 1996, WO
96/39385, published Dec. 12, 1996, and WO 99143663, published Sep.
2, 1999.
Any sorbitol dehydrogenase inhibitor may be used in the
compositions of this invention. The term sorbitol dehydrogenase
inhibitor refers to a compound that inhibits the enzyme sorbitol
dehydrogenase, which catalyzes the oxidation of sorbitol to
fructose. Such inhibition is readily determined by those skilled in
the art according to standard assays (as described in U.S. Pat. No.
5,728,704 and references cited therein). A variety of these
compounds are described and referenced below. However other
sorbitol dehydrogenase inhibitors are available to those skilled in
the art. For example, U.S. Pat. No. 5,728,704 discloses substituted
pyrimidines that inhibit sorbitol dehydrogenase, lower fructose
levels, and/or treat or prevent diabetic complications, such as
diabetic neuropathy, diabetic retinopathy, diabetic nephropathy,
diabetic microangiopathy and diabetic macroangiopathy.
Any known, commercially marketed antidiabetic compound may be used
in the compositions of this invention. A variety of such compounds
are available to those skilled in the art. Examples of such
compounds useful in the compositions and methods of this invention
include, for example, insulin, inhaled insulin, metformin, and
sulfonylureas, such as glipazide (GLUCOTROL.TM.), glyburide
(GLYNASE.TM., MICRONASE.TM.) and chlorpropamide
(DIABINASE.TM.).
Any .beta.-adrenergic agonist may be used in the compositions of
this invention. .beta.-Adrenergic agents have been categorized into
.beta..sub.1, .beta..sub.2, and .beta..sub.3 subtypes. Agonists of
.beta. receptors promote the activation of adenyl cyclase.
Activation of .beta..sub.1 receptors invokes increases in heart
rate. Activation of .beta..sub.2 receptors induces relaxation of
smooth muscle tissue which produces a drop in blood pressure and
the onset of skeletal muscle tremors. Activation of .beta..sub.3
receptors is known to stimulate lipolysis, which is the breakdown
of adipose tissue triglycerides to glycerol and fatty acids.
Activation of .beta..sub.3 receptors also stimulates the metabolic
rate, thereby increasing energy expenditure. Accordingly,
activation of .beta..sub.3 receptors promotes the loss of fat mass.
Compounds that stimulate .beta..sub.3 receptors are therefore
useful as anti-obesity agents. Compounds that are
.beta..sub.3-receptors agonists have hypoglycemic and/or
anti-diabetic activity. Such activity is readily determined by
those skilled in the art according to standard assays
(International Patent Application, Publication No. WO 96/35671).
Several .beta..sub.3-adrenergic agonists are available to those
skilled in the art. For example, International Patent Application,
Publication No. WO 96/35671 discloses compounds, such as
substituted aminopyridines, which are .beta.-adrenergic agonists.
International Patent Application, Publication No. 93/16189
discloses the use of selective .beta..sub.3 receptor agonists in
combination with compounds which modify eating behavior for the
treatment of obesity.
Any thyromimetic antiobesity agent may be used in the compositions
of this invention. These compounds are tissue selective thyroid
hormone agonists. These compounds are able to induce weight loss by
mechanisms other than appetite suppression, e.g., through
stimulation of the metabolic rate in peripheral tissue, which, in
turn, produces weight loss. Such metabolic effect is readily
measured by those skilled in the art according to standard assays.
A variety of these compounds will be known to those skilled in the
art. It is well known to one of ordinary skill in the art that
selectivity of the thermogenic effect is an important requirement
for a useful therapeutic agent in the treatment of, for example,
obesity and related conditions.
Any eating behavior modifying compound may be used in the
compositions of this invention. Compounds that modify eating
behavior include anorectic agents, which are compounds that
diminish the appetite. Such classes and compounds of anorectic
agents are available to one of ordinary skill in the art. For
example, the following are antiobesity agents: phenylpropanolamine,
ephedrine, pseudoephedrine, phentermine, a Neuropeptide Y
(hereinafter also referred to as "NPY") antagonist, a
cholecystokinin-A (hereinafter referred to as CCK-A) agonist, a
monoamine reuptake inhibitor (such as sibutramine), a
sympathomimetic agent, a serotoninergic agent (such as fenfluramine
or fenfluramine), a dopamine agonist (such as bromocriptine), a
melanocyte-stimulating hormone receptor agonist or mimetic, a
melanocyte-stimulating hormone analog, a cannabinoid receptor
antagonist, a melanin concentrating hormone antagonist, the OB
protein (hereinafter referred to as "leptin"), a leptin analog, a
galanin antagonist or a GI lipase inhibitor or decreaser (such as
orlistat). Other antiobesity agents include phosphatase 1B
inhibitors, bombesin agonists, dehydroepiandrosterone or analogs
thereof, glucocorticoid receptor antagonists, orexin receptor
antagonists, urocortin binding protein antagonists or glucagon-like
peptide-1 (insulinotropin) agonists. A particularly preferred
monoamine reuptake inhibitor is sibutramine, which can be prepared
as disclosed in U.S. Pat. No. 4,929,629. Preferred serotoninergic
agents include fenfluramine and fenfluramine, which can be prepared
as disclosed in U.S. Pat. No. 3,198,834. A particularly preferred
dopamine agonist is bromocriptine, which can be prepared as
disclosed in U.S. Pat. Nos. 3,752,814 and 3,752,888. Another
preferred anorectic agent is phentermine, which can be prepared as
disclosed in U.S. Pat. No. 2,408,345.
Any NPY receptors antagonist may be used in the compositions of
this invention. The term NPY receptors antagonist refers to
compounds that interact with NPY receptors and inhibit the activity
of neuropeptide Y at those receptors and thus are useful in
treating disorders associated with neuropeptide Y, such as feeding
disorders, including obesity. Such inhibition is readily determined
by those skilled in the art according to standard assays (such as
those described in International Patent Application, Publication
No. WO 99/07703). A number of NPY receptors antagonist are
available to those skilled in the art. For example, International
Patent Application, Publication No. WO 99107703 discloses certain
4-aminopyrrole (3,2-d) pyrimidines as neuropeptide Y receptor
antagonists. International patent application, Publication No. WO
96/14307, published May 17, 1996; International patent application,
Publication No. WO 96/40660, published Dec. 19, 1996; International
patent application, Publication No. WO 98/03492; International
patent application, Publication No. WO 98/03494; International
patent application, Publication No. WO 98/03493; International
patent application, Publication No. WO 96114307, published May 17,
1996; International patent application, Publication No. WO
96/40660, published Dec. 19, 1996; disclose additional compounds,
such as substituted benzylamine derivatives, which are useful as
neuropeptide Y receptors specific ligands.
Antibodies
The invention also contemplates antibodies that can bind to a STAMP
polypeptide of the invention. In another embodiment, a disease
where glucocorticoid-responsive gene expression is undesirably
active can be treated by administering to a mammal an antibody that
can bind to STAMP polypeptide. Such an antibody may block STAMP
activity or activate STAMP. For example, the antibody can be
directed against a STAMP polypeptide comprising any one of SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:70, or a
combination thereof.
All antibody molecules belong to a family of plasma proteins called
immunoglobulins, whose basic building block, the immunoglobulin
fold or domain, is used in various forms in many molecules of the
immune system and other biological recognition systems. A typical
immunoglobulin has four polypeptide chains, containing an antigen
binding region known as a variable region and a non-varying region
known as the constant region.
Native antibodies and immunoglobulins are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies between the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (VH) followed by a
number of constant domains. Each light chain has a variable domain
at one end (VL) and a constant domain at its other end. The
constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light and heavy chain variable domains (Clothia et al.,
J. Mol. Biol. 186, 651-66, 1985); Novotny and Haber, Proc. Natl.
Acad. Sci. USA 82, 4592-4596 (1985).
Depending on the amino acid sequences of the constant domain of
their heavy chains, immunoglobulins can be assigned to different
classes. There are at least five (5) major classes of
immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these
may be further divided into subclasses (isotypes), e.g. IgG-1,
IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. The heavy chains constant
domains that correspond to the different classes of immunoglobulins
are called alpha (.alpha.), delta (.delta.), epsilon (.epsilon.),
gamma (.gamma.) and mu (.mu.), respectively. The light chains of
antibodies can be assigned to one of two clearly distinct types,
called kappa (.kappa.) and lambda (.lamda.), based on the amino
sequences of their constant domain. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
The term "variable" in the context of variable domain of
antibodies, refers to the fact that certain portions of the
variable domains differ extensively in sequence among antibodies.
The variable domains are for binding and determine the specificity
of each particular antibody for its particular antigen. However,
the variability is not evenly distributed through the variable
domains of antibodies. It is concentrated in three segments called
complementarity determining regions (CDRs) also known as
hypervariable regions both in the light chain and the heavy chain
variable domains.
The more highly conserved portions of variable domains are called
the framework (FR). The variable domains of native heavy and light
chains each comprise four FR regions, largely adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each chain are held together in
close proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen binding site of
antibodies. The constant domains are not involved directly in
binding an antibody to an antigen, but exhibit various effector
function, such as participation of the antibody in
antibody-dependent cellular toxicity.
An antibody that is contemplated for use in the present invention
thus can be in any of a variety of forms, including a whole
immunoglobulin, an antibody fragment such as Fv, Fab, and similar
fragments, a single chain antibody that includes the variable
domain complementarity determining regions (CDR), and the like
forms, all of which fall under the broad term "antibody," as used
herein. The present invention contemplates the use of any
specificity of an antibody, polyclonal or monoclonal, and is not
limited to antibodies that recognize and immunoreact with a
specific epitope. In some embodiments, however, the antibodies of
the invention may react with selected epitopes within the CID, RID
or other domains of the STAMP protein.
The term "antibody fragment" refers to a portion of a full-length
antibody, generally the antigen binding or variable region.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2 and
Fv fragments. Papain digestion of antibodies produces two identical
antigen binding fragments, called the Fab fragment, each with a
single antigen binding site, and a residual "Fc" fragment,
so-called for its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen binding
fragments, which are capable of cross-linking antigen, and a
residual other fragment (which is termed pFc'). Additional
fragments can include diabodies, linear antibodies, single-chain
antibody molecules, and multispecific antibodies formed from
antibody fragments. As used herein, "functional fragment" with
respect to antibodies, refers to Fv, F(ab) and F(ab').sub.2
fragments.
Antibody fragments retain some ability to selectively bind with its
antigen or receptor and are defined as follows:
(1) Fab is the fragment that contains a monovalent antigen-binding
fragment of an antibody molecule. A Fab fragment can be produced by
digestion of whole antibody with the enzyme papain to yield an
intact light chain and a portion of one heavy chain.
(2) Fab' is the fragment of an antibody molecule can be obtained by
treating whole antibody with pepsin, followed by reduction, to
yield an intact light chain and a portion of the heavy chain. Two
Fab' fragments are obtained per antibody molecule. Fab' fragments
differ from Fab fragments by the addition of a few residues at the
carboxyl terminus of the heavy chain CH1 domain including one or
more cysteines from the antibody hinge region.
(3) (Fab').sub.2 is the fragment of an antibody that can be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction. F(ab').sub.2 is a dimer of two Fab' fragments
held together by two disulfide bonds.
(4) Fv is the minimum antibody fragment that contains a complete
antigen recognition and binding site. This region consists of a
dimer of one heavy and one light chain variable domain in a tight,
non-covalent association (V.sub.H-V.sub.L dimer). It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer antigen
binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
(5) Single chain antibody ("SCA"), defined as a genetically
engineered molecule containing the variable region of the light
chain, the variable region of the heavy chain, linked by a suitable
polypeptide linker as a genetically fused single chain molecule.
Such single chain antibodies are also referred to as "single-chain
Fv" or "sFv" antibody fragments. Generally, the Fv polypeptide
further comprises a polypeptide linker between the VH and VL
domains that enables the sFv to form the desired structure for
antigen binding. For a review of sFv see Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds. Springer-Verlag, N.Y., pp. 269-315 (1994).
The term "diabodies" refers to a small antibody fragments with two
antigen-binding sites, which fragments comprise a heavy chain
variable domain (VH) connected to a light chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161, and Hollinger et al., Proc. Natl. Acad Sci. USA 90:
6444-6448 (1993).
The preparation of polyclonal antibodies is well-known to those
skilled in the art. See, for example, Green, et al., Production of
Polyclonal Antisera, in: Immunochemical Protocols (Manson, ed.),
pages 1-5 (Humana Press); Coligan, et al., Production of Polyclonal
Antisera in Rabbits, Rats Mice and Hamsters, in: Current Protocols
in Immunology, section 2.4.1 (1992), which are hereby incorporated
by reference.
The preparation of monoclonal antibodies likewise is conventional.
See, for example, Kohler & Milstein, Nature, 256:495 (1975);
Coligan, et al., sections 2.5.1-2.6.7; and Harlow, et al., in:
Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub.
(1988)), which are hereby incorporated by reference. Methods of in
vitro and in vivo manipulation of monoclonal antibodies are also
available to those skilled in the art. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler and
Milstein, Nature 256, 495 (1975), or they may be made by
recombinant methods, for example, as described in U.S. Pat. No.
4,816,567. The monoclonal antibodies for use with the present
invention may also be isolated from antibody libraries using the
techniques described in Clackson et al. Nature 352: 624-628 (1991),
as well as in Marks et al., J. Mol Biol. 222: 581-597 (1991).
Monoclonal antibodies can be isolated and purified from hybridoma
cultures by a variety of well-established techniques. Such
isolation techniques include affinity chromatography with Protein-A
Sepharose, size-exclusion chromatography, and ion-exchange
chromatography. See, e.g., Coligan, et al., sections 2.7.1-2.7.12
and sections 2.9.1-2.9.3; Barnes, et al., Purification of
Immunoglobulin G (IgG), in: Methods in Molecular Biology, Vol. 10,
pages 79-104 (Humana Press (1992).
Another method for generating antibodies involves a Selected
Lymphocyte Antibody Method (SLAM). The SLAM technology permits the
generation, isolation and manipulation of monoclonal antibodies
without the process of hybridoma generation. The methodology
principally involves the growth of antibody forming cells, the
physical selection of specifically selected antibody forming cells,
the isolation of the genes encoding the antibody and the subsequent
cloning and expression of those genes.
More specifically, an animal is immunized with a source of specific
antigen. The animal can be a rabbit, mouse, rat, or any other
convenient animal. This immunization may consist of purified
protein, in either native or recombinant form, peptides, DNA
encoding the protein of interest or cells expressing the protein of
interest. After a suitable period, during which antibodies can be
detected in the serum of the animal (usually weeks to months),
blood, spleen or other tissues are harvested from the animal.
Lymphocytes are isolated from the blood and cultured under specific
conditions to generate antibody-forming cells, with antibody being
secreted into the culture medium. These cells are detected by any
of several means (complement mediated lysis of antigen-bearing
cells, fluorescence detection or other) and then isolated using
micromanipulation technology. The individual antibody forming cells
are then processed for eventual single cell PCR to obtain the
expressed Heavy and Light chain genes that encode the specific
antibody. Once obtained and sequenced, these genes are cloned into
an appropriate expression vector and recombinant, monoclonal
antibody produced in a heterologous cell system. These antibodies
are then purified via standard methodologies such as the use of
protein A affinity columns. These types of methods are further
described in Babcook, et al., Proc. Natl. Acad. Sci. (USA) 93:
7843-7848 (1996); U.S. Pat. No. 5,627,052; and PCT WO 92/02551 by
Schrader.
Another method involves humanizing a monoclonal antibody by
recombinant means to generate antibodies containing human specific
and recognizable sequences. See, for review, Holmes, et al., J.
Immunol., 158:2192-2201 (1997) and Vaswani, et al., Annals Allergy,
Asthma & Immunol., 81:105-115 (1998). The term "monoclonal
antibody" as used herein refers to an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to conventional polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. In
additional to their specificity, the monoclonal antibodies are
advantageous in that they are synthesized by the hybridoma culture,
uncontaminated by other immunoglobulins. The modifier "monoclonal"
indicates the antibody is obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method.
The monoclonal antibodies herein specifically include "chimeric"
antibodies (immunoglobulins) in which a portion of the heavy and/or
light chain is identical with or homologous to corresponding
sequences in antibodies derived from a particular species or
belonging to a particular antibody class or subclass, while the
remainder of the chain(s) is identical with or homologous to
corresponding sequences in antibodies derived from another species
or belonging to another antibody class or subclass, as well as
fragments of such antibodies, so long as they exhibit the desired
biological activity (U.S. Pat. No. 4,816,567); Morrison et al.
Proc. Natl. Acad Sci. 81, 6851-6855 (1984).
Methods of making antibody fragments are also known in the art (see
for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, (1988), incorporated herein by
reference). Antibody fragments of the present invention can be
prepared by proteolytic hydrolysis of the antibody or by expression
in E. coli of DNA encoding the fragment. Antibody fragments can be
obtained by pepsin or papain digestion of whole antibodies
conventional methods. For example, antibody fragments can be
produced by enzymatic cleavage of antibodies with pepsin to provide
a 5S fragment denoted F(ab').sub.2. This fragment can be further
cleaved using a thiol reducing agent, and optionally a blocking
group for the sulfhydryl groups resulting from cleavage of
disulfide linkages, to produce 3.5 S Fab=monovalent fragments.
Alternatively, an enzymatic cleavage using pepsin produces two
monovalent Fab' fragments and an Fc fragment directly. These
methods are described, for example, in U.S. Pat. No. 4,036,945 and
No. 4,331,647, and references contained therein. These patents are
hereby incorporated in their entireties by reference.
Other methods of cleaving antibodies, such as separation of heavy
chains to form monovalent light-heavy chain fragments, further
cleavage of fragments, or other enzymatic, chemical, or genetic
techniques may also be used, so long as the fragments bind to the
antigen that is recognized by the intact antibody. For example, Fv
fragments comprise an association of V.sub.H and V.sub.L chains.
This association may be noncovalent or the variable chains can be
linked by an intermolecular disulfide bond or cross-linked by
chemicals such as glutaraldehyde. Preferably, the Fv fragments
comprise V.sub.H and V.sub.L chains connected by a peptide linker.
These single-chain antigen binding proteins (sFv) are prepared by
constructing a structural gene comprising DNA sequences encoding
the V.sub.H and V.sub.L domains connected by an oligonucleotide.
The structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are described, for example, by Whitlow, et al., Methods: a
Companion to Methods in Enzymology, Vol. 2, page 97 (1991); Bird,
et al., Science 242:423-426 (1988); Ladner, et al, U.S. Pat. No.
4,946,778; and Pack, et al., Bio/Technology 11:1271-77 (1993).
Another form of an antibody fragment is a peptide coding for a
single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick, et al., Methods: a Companion to
Methods in Enzymology, Vol. 2, page 106 (1991).
The invention further contemplates human and humanized forms of
non-human (e.g. murine) antibodies. Such humanized antibodies can
be chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) that contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementary determining region (CDR) of
the recipient are replaced by residues from a CDR of a nonhuman
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity.
In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are
found neither in the recipient antibody nor in the imported CDR or
framework, sequences. These modifications are made to further
refine and optimize antibody performance. In general, humanized
antibodies can comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the Fv regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see: Jones et al., Nature 321,
522-525 (1986); Reichmann et al., Nature 332, 323-329 (1988);
Presta, Curr. Op. Struct. Biol. 2, 593-596 (1992); Holmes, et al.,
J. Immunol., 158:2192-2201 (1997) and Vaswani, et al., Annals
Allergy, Asthma & Inununol., 81:105-115 (1998); U.S. Pat. Nos.
4,816,567 and 6,331,415; PCT/GB84/00094; PCT/US86/02269;
PCT/US89/00077; PCT/US88/02514; and WO91/09967, each of which is
incorporated herein by reference in its entirety.
The invention also provides methods of mutating antibodies to
optimize their affinity, selectivity, binding strength or other
desirable property. A mutant antibody refers to an amino acid
sequence variant of an antibody. In general, one or more of the
amino acid residues in the mutant antibody is different from what
is present in the reference antibody. Such mutant antibodies
necessarily have less than 100% sequence identity or similarity
with the reference amino acid sequence. In general, mutant
antibodies have at least 75% amino acid sequence identity or
similarity with the amino acid sequence of either the heavy or
light chain variable domain of the reference antibody. Preferably,
mutant antibodies have at least 80%, more preferably at least 85%,
even more preferably at least 90%, and most preferably at least 95%
amino acid sequence identity or similarity with the amino acid
sequence of either the heavy or light chain variable domain of the
reference antibody.
The antibodies of the invention are isolated antibodies. An
isolated antibody is one that has been identified and separated
and/or recovered from a component of the environment in which it
was produced. Contaminant components of its production environment
are materials that would interfere with diagnostic or therapeutic
uses for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. The term "isolated
antibody" also includes antibodies within recombinant cells because
at least one component of the antibody's natural environment will
not be present. Ordinarily, however, isolated antibody will be
prepared by at least one purification step.
If desired, the antibodies of the invention can be purified by any
available procedure. For example, the antibodies can be affinity
purified by binding an antibody preparation to a solid support to
which the antigen used to raise the antibodies is bound. After
washing off contaminants, the antibody can be eluted by known
procedures. Those of skill in the art will know of various
techniques common in the immunology arts for purification and/or
concentration of polyclonal antibodies, as well as monoclonal
antibodies (see for example, Coligan, et al., Unit 9, Current
Protocols in Immunology, Wiley Interscience, 1991, incorporated by
reference).
In some embodiments, the antibody will be purified as measurable by
at least three different methods: 1) to greater than 95% by weight
of antibody as determined by the Lowry method, and most preferably
more than 99% by weight; 2) to a degree sufficient to obtain at
least 15 residues of N-terminal or internal amino acid sequence by
use of a spinning cup sequenator; or 3) to homogeneity by SDS-PAGE
under reducing or non-reducing conditions using Coomassie blue or,
preferably, silver stain.
Expression of STAMP Nucleic Acids
Mammalian expression of STAMP sense, anti-sense, ribozyme, and
siRNA nucleic acids can be accomplished as described in Dijkema et
al., EMBO J. (1985) 4: 761, Gorman et al., Proc. Natl. Acad. Sci.
USA (1982b) 79: 6777, Boshart et al., Cell (1985) 41: 521 and U.S.
Pat. No. 4,399,216. Other features of mammalian expression can be
facilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:
44, Barnes and Sato, Anal. Biochem. (1980) 102: 255, U.S. Pat. Nos.
4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO
87/00195, and U.S. Pat. No. RE 30,985. Use of such STAMP nucleic
acids can augment or inhibit the expression of STAMP
polypeptides.
STAMP nucleic acids can be placed within linear or circular
molecules. They can be placed within autonomously replicating
molecules or within molecules without replication sequences. They
can be regulated by their own or by other regulatory sequences, as
is known in the art.
STAMP nucleic acids can be used in expression cassettes or gene
delivery vehicles, for the purpose of delivering an mRNA or
oligonucleotide (with a sequence from a native mRNA or its
complement), a full-length protein, a fusion protein, a
polypeptide, a ribozyme, a siRNA or a single-chain antibody, into a
cell, preferably a eukaryotic cell. According to the present
invention, a gene delivery vehicle can be, for example, naked
plasmid DNA, a viral expression vector comprising a sense or
anti-sense nucleic acid of the invention, or a sense or anti-sense
nucleic acid of the invention in conjunction with a liposome or a
condensing agent.
STAMP nucleic acids can be introduced into suitable host cells
using a variety of techniques that are available in the art, such
as transferrin-polycation-mediated DNA transfer, transfection with
naked or encapsulated nucleic acids, liposome-mediated DNA
transfer, intracellular transportation of DNA-coated latex beads,
protoplast fusion, viral infection, electroporation, use of nucleic
acid microprojectile procedures and calcium phosphate-mediated
transfection.
In one embodiment of the invention, the gene delivery vehicle
comprises a promoter and one of the STAMP nucleic acids disclosed
herein. Preferred promoters are tissue-specific promoters and
promoters that are activated by cellular proliferation, such as the
thymidine kinase and thymidylate synthase promoters. Other
preferred promoters include promoters that are activated by
infection with a virus, such as the .alpha.- and .beta.-interferon
promoters, and promoters that can be activated by a hormone, such
as estrogen. Other promoters that can be used include the Moloney
virus LTR, the CMV promoter, and the mouse albumin promoter.
A gene delivery vehicle can comprise viral sequences such as a
viral origin of replication or packaging signal. These viral
sequences can be selected from viruses such as astrovirus,
coronavirus, orthomyxovirus, papovavirus, paramyxovirus,
parvovirus, picornavirus, poxvirus, retrovirus, togavirus or
adenovirus. In some embodiments, the gene delivery vehicle is a
recombinant retroviral vector. Recombinant retroviruses and various
uses thereof have been described in numerous references including,
for example, Mann et al., Cell 33:153, 1983, Cane and Mulligan,
Proc. Nat'l. Acad. Sci. USA 81:6349, 1984, Miller et al., Human
Gene Therapy 1:5-14, 1990, U.S. Pat. Nos. 4,405,712, 4,861,719, and
4,980,289, and PCT Application Nos. WO 89/02,468, WO 89/05,349, and
WO 90/02,806. Numerous retroviral gene delivery vehicles can be
utilized in the present invention, including for example those
described in EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698;
WO 93/25234; U.S. Pat. No. 5,219,740; WO 9311230; WO 9310218; Vile
and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer
Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993;
Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al.,
J. Neurosurg. 79:729-735, 1993 (U.S. Pat. No. 4,777,127, GB
2,200,651, EP 0,345,242 and WO91102805).
Examples of retroviruses that can be utilized include avian
leukosis virus (ATCC Nos. VR-535 and VR-247), bovine leukemia virus
(VR-1315), murine leukemia virus (MLV), mink-cell focus-inducing
virus (Koch el al., J. Vir. 49:828, 1984; and Oliff et al., J. Vir.
48:542, 1983), murine sarcoma virus (ATCC Nos. VR-844, 45010 and
45016), reticuloendotheliosis virus (ATCC Nos. VR-994, VR-770 and
45011), Rous sarcoma virus, Mason-Pfizer monkey virus, baboon
endogenous virus, endogenous feline retrovirus (e.g., RD114), and
mouse or rat gL30 sequences used as a retroviral vector. Strains of
MLV from which recombinant retroviruses can be generated include
4070A and 1504A (Hartley and Rowe, J. Vir. 19:19, 1976), Abelson
(ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi (Ru et al., J.
Vir. 67:4722, 1993; and Yantchev Neopksma 26:397, 1979), Gross
(ATCC No. VR-590), Kirsten (Albino et al., J. Exp. Med. 164:1710,
1986), Harvey sarcoma virus (Manly el al., J. Vir. 62:3540, 1988;
and Albino et al., J. Exp. Med. 164:1710, 1986) and Rauscher (ATCC
No. VR-998), and Moloney MLV (ATCC No. VR-190). A non-mouse
retrovirus that can be used is Rous sarcoma virus, for example,
Bratislava (Manly et al., J. Vir. 62:3540, 1988; and Albino et al.,
J. Exp. Med. 164:1710, 1986), Bryan high titer (e.g., ATCC Nos.
VR-334, VR-657, VR-726, VR-659, and VR-728), Bryan standard (ATCC
No. VR-140), Carr-Zilber (Adgighitov et al., Neoplasma 27:159,
1980), Engelbreth-Holm (Laurent et al., Biochem Biophys Acta
908:241, 1987), Harris, Prague (e.g., ATCC Nos. VR-772, and 45033),
or Schmidt-Ruppin (e.g. ATCC Nos. VR-724, VR-725, VR-354)
viruses.
Any of the above retroviruses can be readily utilized in order to
assemble or construct retroviral gene delivery vehicles given the
disclosure provided herein and standard recombinant techniques
(e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,
2.sup.nd Edition (1989), Sambrook et al., Molecular Cloning: A
Laboratory Manual, 3.sup.rd Edition (2001), and Kunkle, Proc. Natl.
Acad. Sci. U.S.A. 82:488, 1985). Portions of retroviral expression
vectors can be derived from different retroviruses. For example,
retrovector LTRs can be derived from a murine sarcoma virus, a tRNA
binding site from a Rous sarcoma virus, a packaging signal from a
murine leukemia virus, and an origin of second strand synthesis
from an avian leukosis virus. These recombinant retroviral vectors
can be used to generate transduction competent retroviral vector
particles by introducing them into appropriate packaging cell lines
(see Ser. No. 071800,921, filed Nov. 29, 1991).
Recombinant retroviruses can be produced that direct the
site-specific integration of the recombinant retroviral genome into
specific regions of the host cell DNA. Such site-specific
integration is useful for mutating the endogenous STAMP gene.
Site-specific integration can be mediated by a chimeric integrase
incorporated into the retroviral particle (see Ser. No. 08/445,466
filed May 22, 1995). It is preferable that the recombinant viral
gene delivery vehicle is a replication-defective recombinant
virus.
Packaging cell lines suitable for use with the above-described
retroviral gene delivery vehicles can be readily prepared (see WO
92/05266) and used to create producer cell lines (also termed
vector cell lines or "VCLs") for production of recombinant viral
particles. In preferred embodiments of the present invention,
packaging cell lines are made from human (e.g., HT1080 cells) or
mink parent cell lines, thereby allowing production of recombinant
retroviral gene delivery vehicles that are capable of surviving
inactivation in human serum. The construction of recombinant
retroviral gene delivery vehicles is described in detail in WO
91/02805. These recombinant retroviral gene delivery vehicles can
be used to generate transduction competent retroviral particles by
introducing them into appropriate packaging cell lines. Similarly,
adenovirus gene delivery vehicles can also be readily prepared and
utilized given the disclosure provided herein (see also Berkner,
Biotechniques 6:616-627, 1988, and Rosenfeld et al., Science
252:431-434, 1991, WO 93/07283, WO 93/06223, and WO 93/07282).
A gene delivery vehicle can also be a recombinant adenoviral gene
delivery vehicle. Such vehicles can be readily prepared and
utilized given the disclosure provided herein (see also Berkner,
Biotechniques 6:616, 1988, and Rosenfeld et al., Science 252:431,
1991, WO 93/07283, WO 93/06223, and WO 93/07282). Adeno-associated
viral gene delivery vehicles can also be constructed and used to
deliver proteins or nucleic acids of the invention to cells in
vitro or in vivo. The use of adeno-associated viral gene delivery
vehicles in vitro is described in Chatteijee et al., Science 258:
1485-1488 (1992), Walsh et al., Proc. Nat'l. Acad. Sci. 89:
7257-7261 (1992), Walsh et al., J. Clin. Invest. 94: 1440-1448
(1994), Flotte et al., J. Biol. Chem. 268: 3781-3790 (1993),
Ponnazhagan et al., J. Exp. Med. 179: 733-738 (1994), Miller et
al., Proc. Nat'l Acad. Sci. 91: 10183-10187 (1994), Einerhand et
al., Gene Ther. 2: 336-343 (1995), Luo et al., Exp. Hematol. 23:
1261-1267 (1995), and Zhou et al., Gene Therapy 3: 223-229 (1996).
In vivo use of these vehicles is described in Flotte et al., Proc.
Nat'l Acad. Sci. 90: 10613-10617(1993), and Kaplitt et al., Nature
Genet. 8:148-153 (1994).
In another embodiment of the invention, a gene delivery vehicle is
derived from a togavirus. Such togaviruses include alphaviruses
such as those described in U.S. Ser. No. 08/405,627, filed Mar. 15,
1995, WO 95/07994. Alpha viruses, including Sindbis and ELVS
viruses can be gene delivery vehicles for nucleic acids of the
invention. Alpha viruses are described in WO 94/21792, WO 92/10578
and WO 95/07994. Several different alphavirus gene delivery vehicle
systems can be constructed and used to deliver nucleic acids to a
cell according to the present invention. Representative examples of
such systems include those described in U.S. Pat. Nos. 5,091,309
and 5,217,879. Preferred alphavirus gene delivery vehicles for use
in the present invention include those that are described in WO
95/07994.
The recombinant viral vehicle can also be a recombinant alphavirus
viral vehicle based on a Sindbis virus. Sindbis constructs, as well
as numerous similar constructs, can be readily prepared. Sindbis
viral gene delivery vehicles typically comprise a 5' sequence
capable of initiating Sindbis virus transcription, a nucleotide
sequence encoding Sindbis non-structural proteins, a viral junction
region inactivated so as to prevent fragment transcription, and a
Sindbis RNA polymerase recognition sequence. Optionally, the viral
junction region can be modified so that nucleic acid transcription
is reduced, increased, or maintained. As will be appreciated by
those in the art, corresponding regions from other alphaviruses can
be used in place of those described above.
The viral junction region of an alphavirus-derived gene delivery
vehicle can comprise a first viral junction region that has been
inactivated in order to prevent transcription of the nucleic acid
and a second viral junction region that has been modified such that
nucleic acid transcription is reduced. An alphavirus-derived
vehicle can also include a 5' promoter capable of initiating
synthesis of viral RNA from cDNA and a 3' sequence that controls
transcription termination.
Other recombinant togaviral gene delivery vehicles that can be
utilized in the present invention include those derived from
Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus
(ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246),
Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250;
ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat. Nos.
5,091,309 and 5,217,879 and in WO 92/10578.
Other viral gene delivery vehicles suitable for use in the present
invention include, for example, those derived from poliovirus
(Evans et al., Nature 339:385, 1989, and Sabin et al., J. Biol.
Standardization 1:115, 1973) (ATCC VR-58); rhinovirus (Arnold et
al., J. Cell. Biochem. L401, 1990) (ATCC VR-1110); pox viruses,
such as canary pox virus or vaccinia virus (Fisher-Hoch et al.,
PROC. NATL. ACAD. SCI. U.S.A. 86:317, 1989; Flexner et al., Ann.
N.Y. Acad. Sci. 569:86, 1989; Flexner et al., Vaccine 8:17, 1990;
U.S. Pat. Nos. 4,603,112 and 4,769,330; WO 89/01973) (ATCC VR-111;
ATCC VR-2010); SV40 (Mulligan et al., Nature 277:108, 1979) (ATCC
VR-305), (Madzak et al., J. Gen. Vir. 73:1533, 1992); influenza
virus (Luytjes et al., Cell 59:1107, 1989; McMicheal et al., The
New England Journal of Medicine 309:13, 1983; and Yap et al.,
Nature 273:238, 1978) (ATCC VR-797); parvovirus such as
adeno-associated virus (Samulski et al., J. Vir. 63:3822, 1989, and
Mendelson et al., Virology 166:154, 1988) (ATCC VR-645); herpes
simplex virus (Kit et al., Adv. Exp. Med. Biol. 215:219, 1989)
(ATCC VR-977; ATCC VR-260); Nature 277: 108, 1979); human
immunodeficiency virus (EPO 386,882, Buchschacher et al., J. Vir.
66:2731, 1992); measles virus (EPO 440,219) (ATCC VR-24); A (ATCC
VR-67; ATCC VR-1247), Aura (ATCC VR-368), Bebaru virus (ATCC
VR-600; ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus
(ATCC VR-64; ATCC VR-1241), Fort Morgan (ATCC VR-924), Getah virus
(ATCC VR-369; ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC
VR-66), Mucambo virus (ATCC VR-580; ATCC VR-1244), Ndumu (ATCC
VR-371), Pixuna virus (ATCC VR-372; ATCC VR-1245), Tonate (ATCC
VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Whataroa (ATCC
VR-926), Y-62-33 (ATCC VR-375), O'Nyong virus, Eastern encephalitis
virus (ATCC VR-65; ATCC VR-1242), Western encephalitis virus (ATCC
VR-70; ATCC VR-1251; ATCC VR-622; ATCC VR-1252), and coronavirus
(Hamre et al., Proc. Soc. Exp. Biol. Med. 121:190, 1966) (ATCC
VR-740).
A nucleic acid of the invention can also be combined with a
condensing agent to form a gene delivery vehicle. In a preferred
embodiment, the condensing agent is a polycation, such as
polylysine, polyarginine, polyornithine, protamine, spermine,
spermidine, and putrescine. Many suitable methods for making such
linkages are known in the art (see, for example, Ser. No.
08/366,787, filed Dec. 30, 1994).
In an alternative embodiment, a nucleic acid is associated with a
liposome to form a gene delivery vehicle. Liposomes are small,
lipid vesicles comprised of an aqueous compartment enclosed by a
lipid bilayer, typically spherical or slightly elongated structures
several hundred Angstroms in diameter. Under appropriate
conditions, a liposome can fuse with the plasma membrane of a cell
or with the membrane of an endocytic vesicle within a cell that has
internalized the liposome, thereby releasing its contents into the
cytoplasm. Prior to interaction with the surface of a cell,
however, the liposome membrane acts as a relatively impermeable
barrier that sequesters and protects its contents, for example,
from degradative enzymes. Additionally, because a liposome is a
synthetic structure, specially designed liposomes can be produced
that incorporate desirable features. See Stryer, Biochemistry, pp.
236-240, 1975 (W. H. Freeman, San Francisco, Calif.); Szoka et al.,
Biochim. Biophys. Acta 600:1, 1980; Bayer et al., Biochim. Biophys.
Acta. 550:464, 1979; Rivnay et al., Meth. Enzymol. 149:119, 1987;
Wang et al., PROC. NATL. ACAD. SCI. U.S.A. 84: 7851, 1987, Plant et
al., Anal. Biochem. 176:420, 1989, and U.S. Pat. No. 4,762,915.
Liposomes can encapsulate a variety of nucleic acid molecules
including DNA, RNA, plasmids, and expression constructs comprising
nucleic acids such those disclosed in the present invention.
Liposomal preparations for use in the present invention include
cationic (positively charged), anionic (negatively charged) and
neutral preparations. Cationic liposomes have been shown to mediate
intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl.
Acad. Sci. USA 84:7413-7416, 1987), mRNA (Malone et al., Proc.
Natl. Acad. Sci. USA 86:6077-6081, 1989), and purified
transcription factors (Debs et al, J. Biol. Chem. 265:10189-10192,
1990), in functional form. Cationic liposomes are readily
available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are available under the trademark Lipofectin.TM., from GIBCO BRL,
Grand Island, N.Y. See also Feigner et al., Proc. Natl. Acad. Sci.
US491: 5148-5152.87, 1994. Other commercially available liposomes
include Transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other
cationic liposomes can be prepared from readily available materials
using techniques well known in the art. See, e.g., Szoka et al.,
Proc. Natl. Acad. Sci. USA 75:4194-4198, 1978; and WO 90/11092 for
descriptions of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
Similarly, anionic and neutral liposomes are readily available,
such as from Avanti Polar Lipids (Birmingham, Ala.), or can be
easily prepared using readily available materials. Such materials
include phosphatidyl choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE) and the like. These materials can also be mixed
with the DOTMA and DOTAP starting materials in appropriate ratios.
Methods for making liposomes using these materials are well known
in the art.
The liposomes can comprise multilamellar vesicles (MLVs), small
unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs).
The various liposome-nucleic acid complexes are prepared using
methods known in the art. See, e.g., Straubinger et al., METHODS OF
IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl.
Acad. Sci. USA 87:3410-3414, 1990; Papahadjopoulos et al., Biochim.
Biophys. Acta 394:483, 1975; Wilson et al., Cell 17:77, 1979;
Deamer and Bangham, Biochim. Biophys. Acta 443:629, 1976; Ostro et
al., Biochem. Biophys. Res. Commun. 76:836, 1977; Fraley et al.,
Proc. Natl. Acad Sci. USA 76:3348, 1979; Enoch and Strittmatter,
Proc. Natl. Acad Sci. USA 76:145, 1979; Fraley et al., J. Biol.
Chem. 255:10431, 1980; Szoka and Papahadjopoulos, Proc. Natl. Acad.
Sci. USA 75:145, 1979; and Schaefer-Ridder et al., Science 215:166,
1982.
In addition, lipoproteins can be included with a nucleic acid of
the invention for delivery to a cell. Examples of such lipoproteins
include chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments,
or fusions of these proteins can also be used. Modifications of
naturally occurring lipoproteins can also be used, such as
acetylated LDL. These lipoproteins can target the delivery of
nucleic acids to cells expressing lipoprotein receptors.
Preferably, if lipoproteins are included with a nucleic acid, no
other targeting ligand is included in the composition.
Receptor-mediated targeted delivery of STAMP nucleic acids to
specific tissues can also be used. Receptor-mediated DNA delivery
techniques are described in, for example, Findeis et al. (1993),
Trends in Biotechnol. 11, 202-05; Chiou et al. (1994), GENE
THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.
A. Wolff, ed.); Wu & Wu (1988), J. Biol. Chem. 263, 621-24; Wu
et al. (1994), J. Biol. Chem. 269, 542-46; Zenke et al. (1990),
Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59; Wu et al. (1991), J.
Biol. Chem. 266, 338-42.
In another embodiment, naked nucleic acid molecules are used as
gene delivery vehicles, as described in WO 90/11092 and U.S. Pat.
No. 5,580,859. Such gene delivery vehicles can be either DNA or RNA
and, in certain embodiments, are linked to killed adenovirus.
Curiel et al., Hum. Gene. Ther. 3:147-154, 1992. Other suitable
vehicles include DNA-ligand (Wu et al., J. Biol. Chem.
264:16985-16987, 1989), lipid-DNA combinations (Feigner et al.,
Proc. Natl. Acad. Sci. USA 84:7413 7417, 1989), liposomes (Wang et
al., Proc. Natl. Acad Sci. 84:7851-7855, 1987) and microprojectiles
(Williams et al., Proc. Natl. Acad. Sci. 88:2726-2730, 1991).
One can increase the efficiency of naked nucleic acid uptake into
cells by coating the nucleic acids onto biodegradable latex beads.
This approach takes advantage of the observation that latex beads,
when incubated with cells in culture, are efficiently transported
and concentrated in the perinuclear region of the cells. The beads
will then be transported into cells when injected into muscle.
Nucleic acid-coated latex beads will be efficiently transported
into cells after endocytosis is initiated by the latex beads and
thus increase gene transfer and expression efficiency. This method
can be improved further by treating the beads to increase their
hydrophobicity, thereby facilitating the disruption of the endosome
and release of nucleic acids into the cytoplasm.
STAMP-specific siRNA, ribozymes and anti-sense nucleic acids can be
introduced into cells in a similar manner. The nucleic acid
construct encoding the siRNA, ribozyme or anti-sense nucleic acid
may include transcriptional regulatory elements, such as a promoter
element, an enhancer or UAS element, and a transcriptional
terminator signal, for controlling transcription of the ribozyme in
the cells. Mechanical methods, such as microinjection,
liposome-mediated transfection, electroporation, or calcium
phosphate precipitation, can be used to introduce the siRNA,
ribozyme or anti-sense DNA construct into cells whose division it
is desired to decrease, as described above. Alternatively, if it is
desired that the cells stably retain the DNA construct, the DNA
construct can be supplied on a plasmid and maintained as a separate
element or integrated into the genome of the cells, as is known in
the art.
Expression of an endogenous STAMP gene in a cell can also be
altered by introducing in frame with the endogenous STAMP gene a
DNA construct comprising a STAMP targeting sequence, a regulatory
sequence, an exon, and an unpaired splice donor site by homologous
recombination, such that a homologous recombinant cell comprising
the DNA construct is formed. The new transcription unit can be used
to turn the STAMP gene on or off as desired. This method of
affecting endogenous gene expression is taught in U.S. Pat. No.
5,641,670.
Integration of a delivered STAMP nucleic acid into the genome of a
cell line or tissue can be monitored by any means known in the art.
For example, Southern blotting of the delivered STAMP nucleic acid
can be performed. A change in the size of the fragments of a
delivered nucleic acid indicates integration. Replication of a
delivered nucleic acid can be monitored inter alia by detecting
incorporation of labeled nucleotides combined with hybridization to
a STAMP probe. Expression of a STAMP nucleic acid can be monitored
by detecting production of STAMP mRNA that hybridizes to the
delivered nucleic acid or by detecting STAMP protein. STAMP protein
can be detected immunologically.
Compositions
The STAMP polypeptides and antibodies of the invention, including
their salts, as well as the STAMP siRNA, ribozymes, sense and
anti-sense nucleic acids are administered to modulate
glucocorticoid responsive gene expression, modulate STAMP activity
or to achieve a reduction in at least one symptom associated with a
condition, indication, infection or disease associated with
inappropriate glucocorticoid-responsive gene expression. Other
agents can be included as described herein such as glucocorticoid
receptors, glucocorticoid agonists, glucocorticoid antagonists
and/or glucocorticoid coactivators.
To achieve the desired effect(s), the STAMP polypeptide, nucleic
acid, antibody, and combinations with other agents thereof, may be
administered as single or divided dosages. For example, STAMP
polypeptides and antibodies can be administered in dosages of at
least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about
0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to
about 100 to 300 mg/kg or at least about 1 mg/kg to about 50 to 100
mg/kg of body weight, although other dosages may provide beneficial
results. The amount administered will vary depending on various
factors including, but not limited to, the polypeptide or antibody
chosen, the disease, the weight, the physical condition, the
health, the age of the mammal, whether prevention or treatment is
to be achieved, and if the polypeptide or antibody is chemically
modified. Such factors can be readily determined by the clinician
employing animal models or other test systems that are available in
the art.
Administration of the therapeutic agents in accordance with the
present invention may be in a single dose, in multiple doses, in a
continuous or intermittent manner, depending, for example, upon the
recipient's physiological condition, whether the purpose of the
administration is therapeutic or prophylactic, and other factors
known to skilled practitioners. The administration of the
polypeptides, nucleic acids and antibodies of the invention may be
essentially continuous over a preselected period of time or may be
in a series of spaced doses. Both local and systemic administration
is contemplated.
To prepare the composition, polypeptides, nucleic acids and
antibodies are synthesized or otherwise obtained, purified as
necessary or desired and then lyophilized and stabilized. The
polypeptide, nucleic acid or antibody can then be adjusted to the
appropriate concentration, and optionally combined with other
agents. The absolute weight of a given polypeptide, nucleic acid or
antibody included in a unit dose can vary widely. For example,
about 0.01 to about 2 g, or about 0.1 to about 500 mg, of at least
one polypeptide, nucleic acid or antibody of the invention, or a
plurality of polypeptides, nucleic acids and antibodies specific
for a particular cell type can be administered. Alternatively, the
unit dosage can vary from about 0.01 g to about 50 g, from about
0.01 g to about 35 g, from about 0.1 g to about 25 g, from about
0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5
g to about 4 g, or from about 0.5 g to about 2 g.
Daily doses of the polypeptides, nucleic acids or antibodies of the
invention can vary as well. Such daily doses can range, for
example, from about 0.1 g/day to about 50 g/day, from about 0.1
g/day to about 25 g/day, from about 0.1 g/day to about 12 g/day,
from about 0.5 g/day to about 8 g/day, from about 0.5 g/day to
about 4 g/day, and from about 0.5 g/day to about 2 g/day.
Thus, one or more suitable unit dosage forms comprising the
therapeutic polypeptides, nucleic acids or antibodies of the
invention can be administered by a variety of routes including
oral, parenteral (including subcutaneous, intravenous,
intramuscular and intraperitoneal), rectal, dermal, transdermal,
intrathoracic, intrapulmonary and intranasal (respiratory) routes.
The therapeutic agents may also be formulated for sustained release
(for example, using microencapsulation, see WO 94/07529, and U.S.
Pat. No. 4,962,091). The formulations may, where appropriate, be
conveniently presented in discrete unit dosage forms and may be
prepared by any of the methods well known to the pharmaceutical
arts. Such methods may include the step of mixing the therapeutic
agent with liquid carriers, solid matrices, semi-solid carriers,
finely divided solid carriers or combinations thereof, and then, if
necessary, introducing or shaping the product into the desired
delivery system.
When the therapeutic agents of the invention are prepared for oral
administration, they are generally combined with a pharmaceutically
acceptable carrier, diluent or excipient to form a pharmaceutical
formulation, or unit dosage form. For oral administration, the
therapeutic agents may be present as a powder, a granular
formulation, a solution, a suspension, an emulsion or in a natural
or synthetic polymer or resin for ingestion of the active
ingredients from a chewing gum. The therapeutic agents may also be
presented as a bolus, electuary or paste. Orally administered
therapeutic agents of the invention can also be formulated for
sustained release, e.g., the therapeutic agents can be coated,
micro-encapsulated, or otherwise placed within a sustained delivery
device. The total active ingredients in such formulations comprise
from 0.001 to 99.9% by weight of the formulation.
By "pharmaceutically acceptable" it is meant a carrier, diluent,
excipient, and/or salt that is compatible with the other
ingredients of the formulation, and not deleterious to the
recipient thereof.
Pharmaceutical formulations containing the therapeutic agents of
the invention can be prepared by procedures known in the art using
well-known and readily available ingredients. For example, the
therapeutic agents can be formulated with common excipients,
diluents, or carriers, and formed into tablets, capsules,
solutions, suspensions, powders, aerosols and the like. Examples of
excipients, diluents, and carriers that are suitable for such
formulations include buffers, as well as fillers and extenders such
as starch, cellulose, sugars, mannitol, and silicic derivatives.
Binding agents can also be included such as carboxymethyl
cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose
and other cellulose derivatives, alginates, gelatin, and
polyvinyl-pyrrolidone. Moisturizing agents can be included such as
glycerol, disintegrating agents such as calcium carbonate and
sodium bicarbonate. Agents for retarding dissolution can also be
included such as paraffin. Resorption accelerators such as
quaternary ammonium compounds can also be included. Surface active
agents such as cetyl alcohol and glycerol monostearate can be
included. Adsorptive carriers such as kaolin and bentonite can be
added. Lubricants such as talc, calcium and magnesium stearate, and
solid polyethyl glycols can also be included. Preservatives may
also be added. The compositions of the invention can also contain
thickening agents such as cellulose and/or cellulose derivatives.
They may also contain gums such as xanthan, guar or carbo gum or
gum arabic, or alternatively polyethylene glycols, bentones and
montmorillonites, and the like.
For example, tablets or caplets containing the therapeutic agents
of the invention can include buffering agents such as calcium
carbonate, magnesium oxide and magnesium carbonate. Caplets and
tablets can also include inactive ingredients such as cellulose,
pregelatinized starch, silicon dioxide, hydroxy propyl methyl
cellulose, magnesium stearate, microcrystalline cellulose, starch,
talc, titanium dioxide, benzoic acid, citric acid, corn starch,
mineral oil, polypropylene glycol, sodium phosphate, zinc stearate,
and the like. Hard or soft gelatin capsules containing at least one
therapeutic agent of the invention can contain inactive ingredients
such as gelatin, microcrystalline cellulose, sodium lauryl sulfate,
starch, talc, and titanium dioxide, and the like, as well as liquid
vehicles such as polyethylene glycols (PEGs) and vegetable oil.
Moreover, enteric-coated caplets or tablets containing one or more
therapeutic agents of the invention are designed to resist
disintegration in the stomach and dissolve in the more neutral to
alkaline environment of the duodenum.
The therapeutic agents of the invention can also be formulated as
elixirs or solutions for convenient oral administration or as
solutions appropriate for parenteral administration, for instance
by intramuscular, subcutaneous, intraperitoneal or intravenous
routes. The pharmaceutical formulations of the therapeutic agents
of the invention can also take the form of an aqueous or anhydrous
solution or dispersion, or alternatively the form of an emulsion or
suspension or salve.
Thus, the therapeutic agents may be formulated for parenteral
administration (e.g., by injection, for example, bolus injection or
continuous infusion) and may be presented in unit dose form in
ampoules, pre-filled syringes, small volume infusion containers or
in multi-dose containers. As noted above, preservatives can be
added to help maintain the shelve life of the dosage form. The
active polypeptides, nucleic acids or antibodies and other
ingredients may form suspensions, solutions, or emulsions in oily
or aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively,
the active polypeptides, nucleic acids or antibodies and other
ingredients may be in powder form, obtained by aseptic isolation of
sterile solid or by lyophilization from solution, for constitution
with a suitable vehicle, e.g., sterile, pyrogen-free water, before
use.
These formulations can contain pharmaceutically acceptable
carriers, vehicles and adjuvants that are well known in the art. It
is possible, for example, to prepare solutions using one or more
organic solvent(s) that is/are acceptable from the physiological
standpoint, chosen, in addition to water, from solvents such as
acetone, ethanol, isopropyl alcohol, glycol ethers such as the
products sold under the name "Dowanol," polyglycols and
polyethylene glycols, C.sub.1-C.sub.4 alkyl esters of short-chain
acids, ethyl or isopropyl lactate, fatty acid triglycerides such as
the products marketed under the name "Miglyol," isopropyl
myristate, animal, mineral and vegetable oils and
polysiloxanes.
It is possible to add, if necessary, an adjuvant chosen from
antioxidants, surfactants, other preservatives, film-forming,
keratolytic or comedolytic agents, perfumes, flavorings and
colorings. Antioxidants such as t-butylhydroquinone, butylated
hydroxyanisole, butylated hydroxytoluene and .alpha.-tocopherol and
its derivatives can be added.
Additionally, the polypeptides or antibodies are well suited to
formulation as sustained release dosage forms and the like. The
formulations can be so constituted that they release the
therapeutic agents, for example, in a particular part of the
intestinal or respiratory tract, possibly over a period of time.
Coatings, envelopes, and protective matrices may be made, for
example, from polymeric substances, such as polylactide-glycolates,
liposomes, microemulsions, microparticles, nanoparticles, or waxes.
These coatings, envelopes, and protective matrices are useful to
coat indwelling devices, e.g., stents, catheters, peritoneal
dialysis tubing, draining devices and the like.
For topical administration, the therapeutic agents may be
formulated as is known in the art for direct application to a
target area. Forms chiefly conditioned for topical application take
the form, for example, of creams, milks, gels, dispersion or
microemulsions, lotions thickened to a greater or lesser extent,
impregnated pads, ointments or sticks, aerosol formulations (e.g.,
sprays or foams), soaps, detergents, lotions or cakes of soap.
Other conventional forms for this purpose include wound dressings,
coated bandages or other polymer coverings, ointments, creams,
lotions, pastes, jellies, sprays, and aerosols. Thus, the
therapeutic agents of the invention can be delivered via patches or
bandages for dermal administration. Alternatively, the polypeptide
or antibody can be formulated to be part of an adhesive polymer,
such as polyacrylate or acrylate/vinyl acetate copolymer. For
long-term applications it might be desirable to use microporous
and/or breathable backing laminates, so hydration or maceration of
the skin can be minimized. The backing layer can be any appropriate
thickness that will provide the desired protective and support
functions. A suitable thickness will generally be from about 10 to
about 200 microns.
Ointments and creams may, for example, be formulated with an
aqueous or oily base with the addition of suitable thickening
and/or gelling agents. Lotions may be formulated with an aqueous or
oily base and will in general also contain one or more emulsifying
agents, stabilizing agents, dispersing agents, suspending agents,
thickening agents, or coloring agents. The therapeutic agents can
also be delivered via iontophoresis, e.g., as disclosed in U.S.
Pat. Nos. 4,140,122; 4,383,529; or 4,051,842. The percent by weight
of a therapeutic agent of the invention present in a topical
formulation will depend on various factors, but generally will be
from 0.001% to 95% of the total weight of the formulation, and
typically 0.01-85% by weight.
Drops, such as eye drops or nose drops, may be formulated with one
or more of the therapeutic agents in an aqueous or non-aqueous base
also comprising one or more dispersing agents, solubilizing agents
or suspending agents. Liquid sprays are conveniently delivered from
pressurized packs. Drops can be delivered via a simple eye
dropper-capped bottle, or via a plastic bottle adapted to deliver
liquid contents dropwise, via a specially shaped closure.
The therapeutic agents may further be formulated for topical
administration in the mouth or throat. For example, the active
ingredients may be formulated as a lozenge further comprising a
flavored base, usually sucrose and acacia or tragacanth; pastilles
comprising the composition in an inert base such as gelatin and
glycerin or sucrose and acacia; and mouthwashes comprising the
composition of the present invention in a suitable liquid
carrier.
The pharmaceutical formulations of the present invention may
include, as optional ingredients, pharmaceutically acceptable
carriers, diluents, solubilizing or emulsifying agents, and salts
of the type that are available in the art. Examples of such
substances include normal saline solutions such as physiologically
buffered saline solutions and water. Specific non-limiting examples
of the carriers and/or diluents that are useful in the
pharmaceutical formulations of the present invention include water
and physiologically acceptable buffered saline solutions such as
phosphate buffered saline solutions pH 7.0-8.0.
The therapeutic agents of the invention can also be administered to
the respiratory tract. Thus, the present invention also provides
aerosol pharmaceutical formulations and dosage forms for use in the
methods of the invention. In general, such dosage forms comprise an
amount of at least one of the agents of the invention effective to
treat or prevent the clinical symptoms of a specific infection,
indication or disease. Any statistically significant attenuation of
one or more symptoms of an infection, indication or disease that
has been treated pursuant to the method of the present invention is
considered to be a treatment of such infection, indication or
disease within the scope of the invention.
Alternatively, for administration by inhalation or insufflation,
the composition may take the form of a dry powder, for example, a
powder mix of the therapeutic agent and a suitable powder base such
as lactose or starch. The powder composition may be presented in
unit dosage form in, for example, capsules or cartridges, or, e.g.,
gelatin or blister packs from which the powder may be administered
with the aid of an inhalator, insufflator, or a metered-dose
inhaler (see, for example, the pressurized metered dose inhaler
(MDI) and the dry powder inhaler disclosed in Newman, S. P. in
Aerosols and the Lung, Clarke, S. W. and Davia, D. eds., pp.
197-224, Butterworths, London, England, 1984).
Therapeutic agents of the present invention can also be
administered in an aqueous solution when administered in an aerosol
or inhaled form. Thus, other aerosol pharmaceutical formulations
may comprise, for example, a physiologically acceptable buffered
saline solution containing between about 0.1 mg/ml and about 100
mg/ml of one or more of the therapeutic agents of the present
invention specific for the indication or disease to be treated. Dry
aerosol in the form of finely divided solid polypeptide, nucleic
acid or antibody particles that are not dissolved or suspended in a
liquid are also useful in the practice of the present invention.
Polypeptides, nucleic acids or antibodies of the present invention
may be formulated as dusting powders and comprise finely divided
particles having an average particle size of between about 1 and 5
.mu.m, alternatively between 2 and 3 .mu.m. Finely divided
particles may be prepared by pulverization and screen filtration
using techniques well known in the art. The particles may be
administered by inhaling a predetermined quantity of the finely
divided material, which can be in the form of a powder. It will be
appreciated that the unit content of active ingredient or
ingredients contained in an individual aerosol dose of each dosage
form need not in itself constitute an effective amount for treating
the particular infection, indication or disease since the necessary
effective amount can be reached by administration of a plurality of
dosage units. Moreover, the effective amount may be achieved using
less than the dose in the dosage form, either individually, or in a
series of administrations.
For administration to the upper (nasal) or lower respiratory tract
by inhalation, the therapeutic agents of the invention are
conveniently delivered from a nebulizer or a pressurized pack or
other convenient means of delivering an aerosol spray. Pressurized
packs may comprise a suitable propellant such as
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol, the dosage unit may be
determined by providing a valve to deliver a metered amount.
Nebulizers include, but are not limited to, those described in U.S.
Pat. Nos. 4,624,251; 3,703,173; 3,561,444; and 4,635,627. Aerosol
delivery systems of the type disclosed herein are available from
numerous commercial sources including Fisons Corporation (Bedford,
Mass.), Schering Corp. (Kenilworth, N.J.) and American Pharmoseal
Co., (Valencia, Calif.). For intra-nasal administration, the
therapeutic agent may also be administered via nose drops, a liquid
spray, such as via a plastic bottle atomizer or metered-dose
inhaler. Typical of atomizers are the Mistometer (Wintrop) and the
Medihaler (Riker).
Furthermore, the active ingredients may also be used in combination
with other therapeutic agents, for example, pain relievers,
anti-inflammatory agents, antihistamines, antimicrobial agents,
bronchodilators and the like, whether for the conditions described
or some other condition.
The present invention further pertains to a packaged pharmaceutical
composition for modulating glucocorticoid responsive gene
expression such as a kit or other container. The kit or container
holds a therapeutically effective amount of a pharmaceutical
composition for modulating glucocorticoid responsive gene
expression and instructions for using the pharmaceutical
composition for modulating glucocorticoid responsive gene
expression. The pharmaceutical composition includes at least one
STAMP polypeptide, siRNA, ribozyme, anti-sense nucleic acid or
antibody of the present invention, in a therapeutically effective
amount such that glucocorticoid responsive gene expression is
modulated. The composition can also contain a glucocorticoid
receptor, a glucocorticoid agonist, a glucocorticoid antagonist
and/or a glucocorticoid coactivator.
In another embodiment, the invention provides a packaged
pharmaceutical composition for modulating STAMP activity. The kit
or container holds a therapeutically effective amount of a
pharmaceutical composition for modulating STAMP activity and
instructions for using the pharmaceutical composition for
modulating STAMP activity. The pharmaceutical composition includes
at least one siRNA, ribozyme, anti-sense nucleic acid or antibody
of the present invention, in a therapeutically effective amount
such that STAMP activity is modulated.
The invention will be further described by reference to the
following detailed examples, which are given for illustration of
the invention, and are not intended to be limiting thereof.
EXAMPLE 1
Materials and Methods
This Example illustrates some of the materials and methods used for
demonstrating the utility of the compositions and methods of the
invention.
Unless otherwise indicated, all operations were performed at
0.degree. C.
Chemicals
Dexamethasone (Dex) and phorbol 12-myristate 13-acetate (PMA) were
purchased from Sigma. Dexamethasone-21-mesylate (Dex-Mes) (Simons
Jr. et al., 1980, J. Org. Chem., 45, 3084-3088) and
Dexamethasone-oxetanone (Dex-Ox) (Pons and Simons Jr., 1981, J.
Org. Chem., 46, 3262-3264) were synthesized as described.
[.sup.35S]methionine and [.sup.32P]dATP were from Amersham.
Restriction enzymes and DNA polymerase were obtained from New
England Biolabs, Amersham Biosciences, or Promega.
Antibodies
Anti-HA mouse monoclonal antibody (Roche), anti-FLAG mouse
monoclonal antibody (Sigma), anti-GR monoclonal antibody (BUBR-2;
Affinity BioReagents), and mouse monoclonal anti-GAL DBD and
anti-VP16 antibodies (Santa Cruz Biotechnology) are commercially
available.
Plasmids and STAMP Cloning
GR, GREtkLUC, TIF2, TIF2.4, TIF2.4m123, SRC1, SRC1-1139C, VP16-GR,
pBAL-GR, GST-TIF2.4, GAL-TIF2.4, GRIP (HA-GRIP) have been described
in He et al. (2002) J. Biol. Chem. 277, 49256-49266. VP16 chimeras
of full length androgen, estrogen (.alpha. and .beta.), thyroid
(.beta.), and retinoid (.alpha.) receptors are described in Chang
et al. (1999) Mol. Cell. Biol 19:8226-39. KIAA0998 plasmid was
donated by Takahiro Nagase (Kazusa DNA Research Institute, Japan).
AP-1-Luc (Inez Rogatsky, UCSF Medical School, San Francisco,
Calif.) and AR, AREtkLUC, VP16/PPAR.gamma., and VP16/RXR.alpha.
(Kai Ge, NIDDK, NIH) were gifts. pSos-TIF2.4 was constructed by
inserting the NcoI/NotI fragment of Gal-TIF2.4 into pSos
(Stratagene). Full length STAMP was constructed by assembling the
BamHI/XbaI fragment of KIAA0998 clone (Japanese Kazusa DNA
Institute) with the EcoRV/BamHI fragment of IMAGE clone 3632160
(Open Biosystems) and inserting the product into the EcoRV/XbaI
sites of pCMV-Sport6 (Invitrogen).
pSos-TIF2.4 was constructed by inserting the NcoI/NotI fragment of
Gal-TIF2.4 into pSos (Stratagene).
Full length STAMP was constructed by assembling the BamHI/XbaI
fragment of KIAA0998 clone (Japanese Kazusa DNA Institute) with the
EcoRV/BamHI fragment of IMAGE clone 3632160 (Open Biosystems) and
inserting the product into the EcoRV/XbaI sites of pCMV-Sport6
(Invitrogen). The following primers were used to construct
different fragments of STAMP or other constructs:
TABLE-US-00027 STAMP 3' PRIMER 2: (SEQ ID NO: 45) 5'-GCT CTA GAT
GCA CCC AGG AGT GGT GAA CAG G-3'. STAMP 5'PRIMER 2: (SEQ ID NO: 46)
5'-AAG ATG AGA CAG GAA TCT GTG CC-3'. 220F: (SEQ ID NO: 47) 5'-TAC
GAT ATC TGA TGG CCC GGG ACC TGG AGG AAA C-3'. 525R: (SEQ ID NO: 48)
5'-AGT AAG AAG GGC TTC AGG-3'. Xb2092R: (SEQ ID NO: 49) 5'-CGT CTA
GAC TAC CAA AGC TGT CAA TGA GGG TG-3'. ER2093F: (SEQ ID NO: 50)
5'-GGA ATT CGA AAA TAC ACC CAA AGA AAA TTC C-3'. Xb2729R: (SEQ ID
NO: 51) 5'-GCT CTA GAC ACC TTT TGC CCC ACT ATC AGA A-3'. ER2728F:
(SEQ ID NO: 52) 5'-GGA ATT CGA TCA CCC TGA GAC TAT AAT GG-3'.
Xb3091R: (SEQ ID NO: 53) 5'-CGT CTA GAG GCC AGT AGG GCT TGG GAT GTT
C-3'. ER3091F: (SEQ ID NO: 54) 5'-GGA ATT CCT GCC ACG CTG TCG ATC
AGG AAG-3'. ER3605F: (SEQ ID NO: 55) 5'-GGA ATT CAC AGG GGT GGT CCC
CCA GCA C-3'. Xb3605R: (SEQ ID NO: 56) 5'-CGT CTA GAT AGC CTG GCT
GTA CAC GTT GTT TTC-3'. TIF2-1140F: (SEQ ID NO: 57) 5'-GGA ATT CGC
ACA AAT GGC CCA GGG TAG C-3'. TIF2-1464R: (SEQ ID NO: 58) 5'-CGG
GAT CCT CAG CAA TAT TTC CGT GTT GTG TC-3'.
HA-pSG5-STAMP (HA/STAMP) was constructed by joining the EcoRV/BamHI
fragment of the 5' STAMP PCR product of 220F and 525R primers with
the BamHI/NotI fragment of KIAA0998 followed by insertion into the
EcoRV/NotI site of a modified HA-pSG5. The modified HA-pSG5 was
prepared by inserting the annealed double-strand oligonucleotide
(GAA TTC CCG GGA TAT CGT CGA CCC ACG CGT CCG GGG CGG CCG CTC TAG
AGT ATC CCT CGA GGA TCC (SEQ ID NO:59) into the EcoRI/BamHI Sites
of HA-pSG5 (from Mike Stallcup, USC, Los Angeles, Calif.), thus
introducing new EcoRV and NotI Sites.
pFlag/STAMP (Flag/STAMP) was constructed by inserting the
EcoRV/XbaI fragment of HA/STAMP into pFlag-CMV2 (Sigma).
GAL/STAMP(623-834) (Gal/6-6CL1) and VP16/6-6CL1 were constructed by
inserting the 1.2 Kb EcoRI fragment of the original 6-6CL1 clone
from the yeast library screening into the PM and VP16 plasmids
(Clontech) respectively.
GAL/STAMP(N623): the PCR amplified fragment prepared with 220F and
Xb2092R primers was then cut and insert into the EcoRV/XbaI sites
of PM plasmid.
GAL/STAMP(N834): the PCR amplified fragment prepared with 220F and
Xb2729R primers was then cut and inserted into the EcoRV/XbaI sites
of PM plasmid.
GAL/STAMP(834C): the PCR amplified fragment prepared with ER2728F
and STAMP 3' primer 2 was then cut and inserted into the EcoR1/XbaI
sites of PM plasmid.
GAL/STAMP(1127C): the PCR amplified fragment prepared with ER3605F
and STAMP 3' primer 2 was then cut and inserted into the EcoR1/XbaI
sites of PM plasmid.
GAL/STAMP(834-956): the PCR amplified fragment prepared with
ER2728F and Xb3091R primers was then cut and inserted into the
EcoR1/XbaI sites of PM plasmid.
GAL/STAMP(956-1127): the PCR amplified fragment prepared with
ER3091F and Xb3605R primers were then cut and inserted into the
EcoR1+XbaI sites of PM plasmid.
GST/STAMP(956C): the PCR amplified fragment prepared with ER3091F
and STAMP 3' primers 2 was then cut and inserted into the
EcoR1/XhoI sites of pGEX6p1 (Amersham Pharmacia).
VP16/SRC(1139C) was constructed by inserting the EcoRI/XbaI
fragment of SRC1-1139C into VP16. VP16/TIF2(1140C) was constructed
by inserting into VP16 the EcoR1/BamHI fragment from the PCR
product of TIF2 with the TIF2-1140F and TIF2-1464R primers.
Cell Culture, Transient Transfection and Reporter Analysis
The culturing and transiently transfection of CV-1 cells was
performed generally as described in He et al. (2002) J. Biol. Chem.
277, 49256-49266 or with some modification of the procedures
described in Rogatsky et al. (2002) Proc. Natl. Acad. Sci. U. S. A.
99, 16701-16706. Triplicate samples of U2OS.rGR cells were seeded
into 24-well plates in DMEM/10% FBS at 20,000 cells per well and
transfected the following day in FBS-free DMEM by using 0.8 .mu.l
of Lipofectamine and 1.6 .mu.l of PLUS reagent (Invitrogen) per
well according to the manufacturer's instructions. The total
transfected DNA was adjusted to 150 ng/well of a 24-well plate with
pBluescriptII SK.sup.+ (Stratagene). The molar amount of plasmids
expressing different protein constructs was kept constant with
added empty plasmid or plasmid expressing human serum albumin (Wang
et al., Mol. Endocrinol. 18: 1376-95 (2004)). phRG-TK Renilla
(Promega) (10 ng/well of a 24-well plate) was included as an
internal control. After transfection (3 h), cells were re-fed with
DMEM/10% FBS, allowed to recover for 3 h, and re-fed with DMEM/10%
FBS containing appropriate hormone dilutions. Twelve hours later,
the cells were lysed and assayed for reporter gene activity using
the Dual Luciferase Assay reagents according to manufacturer's
instruction (Promega, Madison, Wis.). Luciferase activity was
measured by an EG&G Berthhold's luminometer (Microlumat LB
96P). The data were normalized either for total protein or Renilla
null luciferase activity.
Yeast Sos-Ras Two-hybrid Library Screen
The yeast Sos-Recruitment two-hybrid system (Cyto Trap Vector Kit)
and the Human Fetal Brain Cyto Trap Plasmid cDNA Library were
purchased from Stratagene and employed according to the
manufacturer's instructions. Thus, about 50 .mu.g of pSos bait
construct (pSos-TIF2.4) and 50 .mu.g pMyr cDNA plasmid library
(Statagene) were cotransformed into 10 ml of cdc25H yeast competent
cells, spread onto forty 100 mm plates of SD/glu (-UL), and grown
for 2-4 days at 25.degree. C. Colonies were replica plated onto
SD/gal (-UL) plates and grown for 6 days at 37 C. Candidate
"interactor" colonies were selected and "patched" onto SD/glu (-UL)
plates, grown for 2 days at 25.degree. C. for Gal repression. These
colonies were "repatched" onto two serial interaction plates for 4
days at 37.degree. C.: one with SD/glu (-UL) and the other with
SD/gal (-UL). Those colonies that grew on SD/gal (-UL), but not on
SD/glu (-UL), plates were selected for further study. The cDNA
plasmid from these putative colonies was isolated and cotransformed
with bait or empty pSos plasmid into cdc25H yeast cells to see
whether the candidate cDNA clone specifically interacted with the
bait in yeast.
mRNA, Total RNA Extraction and RT-PCR
Human Testis Poly A+ RNA was purchased from BD Clontech. CV-1 cell
total RNA was prepared by growing CV-1 cells to confluence in 60 mm
dishes for 2 days, lysing the cells with TRIzol (Invitrogen)
Reagent, and extracting the total RNA according to the
manufacturer's instructions. First-Strand cDNA was synthesized by
SuperScript II RNase H-Reverse Transcriptase (Invitrogen). As
suggested by the manufacturer, 5 .mu.g of total CV-1 RNA or 0.5
.mu.g human testis mRNA was mixed with 500 ng Oligo (dT)12-18
(Invitrogen), 1 .mu.l 10 mM dNTP, 2 .mu.l 0.1M DTT, 4 .mu.l
5.times. buffer, 1 .mu.l RNaseOUT Ribonuclease Inhibitor
(Invitrogen), 1 .mu.l (200U) SuperScript II Transcriptase at
42.degree. C. for 50 min. PCR was performed with STAMP 5' primer 2
and 3' primer 2 and PfuUltra High-Fidelity DNA Polymerase
(Stratagene) according to the manufacturer's instructions to
amplify the full length STAMP.
Northern Blots
STAMP(2093-2426, SEQ ID NO:60) probe was prepared by cutting the
original 6-6CL1 clone with EcoR1/SalI and purifying the 300 bp
fragment from an agarose (2%) gel. The probe (along with a control
.beta.-actin probe from BD Clontech) was labeled with
[.sup.32P]dATP using Prime-a-Gene Labeling System (Promega)
according to the manufacturer's instructions. Multiple Tissue
Northern Blot (BD Clontech) membranes were prehybridized in 5 ml
ExpressHyb Solution at 68.degree. C. for 30 min according to the
manufacturer's instructions. The ExpressHyb Solution was replaced
with fresh solution containing denatured radio-labeled probe and
the membrane was incubated at 68.degree. C. for 1 hr, rinsed in
Wash Solution 1 several times at room temperature, washed in Wash
Solution 2 for 40 min at 50.degree. C., and subjected to
radio-autography at -70.degree. C.
Mammalian Two-hybrid Assays
These assays were conducted as described (He et al., 2002). In
particular, the recommended procedure for the Mammalian Matchmaker
two-hybrid assay kit (Clontech) was modified slightly by changing
from a chloramphenicol acetyltransferase reporter to a Luciferase
Reporter pFRLuc (Stratagene), which is under the control of five
repeats of the upstream activating sequence for the binding of
GAL4.
Bacterial Expression of Proteins
The pGEX series of plasmids were transformed into Escherichia coli
(BL21; Amersham Biosciences) according to the manufacturer's
recommendations. A single colony was picked and inoculated into 3
ml of LB broth with 100 ng/ml ampicillin. After overnight culture,
1 ml of bacterial culture was diluted into 50 ml of LB broth
containing 100 ng/ml ampicillin, shaken at 37.degree. C. for 2 hr,
adjusted to 0.1 mM isopropyl-1-thio-D-galactopyranoside (IPTG), and
shaken at 37.degree. C. for another 3 hr. The cells were harvested
by centrifugation, washed once with phosphate-buffered saline,
resuspended in 10 ml of phosphate-buffered saline, and sonicated
for 30 cycles at 30% of maximum power. The supernatant was
collected for use after centrifugation (5000.times.g for 20
min).
In Vitro Transcription and Translation Assays
Template DNA plasmids (1 .mu.g), 40 .mu.l of Promega TNT (SP6)
quick coupled transcription/translation system master mixture, and
2 .mu.l of [.sup.35S]methionine (Amersham Biosciences) were brought
up to a total volume of 50 .mu.l with H.sub.2O and incubated at
30.degree. C. for 90 min according to the manufacturer's (Promega)
recommendations. If hormone stimulation is needed, hormones (Dex,
etc) were added into the transcription-translation reaction during
the 30.degree. C. incubation to both increase the stability of the
synthesized GR and to cause activation of the GR-steroid
complexes.
Pull-down Assays
These assays were conducted generally as described (He et al.,
2002). Sonicated bacterial lysates (1 ml) containing overexpressed
GST, GST/TIF2.4, or GST/STAMP-956C were incubated with 160 .mu.l of
glutathione-Sepharose 4B beads for 1 hr at 0.degree. C. The mixture
was centrifuged (12,000.times.g), the supernatant was discarded,
and the pellet was washed with phosphate-buffered saline (3.times.1
ml). Each 20-.mu.l sample of immobilized GST or GST-chimera was
then incubated overnight with 10 .mu.l of .sup.35S-labeled in vitro
translation product. The matrix was washed with phosphate-buffered
saline (4.times.1 ml). The immobilized proteins were removed from
the beads by heating at 90.degree. C. for 5 min in 20 .mu.l of
2.times.SDS loading buffer. The proteins were separated on 8-10%
SDS-PAGE gels and detected by radio-autoradiography.
Immunoprecipitation Assays
Immunoprecipitations were conducted as generally described in Wang
et al. (2004) Mol. Endocrinol. 18: 1476-95, with slight
modifications. The day before transfection, Cos-7 cells were seeded
into 150 mm dishes at 200,000 cells per dish containing 20 ml of
media. On the next day, about 30 .mu.g of DNA/dish was transfected
with 60 .mu.l of FuGene reagent. After 2 days growth, cells were
treated with EtOH.+-.Dex, washed once with 20 ml PBS 2 hr later,
lysed for 5 min at room temperature with 1.6 ml CytoBuster Protein
Extraction Reagent (Novagen) containing protease inhibitor cocktail
(Roche) per 150 mm dish, collected with a cell scraper, and
centrifuged for 5 min at 16,000.times.g (4.degree. C.). Aliquots
(700 .mu.l) of supernatant were incubated on a roller drum (3 rpm)
with either 50 .mu.l of HA-matrix (Roche) (overnight at 4.degree.
C.) or 80 .mu.l or 50% slurry protein-G (Amersham Pharmacia)
(4.degree. C. for 1 hr) and then centrifuged for 1 min at 13,000
rpm (4.degree. C.). The supernatant was incubated with 10 .mu.l of
antibody for 1 hr (4.degree. C. on a roller drum) and then
overnight at 4.degree. C. with 70.mu. of protein-G. On the next
day, the antibody complexes were centrifuged (1 min/13,000 rpm
(16,000.times.g) at 4.degree. C.), washed 3 times with 1 ml lysis
buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 0.1% NP-40), extracted
with 20 .mu.l of 2.times.SDS-loading buffer (95.degree. C. for 3
min), and separated by gel electrophoresis (8-10% SDS-PAGE).
Immunocytochemistry
Cells were cultured in phenol-red free medium with 10%
charcoal/dextran-stripped fetal bovine serum and transiently
transfected in 2-4 chambers per slide (Nalge Nunc). Twenty four to
48 hrs after transfection, the cells were treated with steroid
(some controls received no steroid), washed with 1 ml cold PBS and
fixed with 1 ml of freshly prepared 4% paraformaldehyde (PFA) for
20 min at room temperature. The cells were washed with PBS (5 min),
treated with 1 ml of 100% methanol (10 min), washed with PBS (10
min) and treated with 1 ml of blocking buffer (1.times.PBS, 0.1%
Tween 20, 3% BSA) for 30 min, all at room temperature. After
incubation with 250 .mu.l of the first antibody in blocking buffer
(room temperature for 2 hrs), the cells were washed twice with
PBS+0.05% Tween 20 (each for 10 min) and then treated with 250
.mu.l of the fluorescence-coupled second antibody (anti-mouse;
Jackson ImmunoResearch) in blocking buffer (room temperature for 1
hr in the dark) before being washed (1 ml of PBS+0.05% Tween 20, 10
min) and treated with 1 .mu.g/ml of 4,6-diamidino-2-phenyindole
(DAPI from Sigma) in PBS+0.05% Tween 20 (5 min in the dark). The
cells were washed (PBS+0.05% Tween 20, 10 min) before being
examined with a fluorescent microscope.
ChIP Assays
The basic method of Ma et al. (Ma et al. (2003) Methods Enzymol.
364: 284-2966, was used with modifications. Two days before
harvest, U2OS.rGR cells were seeded into 150 mm dishes at 3,000,000
cells per dish in phenol-red free medium with 10%
charcoal/dextran-stripped fetal bovine serum. Each dish was
transfected on the next day with 15 .mu.g HA-STAMP and 60 .mu.l
Liopfectamine. On the third day, after brief ligand treatment,
formaldehyde (37%) was added directly into the medium to a final
concentration of 1% (37.degree. C., 10 min), which was followed by
glycine to a final concentration of 0.125 M (room temperature, 5
min with shaking). Cells were washed with (4.degree. C.) cold PBS
twice and harvested by scraping into 5 ml of cold (4.degree. C.)
PBS with a protease inhibitor cocktail (Roche). After centrifuge
(1200.times.g, 10 min), the cell pellet was resuspended in 1 ml
hypotonic buffer (0.degree. C., 10 min) and lysed by passing
through a #25 gauge needle five times. The nuclei are collected by
centrifugation (15,000.times.g, 10 min) and resuspended in 250
.mu.l of nuclear lysis buffer per dish (0.degree. C., 10 min). The
nuclear pellet is sonicated with a Fisher Scientific Ultrasonic
Dismembrator (model 500) (3-5 pulses of 15 s pulse on, 45 s pulse
off, 25% power). After centrifugation (15,000.times.g, 10 min), 100
.mu.l of supernatant is diluted into 1 ml of IP dilution buffer and
treated with 20 .mu.l pre-blocked protein G beads (Amersham
Pharmacia) with gentle mixing (4.degree. C., 1 hr on a rotating
drum at 4 rpm). After centrifugation (15,000.times.g, 10 min), the
supernatant was treated with 2 .mu.g of anti-GR (Affinity
BioReagents, PA1-511A) or anti-HA (Santa Cruz, sc7392) antibody
(4.degree. C., overnight). The next morning, 40 .mu.l of
pre-blocked protein G beads were added (4.degree. C., 2 hr). The
pellet was centrifuged (5500 g, 1 min), washed sequentially by 1 ml
each of wash buffers I, II, and III, three times with 1 ml TE, and
then eluted with 2.times.150 .mu.l elution buffer. The eluents were
combined, adjusted to 300 mM NaCl (65.degree. C., overnight, total
volume=300 .mu.l) to reverse the cross-linking and then treated
with 10 .mu.l EDTA (0.5 M), 40 .mu.l Tris-HCl (1 M, pH 6.5) and 2
.mu.l proteinase K (10 .mu.g/.mu.l, Sigma) at 45.degree. C. for 1
hr. The DNA was purified by a Qiagen PCR Purification Kit according
to the manufacture's instructions. The immunoprecipitated DNA was
amplified by PCR using the published primers for the collagenase 3
(Rogatsky et al., EMBO J. 20:6071-83 (2001)) and .beta.-actin (Kim
et al., Mol. Cell 12: 1537-49 (2003)) genes.
siRNA Assays
Four different siRNA oligonucleotides for STAMP were designed and
synthesized by Qiagen:
TABLE-US-00028 1) GCCAGCTGAGTACGCGGAATT; (SEQ ID NO: 39) 2)
CACCCTCTCTGAAGCACAAAA; (SEQ ID NO: 38) 3) CAGCACTGACTATAACCTAAT;
(SEQ ID NO: 37) and 4) GAGGGCAATGAGGCCAAAATA. (SEQ ID NO: 40)
A control .beta.-actin siRNA was also purchased from Qiagen.
U2OS.rGR cells were seeded at 50,000 cells per well in 24 wells
plate the day before transfection. DNA (STAMP plasmid and AP-1/Luc
reporter; total=150 ng) together with 500 ng siRNA were mixed with
1.6 .mu.l Lipofectamine 2000 (Invitrogen), allowed to stand at room
temperature for 15 min, and then added to FBS-free DMEM pre-washed
cells. Four hours later, the cells were re-fed with DMEM/10% FBS
(containing G418), allowed to recover for 2 h, and re-fed with
DMEM/10% FBS (containing G418) containing appropriate hormone
dilutions.+-.PMA. The cells were harvested and Luciferase activity
was determined as described above in the standard transient
transfection studies.
EXAMPLE 2
STAMP Isolation and Characterization
This Example describes the isolation and characterization of the
STAMP factor.
Isolation of a TIF2.4 Binding Protein by Yeast Two-hybrid Library
Screening
The activity of TIF2 and its derivatives (FIG. 1) in modulating
glucocorticoid receptor transactivation properties was assessed by
quantifying the ability of exogenous coactivator to shift the
EC.sub.50 value of the dose-response curve to lower concentrations
of agonist steroid and to increase the partial agonist activity of
an anti-glucocorticoid. Szapary et al. (1996) J. Biol. Chem. 271,
30576-30582; Szapary et al. (1999) Mol. Endocrinol. 13, 2108-2121;
Chen et al. (2000) J. Biol. Chem. 275, 40810-40816; He et al.
(2002) J. Biol. Chem. 277, 49256-49266. Using this assay in CV-1
cells, the inventors previously established that TIF2.4 retains
most of the modulatory activity of TIF2. He et al. (2002) J. Biol.
Chem. 277, 49256-49266. As shown in FIG. 1 of He et al. (2002),
TIF2.37 and TIF2.38 competitively inhibit the actions of TIF2.4,
suggesting that a protein present in CV-1 cells may bind to TIF2.37
and TIF2.38.
These results suggested that TIF2.37 and/or TIF2.38 could be used
to isolate the protein or proteins that bind to these TIF2
fragments. Unfortunately, both TIF2.37 and TIF2.38 show strong
autonomous transactivation activity when used as bait in the
conventional yeast two-hybrid screen (data not shown). Therefore, a
Sos-Ras system (FIG. 1C) was used that involved a membrane-bound
yeast-two hybrid screen that displays no activity with bait but
that has intrinsic transactivation activity on reporter genes. See
also, Aronheim (1997) Nucleic Acids Res 25, 3373-3374. In this
system, TIF2.4 was used as the bait with a human fetal brain
library.
Using the Sos-Ras system with the procedures described in Example
1, a clone was isolated named 6-6CL1 (clone 1 in FIG. 2A), which
had an open reading frame of 214 amino acids. In a mammalian
two-hybrid assay, the 6-6CL1 clone (fused to the VP16 activation
domain) did interact with TIF2.4 but did not interact with equal
amounts of TIF2.4m123 protein (see FIGS. 1 and 2B). These results
indicate that TIF2.4m123 could not block the TIF2.4-induced changes
in GR dose-response curve and partial agonist activity (data not
shown). This may be an indirect effect of the mutated RIDs present
in TIF2.4m 123, because previous results obtained by the inventors
indicate that TIF2.4 fragments which lack the RIDs domains (e.g.
TIF2.37 and TIF2.38 shown in FIG. 1A) do not inhibit the modulatory
actions of TIF2.4 (see He et al. (2002) J. Biol. Chem. 277,
49256-49266). The same two-hybrid assay revealed negligible
association of 6-6CL1 with p300, CBP, and PCAF (data not shown),
which are cofactors known to interact with GR/TIF2 complexes. See
Chen et al., 1997, Cell 90, 569-580; Voegel et al., 1998, EMBO J.
17, 507-519; Ma et al., 1999, Mol. Cell. Biol. 19, 6164-6173; Chen
et al., 2000, J. Biol. Chem. 275, 40810-40816. The 6-6CL1 gene
product also interacted with TIF2.4 when using the opposite
orientation of fusion constructs (FIG. 2C).
SRC-1, like TIF2, can modulate the dose-response curve and partial
agonist activity of GR complexes. Szapary et al., 1999, Mol.
Endocrinol. 13, 2108-2121. The C-terminal 303 amino acid SRC-1
fragment (termed 1139C in FIG. 1) was previously found to possess
the greatest GR modulatory activity of other SRC-1 peptides tested.
He et al., 2002, J. Biol. Chem. 277, 49256-49266. Interestingly,
this same C-terminal portion of SRC-1 strongly interacted with the
6-6CL1 polypeptide in the mammalian two-hybrid assay (FIG. 2D). In
contrast, while the TIF2 and SRC-1 domains are similarly organized
(McKenna and O'Malley, 2002, Cell 108, 465-474; Xu and Li, 2003,
Mol. Endocrinol. 17, 1681-1692), the segment of TIF2 that is
spatially analogous to the 1139C fragment (i.e. the TIF2(1140C)
fragment shown in FIG. 1), does not interact with the 6-6CL1 clone
(FIG. 2E). Western blots showed that such inactivity by the
TIF2(1140C) fragment was not due to poor expression of the protein
(FIG. 2F). These data indicate that distinct (not analogous)
domains of TIF2, and SRC-1 modulate GR transactivation properties.
These data also indicate that the 6-6CL1 polypeptide is part of a
protein that can interact with SRC-1, as well as TIF2. The name
STAMP (SRC-1 and TIF2 associated modulatory protein) was chosen for
the predicted full length protein that contains the 6-6CL1
sequence.
Isolation of the Full Length Clone of STAMP
Examination of the sequences available in GenBank revealed that
6-6CL1 corresponded to part of a human protein of unknown function.
See Nagase et al., 1999, DNA Res 6, 63-70 (GenBank Accession Nos.
AB023215, NM.sub.--015072, BC002766). The presence of an EST
(IMAG:3632160) that extended upstream from the 5' end of those
clones containing 6-6CL1 indicated that the full length clone for
STAMP had not yet been isolated.
Therefore, a full-length STAMP clone was isolated using the primers
described in Example 1. Sequence analysis of the full-length STAMP
sequence indicated that the 1277 amino acid STAMP polypeptide is
encoded in a 4.6 kb cDNA (GenBank Accession No. AY237126) (FIG.
3A). The STAMP polypeptide had a predicted molecular weight of 143
kDa and is encoded in a locus residing on chromosome 14q24.3 with
32 introns (GenBank Accession No. NM.sub.--015072). There is a
weaker Kozak sequence (purine at -3 but no G at +4) (Kozak (1989)
J. Cell Biol. 108, 229-241) for an alternative start site that is 4
amino acids upstream of the predicted major start site (italicized
residues in FIG. 3A). Therefore, there may be heterogeneity in the
amino terminal sequence of the STAMP protein in some tissues. The
presence of multiple stop codons in all reading frames upstream of
the start of translation and several in-frame stop codons at the
end of the open reading frame argue that this clone encodes the
full length of the protein STAMP.
Reverse transcription-Polymerase Chain Reaction (RT-PCR)
amplification of an mRNA preparation from human testis (Clontech)
using internal primers (underlined sequences in FIG. 3A) produced
the correct size for the STAMP cDNA of 4.1 kb (FIG. 3B). These data
indicate that a mRNA encoding the full length STAMP protein exists
in intact cells. Northern blots reveal the existence of the
predicted 4.6 kb mRNA in 12 different tissues (FIG. 3C). The levels
of the STAMP mRNA were highest in heart and skeletal muscle. The
STAMP mRNA levels were usually low in the other tissues examined
suggesting that the STAMP protein levels in these tissues may also
be low. However, these data also indicate that STAMP is a
ubiquitously expressed protein in humans.
RT-PCR of the mRNA from CV-1 cells, followed by sequencing, showed
that monkey cells contain a cDNA (GenBank Accession No. AY383558)
that is 98% identical to the human STAMP cDNA (FIG. 3D; alignment
from BLAST on GCG). This monkey clone is predicted to encode a
protein that is 97% identical to the human STAMP protein (FIG. 3E).
Mice contain a cDNA encoding a protein of unknown function (GenBank
Accession No. XM.sub.--126935) that is 83.3% identical to the
C-terminal 705 amino acids of the human STAMP protein. Thus the
STAMP gene and protein are conserved among these different
species.
Whole Cell Localization of STAMP
Indirect immunofluorescence, using HA-tagged full-length STAMP
revealed that STAMP has a diverse cellular localization in U2OS.rGR
(FIG. 3F1-6) and CV-1 cells (data not shown) in the absence of
dexamethasone (Dex). STAMP was usually observed in the cytoplasm
(red in the original) and associated with glucocorticoid receptors
(GRs, green in the original), as indicated by the lighter shades in
FIG. 3. Twenty minutes after the addition of Dex, STAMP colocalized
with GRs in the nucleus of numerous cells (lighter shades in FIG.
3F). In both cases, the amount of STAMP-GR complexes was variable,
presumably due to different ratios of STAMP to GR in the cells.
Activity of STAMP in GR-mediated Induction
Tests were run to determine whether exogenous STAMP can modulate
the EC.sub.50 of glucocorticoids and the partial agonist activity
of antiglucocorticoids in the transient transfection assays where
GR induced gene expression from a GREtkLUC reporter. See Szapary et
al., 1996, J. Biol. Chem. 271, 30576-30582; Szapary et al., 1999,
Mol. Endocrinol. 13, 2108-2121; Chen et al., 2000, J. Biol. Chem.
275, 40810-40816; He et al., 2002, J. Biol. Chem. 277,
49256-49266.
In the absence of exogenous coactivators and low concentrations of
GR, STAMP caused a weak but statistically significant shift in the
dose-response curve to lower steroid concentrations (1.54.+-.0.12
fold, S.E.M., n=6, P=0.0074) and an increase in the partial agonist
activity of the antagonist Dex-Mes (1.16.+-.0.05 fold higher from
39% to 45% activity, S.E.M., n=6, P=0.023). It should be noted that
STAMP also had a small effect on the total amount of gene product
induced by 1 .mu.M Dex (1.26.+-.0.05 fold increase, S.E.M., n=6)
but there is no change in the fold induction by 1 .mu.M Dex
(0.93.+-.0.08 fold increase, S.E.M., n=6). Addition of TIF2 alone
had a similar, small effect (FIG. 4A1).
However, a significant further left-shift in the dose-response
curve occurs when both STAMP and TIF2 are added (FIG. 4A1, see also
FIG. 4A2). When the fold change in EC.sub.50 from four independent
experiments is normalized to that of GR alone, STAMP plus TIF2
produces a greater change in EC.sub.50 to lower steroid
concentrations than expected from the responses of STAMP and TIF2
alone (FIG. 4B). Similarly, the effect of STAMP plus TIF2 on the
partial agonist activity of Dex-Mes is greater than the sum of the
individual factors (FIG. 4B). This is consistent with STAMP being a
limiting factor in the presence of exogenous GR and TIF2. At the
same time, STAMP increases the fold induction (above basal
activity) with 1 .mu.M Dex both with and without added TIF2 (FIG.
4B).
Activity of STAMP in GR-mediated Repression
Rogatsky et al. have reported that TIF2 augments GR repression of
an AP-1 induced gene. Rogatsky et al., (2002) Proc. Natl. Acad.
Sci. U. S. A. 99, 16701-16706; Rogatsky et al. (2001) EMBO J. 20:
6071-6083. The TIF2 domain identified as having TIF2 activity in GR
repression was the same as used to isolate STAMP. In order to
determine if STAMP was active in GR-mediated gene repression as
well as induction, the ability of STAMP to increase the repressive
activity of GR in the AP-1 responsive whole cell bioassay system
was assessed using U2OS.rGR cells.
As seen in FIG. 5A, the fold induction by PMA was unchanged by the
presence of TIF2, STAMP, or TIF2+STAMP. However, when dexamethasone
was added, the level of GR-related expression (without added TIF2
or STAMP) was reduced by about 5-fold. Addition of TIF2 reduced the
level of GR-related expression even further (about 9-fold). See
also, Rogatsky et al., 2002, Proc. Natl. Acad. Sci. U. S. A. 99,
16701-16706. The presence of exogenous STAMP, without added TIF2,
increased the repression by Dex-bound glucocorticoid receptors to
16-fold. This response may reflect the ability of exogenous STAMP
to supplement limiting concentrations of endogenous STAMP that
interact naturally with endogenous TIF2. Significantly, addition of
STAMP with TIF2 reduced induction of AP-1 expression in the
presence of Dex-bound GRs even further, to about 31-fold (P=0.029;
Mann-Whitney test). These data indicate that STAMP and TIF2 act
cooperatively to modulate glucocorticoid receptor activity.
The enhancement of STAMP activity by TIF2 was investigated by
comparing the fold repression seen with the wild type TIF2 to the
mutant TIF2ml23 (FIG. 1A), which does not interact with GR (Voegel
et al., 1998; Ding et al., 1998; He et al., 2002) and appears not
to bind to STAMP (FIG. 2B). In these experiments, less STAMP (50
vs. 100 ng) was added in order to reduce the fold repression due
STAMP alone. FIG. 5B shows that TIF2m123 was unable to augment
GR-mediated repression either by itself or in combination with
STAMP. Therefore, these results indicate that STAMP activity
requires the RID domains that mediate TIF2 binding to GR and/or
STAMP.
The endogenous collagenase-3 (coll3) gene in U2OS.rGR cells is
repressed by GR. TIF2 potentiates GR repression of coll3 and is
recruited to the coll3 promoter in concert with GR-agonist
complexes (Rogatsky et al., 2001). Because STAMP augments the
ability of TIF2 to increase GR repression of a transiently
transfected gene (FIG. 5A), chromatin immunoprecipitation assays
were used to ascertain whether STAMP is also recruited by GR/TIF2
complexes to the endogenous coll3 gene. FIG. 5C shows that both GR
and STAMP were induced to localize on the coll3 promoter after 20
min of steroid treatment. This is the same time period in which
nuclear colocalization of GR and STAMP is seen (FIG. 6). The
time-dependent promoter binding by both GR and STAMP also indicates
that, in intact cells, this binding is not due simply to increased
protein levels following transient transfection.
Inhibition of STAMP Actions by STAMP siRNA
Experiments were performed to ascertain whether STAMP siRNAs can
reduce STAMP protein levels and reverse the effects of STAMP. Four
siRNAs were prepared that were based upon STAMP cDNA. The
probability that each siRNA would have at least 11 nucleotides in
common with any other gene, and thus silence the expression of a
gene other than STAMP, was exceedingly small. As shown in FIG. 5D1
and 5D2, each STAMP siRNA significantly reduced the level of
overexpressed STAMP protein, with little or no effect on ectopic GR
protein levels, while a non-specific control (.beta.-actin siRNA)
had negligible effects on either protein. Thus, the STAMP siRNAs
were STAMP-specific in that they reduced the levels of expressed
STAMP protein.
As shown in FIG. 5E, each STAMP siRNA was also very effective in
reversing the ability of transfected STAMP to increase the fold
repression by GR of an AP-1 reporter relative to the .beta.-actin
siRNA control. STAMP siRNAs #3 and 4 were then used to lower the
levels of endogenous STAMP. As shown in FIG. 5F, these siRNAs can
reduce the repressive activity of GR-agonist complexes, indicating
that endogenous STAMP is present in U2OS.rGR cells and assists in
GR-mediated inhibition of an AP-1 induced gene.
Identification of Regions of STAMP Interacting with Other
Proteins
FIG. 2 indicates that the clone 6-6CL1, which encompasses amino
acids 623-834 of STAMP, binds to TIF2.4. In order to determine the
specificity of this binding, the interactions of other regions of
STAMP with TIF2.4 were examined in the mammalian two hybrid assay.
FIG. 6A shows that most of the STAMP domain that interacts with
TIF2.4 resides between amino acids 623 and 834 of STAMP. This
interaction domain is called the CID, or coactivator interaction
domain. Western blots (data not shown) and the biological activity
assays (see below) indicated that the weak response of the amino
and carboxyl fragments (N623 and 834C) in FIG. 6A was not due to
poor levels of protein expression. It should be noted that no
segment of STAMP (FIG. 6A), nor the full length STAMP (data not
shown), displayed any intrinsic transactivation activity, as
determined from the inability of each GAL/STAMP chimera to induce
the FRLuc reporter in the absence of TIF2.4.
Interestingly, a region of STAMP more C-terminal than the CID
interacted with GR in a steroid-responsive manner (FIG. 6B). Again,
Western blots (data not shown) and the activity in FIG. 6A argue
that the relative inactivity of GAL/N834 and/623-834 is not due to
inadequate protein expression. The numbers in parentheses above the
bar for VP16/GR (with Dex) represent the fold induction by with and
without Dex. While there was increased association of steroid-free
GR with the STAMP segment of amino acids 834-956, the largest
agonist-induced changes in GR interaction with STAMP occurred when
the C-terminal STAMP domain was used, including amino acids
834-1277 (=834C) (FIG. 6B). There is substantial ligand-independent
association of GR with STAMP(834-956) while the largest
agonist-induced changes in GR interaction with STAMP are restricted
to the two domains of amino acids 956-1127 and 1127-1277 (=1127C)
(FIG. 6B). Neither half of GR (N-terminal [N523] or C-terminal
[407C] half) interacted with STAMP834C in this assay under
conditions that give a strong response with GR407C and the receptor
interaction domain of TIF2 (TIF2.4; data not shown; S3 file
"STAMP-GRdomain/2Hybrid"). Thus, it appears that both N- and
C-terminal domains of GR are required for the interaction of wild
type GR with STAMP.
Importantly, STAMP834C also interacted with agonist-bound, full
length PRs (B-form) and androgen receptors (ARs) but not estrogen
(.alpha. or .beta.), PPAR.gamma.2, thyroid (TR) (.beta.), RXR
.alpha., or retinoid (.alpha.) receptors (FIG. 6C). The lack of
interaction with all but GR, PR, and AR is not due to low
expression of the other receptors as seen by their robust activity
with GAL/TIF2.4 (FIG. 6C). The ability of STAMP to supplement the
whole cell actions of TIF2 with full length AR and TR.beta. was
therefore examined. As shown in FIG. 6D, STAMP augments the
left-shift of the dose-response curve of ARs in the presence of
TIF2 but not when added by itself. Conversely, STAMP increases the
fold induction by AR but has no additional effect with TIF2. In
contrast to AR, added STAMP has little ability with TR.beta. either
to cause a left-shift in the dose-response curve or to increase the
fold induction regardless of whether TIF2 is present (FIG. 6D).
Collectively, these data suggest that STAMP association and
activity with receptors is more discriminatory than that of the
coactivator TIF2 and may be selective for the sub-class of
receptors that bind to a glucocorticoid receptor enhancer (GRE)
(Beato et al. (1989) J. Steroid Biochem. 32:737-48).
The binding of [.sup.35S]methionine-labeled, in vitro translated
STAMP constructs to GST/TIF2.4 in a pulldown assay further
indicates that a weak interaction exists between TIF2.4 and the
full length STAMP, but STAMP fragments encompassing amino acids
623-834 shows strong binding to TIF2.4 (FIG. 7A). This binding is
selective in that neither the amino terminal (N623) nor the
C-terminal (834C) fragments exhibit significantly more binding to
GST/TIF2.4 than to GST itself (FIG. 7A). However, the STAMP 623-824
fragment exhibits strong TIF binding. Similarly, the pulldown
assays that show [.sup.35S]methionine-labeled full length
glucocorticoid receptor (GR) bound to GST/STAMP(956C), although
this binding is steroid-independent (FIG. 7B).
The N-terminal domain of STAMP contains the only previously
characterized, functional domain, which is a tubulin-tyrosine
ligase (TTL) domain at amino acids 113-391 (FIG. 7A). A comparison
of the actions of Flag/STAMP with and without the TTL domain
(STAMP623C) in the presence of transfected GR and TIF2 shows that
STAMP lacking the TTL domain retains 85% of the capacity of full
length STAMP to increase the fold induction with Dex. Moreover,
STAMP lacking the TTL domain retained 40% of its ability to
increase the partial agonist activity of Dex-Mes and decreased the
EC.sub.50 for Dex induction (FIG. 8). Therefore, it appears that
the TTL domain is not essential for any of the identified
activities of STAMP.
Whole Cell Interaction of STAMP with GR and TIF2
The data provided in FIGS. 5 and 7 indicate that the biological
effects of STAMP (see, e.g., FIG. 4-5) are mediated by the binding
of STAMP to TIF2, and possibly to GR. FIGS. 7 and 8 further
indicate that STAMP and TIF2 and/or STAMP and GR functionally
interact with one another. To detect whether STAMP can interact
with TIF2 and/or GR in intact cells, Cos-7 cells were cotransfected
with HA-GRIP 1 (GRIP 1 is the mouse homolog of TIF2), GR, and
Flag-STAMP or Flag itself, and cell lysates were subjected to
immunoprecipitation with various antibodies. Anti-Flag antibody was
used to precipitate Flag-STAMP and associated proteins.
The immunoprecipitation results provided in FIG. 7C show that GRIP1
and GR are both co-immunoprecipitated with STAMP in a manner that
requires the presence of STAMP (in FIG. 7C1 compare lanes 2 and 3
with Flag-STAMP to lane 4 with just Flag). Consistent with the
pulldown data shown in FIG. 7B, the presence or absence of the
agonist Dex does not alter the ability of GR to be
co-immunoprecipitated by STAMP (FIG. 7C3, lane 2 vs. 3). Similarly,
GR and STAMP were co-immunoprecipitated with the chimera HA/GRIP by
anti-HA antibody (data not shown).
Thus, the invention provides novel STAMP polypeptides that interact
with the biologically active domains of TIF2 and SRC-1. The
association of STAMP with TIF2 is mediated by a central domain
within STAMP, while the C-terminal domain of STAMP binds to
glucocorticoid receptors. A mutation of TIF2 (to give TIF2m123)
that prevents TIF2 binding to STAMP and GR also eliminates the
ability of TIF2 to augment GR repressive activity with or without
STAMP. While STAMP can be found in both the cytoplasm and the
nucleus, STAMP and glucocorticoid receptor polypeptides do
co-localize in the nucleus, especially in the presence of
dexamethasone. The addition of STAMP to GR-responsive whole cells
increases the modulatory activity of TIF2 for a GR-induced gene
expression and accentuates the repressive effects of TIF2 for a
GR-suppressed gene activity. STAMP affects multiple aspects of GR
regulated gene expression in whole cells, including both
GR-mediated induction and repression. The repressive activity of
endogenous and exogenous STAMP is largely abrogated by addition of
STAMP siRNAs. These data indicate that STAMP is an important new
factor in the glucocorticoid regulatory network and can act as a
modulator of both GR-mediated gene activation and GR-mediated gene
repression. As illustrated herein GR-mediated gene activation and
repression can be modulated by addition of STAMP polypeptides or
STAMP siRNAs.
Articles
Aronheim, A. (1997). Improved efficiency sos recruitment system:
expression of the mammalian GAP reduces isolation of Ras GTPase
false positives. Nucleic Acids Res 25, 3373-3374.
Beato, M., Chalepakis, G., Schauer, M., and Slater, E. P. (1989).
DNA regulatory elements for steroid hormones. J. Steroid Biochem.
32, 737-748.
Brown, K., Chen, Y., Underhill, T. M., Mymryk, J. S., and Torchia,
J. (2003). The coactivator p/CIP/SRC-3 facilitates retinoic acid
receptor signaling via recruitment of GCN5. J Biol Chem 278,
39402-39412.
Chen, D., Huang, S. M., and Stallcup, M. R. (2000). Synergistic,
p160 coactivator-dependent enhancement of estrogen receptor
function by CARM1 and p300. J. Biol. Chem. 275, 40810-40816.
Chen, D., Ma, H., Hong, H., Koh, S. S., Huang, S.-M., Schurter, B.
T., Aswad, D. W., and Stallcup, M. R. (1999). Regulation of
transcription by a protein methyltransferase. Science 284,
2174-2177.
Chen, H., Lin, R. J., Schiltz, R. L., Chakravarti, D., Nash, A.,
Nagy, L., Privalsky, M. L., Nakatani, Y., and Evans, R. M. (1997).
Nuclear receptor coactivator ACTR is a novel histone
acetyltransferase and forms a multimeric activation complex with
P/CAF and CBP/p300. Cell 90, 569-580.
Chen, S., Sarlis, N. J., and Simons, Jr., S. S. (2000). Evidence
for a common step in three different processes for modulating the
kinetic properties of glucocorticoid receptor-induced gene
transcription. J. Biol. Chem. 275, 30106-30117.
Cheng, S., Brzostek, S., Lee, S. R., Hollenberg, A. N., and Balk,
S. P. (2002). Inhibition of the dihydrotestosterone-activated
androgen receptor by nuclear receptor corepressor. Mol. Endocrinol.
16, 1492-1501.
Cosma, M. P. (2002). Ordered recruitment: gene-specific mechanism
of transcription activation. Mol. Cell 10, 227-236.
Darimont, B. D., Wagner, R. L., Apriletti, J. W., Stallcup, M. R.,
Kushner, P. J., Baxter, J. D., Fletterick, R. J., and Yamamoto, K.
R. (1998). Structure and specificity of nuclear
receptor-coactivator interactions. Genes and Develop. 12,
3343-3356.
Dotzlaw, H., Moehren, U., Mink, S., Cato, A. C., Iniguez, L. J. A.,
and Baniahmad, A. (2002). The amino terminus of the human AR is
target for corepressor action and antihormone agonism. Mol.
Endocrinol. 16, 661-673.
Giannoukos, G., Szapary, D., Smith, C. L., Meeker, J. E. W., and
Simons, Jr., S. S. (2001). New antiprogestins with partial agonist
activity: potential selective progesterone receptor modulators
(SPRMs) and probes for receptor- and coregulator-induced changes in
progesterone receptor induction properties. Mol. Endocrinol. 15,
255-270.
He, Y., Szapary, D., and Simons, Jr., S. S. (2002). Modulation of
induction properties of glucocorticoid receptor-agonist and
-antagonist complexes by coactivators involves binding to receptors
but is independent of ability of coactivators to augment
transactivation. J. Biol. Chem. 277, 49256-49266.
Hong, H., Kohli, K., Trivedi, A., Johnson, D. L., and Stallcup, M.
R. (1996). GRIP1, a novel mouse protein that serves as a
transcriptional coactivator in yeast for the hormone binding
domains of steroid receptors. Proc. Natl. Acad. Sci. USA 93,
4948-4952.
Kalkhoven, E., Valentine, J. E., Heery, D. M., and Parker, M. G.
(1998). Isoforms of steroid receptor co-activator 1 differ in their
ability to potentiate transcription by the oestrogen receptor. EMBO
J. 17, 232-243.
Karin, M., and Chang, L. (2001). AP-1--glucocorticoid receptor
crosstalk taken to a higher level. J Endocrinol 169, 447-451.
Kaul, S., Blackford, Jr., J. A., Cho, S., and Simons, Jr., S. S.
(2002). Ubc9 is a novel modulator of the induction properties of
glucocorticoid receptors. J. Biol. Chem. 277, 12541-12549.
Kim, J. H., Li, H., and Stallcup, M. R. (2003). CoCoA, a nuclear
receptor coactivator which acts through an N-terminal activation
domain of p160 coactivators. Mol Cell 12, 1537-1549.
Koh, S. S., Chen, D., Lee, Y. H., and Stallcup, M. R. (2001).
Synergistic enhancement of nuclear receptor function by p160
coactivators and two coactivators with protein methyltransferase
activities. J. Biol. Chem. 276, 1089-1098.
Kozak, M. (1989). The scanning model of translation: an update. J.
Cell Biol. 108, 229-241.
Lavinsky, R. M., Jepsen, K., Heinzel, T., Torchia, J., Mullen,
T.-M., Schiff, R., Del-Rio, A. L., Ricote, M., Ngo, S., Gemsch, J.,
Hilsenbeck, S. G., Osborne, C. K., Glass, C. K., Rosenfeld, M. G.,
and Rose, D. W. (1998). Diverse signaling pathways modulate nuclear
receptor recruitment of N-CoR and SMRT complexes. Proc. Natl. Acad.
Sci. USA 95, 2920-2925.
Lee, Y. H., Koh, S. S., Zhang, X., Cheng, X., and Stallcup, M. R.
(2002). Synergy among nuclear receptor coactivators: selective
requirement for protein methyltransferase and acetyltransferase
activities. Mol. Cell Biol. 22, 3621-3632.
Ma, H., Hong, H., Huang, S.-M., Irvine, R. A., Webb, P., Kushner,
P. J., Coetzee, G. A., and Stallcup, M. R. (1999). Multiple signal
input and output domains of the 160-kilodalton nuclear receptor
coactivator proteins. Mol. Cell. Biol. 19, 6164-6173.
McInerney, E. M., Rose, D. W., Flynn, S. E., Westin, S., Mullen,
T.-M., Krones, A., Inostroza, J., Torchia, J., Nolte, R. T.,
Assa-Munt, N., Milburn, M. V., Glass, C. K., and Rosenfeld, M. G.
(1998). Determinants of coactivator LXXLL motif specificity in
nuclear receptor transcriptional activation. Genes and Develop. 12,
3357-3368.
McKenna, N. J., Lanz, R. B., and O'Malley, B. W. (1999). Nuclear
receptor coregulators: cellular and molecular biology. Endocrine
Reviews 20, 321-344.
McKenna, N. J., and O'Malley, B. W. (2002). Combinatorial control
of gene expression by nuclear receptors and coregulators. Cell 108,
465-474.
Nagase, T., Ishikawa, K., Suyama, M., Kikuno, R., Hirosawa, M.,
Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., and Ohara, O.
(1999). Prediction of the coding sequences of unidentified human
genes. XIII. The complete sequences of 100 new cDNA clones from
brain which code for large proteins in vitro. DNA Res 6, 63-70.
Onate, S. A., Boonyaratanakornkit, V., Spencer, T. E., Tsai, S. Y.,
Tsai, M.-J., Edwards, D. P., and O'Malley, B. W. (1998). The
steroid receptor coactivator-1 contains multiple receptor
interacting and activation domains that cooperatively enhance the
activation function 1 (AF1) and AF2 domains of steroid receptors.
J. Biol. Chem. 273, 12101-12108.
Onate, S. A., Tsai, S. Y., Tsai, M.-J., and O'Malley, B. W. (1995).
Sequence and characterization of a coactivator for the steroid
hormone receptor superfamily. Science 270, 1354-1357.
Oshima, H., and Simons, Jr., S. S. (1993). Sequence-selective
interactions of transcription factor elements with tandem
glucocorticoid-responsive elements at physiological steroid
concentrations. J. Biol. Chem. 268, 26858-26865.
Pons, M., and Simons, Jr., S. S. (1981). Facile, high yield
synthesis of spiro C-17-steroidal oxetan-3'-ones. J. Org. Chem. 46,
3262-3264.
Robyr, D., Wolffe, A. P., and Wahli, W. (2000). Nuclear hormone
receptor coregulators in action: diversity for shared tasks. Mol.
Endocrinol. 14, 329-347.
Rogatsky, I., Trowbridge, J. M., and Garabedian, M. J. (1997).
Glucocorticoid receptor-mediated cell cycle arrest is achieved
through distinct cell-specific transcriptional regulatory
mechanisms. Mol Cell Biol 17, 3181-3193.
Rogatsky, I., Luecke, H. F., Leitman, D. C., and Yamamoto, K. R.
(2002). Alternate surfaces of transcriptional coregulator GRIP1
function in different glucocorticoid receptor activation and
repression contexts. Proc. Natl. Acad. Sci. U. S. A. 99,
16701-16706.
Rogatsky, I., Zarember, K. A., and Yamamoto, K. R. (2001). Factor
recruitment and TIF2/GRIP1 corepressor activity at a collagenase-3
response element that mediates regulation by phorbol esters and
hormones. EMBO. J. 20, 6071-6083.
Schaaf, M. J., and Cidlowski, J. A. (2002). Molecular mechanisms of
glucocorticoid action and resistance. J Steroid Biochem Mol Biol
83, 37-48.
Schulz, M., Eggert, M., Baniahmad, A., Dostert, A., Heinzel, T.,
and Renkawitz, R. (2002). RU486-induced glucocorticoid receptor
agonism is controlled by the receptor N terminus and by corepressor
binding. J. Biol. Chem. 277, 26238-26243.
Simons, Jr., S. S. (2003). The importance of being varied in
steroid receptor transactivation. TIPS 24, 253-259.
Simons, Jr., S. S., Pons, M., and Johnson, D. F. (1980).
.quadrature.-Keto mesylate: a reactive thiol-specific functional
group. J. Org. Chem. 45, 3084-3088.
Singh, P., Chan, S. W., and Hong, W. (2001). Retinoblastoma protein
is functionally distinct from its homologues in affecting
glucocorticoid receptor-mediated transcription and apoptosis. J.
Biol. Chem. 276, 13762-13770.
Song, L.-N., Huse, B., Rusconi, S., and Simons, Jr., S. S. (2001).
Transactivation specificity of glucocorticoid vs. progesterone
receptors: role of functionally different interactions of
transcription factors with amino- and carboxyl-terminal receptor
domains. J. Biol. Chem. 276, 24806-24816.
Szapary, D., Huang, Y., and Simons, Jr., S. S. (1999). Opposing
effects of corepressor and coactivators in determining the
dose-response curve of agonists, and residual agonist activity of
antagonists, for glucocorticoid receptor regulated gene expression.
Mol. Endocrinol. 13, 2108-2121.
Szapary, D., Xu, M., and Simons, Jr., S. S. (1996). Induction
properties of a transiently transfected glucocorticoid-responsive
gene vary with glucocorticoid receptor concentration. J. Biol.
Chem. 271, 30576-30582.
Torchia, J., Rose, D. W., Inostroza, J., Kamei, Y., Westin, S.,
Glass, C. K., and Rosenfeld, M. G. (1997). The transcriptional
co-activator p/CIP binds CBP and mediates nuclear-receptor
function. Nature 387, 677-684.
Truss, M., and Beato, M. (1993). Steroid hormone receptors:
interaction with deoxyribonucleic acid and transcription factors.
Endocrine Reviews 14, 459-479.
Tsai, M.-J., and O'Malley, B. W. (1994). Molecular mechanisms of
action of steroid/thyroid receptor superfamily members. Annu. Rev.
Biochem. 63, 451-486.
Voegel, J. J., Heine, M. J. S., Tini, M., Vivat, V., Chambon, P.,
and Gronemeyer, H. (1998). The coactivator TIF2 contains three
nuclear receptor-binding motifs and mediates transactivation
through CBP binding-dependent and -independent pathways. EMBO J.
17, 507-519.
Voegel, J. J., Heine, M. J. S., Zechel, C., Chambon, P., and
Gronemeyer, H. (1996). T1F2, a 160 kDa transcriptional mediatior
for the ligand-dependent activation function AF-2 of nuclear
receptors. EMBO J. 15, 3667-3675.
Wagner, B. L., Norris, J. D., Knotts, T. A., Weigel, N. L., and
McDonnell, D. P. (1998). The nuclear corepressors NCoR and SMRT are
key regulators of both ligand- and 8-bromo-cyclic AMP-dependent
transcriptional activity of the human progesterone receptor. Mol.
Cell. Biol. 18, 1369-1378.
Wang, Q., Blackford, Jr., J. A., Song, L.-N., Huang, Y., and
Simons, Jr., S. S. (2004). Equilibrium interactions of corepressors
and coactivators modulate the properties of agonist and antagonist
complexes of glucocorticoid receptors. Mol. Endocrinol. 18,
1376-1395.
Wang, Q., Richter, W. F., Anzick, S. L., Meltzer, P. S., and
Simons, Jr., S. S. (b). Effects of changing concentrations of
receptor, coactivator, and corepressor on the transcriptional
properties of mineralocorticoid and estrogen receptors. Under
Review
Woychik, N. A., and Hampsey, M. (2002). The RNA polymerase II
machinery: structure illuminates function. Cell 108, 453-463.
Wu, Z., Belanger, G., Brennan, B. B., Lum, J. K., Minter, A. R.,
Rowe, S. P., Plachetka, A., Majmudar, C. Y., and Mapp, A. K.
(2003). Targeting the transcriptional machinery with unique
artificial transcriptional activators. J Amer Chem Soc 125,
12390-12391.
Xu, J., and Li, Q. (2003). Review of the in vivo functions of the
p160 steroid receptor coactivator family. Mol. Endocrinol. 17,
1681-1692.
Xu, L., Glass, C. K., and Rosenfeld, M. G. (1999). Coactivator and
corepressor complexes in nuclear receptor function. Current Opin.
Cell Biol. 9, 140-147.
Yao, T.-P., Ku, G., Zhou, N., Scully, R., and Livingston, D. M.
(1996). The nuclear hormone receptor coactivator SRC-1 is a
specific target of p300. Proc. Natl. Acad. Sci. USA 93,
10626-10631.
Zeng, H., Plisov, S. Y., and Simons, Jr., S. S. (2000). Ability of
the glucocorticoid modulatory element (GME) to modify
glucocorticoid receptor transactivation indicates parallel pathways
for the expression of GME and glucocorticoid response element
activities. Mol. Cell. Endo. 162, 221-234.
Zhang, X., Jeyakumar, M., Petukhow, S., and Bagchi, M. K. (1998). A
nuclear receptor corepressor modulates transcriptional activity of
antagonist-occupied steroid hormone receptor. Mol. Endocrinol. 12,
513-524.
All patents and publications referenced or mentioned herein are
indicative of the levels of skill of those skilled in the art to
which the invention pertains, and each such referenced patent or
publication is hereby incorporated by reference to the same extent
as if it had been incorporated by reference in its entirety
individually or set forth herein in its entirety. Applicants
reserve the right to physically incorporate into this specification
any and all materials and information from any such cited patents
or publications.
The specific methods and compositions described herein are
representative of preferred embodiments and are exemplary and not
intended as limitations on the scope of the invention. Other
objects, aspects, and embodiments will occur to those skilled in
the art upon consideration of this specification, and are
encompassed within the spirit of the invention as defined by the
scope of the claims. It will be readily apparent to one skilled in
the art that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the scope and
spirit of the invention. The invention illustratively described
herein suitably may be practiced in the absence of any element or
elements, or limitation or limitations, which is not specifically
disclosed herein as essential. The methods and processes
illustratively described herein suitably may be practiced in
differing orders of steps, and that they are not necessarily
restricted to the orders of steps indicated herein or in the
claims. As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, a reference
to "a host cell" includes a plurality (for example, a culture or
population) of such host cells, and so forth. Under no
circumstances may the patent be interpreted to be limited to the
specific examples or embodiments or methods specifically disclosed
herein. Under no circumstances may the patent be interpreted to be
limited by any statement made by any Examiner or any other official
or employee of the Patent and Trademark Office unless such
statement is specifically and without qualification or reservation
expressly adopted in a responsive writing by Applicants.
The terms and expressions that have been employed are used as terms
of description and not of limitation, and there is no intent in the
use of such terms and expressions to exclude any equivalent of the
features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention as claimed. Thus, it will be understood that
although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted to by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of this invention
as defined by the appended claims.
The invention has been described broadly and generically herein.
Each of the narrower species and subgeneric groupings falling
within the generic disclosure also form part of the invention. This
includes the generic description of the invention with a proviso or
negative limitation removing any subject matter from the genus,
regardless of whether or not the excised material is specifically
recited herein.
Other embodiments are within the following claims. In addition,
where features or aspects of the invention are described in terms
of Markush groups, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group.
SEQUENCE LISTINGS
1
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 74 <210>
SEQ ID NO 1 <211> LENGTH: 1277 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 1 Met Ala
Arg Asp Leu Glu Glu Thr Ala Ser Ser Ser Glu Asp Glu Glu 1 5 10 15
Val Ile Ser Gln Glu Asp His Pro Cys Ile Met Trp Thr Gly Gly Cys 20
25 30 Arg Arg Ile Pro Val Leu Val Phe His Ala Asp Ala Ile Leu Thr
Lys 35 40 45 Asp Asn Asn Ile Arg Val Ile Gly Glu Arg Tyr His Leu
Ser Tyr Lys 50 55 60 Ile Val Arg Thr Asp Ser Arg Leu Val Arg Ser
Ile Leu Thr Ala His 65 70 75 80 Gly Phe His Glu Val His Pro Ser Ser
Thr Asp Tyr Asn Leu Met Trp 85 90 95 Thr Gly Ser His Leu Lys Pro
Phe Leu Leu Arg Thr Leu Ser Glu Ala 100 105 110 Gln Lys Val Asn His
Phe Pro Arg Ser Tyr Glu Leu Thr Arg Lys Asp 115 120 125 Arg Leu Tyr
Lys Asn Ile Ile Arg Met Gln His Thr His Gly Phe Lys 130 135 140 Val
Phe His Ile Leu Pro Gln Thr Phe Leu Leu Pro Ala Glu Tyr Ala 145 150
155 160 Glu Phe Cys Asn Ser Tyr Ser Lys Asp Arg Gly Pro Trp Ile Val
Lys 165 170 175 Pro Val Ala Ser Ser Arg Gly Arg Gly Val Tyr Leu Ile
Asn Asn Pro 180 185 190 Asn Gln Ile Ser Leu Glu Glu Asn Ile Leu Val
Ser Arg Tyr Ile Asn 195 200 205 Asn Pro Leu Leu Ile Asp Asp Phe Lys
Phe Asp Val Arg Leu Tyr Val 210 215 220 Leu Val Thr Ser Tyr Asp Pro
Leu Val Ile Tyr Leu Tyr Glu Glu Gly 225 230 235 240 Leu Ala Arg Phe
Ala Thr Val Arg Tyr Asp Gln Gly Ala Lys Asn Ile 245 250 255 Arg Asn
Gln Phe Met His Leu Thr Asn Tyr Ser Val Asn Lys Lys Ser 260 265 270
Gly Asp Tyr Val Ser Cys Asp Asp Pro Glu Val Glu Asp Tyr Gly Asn 275
280 285 Lys Trp Ser Met Ser Ala Met Leu Arg Tyr Leu Lys Gln Glu Gly
Arg 290 295 300 Asp Thr Thr Ala Leu Met Ala His Val Glu Asp Leu Ile
Ile Lys Thr 305 310 315 320 Ile Ile Ser Ala Glu Leu Ala Ile Ala Thr
Ala Cys Lys Thr Phe Val 325 330 335 Pro His Arg Ser Ser Cys Phe Glu
Leu Tyr Gly Phe Asp Val Leu Ile 340 345 350 Asp Ser Thr Leu Lys Pro
Trp Leu Leu Glu Val Asn Leu Ser Pro Ser 355 360 365 Leu Ala Cys Asp
Ala Pro Leu Asp Leu Lys Ile Lys Ala Ser Met Ile 370 375 380 Ser Asp
Met Phe Thr Val Val Gly Phe Val Cys Gln Asp Pro Ala Gln 385 390 395
400 Arg Ala Ser Thr Arg Pro Ile Tyr Pro Thr Phe Glu Ser Ser Arg Arg
405 410 415 Asn Pro Phe Gln Lys Pro Gln Arg Cys Arg Pro Leu Ser Ala
Ser Asp 420 425 430 Ala Glu Met Lys Asn Leu Val Gly Ser Ala Arg Glu
Lys Gly Pro Gly 435 440 445 Lys Leu Gly Gly Ser Val Leu Gly Leu Ser
Met Glu Glu Ile Lys Val 450 455 460 Leu Arg Arg Val Lys Glu Glu Asn
Asp Arg Arg Gly Gly Phe Ile Arg 465 470 475 480 Ile Phe Pro Thr Ser
Glu Thr Trp Glu Ile Tyr Gly Ser Tyr Leu Glu 485 490 495 His Lys Thr
Ser Met Asn Tyr Met Leu Ala Thr Arg Leu Phe Gln Asp 500 505 510 Arg
Met Thr Ala Asp Gly Ala Pro Glu Leu Lys Ile Glu Ser Leu Asn 515 520
525 Ser Lys Ala Lys Leu His Ala Ala Leu Tyr Glu Arg Lys Leu Leu Ser
530 535 540 Leu Glu Val Arg Lys Arg Arg Arg Arg Ser Ser Arg Leu Arg
Ala Met 545 550 555 560 Arg Pro Lys Tyr Pro Val Ile Thr Gln Pro Ala
Glu Met Asn Val Lys 565 570 575 Thr Glu Thr Glu Ser Glu Glu Glu Glu
Glu Val Ala Leu Asp Asn Glu 580 585 590 Asp Glu Glu Gln Glu Ala Ser
Gln Glu Glu Ser Ala Gly Phe Leu Arg 595 600 605 Glu Asn Gln Ala Lys
Tyr Thr Pro Ser Leu Thr Ala Leu Val Glu Asn 610 615 620 Thr Pro Lys
Glu Asn Ser Met Lys Val Arg Glu Trp Asn Asn Lys Gly 625 630 635 640
Gly His Cys Cys Lys Leu Glu Thr Gln Glu Leu Glu Pro Lys Phe Asn 645
650 655 Leu Met Gln Ile Leu Gln Asp Asn Gly Asn Leu Ser Lys Met Gln
Ala 660 665 670 Arg Ile Ala Phe Ser Ala Tyr Leu Gln His Val Gln Ile
Arg Leu Met 675 680 685 Lys Asp Ser Gly Gly Gln Thr Phe Ser Ala Ser
Trp Ala Ala Lys Glu 690 695 700 Asp Glu Gln Met Glu Leu Val Val Arg
Phe Leu Lys Arg Ala Ser Asn 705 710 715 720 Asn Leu Gln His Ser Leu
Arg Met Val Leu Pro Ser Arg Arg Leu Ala 725 730 735 Leu Leu Glu Arg
Arg Arg Ile Leu Ala His Gln Leu Gly Asp Phe Ile 740 745 750 Ile Val
Tyr Asn Lys Glu Thr Glu Gln Met Ala Glu Lys Lys Ser Lys 755 760 765
Lys Lys Val Glu Glu Glu Glu Glu Asp Gly Val Asn Met Glu Asn Phe 770
775 780 Gln Glu Phe Ile Arg Gln Ala Ser Glu Ala Glu Leu Glu Glu Val
Leu 785 790 795 800 Thr Phe Tyr Thr Gln Lys Asn Lys Ser Ala Ser Val
Phe Leu Gly Thr 805 810 815 His Ser Lys Ile Ser Lys Asn Asn Asn Asn
Tyr Ser Asp Ser Gly Ala 820 825 830 Lys Gly Asp His Pro Glu Thr Ile
Met Glu Glu Val Lys Ile Lys Pro 835 840 845 Pro Lys Gln Gln Gln Thr
Thr Glu Ile His Ser Asp Lys Leu Ser Arg 850 855 860 Phe Thr Thr Ser
Ala Glu Lys Glu Ala Lys Leu Val Tyr Ser Asn Ser 865 870 875 880 Ser
Ser Gly Pro Thr Ala Thr Leu Gln Lys Ile Pro Asn Thr His Leu 885 890
895 Ser Ser Val Thr Thr Ser Asp Leu Ser Pro Gly Pro Cys His His Ser
900 905 910 Ser Leu Ser Gln Ile Pro Ser Ala Ile Pro Ser Met Pro His
Gln Pro 915 920 925 Thr Ile Leu Leu Asn Thr Val Ser Ala Ser Ala Ser
Pro Cys Leu His 930 935 940 Pro Gly Ala Gln Asn Ile Pro Ser Pro Thr
Gly Leu Pro Arg Cys Arg 945 950 955 960 Ser Gly Ser His Thr Ile Gly
Pro Phe Ser Ser Phe Gln Ser Ala Ala 965 970 975 His Ile Tyr Ser Gln
Lys Leu Ser Arg Pro Ser Ser Ala Lys Ala Gly 980 985 990 Ser Cys Tyr
Leu Asn Lys His His Ser Gly Ile Ala Lys Thr Gln Lys 995 1000 1005
Glu Gly Glu Asp Ala Ser Leu Tyr Ser Lys Arg Tyr Asn Gln Ser Met
1010 1015 1020 Val Thr Ala Glu Leu Gln Arg Leu Ala Glu Lys Gln Ala
Ala Arg Gln 1025 1030 1035 1040 Tyr Ser Pro Ser Ser His Ile Asn Leu
Leu Thr Gln Gln Val Thr Asn 1045 1050 1055 Leu Asn Leu Ala Thr Gly
Ile Ile Asn Arg Ser Ser Ala Ser Ala Pro 1060 1065 1070 Pro Thr Leu
Arg Pro Ile Ile Ser Pro Ser Gly Pro Thr Trp Ser Thr 1075 1080 1085
Gln Ser Asp Pro Gln Ala Pro Glu Asn His Ser Ser Ser Pro Gly Ser
1090 1095 1100 Arg Ser Leu Gln Thr Gly Gly Phe Ala Trp Glu Gly Glu
Val Glu Asn 1105 1110 1115 1120 Asn Val Tyr Ser Gln Ala Thr Gly Val
Val Pro Gln His Lys Tyr His 1125 1130 1135 Pro Thr Ala Gly Ser Tyr
Gln Leu Gln Phe Ala Leu Gln Gln Leu Glu 1140 1145 1150 Gln Gln Lys
Leu Gln Ser Arg Gln Leu Leu Asp Gln Ser Arg Ala Arg 1155 1160 1165
His Gln Ala Ile Phe Gly Ser Gln Thr Leu Pro Asn Ser Asn Leu Trp
1170 1175 1180 Thr Met Asn Asn Gly Ala Gly Cys Arg Ile Ser Ser Ala
Thr Ala Ser 1185 1190 1195 1200 Gly Gln Lys Pro Thr Thr Leu Pro Gln
Lys Val Val Pro Pro Pro Ser 1205 1210 1215 Ser Cys Ala Ser Leu Val
Pro Lys Pro Pro Pro Asn His Glu Gln Val 1220 1225 1230 Leu Arg Arg
Ala Thr Ser Gln Lys Ala Ser Lys Gly Ser Ser Ala Glu 1235 1240 1245
Gly Gln Leu Asn Gly Leu Gln Ser Ser Leu Asn Pro Ala Ala Ser Val
1250 1255 1260
Pro Ile Thr Ser Ser Thr Asp Pro Ala His Thr Lys Ile 1265 1270 1275
<210> SEQ ID NO 2 <211> LENGTH: 4692 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2
ggcacgaggg ggaagcagcc gtcggcggct gccctgagcc ttcctgggga aggaggaggg
60 aggtaggcgc agagcgcggt ccacgcctgc tcgccccgaa ccatgggaag
atgagacagg 120 aatctgtgcc atccaaattg cttgatccag tgaatctgct
aggaaaggtc tctgaggccc 180 ccgtctgctg actgcatgac aaaccctaaa
ggaaatgcca atcgtgatgg cccgggacct 240 ggaggaaaca gcatcatcct
cagaggatga ggaggtcata agtcaagagg atcatccatg 300 catcatgtgg
actggaggct gcaggagaat tccagttttg gtattccatg ccgacgctat 360
tcttacaaag gacaacaata ttagagtaat tggagaacgt tatcatttgt cttataagat
420 tgtacgaacg gacagtcgcc tagtacgcag cattctgaca gcccatggat
ttcatgaagt 480 tcacccaagc agcactgact ataacctaat gtggacagga
tcccacctga agcccttctt 540 actgcgcacc ctctctgaag cacaaaaagt
taatcacttt cccaggtctt atgaacttac 600 ccggaaggac cgactgtaca
aaaacattat tcgaatgcag catacacatg gattcaaggt 660 ttttcacatc
ctcccccaga ccttcctcct gccagctgag tacgcggaat tttgtaattc 720
atattcgaag gaccggggac cttggatagt aaaaccagtg gcatcttcaa gggggcgggg
780 cgtctacctg atcaacaatc caaaccagat ctccctggaa gagaacattt
tggtctcccg 840 ttacattaac aaccccctgc tcatagatga tttcaagttt
gacgtgcgcc tctatgtgct 900 cgtgacttcc tatgatcctc ttgtcatcta
tctctatgaa gaaggattgg ctaggtttgc 960 aactgtgcga tatgatcaag
gagccaagaa cattcggaac cagttcatgc atctgacaaa 1020 ctacagtgtc
aacaagaaaa gtggagatta cgtcagttgt gacgatccag aagtggagga 1080
ttatggaaac aaatggagca tgagtgctat gcttaggtac ctgaaacaag aaggcagaga
1140 tacaaccgca ttgatggccc atgtagaaga cctgatcatt aagactataa
tctctgctga 1200 actagctatt gctactgcct gtaaaacctt tgttcctcat
cgcagcagtt gttttgaact 1260 ctatggcttt gacgtgctca tagattctac
tctgaagcca tggttgttgg aagtgaatct 1320 ctctccttct ttggcctgtg
atgcgcctct ggacctaaag attaaagcca gtatgatttc 1380 agatatgttc
actgttgtag gatttgtgtg ccaagatcct gcccagcggg catcaactcg 1440
gccaatttat cccacctttg agtcttccag gcgaaaccct ttccagaaac ctcagcgttg
1500 ccgtccactc tctgccagtg atgcggaaat gaaaaacctc gtgggctcag
cccgggagaa 1560 agggccaggg aagttgggtg gttctgtgct tggtctgtca
atggaggaga tcaaagtttt 1620 acgaagggtg aaggaggaga atgatcggcg
aggtggattt attcgcatat ttcctacatc 1680 tgagacatgg gaaatatatg
ggtcctacct cgagcataag acctcaatga actatatgct 1740 ggcaacacgc
ctcttccagg acagaatgac tgctgatgga gcgccagaat tgaagataga 1800
gagtctgaat tcaaaggcca agctgcatgc tgcactttac gagaggaagc tcctgtctct
1860 ggaggtgcga aaacgtagac gacggagtag cagattgagg gcaatgaggc
caaaataccc 1920 agtgattacc caaccagctg aaatgaatgt taaaactgag
acagagagtg aagaggagga 1980 agaagtcgca ttagataatg aagatgaaga
acaggaggct tcccaggagg agtctgcagg 2040 atttcttaga gaaaatcaag
ccaaatatac accctcattg acagctttgg tagaaaatac 2100 acccaaagaa
aattccatga aagttcgtga atggaataat aaaggtggac actgctgcaa 2160
acttgagact caggagctag agcctaaatt taacctgatg cagattcttc aagataatgg
2220 caatcttagc aaaatgcagg cccgaatagc attctctgcc tatctccagc
atgttcaaat 2280 tcgcctgatg aaagacagtg gcggtcagac gttcagtgcc
agttgggctg ccaaagagga 2340 tgaacagatg gagctggttg ttcgtttcct
caagcgagca tcaaataacc tccagcattc 2400 actgaggatg gtattaccca
gtcgacgatt ggcacttctg gaacgcagaa gaatcctggc 2460 ccaccagctg
ggtgacttta tcattgtata caacaaggaa acagaacaaa tggctgaaaa 2520
gaaatcaaag aagaaagttg aggaagaaga ggaagatggg gtgaatatgg aaaactttca
2580 ggagttcatc agacaagcaa gtgaggctga actggaggag gtgttgactt
tttataccca 2640 aaagaacaag tctgctagtg tcttcctggg gactcactct
aaaatttcta agaacaacaa 2700 caattattct gatagtgggg caaaaggtga
tcaccctgag actataatgg aagaagtgaa 2760 aataaagcca cctaaacagc
aacagacgac agaaattcat tctgataaat tatctcgatt 2820 taccacttca
gcagaaaaag aggcaaaatt agtttatagc aattcctcct ctggtcctac 2880
tgctactctg cagaaaattc ccaacaccca tttgtcatct gttacaacct ctgacctctc
2940 tccagggcct tgccaccatt cttctttatc tcaaattcct tcagctatcc
ccagcatgcc 3000 tcaccagcca acaattttac tgaacacagt ctctgccagt
gcttctccct gcctacatcc 3060 cggggcacag aacatcccaa gccctactgg
cctgccacgc tgtcgatcag gaagtcacac 3120 cattggtccc ttttcttcct
tccaaagtgc tgcacacatc tatagccaga aactgtctcg 3180 tccctcttca
gcaaaggcag gatcgtgcta tctaaacaag catcattcag gaatagccaa 3240
aacacaaaaa gagggagaag atgcttcttt atatagcaaa cggtacaacc aaagtatggt
3300 tacagctgaa cttcagcggc tagctgagaa gcaggcagcg agacagtatt
ctccatccag 3360 ccacatcaac ctcctcaccc aacaggtaac aaacctgaat
ttggcaactg gcatcataaa 3420 cagaagcagt gcttcagctc ccccaaccct
ccgacccatc atcagtccta gtggcccgac 3480 atggtctaca cagtcagacc
cccaagctcc cgagaatcac tccagctctc ctggaagcag 3540 gagcctgcag
acagggggat ttgcctggga aggagaagta gaaaacaacg tgtacagcca 3600
ggctacaggg gtggtccccc agcacaagta tcaccccaca gcaggcagct atcagcttca
3660 atttgccctg cagcaacttg aacaacaaaa acttcagtcc cggcagctcc
tggaccagag 3720 tcgagcccgg caccaggcaa tctttggcag ccagacacta
cctaactcca atttatggac 3780 aatgaataat ggtgcaggtt gtagaatttc
cagtgccaca gctagtggcc agaagccaac 3840 cactctgcca caaaaagtgg
taccacctcc aagttcttgc gcctccctgg ttcccaaacc 3900 cccacccaac
cacgaacaag tgctcagaag ggcaacatcc cagaaagctt ccaaagggtc 3960
ctccgcggaa gggcagctga atggactcca gagcagcctt aaccctgcag cctctgtgcc
4020 catcaccagc tctacagatc ctgctcacac taaaatatga accacaaaca
cacagagaaa 4080 caacctgttc accactcctg ggtgcatgat tgagggtgaa
gcatccacca gcacttcaag 4140 gggtccatag tatttttttt tttgctgcct
caaagtcccc aaagccttcg agcagaagtg 4200 gcagtagatg gttgccaatc
agccaatgca gactttcact gggacaacaa gaaagcagat 4260 cttctgggtt
ttgatggaac ttggcagtgg ggacattcag ctgatgcatt atataccccg 4320
tcagagcaca cttgtatctt ttaccttccc tttgccccat gcccccaaac tgcttaggtc
4380 ttctctgtcc ctttactgct gctgcacaga gatgatataa aagaggctct
ttggctattt 4440 gcattttgct tcctcttctt ttccagatta cagtatgaag
ctttattttc tttgtacaag 4500 cttaaaattt caacatcatc atccgccaaa
gttgttcctc ccttttcgga ggatctaggg 4560 ggaaagagga gcattcatca
caagtttcct agagagagga gacaaatcgg tgtgccattg 4620 acaacatgag
ccagggtaaa ggcacccttt ggaattactg atttcaaaga ttaataaagt 4680
aattctattt tt 4692 <210> SEQ ID NO 3 <211> LENGTH: 212
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 3 Glu Asn Thr Pro Lys Glu Asn Ser Met Lys Val
Arg Glu Trp Asn Asn 1 5 10 15 Lys Gly Gly His Cys Cys Lys Leu Glu
Thr Gln Glu Leu Glu Pro Lys 20 25 30 Phe Asn Leu Met Gln Ile Leu
Gln Asp Asn Gly Asn Leu Ser Lys Met 35 40 45 Gln Ala Arg Ile Ala
Phe Ser Ala Tyr Leu Gln His Val Gln Ile Arg 50 55 60 Leu Met Lys
Asp Ser Gly Gly Gln Thr Phe Ser Ala Ser Trp Ala Ala 65 70 75 80 Lys
Glu Asp Glu Gln Met Glu Leu Val Val Arg Phe Leu Lys Arg Ala 85 90
95 Ser Asn Asn Leu Gln His Ser Leu Arg Met Val Leu Pro Ser Arg Arg
100 105 110 Leu Ala Leu Leu Glu Arg Arg Arg Ile Leu Ala His Gln Leu
Gly Asp 115 120 125 Phe Ile Ile Val Tyr Asn Lys Glu Thr Glu Gln Met
Ala Glu Lys Lys 130 135 140 Ser Lys Lys Lys Val Glu Glu Glu Glu Glu
Asp Gly Val Asn Met Glu 145 150 155 160 Asn Phe Gln Glu Phe Ile Arg
Gln Ala Ser Glu Ala Glu Leu Glu Glu 165 170 175 Val Leu Thr Phe Tyr
Thr Gln Lys Asn Lys Ser Ala Ser Val Phe Leu 180 185 190 Gly Thr His
Ser Lys Ile Ser Lys Asn Asn Asn Asn Tyr Ser Asp Ser 195 200 205 Gly
Ala Lys Gly 210 <210> SEQ ID NO 4 <211> LENGTH: 444
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 4 Gly Asp His Pro Glu Thr Ile Met Glu Glu Val
Lys Ile Lys Pro Pro 1 5 10 15 Lys Gln Gln Gln Thr Thr Glu Ile His
Ser Asp Lys Leu Ser Arg Phe 20 25 30 Thr Thr Ser Ala Glu Lys Glu
Ala Lys Leu Val Tyr Ser Asn Ser Ser 35 40 45 Ser Gly Pro Thr Ala
Thr Leu Gln Lys Ile Pro Asn Thr His Leu Ser 50 55 60 Ser Val Thr
Thr Ser Asp Leu Ser Pro Gly Pro Cys His His Ser Ser 65 70 75 80 Leu
Ser Gln Ile Pro Ser Ala Ile Pro Ser Met Pro His Gln Pro Thr 85 90
95 Ile Leu Leu Asn Thr Val Ser Ala Ser Ala Ser Pro Cys Leu His Pro
100 105 110 Gly Ala Gln Asn Ile Pro Ser Pro Thr Gly Leu Pro Arg Cys
Arg Ser 115 120 125
Gly Ser His Thr Ile Gly Pro Phe Ser Ser Phe Gln Ser Ala Ala His 130
135 140 Ile Tyr Ser Gln Lys Leu Ser Arg Pro Ser Ser Ala Lys Ala Gly
Ser 145 150 155 160 Cys Tyr Leu Asn Lys His His Ser Gly Ile Ala Lys
Thr Gln Lys Glu 165 170 175 Gly Glu Asp Ala Ser Leu Tyr Ser Lys Arg
Tyr Asn Gln Ser Met Val 180 185 190 Thr Ala Glu Leu Gln Arg Leu Ala
Glu Lys Gln Ala Ala Arg Gln Tyr 195 200 205 Ser Pro Ser Ser His Ile
Asn Leu Leu Thr Gln Gln Val Thr Asn Leu 210 215 220 Asn Leu Ala Thr
Gly Ile Ile Asn Arg Ser Ser Ala Ser Ala Pro Pro 225 230 235 240 Thr
Leu Arg Pro Ile Ile Ser Pro Ser Gly Pro Thr Trp Ser Thr Gln 245 250
255 Ser Asp Pro Gln Ala Pro Glu Asn His Ser Ser Ser Pro Gly Ser Arg
260 265 270 Ser Leu Gln Thr Gly Gly Phe Ala Trp Glu Gly Glu Val Glu
Asn Asn 275 280 285 Val Tyr Ser Gln Ala Thr Gly Val Val Pro Gln His
Lys Tyr His Pro 290 295 300 Thr Ala Gly Ser Tyr Gln Leu Gln Phe Ala
Leu Gln Gln Leu Glu Gln 305 310 315 320 Gln Lys Leu Gln Ser Arg Gln
Leu Leu Asp Gln Ser Arg Ala Arg His 325 330 335 Gln Ala Ile Phe Gly
Ser Gln Thr Leu Pro Asn Ser Asn Leu Trp Thr 340 345 350 Met Asn Asn
Gly Ala Gly Cys Arg Ile Ser Ser Ala Thr Ala Ser Gly 355 360 365 Gln
Lys Pro Thr Thr Leu Pro Gln Lys Val Val Pro Pro Pro Ser Ser 370 375
380 Cys Ala Ser Leu Val Pro Lys Pro Pro Pro Asn His Glu Gln Val Leu
385 390 395 400 Arg Arg Ala Thr Ser Gln Lys Ala Ser Lys Gly Ser Ser
Ala Glu Gly 405 410 415 Gln Leu Asn Gly Leu Gln Ser Ser Leu Asn Pro
Ala Ala Ser Val Pro 420 425 430 Ile Thr Ser Ser Thr Asp Pro Ala His
Thr Lys Ile 435 440 <210> SEQ ID NO 5 <211> LENGTH: 655
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 5 Glu Asn Thr Pro Lys Glu Asn Ser Met Lys Val
Arg Glu Trp Asn Asn 1 5 10 15 Lys Gly Gly His Cys Cys Lys Leu Glu
Thr Gln Glu Leu Glu Pro Lys 20 25 30 Phe Asn Leu Met Gln Ile Leu
Gln Asp Asn Gly Asn Leu Ser Lys Met 35 40 45 Gln Ala Arg Ile Ala
Phe Ser Ala Tyr Leu Gln His Val Gln Ile Arg 50 55 60 Leu Met Lys
Asp Ser Gly Gly Gln Thr Phe Ser Ala Ser Trp Ala Ala 65 70 75 80 Lys
Glu Asp Glu Gln Met Glu Leu Val Val Arg Phe Leu Lys Arg Ala 85 90
95 Ser Asn Asn Leu Gln His Ser Leu Arg Met Val Leu Pro Ser Arg Arg
100 105 110 Leu Ala Leu Leu Glu Arg Arg Arg Ile Leu Ala His Gln Leu
Gly Asp 115 120 125 Phe Ile Ile Val Tyr Asn Lys Glu Thr Glu Gln Met
Ala Glu Lys Lys 130 135 140 Ser Lys Lys Lys Val Glu Glu Glu Glu Glu
Asp Gly Val Asn Met Glu 145 150 155 160 Asn Phe Gln Glu Phe Ile Arg
Gln Ala Ser Glu Ala Glu Leu Glu Glu 165 170 175 Val Leu Thr Phe Tyr
Thr Gln Lys Asn Lys Ser Ala Ser Val Phe Leu 180 185 190 Gly Thr His
Ser Lys Ile Ser Lys Asn Asn Asn Asn Tyr Ser Asp Ser 195 200 205 Gly
Ala Lys Gly Asp His Pro Glu Thr Ile Met Glu Glu Val Lys Ile 210 215
220 Lys Pro Pro Lys Gln Gln Gln Thr Thr Glu Ile His Ser Asp Lys Leu
225 230 235 240 Ser Arg Phe Thr Thr Ser Ala Glu Lys Glu Ala Lys Leu
Val Tyr Ser 245 250 255 Asn Ser Ser Ser Gly Pro Thr Ala Thr Leu Gln
Lys Ile Pro Asn Thr 260 265 270 His Leu Ser Ser Val Thr Thr Ser Asp
Leu Ser Pro Gly Pro Cys His 275 280 285 His Ser Ser Leu Ser Gln Ile
Pro Ser Ala Ile Pro Ser Met Pro His 290 295 300 Gln Pro Thr Ile Leu
Leu Asn Thr Val Ser Ala Ser Ala Ser Pro Cys 305 310 315 320 Leu His
Pro Gly Ala Gln Asn Ile Pro Ser Pro Thr Gly Leu Pro Arg 325 330 335
Cys Arg Ser Gly Ser His Thr Ile Gly Pro Phe Ser Ser Phe Gln Ser 340
345 350 Ala Ala His Ile Tyr Ser Gln Lys Leu Ser Arg Pro Ser Ser Ala
Lys 355 360 365 Ala Gly Ser Cys Tyr Leu Asn Lys His His Ser Gly Ile
Ala Lys Thr 370 375 380 Gln Lys Glu Gly Glu Asp Ala Ser Leu Tyr Ser
Lys Arg Tyr Asn Gln 385 390 395 400 Ser Met Val Thr Ala Glu Leu Gln
Arg Leu Ala Glu Lys Gln Ala Ala 405 410 415 Arg Gln Tyr Ser Pro Ser
Ser His Ile Asn Leu Leu Thr Gln Gln Val 420 425 430 Thr Asn Leu Asn
Leu Ala Thr Gly Ile Ile Asn Arg Ser Ser Ala Ser 435 440 445 Ala Pro
Pro Thr Leu Arg Pro Ile Ile Ser Pro Ser Gly Pro Thr Trp 450 455 460
Ser Thr Gln Ser Asp Pro Gln Ala Pro Glu Asn His Ser Ser Ser Pro 465
470 475 480 Gly Ser Arg Ser Leu Gln Thr Gly Gly Phe Ala Trp Glu Gly
Glu Val 485 490 495 Glu Asn Asn Val Tyr Ser Gln Ala Thr Gly Val Val
Pro Gln His Lys 500 505 510 Tyr His Pro Thr Ala Gly Ser Tyr Gln Leu
Gln Phe Ala Leu Gln Gln 515 520 525 Leu Glu Gln Gln Lys Leu Gln Ser
Arg Gln Leu Leu Asp Gln Ser Arg 530 535 540 Ala Arg His Gln Ala Ile
Phe Gly Ser Gln Thr Leu Pro Asn Ser Asn 545 550 555 560 Leu Trp Thr
Met Asn Asn Gly Ala Gly Cys Arg Ile Ser Ser Ala Thr 565 570 575 Ala
Ser Gly Gln Lys Pro Thr Thr Leu Pro Gln Lys Val Val Pro Pro 580 585
590 Pro Ser Ser Cys Ala Ser Leu Val Pro Lys Pro Pro Pro Asn His Glu
595 600 605 Gln Val Leu Arg Arg Ala Thr Ser Gln Lys Ala Ser Lys Gly
Ser Ser 610 615 620 Ala Glu Gly Gln Leu Asn Gly Leu Gln Ser Ser Leu
Asn Pro Ala Ala 625 630 635 640 Ser Val Pro Ile Thr Ser Ser Thr Asp
Pro Ala His Thr Lys Ile 645 650 655 <210> SEQ ID NO 6
<211> LENGTH: 760 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 6 Gly Ala Pro Glu Leu Lys Ile
Glu Ser Leu Asn Ser Lys Ala Lys Leu 1 5 10 15 His Ala Ala Leu Tyr
Glu Arg Lys Leu Leu Ser Leu Glu Val Arg Lys 20 25 30 Arg Arg Arg
Arg Ser Ser Arg Leu Arg Ala Met Arg Pro Lys Tyr Pro 35 40 45 Val
Ile Thr Gln Pro Ala Glu Met Asn Val Lys Thr Glu Thr Glu Ser 50 55
60 Glu Glu Glu Glu Glu Val Ala Leu Asp Asn Glu Asp Glu Glu Gln Glu
65 70 75 80 Ala Ser Gln Glu Glu Ser Ala Gly Phe Leu Arg Glu Asn Gln
Ala Lys 85 90 95 Tyr Thr Pro Ser Leu Thr Ala Leu Val Glu Asn Thr
Pro Lys Glu Asn 100 105 110 Ser Met Lys Val Arg Glu Trp Asn Asn Lys
Gly Gly His Cys Cys Lys 115 120 125 Leu Glu Thr Gln Glu Leu Glu Pro
Lys Phe Asn Leu Met Gln Ile Leu 130 135 140 Gln Asp Asn Gly Asn Leu
Ser Lys Met Gln Ala Arg Ile Ala Phe Ser 145 150 155 160 Ala Tyr Leu
Gln His Val Gln Ile Arg Leu Met Lys Asp Ser Gly Gly 165 170 175 Gln
Thr Phe Ser Ala Ser Trp Ala Ala Lys Glu Asp Glu Gln Met Glu 180 185
190 Leu Val Val Arg Phe Leu Lys Arg Ala Ser Asn Asn Leu Gln His Ser
195 200 205 Leu Arg Met Val Leu Pro Ser Arg Arg Leu Ala Leu Leu Glu
Arg Arg 210 215 220 Arg Ile Leu Ala His Gln Leu Gly Asp Phe Ile Ile
Val Tyr Asn Lys 225 230 235 240 Glu Thr Glu Gln Met Ala Glu Lys Lys
Ser Lys Lys Lys Val Glu Glu 245 250 255 Glu Glu Glu Asp Gly Val Asn
Met Glu Asn Phe Gln Glu Phe Ile Arg 260 265 270 Gln Ala Ser Glu Ala
Glu Leu Glu Glu Val Leu Thr Phe Tyr Thr Gln
275 280 285 Lys Asn Lys Ser Ala Ser Val Phe Leu Gly Thr His Ser Lys
Ile Ser 290 295 300 Lys Asn Asn Asn Asn Tyr Ser Asp Ser Gly Ala Lys
Gly Asp His Pro 305 310 315 320 Glu Thr Ile Met Glu Glu Val Lys Ile
Lys Pro Pro Lys Gln Gln Gln 325 330 335 Thr Thr Glu Ile His Ser Asp
Lys Leu Ser Arg Phe Thr Thr Ser Ala 340 345 350 Glu Lys Glu Ala Lys
Leu Val Tyr Ser Asn Ser Ser Ser Gly Pro Thr 355 360 365 Ala Thr Leu
Gln Lys Ile Pro Asn Thr His Leu Ser Ser Val Thr Thr 370 375 380 Ser
Asp Leu Ser Pro Gly Pro Cys His His Ser Ser Leu Ser Gln Ile 385 390
395 400 Pro Ser Ala Ile Pro Ser Met Pro His Gln Pro Thr Ile Leu Leu
Asn 405 410 415 Thr Val Ser Ala Ser Ala Ser Pro Cys Leu His Pro Gly
Ala Gln Asn 420 425 430 Ile Pro Ser Pro Thr Gly Leu Pro Arg Cys Arg
Ser Gly Ser His Thr 435 440 445 Ile Gly Pro Phe Ser Ser Phe Gln Ser
Ala Ala His Ile Tyr Ser Gln 450 455 460 Lys Leu Ser Arg Pro Ser Ser
Ala Lys Ala Gly Ser Cys Tyr Leu Asn 465 470 475 480 Lys His His Ser
Gly Ile Ala Lys Thr Gln Lys Glu Gly Glu Asp Ala 485 490 495 Ser Leu
Tyr Ser Lys Arg Tyr Asn Gln Ser Met Val Thr Ala Glu Leu 500 505 510
Gln Arg Leu Ala Glu Lys Gln Ala Ala Arg Gln Tyr Ser Pro Ser Ser 515
520 525 His Ile Asn Leu Leu Thr Gln Gln Val Thr Asn Leu Asn Leu Ala
Thr 530 535 540 Gly Ile Ile Asn Arg Ser Ser Ala Ser Ala Pro Pro Thr
Leu Arg Pro 545 550 555 560 Ile Ile Ser Pro Ser Gly Pro Thr Trp Ser
Thr Gln Ser Asp Pro Gln 565 570 575 Ala Pro Glu Asn His Ser Ser Ser
Pro Gly Ser Arg Ser Leu Gln Thr 580 585 590 Gly Gly Phe Ala Trp Glu
Gly Glu Val Glu Asn Asn Val Tyr Ser Gln 595 600 605 Ala Thr Gly Val
Val Pro Gln His Lys Tyr His Pro Thr Ala Gly Ser 610 615 620 Tyr Gln
Leu Gln Phe Ala Leu Gln Gln Leu Glu Gln Gln Lys Leu Gln 625 630 635
640 Ser Arg Gln Leu Leu Asp Gln Ser Arg Ala Arg His Gln Ala Ile Phe
645 650 655 Gly Ser Gln Thr Leu Pro Asn Ser Asn Leu Trp Thr Met Asn
Asn Gly 660 665 670 Ala Gly Cys Arg Ile Ser Ser Ala Thr Ala Ser Gly
Gln Lys Pro Thr 675 680 685 Thr Leu Pro Gln Lys Val Val Pro Pro Pro
Ser Ser Cys Ala Ser Leu 690 695 700 Val Pro Lys Pro Pro Pro Asn His
Glu Gln Val Leu Arg Arg Ala Thr 705 710 715 720 Ser Gln Lys Ala Ser
Lys Gly Ser Ser Ala Glu Gly Gln Leu Asn Gly 725 730 735 Leu Gln Ser
Ser Leu Asn Pro Ala Ala Ser Val Pro Ile Thr Ser Ser 740 745 750 Thr
Asp Pro Ala His Thr Lys Ile 755 760 <210> SEQ ID NO 7
<211> LENGTH: 808 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 7 Met Lys Asn Leu Val Gly Ser
Ala Arg Glu Lys Gly Pro Gly Lys Leu 1 5 10 15 Gly Gly Ser Val Leu
Gly Leu Ser Met Glu Glu Ile Lys Val Leu Arg 20 25 30 Arg Val Lys
Glu Glu Asn Asp Arg Arg Gly Gly Phe Ile Arg Ile Phe 35 40 45 Pro
Thr Ser Glu Thr Trp Glu Ile Tyr Gly Ser Tyr Leu Glu His Lys 50 55
60 Thr Ser Met Asn Tyr Met Leu Ala Thr Arg Leu Phe Gln Asp Arg Met
65 70 75 80 Thr Ala Asp Gly Ala Pro Glu Leu Lys Ile Glu Ser Leu Asn
Ser Lys 85 90 95 Ala Lys Leu His Ala Ala Leu Tyr Glu Arg Lys Leu
Leu Ser Leu Glu 100 105 110 Val Arg Lys Arg Arg Arg Arg Ser Ser Arg
Leu Arg Ala Met Arg Pro 115 120 125 Lys Tyr Pro Val Ile Thr Gln Pro
Ala Glu Met Asn Val Lys Thr Glu 130 135 140 Thr Glu Ser Glu Glu Glu
Glu Glu Val Ala Leu Asp Asn Glu Asp Glu 145 150 155 160 Glu Gln Glu
Ala Ser Gln Glu Glu Ser Ala Gly Phe Leu Arg Glu Asn 165 170 175 Gln
Ala Lys Tyr Thr Pro Ser Leu Thr Ala Leu Val Glu Asn Thr Pro 180 185
190 Lys Glu Asn Ser Met Lys Val Arg Glu Trp Asn Asn Lys Gly Gly His
195 200 205 Cys Cys Lys Leu Glu Thr Gln Glu Leu Glu Pro Lys Phe Asn
Leu Met 210 215 220 Gln Ile Leu Gln Asp Asn Gly Asn Leu Ser Lys Met
Gln Ala Arg Ile 225 230 235 240 Ala Phe Ser Ala Tyr Leu Gln His Val
Gln Ile Arg Leu Met Lys Asp 245 250 255 Ser Gly Gly Gln Thr Phe Ser
Ala Ser Trp Ala Ala Lys Glu Asp Glu 260 265 270 Gln Met Glu Leu Val
Val Arg Phe Leu Lys Arg Ala Ser Asn Asn Leu 275 280 285 Gln His Ser
Leu Arg Met Val Leu Pro Ser Arg Arg Leu Ala Leu Leu 290 295 300 Glu
Arg Arg Arg Ile Leu Ala His Gln Leu Gly Asp Phe Ile Ile Val 305 310
315 320 Tyr Asn Lys Glu Thr Glu Gln Met Ala Glu Lys Lys Ser Lys Lys
Lys 325 330 335 Val Glu Glu Glu Glu Glu Asp Gly Val Asn Met Glu Asn
Phe Gln Glu 340 345 350 Phe Ile Arg Gln Ala Ser Glu Ala Glu Leu Glu
Glu Val Leu Thr Phe 355 360 365 Tyr Thr Gln Lys Asn Lys Ser Ala Ser
Val Phe Leu Gly Thr His Ser 370 375 380 Lys Ile Ser Lys Asn Asn Asn
Asn Tyr Ser Asp Ser Gly Ala Lys Gly 385 390 395 400 Asp His Pro Glu
Thr Ile Met Glu Glu Val Lys Ile Lys Pro Pro Lys 405 410 415 Gln Gln
Gln Thr Thr Glu Ile His Ser Asp Lys Leu Ser Arg Phe Thr 420 425 430
Thr Ser Ala Glu Lys Glu Ala Lys Leu Val Tyr Ser Asn Ser Ser Ser 435
440 445 Gly Pro Thr Ala Thr Leu Gln Lys Ile Pro Asn Thr His Leu Ser
Ser 450 455 460 Val Thr Thr Ser Asp Leu Ser Pro Gly Pro Cys His His
Ser Ser Leu 465 470 475 480 Ser Gln Ile Pro Ser Ala Ile Pro Ser Met
Pro His Gln Pro Thr Ile 485 490 495 Leu Leu Asn Thr Val Ser Ala Ser
Ala Ser Pro Cys Leu His Pro Gly 500 505 510 Ala Gln Asn Ile Pro Ser
Pro Thr Gly Leu Pro Arg Cys Arg Ser Gly 515 520 525 Ser His Thr Ile
Gly Pro Phe Ser Ser Phe Gln Ser Ala Ala His Ile 530 535 540 Tyr Ser
Gln Lys Leu Ser Arg Pro Ser Ser Ala Lys Ala Gly Ser Cys 545 550 555
560 Tyr Leu Asn Lys His His Ser Gly Ile Ala Lys Thr Gln Lys Glu Gly
565 570 575 Glu Asp Ala Ser Leu Tyr Ser Lys Arg Tyr Asn Gln Ser Met
Val Thr 580 585 590 Ala Glu Leu Gln Arg Leu Ala Glu Lys Gln Ala Ala
Arg Gln Tyr Ser 595 600 605 Pro Ser Ser His Ile Asn Leu Leu Thr Gln
Gln Val Thr Asn Leu Asn 610 615 620 Leu Ala Thr Gly Ile Ile Asn Arg
Ser Ser Ala Ser Ala Pro Pro Thr 625 630 635 640 Leu Arg Pro Ile Ile
Ser Pro Ser Gly Pro Thr Trp Ser Thr Gln Ser 645 650 655 Asp Pro Gln
Ala Pro Glu Asn His Ser Ser Ser Pro Gly Ser Arg Ser 660 665 670 Leu
Gln Thr Gly Gly Phe Ala Trp Glu Gly Glu Val Glu Asn Asn Val 675 680
685 Tyr Ser Gln Ala Thr Gly Val Val Pro Gln His Lys Tyr His Pro Thr
690 695 700 Ala Gly Ser Tyr Gln Leu Gln Phe Ala Leu Gln Gln Leu Glu
Gln Gln 705 710 715 720 Lys Leu Gln Ser Arg Gln Leu Leu Asp Gln Ser
Arg Ala Arg His Gln 725 730 735 Ala Ile Phe Gly Ser Gln Thr Leu Pro
Asn Ser Asn Leu Trp Thr Met 740 745 750 Asn Asn Gly Ala Gly Cys Arg
Ile Ser Ser Ala Thr Ala Ser Gly Gln 755 760 765 Lys Pro Thr Thr Leu
Pro Gln Lys Val Val Pro Pro Pro Ser Ser Cys 770 775 780 Ala Ser Leu
Val Pro Lys Pro Pro Pro Asn His Glu Gln Val Leu Arg 785 790 795 800
Arg Ala Thr Ser Gln Lys Ala Ser
805 <210> SEQ ID NO 8 <211> LENGTH: 669 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
8 Lys Ile Lys Ala Ser Met Ile Ser Asp Met Phe Thr Val Val Gly Phe 1
5 10 15 Val Cys Gln Asp Pro Ala Gln Arg Ala Ser Thr Arg Pro Ile Tyr
Pro 20 25 30 Thr Phe Glu Ser Ser Arg Arg Asn Pro Phe Gln Lys Pro
Gln Arg Cys 35 40 45 Arg Pro Leu Ser Ala Ser Asp Ala Glu Met Lys
Asn Leu Val Gly Ser 50 55 60 Ala Arg Glu Lys Gly Pro Gly Lys Leu
Gly Gly Ser Val Leu Gly Leu 65 70 75 80 Ser Met Glu Glu Ile Lys Val
Leu Arg Arg Val Lys Glu Glu Asn Asp 85 90 95 Arg Arg Gly Gly Phe
Ile Arg Ile Phe Pro Thr Ser Glu Thr Trp Glu 100 105 110 Ile Tyr Gly
Ser Tyr Leu Glu His Lys Thr Ser Met Asn Tyr Met Leu 115 120 125 Ala
Thr Arg Leu Phe Gln Asp Arg Met Thr Ala Asp Gly Ala Pro Glu 130 135
140 Leu Lys Ile Glu Ser Leu Asn Ser Lys Ala Lys Leu His Ala Ala Leu
145 150 155 160 Tyr Glu Arg Lys Leu Leu Ser Leu Glu Val Arg Lys Arg
Arg Arg Arg 165 170 175 Ser Ser Arg Leu Arg Ala Met Arg Pro Lys Tyr
Pro Val Ile Thr Gln 180 185 190 Pro Ala Glu Met Asn Val Lys Thr Glu
Thr Glu Ser Glu Glu Glu Glu 195 200 205 Glu Val Ala Leu Asp Asn Glu
Asp Glu Glu Gln Glu Ala Ser Gln Glu 210 215 220 Glu Ser Ala Gly Phe
Leu Arg Glu Asn Gln Ala Lys Tyr Thr Pro Ser 225 230 235 240 Leu Thr
Ala Leu Val Glu Asn Thr Pro Lys Glu Asn Ser Met Lys Val 245 250 255
Arg Glu Trp Asn Asn Lys Gly Gly His Cys Cys Lys Leu Glu Thr Gln 260
265 270 Glu Leu Glu Pro Lys Phe Asn Leu Met Gln Ile Leu Gln Asp Asn
Gly 275 280 285 Asn Leu Ser Lys Met Gln Ala Arg Ile Ala Phe Ser Ala
Tyr Leu Gln 290 295 300 His Val Gln Ile Arg Leu Met Lys Asp Ser Gly
Gly Gln Thr Phe Ser 305 310 315 320 Ala Ser Trp Ala Ala Lys Glu Asp
Glu Gln Met Glu Leu Val Val Arg 325 330 335 Phe Leu Lys Arg Ala Ser
Asn Asn Leu Gln His Ser Leu Arg Met Val 340 345 350 Leu Pro Ser Arg
Arg Leu Ala Leu Leu Glu Arg Arg Arg Ile Leu Ala 355 360 365 His Gln
Leu Gly Asp Phe Ile Ile Val Tyr Asn Lys Glu Thr Glu Gln 370 375 380
Met Ala Glu Lys Lys Ser Lys Lys Lys Val Glu Glu Glu Glu Glu Asp 385
390 395 400 Gly Val Asn Met Glu Asn Phe Gln Glu Phe Ile Arg Gln Ala
Ser Glu 405 410 415 Ala Glu Leu Glu Glu Val Leu Thr Phe Tyr Thr Gln
Lys Asn Lys Ser 420 425 430 Ala Ser Val Phe Leu Gly Thr His Ser Lys
Ile Ser Lys Asn Asn Asn 435 440 445 Asn Tyr Ser Asp Ser Gly Ala Lys
Gly Asp His Pro Glu Thr Ile Met 450 455 460 Glu Glu Val Lys Ile Lys
Pro Pro Lys Gln Gln Gln Thr Thr Glu Ile 465 470 475 480 His Ser Asp
Lys Leu Ser Arg Phe Thr Thr Ser Ala Glu Lys Glu Ala 485 490 495 Lys
Leu Val Tyr Ser Asn Ser Ser Ser Gly Pro Thr Ala Thr Leu Gln 500 505
510 Lys Ile Pro Asn Thr His Leu Ser Ser Val Thr Thr Ser Asp Leu Ser
515 520 525 Pro Gly Pro Cys His His Ser Ser Leu Ser Gln Ile Pro Ser
Ala Ile 530 535 540 Pro Ser Met Pro His Gln Pro Thr Ile Leu Leu Asn
Thr Val Ser Ala 545 550 555 560 Ser Ala Ser Pro Cys Leu His Pro Gly
Ala Gln Asn Ile Pro Ser Pro 565 570 575 Thr Gly Leu Pro Arg Cys Arg
Ser Gly Ser His Thr Ile Gly Pro Phe 580 585 590 Ser Ser Phe Gln Ser
Ala Ala His Ile Tyr Ser Gln Lys Leu Ser Arg 595 600 605 Pro Ser Ser
Ala Lys Ala Gly Ser Cys Tyr Leu Asn Lys His His Ser 610 615 620 Gly
Ile Ala Lys Thr Gln Lys Glu Gly Glu Asp Ala Ser Leu Tyr Ser 625 630
635 640 Lys Arg Tyr Asn Gln Ser Met Val Thr Ala Glu Leu Gln Arg Leu
Ala 645 650 655 Glu Lys Gln Ala Ala Arg Gln Tyr Ser Pro Ser Ser His
660 665 <210> SEQ ID NO 9 <211> LENGTH: 406 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
9 Ser Ala Ser Asp Ala Glu Met Lys Asn Leu Val Gly Ser Ala Arg Glu 1
5 10 15 Lys Gly Pro Gly Lys Leu Gly Gly Ser Val Leu Gly Leu Ser Met
Glu 20 25 30 Glu Ile Lys Val Leu Arg Arg Val Lys Glu Glu Asn Asp
Arg Arg Gly 35 40 45 Gly Phe Ile Arg Ile Phe Pro Thr Ser Glu Thr
Trp Glu Ile Tyr Gly 50 55 60 Ser Tyr Leu Glu His Lys Thr Ser Met
Asn Tyr Met Leu Ala Thr Arg 65 70 75 80 Leu Phe Gln Asp Arg Met Thr
Ala Asp Gly Ala Pro Glu Leu Lys Ile 85 90 95 Glu Ser Leu Asn Ser
Lys Ala Lys Leu His Ala Ala Leu Tyr Glu Arg 100 105 110 Lys Leu Leu
Ser Leu Glu Val Arg Lys Arg Arg Arg Arg Ser Ser Arg 115 120 125 Leu
Arg Ala Met Arg Pro Lys Tyr Pro Val Ile Thr Gln Pro Ala Glu 130 135
140 Met Asn Val Lys Thr Glu Thr Glu Ser Glu Glu Glu Glu Glu Val Ala
145 150 155 160 Leu Asp Asn Glu Asp Glu Glu Gln Glu Ala Ser Gln Glu
Glu Ser Ala 165 170 175 Gly Phe Leu Arg Glu Asn Gln Ala Lys Tyr Thr
Pro Ser Leu Thr Ala 180 185 190 Leu Val Glu Asn Thr Pro Lys Glu Asn
Ser Met Lys Val Arg Glu Trp 195 200 205 Asn Asn Lys Gly Gly His Cys
Cys Lys Leu Glu Thr Gln Glu Leu Glu 210 215 220 Pro Lys Phe Asn Leu
Met Gln Ile Leu Gln Asp Asn Gly Asn Leu Ser 225 230 235 240 Lys Met
Gln Ala Arg Ile Ala Phe Ser Ala Tyr Leu Gln His Val Gln 245 250 255
Ile Arg Leu Met Lys Asp Ser Gly Gly Gln Thr Phe Ser Ala Ser Trp 260
265 270 Ala Ala Lys Glu Asp Glu Gln Met Glu Leu Val Val Arg Phe Leu
Lys 275 280 285 Arg Ala Ser Asn Asn Leu Gln His Ser Leu Arg Met Val
Leu Pro Ser 290 295 300 Arg Arg Leu Ala Leu Leu Glu Arg Arg Arg Ile
Leu Ala His Gln Leu 305 310 315 320 Gly Asp Phe Ile Ile Val Tyr Asn
Lys Glu Thr Glu Gln Met Ala Glu 325 330 335 Lys Lys Ser Lys Lys Lys
Val Glu Glu Glu Glu Glu Asp Gly Val Asn 340 345 350 Met Glu Asn Phe
Gln Glu Phe Ile Arg Gln Ala Ser Glu Ala Glu Leu 355 360 365 Glu Glu
Val Leu Thr Phe Tyr Thr Gln Lys Asn Lys Ser Ala Ser Val 370 375 380
Phe Leu Gly Thr His Ser Lys Ile Ser Lys Asn Asn Asn Asn Tyr Ser 385
390 395 400 Asp Ser Gly Ala Lys Gly 405 <210> SEQ ID NO 10
<211> LENGTH: 317 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 10 Gly Ala Pro Glu Leu Lys Ile
Glu Ser Leu Asn Ser Lys Ala Lys Leu 1 5 10 15 His Ala Ala Leu Tyr
Glu Arg Lys Leu Leu Ser Leu Glu Val Arg Lys 20 25 30 Arg Arg Arg
Arg Ser Ser Arg Leu Arg Ala Met Arg Pro Lys Tyr Pro 35 40 45 Val
Ile Thr Gln Pro Ala Glu Met Asn Val Lys Thr Glu Thr Glu Ser 50 55
60 Glu Glu Glu Glu Glu Val Ala Leu Asp Asn Glu Asp Glu Glu Gln Glu
65 70 75 80 Ala Ser Gln Glu Glu Ser Ala Gly Phe Leu Arg Glu Asn Gln
Ala Lys 85 90 95 Tyr Thr Pro Ser Leu Thr Ala Leu Val Glu Asn Thr
Pro Lys Glu Asn 100 105 110
Ser Met Lys Val Arg Glu Trp Asn Asn Lys Gly Gly His Cys Cys Lys 115
120 125 Leu Glu Thr Gln Glu Leu Glu Pro Lys Phe Asn Leu Met Gln Ile
Leu 130 135 140 Gln Asp Asn Gly Asn Leu Ser Lys Met Gln Ala Arg Ile
Ala Phe Ser 145 150 155 160 Ala Tyr Leu Gln His Val Gln Ile Arg Leu
Met Lys Asp Ser Gly Gly 165 170 175 Gln Thr Phe Ser Ala Ser Trp Ala
Ala Lys Glu Asp Glu Gln Met Glu 180 185 190 Leu Val Val Arg Phe Leu
Lys Arg Ala Ser Asn Asn Leu Gln His Ser 195 200 205 Leu Arg Met Val
Leu Pro Ser Arg Arg Leu Ala Leu Leu Glu Arg Arg 210 215 220 Arg Ile
Leu Ala His Gln Leu Gly Asp Phe Ile Ile Val Tyr Asn Lys 225 230 235
240 Glu Thr Glu Gln Met Ala Glu Lys Lys Ser Lys Lys Lys Val Glu Glu
245 250 255 Glu Glu Glu Asp Gly Val Asn Met Glu Asn Phe Gln Glu Phe
Ile Arg 260 265 270 Gln Ala Ser Glu Ala Glu Leu Glu Glu Val Leu Thr
Phe Tyr Thr Gln 275 280 285 Lys Asn Lys Ser Ala Ser Val Phe Leu Gly
Thr His Ser Lys Ile Ser 290 295 300 Lys Asn Asn Asn Asn Tyr Ser Asp
Ser Gly Ala Lys Gly 305 310 315 <210> SEQ ID NO 11
<211> LENGTH: 322 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 11 Leu Pro Arg Cys Arg Ser Gly
Ser His Thr Ile Gly Pro Phe Ser Ser 1 5 10 15 Phe Gln Ser Ala Ala
His Ile Tyr Ser Gln Lys Leu Ser Arg Pro Ser 20 25 30 Ser Ala Lys
Ala Gly Ser Cys Tyr Leu Asn Lys His His Ser Gly Ile 35 40 45 Ala
Lys Thr Gln Lys Glu Gly Glu Asp Ala Ser Leu Tyr Ser Lys Arg 50 55
60 Tyr Asn Gln Ser Met Val Thr Ala Glu Leu Gln Arg Leu Ala Glu Lys
65 70 75 80 Gln Ala Ala Arg Gln Tyr Ser Pro Ser Ser His Ile Asn Leu
Leu Thr 85 90 95 Gln Gln Val Thr Asn Leu Asn Leu Ala Thr Gly Ile
Ile Asn Arg Ser 100 105 110 Ser Ala Ser Ala Pro Pro Thr Leu Arg Pro
Ile Ile Ser Pro Ser Gly 115 120 125 Pro Thr Trp Ser Thr Gln Ser Asp
Pro Gln Ala Pro Glu Asn His Ser 130 135 140 Ser Ser Pro Gly Ser Arg
Ser Leu Gln Thr Gly Gly Phe Ala Trp Glu 145 150 155 160 Gly Glu Val
Glu Asn Asn Val Tyr Ser Gln Ala Thr Gly Val Val Pro 165 170 175 Gln
His Lys Tyr His Pro Thr Ala Gly Ser Tyr Gln Leu Gln Phe Ala 180 185
190 Leu Gln Gln Leu Glu Gln Gln Lys Leu Gln Ser Arg Gln Leu Leu Asp
195 200 205 Gln Ser Arg Ala Arg His Gln Ala Ile Phe Gly Ser Gln Thr
Leu Pro 210 215 220 Asn Ser Asn Leu Trp Thr Met Asn Asn Gly Ala Gly
Cys Arg Ile Ser 225 230 235 240 Ser Ala Thr Ala Ser Gly Gln Lys Pro
Thr Thr Leu Pro Gln Lys Val 245 250 255 Val Pro Pro Pro Ser Ser Cys
Ala Ser Leu Val Pro Lys Pro Pro Pro 260 265 270 Asn His Glu Gln Val
Leu Arg Arg Ala Thr Ser Gln Lys Ala Ser Lys 275 280 285 Gly Ser Ser
Ala Glu Gly Gln Leu Asn Gly Leu Gln Ser Ser Leu Asn 290 295 300 Pro
Ala Ala Ser Val Pro Ile Thr Ser Ser Thr Asp Pro Ala His Thr 305 310
315 320 Lys Ile <210> SEQ ID NO 12 <211> LENGTH: 172
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12 Leu Pro Arg Cys Arg Ser Gly Ser His Thr
Ile Gly Pro Phe Ser Ser 1 5 10 15 Phe Gln Ser Ala Ala His Ile Tyr
Ser Gln Lys Leu Ser Arg Pro Ser 20 25 30 Ser Ala Lys Ala Gly Ser
Cys Tyr Leu Asn Lys His His Ser Gly Ile 35 40 45 Ala Lys Thr Gln
Lys Glu Gly Glu Asp Ala Ser Leu Tyr Ser Lys Arg 50 55 60 Tyr Asn
Gln Ser Met Val Thr Ala Glu Leu Gln Arg Leu Ala Glu Lys 65 70 75 80
Gln Ala Ala Arg Gln Tyr Ser Pro Ser Ser His Ile Asn Leu Leu Thr 85
90 95 Gln Gln Val Thr Asn Leu Asn Leu Ala Thr Gly Ile Ile Asn Arg
Ser 100 105 110 Ser Ala Ser Ala Pro Pro Thr Leu Arg Pro Ile Ile Ser
Pro Ser Gly 115 120 125 Pro Thr Trp Ser Thr Gln Ser Asp Pro Gln Ala
Pro Glu Asn His Ser 130 135 140 Ser Ser Pro Gly Ser Arg Ser Leu Gln
Thr Gly Gly Phe Ala Trp Glu 145 150 155 160 Gly Glu Val Glu Asn Asn
Val Tyr Ser Gln Ala Thr 165 170 <210> SEQ ID NO 13
<211> LENGTH: 151 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 13 Thr Gly Val Val Pro Gln His
Lys Tyr His Pro Thr Ala Gly Ser Tyr 1 5 10 15 Gln Leu Gln Phe Ala
Leu Gln Gln Leu Glu Gln Gln Lys Leu Gln Ser 20 25 30 Arg Gln Leu
Leu Asp Gln Ser Arg Ala Arg His Gln Ala Ile Phe Gly 35 40 45 Ser
Gln Thr Leu Pro Asn Ser Asn Leu Trp Thr Met Asn Asn Gly Ala 50 55
60 Gly Cys Arg Ile Ser Ser Ala Thr Ala Ser Gly Gln Lys Pro Thr Thr
65 70 75 80 Leu Pro Gln Lys Val Val Pro Pro Pro Ser Ser Cys Ala Ser
Leu Val 85 90 95 Pro Lys Pro Pro Pro Asn His Glu Gln Val Leu Arg
Arg Ala Thr Ser 100 105 110 Gln Lys Ala Ser Lys Gly Ser Ser Ala Glu
Gly Gln Leu Asn Gly Leu 115 120 125 Gln Ser Ser Leu Asn Pro Ala Ala
Ser Val Pro Ile Thr Ser Ser Thr 130 135 140 Asp Pro Ala His Thr Lys
Ile 145 150 <210> SEQ ID NO 14 <211> LENGTH: 834
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 14 Met Ala Arg Asp Leu Glu Glu Thr Ala Ser
Ser Ser Glu Asp Glu Glu 1 5 10 15 Val Ile Ser Gln Glu Asp His Pro
Cys Ile Met Trp Thr Gly Gly Cys 20 25 30 Arg Arg Ile Pro Val Leu
Val Phe His Ala Asp Ala Ile Leu Thr Lys 35 40 45 Asp Asn Asn Ile
Arg Val Ile Gly Glu Arg Tyr His Leu Ser Tyr Lys 50 55 60 Ile Val
Arg Thr Asp Ser Arg Leu Val Arg Ser Ile Leu Thr Ala His 65 70 75 80
Gly Phe His Glu Val His Pro Ser Ser Thr Asp Tyr Asn Leu Met Trp 85
90 95 Thr Gly Ser His Leu Lys Pro Phe Leu Leu Arg Thr Leu Ser Glu
Ala 100 105 110 Gln Lys Val Asn His Phe Pro Arg Ser Tyr Glu Leu Thr
Arg Lys Asp 115 120 125 Arg Leu Tyr Lys Asn Ile Ile Arg Met Gln His
Thr His Gly Phe Lys 130 135 140 Val Phe His Ile Leu Pro Gln Thr Phe
Leu Leu Pro Ala Glu Tyr Ala 145 150 155 160 Glu Phe Cys Asn Ser Tyr
Ser Lys Asp Arg Gly Pro Trp Ile Val Lys 165 170 175 Pro Val Ala Ser
Ser Arg Gly Arg Gly Val Tyr Leu Ile Asn Asn Pro 180 185 190 Asn Gln
Ile Ser Leu Glu Glu Asn Ile Leu Val Ser Arg Tyr Ile Asn 195 200 205
Asn Pro Leu Leu Ile Asp Asp Phe Lys Phe Asp Val Arg Leu Tyr Val 210
215 220 Leu Val Thr Ser Tyr Asp Pro Leu Val Ile Tyr Leu Tyr Glu Glu
Gly 225 230 235 240 Leu Ala Arg Phe Ala Thr Val Arg Tyr Asp Gln Gly
Ala Lys Asn Ile 245 250 255 Arg Asn Gln Phe Met His Leu Thr Asn Tyr
Ser Val Asn Lys Lys Ser 260 265 270 Gly Asp Tyr Val Ser Cys Asp Asp
Pro Glu Val Glu Asp Tyr Gly Asn 275 280 285 Lys Trp Ser Met Ser Ala
Met Leu Arg Tyr Leu Lys Gln Glu Gly Arg
290 295 300 Asp Thr Thr Ala Leu Met Ala His Val Glu Asp Leu Ile Ile
Lys Thr 305 310 315 320 Ile Ile Ser Ala Glu Leu Ala Ile Ala Thr Ala
Cys Lys Thr Phe Val 325 330 335 Pro His Arg Ser Ser Cys Phe Glu Leu
Tyr Gly Phe Asp Val Leu Ile 340 345 350 Asp Ser Thr Leu Lys Pro Trp
Leu Leu Glu Val Asn Leu Ser Pro Ser 355 360 365 Leu Ala Cys Asp Ala
Pro Leu Asp Leu Lys Ile Lys Ala Ser Met Ile 370 375 380 Ser Asp Met
Phe Thr Val Val Gly Phe Val Cys Gln Asp Pro Ala Gln 385 390 395 400
Arg Ala Ser Thr Arg Pro Ile Tyr Pro Thr Phe Glu Ser Ser Arg Arg 405
410 415 Asn Pro Phe Gln Lys Pro Gln Arg Cys Arg Pro Leu Ser Ala Ser
Asp 420 425 430 Ala Glu Met Lys Asn Leu Val Gly Ser Ala Arg Glu Lys
Gly Pro Gly 435 440 445 Lys Leu Gly Gly Ser Val Leu Gly Leu Ser Met
Glu Glu Ile Lys Val 450 455 460 Leu Arg Arg Val Lys Glu Glu Asn Asp
Arg Arg Gly Gly Phe Ile Arg 465 470 475 480 Ile Phe Pro Thr Ser Glu
Thr Trp Glu Ile Tyr Gly Ser Tyr Leu Glu 485 490 495 His Lys Thr Ser
Met Asn Tyr Met Leu Ala Thr Arg Leu Phe Gln Asp 500 505 510 Arg Met
Thr Ala Asp Gly Ala Pro Glu Leu Lys Ile Glu Ser Leu Asn 515 520 525
Ser Lys Ala Lys Leu His Ala Ala Leu Tyr Glu Arg Lys Leu Leu Ser 530
535 540 Leu Glu Val Arg Lys Arg Arg Arg Arg Ser Ser Arg Leu Arg Ala
Met 545 550 555 560 Arg Pro Lys Tyr Pro Val Ile Thr Gln Pro Ala Glu
Met Asn Val Lys 565 570 575 Thr Glu Thr Glu Ser Glu Glu Glu Glu Glu
Val Ala Leu Asp Asn Glu 580 585 590 Asp Glu Glu Gln Glu Ala Ser Gln
Glu Glu Ser Ala Gly Phe Leu Arg 595 600 605 Glu Asn Gln Ala Lys Tyr
Thr Pro Ser Leu Thr Ala Leu Val Glu Asn 610 615 620 Thr Pro Lys Glu
Asn Ser Met Lys Val Arg Glu Trp Asn Asn Lys Gly 625 630 635 640 Gly
His Cys Cys Lys Leu Glu Thr Gln Glu Leu Glu Pro Lys Phe Asn 645 650
655 Leu Met Gln Ile Leu Gln Asp Asn Gly Asn Leu Ser Lys Met Gln Ala
660 665 670 Arg Ile Ala Phe Ser Ala Tyr Leu Gln His Val Gln Ile Arg
Leu Met 675 680 685 Lys Asp Ser Gly Gly Gln Thr Phe Ser Ala Ser Trp
Ala Ala Lys Glu 690 695 700 Asp Glu Gln Met Glu Leu Val Val Arg Phe
Leu Lys Arg Ala Ser Asn 705 710 715 720 Asn Leu Gln His Ser Leu Arg
Met Val Leu Pro Ser Arg Arg Leu Ala 725 730 735 Leu Leu Glu Arg Arg
Arg Ile Leu Ala His Gln Leu Gly Asp Phe Ile 740 745 750 Ile Val Tyr
Asn Lys Glu Thr Glu Gln Met Ala Glu Lys Lys Ser Lys 755 760 765 Lys
Lys Val Glu Glu Glu Glu Glu Asp Gly Val Asn Met Glu Asn Phe 770 775
780 Gln Glu Phe Ile Arg Gln Ala Ser Glu Ala Glu Leu Glu Glu Val Leu
785 790 795 800 Thr Phe Tyr Thr Gln Lys Asn Lys Ser Ala Ser Val Phe
Leu Gly Thr 805 810 815 His Ser Lys Ile Ser Lys Asn Asn Asn Asn Tyr
Ser Asp Ser Gly Ala 820 825 830 Lys Gly <210> SEQ ID NO 15
<211> LENGTH: 623 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 15 Met Ala Arg Asp Leu Glu Glu
Thr Ala Ser Ser Ser Glu Asp Glu Glu 1 5 10 15 Val Ile Ser Gln Glu
Asp His Pro Cys Ile Met Trp Thr Gly Gly Cys 20 25 30 Arg Arg Ile
Pro Val Leu Val Phe His Ala Asp Ala Ile Leu Thr Lys 35 40 45 Asp
Asn Asn Ile Arg Val Ile Gly Glu Arg Tyr His Leu Ser Tyr Lys 50 55
60 Ile Val Arg Thr Asp Ser Arg Leu Val Arg Ser Ile Leu Thr Ala His
65 70 75 80 Gly Phe His Glu Val His Pro Ser Ser Thr Asp Tyr Asn Leu
Met Trp 85 90 95 Thr Gly Ser His Leu Lys Pro Phe Leu Leu Arg Thr
Leu Ser Glu Ala 100 105 110 Gln Lys Val Asn His Phe Pro Arg Ser Tyr
Glu Leu Thr Arg Lys Asp 115 120 125 Arg Leu Tyr Lys Asn Ile Ile Arg
Met Gln His Thr His Gly Phe Lys 130 135 140 Val Phe His Ile Leu Pro
Gln Thr Phe Leu Leu Pro Ala Glu Tyr Ala 145 150 155 160 Glu Phe Cys
Asn Ser Tyr Ser Lys Asp Arg Gly Pro Trp Ile Val Lys 165 170 175 Pro
Val Ala Ser Ser Arg Gly Arg Gly Val Tyr Leu Ile Asn Asn Pro 180 185
190 Asn Gln Ile Ser Leu Glu Glu Asn Ile Leu Val Ser Arg Tyr Ile Asn
195 200 205 Asn Pro Leu Leu Ile Asp Asp Phe Lys Phe Asp Val Arg Leu
Tyr Val 210 215 220 Leu Val Thr Ser Tyr Asp Pro Leu Val Ile Tyr Leu
Tyr Glu Glu Gly 225 230 235 240 Leu Ala Arg Phe Ala Thr Val Arg Tyr
Asp Gln Gly Ala Lys Asn Ile 245 250 255 Arg Asn Gln Phe Met His Leu
Thr Asn Tyr Ser Val Asn Lys Lys Ser 260 265 270 Gly Asp Tyr Val Ser
Cys Asp Asp Pro Glu Val Glu Asp Tyr Gly Asn 275 280 285 Lys Trp Ser
Met Ser Ala Met Leu Arg Tyr Leu Lys Gln Glu Gly Arg 290 295 300 Asp
Thr Thr Ala Leu Met Ala His Val Glu Asp Leu Ile Ile Lys Thr 305 310
315 320 Ile Ile Ser Ala Glu Leu Ala Ile Ala Thr Ala Cys Lys Thr Phe
Val 325 330 335 Pro His Arg Ser Ser Cys Phe Glu Leu Tyr Gly Phe Asp
Val Leu Ile 340 345 350 Asp Ser Thr Leu Lys Pro Trp Leu Leu Glu Val
Asn Leu Ser Pro Ser 355 360 365 Leu Ala Cys Asp Ala Pro Leu Asp Leu
Lys Ile Lys Ala Ser Met Ile 370 375 380 Ser Asp Met Phe Thr Val Val
Gly Phe Val Cys Gln Asp Pro Ala Gln 385 390 395 400 Arg Ala Ser Thr
Arg Pro Ile Tyr Pro Thr Phe Glu Ser Ser Arg Arg 405 410 415 Asn Pro
Phe Gln Lys Pro Gln Arg Cys Arg Pro Leu Ser Ala Ser Asp 420 425 430
Ala Glu Met Lys Asn Leu Val Gly Ser Ala Arg Glu Lys Gly Pro Gly 435
440 445 Lys Leu Gly Gly Ser Val Leu Gly Leu Ser Met Glu Glu Ile Lys
Val 450 455 460 Leu Arg Arg Val Lys Glu Glu Asn Asp Arg Arg Gly Gly
Phe Ile Arg 465 470 475 480 Ile Phe Pro Thr Ser Glu Thr Trp Glu Ile
Tyr Gly Ser Tyr Leu Glu 485 490 495 His Lys Thr Ser Met Asn Tyr Met
Leu Ala Thr Arg Leu Phe Gln Asp 500 505 510 Arg Met Thr Ala Asp Gly
Ala Pro Glu Leu Lys Ile Glu Ser Leu Asn 515 520 525 Ser Lys Ala Lys
Leu His Ala Ala Leu Tyr Glu Arg Lys Leu Leu Ser 530 535 540 Leu Glu
Val Arg Lys Arg Arg Arg Arg Ser Ser Arg Leu Arg Ala Met 545 550 555
560 Arg Pro Lys Tyr Pro Val Ile Thr Gln Pro Ala Glu Met Asn Val Lys
565 570 575 Thr Glu Thr Glu Ser Glu Glu Glu Glu Glu Val Ala Leu Asp
Asn Glu 580 585 590 Asp Glu Glu Gln Glu Ala Ser Gln Glu Glu Ser Ala
Gly Phe Leu Arg 595 600 605 Glu Asn Gln Ala Lys Tyr Thr Pro Ser Leu
Thr Ala Leu Val Glu 610 615 620 <210> SEQ ID NO 16
<211> LENGTH: 123 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 16 Gly Asp His Pro Glu Thr Ile
Met Glu Glu Val Lys Ile Lys Pro Pro 1 5 10 15 Lys Gln Gln Gln Thr
Thr Glu Ile His Ser Asp Lys Leu Ser Arg Phe 20 25 30 Thr Thr Ser
Ala Glu Lys Glu Ala Lys Leu Val Tyr Ser Asn Ser Ser 35 40 45 Ser
Gly Pro Thr Ala Thr Leu Gln Lys Ile Pro Asn Thr His Leu Ser 50 55
60 Ser Val Thr Thr Ser Asp Leu Ser Pro Gly Pro Cys His His Ser Ser
65 70 75 80
Leu Ser Gln Ile Pro Ser Ala Ile Pro Ser Met Pro His Gln Pro Thr 85
90 95 Ile Leu Leu Asn Thr Val Ser Ala Ser Ala Ser Pro Cys Leu His
Pro 100 105 110 Gly Ala Gln Asn Ile Pro Ser Pro Thr Gly Leu 115 120
<210> SEQ ID NO 17 <211> LENGTH: 21 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic siRNA <400>
SEQUENCE: 17 aagcagccgu cggcggcugu u 21 <210> SEQ ID NO 18
<211> LENGTH: 21 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic siRNA <400> SEQUENCE: 18 aaccauggga
agaugagacu u 21 <210> SEQ ID NO 19 <211> LENGTH: 21
<212> TYPE: RNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
siRNA <400> SEQUENCE: 19 aaucugugcc auccaaauuu u 21
<210> SEQ ID NO 20 <211> LENGTH: 21 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic siRNA <400>
SEQUENCE: 20 aaaggucucu gaggcccccu u 21 <210> SEQ ID NO 21
<211> LENGTH: 21 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic siRNA <400> SEQUENCE: 21 aaacccuaaa
ggaaaugccu u 21 <210> SEQ ID NO 22 <211> LENGTH: 21
<212> TYPE: RNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
siRNA <400> SEQUENCE: 22 aaacagcauc auccucagau u 21
<210> SEQ ID NO 23 <211> LENGTH: 21 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic siRNA <400>
SEQUENCE: 23 aaaaaguuaa ucacuuuccu u 21 <210> SEQ ID NO 24
<211> LENGTH: 21 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic siRNA <400> SEQUENCE: 24 aaaacauuau
ucgaaugcau u 21 <210> SEQ ID NO 25 <211> LENGTH: 21
<212> TYPE: RNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
siRNA <400> SEQUENCE: 25 aagguuuuuc acauccuccu u 21
<210> SEQ ID NO 26 <211> LENGTH: 21 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic siRNA <400>
SEQUENCE: 26 aauuuuguaa uucauauucu u 21 <210> SEQ ID NO 27
<211> LENGTH: 21 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic siRNA <400> SEQUENCE: 27 aaggaccggg
gaccuuggau u 21 <210> SEQ ID NO 28 <211> LENGTH: 21
<212> TYPE: RNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
siRNA <400> SEQUENCE: 28 aaaccagugg caucuucaau u 21
<210> SEQ ID NO 29 <211> LENGTH: 21 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic siRNA <400>
SEQUENCE: 29 aacaauccaa accagaucuu u 21 <210> SEQ ID NO 30
<211> LENGTH: 21 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic siRNA <400> SEQUENCE: 30 aagagaacau
uuuggucucu u 21 <210> SEQ ID NO 31 <211> LENGTH: 22
<212> TYPE: RNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
siRNA <400> SEQUENCE: 31 aacccccugc ucauagauga uu 22
<210> SEQ ID NO 32 <211> LENGTH: 22 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic siRNA <400>
SEQUENCE: 32 aagaaggauu ggcuagguuu uu 22 <210> SEQ ID NO 33
<211> LENGTH: 23 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic siRNA <400> SEQUENCE: 33 cagcacugac
uauaaccuaa uuu 23 <210> SEQ ID NO 34 <211> LENGTH: 23
<212> TYPE: RNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
siRNA <400> SEQUENCE: 34 cacccucucu gaagcacaaa auu 23
<210> SEQ ID NO 35 <211> LENGTH: 23 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic siRNA <400>
SEQUENCE: 35 gccagcugag uacgcggaau uuu 23 <210> SEQ ID NO 36
<211> LENGTH: 23 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic siRNA <400> SEQUENCE: 36 gagggcaaug
aggccaaaau auu 23 <210> SEQ ID NO 37
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 37 cagcactgac tataacctaa t 21
<210> SEQ ID NO 38 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 38
caccctctct gaagcacaaa a 21 <210> SEQ ID NO 39 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 39 gccagctgag tacgcggaat t 21 <210> SEQ
ID NO 40 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 40 gagggcaatg
aggccaaaat a 21 <210> SEQ ID NO 41 <211> LENGTH: 6
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 41 tgttct 6 <210> SEQ ID NO 42
<211> LENGTH: 1464 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 42 Met Ser Gly Met Gly
Glu Asn Thr Ser Asp Pro Ser Arg Ala Glu Thr 1 5 10 15 Arg Lys Arg
Lys Glu Cys Pro Asp Gln Leu Gly Pro Ser Pro Lys Arg 20 25 30 Asn
Thr Glu Lys Arg Asn Arg Glu Gln Glu Asn Lys Tyr Ile Glu Glu 35 40
45 Leu Ala Glu Leu Ile Phe Ala Asn Phe Asn Asp Ile Asp Asn Phe Asn
50 55 60 Phe Lys Pro Asp Lys Cys Ala Ile Leu Lys Glu Thr Val Lys
Gln Ile 65 70 75 80 Arg Gln Ile Lys Glu Gln Glu Lys Ala Ala Ala Ala
Asn Ile Asp Glu 85 90 95 Val Gln Lys Ser Asp Val Ser Ser Thr Gly
Gln Gly Val Ile Asp Lys 100 105 110 Asp Ala Leu Gly Pro Met Met Leu
Glu Ala Leu Asp Gly Phe Phe Phe 115 120 125 Val Val Asn Leu Glu Gly
Asn Val Val Phe Val Ser Glu Asn Val Thr 130 135 140 Gln Tyr Leu Arg
Tyr Asn Gln Glu Glu Leu Met Asn Lys Ser Val Tyr 145 150 155 160 Ser
Ile Leu His Val Gly Asp His Thr Glu Phe Val Lys Asn Leu Leu 165 170
175 Pro Lys Ser Ile Val Asn Gly Gly Ser Trp Ser Gly Glu Pro Pro Arg
180 185 190 Arg Asn Ser His Thr Phe Asn Cys Arg Met Leu Val Lys Pro
Leu Pro 195 200 205 Asp Ser Glu Glu Glu Gly His Asp Asn Gln Glu Ala
His Gln Lys Tyr 210 215 220 Glu Thr Met Gln Cys Phe Ala Val Ser Gln
Pro Lys Ser Ile Lys Glu 225 230 235 240 Glu Gly Glu Asp Leu Gln Ser
Cys Leu Ile Cys Val Ala Arg Arg Val 245 250 255 Pro Met Lys Glu Arg
Pro Val Leu Pro Ser Ser Glu Ser Phe Thr Thr 260 265 270 Arg Gln Asp
Leu Gln Gly Lys Ile Thr Ser Leu Asp Thr Ser Thr Met 275 280 285 Arg
Ala Ala Met Lys Pro Gly Trp Glu Asp Leu Val Arg Arg Cys Ile 290 295
300 Gln Lys Phe His Ala Gln His Glu Gly Glu Ser Val Ser Tyr Ala Lys
305 310 315 320 Arg His His His Glu Val Leu Arg Gln Gly Leu Ala Phe
Ser Gln Ile 325 330 335 Tyr Arg Phe Ser Leu Ser Asp Gly Thr Leu Val
Ala Ala Gln Thr Lys 340 345 350 Ser Lys Leu Ile Arg Ser Gln Thr Thr
Asn Glu Pro Gln Leu Val Ile 355 360 365 Ser Leu His Met Leu His Arg
Glu Gln Asn Val Cys Val Met Asn Pro 370 375 380 Asp Leu Thr Gly Gln
Thr Met Gly Lys Pro Leu Asn Pro Ile Ser Ser 385 390 395 400 Asn Ser
Pro Ala His Gln Ala Leu Cys Ser Gly Asn Pro Gly Gln Asp 405 410 415
Met Thr Leu Ser Ser Asn Ile Asn Phe Pro Ile Asn Gly Pro Lys Glu 420
425 430 Gln Met Gly Met Pro Met Gly Arg Phe Gly Gly Ser Gly Gly Met
Asn 435 440 445 His Val Ser Gly Met Gln Ala Thr Thr Pro Gln Gly Ser
Asn Tyr Ala 450 455 460 Leu Lys Met Asn Ser Pro Ser Gln Ser Ser Pro
Gly Met Asn Pro Gly 465 470 475 480 Gln Pro Thr Ser Met Leu Ser Pro
Arg His Arg Met Ser Pro Gly Val 485 490 495 Ala Gly Ser Pro Arg Ile
Pro Pro Ser Gln Phe Ser Pro Ala Gly Ser 500 505 510 Leu His Ser Pro
Val Gly Val Cys Ser Ser Thr Gly Asn Ser His Ser 515 520 525 Tyr Thr
Asn Ser Ser Leu Asn Ala Leu Gln Ala Leu Ser Glu Gly His 530 535 540
Gly Val Ser Leu Gly Ser Ser Leu Ala Ser Pro Asp Leu Lys Met Gly 545
550 555 560 Asn Leu Gln Asn Ser Pro Val Asn Met Asn Pro Pro Pro Leu
Ser Lys 565 570 575 Met Gly Ser Leu Asp Ser Lys Asp Cys Phe Gly Leu
Tyr Gly Glu Pro 580 585 590 Ser Glu Gly Thr Thr Gly Gln Ala Glu Ser
Ser Cys His Pro Gly Glu 595 600 605 Gln Lys Glu Thr Asn Asp Pro Asn
Leu Pro Pro Ala Val Ser Ser Glu 610 615 620 Arg Ala Asp Gly Gln Ser
Arg Leu His Asp Ser Lys Gly Gln Thr Lys 625 630 635 640 Leu Leu Gln
Leu Leu Thr Thr Lys Ser Asp Gln Met Glu Pro Ser Pro 645 650 655 Leu
Ala Ser Ser Leu Ser Asp Thr Asn Lys Asp Ser Thr Gly Ser Leu 660 665
670 Pro Gly Ser Gly Ser Thr His Gly Thr Ser Leu Lys Glu Lys His Lys
675 680 685 Ile Leu His Arg Leu Leu Gln Asp Ser Ser Ser Pro Val Asp
Leu Ala 690 695 700 Lys Leu Thr Ala Glu Ala Thr Gly Lys Asp Leu Ser
Gln Glu Ser Ser 705 710 715 720 Ser Thr Ala Pro Gly Ser Glu Val Thr
Ile Lys Gln Glu Pro Val Ser 725 730 735 Pro Lys Lys Lys Glu Asn Ala
Leu Leu Arg Tyr Leu Leu Asp Lys Asp 740 745 750 Asp Thr Lys Asp Ile
Gly Leu Pro Glu Ile Thr Pro Lys Leu Glu Arg 755 760 765 Leu Asp Ser
Lys Thr Asp Pro Ala Ser Asn Thr Lys Leu Ile Ala Met 770 775 780 Lys
Thr Glu Lys Glu Glu Met Ser Phe Glu Pro Gly Asp Gln Pro Gly 785 790
795 800 Ser Glu Leu Asp Asn Leu Glu Glu Ile Leu Asp Asp Leu Gln Asn
Ser 805 810 815 Gln Leu Pro Gln Leu Phe Pro Asp Thr Arg Pro Gly Ala
Pro Ala Gly 820 825 830 Ser Val Asp Lys Gln Ala Ile Ile Asn Asp Leu
Met Gln Leu Thr Ala 835 840 845 Glu Asn Ser Pro Val Thr Pro Val Gly
Ala Gln Lys Thr Ala Leu Arg 850 855 860 Ile Ser Gln Ser Thr Phe Asn
Asn Pro Arg Pro Gly Gln Leu Gly Arg 865 870 875 880 Leu Leu Pro Asn
Gln Asn Leu Pro Leu Asp Ile Thr Leu Gln Ser Pro 885 890 895 Thr Gly
Ala Gly Pro Phe Pro Pro Ile Arg Asn Ser Ser Pro Tyr Ser 900 905 910
Val Ile Pro Gln Pro Gly Met Met Gly Asn Gln Gly Met Ile Gly Asn 915
920 925 Gln Gly Asn Leu Gly Asn Ser Ser Thr Gly Met Ile Gly Asn Ser
Ala 930 935 940 Ser Arg Pro Thr Met Pro Ser Gly Glu Trp Ala Pro Gln
Ser Ser Ala 945 950 955 960 Val Arg Val Thr Cys Ala Ala Thr Thr Ser
Ala Met Asn Arg Pro Val 965 970 975 Gln Gly Gly Met Ile Arg Asn Pro
Ala Ala Ser Ile Pro Met Arg Pro 980 985 990 Ser Ser Gln Pro Gly Gln
Arg Gln Thr Leu Gln Ser Gln Val Met Asn 995 1000 1005 Ile Gly Pro
Ser Glu Leu Glu Met Asn Met Gly Gly Pro Gln Tyr Ser 1010 1015 1020
Gln Gln Gln Ala Pro Pro Asn Gln Thr Ala Pro Trp Pro Glu Ser Ile
1025 1030 1035 1040
Leu Pro Ile Asp Gln Ala Ser Phe Ala Ser Gln Asn Arg Gln Pro Phe
1045 1050 1055 Gly Ser Ser Pro Asp Asp Leu Leu Cys Pro His Pro Ala
Ala Glu Ser 1060 1065 1070 Pro Ser Asp Glu Gly Ala Leu Leu Asp Gln
Leu Tyr Leu Ala Leu Arg 1075 1080 1085 Asn Phe Asp Gly Leu Glu Glu
Ile Asp Arg Ala Leu Gly Ile Pro Glu 1090 1095 1100 Leu Val Ser Gln
Ser Gln Ala Val Asp Pro Glu Gln Phe Ser Ser Gln 1105 1110 1115 1120
Asp Ser Asn Ile Met Leu Glu Gln Lys Ala Pro Val Phe Pro Gln Gln
1125 1130 1135 Tyr Ala Ser Gln Ala Gln Met Ala Gln Gly Ser Tyr Ser
Pro Met Gln 1140 1145 1150 Asp Pro Asn Phe His Thr Met Gly Gln Arg
Pro Ser Tyr Ala Thr Leu 1155 1160 1165 Arg Met Gln Pro Arg Pro Gly
Leu Arg Pro Thr Gly Leu Val Gln Asn 1170 1175 1180 Gln Pro Asn Gln
Leu Arg Leu Gln Leu Gln His Arg Leu Gln Ala Gln 1185 1190 1195 1200
Gln Asn Arg Gln Pro Leu Met Asn Gln Ile Ser Asn Val Ser Asn Val
1205 1210 1215 Asn Leu Thr Leu Arg Pro Gly Val Pro Thr Gln Ala Pro
Ile Asn Ala 1220 1225 1230 Gln Met Leu Ala Gln Arg Gln Arg Glu Ile
Leu Asn Gln His Leu Arg 1235 1240 1245 Gln Arg Gln Met His Gln Gln
Gln Gln Val Gln Gln Arg Thr Leu Met 1250 1255 1260 Met Arg Gly Gln
Gly Leu Asn Met Thr Pro Ser Met Val Ala Pro Ser 1265 1270 1275 1280
Gly Met Pro Ala Thr Met Ser Asn Pro Arg Ile Pro Gln Ala Asn Ala
1285 1290 1295 Gln Gln Phe Pro Phe Pro Pro Asn Tyr Gly Ile Ser Gln
Gln Pro Asp 1300 1305 1310 Pro Gly Phe Thr Gly Ala Thr Thr Pro Gln
Ser Pro Leu Met Ser Pro 1315 1320 1325 Arg Met Ala His Thr Gln Ser
Pro Met Met Gln Gln Ser Gln Ala Asn 1330 1335 1340 Pro Ala Tyr Gln
Ala Pro Ser Asp Ile Asn Gly Trp Ala Gln Gly Asn 1345 1350 1355 1360
Met Gly Gly Asn Ser Met Phe Ser Gln Gln Ser Pro Pro His Phe Gly
1365 1370 1375 Gln Gln Ala Asn Thr Ser Met Tyr Ser Asn Asn Met Asn
Ile Asn Val 1380 1385 1390 Ser Met Ala Thr Asn Thr Gly Gly Met Ser
Ser Met Asn Gln Met Thr 1395 1400 1405 Gly Gln Ile Ser Met Thr Ser
Val Thr Ser Val Pro Thr Ser Gly Leu 1410 1415 1420 Ser Ser Met Gly
Pro Glu Gln Val Asn Asp Pro Ala Leu Arg Gly Gly 1425 1430 1435 1440
Asn Leu Phe Pro Asn Gln Leu Pro Gly Met Asp Met Ile Lys Gln Glu
1445 1450 1455 Gly Asp Thr Thr Arg Lys Tyr Cys 1460 <210> SEQ
ID NO 43 <211> LENGTH: 1440 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 43 Met Ser Gly Leu Gly
Asp Ser Ser Ser Asp Pro Ala Asn Pro Asp Ser 1 5 10 15 His Lys Arg
Lys Gly Ser Pro Cys Asp Thr Leu Ala Ser Ser Thr Glu 20 25 30 Lys
Arg Arg Arg Glu Gln Glu Asn Lys Tyr Leu Glu Glu Leu Ala Glu 35 40
45 Leu Leu Ser Ala Asn Ile Ser Asp Ile Asp Ser Leu Ser Val Lys Pro
50 55 60 Asp Lys Cys Lys Ile Leu Lys Lys Thr Val Asp Gln Ile Gln
Leu Met 65 70 75 80 Lys Arg Met Glu Gln Glu Lys Ser Thr Thr Asp Asp
Asp Val Gln Lys 85 90 95 Ser Asp Ile Ser Ser Ser Ser Gln Gly Val
Ile Glu Lys Glu Ser Leu 100 105 110 Gly Pro Leu Leu Leu Glu Ala Leu
Asp Gly Phe Phe Phe Val Val Asn 115 120 125 Cys Glu Gly Arg Ile Val
Phe Val Ser Glu Asn Val Thr Ser Tyr Leu 130 135 140 Gly Tyr Asn Gln
Glu Glu Leu Met Asn Thr Ser Val Tyr Ser Ile Leu 145 150 155 160 His
Val Gly Asp His Ala Glu Phe Val Lys Asn Leu Leu Pro Lys Ser 165 170
175 Leu Val Asn Gly Val Pro Trp Pro Gln Glu Ala Thr Arg Arg Asn Ser
180 185 190 His Thr Phe Asn Cys Arg Met Leu Ile His Pro Pro Asp Glu
Pro Gly 195 200 205 Thr Glu Asn Gln Glu Ala Cys Gln Arg Tyr Glu Val
Met Gln Cys Phe 210 215 220 Thr Val Ser Gln Pro Lys Ser Ile Gln Glu
Asp Gly Glu Asp Phe Gln 225 230 235 240 Ser Cys Leu Ile Cys Ile Ala
Arg Arg Leu Pro Arg Pro Pro Ala Ile 245 250 255 Thr Gly Val Glu Ser
Phe Met Thr Lys Gln Asp Thr Thr Gly Lys Ile 260 265 270 Ile Ser Ile
Asp Thr Ser Ser Leu Arg Ala Ala Gly Arg Thr Gly Trp 275 280 285 Glu
Asp Leu Val Arg Lys Cys Ile Tyr Ala Phe Phe Gln Pro Gln Gly 290 295
300 Arg Glu Pro Ser Tyr Ala Arg Gln Leu Phe Gln Glu Val Met Thr Arg
305 310 315 320 Gly Thr Ala Ser Ser Pro Ser Tyr Arg Phe Ile Leu Asn
Asp Gly Thr 325 330 335 Met Leu Ser Ala His Thr Lys Cys Lys Leu Cys
Tyr Pro Gln Ser Pro 340 345 350 Asp Met Gln Pro Phe Ile Met Gly Ile
His Ile Ile Asp Arg Glu His 355 360 365 Ser Gly Leu Ser Pro Gln Asp
Asp Thr Asn Ser Gly Met Ser Ile Pro 370 375 380 Arg Val Asn Pro Ser
Val Asn Pro Ser Ile Ser Pro Ala His Gly Val 385 390 395 400 Ala Arg
Ser Ser Thr Leu Pro Pro Ser Asn Ser Asn Met Val Ser Thr 405 410 415
Arg Ile Asn Arg Gln Gln Ser Ser Asp Leu His Ser Ser Ser His Ser 420
425 430 Asn Ser Ser Asn Ser Gln Gly Ser Phe Gly Cys Ser Pro Gly Ser
Gln 435 440 445 Ile Val Ala Asn Val Ala Leu Asn Gln Gly Gln Ala Ser
Ser Gln Ser 450 455 460 Ser Asn Pro Ser Leu Asn Leu Asn Asn Ser Pro
Met Glu Gly Thr Gly 465 470 475 480 Ile Ser Leu Ala Gln Phe Met Ser
Pro Arg Arg Gln Val Thr Ser Gly 485 490 495 Leu Ala Thr Arg Pro Arg
Met Pro Asn Asn Ser Phe Pro Pro Asn Ile 500 505 510 Ser Thr Leu Ser
Ser Pro Val Gly Met Thr Ser Ser Ala Cys Asn Asn 515 520 525 Asn Asn
Arg Ser Tyr Ser Asn Ile Pro Val Thr Ser Leu Gln Gly Met 530 535 540
Asn Glu Gly Pro Asn Asn Ser Val Gly Phe Ser Ala Ser Ser Pro Val 545
550 555 560 Leu Arg Gln Met Ser Ser Gln Asn Ser Pro Ser Arg Leu Asn
Ile Gln 565 570 575 Pro Ala Lys Ala Glu Ser Lys Asp Asn Lys Glu Ile
Ala Ser Ile Leu 580 585 590 Asn Glu Met Ile Gln Ser Asp Asn Ser Ser
Ser Asp Gly Lys Pro Leu 595 600 605 Asp Ser Gly Leu Leu His Asn Asn
Asp Arg Leu Ser Asp Gly Asp Ser 610 615 620 Lys Tyr Ser Gln Thr Ser
His Lys Leu Val Gln Leu Leu Thr Thr Thr 625 630 635 640 Ala Glu Gln
Gln Leu Arg His Ala Asp Ile Asp Thr Ser Cys Lys Asp 645 650 655 Val
Leu Ser Cys Thr Gly Thr Ser Asn Ser Ala Ser Ala Asn Ser Ser 660 665
670 Gly Gly Ser Cys Pro Ser Ser His Ser Ser Leu Thr Glu Arg His Lys
675 680 685 Ile Leu His Arg Leu Leu Gln Glu Gly Ser Pro Ser Asp Ile
Thr Thr 690 695 700 Leu Ser Val Glu Pro Asp Lys Lys Asp Ser Ala Ser
Thr Ser Val Ser 705 710 715 720 Val Thr Gly Gln Val Gln Gly Asn Ser
Ser Ile Lys Leu Glu Leu Asp 725 730 735 Ala Ser Lys Lys Lys Glu Ser
Lys Asp His Gln Leu Leu Arg Tyr Leu 740 745 750 Leu Asp Lys Asp Glu
Lys Asp Leu Arg Ser Thr Pro Asn Leu Ser Leu 755 760 765 Asp Asp Val
Lys Val Lys Val Glu Lys Lys Glu Gln Met Asp Pro Cys 770 775 780 Asn
Thr Asn Pro Thr Pro Met Thr Lys Pro Thr Pro Glu Glu Ile Lys 785 790
795 800 Leu Glu Ala Gln Ser Gln Phe Thr Ala Asp Leu Asp Gln Phe Asp
Gln 805 810 815 Leu Leu Pro Thr Leu Glu Lys Ala Ala Gln Leu Pro Gly
Leu Cys Glu 820 825 830 Thr Asp Arg Met Asp Gly Ala Val Thr Ser Val
Thr Ile Lys Ser Glu 835 840 845 Ile Leu Pro Ala Ser Leu Gln Ser Ala
Thr Ala Arg Pro Thr Ser Arg 850 855 860
Leu Asn Arg Leu Pro Glu Leu Glu Leu Glu Ala Ile Asp Asn Gln Phe 865
870 875 880 Gly Gln Pro Gly Thr Gly Asp Gln Ile Pro Trp Thr Asn Asn
Thr Val 885 890 895 Thr Ala Ile Asn Gln Ser Lys Ser Glu Asp Gln Cys
Ile Ser Ser Gln 900 905 910 Leu Asp Glu Leu Leu Cys Pro Pro Thr Thr
Val Glu Gly Arg Asn Asp 915 920 925 Glu Lys Ala Leu Leu Glu Gln Leu
Val Ser Phe Leu Ser Gly Lys Asp 930 935 940 Glu Thr Glu Leu Ala Glu
Leu Asp Arg Ala Leu Gly Ile Asp Lys Leu 945 950 955 960 Val Gln Gly
Gly Gly Leu Asp Val Leu Ser Glu Arg Phe Pro Pro Gln 965 970 975 Gln
Ala Thr Pro Pro Leu Ile Met Glu Glu Arg Pro Asn Leu Tyr Ser 980 985
990 Gln Pro Tyr Ser Ser Pro Ser Pro Thr Ala Asn Leu Pro Ser Pro Phe
995 1000 1005 Gln Gly Met Val Arg Gln Lys Pro Ser Leu Gly Thr Met
Pro Val Gln 1010 1015 1020 Val Thr Pro Pro Arg Gly Ala Phe Ser Pro
Gly Met Gly Met Gln Pro 1025 1030 1035 1040 Arg Gln Thr Leu Asn Arg
Pro Pro Ala Ala Pro Asn Gln Leu Arg Leu 1045 1050 1055 Gln Leu Gln
Gln Arg Leu Gln Gly Gln Gln Gln Leu Ile His Gln Asn 1060 1065 1070
Arg Gln Ala Ile Leu Asn Gln Phe Ala Ala Thr Ala Pro Val Gly Ile
1075 1080 1085 Asn Met Arg Ser Gly Met Gln Gln Gln Ile Thr Pro Gln
Pro Pro Leu 1090 1095 1100 Asn Ala Gln Met Leu Ala Gln Arg Gln Arg
Glu Leu Tyr Ser Gln Gln 1105 1110 1115 1120 His Arg Gln Arg Gln Leu
Ile Gln Gln Gln Arg Ala Met Leu Met Arg 1125 1130 1135 Gln Gln Ser
Phe Gly Asn Asn Leu Pro Pro Ser Ser Gly Leu Pro Val 1140 1145 1150
Gln Met Gly Asn Pro Arg Leu Pro Gln Gly Ala Pro Gln Gln Phe Pro
1155 1160 1165 Tyr Pro Pro Asn Tyr Gly Thr Asn Pro Gly Thr Pro Pro
Ala Ser Thr 1170 1175 1180 Ser Pro Phe Ser Gln Leu Ala Ala Asn Pro
Glu Ala Ser Leu Ala Asn 1185 1190 1195 1200 Arg Asn Ser Met Val Ser
Arg Gly Met Thr Gly Asn Ile Gly Gly Gln 1205 1210 1215 Phe Gly Thr
Gly Ile Asn Pro Gln Met Gln Gln Asn Val Phe Gln Tyr 1220 1225 1230
Pro Gly Ala Gly Met Val Pro Gln Gly Glu Ala Asn Phe Ala Pro Ser
1235 1240 1245 Leu Ser Pro Gly Ser Ser Met Val Pro Met Pro Ile Pro
Pro Pro Gln 1250 1255 1260 Ser Ser Leu Leu Gln Gln Thr Pro Pro Ala
Ser Gly Tyr Gln Ser Pro 1265 1270 1275 1280 Asp Met Lys Ala Trp Gln
Gln Gly Ala Ile Gly Asn Asn Asn Val Phe 1285 1290 1295 Ser Gln Ala
Val Gln Asn Gln Pro Thr Pro Ala Gln Pro Gly Val Tyr 1300 1305 1310
Asn Asn Met Ser Ile Thr Val Ser Met Ala Gly Gly Asn Thr Asn Val
1315 1320 1325 Gln Asn Met Asn Pro Met Met Ala Gln Met Gln Met Ser
Ser Leu Gln 1330 1335 1340 Met Pro Gly Met Asn Thr Val Cys Pro Glu
Gln Ile Asn Asp Pro Ala 1345 1350 1355 1360 Leu Arg His Thr Gly Leu
Tyr Cys Asn Gln Leu Ser Ser Thr Asp Leu 1365 1370 1375 Leu Lys Thr
Glu Ala Asp Gly Thr Gln Val Gln Gln Val Gln Val Phe 1380 1385 1390
Ala Asp Val Gln Cys Thr Val Asn Leu Val Gly Gly Asp Pro Tyr Leu
1395 1400 1405 Asn Gln Pro Gly Pro Leu Gly Thr Gln Lys Pro Thr Ser
Gly Pro Gln 1410 1415 1420 Thr Pro Gln Ala Gln Gln Lys Ser Leu Leu
Gln Gln Leu Leu Thr Glu 1425 1430 1435 1440 <210> SEQ ID NO
44 <211> LENGTH: 777 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 44 Met Asp Ser Lys Glu
Ser Leu Thr Pro Gly Arg Glu Glu Asn Pro Ser 1 5 10 15 Ser Val Leu
Ala Gln Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr 20 25 30 Leu
Arg Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu 35 40
45 Ala Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp
50 55 60 Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu
Ser Lys 65 70 75 80 Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu
Thr Glu Thr Lys 85 90 95 Val Met Gly Asn Asp Leu Gly Phe Pro Gln
Gln Gly Gln Ile Ser Leu 100 105 110 Ser Ser Gly Glu Thr Asp Leu Lys
Leu Leu Glu Glu Ser Ile Ala Asn 115 120 125 Leu Asn Arg Ser Thr Ser
Val Pro Glu Asn Pro Lys Ser Ser Ala Ser 130 135 140 Thr Ala Val Ser
Ala Ala Pro Thr Glu Lys Glu Phe Pro Lys Thr His 145 150 155 160 Ser
Asp Val Ser Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr 165 170
175 Asn Gly Gly Asn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp
180 185 190 Ile Leu Gln Asp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys
Glu Thr 195 200 205 Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp
Glu Asn Cys Leu 210 215 220 Leu Ser Pro Leu Ala Gly Glu Asp Asp Ser
Phe Leu Leu Glu Gly Asn 225 230 235 240 Ser Asn Glu Asp Cys Lys Pro
Leu Ile Leu Pro Asp Thr Lys Pro Lys 245 250 255 Ile Lys Asp Asn Gly
Asp Leu Val Leu Ser Ser Pro Ser Asn Val Thr 260 265 270 Leu Pro Gln
Val Lys Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr 275 280 285 Pro
Gly Val Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala 290 295
300 Ser Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser
305 310 315 320 Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His
Tyr Asp Met 325 330 335 Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln
Lys Pro Ile Phe Asn 340 345 350 Val Ile Pro Pro Ile Pro Val Gly Ser
Glu Asn Trp Asn Arg Cys Gln 355 360 365 Gly Ser Gly Asp Asp Asn Leu
Thr Ser Leu Gly Thr Leu Asn Phe Pro 370 375 380 Gly Arg Thr Val Phe
Ser Asn Gly Tyr Ser Ser Pro Ser Met Arg Pro 385 390 395 400 Asp Val
Ser Ser Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly Pro 405 410 415
Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His 420
425 430 Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg
Ala 435 440 445 Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn
Asp Cys Ile 450 455 460 Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala
Cys Arg Tyr Arg Lys 465 470 475 480 Cys Leu Gln Ala Gly Met Asn Leu
Glu Ala Arg Lys Thr Lys Lys Lys 485 490 495 Ile Lys Gly Ile Gln Gln
Ala Thr Thr Gly Val Ser Gln Glu Thr Ser 500 505 510 Glu Asn Pro Gly
Asn Lys Thr Ile Val Pro Ala Thr Leu Pro Gln Leu 515 520 525 Thr Pro
Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu 530 535 540
Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Thr Trp Arg Ile Met 545
550 555 560 Thr Thr Leu Asn Met Leu Gly Gly Arg Gln Val Ile Ala Ala
Val Lys 565 570 575 Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn Leu His
Leu Asp Asp Gln 580 585 590 Met Thr Leu Leu Gln Tyr Ser Trp Met Phe
Leu Met Ala Phe Ala Leu 595 600 605 Gly Trp Arg Ser Tyr Arg Gln Ser
Ser Ala Asn Leu Leu Cys Phe Ala 610 615 620 Pro Asp Leu Ile Ile Asn
Glu Gln Arg Met Thr Leu Pro Cys Met Tyr 625 630 635 640 Asp Gln Cys
Lys His Met Leu Tyr Val Ser Ser Glu Leu His Arg Leu 645 650 655 Gln
Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu Leu Leu Leu 660 665
670 Ser Ser Val Pro Lys Asp Gly Leu Lys Ser Gln Glu Leu Phe Asp Glu
675 680 685 Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile Val
Lys Arg 690 695 700 Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg Phe Tyr
Gln Leu Thr Lys 705 710 715 720
Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu Asn Tyr Cys 725
730 735 Phe Gln Thr Phe Leu Asp Lys Thr Met Ser Ile Glu Phe Pro Glu
Met 740 745 750 Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro Lys Tyr Ser
Asn Gly Asn 755 760 765 Ile Lys Lys Leu Leu Phe His Gln Lys 770 775
<210> SEQ ID NO 45 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 45 gctctagatg cacccaggag tggtgaacag g 31 <210> SEQ
ID NO 46 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 46
aagatgagac aggaatctgt gcc 23 <210> SEQ ID NO 47 <211>
LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: A
synthetic primer <400> SEQUENCE: 47 tacgatatct gatggcccgg
gacctggagg aaac 34 <210> SEQ ID NO 48 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
primer <400> SEQUENCE: 48 agtaagaagg gcttcagg 18 <210>
SEQ ID NO 49 <211> LENGTH: 32 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 49 cgtctagact accaaagctg tcaatgaggg tg 32 <210> SEQ
ID NO 50 <211> LENGTH: 31 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 50
ggaattcgaa aatacaccca aagaaaattc c 31 <210> SEQ ID NO 51
<211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic primer <400> SEQUENCE: 51 gctctagaca
ccttttgccc cactatcaga a 31 <210> SEQ ID NO 52 <211>
LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: A
synthetic primer <400> SEQUENCE: 52 ggaattcgat caccctgaga
ctataatgg 29 <210> SEQ ID NO 53 <211> LENGTH: 31
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
primer <400> SEQUENCE: 53 cgtctagagg ccagtagggc ttgggatgtt c
31 <210> SEQ ID NO 54 <211> LENGTH: 30 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: A synthetic primer
<400> SEQUENCE: 54 ggaattcctg ccacgctgtc gatcaggaag 30
<210> SEQ ID NO 55 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: A synthetic primer <400>
SEQUENCE: 55 ggaattcaca ggggtggtcc cccagcac 28 <210> SEQ ID
NO 56 <211> LENGTH: 33 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 56
cgtctagata gcctggctgt acacgttgtt ttc 33 <210> SEQ ID NO 57
<211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A synthetic primer <400> SEQUENCE: 57 ggaattcgca
caaatggccc agggtagc 28 <210> SEQ ID NO 58 <211> LENGTH:
32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A synthetic
primer <400> SEQUENCE: 58 cgggatcctc agcaatattt ccgtgttgtg tc
32 <210> SEQ ID NO 59 <211> LENGTH: 66 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: A synthetic oligonucleotide
<400> SEQUENCE: 59 gaattcccgg gatatcgtcg acccacgcgt
ccggggcggc cgctctagag tatccctcga 60 ggatcc 66 <210> SEQ ID NO
60 <211> LENGTH: 336 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 60 tagaaaatac
acccaaagaa aattccatga aagttcgtga atggaataat aaaggtggac 60
actgctgcaa acttgagact caggagctag agcctaaatt taacctgatg cagattcttc
120 aagataatgg caatcttagc aaaatgcagg cccgaatagc attctctgcc
tatctccagc 180 atgttcaaat tcgcctgatg aaagacagtg gcggtcagac
gttcagtgcc agttgggctg 240 ccaaagagga tgaacagatg gagctggttg
ttcgtttcct caagcgagca tcaaataacc 300 tccagcattc actgaggatg
gtattaccca gtcgac 336 <210> SEQ ID NO 61 <211> LENGTH:
4021 <212> TYPE: DNA <213> ORGANISM: Cercopithecus
aethiops <400> SEQUENCE: 61 tgaatctgct aggaaaggtc tctgaggccc
ccgtctgccg actgcatgac aaaccctaaa 60 ggaaatgcca gtcgtgatgg
cccgggacct ggaggaaaca gcatcatcct cagaggatga 120 ggaggtcata
agtcaagagg atcatccatg catcatgtgg actggaggct gtaggagaat 180
tccagttttg gtattccatg ccgacgctat tcttacaaag gacaacaata ttagagtaat
240 tggagaacgt tatcatttgt cttataagat tgtacgaacg gacagtcgcc
tagtacgcag 300 cattctgaca gcccatggat ttcatgaagt tcacccaagc
agcactgact ataacctaat 360 gtggacagga tcccacctga agcccttctt
actgcgcacc ctctctgaag cacaaaaagt 420 taatcacttt cccaggtctt
atgaacttac ccggaaggac cgactgtaca aaaacattat 480 tcgaatgcag
catacacatg gattcaaggc ttttcacatc ctcccccaga ccttcctcct 540
gccagctgag tacgcggaat tttgtaattc atattcgaag gaccggggac cttggatagt
600 aaaaccagtg gcatcttcta gggggcgggg cgtctacctg atcaacaatc
caaaccagat 660 ttccctggaa gaaaacattc tggtctcccg ttatattaac
aaccccctgc tcatagatga 720 tttcaagttt gatgtgcgcc tctatgtgct
ggtgacttcc tatgatcctc ttgtcatcta 780 tctctatgaa gaaggattgg
ctaggtttgc aactgtgcga tatgatcaag gagccaagaa 840 cattcggaac
cagttcatgc atctgacaaa ctacagtgtg aacaagaaga gtggagacta 900
cgtcagttgt gatgatccag aagtggagga ctatggaaac aaatggagca tgagtgctat
960 gcttaggtac ctgaaacaag aaggcagaga tacaactgca ttgatggccc
atgtagaaga 1020 cctgatcatt aagactataa tctctgctga actagctatt
gctactgcct gtaaaacctt 1080 tgttcctcat cgcagcagtt gttttgaact
ctatggcttt gacgtgctca tagatgctac 1140 tctgaagcca tggttgttgg
aagtgaatct ctctccttct ttggcctgtg atgcacctct 1200 ggacctaaag
attaaagcca gtatgatttc agatatgttc actgttgttg gatttgtgtg 1260
ccaagatcct gcccagcggg catcaacccg gccaatttat cccacctttg agtcttccag
1320 gcgaaaccct ttccagaaac ctcagcgtcc acttccagca cagtttcatt
catcagagcc 1380 aaagcagcgt tcccgtccac tctctgccag tgatgcggaa
atgaaaaacc tcgtgggctc 1440 agcccgggag aaagggccag ggaagttggg
tggttctgtg cttggtctgt caatggagga 1500 gatcaaagtt ttacggaggg
tgaaggagga gaatgatcgg agaggtggat ttattcgcat 1560 atttcctaca
tctgagacat gggaaatata tgggtcctac ctcgagcata agacctcaat 1620
gaactatatg ctggcaacac gcctcttcca ggacagaatg actgctgatg gagcaccaga
1680 attgaagata gagggcctga attcaaaggc caagctgcat gctgcacttt
acgagaggaa 1740 gctcctgtct ctggaggtgc gaaaacgtag acgacggagt
agcagattga gggcaatgag 1800 gccaaaatac ccagtgatta cccaaccagc
tgaaatgaat gttaaaactg agacagagag 1860 tgaagaggag gaagaagtcg
cattagacaa tgaagatgaa gagcaggaag cttcccagga 1920 ggagtctgca
ggatttctta gagaaaatca agccaaagat acaccctcat tgacaacttt 1980
ggtagaaaat acacccaaag aaaattccgt gaaagttcgt gaatggagta aaaaaggtga
2040 acggtgctgc aaacttgaga ctcaggagct ggagcctaaa tttaacctga
tgcaggttct 2100 tcaagataac ggcaatctta gcaaagtgca ggcccgaata
gcattctcta cctatctcca 2160 gcatgttcaa attcgcctga tgaaagacag
tggaggtcag acgttcagtg ccagttgggc 2220 tgccaaagag gatgaacaga
tggagctggt cgttcgtttc ctcaagcgag catcaaataa 2280 ccttcagcag
tcactgagga tggtattacc cagccgacga ttggcacttc tggaacgcag 2340
aagaatcctg gcccaccagc tgggtgactt tatcattgta tacaacaagg aaacagaaca
2400 aatggctgaa aagaaatcaa agaagaaagt tgaagaagaa gaggaggatg
gagtgaatat 2460 ggaaaacttt caggagttca tcagacaagc aagtgaggct
gaactggagg aggtgttgac 2520 tttttatacc caaaagaaca agtctgctag
tgtcttcctg gggactcact ctaaaagttc 2580 taagaacaac aacagttatt
ctgatagtgg ggcaaaaggt gatcaccctg agactgtaat 2640 ggaagaagcg
aaaatgaagc cgcctaaaca gcaacagaca acagaaattc actctgataa 2700
attatctcga tttaccactt cagcagaaaa agaggcaaaa ttagtttata ccagttcttc
2760 gtcgactcct ttctctggtc ctactgctac tctgcagaaa attcccaaca
cccatttgtc 2820 atctgttaca acctcagacc tctctccagg gcctggccac
cattcttctt tatctcaaat 2880 tccttcagct atccccagca tgcctcacca
gccaacaatt ttactgaaca cagtctctgc 2940 cagtgcttct ccctccctac
atcctgggac acagaacatc ccaagccctg ctggcctgcc 3000 tcgctgtcga
tcaggaagtc acaccattgg ctccttttct tccttccaaa gtgctgcaca 3060
catctatagc cagaaactgt ctcgtccctc ttcagcaaag gcaggatcgt gctatctaaa
3120 caagcatcat tcaggaatag ccaaaacaca acaagaggga gaagatgctt
ctttatatag 3180 caaacggtac aaccaaagta tggttacagc tgaacttcag
cggctagctg agaagcaggc 3240 agcgagacag tattctccat ccagccacat
caacctcctc acccaacagg tgacaaactt 3300 gaatttggcc actggcatca
taaacagaag cagtgcttca actcccccca ccctccaacc 3360 catcatcagc
cctagtggcc ccacatggtt ggtgcagtcg gaccctcaag ctcctgagaa 3420
tcactccagc cctcccagaa gcaggagcct ccagacaggt gggtttgcct gggaaggaga
3480 ggtagaaaac aacgtgtaca gcaaggctac cggggtggtc ccccagcaca
agtatcaccc 3540 cacagcaggc agctatcagc tccattttgc cctgcagcaa
cttgaacaac aaaaacttca 3600 gtcccggcag ctcctggacc agagtcgagc
ccggcaccag gcaatctttg gcagccagac 3660 actacctaac tccaatttat
ggacaatgaa taatggtgca ggttgtagaa tttccagtgc 3720 cacagctagt
ggccagaagc caaccactct gccacaaaaa gcagtaccac ctccaagctc 3780
ttgcgcctcc ctggtcccca aaccccctcc caaccacaaa caagtgctca gaagggcaac
3840 atcccagagg gcttccaaag ggtcctcggc atatgcgcag ctgaatggac
tccagagcag 3900 ccttaaccct gcagcctctg tgcccatcac cagctccaca
gatcctgctc acactaaaag 3960 atgaaccaca aacacacaga gaaacgacct
gttcaccact cctggggtgc atctagagca 4020 t 4021 <210> SEQ ID NO
62 <211> LENGTH: 1299 <212> TYPE: PRT <213>
ORGANISM: Cercopithecus aethiops <400> SEQUENCE: 62 Met Pro
Val Val Met Ala Arg Asp Leu Glu Glu Thr Ala Ser Ser Ser 1 5 10 15
Glu Asp Glu Glu Val Ile Ser Gln Glu Asp His Pro Cys Ile Met Trp 20
25 30 Thr Gly Gly Cys Arg Arg Ile Pro Val Leu Val Phe His Ala Asp
Ala 35 40 45 Ile Leu Thr Lys Asp Asn Asn Ile Arg Val Ile Gly Glu
Arg Tyr His 50 55 60 Leu Ser Tyr Lys Ile Val Arg Thr Asp Ser Arg
Leu Val Arg Ser Ile 65 70 75 80 Leu Thr Ala His Gly Phe His Glu Val
His Pro Ser Ser Thr Asp Tyr 85 90 95 Asn Leu Met Trp Thr Gly Ser
His Leu Lys Pro Phe Leu Leu Arg Thr 100 105 110 Leu Ser Glu Ala Gln
Lys Val Asn His Phe Pro Arg Ser Tyr Glu Leu 115 120 125 Thr Arg Lys
Asp Arg Leu Tyr Lys Asn Ile Ile Arg Met Gln His Thr 130 135 140 His
Gly Phe Lys Ala Phe His Ile Leu Pro Gln Thr Phe Leu Leu Pro 145 150
155 160 Ala Glu Tyr Ala Glu Phe Cys Asn Ser Tyr Ser Lys Asp Arg Gly
Pro 165 170 175 Trp Ile Val Lys Pro Val Ala Ser Ser Arg Gly Arg Gly
Val Tyr Leu 180 185 190 Ile Asn Asn Pro Asn Gln Ile Ser Leu Glu Glu
Asn Ile Leu Val Ser 195 200 205 Arg Tyr Ile Asn Asn Pro Leu Leu Ile
Asp Asp Phe Lys Phe Asp Val 210 215 220 Arg Leu Tyr Val Leu Val Thr
Ser Tyr Asp Pro Leu Val Ile Tyr Leu 225 230 235 240 Tyr Glu Glu Gly
Leu Ala Arg Phe Ala Thr Val Arg Tyr Asp Gln Gly 245 250 255 Ala Lys
Asn Ile Arg Asn Gln Phe Met His Leu Thr Asn Tyr Ser Val 260 265 270
Asn Lys Lys Ser Gly Asp Tyr Val Ser Cys Asp Asp Pro Glu Val Glu 275
280 285 Asp Tyr Gly Asn Lys Trp Ser Met Ser Ala Met Leu Arg Tyr Leu
Lys 290 295 300 Gln Glu Gly Arg Asp Thr Thr Ala Leu Met Ala His Val
Glu Asp Leu 305 310 315 320 Ile Ile Lys Thr Ile Ile Ser Ala Glu Leu
Ala Ile Ala Thr Ala Cys 325 330 335 Lys Thr Phe Val Pro His Arg Ser
Ser Cys Phe Glu Leu Tyr Gly Phe 340 345 350 Asp Val Leu Ile Asp Ala
Thr Leu Lys Pro Trp Leu Leu Glu Val Asn 355 360 365 Leu Ser Pro Ser
Leu Ala Cys Asp Ala Pro Leu Asp Leu Lys Ile Lys 370 375 380 Ala Ser
Met Ile Ser Asp Met Phe Thr Val Val Gly Phe Val Cys Gln 385 390 395
400 Asp Pro Ala Gln Arg Ala Ser Thr Arg Pro Ile Tyr Pro Thr Phe Glu
405 410 415 Ser Ser Arg Arg Asn Pro Phe Gln Lys Pro Gln Arg Pro Leu
Pro Ala 420 425 430 Gln Phe His Ser Ser Glu Pro Lys Gln Arg Ser Arg
Pro Leu Ser Ala 435 440 445 Ser Asp Ala Glu Met Lys Asn Leu Val Gly
Ser Ala Arg Glu Lys Gly 450 455 460 Pro Gly Lys Leu Gly Gly Ser Val
Leu Gly Leu Ser Met Glu Glu Ile 465 470 475 480 Lys Val Leu Arg Arg
Val Lys Glu Glu Asn Asp Arg Arg Gly Gly Phe 485 490 495 Ile Arg Ile
Phe Pro Thr Ser Glu Thr Trp Glu Ile Tyr Gly Ser Tyr 500 505 510 Leu
Glu His Lys Thr Ser Met Asn Tyr Met Leu Ala Thr Arg Leu Phe 515 520
525 Gln Asp Arg Met Thr Ala Asp Gly Ala Pro Glu Leu Lys Ile Glu Gly
530 535 540 Leu Asn Ser Lys Ala Lys Leu His Ala Ala Leu Tyr Glu Arg
Lys Leu 545 550 555 560 Leu Ser Leu Glu Val Arg Lys Arg Arg Arg Arg
Ser Ser Arg Leu Arg 565 570 575 Ala Met Arg Pro Lys Tyr Pro Val Ile
Thr Gln Pro Ala Glu Met Asn 580 585 590 Val Lys Thr Glu Thr Glu Ser
Glu Glu Glu Glu Glu Val Ala Leu Asp 595 600 605 Asn Glu Asp Glu Glu
Gln Glu Ala Ser Gln Glu Glu Ser Ala Gly Phe 610 615 620 Leu Arg Glu
Asn Gln Ala Lys Asp Thr Pro Ser Leu Thr Thr Leu Val 625 630 635 640
Glu Asn Thr Pro Lys Glu Asn Ser Val Lys Val Arg Glu Trp Ser Lys 645
650 655 Lys Gly Glu Arg Cys Cys Lys Leu Glu Thr Gln Glu Leu Glu Pro
Lys 660 665 670 Phe Asn Leu Met Gln Val Leu Gln Asp Asn Gly Asn Leu
Ser Lys Val 675 680 685 Gln Ala Arg Ile Ala Phe Ser Thr Tyr Leu Gln
His Val Gln Ile Arg 690 695 700 Leu Met Lys Asp Ser Gly Gly Gln Thr
Phe Ser Ala Ser Trp Ala Ala 705 710 715 720 Lys Glu Asp Glu Gln Met
Glu Leu Val Val Arg Phe Leu Lys Arg Ala
725 730 735 Ser Asn Asn Leu Gln Gln Ser Leu Arg Met Val Leu Pro Ser
Arg Arg 740 745 750 Leu Ala Leu Leu Glu Arg Arg Arg Ile Leu Ala His
Gln Leu Gly Asp 755 760 765 Phe Ile Ile Val Tyr Asn Lys Glu Thr Glu
Gln Met Ala Glu Lys Lys 770 775 780 Ser Lys Lys Lys Val Glu Glu Glu
Glu Glu Asp Gly Val Asn Met Glu 785 790 795 800 Asn Phe Gln Glu Phe
Ile Arg Gln Ala Ser Glu Ala Glu Leu Glu Glu 805 810 815 Val Leu Thr
Phe Tyr Thr Gln Lys Asn Lys Ser Ala Ser Val Phe Leu 820 825 830 Gly
Thr His Ser Lys Ser Ser Lys Asn Asn Asn Ser Tyr Ser Asp Ser 835 840
845 Gly Ala Lys Gly Asp His Pro Glu Thr Val Met Glu Glu Ala Lys Met
850 855 860 Lys Pro Pro Lys Gln Gln Gln Thr Thr Glu Ile His Ser Asp
Lys Leu 865 870 875 880 Ser Arg Phe Thr Thr Ser Ala Glu Lys Glu Ala
Lys Leu Val Tyr Thr 885 890 895 Ser Ser Ser Ser Thr Pro Phe Ser Gly
Pro Thr Ala Thr Leu Gln Lys 900 905 910 Ile Pro Asn Thr His Leu Ser
Ser Val Thr Thr Ser Asp Leu Ser Pro 915 920 925 Gly Pro Gly His His
Ser Ser Leu Ser Gln Ile Pro Ser Ala Ile Pro 930 935 940 Ser Met Pro
His Gln Pro Thr Ile Leu Leu Asn Thr Val Ser Ala Ser 945 950 955 960
Ala Ser Pro Ser Leu His Pro Gly Thr Gln Asn Ile Pro Ser Pro Ala 965
970 975 Gly Leu Pro Arg Cys Arg Ser Gly Ser His Thr Ile Gly Ser Phe
Ser 980 985 990 Ser Phe Gln Ser Ala Ala His Ile Tyr Ser Gln Lys Leu
Ser Arg Pro 995 1000 1005 Ser Ser Ala Lys Ala Gly Ser Cys Tyr Leu
Asn Lys His His Ser Gly 1010 1015 1020 Ile Ala Lys Thr Gln Gln Glu
Gly Glu Asp Ala Ser Leu Tyr Ser Lys 1025 1030 1035 1040 Arg Tyr Asn
Gln Ser Met Val Thr Ala Glu Leu Gln Arg Leu Ala Glu 1045 1050 1055
Lys Gln Ala Ala Arg Gln Tyr Ser Pro Ser Ser His Ile Asn Leu Leu
1060 1065 1070 Thr Gln Gln Val Thr Asn Leu Asn Leu Ala Thr Gly Ile
Ile Asn Arg 1075 1080 1085 Ser Ser Ala Ser Thr Pro Pro Thr Leu Gln
Pro Ile Ile Ser Pro Ser 1090 1095 1100 Gly Pro Thr Trp Leu Val Gln
Ser Asp Pro Gln Ala Pro Glu Asn His 1105 1110 1115 1120 Ser Ser Pro
Pro Arg Ser Arg Ser Leu Gln Thr Gly Gly Phe Ala Trp 1125 1130 1135
Glu Gly Glu Val Glu Asn Asn Val Tyr Ser Lys Ala Thr Gly Val Val
1140 1145 1150 Pro Gln His Lys Tyr His Pro Thr Ala Gly Ser Tyr Gln
Leu His Phe 1155 1160 1165 Ala Leu Gln Gln Leu Glu Gln Gln Lys Leu
Gln Ser Arg Gln Leu Leu 1170 1175 1180 Asp Gln Ser Arg Ala Arg His
Gln Ala Ile Phe Gly Ser Gln Thr Leu 1185 1190 1195 1200 Pro Asn Ser
Asn Leu Trp Thr Met Asn Asn Gly Ala Gly Cys Arg Ile 1205 1210 1215
Ser Ser Ala Thr Ala Ser Gly Gln Lys Pro Thr Thr Leu Pro Gln Lys
1220 1225 1230 Ala Val Pro Pro Pro Ser Ser Cys Ala Ser Leu Val Pro
Lys Pro Pro 1235 1240 1245 Pro Asn His Lys Gln Val Leu Arg Arg Ala
Thr Ser Gln Arg Ala Ser 1250 1255 1260 Lys Gly Ser Ser Ala Tyr Ala
Gln Leu Asn Gly Leu Gln Ser Ser Leu 1265 1270 1275 1280 Asn Pro Ala
Ala Ser Val Pro Ile Thr Ser Ser Thr Asp Pro Ala His 1285 1290 1295
Thr Lys Arg <210> SEQ ID NO 63 <211> LENGTH: 1281
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 63 Met Pro Ile Val Met Ala Arg Asp Leu Glu
Glu Thr Ala Ser Ser Ser 1 5 10 15 Glu Asp Glu Glu Val Ile Ser Gln
Glu Asp His Pro Cys Ile Met Trp 20 25 30 Thr Gly Gly Cys Arg Arg
Ile Pro Val Leu Val Phe His Ala Asp Ala 35 40 45 Ile Leu Thr Lys
Asp Asn Asn Ile Arg Val Ile Gly Glu Arg Tyr His 50 55 60 Leu Ser
Tyr Lys Ile Val Arg Thr Asp Ser Arg Leu Val Arg Ser Ile 65 70 75 80
Leu Thr Ala His Gly Phe His Glu Val His Pro Ser Ser Thr Asp Tyr 85
90 95 Asn Leu Met Trp Thr Gly Ser His Leu Lys Pro Phe Leu Leu Arg
Thr 100 105 110 Leu Ser Glu Ala Gln Lys Val Asn His Phe Pro Arg Ser
Tyr Glu Leu 115 120 125 Thr Arg Lys Asp Arg Leu Tyr Lys Asn Ile Ile
Arg Met Gln His Thr 130 135 140 His Gly Phe Lys Ala Phe His Ile Leu
Pro Gln Thr Phe Leu Leu Pro 145 150 155 160 Ala Glu Tyr Ala Glu Phe
Cys Asn Ser Tyr Ser Lys Asp Arg Gly Pro 165 170 175 Trp Ile Val Lys
Pro Val Ala Ser Ser Arg Gly Arg Gly Val Tyr Leu 180 185 190 Ile Asn
Asn Pro Asn Gln Ile Ser Leu Glu Glu Asn Ile Leu Val Ser 195 200 205
Arg Tyr Ile Asn Asn Pro Leu Leu Ile Asp Asp Phe Lys Phe Asp Val 210
215 220 Arg Leu Tyr Val Leu Val Thr Ser Tyr Asp Pro Leu Val Ile Tyr
Leu 225 230 235 240 Tyr Glu Glu Gly Leu Ala Arg Phe Ala Thr Val Arg
Tyr Asp Gln Gly 245 250 255 Ala Lys Asn Ile Arg Asn Gln Phe Met His
Leu Thr Asn Tyr Ser Val 260 265 270 Asn Lys Lys Ser Gly Asp Tyr Val
Ser Cys Asp Asp Pro Glu Val Glu 275 280 285 Asp Tyr Gly Asn Lys Trp
Ser Met Ser Ala Met Leu Arg Tyr Leu Lys 290 295 300 Gln Glu Gly Arg
Asp Thr Thr Ala Leu Met Ala His Val Glu Asp Leu 305 310 315 320 Ile
Ile Lys Thr Ile Ile Ser Ala Glu Leu Ala Ile Ala Thr Ala Cys 325 330
335 Lys Thr Phe Val Pro His Arg Ser Ser Cys Phe Glu Leu Tyr Gly Phe
340 345 350 Asp Val Leu Ile Asp Ser Thr Leu Lys Pro Trp Leu Leu Glu
Val Asn 355 360 365 Leu Ser Pro Ser Leu Ala Cys Asp Ala Pro Leu Asp
Leu Lys Ile Lys 370 375 380 Ala Ser Met Ile Ser Asp Met Phe Thr Val
Val Gly Phe Val Cys Gln 385 390 395 400 Asp Pro Ala Gln Arg Ala Ser
Thr Arg Pro Ile Tyr Pro Thr Phe Glu 405 410 415 Ser Ser Arg Arg Asn
Pro Phe Gln Lys Pro Gln Arg Cys Arg Pro Leu 420 425 430 Ser Ala Ser
Asp Ala Glu Met Lys Asn Leu Val Gly Ser Ala Arg Glu 435 440 445 Lys
Gly Pro Gly Lys Leu Gly Gly Ser Val Leu Gly Leu Ser Met Glu 450 455
460 Glu Ile Lys Val Leu Arg Arg Val Lys Glu Glu Asn Asp Arg Arg Gly
465 470 475 480 Gly Phe Ile Arg Ile Phe Pro Thr Ser Glu Thr Trp Glu
Ile Tyr Gly 485 490 495 Ser Tyr Leu Glu His Lys Thr Ser Met Asn Tyr
Met Leu Ala Thr Arg 500 505 510 Leu Phe Gln Asp Arg Met Thr Ala Asp
Gly Ala Pro Glu Leu Lys Ile 515 520 525 Glu Ser Leu Asn Ser Lys Ala
Lys Leu His Ala Ala Leu Tyr Glu Arg 530 535 540 Lys Leu Leu Ser Leu
Glu Val Arg Lys Arg Arg Arg Arg Ser Ser Arg 545 550 555 560 Leu Arg
Ala Met Arg Pro Lys Tyr Pro Val Ile Thr Gln Pro Ala Glu 565 570 575
Met Asn Val Lys Thr Glu Thr Glu Ser Glu Glu Glu Glu Glu Val Ala 580
585 590 Leu Asp Asn Glu Asp Glu Glu Gln Glu Ala Ser Gln Glu Glu Ser
Ala 595 600 605 Gly Phe Leu Arg Glu Asn Gln Ala Lys Tyr Thr Pro Ser
Leu Thr Ala 610 615 620 Leu Val Glu Asn Thr Pro Lys Glu Asn Ser Met
Lys Val Arg Glu Trp 625 630 635 640 Asn Asn Lys Gly Gly His Cys Cys
Lys Leu Glu Thr Gln Glu Leu Glu 645 650 655 Pro Lys Phe Asn Leu Met
Gln Ile Leu Gln Asp Asn Gly Asn Leu Ser 660 665 670 Lys Met Gln Ala
Arg Ile Ala Phe Ser Ala Tyr Leu Gln His Val Gln 675 680 685 Ile Arg
Leu Met Lys Asp Ser Gly Gly Gln Thr Phe Ser Ala Ser Trp 690 695 700
Ala Ala Lys Glu Asp Glu Gln Met Glu Leu Val Val Arg Phe Leu Lys 705
710 715 720
Arg Ala Ser Asn Asn Leu Gln His Ser Leu Arg Met Val Leu Pro Ser 725
730 735 Arg Arg Leu Ala Leu Leu Glu Arg Arg Arg Ile Leu Ala His Gln
Leu 740 745 750 Gly Asp Phe Ile Ile Val Tyr Asn Lys Glu Thr Glu Gln
Met Ala Glu 755 760 765 Lys Lys Ser Lys Lys Lys Val Glu Glu Glu Glu
Glu Asp Gly Val Asn 770 775 780 Met Glu Asn Phe Gln Glu Phe Ile Arg
Gln Ala Ser Glu Ala Glu Leu 785 790 795 800 Glu Glu Val Leu Thr Phe
Tyr Thr Gln Lys Asn Lys Ser Ala Ser Val 805 810 815 Phe Leu Gly Thr
His Ser Lys Ile Ser Lys Asn Asn Asn Asn Tyr Ser 820 825 830 Asp Ser
Gly Ala Lys Gly Asp His Pro Glu Thr Ile Met Glu Glu Val 835 840 845
Lys Ile Lys Pro Pro Lys Gln Gln Gln Thr Thr Glu Ile His Ser Asp 850
855 860 Lys Leu Ser Arg Phe Thr Thr Ser Ala Glu Lys Glu Ala Lys Leu
Val 865 870 875 880 Tyr Ser Asn Ser Ser Ser Gly Pro Thr Ala Thr Leu
Gln Lys Ile Pro 885 890 895 Asn Thr His Leu Ser Ser Val Thr Thr Ser
Asp Leu Ser Pro Gly Pro 900 905 910 Cys His His Ser Ser Leu Ser Gln
Ile Pro Ser Ala Ile Pro Ser Met 915 920 925 Pro His Gln Pro Thr Ile
Leu Leu Asn Thr Val Ser Ala Ser Ala Ser 930 935 940 Pro Cys Leu His
Pro Gly Ala Gln Asn Ile Pro Ser Pro Thr Gly Leu 945 950 955 960 Pro
Arg Cys Arg Ser Gly Ser His Thr Ile Gly Pro Phe Ser Ser Phe 965 970
975 Gln Ser Ala Ala His Ile Tyr Ser Gln Lys Leu Ser Arg Pro Ser Ser
980 985 990 Ala Lys Ala Gly Ser Cys Tyr Leu Asn Lys His His Ser Gly
Ile Ala 995 1000 1005 Lys Thr Gln Lys Glu Gly Glu Asp Ala Ser Leu
Tyr Ser Lys Arg Tyr 1010 1015 1020 Asn Gln Ser Met Val Thr Ala Glu
Leu Gln Arg Leu Ala Glu Lys Gln 1025 1030 1035 1040 Ala Ala Arg Gln
Tyr Ser Pro Ser Ser His Ile Asn Leu Leu Thr Gln 1045 1050 1055 Gln
Val Thr Asn Leu Asn Leu Ala Thr Gly Ile Ile Asn Arg Ser Ser 1060
1065 1070 Ala Ser Ala Pro Pro Thr Leu Arg Pro Ile Ile Ser Pro Ser
Gly Pro 1075 1080 1085 Thr Trp Ser Thr Gln Ser Asp Pro Gln Ala Pro
Glu Asn His Ser Ser 1090 1095 1100 Ser Pro Gly Ser Arg Ser Leu Gln
Thr Gly Gly Phe Ala Trp Glu Gly 1105 1110 1115 1120 Glu Val Glu Asn
Asn Val Tyr Ser Gln Ala Thr Gly Val Val Pro Gln 1125 1130 1135 His
Lys Tyr His Pro Thr Ala Gly Ser Tyr Gln Leu Gln Phe Ala Leu 1140
1145 1150 Gln Gln Leu Glu Gln Gln Lys Leu Gln Ser Arg Gln Leu Leu
Asp Gln 1155 1160 1165 Ser Arg Ala Arg His Gln Ala Ile Phe Gly Ser
Gln Thr Leu Pro Asn 1170 1175 1180 Ser Asn Leu Trp Thr Met Asn Asn
Gly Ala Gly Cys Arg Ile Ser Ser 1185 1190 1195 1200 Ala Thr Ala Ser
Gly Gln Lys Pro Thr Thr Leu Pro Gln Lys Val Val 1205 1210 1215 Pro
Pro Pro Ser Ser Cys Ala Ser Leu Val Pro Lys Pro Pro Pro Asn 1220
1225 1230 His Glu Gln Val Leu Arg Arg Ala Thr Ser Gln Lys Ala Ser
Lys Gly 1235 1240 1245 Ser Ser Ala Glu Gly Gln Leu Asn Gly Leu Gln
Ser Ser Leu Asn Pro 1250 1255 1260 Ala Ala Phe Val Pro Ile Thr Ser
Ser Thr Asp Pro Ala His Thr Lys 1265 1270 1275 1280 Ile <210>
SEQ ID NO 64 <211> LENGTH: 1299 <212> TYPE: PRT
<213> ORGANISM: Pongo pygmaeus <400> SEQUENCE: 64 Met
Pro Ile Val Met Ala Arg Asp Leu Glu Glu Thr Ala Ser Ser Ser 1 5 10
15 Glu Asp Glu Glu Val Ile Ser Gln Glu Asp His Pro Cys Ile Met Trp
20 25 30 Thr Gly Gly Cys Arg Arg Ile Pro Val Leu Val Phe His Ala
Asp Ala 35 40 45 Ile Leu Thr Lys Asp Asn Asn Ile Arg Val Ile Gly
Glu Arg Tyr His 50 55 60 Leu Ser Tyr Lys Ile Val Arg Thr Asp Ser
Arg Leu Val Arg Ser Ile 65 70 75 80 Leu Thr Ala His Gly Phe His Glu
Val His Pro Ser Ser Thr Asp Tyr 85 90 95 Asn Leu Met Trp Thr Gly
Ser His Leu Lys Pro Phe Leu Leu Arg Thr 100 105 110 Leu Ser Glu Ala
Gln Lys Val Asn His Phe Pro Arg Ser Tyr Glu Leu 115 120 125 Thr Arg
Lys Asp Arg Leu Tyr Lys Asn Ile Ile Arg Met Gln His Thr 130 135 140
His Gly Phe Lys Ala Phe His Ile Leu Pro Gln Thr Phe Leu Leu Pro 145
150 155 160 Ala Glu Tyr Ala Glu Phe Cys Asn Ser Tyr Ser Lys Asp Arg
Gly Pro 165 170 175 Trp Ile Val Lys Pro Val Ala Ser Ser Arg Gly Arg
Gly Val Tyr Leu 180 185 190 Ile Asn Asn Pro Asn Gln Ile Ser Leu Glu
Glu Asn Ile Leu Val Ser 195 200 205 Arg Tyr Ile Asn Asn Pro Leu Leu
Ile Asp Asp Phe Lys Phe Asp Val 210 215 220 Arg Leu Tyr Val Leu Val
Thr Ser Tyr Asp Pro Leu Val Ile Tyr Leu 225 230 235 240 Tyr Glu Glu
Gly Leu Ala Arg Phe Ala Thr Val Arg Tyr Asp Gln Gly 245 250 255 Ala
Lys Asn Ile Arg Asn Gln Phe Met His Leu Thr Asn Tyr Ser Val 260 265
270 Asn Lys Lys Ser Gly Asp Tyr Val Ser Cys Asp Asp Pro Glu Val Glu
275 280 285 Asp Tyr Gly Asn Lys Trp Ser Met Ser Ala Met Leu Arg Tyr
Leu Lys 290 295 300 Gln Glu Gly Arg Asp Thr Thr Ala Leu Met Ala His
Val Glu Asp Leu 305 310 315 320 Ile Ile Lys Thr Ile Ile Ser Ala Glu
Leu Ala Ile Ala Thr Ala Cys 325 330 335 Lys Thr Phe Val Pro His Arg
Ser Ser Cys Phe Glu Leu Tyr Gly Phe 340 345 350 Asp Val Leu Ile Asp
Ser Thr Leu Lys Pro Trp Leu Leu Glu Val Asn 355 360 365 Leu Ser Pro
Ser Leu Ala Cys Asp Ala Pro Leu Asp Leu Lys Ile Lys 370 375 380 Ala
Ser Met Ile Ser Asp Met Phe Thr Val Val Gly Phe Val Cys Gln 385 390
395 400 Asp Pro Ala Gln Arg Ala Ser Thr Arg Pro Ile Tyr Pro Thr Phe
Glu 405 410 415 Ser Ser Arg Arg Asn Pro Phe Gln Lys Pro Gln Arg Cys
Arg Pro Leu 420 425 430 Ser Ala Ser Asp Ala Glu Met Lys Asn Leu Val
Gly Ser Ala Arg Glu 435 440 445 Lys Gly Pro Gly Lys Leu Gly Gly Ser
Val Leu Gly Leu Ser Met Glu 450 455 460 Glu Ile Lys Val Leu Arg Arg
Val Lys Glu Glu Asn Asp Arg Arg Gly 465 470 475 480 Gly Phe Ile Arg
Ile Phe Pro Thr Ser Glu Thr Trp Glu Ile Tyr Gly 485 490 495 Ser Tyr
Leu Glu His Lys Thr Ser Met Asn Tyr Met Leu Ala Thr Arg 500 505 510
Leu Phe Gln Asp Arg Gly Asn Pro Arg Arg Ser Leu Leu Thr Gly Arg 515
520 525 Thr Arg Met Thr Ala Asp Gly Ala Pro Glu Leu Lys Ile Glu Ser
Leu 530 535 540 Asn Ser Lys Ala Lys Leu His Ala Ala Leu Tyr Glu Arg
Lys Leu Leu 545 550 555 560 Ser Leu Glu Val Arg Lys Arg Arg Arg Arg
Ser Ser Arg Leu Arg Ala 565 570 575 Met Arg Pro Lys Tyr Pro Val Ile
Thr Gln Pro Ala Glu Met Asn Val 580 585 590 Lys Thr Glu Thr Glu Ser
Glu Glu Glu Glu Glu Val Ala Leu Asp Asn 595 600 605 Glu Glu Glu Glu
Gln Glu Ala Ser Gln Glu Glu Ser Ala Gly Phe Leu 610 615 620 Arg Glu
Asn Gln Ala Lys Tyr Thr Pro Ser Leu Thr Ala Leu Val Glu 625 630 635
640 Asn Thr Pro Lys Glu His Ser Met Lys Val Arg Glu Trp Asn Asn Lys
645 650 655 Gly Gly His Cys Cys Lys Leu Glu Thr Gln Glu Leu Glu Pro
Lys Phe 660 665 670 Asn Leu Val Gln Ile Leu Gln Asp Asn Gly Asn Leu
Ser Lys Val Gln 675 680 685 Ala Arg Ile Ala Phe Ser Ala Tyr Leu Gln
His Val Gln Ile Arg Leu 690 695 700 Met Lys Asp Ser Gly Gly Gln Thr
Phe Ser Ala Ser Trp Ala Ala Lys 705 710 715 720
Glu Asp Glu Gln Met Glu Leu Val Val Arg Phe Leu Lys Arg Ala Ser 725
730 735 Asn Asn Leu Gln His Ser Leu Arg Met Val Leu Pro Ser Arg Arg
Leu 740 745 750 Ala Leu Leu Glu Arg Arg Arg Ile Leu Ala His Gln Leu
Gly Asp Phe 755 760 765 Ile Ile Val Tyr Asn Lys Glu Thr Glu Gln Met
Ala Glu Lys Lys Ser 770 775 780 Lys Lys Lys Val Glu Glu Glu Glu Glu
Asp Gly Val Asn Met Glu Asn 785 790 795 800 Phe Gln Glu Phe Ile Arg
Gln Ala Ser Glu Ala Glu Leu Glu Glu Val 805 810 815 Leu Thr Phe Tyr
Thr Gln Lys Asn Lys Ser Ala Ser Val Phe Leu Gly 820 825 830 Thr His
Ser Lys Ser Ser Lys Asn Asn Asn Ser Tyr Ser Asp Ser Gly 835 840 845
Ala Lys Gly Asp His Pro Glu Thr Ile Met Glu Glu Val Lys Ile Lys 850
855 860 Pro Pro Lys Gln Gln Gln Thr Thr Glu Ile His Ser Asp Lys Leu
Ser 865 870 875 880 Arg Phe Thr Thr Ser Ala Glu Lys Glu Ala Lys Leu
Val Tyr Ser Asn 885 890 895 Ser Ser Ser Thr Pro Phe Ser Gly Pro Thr
Ala Thr Leu Gln Lys Ile 900 905 910 Pro Asn Thr His Leu Ser Ser Val
Thr Thr Ser Asp Leu Ser Pro Gly 915 920 925 Pro Gly His His Ser Ser
Leu Ser Gln Ile Pro Ser Ala Ile Pro Ser 930 935 940 Met Pro His Gln
Pro Thr Val Leu Leu Asn Thr Val Ser Ala Ser Ala 945 950 955 960 Ser
Pro Cys Leu His Thr Gly Thr Gln Asn Ile Pro Asn Pro Ala Gly 965 970
975 Leu Pro Arg Cys Arg Ser Gly Ser His Thr Ile Gly Pro Phe Ser Ser
980 985 990 Phe Gln Ser Ala Ala His Ile Tyr Ser Gln Lys Leu Ser Arg
Pro Ser 995 1000 1005 Ser Ala Lys Ala Ala Gly Ser Cys Tyr Leu Asn
Lys His His Ser Gly 1010 1015 1020 Ile Ala Lys Thr Gln Lys Glu Gly
Glu Asp Ala Ser Ser Tyr Ser Lys 1025 1030 1035 1040 Arg Tyr Asn Gln
Ser Met Val Thr Ala Glu Leu Gln Arg Leu Ala Glu 1045 1050 1055 Lys
Gln Ala Ala Arg Gln Tyr Ser Pro Ser Ser His Ile Asn Leu Leu 1060
1065 1070 Thr Gln Gln Val Thr Asn Leu Asn Leu Ala Thr Gly Ile Ile
Asn Arg 1075 1080 1085 Ser Ser Ala Ser Thr Pro Pro Thr Leu Arg Pro
Ile Ile Ser Pro Ser 1090 1095 1100 Gly Pro Thr Trp Ser Thr Gln Ser
Asp Pro Gln Ala Pro Glu Asn His 1105 1110 1115 1120 Ser Ser Pro Pro
Gly Ser Arg Ser Leu Gln Thr Gly Val Phe Ala Trp 1125 1130 1135 Glu
Gly Glu Val Glu Asn Asn Val Tyr Ser Lys Ala Thr Gly Val Val 1140
1145 1150 Pro Gln His Lys Tyr His Pro Thr Ala Gly Ser Tyr Gln Leu
His Phe 1155 1160 1165 Ala Leu Gln Gln Leu Glu Gln Gln Lys Leu Gln
Ser Arg Gln Leu Leu 1170 1175 1180 Asp Gln Ser Arg Ala Arg His Gln
Ala Ile Phe Gly Ser Gln Thr Leu 1185 1190 1195 1200 Pro Asn Ser Asn
Leu Trp Thr Met Asn Asn Gly Ala Gly Cys Arg Ile 1205 1210 1215 Ser
Ser Ala Thr Ala Ser Gly Gln Lys Pro Thr Thr Leu Pro Gln Lys 1220
1225 1230 Val Val Pro Pro Pro Ser Ser Cys Ala Ser Leu Val Pro Lys
Pro Pro 1235 1240 1245 Pro Asn His Lys Gln Val Leu Arg Arg Ala Thr
Ser Gln Arg Ala Ser 1250 1255 1260 Lys Gly Ser Ser Ala Glu Gly Gln
Leu Asn Gly Leu Gln Ser Ser Leu 1265 1270 1275 1280 Asn Pro Ala Ala
Phe Val Pro Ile Thr Ser Ser Thr Asp Pro Ala His 1285 1290 1295 Thr
Lys Ile <210> SEQ ID NO 65 <400> SEQUENCE: 65 000
<210> SEQ ID NO 66 <211> LENGTH: 12 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 66
atgccaatcg tg 12 <210> SEQ ID NO 67 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 67 Met Pro Ile Val 1 <210> SEQ ID NO 68
<211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 68 atggcccggg ac 12 <210>
SEQ ID NO 69 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 69 Met Ala
Arg Asp 1 <210> SEQ ID NO 70 <211> LENGTH: 1281
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 70 Met Pro Ile Val Met Ala Arg Asp Leu Glu
Glu Thr Ala Ser Ser Ser 1 5 10 15 Glu Asp Glu Glu Val Ile Ser Gln
Glu Asp His Pro Cys Ile Met Trp 20 25 30 Thr Gly Gly Cys Arg Arg
Ile Pro Val Leu Val Phe His Ala Asp Ala 35 40 45 Ile Leu Thr Lys
Asp Asn Asn Ile Arg Val Ile Gly Glu Arg Tyr His 50 55 60 Leu Ser
Tyr Lys Ile Val Arg Thr Asp Ser Arg Leu Val Arg Ser Ile 65 70 75 80
Leu Thr Ala His Gly Phe His Glu Val His Pro Ser Ser Thr Asp Tyr 85
90 95 Asn Leu Met Trp Thr Gly Ser His Leu Lys Pro Phe Leu Leu Arg
Thr 100 105 110 Leu Ser Glu Ala Gln Lys Val Asn His Phe Pro Arg Ser
Tyr Glu Leu 115 120 125 Thr Arg Lys Asp Arg Leu Tyr Lys Asn Ile Ile
Arg Met Gln His Thr 130 135 140 His Gly Phe Lys Val Phe His Ile Leu
Pro Gln Thr Phe Leu Leu Pro 145 150 155 160 Ala Glu Tyr Ala Glu Phe
Cys Asn Ser Tyr Ser Lys Asp Arg Gly Pro 165 170 175 Trp Ile Val Lys
Pro Val Ala Ser Ser Arg Gly Arg Gly Val Tyr Leu 180 185 190 Ile Asn
Asn Pro Asn Gln Ile Ser Leu Glu Glu Asn Ile Leu Val Ser 195 200 205
Arg Tyr Ile Asn Asn Pro Leu Leu Ile Asp Asp Phe Lys Phe Asp Val 210
215 220 Arg Leu Tyr Val Leu Val Thr Ser Tyr Asp Pro Leu Val Ile Tyr
Leu 225 230 235 240 Tyr Glu Glu Gly Leu Ala Arg Phe Ala Thr Val Arg
Tyr Asp Gln Gly 245 250 255 Ala Lys Asn Ile Arg Asn Gln Phe Met His
Leu Thr Asn Tyr Ser Val 260 265 270 Asn Lys Lys Ser Gly Asp Tyr Val
Ser Cys Asp Asp Pro Glu Val Glu 275 280 285 Asp Tyr Gly Asn Lys Trp
Ser Met Ser Ala Met Leu Arg Tyr Leu Lys 290 295 300 Gln Glu Gly Arg
Asp Thr Thr Ala Leu Met Ala His Val Glu Asp Leu 305 310 315 320 Ile
Ile Lys Thr Ile Ile Ser Ala Glu Leu Ala Ile Ala Thr Ala Cys 325 330
335 Lys Thr Phe Val Pro His Arg Ser Ser Cys Phe Glu Leu Tyr Gly Phe
340 345 350 Asp Val Leu Ile Asp Ser Thr Leu Lys Pro Trp Leu Leu Glu
Val Asn 355 360 365 Leu Ser Pro Ser Leu Ala Cys Asp Ala Pro Leu Asp
Leu Lys Ile Lys 370 375 380 Ala Ser Met Ile Ser Asp Met Phe Thr Val
Val Gly Phe Val Cys Gln 385 390 395 400 Asp Pro Ala Gln Arg Ala Ser
Thr Arg Pro Ile Tyr Pro Thr Phe Glu 405 410 415 Ser Ser Arg Arg Asn
Pro Phe Gln Lys Pro Gln Arg Cys Arg Pro Leu 420 425 430 Ser Ala Ser
Asp Ala Glu Met Lys Asn Leu Val Gly Ser Ala Arg Glu 435 440 445
Lys Gly Pro Gly Lys Leu Gly Gly Ser Val Leu Gly Leu Ser Met Glu 450
455 460 Glu Ile Lys Val Leu Arg Arg Val Lys Glu Glu Asn Asp Arg Arg
Gly 465 470 475 480 Gly Phe Ile Arg Ile Phe Pro Thr Ser Glu Thr Trp
Glu Ile Tyr Gly 485 490 495 Ser Tyr Leu Glu His Lys Thr Ser Met Asn
Tyr Met Leu Ala Thr Arg 500 505 510 Leu Phe Gln Asp Arg Met Thr Ala
Asp Gly Ala Pro Glu Leu Lys Ile 515 520 525 Glu Ser Leu Asn Ser Lys
Ala Lys Leu His Ala Ala Leu Tyr Glu Arg 530 535 540 Lys Leu Leu Ser
Leu Glu Val Arg Lys Arg Arg Arg Arg Ser Ser Arg 545 550 555 560 Leu
Arg Ala Met Arg Pro Lys Tyr Pro Val Ile Thr Gln Pro Ala Glu 565 570
575 Met Asn Val Lys Thr Glu Thr Glu Ser Glu Glu Glu Glu Glu Val Ala
580 585 590 Leu Asp Asn Glu Asp Glu Glu Gln Glu Ala Ser Gln Glu Glu
Ser Ala 595 600 605 Gly Phe Leu Arg Glu Asn Gln Ala Lys Tyr Thr Pro
Ser Leu Thr Ala 610 615 620 Leu Val Glu Asn Thr Pro Lys Glu Asn Ser
Met Lys Val Arg Glu Trp 625 630 635 640 Asn Asn Lys Gly Gly His Cys
Cys Lys Leu Glu Thr Gln Glu Leu Glu 645 650 655 Pro Lys Phe Asn Leu
Met Gln Ile Leu Gln Asp Asn Gly Asn Leu Ser 660 665 670 Lys Met Gln
Ala Arg Ile Ala Phe Ser Ala Tyr Leu Gln His Val Gln 675 680 685 Ile
Arg Leu Met Lys Asp Ser Gly Gly Gln Thr Phe Ser Ala Ser Trp 690 695
700 Ala Ala Lys Glu Asp Glu Gln Met Glu Leu Val Val Arg Phe Leu Lys
705 710 715 720 Arg Ala Ser Asn Asn Leu Gln His Ser Leu Arg Met Val
Leu Pro Ser 725 730 735 Arg Arg Leu Ala Leu Leu Glu Arg Arg Arg Ile
Leu Ala His Gln Leu 740 745 750 Gly Asp Phe Ile Ile Val Tyr Asn Lys
Glu Thr Glu Gln Met Ala Glu 755 760 765 Lys Lys Ser Lys Lys Lys Val
Glu Glu Glu Glu Glu Asp Gly Val Asn 770 775 780 Met Glu Asn Phe Gln
Glu Phe Ile Arg Gln Ala Ser Glu Ala Glu Leu 785 790 795 800 Glu Glu
Val Leu Thr Phe Tyr Thr Gln Lys Asn Lys Ser Ala Ser Val 805 810 815
Phe Leu Gly Thr His Ser Lys Ile Ser Lys Asn Asn Asn Asn Tyr Ser 820
825 830 Asp Ser Gly Ala Lys Gly Asp His Pro Glu Thr Ile Met Glu Glu
Val 835 840 845 Lys Ile Lys Pro Pro Lys Gln Gln Gln Thr Thr Glu Ile
His Ser Asp 850 855 860 Lys Leu Ser Arg Phe Thr Thr Ser Ala Glu Lys
Glu Ala Lys Leu Val 865 870 875 880 Tyr Ser Asn Ser Ser Ser Gly Pro
Thr Ala Thr Leu Gln Lys Ile Pro 885 890 895 Asn Thr His Leu Ser Ser
Val Thr Thr Ser Asp Leu Ser Pro Gly Pro 900 905 910 Cys His His Ser
Ser Leu Ser Gln Ile Pro Ser Ala Ile Pro Ser Met 915 920 925 Pro His
Gln Pro Thr Ile Leu Leu Asn Thr Val Ser Ala Ser Ala Ser 930 935 940
Pro Cys Leu His Pro Gly Ala Gln Asn Ile Pro Ser Pro Thr Gly Leu 945
950 955 960 Pro Arg Cys Arg Ser Gly Ser His Thr Ile Gly Pro Phe Ser
Ser Phe 965 970 975 Gln Ser Ala Ala His Ile Tyr Ser Gln Lys Leu Ser
Arg Pro Ser Ser 980 985 990 Ala Lys Ala Gly Ser Cys Tyr Leu Asn Lys
His His Ser Gly Ile Ala 995 1000 1005 Lys Thr Gln Lys Glu Gly Glu
Asp Ala Ser Leu Tyr Ser Lys Arg Tyr 1010 1015 1020 Asn Gln Ser Met
Val Thr Ala Glu Leu Gln Arg Leu Ala Glu Lys Gln 1025 1030 1035 1040
Ala Ala Arg Gln Tyr Ser Pro Ser Ser His Ile Asn Leu Leu Thr Gln
1045 1050 1055 Gln Val Thr Asn Leu Asn Leu Ala Thr Gly Ile Ile Asn
Arg Ser Ser 1060 1065 1070 Ala Ser Ala Pro Pro Thr Leu Arg Pro Ile
Ile Ser Pro Ser Gly Pro 1075 1080 1085 Thr Trp Ser Thr Gln Ser Asp
Pro Gln Ala Pro Glu Asn His Ser Ser 1090 1095 1100 Ser Pro Gly Ser
Arg Ser Leu Gln Thr Gly Gly Phe Ala Trp Glu Gly 1105 1110 1115 1120
Glu Val Glu Asn Asn Val Tyr Ser Gln Ala Thr Gly Val Val Pro Gln
1125 1130 1135 His Lys Tyr His Pro Thr Ala Gly Ser Tyr Gln Leu Gln
Phe Ala Leu 1140 1145 1150 Gln Gln Leu Glu Gln Gln Lys Leu Gln Ser
Arg Gln Leu Leu Asp Gln 1155 1160 1165 Ser Arg Ala Arg His Gln Ala
Ile Phe Gly Ser Gln Thr Leu Pro Asn 1170 1175 1180 Ser Asn Leu Trp
Thr Met Asn Asn Gly Ala Gly Cys Arg Ile Ser Ser 1185 1190 1195 1200
Ala Thr Ala Ser Gly Gln Lys Pro Thr Thr Leu Pro Gln Lys Val Val
1205 1210 1215 Pro Pro Pro Ser Ser Cys Ala Ser Leu Val Pro Lys Pro
Pro Pro Asn 1220 1225 1230 His Glu Gln Val Leu Arg Arg Ala Thr Ser
Gln Lys Ala Ser Lys Gly 1235 1240 1245 Ser Ser Ala Glu Gly Gln Leu
Asn Gly Leu Gln Ser Ser Leu Asn Pro 1250 1255 1260 Ala Ala Ser Val
Pro Ile Thr Ser Ser Thr Asp Pro Ala His Thr Lys 1265 1270 1275 1280
Ile <210> SEQ ID NO 71 <211> LENGTH: 3996 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
71 tgaatctgct aggaaaggtc tctgaggccc ccgtctgctg actgcatgac
aaaccctaaa 60 ggaaatgcca atcgtgatgg cccgggacct ggaggaaaca
gcatcatcct cagaggatga 120 ggaggtcata agtcaagagg atcatccatg
catcatgtgg actggaggct gcaggagaat 180 tccagttttg gtattccatg
ccgacgctat tcttacaaag gacaacaata ttagagtaat 240 tggagaacgt
tatcatttgt cttataagat tgtacgaacg gacagtcgcc tagtacgcag 300
cattctgaca gcccatggat ttcatgaagt tcacccaagc agcactgact ataacctaat
360 gtggacagga tcccacctga agcccttctt actgcgcacc ctctctgaag
cacaaaaagt 420 taatcacttt cccaggtctt atgaacttac ccggaaggac
cgactgtaca aaaacattat 480 tcgaatgcag catacacatg gattcaaggt
ttttcacatc ctcccccaga ccttcctcct 540 gccagctgag tacgcggaat
tttgtaattc atattcgaag gaccggggac cttggatagt 600 aaaaccagtg
gcatcttcaa gggggcgggg cgtctacctg atcaacaatc caaaccagat 660
ctccctggaa gagaacattt tggtctcccg ttacattaac aaccccctgc tcatagatga
720 tttcaagttt gacgtgcgcc tctatgtgct cgtgacttcc tatgatcctc
ttgtcatcta 780 tctctatgaa gaaggattgg ctaggtttgc aactgtgcga
tatgatcaag gagccaagaa 840 cattcggaac cagttcatgc atctgacaaa
ctacagtgtc aacaagaaaa gtggagatta 900 cgtcagttgt gacgatccag
aagtggagga ttatggaaac aaatggagca tgagtgctat 960 gcttaggtac
ctgaaacaag aaggcagaga tacaaccgca ttgatggccc atgtagaaga 1020
cctgatcatt aagactataa tctctgctga actagctatt gctactgcct gtaaaacctt
1080 tgttcctcat cgcagcagtt gttttgaact ctatggcttt gacgtgctca
tagattctac 1140 tctgaagcca tggttgttgg aagtgaatct ctctccttct
ttggcctgtg atgcgcctct 1200 ggacctaaag attaaagcca gtatgatttc
agatatgttc actgttgtag gatttgtgtg 1260 ccaagatcct gcccagcggg
catcaactcg gccaatttat cccacctttg agtcttccag 1320 gcgaaaccct
ttccagaaac ctcagcgttg ccgtccactc tctgccagtg atgcggaaat 1380
gaaaaacctc gtgggctcag cccgggagaa agggccaggg aagttgggtg gttctgtgct
1440 tggtctgtca atggaggaga tcaaagtttt acgaagggtg aaggaggaga
atgatcggcg 1500 aggtggattt attcgcatat ttcctacatc tgagacatgg
gaaatatatg ggtcctacct 1560 cgagcataag acctcaatga actatatgct
ggcaacacgc ctcttccagg acagaatgac 1620 tgctgatgga gcgccagaat
tgaagataga gagtctgaat tcaaaggcca agctgcatgc 1680 tgcactttac
gagaggaagc tcctgtctct ggaggtgcga aaacgtagac gacggagtag 1740
cagattgagg gcaatgaggc caaaataccc agtgattacc caaccagctg aaatgaatgt
1800 taaaactgag acagagagtg aagaggagga agaagtcgca ttagataatg
aagatgaaga 1860 acaggaggct tcccaggagg agtctgcagg atttcttaga
gaaaatcaag ccaaatatac 1920 accctcattg acagctttgg tagaaaatac
acccaaagaa aattccatga aagttcgtga 1980 atggaataat aaaggtggac
actgctgcaa acttgagact caggagctag agcctaaatt 2040 taacctgatg
cagattcttc aagataatgg caatcttagc aaaatgcagg cccgaatagc 2100
attctctgcc tatctccagc atgttcaaat tcgcctgatg aaagacagtg gcggtcagac
2160 gttcagtgcc agttgggctg ccaaagagga tgaacagatg gagctggttg
ttcgtttcct 2220 caagcgagca tcaaataacc tccagcattc actgaggatg
gtattaccca gtcgacgatt 2280 ggcacttctg gaacgcagaa gaatcctggc
ccaccagctg ggtgacttta tcattgtata 2340 caacaaggaa acagaacaaa
tggctgaaaa gaaatcaaag aagaaagttg aggaagaaga 2400 ggaagatggg
gtgaatatgg aaaactttca ggagttcatc agacaagcaa gtgaggctga 2460
actggaggag gtgttgactt tttataccca aaagaacaag tctgctagtg tcttcctggg
2520 gactcactct aaaatttcta agaacaacaa caattattct gatagtgggg
caaaaggtga 2580
tcaccctgag actataatgg aagaagtgaa aataaagcca cctaaacagc aacagacgac
2640 agaaattcat tctgataaat tatctcgatt taccacttca gcagaaaaag
aggcaaaatt 2700 agtttatagc aattcctcct ctggtcctac tgctactctg
cagaaaattc ccaacaccca 2760 tttgtcatct gttacaacct ctgacctctc
tccagggcct tgccaccatt cttctttatc 2820 tcaaattcct tcagctatcc
ccagcatgcc tcaccagcca acaattttac tgaacacagt 2880 ctctgccagt
gcttctccct gcctacatcc cggggcacag aacatcccaa gccctactgg 2940
cctgccacgc tgtcgatcag gaagtcacac cattggtccc ttttcttcct tccaaagtgc
3000 tgcacacatc tatagccaga aactgtctcg tccctcttca gcaaaggcag
gatcgtgcta 3060 tctaaacaag catcattcag gaatagccaa aacacaaaaa
gagggagaag atgcttcttt 3120 atatagcaaa cggtacaacc aaagtatggt
tacagctgaa cttcagcggc tagctgagaa 3180 gcaggcagcg agacagtatt
ctccatccag ccacatcaac ctcctcaccc aacaggtaac 3240 aaacctgaat
ttggcaactg gcatcataaa cagaagcagt gcttcagctc ccccaaccct 3300
ccgacccatc atcagtccta gtggcccgac atggtctaca cagtcagacc cccaagctcc
3360 cgagaatcac tccagctctc ctggaagcag gagcctgcag acagggggat
ttgcctggga 3420 aggagaagta gaaaacaacg tgtacagcca ggctacaggg
gtggtccccc agcacaagta 3480 tcaccccaca gcaggcagct atcagcttca
atttgccctg cagcaacttg aacaacaaaa 3540 acttcagtcc cggcagctcc
tggaccagag tcgagcccgg caccaggcaa tctttggcag 3600 ccagacacta
cctaactcca atttatggac aatgaataat ggtgcaggtt gtagaatttc 3660
cagtgccaca gctagtggcc agaagccaac cactctgcca caaaaagtgg taccacctcc
3720 aagttcttgc gcctccctgg ttcccaaacc cccacccaac cacgaacaag
tgctcagaag 3780 ggcaacatcc cagaaagctt ccaaagggtc ctccgcggaa
gggcagctga atggactcca 3840 gagcagcctt aaccctgcag cctctgtgcc
catcaccagc tctacagatc ctgctcacac 3900 taaaatatga accacaaaca
cacagagaaa caacctgttc accactcctg ggtgcatgat 3960 tgagggtgaa
gcatccacca gcacttcaag gggtcc 3996 <210> SEQ ID NO 72
<211> LENGTH: 1281 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 72 Met Pro Ile Val Met
Ala Arg Asp Leu Glu Glu Thr Ala Ser Ser Ser 1 5 10 15 Glu Asp Glu
Glu Val Ile Ser Gln Glu Asp His Pro Cys Ile Met Trp 20 25 30 Thr
Gly Gly Cys Arg Arg Ile Pro Val Leu Val Phe His Ala Asp Ala 35 40
45 Ile Leu Thr Lys Asp Asn Asn Ile Arg Val Ile Gly Glu Arg Tyr His
50 55 60 Leu Ser Tyr Lys Ile Val Arg Thr Asp Ser Arg Leu Val Arg
Ser Ile 65 70 75 80 Leu Thr Ala His Gly Phe His Glu Val His Pro Ser
Ser Thr Asp Tyr 85 90 95 Asn Leu Met Trp Thr Gly Ser His Leu Lys
Pro Phe Leu Leu Arg Thr 100 105 110 Leu Ser Glu Ala Gln Lys Val Asn
His Phe Pro Arg Ser Tyr Glu Leu 115 120 125 Thr Arg Lys Asp Arg Leu
Tyr Lys Asn Ile Ile Arg Met Gln His Thr 130 135 140 His Gly Phe Lys
Val Phe His Ile Leu Pro Gln Thr Phe Leu Leu Pro 145 150 155 160 Ala
Glu Tyr Ala Glu Phe Cys Asn Ser Tyr Ser Lys Asp Arg Gly Pro 165 170
175 Trp Ile Val Lys Pro Val Ala Ser Ser Arg Gly Arg Gly Val Tyr Leu
180 185 190 Ile Asn Asn Pro Asn Gln Ile Ser Leu Glu Glu Asn Ile Leu
Val Ser 195 200 205 Arg Tyr Ile Asn Asn Pro Leu Leu Ile Asp Asp Phe
Lys Phe Asp Val 210 215 220 Arg Leu Tyr Val Leu Val Thr Ser Tyr Asp
Pro Leu Val Ile Tyr Leu 225 230 235 240 Tyr Glu Glu Gly Leu Ala Arg
Phe Ala Thr Val Arg Tyr Asp Gln Gly 245 250 255 Ala Lys Asn Ile Arg
Asn Gln Phe Met His Leu Thr Asn Tyr Ser Val 260 265 270 Asn Lys Lys
Ser Gly Asp Tyr Val Ser Cys Asp Asp Pro Glu Val Glu 275 280 285 Asp
Tyr Gly Asn Lys Trp Ser Met Ser Ala Met Leu Arg Tyr Leu Lys 290 295
300 Gln Glu Gly Arg Asp Thr Thr Ala Leu Met Ala His Val Glu Asp Leu
305 310 315 320 Ile Ile Lys Thr Ile Ile Ser Ala Glu Leu Ala Ile Ala
Thr Ala Cys 325 330 335 Lys Thr Phe Val Pro His Arg Ser Ser Cys Phe
Glu Leu Tyr Gly Phe 340 345 350 Asp Val Leu Ile Asp Ser Thr Leu Lys
Pro Trp Leu Leu Glu Val Asn 355 360 365 Leu Ser Pro Ser Leu Ala Cys
Asp Ala Pro Leu Asp Leu Lys Ile Lys 370 375 380 Ala Ser Met Ile Ser
Asp Met Phe Thr Val Val Gly Phe Val Cys Gln 385 390 395 400 Asp Pro
Ala Gln Arg Ala Ser Thr Arg Pro Ile Tyr Pro Thr Phe Glu 405 410 415
Ser Ser Arg Arg Asn Pro Phe Gln Lys Pro Gln Arg Cys Arg Pro Leu 420
425 430 Ser Ala Ser Asp Ala Glu Met Lys Asn Leu Val Gly Ser Ala Arg
Glu 435 440 445 Lys Gly Pro Gly Lys Leu Gly Gly Ser Val Leu Gly Leu
Ser Met Glu 450 455 460 Glu Ile Lys Val Leu Arg Arg Val Lys Glu Glu
Asn Asp Arg Arg Gly 465 470 475 480 Gly Phe Ile Arg Ile Phe Pro Thr
Ser Glu Thr Trp Glu Ile Tyr Gly 485 490 495 Ser Tyr Leu Glu His Lys
Thr Ser Met Asn Tyr Met Leu Ala Thr Arg 500 505 510 Leu Phe Gln Asp
Arg Met Thr Ala Asp Gly Ala Pro Glu Leu Lys Ile 515 520 525 Glu Ser
Leu Asn Ser Lys Ala Lys Leu His Ala Ala Leu Tyr Glu Arg 530 535 540
Lys Leu Leu Ser Leu Glu Val Arg Lys Arg Arg Arg Arg Ser Ser Arg 545
550 555 560 Leu Arg Ala Met Arg Pro Lys Tyr Pro Val Ile Thr Gln Pro
Ala Glu 565 570 575 Met Asn Val Lys Thr Glu Thr Glu Ser Glu Glu Glu
Glu Glu Val Ala 580 585 590 Leu Asp Asn Glu Asp Glu Glu Gln Glu Ala
Ser Gln Glu Glu Ser Ala 595 600 605 Gly Phe Leu Arg Glu Asn Gln Ala
Lys Tyr Thr Pro Ser Leu Thr Ala 610 615 620 Leu Val Glu Asn Thr Pro
Lys Glu Asn Ser Met Lys Val Arg Glu Trp 625 630 635 640 Asn Asn Lys
Gly Gly His Cys Cys Lys Leu Glu Thr Gln Glu Leu Glu 645 650 655 Pro
Lys Phe Asn Leu Met Gln Ile Leu Gln Asp Asn Gly Asn Leu Ser 660 665
670 Lys Met Gln Ala Arg Ile Ala Phe Ser Ala Tyr Leu Gln His Val Gln
675 680 685 Ile Arg Leu Met Lys Asp Ser Gly Gly Gln Thr Phe Ser Ala
Ser Trp 690 695 700 Ala Ala Lys Glu Asp Glu Gln Met Glu Leu Val Val
Arg Phe Leu Lys 705 710 715 720 Arg Ala Ser Asn Asn Leu Gln His Ser
Leu Arg Met Val Leu Pro Ser 725 730 735 Arg Arg Leu Ala Leu Leu Glu
Arg Arg Arg Ile Leu Ala His Gln Leu 740 745 750 Gly Asp Phe Ile Ile
Val Tyr Asn Lys Glu Thr Glu Gln Met Ala Glu 755 760 765 Lys Lys Ser
Lys Lys Lys Val Glu Glu Glu Glu Glu Asp Gly Val Asn 770 775 780 Met
Glu Asn Phe Gln Glu Phe Ile Arg Gln Ala Ser Glu Ala Glu Leu 785 790
795 800 Glu Glu Val Leu Thr Phe Tyr Thr Gln Lys Asn Lys Ser Ala Ser
Val 805 810 815 Phe Leu Gly Thr His Ser Lys Ile Ser Lys Asn Asn Asn
Asn Tyr Ser 820 825 830 Asp Ser Gly Ala Lys Gly Asp His Pro Glu Thr
Ile Met Glu Glu Val 835 840 845 Lys Ile Lys Pro Pro Lys Gln Gln Gln
Thr Thr Glu Ile His Ser Asp 850 855 860 Lys Leu Ser Arg Phe Thr Thr
Ser Ala Glu Lys Glu Ala Lys Leu Val 865 870 875 880 Tyr Ser Asn Ser
Ser Ser Gly Pro Thr Ala Thr Leu Gln Lys Ile Pro 885 890 895 Asn Thr
His Leu Ser Ser Val Thr Thr Ser Asp Leu Ser Pro Gly Pro 900 905 910
Cys His His Ser Ser Leu Ser Gln Ile Pro Ser Ala Ile Pro Ser Met 915
920 925 Pro His Gln Pro Thr Ile Leu Leu Asn Thr Val Ser Ala Ser Ala
Ser 930 935 940 Pro Cys Leu His Pro Gly Ala Gln Asn Ile Pro Ser Pro
Thr Gly Leu 945 950 955 960 Pro Arg Cys Arg Ser Gly Ser His Thr Ile
Gly Pro Phe Ser Ser Phe 965 970 975 Gln Ser Ala Ala His Ile Tyr Ser
Gln Lys Leu Ser Arg Pro Ser Ser 980 985 990 Ala Lys Ala Gly Ser Cys
Tyr Leu Asn Lys His His Ser Gly Ile Ala 995 1000 1005 Lys Thr Gln
Lys Glu Gly Glu Asp Ala Ser Leu Tyr Ser Lys Arg Tyr 1010 1015 1020
Asn Gln Ser Met Val Thr Ala Glu Leu Gln Arg Leu Ala Glu Lys Gln
1025 1030 1035 1040
Ala Ala Arg Gln Tyr Ser Pro Ser Ser His Ile Asn Leu Leu Thr Gln
1045 1050 1055 Gln Val Thr Asn Leu Asn Leu Ala Thr Gly Ile Ile Asn
Arg Ser Ser 1060 1065 1070 Ala Ser Ala Pro Pro Thr Leu Arg Pro Ile
Ile Ser Pro Ser Gly Pro 1075 1080 1085 Thr Trp Ser Thr Gln Ser Asp
Pro Gln Ala Pro Glu Asn His Ser Ser 1090 1095 1100 Ser Pro Gly Ser
Arg Ser Leu Gln Thr Gly Gly Phe Ala Trp Glu Gly 1105 1110 1115 1120
Glu Val Glu Asn Asn Val Tyr Ser Gln Ala Thr Gly Val Val Pro Gln
1125 1130 1135 His Lys Tyr His Pro Thr Ala Gly Ser Tyr Gln Leu Gln
Phe Ala Leu 1140 1145 1150 Gln Gln Leu Glu Gln Gln Lys Leu Gln Ser
Arg Gln Leu Leu Asp Gln 1155 1160 1165 Ser Arg Ala Arg His Gln Ala
Ile Phe Gly Ser Gln Thr Leu Pro Asn 1170 1175 1180 Ser Asn Leu Trp
Thr Met Asn Asn Gly Ala Gly Cys Arg Ile Ser Ser 1185 1190 1195 1200
Ala Thr Ala Ser Gly Gln Lys Pro Thr Thr Leu Pro Gln Lys Val Val
1205 1210 1215 Pro Pro Pro Ser Ser Cys Ala Ser Leu Val Pro Lys Pro
Pro Pro Asn 1220 1225 1230 His Glu Gln Val Leu Arg Arg Ala Thr Ser
Gln Lys Ala Ser Lys Gly 1235 1240 1245 Ser Ser Ala Glu Gly Gln Leu
Asn Gly Leu Gln Ser Ser Leu Asn Pro 1250 1255 1260 Ala Ala Ser Val
Pro Ile Thr Ser Ser Thr Asp Pro Ala His Thr Lys 1265 1270 1275 1280
Ile <210> SEQ ID NO 73 <400> SEQUENCE: 73 000
<210> SEQ ID NO 74 <211> LENGTH: 4691 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 74
gcacgagggg gaagcagccg tcggcggctg ccctgagcct tcctggggaa ggaggaggga
60 ggtaggcgca gagcgcggtc cacgcctgct cgccccgaac catgggaaga
tgagacagga 120 atctgtgcca tccaaattgc ttgatccagt gaatctgcta
ggaaaggtct ctgaggcccc 180 cgtctgctga ctgcatgaca aaccctaaag
gaaatgccaa tcgtgatggc ccgggacctg 240 gaggaaacag catcatcctc
agaggatgag gaggtcataa gtcaagagga tcatccatgc 300 atcatgtgga
ctggaggctg caggagaatt ccagttttgg tattccatgc cgacgctatt 360
cttacaaagg acaacaatat tagagtaatt ggagaacgtt atcatttgtc ttataagatt
420 gtacgaacgg acagtcgcct agtacgcagc attctgacag cccatggatt
tcatgaagtt 480 cacccaagca gcactgacta taacctaatg tggacaggat
cccacctgaa gcccttctta 540 ctgcgcaccc tctctgaagc acaaaaagtt
aatcactttc ccaggtctta tgaacttacc 600 cggaaggacc gactgtacaa
aaacattatt cgaatgcagc atacacatgg attcaaggtt 660 tttcacatcc
tcccccagac cttcctcctg ccagctgagt acgcggaatt ttgtaattca 720
tattcgaagg accggggacc ttggatagta aaaccagtgg catcttcaag ggggcggggc
780 gtctacctga tcaacaatcc aaaccagatc tccctggaag agaacatttt
ggtctcccgt 840 tacattaaca accccctgct catagatgat ttcaagtttg
acgtgcgcct ctatgtgctc 900 gtgacttcct atgatcctct tgtcatctat
ctctatgaag aaggattggc taggtttgca 960 actgtgcgat atgatcaagg
agccaagaac attcggaacc agttcatgca tctgacaaac 1020 tacagtgtca
acaagaaaag tggagattac gtcagttgtg acgatccaga agtggaggat 1080
tatggaaaca aatggagcat gagtgctatg cttaggtacc tgaaacaaga aggcagagat
1140 acaaccgcat tgatggccca tgtagaagac ctgatcatta agactataat
ctctgctgaa 1200 ctagctattg ctactgcctg taaaaccttt gttcctcatc
gcagcagttg ttttgaactc 1260 tatggctttg acgtgctcat agattctact
ctgaagccat ggttgttgga agtgaatctc 1320 tctccttctt tggcctgtga
tgcgcctctg gacctaaaga ttaaagccag tatgatttca 1380 gatatgttca
ctgttgtagg atttgtgtgc caagatcctg cccagcgggc atcaactcgg 1440
ccaatttatc ccacctttga gtcttccagg cgaaaccctt tccagaaacc tcagcgttgc
1500 cgtccactct ctgccagtga tgcggaaatg aaaaacctcg tgggctcagc
ccgggagaaa 1560 gggccaggga agttgggtgg ttctgtgctt ggtctgtcaa
tggaggagat caaagtttta 1620 cgaagggtga aggaggagaa tgatcggcga
ggtggattta ttcgcatatt tcctacatct 1680 gagacatggg aaatatatgg
gtcctacctc gagcataaga cctcaatgaa ctatatgctg 1740 gcaacacgcc
tcttccagga cagaatgact gctgatggag cgccagaatt gaagatagag 1800
agtctgaatt caaaggccaa gctgcatgct gcactttacg agaggaagct cctgtctctg
1860 gaggtgcgaa aacgtagacg acggagtagc agattgaggg caatgaggcc
aaaataccca 1920 gtgattaccc aaccagctga aatgaatgtt aaaactgaga
cagagagtga agaggaggaa 1980 gaagtcgcat tagataatga agatgaagaa
caggaggctt cccaggagga gtctgcagga 2040 tttcttagag aaaatcaagc
caaatataca ccctcattga cagctttggt agaaaataca 2100 cccaaagaaa
attccatgaa agttcgtgaa tggaataata aaggtggaca ctgctgcaaa 2160
cttgagactc aggagctaga gcctaaattt aacctgatgc agattcttca agataatggc
2220 aatcttagca aaatgcaggc ccgaatagca ttctctgcct atctccagca
tgttcaaatt 2280 cgcctgatga aagacagtgg cggtcagacg ttcagtgcca
gttgggctgc caaagaggat 2340 gaacagatgg agctggttgt tcgtttcctc
aagcgagcat caaataacct ccagcattca 2400 ctgaggatgg tattacccag
tcgacgattg gcacttctgg aacgcagaag aatcctggcc 2460 caccagctgg
gtgactttat cattgtatac aacaaggaaa cagaacaaat ggctgaaaag 2520
aaatcaaaga agaaagttga ggaagaagag gaagatgggg tgaatatgga aaactttcag
2580 gagttcatca gacaagcaag tgaggctgaa ctggaggagg tgttgacttt
ttatacccaa 2640 aagaacaagt ctgctagtgt cttcctgggg actcactcta
aaatttctaa gaacaacaac 2700 aattattctg atagtggggc aaaaggtgat
caccctgaga ctataatgga agaagtgaaa 2760 ataaagccac ctaaacagca
acagacgaca gaaattcatt ctgataaatt atctcgattt 2820 accacttcag
cagaaaaaga ggcaaaatta gtttatagca attcctcctc tggtcctact 2880
gctactctgc agaaaattcc caacacccat ttgtcatctg ttacaacctc tgacctctct
2940 ccagggcctt gccaccattc ttctttatct caaattcctt cagctatccc
cagcatgcct 3000 caccagccaa caattttact gaacacagtc tctgccagtg
cttctccctg cctacatccc 3060 ggggcacaga acatcccaag ccctactggc
ctgccacgct gtcgatcagg aagtcacacc 3120 attggtccct tttcttcctt
ccaaagtgct gcacacatct atagccagaa actgtctcgt 3180 ccctcttcag
caaaggcagg atcgtgctat ctaaacaagc atcattcagg aatagccaaa 3240
acacaaaaag agggagaaga tgcttcttta tatagcaaac ggtacaacca aagtatggtt
3300 acagctgaac ttcagcggct agctgagaag caggcagcga gacagtattc
tccatccagc 3360 cacatcaacc tcctcaccca acaggtaaca aacctgaatt
tggcaactgg catcataaac 3420 agaagcagtg cttcagctcc cccaaccctc
cgacccatca tcagtcctag tggcccgaca 3480 tggtctacac agtcagaccc
ccaagctccc gagaatcact ccagctctcc tggaagcagg 3540 agcctgcaga
cagggggatt tgcctgggaa ggagaagtag aaaacaacgt gtacagccag 3600
gctacagggg tggtccccca gcacaagtat caccccacag caggcagcta tcagcttcaa
3660 tttgccctgc agcaacttga acaacaaaaa cttcagtccc ggcagctcct
ggaccagagt 3720 cgagcccggc accaggcaat ctttggcagc cagacactac
ctaactccaa tttatggaca 3780 atgaataatg gtgcaggttg tagaatttcc
agtgccacag ctagtggcca gaagccaacc 3840 actctgccac aaaaagtggt
accacctcca agttcttgcg cctccctggt tcccaaaccc 3900 ccacccaacc
acgaacaagt gctcagaagg gcaacatccc agaaagcttc caaagggtcc 3960
tccgcggaag ggcagctgaa tggactccag agcagcctta accctgcagc ctctgtgccc
4020 atcaccagct ctacagatcc tgctcacact aaaatatgaa ccacaaacac
acagagaaac 4080 aacctgttca ccactcctgg gtgcatgatt gagggtgaag
catccaccag cacttcaagg 4140 ggtccatagt attttttttt ttgctgcctc
aaagtcccca aagccttcga gcagaagtgg 4200 cagtagatgg ttgccaatca
gccaatgcag actttcactg ggacaacaag aaagcagatc 4260 ttctgggttt
tgatggaact tggcagtggg gacattcagc tgatgcatta tataccccgt 4320
cagagcacac ttgtatcttt taccttccct ttgccccatg cccccaaact gcttaggtct
4380 tctctgtccc tttactgctg ctgcacagag atgatataaa agaggctctt
tggctatttg 4440 cattttgctt cctcttcttt tccagattac agtatgaagc
tttattttct ttgtacaagc 4500 ttaaaatttc aacatcatca tccgccaaag
ttgttcctcc cttttcggag gatctagggg 4560 gaaagaggag cattcatcac
aagtttccta gagagaggag acaaatcggt gtgccattga 4620 caacatgagc
cagggtaaag gcaccctttg gaattactga tttcaaagat taataaagta 4680
attctatttt t 4691
* * * * *