U.S. patent application number 12/707700 was filed with the patent office on 2010-08-26 for cancer markers.
Invention is credited to William Thomas Melvin, Graeme Ian Murray, Colin Matheson Telfer.
Application Number | 20100216156 12/707700 |
Document ID | / |
Family ID | 32966938 |
Filed Date | 2010-08-26 |
United States Patent
Application |
20100216156 |
Kind Code |
A1 |
Murray; Graeme Ian ; et
al. |
August 26, 2010 |
CANCER MARKERS
Abstract
Provided are previously uncharacterized markers of cancers, for
example colorectal cancers, and uses of these as diagnostic and
prognostic markers of cancers, and in particular colorectal
cancers. The markers are SEQ ID NO:1--hnRNP-K; SEQ ID NO:2--HMG-1;
SEQ ID NO:3--proteasome subunit alpha type 1; SEQ ID
NO:4--bifunctional purine biosynthesis protein; SEQ ID NO:5--STI1;
SEQ ID NO:6--annex in IV; SEQ ID NO:7--60 kDa heat shock protein;
SEQ ID NO:8--T complex protein 1 beta subunit; SEQ ID NO:9--T
complex protein 1 epsilon subunit; SEQ ID NO:10--mortalin; and SEQ
ID NO:11--TER-ATPase. The invention further provides related
methods and materials for the use of the markers in therapeutic
intervention in colorectal and other cancers e.g. to specifically
target neoplastic cells without causing significant toxicity in
healthy tissues, and to provide methods for the evaluation of the
ability of candidate therapeutic compounds to modulate the
biological activity of cancerous cells from the colon, rectum and
other tissues.
Inventors: |
Murray; Graeme Ian;
(Aberdeen, GB) ; Telfer; Colin Matheson;
(Aberdeen, GB) ; Melvin; William Thomas;
(Aberdeen, GB) |
Correspondence
Address: |
DANN, DORFMAN, HERRELL & SKILLMAN
1601 MARKET STREET, SUITE 2400
PHILADELPHIA
PA
19103-2307
US
|
Family ID: |
32966938 |
Appl. No.: |
12/707700 |
Filed: |
February 18, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10548694 |
Sep 8, 2005 |
7700307 |
|
|
PCT/GB2004/000981 |
Mar 8, 2004 |
|
|
|
12707700 |
|
|
|
|
Current U.S.
Class: |
435/7.1 ;
435/29 |
Current CPC
Class: |
G01N 33/57419 20130101;
G01N 2800/52 20130101 |
Class at
Publication: |
435/7.1 ;
435/29 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C12Q 1/02 20060101 C12Q001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 8, 2003 |
GB |
0305342.8 |
Mar 8, 2003 |
GB |
0305345.1 |
Mar 8, 2003 |
GB |
0305346.9 |
Mar 8, 2003 |
GB |
0305347.7 |
Mar 8, 2003 |
GB |
0305348.5 |
Mar 8, 2003 |
GB |
0305350.1 |
Mar 8, 2003 |
GB |
0305351.9 |
Mar 8, 2003 |
GB |
0305353.5 |
Mar 8, 2003 |
GB |
0305382.4 |
Mar 8, 2003 |
GB |
0305385.7 |
Mar 8, 2003 |
GB |
0305387.3 |
Claims
1. A method of discriminating cancer cells from normal cells, which
method comprises determining whether a target protein is
over-expressed in said cells, said target protein being the 60 kDa
heat shock protein of SEQ ID NO:7, or a variant having at least 95%
homology therewith.
2. A method as claimed in claim 1 wherein said method is performed
on an individual in whom said cells are present or from whom said
cells have been derived, and the determination of protein
over-expression is used in diagnosing or predicting the onset of
cancer.
3. A method for diagnosing or predicting the onset of a cancer in a
tissue of an individual, which method comprises the steps of: (a)
determining the expression of a target protein in a sample of the
tissue from the individual, said target protein being the 60 kDa
heat shock protein of SEQ ID NO: 7, or a variant having at least
95% homology therewith, and (b) comparing the pattern or level of
expression observed with the pattern or level of expression of the
same protein in a second clinically normal tissue sample from the
same individual or a second healthy individual, wherein a
difference in the expression patterns or levels observed is
correlated with the presence of cancer cells in the sample.
4. (canceled)
5. A method as claimed in claim 3, wherein the target protein is
detected using a recognition compound which is a binding moiety
capable of specifically binding the target protein, which binding
moiety is optionally linked to a detectable label.
6. A method as claimed in claim 5 wherein the method comprises the
steps of (a) obtaining from a patient a tissue sample to be tested
for the presence of cancer cells; (b) producing a prepared sample
in a sample preparation process; (c) contacting the prepared sample
with the recognition compound that binds to the target protein; and
(d) detecting binding of the recognition compound to the target
protein, if present, in the prepared sample.
7. A method as claimed in claim 5 wherein the recognition compound
is an antibody.
8.-39. (canceled)
40. A method as claimed in claim 1 wherein the cancer is colorectal
cancer.
41. A method as claimed in claim 3 wherein the sample is derived
from tissues of the colon and/or rectum.
42. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/548,694, filed Sep. 8, 2005, which is the U.S. National
Stage of International Application No. PCT/GB2004/000981, filed
Mar. 8, 2004. The entire disclosure of each the aforesaid
applications is incorporated by reference in the present
application.
BACKGROUND TO THE INVENTION
[0002] Cancer remains one of the leading causes of death in the
Western world. Clinically, the treatment of human cancer currently
involves the use of a broad variety of medical approaches,
including surgery, radiation therapy and chemotherapeutic drug
therapy (see, for example, the Oxford Textbook of Oncology, Souhami
R L, Tannock I, Hohenberger P, and Horiot J-C (ed. s), 2nd edition,
New York, N.Y., Oxford University Press, 2002).
[0003] A diverse group of chemotherapeutic agents are used in the
treatment of human cancer, including the taxanes paclitaxel and
docetaxel, the topoisomerase inhibitors etoposide, topotecan and
irinotecan, the antimetabolites methotrexate, 5-fluorouracil,
5-fluorodeoxyuridine, 6-mercaptopurine, 6-thioguanine, cytosine
arabinoside, 5-aza-cytidine and hydroxyurea; the alkylating agents
cyclophosphamide, melphalan, busulfan, CCNU, MeCCNU, BCNU,
streptozotocin, chlorambucil, bis-diamminedichloroplatinum,
azetidinylbenzoquinone; the plant alkaloids vincristine,
vinblastine, vindesine, and VM-26;the antibiotics actinomycin-D,
doxorubicin, daunorubicin, mithramycin, mitomycin C and bleomycin;
and miscellaneous agents such as dacarbazine, mAMSA and
mitoxantrone. However, some neoplastic cells develop resistance to
specific chemotherapeutic agents or even to multiple
chemotherapeutic agents, and some tumours are intrinsically
resistant to certain chemotherapeutic agents. Such drug resistance
or multiple drug resistance can theoretically arise from expression
of genes that confer resistance to the agent, or from lack of
expression of genes that make the cells sensitive to a particular
anticancer drug.
[0004] It is well established that certain pathological conditions,
including cancer, are characterized by the abnormal expression of
certain molecules, and these molecules thus serve as "markers" for
a particular pathological condition.
[0005] Apart from their use as diagnostic "targets", i.e. abnormal
components that can be identified to diagnose the pathological
condition, the molecules serve as reagents which can be used to
generate diagnostic and/or therapeutic agents. An example of this,
which is not intended to be limiting, is the use of markers of
cancer to produce antibodies specific to a particular marker. A
further non-limiting example is the use of a peptide which
complexes with an MHC molecule, to generate cytolytic T cells
against cells expressing the marker.
[0006] One particular cancer target of interest is colorectal
cancer. Colorectal cancers are the third most common malignancies
in the world, and amongst men in the European Union it is the
second most common cause of cancer death after lung cancer.
Although more than 90% of cases are curable when diagnosed at an
early stage in development, the majority of patients with
colorectal cancer present clinically when the tumour is at an
advanced, metastatic stage. Consequently, the disease kills around
98,500 people every year in the EU (where less than 50% of patients
survive 5 years after an initial diagnosis of colorectal cancer)
and an estimated 437,000 people per annum worldwide. This problem
of late diagnosis is compounded by the resistance of some patients'
tumours to currently available chemotherapy; leading to a failure
to respond to treatment. Such patients require earlier detection
and more successful treatment of their illness, and to this end it
is desirable to identify proteins whose expression is associated
with cancerous cells, which may serve as diagnostic markers,
prognostic indicators and therapeutic targets.
[0007] Colorectal cancer is a consequence of pathologic
transformation of normal cells of the colonic epithelium to an
invasive cancer, and may result from inherited mutation,
spontaneous mutation or exposure to carcinogens in the bowel
contents. The majority of cancers of the colorectum are
adenocarcinomas (Jass & Morson, J. Clin. Pathol. 40: 1016-23,
1987), but questions remain concerning the true origins of
colorectal carcinomas. Such carcinomas may arise both from within
existing benign neoplasms ("adenomas"), in what has been termed the
adenoma to carcinoma sequence (Muto et al, Cancer 30: 2251-70,
1975), but the majority of adenomas do not appear to progress to
carcinoma and indeed may even regress (Knoemschild, Surg. Forum
XIV: 137-8, 1963). Alternatively carcinomas may arise de novo from
areas of generalised dysplasia without an adenomatous stage.
Clinical evidence supports the identification of environment, diet,
age and sex as risk factors for colorectal cancer, but the lack of
confirmed involvement of these factors in all cases suggests an
underlying genetic basis for colorectal tumour formation. Several
genetic alterations have been implicated in development of
colorectal cancer, including mutations in tumour-suppressor genes,
proto-oncogenes and DNA repair genes (reviewed by Robbins &
Itzkowitz, Med Clin North Am 86: 1467-95, 2002 ; Fearnhead et al,
Br Med Bull 64: 27-43, 2002). For example, WO 0077252 identifies
the Barx2 gene as a candidate tumour suppressor implicated in
ovarian and colorectal cancer.
[0008] One of the earliest detectable events, which may be the
initiating event in colorectal tumourigenesis, is inactivating
mutation of both alleles of the adenomatous polyposis coli (APC)
tumour suppressor gene. Other implicated genes include MCC, p53,
DCC (deleted in colorectal carcinoma), and genes in the TGF-beta
signalling pathway. Tumour specific patterns of expression have
also been demonstrated for a number of proteins in colorectal
tissues, and these proteins are undergoing evaluation as diagnostic
and therapeutic targets. One such protein is carcinoembryonic
antigen (CEA), which is detectable in the majority of colorectal
cancers but not in normal tissues (reviewed by Hammarstrom, Semin
Cancer Biol 9: 67-81, 1999). CEA is immunologically detectable in
the serum of colorectal cancer patients, and detection of CEA mRNA
by RT-PCR can identify lymph node micrometastases, which are a
prognostic indicator of a reduced chance of survival in colorectal
cancers (Liefers et al, New England J. of Med. 339: 223-8, 1998).
Another promising marker for colorectal cancer is minichromosome
maintenance protein 2 (MCM2), which is being developed as a target
for diagnosis from stool samples (Davies et al, Lancet 359: 1917-9,
2002).
[0009] At the present time, none of the protein markers under
investigation are in routine clinical use, and further targets for
diagnosis, prognosis and treatment are desirable. The current
routine diagnostic test for colorectal cancer is the FOBT (Faecal
Occult Blood Test), which is lacking in sensitivity and
specificity. Evaluation of the effectiveness of this test indicates
that it may fail to detect as many as 76% of suspicious growths
(Lieberman et al, N Engl J Med; 345: 555-60, 2001). It also results
in a large number of false positives and these patients require to
undergo the unpleasant, invasive procedure of colonoscopy. Even
when administered together these two procedures have been found to
miss 24% of tumours and precancerous polyps. Currently the best
candidates for new diagnostic tests are based on DNA analysis, but
these have at best a 50% detection rate.
[0010] It will be appreciated from the forgoing that the provision
of novel specific, reliable markers that are differentially
expressed in normal and transformed tissues (such as colorectal
tissue) would provide a useful contribution to the art. Such
markers could be used inter alia in the diagnosis of cancers such
as colorectal cancer, the prediction of the onset of cancers such
as colorectal cancer, or the treatment of cancers such as
colorectal cancer.
SUMMARY OF THE INVENTION
[0011] The present inventors have used specific proteomics
approaches to identify proteins that are expressed in cancer cells
but not in normal tissues. The target proteins of the present
invention are listed in Table 2 and discussed in Example 2
herein.
[0012] Each marker has been identified by up-regulation of
expression in colorectal tumour samples, an observation not
previously made in this tissue type for any of these markers.
[0013] Previously, proteome studies have proven to be of limited
success in identifying such markers. WO9842736 and WO9843091 do
disclose certain differentially-expressed protein markers
identified by proteomic analysis of clinical samples following
laborious processing intended to enrich tumour epithelial cells by
removing stromal cells and connective tissue contaminants. However,
more recently, a proteomic comparison of murine normal and
neoplastic colon tissues identified no statistically significant
differences in protein expression patterns (Cole et al,
Electrophoresis 21: 1772-81, 2000).
[0014] After the presently claimed priority date, the following
studies were published relating to some of the markers disclosed
herein: Kuniyasu H, Chihara Y, Kondo H, Ohmori H, Ukai R (2003)
Amphoterin induction in prostatic stromal cells by androgen
deprivation is associated with metastatic prostate cancer. Oncol
Rep. 10(6): 1863-8; Cappello F, Bellafiore M, Palma A, David S,
Marciano V, Bartolotta T, Sciume C, Modica G, Farina F, Zummo G,
Bucchieri F. (2003) 60 KDa chaperonin (HSP60) is over-expressed
during colorectal carcinogenesis. European Journal of
Histochemistry 47(2): 105-10; Yamamoto S, Tomita Y, Hoshida Y,
Sakon M, Kameyama M, Imaoka S, Sekimoto M, Nakamori S, Monden M,
Aozasa K. (2004) Expression of valosin-containing protein in
colorectal carcinomas as a predictor for disease recurrence and
prognosis. Clinical Cancer Research 10 (2): 651-7.
[0015] Other publications concerning the present markers are
discussed in Example 2 herein.
[0016] Accordingly, the present invention describes the use of the
target proteins listed in Table 2 (which may be referred to
hereinafter as "the target proteins of the present invention") as
markers of cancer, and provides methods for their use in such
applications.
[0017] As discussed in detail below, the target proteins of the
present invention are of particular use inter alia as diagnostic
and prognostic markers of cancers, and in particular colorectal
cancers. As with known markers, they may be used for example to
assist diagnosing the presence of cancer at an early stage in the
progression of the disease and predicting the likelihood of
clinically successful outcome, particularly with regard to the
sensitivity or resistance of a particular patient's tumour to a
chemotherapeutic agent or combinations of chemotherapeutic agents.
Furthermore these targets can be used for therapeutic intervention
in colorectal and other cancers e.g. to specifically target
neoplastic cells without causing significant toxicity in healthy
tissues, and to provide methods for the evaluation of the ability
of candidate therapeutic compounds to modulate the biological
activity of cancerous cells from the colon, rectum and other
tissues. Thus the present invention relates to the diagnosis and
treatment of cancer, and specifically to the discrimination of
neoplastic cells from normal cells on the basis of over-expression
of specific tumour antigens and the targeting of treatment through
exploitation of the differential expression of these antigens
within neoplastic cells. The invention specifically relates to the
detection of one or more proteins ("target proteins") that are
over-expressed in neoplastic cells compared with the expression in
pathologically normal cells (see Table 2). Furthermore the
invention provides evidence for up-regulation of expression of this
target in tumour cells where this has not previously been reported.
Accordingly, this protein, as well as nucleic acid sequences
encoding this protein, or sequences complementary thereto, can be
used as a cancer marker useful in diagnosing or predicting the
onset of a cancer such as colorectal cancer, monitoring the
efficacy of a cancer therapy and/or as a target of such a
therapy.
[0018] The invention in particular relates to the discrimination of
neoplastic cells from normal cells on the basis of the
over-expression of a target protein of the present invention, or
the gene that encodes this protein. To enable this identification,
the invention provides a pattern of expression of a specific
protein, the expression of which is increased in neoplastic cells
in comparison to normal cells. The invention provides a variety of
methods for detecting this protein and the expression pattern of
this protein and using this information for the diagnosis and
treatment of cancer.
[0019] Furthermore, it is contemplated that the skilled artisan may
produce novel therapeutics for treating colorectal cancer which
include, for example: antibodies which can be administered to an
individual that bind to and reduce or eliminate the biological
activity of the target protein in vivo; nucleic acid or peptidyl
nucleic acid sequences which hybridize with genes or gene
transcripts encoding the target proteins thereby to reduce
expression of the target proteins in vivo; or small molecules, for
example, organic molecules which interact with the target proteins
or other cellular moieties, for example, receptors for the target
protein, thereby to reduce or eliminate the biological activity of
the target protein.
[0020] The invention therefore further provides methods for
targeting of therapeutic treatments for cancers by directing
treatment against this over-expressed protein. Methods for
achieving this targeting may include, but are not limited to;
[0021] i) conjugation of therapeutic drugs to a moiety such as an
immunoglobulin or aptamer that specifically recognises the
molecular structure of the target protein,
[0022] (ii) exposure of the host immune system to the target
protein or fragments thereof by immunisation using proteins,
polypeptides, expression vectors or DNA vaccine constructs in order
to direct the host immune system against neoplastic cells in which
the target protein is over-expressed,
[0023] (iii) modification of the biological activity of the target
protein by small molecule ligands,
[0024] (iv) exploitation of the biological activity of the target
protein to activate prodrugs,
[0025] (v) modulation of the expression of the target protein in
cells by methods such as antisense gene silencing, use of small
interfering RNA molecules, or the targeting of regulatory elements
in the gene encoding the target protein or regulatory proteins that
bind to these elements,
[0026] (vi) specific modulation of the physical interaction of the
target protein with other components of the cell, with for example
a small molecule ligand or an immunoglobulin, in order to exert a
therapeutic benefit.
[0027] The present invention thereby provides a wide range of novel
methods for the diagnosis, prognosis and treatment of cancers,
including colorectal cancer, on the basis of the differential
expression of the target protein. These and other numerous
additional aspects and advantages of the invention will become
apparent to the skilled artisan upon consideration of the following
detailed description of the invention.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0028] In the studies disclosed herein, proteomic analysis was
applied to colorectal samples from a clinical tissue bank into
which have been collected both tumour and pathologically normal
(disease-free) tissues from each individual donor. Using a process
whereby proteins are recovered from frozen sections of donor
tissues selected from a bank of fresh frozen tissues on the basis
of optimal tumour histology and cellularity, it was possible to
derive the protein expression profiles of carefully selected sets
of normal colon tissue and advanced (Duke's C stage) colorectal
carcinomas selected from 16 patients. Comparison of the protein
expression "fingerprints" of the tumour and normal tissue sets
revealed differences that arise as a result of the disease process.
Table 1 provides details of proteins identified by these means
whose up-regulation in colorectal tumour tissues has previously
been reported. Table 2 provides details of proteins identified by
these means whose up-regulation in colorectal tumour tissues has
not been previously demonstrated and thereby provides the basis of
the present invention.
[0029] The objective of the present study was to identify new
targets for cancer diagnosis and therapy. Accordingly, a first
aspect of the present invention provides a method for the
identification of cancer cells, which method comprises determining
the expression of the target protein of the invention in a sample
of tissue from a first individual and comparing the pattern of
expression observed with the pattern of expression of the same
protein in a second clinically normal tissue sample from the same
individual or a second healthy individual, with the presence of
tumour cells in the sample from the first individual indicated by a
difference in the expression patterns observed.
[0030] More specifically, the invention provides a diagnostic assay
for characterising tumours and neoplastic cells, particularly human
neoplastic cells, by the differential expression of the target
protein whereby the neoplastic phenotype is associated with,
identified by and can be diagnosed on the basis thereof. This
diagnostic assay comprises detecting, qualitatively or preferably
quantitatively, the expression level of the target protein and
making a diagnosis of cancer on the basis of this expression
level.
[0031] In this context, "determining the expression" means
qualitative and/or quantitative determinations, of the presence of
the target protein of the invention including measuring an amount
of biological activity of the target protein in terms of units of
activity or units activity per unit time, and so forth.
[0032] As used herein, the term "expression" generally refers to
the cellular processes by which a polypeptide is produced from
RNA.
[0033] As used herein, the term "cancer" encompasses cancers in all
forms, including polyps, neoplastic cells and preneoplastic cells
and includes sarcomas and carcinomas. Exemplary sarcomas and
carcinomas include, but are not limited to, fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumour, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms'tumour, cervical cancer, testicular tumour, lung
carcinoma (including small cell lung carcinoma and non-small cell
lung carcinoma), bladder carcinoma, epithelial carcinoma, glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
meningioma, melanoma, neuroblastoma, retinoblastoma; leukaemias,
e.g., acute lymphocytic leukaemia and acute myelocytic leukaemia
(myeloblastic, promyelocytic, myelomonocytic, monocytic and
erythroleukaemia); chronic leukaemia (chronic myelocytic
(granulocytic) leukaemia and chronic lymphocytic leukaemia); and
polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's
disease), multiple myeloma, Waldenstroom's macroglobulinemia, and
heavy chain disease.
[0034] In a preferred embodiment of the present invention, this
method may be applied to diagnosis of colorectal cancer. The terms
"colon cancer", "rectal cancer", and "colorectal cancer" are used
interchangeably herein.
[0035] Species variants are also encompassed by this invention
where the patient is a non-human mammal, as are allelic or other
variants of the proteins described in Table 2, and any reference to
the proteins in that table will be understood to embrace, alleles,
homologues or other naturally occurring variants.
[0036] Thus included within the definition of the target protein of
the invention are amino acid variants of the naturally occurring
sequence as provided in any of SEQ ID NOs:1-11. Preferably, variant
sequences are at least 75% homologous to the wild-type sequence,
more preferably at least 80% homologous, even more preferably at
least 85% homologous, yet more preferably at least 90% homologous
or most preferably at least 95% homologous to at least a portion of
the reference sequence supplied (SEQ ID NOs:1-11). In some
embodiments the homology will be as high as 94 to 96 or 98%.
Homology in this context means sequence similarity or identity,
with identity being preferred. To determine whether a candidate
peptide region has the requisite percentage similarity or identity
to a reference polypeptide or peptide oligomer, the candidate amino
acid sequence and the reference amino acid sequence are first
aligned using a standard computer programme such as are
commercially available and widely used by those skilled in the art.
In a preferred embodiment the NCBI BLAST method is used
(www.ncbi.nlm.nih.gov/BLAST/). Once the two sequences have been
aligned, a percent similarity score may be calculated. In all
instances, variants of the naturally-occurring sequence, as
detailed in SEQ ID NO:1-11 herein, must be confirmed for their
function as marker proteins. Specifically, their presence or
absence in a particular form or in a particular biological
compartment must be indicative of the presence or absence of cancer
in an individual. This routine experimentation can be carried out
by using standard methods known in the art in the light of the
disclosure herein.
[0037] In one aspect of the present invention, the target protein
can be detected using a binding moiety capable of specifically
binding the marker protein. By way of example, the binding moiety
may comprise a member of a ligand-receptor pair, i.e. a pair of
molecules capable of having a specific binding interaction. The
binding moiety may comprise, for example, a member of a specific
binding pair, such as antibody-antigen, enzyme-substrate, nucleic
acid-nucleic acid, protein-nucleic acid, protein-protein, or other
specific binding pair known in the art. Binding proteins may be
designed which have enhanced affinity for the target protein of the
invention. Optionally, the binding moiety may be linked with a
detectable label, such as an enzymatic, fluorescent, radioactive,
phosphorescent, coloured particle label or spin label. The labelled
complex may be detected, for example, visually or with the aid of a
spectrophotometer or other detector.
[0038] A preferred embodiment of the present invention involves the
use of a recognition agent, for example an antibody recognising the
target protein of the invention, to contact a sample of tissues,
cells, blood or body product, or samples derived therefrom, and
screening for a positive response. The positive response may for
example be indicated by an agglutination reaction or by a
visualisable change such as a colour change or fluorescence, e. g.
immunostaining, or by a quantitative method such as in use of
radio-immunological methods or enzyme-linked antibody methods.
[0039] The method therefore typically includes the steps of (a)
obtaining from a patient a tissue sample to be tested for the
presence of cancer cells; (b) producing a prepared sample in a
sample preparation process; (c) contacting the prepared sample with
a recognition agent, such as an antibody, that reacts with the
target protein of the invention; and (d) detecting binding of the
recognition agent to the target protein, if present, in the
prepared sample. The human tissue sample can be from the colon or
any other tissue in which tumour-specific expression of the
appropriate protein can be demonstrated. The sample may further
comprise sections cut from patient tissues or it may contain whole
cells or it may be, for example, a body fluid sample selected from
the group consisting of : blood; serum; plasma; fecal matter;
urine; vaginal secretion; breast exudate; spinal fluid; saliva;
ascitic fluid; peritoneal fluid; sputum; and colorectal exudate, or
an effusion, where the sample may contain cells, or may contain
shed antigen. A preferred sample preparation process includes
tissue fixation and production of a thin section. The thin section
can then be subjected to immunohistochemical analysis to detect
binding of the recognition agent to the target protein. Preferably,
the immunohistochemical analysis includes a conjugated enzyme
labelling technique. A preferred thin section preparation method
includes formalin fixation and wax embedding. Alternative sample
preparation processes include tissue homogenisation. When sample
preparation includes tissue homogenisation, a preferred method for
detecting binding of the antibody to the target protein is Western
blot analysis. Alternatively, an immunoassay can be used to detect
binding of the antibody to the target protein. Examples of
immunoassays are antibody capture assays, two-antibody sandwich
assays, and antigen capture assays. In a sandwich immunoassay, two
antibodies capable of binding the marker protein generally are
used, e. g. one immobilised onto a solid support, and one free in
solution and labelled with a detectable chemical compound. Examples
of chemical labels that may be used for the second antibody include
radioisotopes, fluorescent compounds, spin labels, coloured
particles such as colloidal gold and coloured latex, and enzymes or
other molecules that generate coloured or electrochemically active
products when exposed to a reactant or enzyme substrate. When a
sample containing the marker protein is placed in this system, the
marker protein binds to both the immobilised antibody and the
labelled antibody, to form a "sandwich" immune complex on the
support's surface. The complexed protein is detected by washing
away non-bound sample components and excess labelled antibody, and
measuring the amount of labelled antibody complexed to protein on
the support's surface. Alternatively, the antibody free in
solution, which can be labelled with a chemical moiety, for
example, a hapten, may be detected by a third antibody labelled
with a detectable moiety which binds the free antibody or, for
example, the hapten coupled thereto. Preferably, the immunoassay is
a solid support-based immunoassay. Alternatively, the immunoassay
may be one of the immunoprecipitation techniques known in the art,
such as, for example, a nephelometric immunoassay or a
turbidimetric immunoassay. When Western blot analysis or an
immunoassay is used, preferably it includes a conjugated enzyme
labelling technique.
[0040] Although the recognition agent will conveniently be an
antibody, other recognition agents are known or may become
available, and can be used in the present invention. For example,
antigen binding domain fragments of antibodies, such as Fab
fragments, can be used. Also, so-called RNA aptamers may be used.
Therefore, unless the context specifically indicates otherwise, the
term "antibody" as used herein is intended to include other
recognition agents. Where antibodies are used, they may be
polyclonal or monoclonal. Optionally, the antibody can be produced
by a method such that it recognizes a preselected epitope from the
target protein of the invention.
[0041] The isolated target protein of the invention may be used for
the development of diagnostic and other tissue evaluation kits and
assays to monitor the level of the proteins in a tissue or fluid
sample. For example, the kit may include antibodies or other
specific binding moieties which bind specifically to the target
protein which permit the presence and/or concentration of the
colorectal cancer-associated proteins to be detected and/or
quantified in a tissue or fluid sample.
[0042] Accordingly, the invention further provides for the
production of suitable kits for detecting the target protein, which
may for example include a receptacle or other means for receiving a
sample to be evaluated, and a means for detecting the presence
and/or quantity in the sample of the target protein of the
invention and optionally instructions for performing such an
assay.
[0043] In a further aspect of the present invention is provided
herein a method of evaluating the effect of a candidate therapeutic
drug for the treatment of cancer, said method comprising
administering said drug to a patient, removing a cell sample from
said patient; and determining the expression profile of the target
protein of the invention in said cell sample. This method may
further comprise comparing said expression profile to an expression
profile of a healthy individual. In a preferred embodiment, said
patient is receiving treatment for colorectal cancer and said cell
sample is derived from tissues of the colon and/or rectum. In a
further preferred embodiment the present invention further provides
a method for determine the efficacy of a therapeutic regime at one
or more timepoints, said method comprising determining a baseline
value for the expression of the protein being tested in a given
individual within a given tissue such as a tumour, administering a
given therapeutic drug, and then redetermining expression levels of
the protein within that given tissue at one or more instances
thereafter, observing changes in protein levels as an indication of
the efficacy of the therapeutic regime.
[0044] In a further aspect of the present invention the target
protein of the invention provides a mechanism for the selective
targeting of anti-cancer drugs based on metabolism by the target
protein within tumours. The present invention therefore provides
for the design of, or screening for, drugs that undergo specific
metabolism in tumours mediated by the target protein of the
invention, whereby this metabolism converts a non-toxic moiety into
a toxic one, which kills or inhibits the tumour or makes it more
susceptible to other agents. In a further preferred embodiment of
the present invention, a method of treating colorectal cancer is
provided, said method comprising use of a drug that is specifically
metabolised to an active form by contact with the target protein of
the invention.
[0045] A further aspect of the invention provides for the targeting
of cytotoxic drugs or other therapeutic agents, or the targeting of
imaging agents, by virtue of their recognition of epitopes derived
from the target protein of the invention on the surface of a tumour
cell, whether as part of the complete target protein itself or in
some degraded form such as in the presentation on the surface of a
cell bound to a MHC protein.
[0046] A further embodiment of the present invention is the
development of therapies for treatment of conditions which are
characterized by over-expression of the target protein of the
invention via immunotherapeutic approaches. More specifically, the
invention provides methods for stimulation of the immune system of
cancer patients, for example by activating cytotoxic or helper
T-cells which recognise epitopes derived from the protein of the
invention so as to implement a cell-mediated or humoral immune
response against the tumour. By way of example, the activation of
the immune system can be achieved by immunisation with sequences
derived from the target protein of the invention in an amount
sufficient to provoke or augment an immune response. By way of
further example, which is specifically not intended to limit the
scope of the invention, these may be administered as naked
peptides, as peptides conjugated or encapsulated in one or more
additional molecules (e.g. liposomes) such that a pharmacological
parameter (e.g. tissue permeability, resistance to endogenous
proteolysis, circulating half-life etc) is improved, or in a
suitable expression vector which causes the expression of the
sequences at an appropriate site within the body to provoke an
immune response. The proteins or peptides may be combined with one
or more of the known immune adjuvants, such as saponins, GM-CSF,
interleukins, and so forth. Peptides that are too small to generate
a sufficient immune response when administered alone can be coupled
to one or more of the various conjugates used to stimulate such
responses which are well known in the art. Furthermore, peptides
which form non-covalent complexes with MHC molecules within cells
of the host immune system may be used to elicit proliferation of
cytolytic T cells against any such complexes in the subject. Such
peptides may be administered endogenously or may be administered to
isolated T-cells ex-vivo and then reperfused into the subject being
treated. Alternatively, the generation of a host immune response
can be accomplished by administration of cells, preferably rendered
non-proliferative by standard methods, which present relevant T
cell or B cell epitopes to trigger the required response.
[0047] Because up-regulation of expression of the target protein of
the invention is associated with tumour cells, it is likely that
these proteins in some way contribute to the process of
tumourigenesis or the persistence of tumour cells. Consequently,
the present invention provides for the reduction of the expression
level of the target protein in tumour cells, for example by the use
of suicide inhibitors or by using antisense RNA methods to decrease
the synthesis of the protein. Similarly, this reduction in
expression levels could also be achieved by down-regulation of the
corresponding gene promoter. A preferred method comprises the step
of administering to a patient diagnosed as having cancer, such as
colorectal cancer, a therapeutically-effective amount of a compound
which reduces in vivo the expression of the target protein. In a
preferred embodiment, the compound is a polynucleotide, for
example, an anti-sense nucleic acid sequence or a peptidyl nucleic
acid (PNA), more preferably from 10 to 100 nucleotides in length,
capable of binding to and reducing the expression (for example,
transcription or translation) of a nucleic acid encoding at least a
portion of the target protein of the invention. After
administration, the anti-sense nucleic acid sequence or the
anti-sense PNA molecule binds to the nucleic acid sequences
encoding, at least in part, the target protein thereby to reduce in
vivo expression of the target protein. By way of further example,
constructs of the present invention capable of reducing expression
of the target protein can be administered to the subject either as
a naked polynucleotide or formulated with a carrier, such as a
liposome, to facilitate incorporation into a cell. Such constructs
can also be incorporated into appropriate vaccines, such as in
viral vectors (e.g. vaccinia), bacterial constructs, such as
variants of the well known BCG vaccine, and so forth.
[0048] A particularly useful therapeutic embodiment of the present
invention provides an oligonucleotide or peptidyl nucleic acid
sequence complementary and capable of hybridizing under
physiological conditions to part, or all, of the gene encoding the
target protein or to part, or all, of the transcript encoding the
target protein thereby to reduce or inhibit transcription and/or
translation of the target protein gene.
[0049] Anti-sense oligonucleotides have been used extensively to
inhibit gene expression in normal and abnormal cells. For a recent
review, see Phillips, ed., Antisense Technology, in Methods in
Enzymology, vols. 313-314, Academic Press; Hartmann, ed., 1999. In
addition, the synthesis and use of peptidyl nucleic acids as
anti-sense-based therapeutics are described in PCT publications
PCT/EP92/01219, PCT/US92/1092, and PCT/US94/013523. Accordingly,
the anti-sense-based therapeutics may be used as part of
chemotherapy, either alone or in combination with other
therapies.
[0050] Double stranded RNA (dsRNA) has been found to be even more
effective in gene silencing than both sense or antisense strands
alone (Fire A. et al Nature, Vol 391, (1998)). dsRNA mediated
silencing is gene specific and is often termed RNA interference
(RNAi) (See also Fire (1999) Trends Genet. 15: 358-363, Sharp
(2001) Genes Dev. 15: 485-490, Hammond et al. (2001) Nature Rev.
Genes 2: 1110-1119 and Tuschl (2001) Chem. Biochem. 2:
239-245).
[0051] RNA interference is a two step process. First, dsRNA is
cleaved within the cell to yield short interfering RNAs (siRNAs) of
about 21-23nt length with 5' terminal phosphate and 3' short
overhangs (.about.2nt) The siRNAs target the corresponding mRNA
sequence specifically for destruction (Zamore P. D. Nature
Structural Biology, 8,9, 746-750, (2001). Thus in one embodiment,
the invention provides double stranded RNA comprising a sequence
encoding a target protein of the present invention, which may for
example be a "long" double stranded RNA (which will be processed to
siRNA, e.g., as described above). These RNA products may be
synthesised in vitro, e.g., by conventional chemical synthesis
methods.
[0052] RNAi may be also be efficiently induced using chemically
synthesized siRNA duplexes of the same structure with 3'-overhang
ends (Zamore PD et al Cell, 101, 25-33, (2000)). Synthetic siRNA
duplexes have been shown to specifically suppress expression of
endogenous and heterologous genes in a wide range of mammalian cell
lines (Elbashir S M. et al. Nature, 411,494-498, (2001)). Thus
siRNA duplexes containing between 20 and 25 bps, more preferably
between 21 and 23 bps, of the sequence encoding a target protein of
the present invention form one aspect of the invention e.g. as
produced synthetically, optionally in protected form to prevent
degradation. Alternatively siRNA may be produced from a vector, in
vitro (for recovery and use) or in vivo.
[0053] Accordingly, the vector may comprise a nucleic acid sequence
encoding a target protein of the present invention (including a
nucleic acid sequence encoding a variant or fragment thereof),
suitable for introducing an siRNA into the cell in any of the ways
known in the art, for example, as described in any of references
cited herein, which references are specifically incorporated herein
by reference.
[0054] In one embodiment, the vector may comprise a nucleic acid
sequence according to the invention in both the sense and antisense
orientation, such that when expressed as RNA the sense and
antisense sections will associate to form a double stranded RNA.
This may for example be a long double stranded RNA (e.g., more than
23nts) which may be processed in the cell to produce siRNAs (see
for example Myers (2003) Nature Biotechnology 21: 324-328).
[0055] Alternatively, the double stranded RNA may directly encode
the sequences which form the siRNA duplex, as described above. In
another embodiment, the sense and antisense sequences are provided
on different vectors. These vectors and RNA products may be useful
for example to inhibit de novo production of the protein of the
present invention in a cell. They may be used analogously to the
expression vectors in the various embodiments of the invention
discussed herein.
[0056] In particular there is provided double-stranded RNA which
comprises an RNA sequence encoding a target protein of the present
invention or a fragment thereof, which may be an siRNA duplex
consisting of between 20 and 25 bps. Also provided are vectors
encoding said dsRNA or siRNA duplexes. Also provided are methods of
producing said siRNA duplexes comprising introducing such vectors
into a host cell and causing or allowing transcription from the
vector in the cell. Separate vectors may encode: (i) the sense
sequence of the siRNA duplex, and (ii) the anti-sense sequence of
the siRNA duplex.
[0057] An additional DNA based therapeutic approach provided by the
present invention is the use of a vector which comprises one or
more nucleotide sequences, preferably a plurality of these, each of
which encodes an immunoreactive peptide derived from the target
protein of the invention. Alternatively, a further method of the
invention involves combining one or more of these nucleotide
sequences encoding peptides derived from the target protein of the
invention in combination with nucleotide sequences encoding
peptides derived from other tumour markers known in the art to be
expressed by cancer cells, and encompasses inclusion of such
sequences in all possible variations, such as one from each
protein, several from one or more protein and one from each of one
or more additional proteins, and so forth.
[0058] A further aspect of the present invention provides novel
methods for screening for compositions that modulate the expression
or biological activity of the target protein of the invention. As
used herein, the term "biological activity" means any observable
effect resulting from interaction between the target protein and a
ligand or binding partner. Representative, but non-limiting,
examples of biological activity in the context of the present
invention include association of the target protein of the
invention with a ligand, such as any of those shown in Table 4.
[0059] The term "biological activity" also encompasses both the
inhibition and the induction of the expression of the target
protein of the invention. Further, the term "biological activity"
encompasses any and all effects resulting from the binding of a
ligand or other in vivo binding partner by a polypeptide derivative
of the protein of the invention. In one embodiment, a method of
screening drug candidates comprises providing a cell that expresses
the target protein of the invention, adding a candidate therapeutic
compound to said cell and determining the effect of said compound
on the expression or biological activity of said protein. In a
further embodiment, the method of screening candidate therapeutic
compounds includes comparing the level of expression or biological
activity of the protein in the absence of said candidate
therapeutic compound to the level of expression or biological
activity in the presence of said candidate therapeutic compound.
Where said candidate therapeutic compound is present its
concentration may be varied, and said comparison of expression
level or biological activity may occur after addition or removal of
the candidate therapeutic compound. The expression level or
biological activity of said target protein may show an increase or
decrease in response to treatment with the candidate therapeutic
compound.
[0060] Candidate therapeutic molecules of the present invention may
include, by way of example, peptides produced by expression of an
appropriate nucleic acid sequence in a host cell or using synthetic
organic chemistries, or non-peptide small molecules produced using
conventional synthetic organic chemistries well known in the art.
Screening assays may be automated in order to facilitate the
screening of a large number of small molecules at the same
time.
[0061] As used herein, the terms "candidate therapeutic compound"
refers to a substance that is believed to interact with the target
protein of the invention (or a fragment thereof), and which can be
subsequently evaluated for such an interaction. Representative
candidate therapeutic compounds include "xenobiotics", such as
drugs and other therapeutic agents, natural products and extracts,
carcinogens and environmental pollutants, as well as "endobiotics"
such as steroids, fatty acids and prostaglandins. Other examples of
candidate compounds that can be investigated using the methods of
the present invention include, but are not restricted to, agonists
and antagonists of the target protein of the invention, toxins and
venoms, viral epitopes, hormones (e.g., opioid peptides, steroids,
etc.), hormone receptors, peptides, enzymes, enzyme substrates,
co-factors, lectins, sugars, oligonucleotides or nucleic acids,
oligosaccharides, proteins, small molecules and monoclonal
antibodies.
[0062] In one preferred embodiment the present invention provides a
method of drug screening utilising eukaryotic or prokaryotic host
cells stably transformed with recombinant polynucleotides
expressing the target protein of the invention or a fragment
thereof, preferably in competitive binding assays. Such cells,
either in viable or fixed form, can be used for standard binding
assays. For example, the assay may measure the formation of
complexes between a target protein and the agent being tested, or
examine the degree to which the formation of a complex between the
target protein or fragment thereof and a known ligand or binding
partner is interfered with by the agent being tested. Thus, the
present invention provides methods of screening for drugs
comprising contacting such an agent with the target protein of the
invention or a fragment thereof or a variant thereof found in a
tumour cell and assaying (i) for the presence of a complex between
the agent and the target protein, fragment or variant thereof, or
(ii) for the presence of a complex between the target protein,
fragment or variant and a ligand or binding partner. In such
competitive binding assays the target protein or fragment or
variant is typically labelled. Free target protein, fragment or
variant thereof is separated from that present in a protein:
protein complex and the amount of free (i.e. uncomplexed) label is
a measure of the binding of the agent being tested to the target
protein or its interference with binding of the target protein to a
ligand or binding partner, respectively.
[0063] Alternatively, an assay of the invention may measure the
influence of the agent being tested on a biological activity of the
target protein. Thus, the present invention provides methods of
screening for drugs comprising contacting such an agent with the
target protein of the invention or a fragment thereof or a variant
thereof found in a tumour cell and assaying for the influence of
such an agent on a biological activity of the target protein, by
methods well known in the art. In such activity assays the
biological activity of the target protein, fragment or variant
thereof is typically monitored by provision of a reporter system.
For example, this may involve provision of a natural or synthetic
substrate that generates a detectable signal in proportion to the
degree to which it is acted upon by the biological activity of the
target molecule.
[0064] It is contemplated that, once candidate therapeutic
compounds have been elucidated, rational drug design methodologies
well known in the art may be employed to enhance their efficacy.
The goal of rational drug design is to produce structural analogues
of biologically active polypeptides of interest or of small
molecules with which they interact (e. g. agonists, antagonists,
inhibitors) in order to fashion drugs which are, for example, more
active or stable forms of the polypeptide, or which, for example,
enhance or interfere with the function of a polypeptide in vivo. In
one approach, one first determines the three-dimensional structure
of a protein of interest, such as the target protein of the
invention or, for example, of the target protein in complex with a
ligand, by x-ray crystallography, by computer modelling or most
typically, by a combination of approaches. For example, the skilled
artisan may use a variety of computer programmes which assist in
the development of quantitative structure activity relationships
(QSAR) that act as a guide in the design of novel, improved
candidate therapeutic molecules. Less often, useful information
regarding the structure of a polypeptide may be gained by modelling
based on the structure of homologous proteins. In addition,
peptides can be analysed by alanine scanning (Wells, Methods
Enzymol. 202: 390-411, 1991), in which each amino acid residue of
the peptide is sequentially replaced by an alanine residue, and its
effect on the peptide's activity is determined in order to
determine the important regions of the peptide. It is also possible
to design drugs based on a pharmacophore derived from the crystal
structure of a target-specific antibody selected by a functional
assay. It is further possible to avoid the use of protein
crystallography by generating anti-idiotypic antibodies to such a
functional, target-specific antibody, which have the same
three-dimensional conformation as the original target protein.
These anti-idiotypic antibodies can subsequently be used to
identify and isolate peptides from libraries, which themselves act
as pharmacophores for further use in rational drug design.
[0065] For use as a medicament in vivo, candidate therapeutic
compounds so identified may be combined with a suitable
pharmaceutically acceptable carrier, such as physiological saline
or one of the many other useful carriers well characterized in the
medical art. Such pharmaceutical compositions may be provided
directly to malignant cells, for example, by direct injection, or
may be provided systemically, provided the formulation chosen
permits delivery of the therapeutically effective molecule to
tumour cells containing the target protein of the invention.
Suitable dose ranges and cell toxicity levels may be assessed using
standard dose ranging methodology. Dosages administered may vary
depending, for example, on the nature of the malignancy, the age,
weight and health of the individual, as well as other factors.
[0066] A further aspect of the present invention provides for cells
and animals which express the target protein of the invention and
can be used as model systems to study and test for substances which
have potential as therapeutic agents.
[0067] Such cells may be isolated from individuals with mutations,
either somatic or germline, in the gene encoding the target protein
of the invention, or can be engineered to express or over-express
the target protein or a variant thereof, using methods well known
in the art. After a test substance is applied to the cells, any
relevant trait of the cells can be assessed, including by way of
example growth, viability, tumourigenicity in nude mice,
invasiveness of cells, and growth factor dependence, assays for
each of which traits are known in the art.
[0068] Animals for testing candidate therapeutic agents can be
selected after mutagenesis of whole animals or after treatment of
germline cells or zygotes. As discussed in more detail below, by
way of example, such treatments can include insertion of genes
encoding the target protein of the invention in wild-type or
variant form, typically from a second animal species, as well as
insertion of disrupted homologous genes. Alternatively, the
endogenous target protein gene (s) of the animals may be disrupted
by insertion or deletion mutation or other genetic alterations
using conventional techniques that are well known in the art. After
test substances have been administered to the animals, the growth
of tumours can be assessed. If the test substance prevents or
suppresses the growth of tumours, then the test substance is a
candidate therapeutic agent for the treatment of those cancers
expressing the target protein of the invention, for example of
colorectal cancers. These animal models provide an extremely
important testing vehicle for potential therapeutic compounds.
[0069] Thus the present invention thus provides a transgenic
non-human animal, particularly a rodent, which comprises an
inactive copy of the gene encoding a target protein of the present
invention.
[0070] The invention further provides a method of testing a
putative therapeutic of the invention which comprises administering
said therapeutic to an animal according to the invention and
determining the effect of the therapeutic.
[0071] For the purposes of the present invention, it will be
understood that reference to an inactive copy of the gene encoding
a target protein of the present invention includes any
non-wild-type variant of the gene which results in knock out or
down regulation of the gene, and optionally in a cancer phenotype.
Thus the gene may be deleted in its entirety, or mutated such that
the animal produces a truncated protein, for example by
introduction of a stop codon and optionally upstream coding
sequences into the open reading frame of the gene encoding a target
protein of the present invention. Equally, the open reading frame
may be intact and the inactive copy of the gene provided by
mutations in promoter regions.
[0072] Generally, inactivation of the gene may be made by targeted
homologous recombination. Techniques for this are known as such in
the art.
[0073] This may be achieved in a variety of ways. A typical
strategy is to use targeted homologous recombination to replace,
modify or delete the wild-type gene in an embryonic stem (ES) cell.
A targeting vector comprising a modified target gene is introduced
into ES cells by electroporation, lipofection or microinjection. In
a few ES cells, the targeting vector pairs with the cognate
chromosomal DNA sequence and transfers the desired mutation carried
by the vector into the genome by homologous recombination.
Screening or enrichment procedures are used to identify the
transfected cells, and a transfected cell is cloned and maintained
as a pure population. Next, the altered ES cells are injected into
the blastocyst of a preimplantation mouse embryo or alternatively
an aggregation chimera is prepared in which the ES cells are placed
between two blastocysts which, with the ES cells, merge to form a
single chimeric blastocyst. The chimeric blastocyst is surgically
transferred into the uterus of a foster mother where the
development is allowed to progress to term. The resulting animal
will be a chimera of normal and donor cells. Typically the donor
cells will be from an animal with a clearly distinguishable
phenotype such as skin colour, so that the chimeric progeny is
easily identified. The progeny is then bred and its descendants
cross-bred, giving rise to heterozygotes and homozygotes for the
targeted mutation. The production of transgenic animals is
described further by Capecchi, M. R., 1989, Science 244; 1288-1292;
Valancius and Smithies, 1991, Mol. Cell. Biol. 11; 1402-1408; and
Hasty et al, 1991, Nature 350; 243-246, the disclosures of which
are incorporated herein by reference.
[0074] Homologous recombination in gene targeting may be used to
replace the wild-type gene encoding a target protein of the present
invention with a specifically defined mutant form (e.g. truncated
or containing one or more substitutions).
[0075] The inactive gene may also be one in which its expression
may be selectively blocked either permanently or temporarily.
Permanent blocking may be achieved by supplying means to delete the
gene in response to a signal. An example of such a means is the
cre-lox system where phage lox sites are provided at either end of
the transgene, or at least between a sufficient portion thereof
(e.g. in two exons located either side or one or more introns).
Expression of a cre recombinase causes excision and circularisation
of the nuclei acid between the two lox sites. Various lines of
transgenic animals, particularly mice, are currently available in
the art which express cre recombinase in a developmentally or
tissue restricted manner, see for example Tsien, Cell, Vol. 87(7):
1317-1326, (1996) and Betz, Current Biology, Vol. 6(10): 1307-1316
(1996). These animals may be crossed with lox transgenic animals of
the invention to examine the function of the gene encoding a target
protein of the present invention. An alternative mechanism of
control is to supply a promoter from a tetracycline resistance
gene, tet, to the control regions of the target gene locus such
that addition of tetracycline to a cell binds to the promoter and
blocks expression of the gene encoding a target protein of the
present invention. Alternatively GAL4, VP16 and other
transactivators could be used to modulate gene expression including
that of a transgene containing the gene encoding a target protein
of the present invention. Furthermore, the target gene could also
be expressed in ectopic sites, that is in sites where the gene is
not normally expressed in time or space.
[0076] Transgenic targeting techniques may also be used to delete
the gene encoding a target protein of the present invention.
Methods of targeted gene deletion are described by Brenner et al,
WO94/21787 (Cell Genesys), the disclosure of which is incorporated
herein by reference.
[0077] In a further embodiment of the invention, there is provided
a non-human animal which expresses the gene encoding a target
protein of the present invention at a higher than wild-type level.
Preferably this means that the gene encoding a target protein of
the present invention is expressed at least 120-200% of the level
found in wild-type animals of the same species, when cells which
express the gene are compared. Also, this gene could be expressed
in an ectopic location where the target gene is not normally
expressed in time or space. Comparisons may be conveniently done by
northern blotting and quantification of the transcript level. The
higher level of expression may be due to the presence of one or
more, for example two or three, additional copies of the target
gene or by modification to the gene encoding a target protein of
the present inventions to provide over-expression, for example by
introduction of a strong promoter or enhancer in operable linkage
with the wild-type gene. The provision of animals with additional
copies of genes may be achieved using the techniques described
herein for the provision of "knock-out" animals.
[0078] In another aspect, animals are provided in which the gene
encoding a target protein of the present invention is expressed at
an ectopic location. This means that the gene is expressed in a
location or at a time during development which does not occur in a
wild-type animal. For example, the gene may be linked to a
developmentally regulated promoter such as Wnt-1 and others
(Echeland, Y. Et al., Development 120, 2213-2224, 1998;
Rinkenberger, J. C. et al., Dev. Genet. 21,6- 10, 1997, or a tissue
specific promoter such as HoxB (Machonochie, M. K. et al, Genes
& Dev 11, 1885-1895, 1997).
[0079] Non-human mammalian animals include non-human primates,
rodents, rabbits, sheep, cattle, goats, pigs. Rodents include mice,
rats, and guinea pigs. Amphibians include frogs. Fish such as zebra
fish, may also be used. Transgenic non-human mammals of the
invention may be used for experimental purposes in studying cancer,
and in the development of therapies designed to alleviate the
symptoms or progression of cancer. By "experimental" it is meant
permissible for use in animal experimentation or testing purposes
under prevailing legislation applicable to the research facility
where such experimentation occurs.
[0080] Other features of the invention will be clear to the skilled
artisan, and need not be repeated here. The terms and expressions
employed herein are used as terms of description and not of
limitation; there is no intention in the use of such terms and
expressions to exclude any equivalents of the features shown and
described or portions thereof, it being recognized that various
modifications are possible within the scope of the invention.
[0081] The disclosure of all references cited herein, inasmuch as
it may be used by those skilled in the art to carry out the
invention, is hereby specifically incorporated herein by
cross-reference.
[0082] Tables and Sequences
[0083] Table 1: differentially expressed proteins identified in the
present study and already known to be upregulated in colorectal
cancer.
[0084] Table 2: protein detectable in colorectal cancer samples but
not normal colon tissue controls.
[0085] Table 3: Clinicopathological characteristics of the cases
used for proteome analysis. All the cases were Dukes C colorectal
cancers.
[0086] Sequence Annex I:
[0087] Seq ID No 1: wild-type amino acid sequence of hnRNP-K as
sourced from the public SwissProt protein sequence database
(SwissProt primary accession number Q07244).
[0088] Seq ID No 2: wild-type amino acid sequence of HMG-1 as
sourced from the public SwissProt protein sequence database
(SwissProt primary accession number P09429).
[0089] Seq ID No 3: wild-type amino acid sequence of proteasome
subunit alpha type 1 as sourced from the public SwissProt protein
sequence database (SwissProt primary accession number P25786).
[0090] Seq ID No 4: wild-type amino acid sequence of bifunctional
purine biosynthesis protein as sourced from the public SwissProt
protein sequence database (SwissProt primary accession number
P31939).
[0091] Seq ID No 5: wild-type amino acid sequence of STI1 as
sourced from the public SwissProt protein sequence database
(SwissProt primary accession number P31948).
[0092] Seq ID No 6: wild-type amino acid sequence of annexin IV as
sourced from the public SwissProt protein sequence database
(SwissProt primary accession number P09525).
[0093] Seq ID No 7: wild-type amino acid sequence of 60 kDa heat
shock protein as sourced from the public SwissProt protein sequence
database (SwissProt primary accession number P10809).
[0094] Seq ID No 8: wild-type amino acid sequence of T complex
protein 1 beta subunit as sourced from the public SwissProt protein
sequence database (SwissProt primary accession number P78371).
[0095] Seq ID No 9: wild-type amino acid sequence of T complex
protein 1 epsilon subunit as sourced from the public SwissProt
protein sequence database (SwissProt primary accession number
P48643).
[0096] Seq ID No 10: wild-type amino acid sequence of mortalin as
sourced from the public SwissProt protein sequence database
(SwissProt primary accession number P38646).
[0097] Seq ID No 11: wild-type amino acid sequence of TER-ATPase as
sourced from the public SwissProt protein sequence database
(SwissProt primary accession number P55072).
Example 1
Identification of Novel Markers
[0098] Proteins exhibiting differential expression in clinically
resected colorectal tumours and normal colon tissues were
identified as follows;
[0099] Tissue Samples
[0100] Proteomic analysis was performed on fresh frozen tissue
samples obtained from primary colorectal cancer resections and
which had been stored in the Aberdeen colorectal cancer tissue
bank. None of the patients in this study disclosed herein had
received chemotherapy or radiotherapy prior to surgery.
Representative samples of viable tumour and normal colorectal
mucosa (obtained at a distance of at least 5 cm from tumour) were
dissected from colorectal cancer excision specimens within 30
minutes of surgical removal, and these dissected samples were
immediately frozen in liquid nitrogen and stored at -80.degree. C.
prior to analysis. Proteomic analysis was performed in duplicate on
16 matched pairs of frozen tumour and normal colorectal tissue
samples. All cases selected were Dukes C colorectal cancers (see
Table 3).
[0101] Two-dimensional Gel Electrophoresis
[0102] Lysis buffer was prepared according to our established
protocols [Lawrie L, Curran S, McLeod H L, Fothergill J E, Murray G
I. (2001) Application of laser capture microdissection and
proteomics in colon cancer. Molecular Pathology 54: 253-258.] and
contained urea (42% w/v); thiourea (15% w/v); Chaps
[3-(3-cholamidopropyl) dimethylammonio-1-propanesulfonate] (4%
w/v); N-decanoyl-N-methylglucamine (Mega 10, 1% w/v),
1-0-Octyl-p-D-glucopyranoside (OBG, 1% w/v), Triton X-100
(polyoxyethylene-p-isooctylphenol) (0.5% v/v); Tris [Tris
(hydroxymethyl)aminomethane] (0.5% w/v); DTT (dithiothreitol) (0.8%
w/v); IPG 3-10 NL (immobilised pH gradient) buffer (1% v/v),
.beta.-mercaptoethanol (1% v/v), tributylphosphine (0.2% v/v). All
chemicals were obtained from Amersham Biosciences, UK, with the
exception of OBG (Aldrich, UK) and Mega 10 (Sigma, UK).
[0103] Frozen sections (10 .mu.m in thickness) of tumour and normal
were cut using a cryostat and thirty 10 .mu.m sections of normal
tissue and thirty 10 .mu.m sections of tumour tissue were
solubilised in 350 .mu.l and 500 .mu.l of lysis buffer respectively
[Lawrie L, Curran S, McLeod H L, Fothergill J E, Murray G I. (2001)
Application of laser capture microdissection and proteomics in
colon cancer. Molecular Pathology 54: 253-258]. One section from
each sample was stained with haematoxylin and eosin to confirm the
diagnosis of each tumour and normal sample; an assessment of tumour
cellularity was also made of the tumour sample. 500 .mu.g of normal
and tumour sample were loaded, in duplicate, into Immobiline. Dry
strip holders and Immobiline Drystrips, pI 3-10 NL, (Amersham
Biosciences) were placed into the strip holders. The strips were
incubated overnight at room temperature to allow the strips to
absorb the samples. After incubation the strips were removed, the
strip holders cleaned, and small pieces of dampened electrode
strips were then placed over the electrodes in the strip holders to
help absorb excess salt during the 1st dimension focusing stage.
The strips were then placed back into the strip holders and covered
with dry strip cover fluid (Amersham Biosciences). The 1st
dimension focusing was carried out on an IPGPhor system under the
following conditions; 30 min at 20V, 1.5 hr at 200V, 1.5 hr
gradient to 3500V, 35 hr at 3500V, at 15.degree. C. After
completion of focusing the strips were equilibrated for 30 min in
equilibration buffer containing urea (36% w/v); 0.5M Tris-HCl, pH
6.9 (20% v/v); 20% SDS (dodecyl sulphate, sodium salt) (20% v/v);
DTT (0. 4% w/v); glycerol (30% v/v). Strips were equilibrated for a
further 30 min in equilibration buffer where DTT was replaced by
iodoacetamide (1% w/v). All chemicals were obtained from Amersham
Biosciences.
[0104] Proteins were separated in the 2nd dimension according to
their molecular weight on a 7 cm NuPAGE 4-12%, 1 well, Bis-Tris gel
(Invitrogen, Paisley, UK). 1st dimension strips were attached to
the 2nd dimension gel with a 4% low melting point agarose solution
(Amersham Biosciences). Normal and tumour samples from the same
patient were run in the same gel tank to account for any
differences caused by the gel running process. Gels were run at a
constant 120V until the bromophenol dye front reached the end of
the gel.
[0105] Proteins were visualised using a Colloidal Blue Staining Kit
(Invitrogen, Paisley, UK). Gels were fixed in a solution containing
methanol (50% v/v), acetic acid (10% v/v) for 30 min, then
transferred to a staining solution containing methanol (20% v/v),
Stainer A (20% v/v), Stainer B (5% v/v) for overnight staining to
visualise the proteins. Gels were destained using HPLC-grade water
with microwave heating.
[0106] Detection of Differential Protein Expression
[0107] Destained gels were immediately photographed to produce a
black and white image. Gel photographs were scanned to produce a
computer image which was then enlarged and printed onto sheets of
acetate. Overlaying the normal and tumour acetate gel pictures
allowed proteins which were differentially expressed to be
detected. Differentially expressed protein spots were cut from the
gel in preparation for identification by mass spectrometry.
[0108] Identification of Proteins from Gel
[0109] Individual proteins were identified by peptide mass mapping.
Protein spots were cut from the gel, washed to remove Coomassie
stain, reduced with DTT and alkylated with iodoacetamide then
digested with trypsin. Trypsin cleaves proteins (at peptide bonds)
after arginine and lysine residues. This action produced a set of
tryptic fragments unique to each protein. The resultant tryptic
peptides were extracted from the gel pieces under full automation
(Pro-Gest Robot, Genomic Solutions). The tryptic fragments were
desalted using micro porous tips (Millipore), and deposited onto a
sample plate along with a matrix chemical
(.alpha.-cyano-4-hydroxycinnamic acid) under full automation
(Pro-MS, Genomic Solutions). The masses of the tryptic fragments
were then determined by Matrix Assisted Laser Desorption Ionisation
Time of Flight Mass Spectrometry (MALDI-TOF MS) using a PerSeptive
Biosystems Voyager-DE STR mass spectrometer.
[0110] To identify the original protein, the masses of the tryptic
peptides were entered into the MS-Fit database-searching program.
Database-searching programs attempt to match the experimentally
obtained masses of tryptic peptides with the theoretically
calculated masses of tryptic peptides derived from all proteins
within a database. The database search was restricted to search
only for human proteins, no restriction was placed on either the
molecular weight or the isoelectric point of the protein. To be
confident that the correct protein was identified, a clear
difference in statistical score between the proteins ranked first
and second in the results list had to be obtained.
[0111] The study disclosed herein identified as
differentially-expressed a number of proteins that have previously
been reported as up-regulated in colorectal tumours, thereby
validating our experimental methodology and supporting our novel
findings. These are shown in Table 1.
[0112] Increased expression of both calgranulin A (calcium binding
protein S100A8, SwissProt Accession Number P05109) and calgranulin
B (calcium binding protein S100A9, SwissProt Accession Number
P06702) has been previously reported in colorectal tissues (Stulik
J. et al, Electrophoresis 20: 1047-54, 1999). These authors
examined 23 matched sets of colorectal carcinoma and normal colon
mucosa, and found a significant increase in calgranulin A and B
expression in malignant tissues of 70% of donors.
[0113] Nucleoside diphosphate kinase A (nm23, SwissProt Accession
Number P15531) is widely regarded as a tumour marker in a variety
of cancers, but is the subject of some controversy. In certain
tumours such as metastatic ovarian carcinoma (Viel et al, Cancer
Res 55: 2645-2650, 1995), malignant melanoma (Florenes et al,
Cancer Res 52:
[0114] 6088-91, 1992), hepatocellular carcinoma (Kodera et al,
Cancer 73: 259-65, 1994) and prostate cancer (Fishman et al. J
Urol. 152: 202-7, 1994) low levels of expression of nm23 correlate
with a highly metastatic phenotype, suggesting a role for the
protein in inhibiting the process of metastasis. However, among
other tumour types, nm23 expression has no apparent relationship to
metastatic potential, and may even correlate directly with severity
in some of these cancers. For example, a 2-fold increase in nm23
expression is observed in advanced stages of thyroid carcinoma,
suggesting a direct correlation of nm23 expression with rapid cell
proliferation in thyroid cancer (Zou et al, Br J Cancer 68: 385-8,
1993). Results in colorectal tumours are confusing and
contradictory; some researchers report no significant correlation
between nm23 expression and colorectal tumour histology, serosal
invasion, lymphatic invasion, venous invasion, or lymph node
metastasis (Yamaguchi et al, Br J Cancer 68: 1020-4, 1993) whilst
others find that nm23 expression increases with local colorectal
tumour severity, and reaches even higher levels in liver metastases
(Zeng et al, Br J Cancer 70: 1025-30, 1994).
[0115] Over-expression of prohibitin (SwissProt Accession Number
P35232) has been reported in tumours from a variety of tissue
sources, including colon (Coates et al, Exp Cell Res 265: 262-73,
2001) as well as in breast cancer cell lines (Williams et al,
Electrophoresis 19: 333-43, 1998).
Example 2
Discussion of Novel Markers
[0116] TABLE 2 shows 11 novel markers not detectable in normal
tissue controls, but found in tumour samples analysed: Name:
hnRNP-K hnRNP-K is a member of the poly(C) binding proteins
(PCBPs), which are involved in mRNA stabilization, translational
activation, and translational silencing. It binds tightly to
poly(C) sequences, and is likely to play a role in the nuclear
metabolism of hnRNAs, particularly for pre-mRNAs that contain
cytidine-rich sequences. It is known to be upregulated in SV-40
transformed human keratinocytes (Dejgaard et al, J Mol Biol 236:
33-48, 1994), and to be present at higher levels in samples from
grade III human breast cancer than in samples from grade II cancer
(Mandal et al, J Biol Chem 276: 9699-704, 2001). Over-expression of
the related protein hnRNP A2/B1 (SwissProt P22626) is associated
with tumours, for example in the lung (Mulshine et al, Clin Chest
Med. 23: 37-48,2002) and pancreas (Yan-Sanders et al, Cancer Lett
183: 215-20, 2002), but hnRNP-K has not previously been recognised
as a marker of colorectal cancer. U.S. Pat. No. 6,358,683 discloses
the elevated expression of hnRNP-K in breast cancer cells and
diagnosis of breast cancer from patient blood samples by assaying
hnRNP-K amongst other markers.
[0117] As shown in Table 2, this protein was not detectable in
normal tissue controls, but was found in 14 of the 16 colorectal
tumour samples analysed. The amino acid sequence of this protein,
identified as hnRNP-K by comparison of the experimentally-derived
tryptic peptide fingerprint with entries in the MS-Fit database, is
referred to herein as SEQ ID NO:1. hnRNP-K was detected in the
tumour of six out of the eight individuals in the good survival
cohort compared with all eight of the individuals in the poor
survival cohort.
[0118] Name: HMG-1
[0119] HMG-1 ("HMGB1" in the new nomenclature, also called
amphoterin) is a nuclear architectural chromatin-binding factor
that bends DNA and promotes protein assembly on specific
recognition sequences. However, HMG-1 is also secreted by activated
monocytes and macrophages, and is passively released by necrotic or
damaged cells into their environment, where it binds with high
affinity to RAGE (the receptor for advanced glycation end products)
and is a potent mediator of inflammation and immune response. In
apoptotic cells generalized underacetylation of histone prevents
the release of HMGB1 even after partial autolysis, and thus fail to
promote inflammation even if not cleared promptly by phagocytic
cells. In this way apoptotic cells are prevented from generating
the signal that is broadcast by cells damaged or killed by trauma
(Scaffidi et al, Nature 418: 191-5, 2002). Overexpression of HMGB1
induced by steroid hormones protects chromatin/platin adducts from
the nuclear DNA repair apparatus (He et al; PNAS 97: 5768-72,
2000). HMGB1 is found in many cell types and described as a
ubiquitous nuclear protein present at a copy number of
.about.10.sup.6 per typical mammalian cell, but its expression may
be associated with a dividing, DNA-replicating phenotype. As the
study disclosed herein involves an enrichment of patient samples
for cytoplasmic proteins we might expect not to see HMGB1 in normal
samples, and it may be that in tumour cells HMGB1 is released from
nuclear sequestration and is detected by proteomic examination of
the cytoplasm. The related HMGA proteins (e.g. HMGI(Y)) are known
tumour antigens; HMGI(Y) protein has been shown to be overexpressed
in various human malignancies, including colon, prostate and
thyroid carcinomas (Chiappetta et al, Int. J. Can 91: 147-51,
2001). HMGB1 has not previously been reported as a tumour antigen,
and its use in diagnosis of cancers is not described in the patent
literature. Modulation of HMGB's interaction with the RAGE
receptor, which is known to stimulate cell mobility and replication
and to trigger inflammatory responses, is the subject of several
patent applications (e.g. WO 0047104, WO 02070473, WO 02069965, WO
0192210).
[0120] As shown in Table 2, this protein was not detectable in
normal tissue controls, but was found in 14 of the 16 colorectal
tumour samples analysed. The amino acid sequence of this protein,
identified as HMG-1 by comparison of the experimentally-derived
tryptic peptide fingerprint with entries in the MS-Fit database, is
referred to herein as SEQ ID NO:2. HMG-1 was detected in the
tumours of seven of the eight individuals in each of the good
survival and poor survival cohorts.
[0121] Name: Proteasome Subunit Alpha Type 1
[0122] Proteasome subunit alpha type 1 (also "proteasome component
C2") is a component of the 26S proteasome, a large multicatalytic
protease complex (reviewed by Naujokat & Hoffmann, Lab Invest
82: 965-80, 2002, and Coux, Prog Mol Subcell Biol. 29: 85-107,
2002). This complex, which is found in the cytoplasm and nucleus of
all eukaryotic cells, is the terminal step in the
ubiquitin-protease mechanism, which regulates basic cellular
processes through targeted degradation of regulatory proteins such
as those governing the cell cycle. The 26S proteasome complex is
composed of the barrel-shaped 20S catalytic core unit capped at
each end by the 19S regulatory complex. The non-catalytic alpha
subunits form the outer surface of the 26S barrel, and mediate
substrate translocation into the central cavity and interaction
between the 20S and 19S subunits. Whilst derangement of proteasome
function is a known feature of certain diseases, including cancer
and neurodegenerative conditions, and inhibition of proteasome
function is a therapeutic goal of cancer research (see for example
Shah et al, Surgery 131: 595-600, 2002), up-regulation of
expression of subunit alpha type 1 has not previously been observed
in cancer. U.S. Pat. No. 5,843,715 teaches the use of
genetically-engineered variant proteasome subunits to direct
antigen processing and presentation towards desired antigens (e.g.
tumour antigens, Alzheimer's proteins etc) for therapeutic
benefit.
[0123] As shown in Table 2, this protein was not detectable in
normal tissue controls, but was found in 13 of the 16 colorectal
tumour samples analysed. The amino acid sequence of this protein,
identified as proteasome subunit alpha type 1 by comparison of the
experimentally-derived tryptic peptide fingerprint with entries in
the MS-Fit database, is referred to herein as SEQ ID NO:3.
[0124] Proteasome subunit alpha type 1 was detected in the tumours
of six of the eight individuals in the good survival cohort and
seven of the eight individuals in the poor survival cohort.
[0125] Name: Bifunctional Purine Biosynthesis Protein
[0126] The human purH gene encodes this 591-amino acid bifunctional
protein which exhibits the final two activities of the purine
nucleotide biosynthetic pathway, AICARFT and IMPCH, located within
the C-terminal and N-terminal regions, respectively (Rayl et al., J
Biol Chem 271: 2225-33, 1996; Sugita et al, J Biochem (Tokyo) 122:
309-13 1997). As with another enzymatic activity earlier in the
pathway, glycinamide ribonucleotide formyltransferase (GARFT), it
requires a reduced folate cofactor, 10-formyltetrahydrofolate.
AICARFT inhibition is thought to be the origin of the anti-purine
effects of anti-folates such as methotrexate whose primary target
is dihydrofolate reductase (Budzik et al, Life Sci 66: 2297-307,
2000). Due to the central role played by this pathway in the
synthesis of nucleotides for DNA replication, its component enzymes
are of interest as targets for chemotherapy (see for example
Greasley et al, Nat Struct Biol 8: 402-6). Inhibitors of GARFT are
currently in clinical trials as anti-neoplastic agents (e.g.
Tularik Inc.'s T64/lometrexol, Eli Lilly's LY309887, Agouron
Pharmaceuticals' AG2037). WO0056924 discloses an association
between biallelic markers of a PURH gene and cancer, particularly
prostate cancer, and provides means to determine the predisposition
of individuals to cancer as well as means for the diagnosis of
cancer and for the prognosis/detection of an eventual treatment
response to agents acting on cancer. WO9413295 and WO0013688
disclose inhibitors of GARFT and AICARFT, and their use as
antiproliferative agents.
[0127] As shown in Table 2, this protein was not detectable in
normal tissue controls, but was found in 12 of the 16 colorectal
tumour samples analysed. The amino acid sequence of this protein,
identified as bifunctional purine biosynthesis protein by
comparison of the experimentally-derived tryptic peptide
fingerprint with entries in the MS-Fit database, is referred to
herein as SEQ ID NO:4. Bifunctional purine biosynthesis protein was
detected in the tumours of five of the eight individuals in the
good survival cohort compared with seven of the eight individuals
in the poor survival cohort.
[0128] Name: STI-1
[0129] The molecular chaperone Hsp90 plays an essential role in the
folding and activation of a set of client proteins involved in cell
cycle regulation, signal transduction, and responsiveness to
steroid hormone, in a manner dependent on its own endogenous ATPase
activity (reviewed by Pearl & Prodromou, Curr Opin Struct Biol
10: 46-51, 2000). From experiments in yeast it is known that for
some client proteins the co-chaperone stress-inducible protein 1
(STI1, also called Hsp70/Hsp90 organizing protein (Hop) or p60)
acts as a scaffold for assembly of the Hsp70/Hsp90 chaperone
heterocomplex, recruiting Hsp70 and the bound client to Hsp90 and
sterically inhibiting Hsp90 ATPase activity during assembly
(Johnson et al, J Biol Chem 273: 3679-86,1998; Richter et al, J
Biol Chem. Jan 13th 2003 [e-publication ahead of print]). The
phosphorylation of murine STI1 by casein kinase II (CKII) at 5189
and by cdc2 kinase (p34cdc2) at T198 has been implicated as a
potential cell cycle checkpoint (Longshaw et al, Biol Chem 381:
1133-8, 2000). STI1 has also been observed to bind TCP-1, subunits
beta and epsilon of which were also identified as cancer markers in
the present study, stimulating its nucleotide exchange activity
(Gebauer et al, J Biol Chem 273: 29475-80, 1998).
[0130] As shown in Table 2, this protein was not detectable in
normal tissue controls, but was found in 12 of the 16 colorectal
tumour samples analysed. The amino acid sequence of this protein,
identified as STI1 by comparison of the experimentally-derived
tryptic peptide fingerprint with entries in the MS-Fit database, is
referred to herein as SEQ ID NO:5. STI1 was detected in the tumours
of five of the eight individuals in the good survival cohort and
seven of the eight individuals in the poor survival cohort.
[0131] Name: Annexin IV
[0132] Annexin IV (also called annexin A4) is a member of the
annexin family of Ca2+- and phospholipid-binding proteins, which
have poorly defined functions in membrane-related events along
exocytotic and endocytotic pathways. Annexin IV is threonine
phosphorylated by protein kinase C (Kaetzel et al, Biochemistry 40:
4192-9, 2001), binds to surfactant protein A in a
CA.sup.2+-dependent manner (Sohma et al, Biochem. J. 312: 175-81,
1995) and may bind to glycosylphosphatidylinositol-anchored
glycoprotein GP-2, a major component of the zymogen granule
membrane (Tsujii-Hayashi et al, J Biol Chem 277: 47493-9, 2002).
IHC analysis in a broad variety of human tissues indicates that
annexin IV is almost exclusively found in epithelial cells (Dreier
et al, Histochem Cell Biol 110: 137-48, 1998). The over-expression
of annexin IV in C6 cells by transfection with annexin IV-DNA
induces activation of NFkappaB (Ohkawa et al, Biochim Biophys Acta
1588: 217, 2002), while the Fas-induced cell death of Jurkat
T-lymphocytes is accompanied by translocation of annexin IV from
the nucleus to the cytosol (Gerner et al, J Biol Chem 275:
39018-26, 2000). The protein also plays a role in the paclitaxel
resistant phenotype of the H460/T800 cell line and is among the
earliest proteins induced in cells in response to cytotoxic stress
such as antimitotic drug treatment (Han et al, Br J Cancer 83:
83-8, 2000). Elevated levels of annexin IV have been demonstrated
by specific double-antibody radioimmunoassay in the sera of 47.3%
(35 of 74) cervical cancer patients and 41.9% (18 of 43)
endometrial cancer patients (Gocze et al, Strahlenther Onkol 167:
538-44, 1991). The related protein annexin II appears to be
overexpressed in advanced colorectal carcinoma and may be related
to the progression and metastatic spread of the disease (Emoto et
al, Cancer 92: 1419-26, 2001) but annexin IV has not previously
been identified as a marker of colorectal tumours. WO0111372
discusses the diagnosis of cancers by means of detecting increased
expression of annexin proteins in biological samples, but provides
examples of annexins I and II only, whilst WO0012547 discusses use
of annexin V as a marker in the diagnosis of cancer. U.S. Pat. No.
5,316,915 describes the production of an antibody capture assay to
detect antibodies against annexins (including annexin IV) within
human body fluids.
[0133] As shown in Table 2, this protein was not detectable in
normal tissue controls, but was found in 11 of the 15 colorectal
tumour samples analysed. The amino acid sequence of this protein,
identified as annexin IV by comparison of the
experimentally-derived tryptic peptide fingerprint with entries in
the MS-Fit database, is referred to herein as SEQ ID NO:6. Annexin
IV was detected in the tumours of four of the eight individuals in
the good survival cohort compared with seven of the seven
individuals in the poor survival cohort, showing that this
particular protein constitutes a useful prognostic indicator in
certain cancers such as colorectal carcinoma.
[0134] Name: 60 kDa Heat Shock Protein
[0135] 60 kDa heat shock protein (hsp60) is abundant in a variety
of mammalian cells under normal conditions (Welch et al, Physiol.
Rev. 72: 1063-81, 1992), where its major functions are protein
chaperoning and protein folding (reviewed by Bukau & Horwich,
Cell 92: 351-66, 1998). Both processes are co-regulated by hsp10.
hsp60 has been shown to bind a diverse range of cellular protein
components; recently identified binding partners include the
calcineurin B regulatory subunit of the
Ca.sup.2+/calmodulin-dependent protein phosphatase calcineurin (Li
& Handschumacher, Biochim Biophys Acta 1599: 72-81, 2002), the
human hepatitis B virus polymerase (Park & Jung, J. Virol. 75:
6962-8, 2001), integrin alpha 3 beta 1 (Barazi et al, Cancer Res
62: 1541-8, 2002), the infectious prion protein PrP (Stockel &
Hartl, J Mol Biol 313: 861-7, 2001) and a receptor molecule on
macrophages found to be distinct from receptors for other members
of the heat shock protein family (Habich et al, J Immunol 168:
569-76, 2002). Whereas aberrant expression of hsp60 has been
associated with autoimmune disease, hsp60 has a role together with
hsp70 in antigen presentation in malignant diseases (Multhoff et
al, Int. J. Cancer 61: 272-279, 1995), with enhanced hsp60
expression reported in myeloid leukaemia (Chant et al, Br. J.
Haematol., 90: 163-8, 1995), breast carcinoma (Franzen et al,
Electrophoresis, 18: 582-7, 1997 ; Bini et al, Electrophoresis 18:
2832-41,1997) and prostate cancers (Cornford et al, Cancer Res 60:
7099-105, 2000). Two proteins of 40 kDa and 47 kDa have been
detected in colorectal tumours and not in normal tissue via
immunoblotting with an antibody to hsp60 (Otaka et al, J Clin
Gastroenterol 21: 224-9, 1995), but up-regulation of hsp60
expression in colorectal tumours has not previously been
definitively observed. U.S. Pat. No. 5,434,046 discusses prognosis
of ovarian cancer treated with cis-platin by measurement of Hsp60
expression. As shown in Table 2, this protein was not detectable in
normal tissue controls, but was found in 11 of the 16 colorectal
tumour samples analysed. The amino acid sequence of this protein,
identified as 60 kDa heat shock protein by comparison of the
experimentally-derived tryptic peptide fingerprint with entries in
the MS-Fit database, is referred to herein as SEQ ID NO:7.60 kDa
heat shock protein was detected in the tumours of four of the eight
individuals in the good survival cohort compared with seven of the
eight individuals in the poor survival cohort, showing that this
particular protein constitutes a useful prognostic indicator in
certain cancers such as colorectal carcinoma.
[0136] Name: T Complex Protein 1 Beta Subunit
[0137] The TCP-1 ring complex (TRiC; also called CCT, for
chaperonin containing TCP-1) is a large (approximately 900 kDa)
ring-shaped multisubunit complex consisting of eight different, yet
homologous, subunits ranging between 50 and 60 kDa, which include
the TCP-1 beta species (Frydman et al, EMBO J. 11: 4767-78, 1992 ;
Gao et al, Cell 69: 1043-50, 1992; Lewis et al, Nature 358:
249-252, 1992). TCP-1 mediates protein folding of an as yet poorly
defined physiological substrate spectrum in the eukaryotic cytosol,
binding client proteins within its central cavity and inducing
their folding by an ATP-dependent mechanism. Genetic and
biochemical data show that it is required for the folding of the
cytoskeletal proteins actin and tubulin, and TCP-1 was originally
proposed to be a chaperone specialized for the folding of these
proteins (Lewis et al, J. Cell Biol. 132: 1-4, 1996). However,
recent experiments suggest a broader substrate spectrum of distinct
proteins that require TCP-1 for proper folding in vivo, including
the Von Hippel-Lindau tumour suppressor protein and cyclin E (Dunn
et al, J Struct Biol 135: 176-84, 2001). Analysis of
auto-antibodies in sera from astrocytoma patients identified TCP-1
as a potential preferentially-expressed antigen in such tumours
(Schmits R et al, Int J Cancer 98: 73-7, 2002), and expression of
the TCP-1 subunit has been linked with the S to G2/M phase
transition of the cell cycle (Dittmar et al, Cell Biol Int 21:
383-91, 1997).
[0138] As shown in Table 2, this protein was not detectable in
normal tissue controls, but was found in 11 of the 16 colorectal
tumour samples analysed. The amino acid sequence of this protein,
identified as T complex protein 1 beta subunit by comparison of the
experimentally-derived tryptic peptide fingerprint with entries in
the MS-Fit database, is referred to herein as SEQ ID NO:8. T
complex protein 1 beta subunit was detected in the tumours of five
of the eight individuals in the good survival cohort and six of the
eight individuals in the poor survival cohort.
[0139] Name: T Complex Protein 1 Epsilon Subunit
[0140] The TCP-1 ring complex (TRiC; also called CCT, for
chaperonin containing TCP-1) is a large (approximately 900 kDa)
ring-shaped multisubunit complex consisting of eight different, yet
homologous, subunits ranging between 50 and 60 kDa, which include
the TCP-1 epsilon species (Frydman et al, EMBO J. 11: 4767-78,1992;
Gao et al, Cell 69: 1043-50, 1992; Lewis et al, Nature 358:
249-252, 1992). TCP-1 mediates protein folding of an as yet poorly
defined physiological substrate spectrum in the eukaryotic cytosol,
binding client proteins within its central cavity and inducing
their folding by an ATP-dependent mechanism. Genetic and
biochemical data show that it is required for the folding of the
cytoskeletal proteins actin and tubulin, and TCP-1 was originally
proposed to be a chaperone specialized for the folding of these
proteins (Lewis et al, J. Cell Biol. 132: 1-4, 1996). However,
recent experiments suggest a broader substrate spectrum of distinct
proteins that require TCP-1 for proper folding in vivo, including
the Von Hippel-Lindau tumour suppressor protein and cyclin E (Dunn
et al, J Struct Biol 135: 176-84, 2001). Epstein-Barr virus-encoded
nuclear protein EBNA-3 is known to interact with the TCP-1
epsilon-subunit (Kashuba et al, J Hum Virol. 2: 33-7, 1999).
Analysis of auto-antibodies in sera from astrocytoma patients
identified TCP-1 as a potential preferentially-expressed antigen in
such tumours (Schmits R et al, Int J Cancer 98: 73-7, 2002), and
expression of the TCP-1 subunit has been linked with the S to G2/M
phase transition of the cell cycle (Dittmar et al, Cell Biol Int
21: 383-91, 1997).
[0141] As shown in Table 2, this protein was not detectable in
normal tissue controls, but was found in 11 of the 16 colorectal
tumour samples analysed. The amino acid sequence of this protein,
identified as T complex protein 1 epsilon subunit by comparison of
the experimentally-derived tryptic peptide fingerprint with entries
in the MS-Fit database, is referred to herein as SEQ ID NO:9. T
complex protein 1 epsilon subunit was detected in the tumour of
only four of the eight individuals in the good survival cohort
compared with seven of the eight individuals in the poor survival
cohort, showing that this particular protein constitutes a useful
prognostic indicator in certain cancers such as colorectal
carcinoma.
[0142] Name: Mortalin
[0143] Human mortalin (also called stress-70 protein) is a member
of the hsp 70 family of proteins initially identified by virtue of
its association with a cellular mortal phenotype. The subcellular
localisation of mortalin is distinct in normal and immortalised
cells, with cytosolic and perinuclear distribution patterns
distinguishing the mortal phenotype from the immortal,
respectively. Consistently, the cytosolic mortalin is seen to have
a senescence-inducing function in contrast to the perinuclear
mortalin which has no detectable effect on cellular phenotype
(Wadhwa et al, Histol Histopathol 17: 1173-7, 2002; Kaul et al, Exp
Gerontol 37: 1157-64, 2002). The human mortalin gene, HSPA9, has
been localized to chromosome 5, band q31, a region that is
frequently deleted in myeloid leukaemias and myelodysplasia, making
it a candidate tumour suppressor gene, and has presumed functions
in the stress response, intracellular trafficking, antigen
processing, control of cell proliferation, differentiation and
tumourigenesis. Transfection of murine 3T3 cells with human
mortalin cDNA results in their malignant transformation (Wadhwa et
al, J Biol Chem 268: 6615-21, 1993), as does transfection with the
murine homologue mot-2 (Kaul et al, Oncogene 17: 907-11, 1998),
which is known to bind and inactivate the tumour suppressor protein
p53 (Wadhwa et al, J. Biol. Chem 273: 29586-91, 1998). A number of
binding partners for mortalin have confirmed, including glucose
regulated protein 94 (Takano et al, Biochem J. 357: 393-8, 2001),
fibroblast growth factor 1 (Mizukoshi et al, Biochem J. 343: 461-6,
1999) and the interleukin 1 receptor type 1 (Sacht et al,
Biofactors 9: 49-60, 1999). Proteomic analysis of human breast
ductal carcinoma and histologically normal tissue has identified
mortalin as highly expressed in all carcinoma specimens, and less
intense and occasionally undetectable in normal tissue (Bini et al,
Electrophoresis 18: 2832-41, 1997). Mortalin expression is also
elevated in a variety of neurological tumours, including low-grade
astrocytoma, anaplastic astrocytoma, glioblastoma, meningiomas,
neurinomas, pituitary adenomas, and metastases thereof (Takano et
al, Exp Cell Res 237: 38-45, 1997). WO0144807 describes a screening
system for drugs that disrupt interaction of p53 with mortalin.
[0144] US 56270394 discusses characterisation of intracellular
mortalin expression with an anti-mortalin antibody and comparison
with the "complementation group" of immortalised cells, which is
described as an indicator of cellular mortality/immortality
phenotype.
[0145] As shown in Table 2, this protein was not detectable in
normal tissue controls, but was found in 9 of the 16 colorectal
tumour samples analysed. The amino acid sequence of this protein,
identified as mortalin by comparison of the experimentally-derived
tryptic peptide fingerprint with entries in the MS-Fit database, is
referred to herein as SEQ ID NO:10. Mortalin was detected in the
tumour of only three of the eight individuals in the good survival
cohort compared with six of the eight individuals in the poor
survival cohort, showing that this particular protein constitutes a
useful prognostic indicator in certain cancers such as colorectal
carcinoma.
[0146] Name: TER-ATPase TER ATPase, also called valosin-containing
protein (VCP), is the mammalian homologue of the Saccharomyces
cerevisiae cell cycle control protein cdc48p (reviewed by Wojcik,
Trends Cell Biol 12: 212, 2002). It is a homohexameric protein
exhibiting a nuclear and cytoplasmic distribution, which has
multiple biological functions and has been shown in murine cells to
be tyrosine phosphorylated in response to T cell antigen receptor
activation, which may provide a link. between TCR ligand binding
and cell cycle control (Egerton et al, EMBO J 11: 3533-40, 1992).
TER ATPase acts as a chaperone in membrane fusions involved in the
transfer of transition vesicles from the transitional endoplasmic
reticulum to the Golgi apparatus. It also physically targets
ubiquitinated nuclear factor kappaB inhibitor to the proteasome for
degradation (Dai et al, J Biol Chem 273: 3562-73, 1998). Loss of
TER ATPase function results in an inhibition of Ub-Pr- mediated
degradation and an accumulation of ubiquitinated proteins,
suggesting a role in the degradation of multiple Ub-Pr pathway
substrates (Dai & Li, Nat Cell Biol 3: 740-4, 2001). TER ATPase
is reported to bind residues 303-625 of the BRCA1 protein via its
N-terminal region, and may thereby participate as an ATP
transporter in DNA damage-repair functions (Zhang et al, DNA Cell
Biol 19: 253-63, 2000). Stimulation of TER ATPase-transfected Dunn
cells (a murine osteosarcoma cell line) with TNFalpha induced
persistent activation of NFkappaB via enhanced cytoplasmic
degradation of p-IkappaBalpha, a reduced rate of apoptosis and
increased metastatic potential when used to inoculate male C3H mice
(Asai et al, Jpn J Cancer Res 93: 296-304, 2002). Hepatocellular
carcinoma (HCC) patients who had more TER ATPase in their tumours
than in normal endothelial tissue showed a higher rate of portal
vein invasion in the tumour and poorer disease-free and overall
survival than patients in whose tumour cells the staining intensity
was weaker than in normal tissue, making TER ATPase a prognostic
indicator in patients with HCC (Yamamoto et al, J Clin Oncol 21:
447-52, 2003). WO0216938 discusses a method of screening for
compounds that treat neurodegenerative diseases by inhibiting
binding of TER ATPase to an abnormal protein substrate. As shown in
Table 2, this protein was not detectable in normal tissue controls,
but was found in 7 of the 16 colorectal tumour samples analysed.
The amino acid sequence of this protein, identified as TER ATPase
by comparison of the experimentally-derived tryptic peptide
fingerprint with entries in the MS-Fit database, is referred to
herein as SEQ ID NO:11. TER ATPase was detected in the tumour of
only one of the eight individuals in the good survival cohort
compared with six of the eight individuals in the poor survival
cohort, showing that this particular protein constitutes a useful
prognostic indicator in certain cancers such as colorectal
carcinoma.
TABLE-US-00001 TABLE 1 Differentially expressed proteins identified
in the present study and already known to be upregulated in
colorectal cancer Abbreviated SwissProt frequency of name Accession
No. full name up-regulation calgranulin A P05109 S100 calcium
binding 10/13 (77%) protein A8 calgranulin B P06702 S100 calcium
binding 12/16 (75%) protein A9 nm-23 P15531 nucleoside diphosphate
12/16 (75%) kinase A -- P35232 prohibitin 9/16 (56%)
TABLE-US-00002 TABLE 2 Proteins detectable in colorectal cancer
samples but not normal colon tissue controls: novel findings of the
present study SwissProt Abbreviated Accession frequency of name No.
full name up-regulation hnRNP K Q07244 heterogeneous nuclear 14/16
(88%) riboprotein K HMGB1 P09429 high mobility group protein 1
14/16 (88%) (amphoterin) -- P25786 proteasome subunit alpha type 1
13/16 (81%) PURH P31939 bifunctional purine 12/16 (75%)
biosynthesis protein STI1 P31948 stress-induced phosphoprotein 1
12/16 (75%) -- P09525 annexin IV (annexin A4) 11/15 (73%) Hsp60
P10809 60 kDa heat shock protein 11/16 (69%) TCP-1.beta. P78371
T-complex protein 1, beta 11/16 (69%) subunit TCP-1.epsilon. P48643
T-complex protein 1, epsilon 11/69 (69%) subunit mortalin P38646
mitochondrial stress-70 protein 9/16 (56%) TER ATPase P55072
transitional endoplasmic 7/16 (44) reticulum ATPase
TABLE-US-00003 TABLE 3 Clinicopathological characteristics of the
cases used for proteome analysis. All the cases were Dukes C
colorectal cancers. characteristics Sex Male: n = 8 Female: n = 8
Age <55 years: n = 6 .gtoreq.55 years: n = 10 Site Proximal: n =
8 Distal: n = 8 Tumour differentiation Well: n = 0 Moderate: n = 15
Poor: n = 1
TABLE-US-00004 TABLE 4 full name example ligands heterogeneous
cytidine-rich polyribonucleotides nuclear riboprotein K high
mobility group Chromatin protein 1 (amphoterin) proteasome subunit
other components of the 26S proteasome, including the 20S alpha and
19S subunits type 1 bifunctional purine the purine synthesis
intermediates AICAR (aminoimidazole biosynthesis protein
carboxamide ribonucleotide) and FAICAR (formylaminoimidazole
carboxamide ribonucleotide) stress-induced 70 kDa heat shock
protein, 90 kDa heat shock protein, case phosphoprotein 1 in kinase
II, cdc2 kinase or T complex protein 1 annexin IV (annexin protein
kinase C, surfactant protein A or A4)
glycosylphosphatidylinositol-anchored glycoprotein GP-2. 60 kDa
heat shock protein calcineurin B, the human hepatitis B virus
polymerase, integrin alpha 3 beta 1 or the infectious prion protein
PrP T-complex protein 1, actin, tubulin, the Von Hippel-Lindau
tumour suppressor beta subunit protein or cyclin E T-complex
protein 1, actin, tubulin, the Von Hippel-Lindau tumour suppressor
epsilon subunit protein, cyclin E or the Epstein-Barr virus-encoded
nuclear protein EBNA-3 mitochondrial Glucose regulated protein 94,
fibroblast growth factor 1 stress-70 or the interleukin 1 receptor
type 1 protein transitional nuclear factor kappa B inhibitor
endoplasmic reticulum ATPase
Sequence CWU 1
1
111463PRTHomo sapienshnRNP-K 1Met Glu Thr Glu Gln Pro Glu Glu Thr
Phe Pro Asn Thr Glu Thr Asn1 5 10 15Gly Glu Phe Gly Lys Arg Pro Ala
Glu Asp Met Glu Glu Glu Gln Ala 20 25 30Phe Lys Arg Ser Arg Asn Thr
Asp Glu Met Val Glu Leu Arg Ile Leu 35 40 45Leu Gln Ser Lys Asn Ala
Gly Ala Val Ile Gly Lys Gly Gly Lys Asn 50 55 60Ile Lys Ala Leu Arg
Thr Asp Tyr Asn Ala Ser Val Ser Val Pro Asp65 70 75 80Ser Ser Gly
Pro Glu Arg Ile Leu Ser Ile Ser Ala Asp Ile Glu Thr 85 90 95Ile Gly
Glu Ile Leu Lys Lys Ile Ile Pro Thr Leu Glu Glu Gly Leu 100 105
110Gln Leu Pro Ser Pro Thr Ala Thr Ser Gln Leu Pro Leu Glu Ser Asp
115 120 125Ala Val Glu Cys Leu Asn Tyr Gln His Tyr Lys Gly Ser Asp
Phe Asp 130 135 140Cys Glu Leu Arg Leu Leu Ile His Gln Ser Leu Ala
Gly Gly Ile Ile145 150 155 160Gly Val Lys Gly Ala Lys Ile Lys Glu
Leu Arg Glu Asn Thr Gln Thr 165 170 175Thr Ile Lys Leu Phe Gln Glu
Cys Cys Pro His Ser Thr Asp Arg Val 180 185 190Val Leu Ile Gly Gly
Lys Pro Asp Arg Val Val Glu Cys Ile Lys Ile 195 200 205Ile Leu Asp
Leu Ile Ser Glu Ser Pro Ile Lys Gly Arg Ala Gln Pro 210 215 220Tyr
Asp Pro Asn Phe Tyr Asp Glu Thr Tyr Asp Tyr Gly Gly Phe Thr225 230
235 240Met Met Phe Asp Asp Arg Arg Gly Arg Pro Val Gly Phe Pro Met
Arg 245 250 255Gly Arg Gly Gly Phe Asp Arg Met Pro Pro Gly Arg Gly
Gly Arg Pro 260 265 270Met Pro Pro Ser Arg Arg Asp Tyr Asp Asp Met
Ser Pro Arg Arg Gly 275 280 285Pro Pro Pro Pro Pro Pro Gly Arg Gly
Gly Arg Gly Gly Ser Arg Ala 290 295 300Arg Asn Leu Pro Leu Pro Pro
Pro Pro Pro Pro Arg Gly Gly Asp Leu305 310 315 320Met Ala Tyr Asp
Arg Arg Gly Arg Pro Gly Asp Arg Tyr Asp Gly Met 325 330 335Val Gly
Phe Ser Ala Asp Glu Thr Trp Asp Ser Ala Ile Asp Thr Trp 340 345
350Ser Pro Ser Glu Trp Gln Met Ala Tyr Glu Pro Gln Gly Gly Ser Gly
355 360 365Tyr Asp Tyr Ser Tyr Ala Gly Gly Arg Gly Ser Tyr Gly Asp
Leu Gly 370 375 380Gly Pro Ile Ile Thr Thr Gln Val Thr Ile Pro Lys
Asp Leu Ala Gly385 390 395 400Ser Ile Ile Gly Lys Gly Gly Gln Arg
Ile Lys Gln Ile Arg His Glu 405 410 415Ser Gly Ala Ser Ile Lys Ile
Asp Glu Pro Leu Glu Gly Ser Glu Asp 420 425 430Arg Ile Ile Thr Ile
Thr Gly Thr Gln Asp Gln Ile Gln Asn Ala Gln 435 440 445Tyr Leu Leu
Gln Asn Ser Val Lys Gln Tyr Ser Gly Lys Phe Phe 450 455
4602214PRTHomo sapiensHMG-1 2Gly Lys Gly Asp Pro Lys Lys Pro Arg
Gly Lys Met Ser Ser Tyr Ala1 5 10 15Phe Phe Val Gln Thr Cys Arg Glu
Glu His Lys Lys Lys His Pro Asp 20 25 30Ala Ser Val Asn Phe Ser Glu
Phe Ser Lys Lys Cys Ser Glu Arg Trp 35 40 45Lys Thr Met Ser Ala Lys
Glu Lys Gly Lys Phe Glu Asp Met Ala Lys 50 55 60Ala Asp Lys Ala Arg
Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro Pro65 70 75 80Lys Gly Glu
Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg 85 90 95Pro Pro
Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys Ile 100 105
110Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys Leu
115 120 125Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro
Tyr Glu 130 135 140Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys
Asp Ile Ala Ala145 150 155 160Tyr Arg Ala Lys Gly Lys Pro Asp Ala
Ala Lys Lys Gly Val Val Lys 165 170 175Ala Glu Lys Ser Lys Lys Lys
Lys Glu Glu Glu Glu Asp Glu Glu Asp 180 185 190Glu Glu Asp Glu Glu
Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu Glu 195 200 205Glu Asp Asp
Asp Asp Glu 2103263PRTHomo sapiensproteasome subunit alpha type 1
3Met Phe Arg Asn Gln Tyr Asp Asn Asp Val Thr Val Trp Ser Pro Gln1 5
10 15Gly Arg Ile His Gln Ile Glu Tyr Ala Met Glu Ala Val Lys Gln
Gly 20 25 30Ser Ala Thr Val Gly Leu Lys Ser Lys Thr His Ala Val Leu
Val Ala 35 40 45Leu Lys Arg Ala Gln Ser Glu Leu Ala Ala His Gln Lys
Lys Ile Leu 50 55 60His Val Asp Asn His Ile Gly Ile Ser Ile Ala Gly
Leu Thr Ala Asp65 70 75 80Ala Arg Leu Leu Cys Asn Phe Met Arg Gln
Glu Cys Leu Asp Ser Arg 85 90 95Phe Val Phe Asp Arg Pro Leu Pro Val
Ser Arg Leu Val Ser Leu Ile 100 105 110Gly Ser Lys Thr Gln Ile Pro
Thr Gln Arg Tyr Gly Arg Arg Pro Tyr 115 120 125Gly Val Gly Leu Leu
Ile Ala Gly Tyr Asp Asp Met Gly Pro His Ile 130 135 140Phe Gln Thr
Cys Pro Ser Ala Asn Tyr Phe Asp Cys Arg Ala Met Ser145 150 155
160Ile Gly Ala Arg Ser Gln Ser Ala Arg Thr Tyr Leu Glu Arg His Met
165 170 175Ser Glu Phe Met Glu Cys Asn Leu Asn Glu Leu Val Lys His
Gly Leu 180 185 190Arg Ala Leu Arg Glu Thr Leu Pro Ala Glu Gln Asp
Leu Thr Thr Lys 195 200 205Asn Val Ser Ile Gly Ile Val Gly Lys Asp
Leu Glu Phe Thr Ile Tyr 210 215 220Asp Asp Asp Asp Val Ser Pro Phe
Leu Glu Gly Leu Glu Glu Arg Pro225 230 235 240Gln Arg Lys Ala Gln
Pro Ala Gln Pro Ala Asp Glu Pro Ala Glu Lys 245 250 255Ala Asp Glu
Pro Met Glu His 2604592PRTHomo sapiensbifunctional purine
biosynthesis protein 4Met Ala Pro Gly Gln Leu Ala Leu Phe Ser Val
Ser Asp Lys Thr Gly1 5 10 15Leu Val Glu Phe Ala Arg Asn Leu Thr Ala
Leu Gly Leu Asn Leu Val 20 25 30Ala Ser Gly Gly Thr Ala Lys Ala Leu
Arg Asp Ala Gly Leu Ala Val 35 40 45Arg Asp Val Ser Glu Leu Thr Gly
Phe Pro Glu Met Leu Gly Gly Arg 50 55 60Val Lys Thr Leu His Pro Ala
Val His Ala Gly Ile Leu Ala Arg Asn65 70 75 80Ile Pro Glu Asp Asn
Ala Asp Met Ala Arg Leu Asp Phe Asn Leu Ile 85 90 95Arg Val Val Ala
Cys Asn Leu Tyr Pro Phe Val Lys Thr Val Ala Ser 100 105 110Pro Gly
Val Thr Val Glu Glu Ala Val Glu Gln Ile Asp Ile Gly Gly 115 120
125Val Thr Leu Leu Arg Ala Ala Ala Lys Asn His Ala Arg Val Thr Val
130 135 140Val Cys Glu Pro Glu Asp Tyr Val Val Val Ser Thr Glu Met
Gln Ser145 150 155 160Ser Glu Ser Lys Asp Thr Ser Leu Glu Thr Arg
Arg Gln Leu Ala Leu 165 170 175Lys Ala Phe Thr His Thr Ala Gln Tyr
Asp Glu Ala Ile Ser Asp Tyr 180 185 190Phe Arg Lys Gln Tyr Ser Lys
Gly Val Ser Gln Met Pro Leu Arg Tyr 195 200 205Gly Met Asn Pro His
Gln Thr Pro Ala Gln Leu Tyr Thr Leu Gln Pro 210 215 220Lys Leu Pro
Ile Thr Val Leu Asn Gly Ala Pro Gly Phe Ile Asn Leu225 230 235
240Cys Asp Ala Leu Asn Ala Trp Gln Leu Val Lys Glu Leu Lys Glu Ala
245 250 255Leu Gly Ile Pro Ala Ala Ala Ser Phe Lys His Val Ser Pro
Ala Gly 260 265 270Ala Ala Val Gly Ile Pro Leu Ser Glu Asp Glu Ala
Lys Val Cys Met 275 280 285Val Tyr Asp Leu Tyr Lys Thr Leu Thr Pro
Ile Ser Ala Ala Tyr Ala 290 295 300Arg Ala Arg Gly Ala Asp Arg Met
Ser Ser Phe Gly Asp Phe Val Ala305 310 315 320Leu Ser Asp Val Cys
Asp Val Pro Thr Ala Lys Ile Ile Ser Arg Glu 325 330 335Val Ser Asp
Gly Ile Ile Ala Pro Gly Tyr Glu Glu Glu Ala Leu Thr 340 345 350Ile
Leu Ser Lys Lys Lys Asn Gly Asn Tyr Cys Val Leu Gln Met Asp 355 360
365Gln Ser Tyr Lys Pro Asp Glu Asn Glu Val Arg Thr Leu Phe Gly Leu
370 375 380His Leu Ser Gln Lys Arg Asn Asn Gly Val Val Asp Lys Ser
Leu Phe385 390 395 400Ser Asn Val Val Thr Lys Asn Lys Asp Leu Pro
Glu Ser Ala Leu Arg 405 410 415Asp Leu Ile Val Ala Thr Ile Ala Val
Lys Tyr Thr Gln Ser Asn Ser 420 425 430Val Cys Tyr Ala Lys Asn Gly
Gln Val Ile Gly Ile Gly Ala Gly Gln 435 440 445Gln Ser Arg Ile His
Cys Thr Arg Leu Ala Gly Asp Lys Ala Asn Tyr 450 455 460Trp Trp Leu
Arg His His Pro Gln Val Leu Ser Met Lys Phe Lys Thr465 470 475
480Gly Val Lys Arg Ala Glu Ile Ser Asn Ala Ile Asp Gln Tyr Val Thr
485 490 495Gly Thr Ile Gly Glu Asp Glu Asp Leu Ile Lys Trp Lys Ala
Leu Phe 500 505 510Glu Glu Val Pro Glu Leu Leu Thr Glu Ala Glu Lys
Lys Glu Trp Val 515 520 525Glu Lys Leu Thr Glu Val Ser Ile Ser Ser
Asp Ala Phe Phe Pro Phe 530 535 540Arg Asp Asn Val Asp Arg Ala Lys
Arg Ser Gly Val Ala Tyr Ile Ala545 550 555 560Ala Pro Ser Gly Ser
Ala Ala Asp Lys Val Val Ile Glu Ala Cys Asp 565 570 575Glu Leu Gly
Ile Ile Leu Ala His Thr Asn Leu Arg Leu Phe His His 580 585
5905543PRTHomo sapiensSTI1 5Met Glu Gln Val Asn Glu Leu Lys Glu Lys
Gly Asn Lys Ala Leu Ser1 5 10 15Val Gly Asn Ile Asp Asp Ala Leu Gln
Cys Tyr Ser Glu Ala Ile Lys 20 25 30Leu Asp Pro His Asn His Val Leu
Tyr Ser Asn Arg Ser Ala Ala Tyr 35 40 45Ala Lys Lys Gly Asp Tyr Gln
Lys Ala Tyr Glu Asp Gly Cys Lys Thr 50 55 60Val Asp Leu Lys Pro Asp
Trp Gly Lys Gly Tyr Ser Arg Lys Ala Ala65 70 75 80Ala Leu Glu Phe
Leu Asn Arg Phe Glu Glu Ala Lys Arg Thr Tyr Glu 85 90 95Glu Gly Leu
Lys His Glu Ala Asn Asn Pro Gln Leu Lys Glu Gly Leu 100 105 110Gln
Asn Met Glu Ala Arg Leu Ala Glu Arg Lys Phe Met Asn Pro Phe 115 120
125Asn Met Pro Asn Leu Tyr Gln Lys Leu Glu Ser Asp Pro Arg Thr Arg
130 135 140Thr Leu Leu Ser Asp Pro Thr Tyr Arg Glu Leu Ile Glu Gln
Leu Arg145 150 155 160Asn Lys Pro Ser Asp Leu Gly Thr Lys Leu Gln
Asp Pro Arg Ile Met 165 170 175Thr Thr Leu Ser Val Leu Leu Gly Val
Asp Leu Gly Ser Met Asp Glu 180 185 190Glu Glu Glu Ile Ala Thr Pro
Pro Pro Pro Pro Pro Pro Lys Lys Glu 195 200 205Thr Lys Pro Glu Pro
Met Glu Glu Asp Leu Pro Glu Asn Lys Lys Gln 210 215 220Ala Leu Lys
Glu Lys Glu Leu Gly Asn Asp Ala Tyr Lys Lys Lys Asp225 230 235
240Phe Asp Thr Ala Leu Lys His Tyr Asp Lys Ala Lys Glu Leu Asp Pro
245 250 255Thr Asn Met Thr Tyr Ile Thr Asn Gln Ala Ala Val Tyr Phe
Glu Lys 260 265 270Gly Asp Tyr Asn Lys Cys Arg Glu Leu Cys Glu Lys
Ala Ile Glu Val 275 280 285Gly Arg Glu Asn Arg Glu Asp Tyr Arg Gln
Ile Ala Lys Ala Tyr Ala 290 295 300Arg Ile Gly Asn Ser Tyr Phe Lys
Glu Glu Lys Tyr Lys Asp Ala Ile305 310 315 320His Phe Tyr Asn Lys
Ser Leu Ala Glu His Arg Thr Pro Asp Val Leu 325 330 335Lys Lys Cys
Gln Gln Ala Glu Lys Ile Leu Lys Glu Gln Glu Arg Leu 340 345 350Ala
Tyr Ile Asn Pro Asp Leu Ala Leu Glu Glu Lys Asn Lys Gly Asn 355 360
365Glu Cys Phe Gln Lys Gly Asp Tyr Pro Gln Ala Met Lys His Tyr Thr
370 375 380Glu Ala Ile Lys Arg Asn Pro Lys Asp Ala Lys Leu Tyr Ser
Asn Arg385 390 395 400Ala Ala Cys Tyr Thr Lys Leu Leu Glu Phe Gln
Leu Ala Leu Lys Asp 405 410 415Cys Glu Glu Cys Ile Gln Leu Glu Pro
Thr Phe Ile Lys Gly Tyr Thr 420 425 430Arg Lys Ala Ala Ala Leu Glu
Ala Met Lys Asp Tyr Thr Lys Ala Met 435 440 445Asp Val Tyr Gln Lys
Ala Leu Asp Leu Asp Ser Ser Cys Lys Glu Ala 450 455 460Ala Asp Gly
Tyr Gln Arg Cys Met Met Ala Gln Tyr Asn Arg His Asp465 470 475
480Ser Pro Glu Asp Val Lys Arg Arg Ala Met Ala Asp Pro Glu Val Gln
485 490 495Gln Ile Met Ser Asp Pro Ala Met Arg Leu Ile Leu Glu Gln
Met Gln 500 505 510Lys Asp Pro Gln Ala Leu Ser Glu His Leu Lys Asn
Pro Val Ile Ala 515 520 525Gln Lys Ile Gln Lys Leu Met Asp Val Gly
Leu Ile Ala Ile Arg 530 535 5406318PRTHomo sapiensannexin IV 6Ala
Thr Lys Gly Gly Thr Val Lys Ala Ala Ser Gly Phe Asn Ala Met1 5 10
15Glu Asp Ala Gln Thr Leu Arg Lys Ala Met Lys Gly Leu Gly Thr Asp
20 25 30Glu Asp Ala Ile Ile Ser Val Leu Ala Tyr Arg Asn Thr Ala Gln
Arg 35 40 45Gln Glu Ile Arg Thr Ala Tyr Lys Ser Thr Ile Gly Arg Asp
Leu Ile 50 55 60Asp Asp Leu Lys Ser Glu Leu Ser Gly Asn Phe Glu Gln
Val Ile Val65 70 75 80Gly Met Met Thr Pro Thr Val Leu Tyr Asp Val
Gln Glu Leu Arg Arg 85 90 95Ala Met Lys Gly Ala Gly Thr Asp Glu Gly
Cys Leu Ile Glu Ile Leu 100 105 110Ala Ser Arg Thr Pro Glu Glu Ile
Arg Arg Ile Ser Gln Thr Tyr Gln 115 120 125Gln Gln Tyr Gly Arg Ser
Leu Glu Asp Asp Ile Arg Ser Asp Thr Ser 130 135 140Phe Met Phe Gln
Arg Val Leu Val Ser Leu Ser Ala Gly Gly Arg Asp145 150 155 160Glu
Gly Asn Tyr Leu Asp Asp Ala Leu Val Arg Gln Asp Ala Gln Asp 165 170
175Leu Tyr Glu Ala Gly Glu Lys Lys Trp Gly Thr Asp Glu Val Lys Phe
180 185 190Leu Thr Val Leu Cys Ser Arg Asn Arg Asn His Leu Leu His
Val Phe 195 200 205Asp Glu Tyr Lys Arg Ile Ser Gln Lys Asp Ile Glu
Gln Ser Ile Lys 210 215 220Ser Glu Thr Ser Gly Ser Phe Glu Asp Ala
Leu Leu Ala Ile Val Lys225 230 235 240Cys Met Arg Asn Lys Ser Ala
Tyr Phe Ala Glu Lys Leu Tyr Lys Ser 245 250 255Met Lys Gly Leu Gly
Thr Asp Asp Asn Thr Leu Ile Arg Val Met Val 260 265 270Ser Arg Ala
Glu Ile Asp Met Leu Asp Ile Arg Ala His Phe Lys Arg 275 280 285Leu
Tyr Gly Lys Ser Leu Tyr Ser Phe Ile Lys Gly Asp Thr Ser Gly 290 295
300Asp Tyr Arg Lys Val Leu Leu Val Leu Cys Gly Gly Asp Asp305 310
3157573PRTHomo sapiens60 kDa heat shock protein 7Met Leu Arg Leu
Pro Thr Val Phe Arg Gln Met Arg Pro Val Ser Arg1 5 10 15Val Leu Ala
Pro His Leu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe 20 25 30Gly Ala
Asp Ala Arg Ala Leu Met Leu Gln Gly Val Asp Leu Leu Ala 35 40 45Asp
Ala Val Ala Val Thr Met Gly
Pro Lys Gly Arg Thr Val Ile Ile 50 55 60Glu Gln Ser Trp Gly Ser Pro
Lys Val Thr Lys Asp Gly Val Thr Val65 70 75 80Ala Lys Ser Ile Asp
Leu Lys Asp Lys Tyr Lys Asn Ile Gly Ala Lys 85 90 95Leu Val Gln Asp
Val Ala Asn Asn Thr Asn Glu Glu Ala Gly Asp Gly 100 105 110Thr Thr
Thr Ala Thr Val Leu Ala Arg Ser Ile Ala Lys Glu Gly Phe 115 120
125Glu Lys Ile Ser Lys Gly Ala Asn Pro Val Glu Ile Arg Arg Gly Val
130 135 140Met Leu Ala Val Asp Ala Val Ile Ala Glu Leu Lys Lys Gln
Ser Lys145 150 155 160Pro Val Thr Thr Pro Glu Glu Ile Ala Gln Val
Ala Thr Ile Ser Ala 165 170 175Asn Gly Asp Lys Glu Ile Gly Asn Ile
Ile Ser Asp Ala Met Lys Lys 180 185 190Val Gly Arg Lys Gly Val Ile
Thr Val Lys Asp Gly Lys Thr Leu Asn 195 200 205Asp Glu Leu Glu Ile
Ile Glu Gly Met Lys Phe Asp Arg Gly Tyr Ile 210 215 220Ser Pro Tyr
Phe Ile Asn Thr Ser Lys Gly Gln Lys Cys Glu Phe Gln225 230 235
240Asp Ala Tyr Val Leu Leu Ser Glu Lys Lys Ile Ser Ser Ile Gln Ser
245 250 255Ile Val Pro Ala Leu Glu Ile Ala Asn Ala His Arg Lys Pro
Leu Val 260 265 270Ile Ile Ala Glu Asp Val Asp Gly Glu Ala Leu Ser
Thr Leu Val Leu 275 280 285Asn Arg Leu Lys Val Gly Leu Gln Val Val
Ala Val Lys Ala Pro Gly 290 295 300Phe Gly Asp Asn Arg Lys Asn Gln
Leu Lys Asp Met Ala Ile Ala Thr305 310 315 320Gly Gly Ala Val Phe
Gly Glu Glu Gly Leu Thr Leu Asn Leu Glu Asp 325 330 335Val Gln Pro
His Asp Leu Gly Lys Val Gly Glu Val Ile Val Thr Lys 340 345 350Asp
Asp Ala Met Leu Leu Lys Gly Lys Gly Asp Lys Ala Gln Ile Glu 355 360
365Lys Arg Ile Gln Glu Ile Ile Glu Gln Leu Asp Val Thr Thr Ser Glu
370 375 380Tyr Glu Lys Glu Lys Leu Asn Glu Arg Leu Ala Lys Leu Ser
Asp Gly385 390 395 400Val Ala Val Leu Lys Val Gly Gly Thr Ser Asp
Val Glu Val Asn Glu 405 410 415Lys Lys Asp Arg Val Thr Asp Ala Leu
Asn Ala Thr Arg Ala Ala Val 420 425 430Glu Glu Gly Ile Val Leu Gly
Gly Gly Cys Ala Leu Leu Arg Cys Ile 435 440 445Pro Ala Leu Asp Ser
Leu Thr Pro Ala Asn Glu Asp Gln Lys Ile Gly 450 455 460Ile Glu Ile
Ile Lys Arg Thr Leu Lys Ile Pro Ala Met Thr Ile Ala465 470 475
480Lys Asn Ala Gly Val Glu Gly Ser Leu Ile Val Glu Lys Ile Met Gln
485 490 495Ser Ser Ser Glu Val Gly Tyr Asp Ala Met Ala Gly Asp Phe
Val Asn 500 505 510Met Val Glu Lys Gly Ile Ile Asp Pro Thr Lys Val
Val Arg Thr Ala 515 520 525Leu Leu Asp Ala Ala Gly Val Ala Ser Leu
Leu Thr Thr Ala Glu Val 530 535 540Val Val Thr Glu Ile Pro Lys Glu
Glu Lys Asp Pro Gly Met Gly Ala545 550 555 560Met Gly Gly Met Gly
Gly Gly Met Gly Gly Gly Met Phe 565 5708535PRTHomo sapiensT complex
protein 1 beta subunit 8Met Ala Ser Leu Ser Leu Ala Pro Val Asn Ile
Phe Lys Ala Gly Ala1 5 10 15Asp Glu Glu Arg Ala Glu Thr Ala Arg Leu
Thr Ser Phe Ile Gly Ala 20 25 30Ile Ala Ile Gly Asp Leu Val Lys Ser
Thr Leu Gly Pro Lys Gly Met 35 40 45Asp Lys Ile Leu Leu Ser Ser Gly
Arg Asp Ala Ser Leu Met Val Thr 50 55 60Asn Asp Gly Ala Thr Ile Leu
Lys Asn Ile Gly Val Asp Asn Pro Ala65 70 75 80Ala Lys Val Leu Val
Asp Met Ser Arg Val Gln Asp Asp Glu Val Gly 85 90 95Asp Gly Thr Thr
Ser Val Thr Val Leu Ala Ala Glu Leu Leu Arg Glu 100 105 110Ala Glu
Ser Leu Ile Ala Lys Lys Ile His Pro Gln Thr Ile Ile Ala 115 120
125Gly Trp Arg Glu Ala Thr Lys Ala Ala Arg Glu Ala Leu Leu Ser Ser
130 135 140Ala Val Asp His Gly Ser Asp Glu Val Lys Phe Arg Gln Asp
Leu Met145 150 155 160Asn Ile Ala Gly Thr Thr Leu Ser Ser Lys Leu
Leu Thr His His Lys 165 170 175Asp His Phe Thr Lys Leu Ala Val Glu
Ala Val Leu Arg Leu Lys Gly 180 185 190Ser Gly Asn Leu Glu Ala Ile
His Ile Ile Lys Lys Leu Gly Gly Ser 195 200 205Leu Ala Asp Ser Tyr
Leu Asp Glu Gly Phe Leu Leu Asp Lys Lys Ile 210 215 220Gly Val Asn
Gln Pro Lys Arg Ile Glu Asn Ala Lys Ile Leu Ile Ala225 230 235
240Asn Thr Gly Met Asp Thr Asp Lys Ile Lys Ile Phe Gly Ser Arg Val
245 250 255Arg Val Asp Ser Thr Ala Lys Val Ala Glu Ile Glu His Ala
Glu Lys 260 265 270Glu Lys Met Lys Glu Lys Val Glu Arg Ile Leu Lys
His Gly Ile Asn 275 280 285Cys Phe Ile Asn Arg Gln Leu Ile Tyr Asn
Tyr Pro Glu Gln Leu Phe 290 295 300Gly Ala Ala Gly Val Met Ala Ile
Glu His Ala Asp Phe Ala Gly Val305 310 315 320Glu Arg Leu Ala Leu
Val Thr Gly Gly Glu Ile Ala Ser Thr Phe Asp 325 330 335His Pro Glu
Leu Val Lys Leu Gly Ser Cys Lys Leu Ile Glu Glu Val 340 345 350Met
Ile Gly Glu Asp Lys Leu Ile His Phe Ser Gly Val Ala Leu Gly 355 360
365Glu Ala Cys Thr Ile Val Leu Arg Gly Ala Thr Gln Gln Ile Leu Asp
370 375 380Glu Ala Glu Arg Ser Leu His Asp Ala Leu Cys Val Leu Ala
Gln Thr385 390 395 400Val Lys Asp Ser Arg Thr Val Tyr Gly Gly Gly
Cys Ser Glu Met Leu 405 410 415Met Ala His Ala Val Thr Gln Leu Ala
Asn Arg Thr Pro Gly Lys Glu 420 425 430Ala Val Ala Met Glu Ser Tyr
Ala Lys Ala Leu Arg Met Leu Pro Thr 435 440 445Ile Ile Ala Asp Asn
Ala Gly Tyr Asp Ser Ala Asp Leu Val Ala Gln 450 455 460Leu Arg Ala
Ala His Ser Glu Gly Asn Thr Thr Ala Gly Leu Asp Met465 470 475
480Arg Glu Gly Thr Ile Gly Asp Met Ala Ile Leu Gly Ile Thr Glu Ser
485 490 495Phe Gln Val Lys Arg Gln Val Leu Leu Ser Ala Ala Glu Ala
Ala Glu 500 505 510Val Ile Leu Arg Val Asp Asn Ile Ile Lys Ala Ala
Pro Arg Lys Arg 515 520 525Val Pro Asp His His Pro Cys 530
5359541PRTHomo sapiensT complex protein 1 epsilon subunit 9Met Ala
Ser Met Gly Thr Leu Ala Phe Asp Glu Tyr Gly Arg Pro Phe1 5 10 15Leu
Ile Ile Lys Asp Gln Asp Arg Lys Ser Arg Leu Met Gly Leu Glu 20 25
30Ala Leu Lys Ser His Ile Met Ala Ala Lys Ala Val Ala Asn Thr Met
35 40 45Arg Thr Ser Leu Gly Pro Asn Gly Leu Asp Lys Met Met Val Asp
Lys 50 55 60Asp Gly Asp Val Thr Val Thr Asn Asp Gly Ala Thr Ile Leu
Ser Met65 70 75 80Met Asp Val Asp His Gln Ile Ala Lys Leu Met Val
Glu Leu Ser Lys 85 90 95Ser Gln Asp Asp Glu Ile Gly Asp Gly Thr Thr
Gly Val Val Val Leu 100 105 110Ala Gly Ala Leu Leu Glu Glu Ala Glu
Gln Leu Leu Asp Arg Gly Ile 115 120 125His Pro Ile Arg Ile Ala Asp
Gly Tyr Glu Gln Ala Ala Arg Val Ala 130 135 140Ile Glu His Leu Asp
Lys Ile Ser Asp Ser Val Leu Val Asp Ile Lys145 150 155 160Asp Thr
Glu Pro Leu Ile Gln Thr Ala Lys Thr Thr Leu Gly Ser Lys 165 170
175Val Val Asn Ser Cys His Arg Gln Met Ala Glu Ile Ala Val Asn Ala
180 185 190Val Leu Thr Val Ala Asp Met Glu Arg Arg Asp Val Asp Phe
Glu Leu 195 200 205Ile Lys Val Glu Gly Lys Val Gly Gly Arg Leu Glu
Asp Thr Lys Leu 210 215 220Ile Lys Gly Val Ile Val Asp Lys Asp Phe
Ser His Pro Gln Met Pro225 230 235 240Lys Lys Val Glu Asp Ala Lys
Ile Ala Ile Leu Thr Cys Pro Phe Glu 245 250 255Pro Pro Lys Pro Lys
Thr Lys His Lys Leu Asp Val Thr Ser Val Glu 260 265 270Asp Tyr Lys
Ala Leu Gln Lys Tyr Glu Lys Glu Lys Phe Glu Glu Met 275 280 285Ile
Gln Gln Ile Lys Glu Thr Gly Ala Asn Leu Ala Ile Cys Gln Trp 290 295
300Gly Phe Asp Asp Glu Ala Asn His Leu Leu Leu Gln Asn Asn Leu
Pro305 310 315 320Ala Val Arg Trp Val Gly Gly Pro Glu Ile Glu Leu
Ile Ala Ile Ala 325 330 335Thr Gly Gly Arg Ile Val Pro Arg Phe Ser
Glu Leu Thr Ala Glu Lys 340 345 350Leu Gly Phe Ala Gly Leu Val Gln
Glu Ile Ser Phe Gly Thr Thr Lys 355 360 365Asp Lys Met Leu Val Ile
Glu Gln Cys Lys Asn Ser Arg Ala Val Thr 370 375 380Ile Phe Ile Arg
Gly Gly Asn Lys Met Ile Ile Glu Glu Ala Lys Arg385 390 395 400Ser
Leu His Asp Ala Leu Cys Val Ile Arg Asn Leu Ile Arg Asp Asn 405 410
415Arg Val Val Tyr Gly Gly Gly Ala Ala Glu Ile Ser Cys Ala Leu Ala
420 425 430Val Ser Gln Glu Ala Asp Lys Cys Pro Thr Leu Glu Gln Tyr
Ala Met 435 440 445Arg Ala Phe Ala Asp Ala Leu Glu Val Ile Pro Met
Ala Leu Ser Glu 450 455 460Asn Ser Gly Met Asn Pro Ile Gln Thr Met
Thr Glu Val Arg Ala Arg465 470 475 480Gln Val Lys Glu Met Asn Pro
Ala Leu Gly Ile Asp Cys Leu His Lys 485 490 495Gly Thr Asn Asp Met
Lys Gln Gln His Val Ile Glu Thr Leu Ile Gly 500 505 510Lys Lys Gln
Gln Ile Ser Leu Ala Thr Gln Met Val Arg Met Ile Leu 515 520 525Lys
Ile Asp Asp Ile Arg Lys Pro Gly Glu Ser Glu Glu 530 535
54010679PRTHomo sapiensmortalin 10Met Ile Ser Ala Ser Arg Ala Ala
Ala Ala Arg Leu Val Gly Ala Ala1 5 10 15Ala Ser Arg Gly Pro Thr Ala
Ala Arg His Gln Asp Ser Trp Asn Gly 20 25 30Leu Ser His Glu Ala Phe
Arg Leu Val Ser Arg Arg Asp Tyr Ala Ser 35 40 45Glu Ala Ile Lys Gly
Ala Val Val Gly Ile Asp Leu Gly Thr Thr Asn 50 55 60Ser Cys Val Ala
Val Met Glu Gly Lys Gln Ala Lys Val Leu Glu Asn65 70 75 80Ala Glu
Gly Ala Arg Thr Thr Pro Ser Val Val Ala Phe Thr Ala Asp 85 90 95Gly
Glu Arg Leu Val Gly Met Pro Ala Lys Arg Gln Ala Val Thr Asn 100 105
110Pro Asn Asn Thr Phe Tyr Ala Thr Lys Arg Leu Ile Gly Arg Arg Tyr
115 120 125Asp Asp Pro Glu Val Gln Lys Asp Ile Lys Asn Val Pro Phe
Lys Ile 130 135 140Val Arg Ala Ser Asn Gly Asp Ala Trp Val Glu Ala
His Gly Lys Leu145 150 155 160Tyr Ser Pro Ser Gln Ile Gly Ala Phe
Val Leu Met Lys Met Lys Glu 165 170 175Thr Ala Glu Asn Tyr Leu Gly
His Thr Ala Lys Asn Ala Val Ile Thr 180 185 190Val Pro Ala Tyr Phe
Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp Ala 195 200 205Gly Gln Ile
Ser Gly Leu Asn Val Leu Arg Val Ile Asn Glu Pro Thr 210 215 220Ala
Ala Ala Leu Ala Tyr Gly Leu Asp Lys Ser Glu Asp Lys Val Ile225 230
235 240Ala Val Tyr Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser Ile Leu
Glu 245 250 255Ile Gln Lys Gly Val Phe Glu Val Lys Ser Thr Asn Gly
Asp Thr Phe 260 265 270Leu Gly Gly Glu Asp Phe Asp Gln Ala Leu Leu
Arg His Ile Val Lys 275 280 285Glu Phe Lys Arg Glu Thr Gly Val Asp
Leu Thr Lys Asp Asn Met Ala 290 295 300Leu Gln Arg Val Arg Glu Ala
Ala Glu Lys Ala Lys Cys Glu Leu Ser305 310 315 320Ser Ser Val Gln
Thr Asp Ile Asn Leu Pro Tyr Leu Thr Met Asp Ser 325 330 335Ser Gly
Pro Lys His Leu Asn Met Lys Leu Thr Arg Ala Gln Phe Glu 340 345
350Gly Ile Val Thr Asp Leu Ile Arg Arg Thr Ile Ala Pro Cys Gln Lys
355 360 365Ala Met Gln Asp Ala Glu Val Ser Lys Ser Asp Ile Gly Glu
Val Ile 370 375 380Leu Val Gly Gly Met Thr Arg Met Pro Lys Val Gln
Gln Thr Val Gln385 390 395 400Asp Leu Phe Gly Arg Ala Pro Ser Lys
Ala Val Asn Pro Asp Glu Ala 405 410 415Val Ala Ile Gly Ala Ala Ile
Gln Gly Gly Val Leu Ala Gly Asp Val 420 425 430Thr Asp Val Leu Leu
Leu Asp Val Thr Pro Leu Ser Leu Gly Ile Glu 435 440 445Thr Leu Gly
Gly Val Phe Thr Lys Leu Ile Asn Arg Asn Thr Thr Ile 450 455 460Pro
Thr Lys Lys Ser Gln Val Phe Ser Thr Ala Ala Asp Gly Gln Thr465 470
475 480Gln Val Glu Ile Lys Val Cys Gln Gly Glu Arg Glu Met Ala Gly
Asp 485 490 495Asn Lys Leu Leu Gly Gln Phe Thr Leu Ile Gly Ile Pro
Pro Ala Pro 500 505 510Arg Gly Val Pro Gln Ile Glu Val Thr Phe Asp
Ile Asp Ala Asn Gly 515 520 525Ile Val His Val Ser Ala Lys Asp Lys
Gly Thr Gly Arg Glu Gln Gln 530 535 540Ile Val Ile Gln Ser Ser Gly
Gly Leu Ser Lys Asp Asp Ile Glu Asn545 550 555 560Met Val Lys Asn
Ala Glu Lys Tyr Ala Glu Glu Asp Arg Arg Lys Lys 565 570 575Glu Arg
Val Glu Ala Val Asn Met Ala Glu Gly Ile Ile His Asp Thr 580 585
590Glu Thr Lys Met Glu Glu Phe Lys Asp Gln Leu Pro Ala Asp Glu Cys
595 600 605Asn Lys Leu Lys Glu Glu Ile Ser Lys Met Arg Glu Leu Leu
Ala Arg 610 615 620Lys Asp Ser Glu Thr Gly Glu Asn Ile Arg Gln Ala
Ala Ser Ser Leu625 630 635 640Gln Gln Ala Ser Leu Lys Leu Phe Glu
Met Ala Tyr Lys Lys Met Ala 645 650 655Ser Glu Arg Glu Gly Ser Gly
Ser Ser Gly Thr Gly Glu Gln Lys Glu 660 665 670Asp Gln Lys Glu Glu
Lys Gln 67511806PRTHomo sapiensTER-ATPase 11Met Ala Ser Gly Ala Asp
Ser Lys Gly Asp Asp Leu Ser Thr Ala Ile1 5 10 15Leu Lys Gln Lys Asn
Arg Pro Asn Arg Leu Ile Val Asp Glu Ala Ile 20 25 30Asn Glu Asp Asn
Ser Val Val Ser Leu Ser Gln Pro Lys Met Asp Glu 35 40 45Leu Gln Leu
Phe Arg Gly Asp Thr Val Leu Leu Lys Gly Lys Lys Arg 50 55 60Arg Glu
Ala Val Cys Ile Val Leu Ser Asp Asp Thr Cys Ser Asp Glu65 70 75
80Lys Ile Arg Met Asn Arg Val Val Arg Asn Asn Leu Arg Val Arg Leu
85 90 95Gly Asp Val Ile Ser Ile Gln Pro Cys Pro Asp Val Lys Tyr Gly
Lys 100 105 110Arg Ile His Val Leu Pro Ile Asp Asp Thr Val Glu Gly
Ile Thr Gly 115 120 125Asn Leu Phe Glu Val Tyr Leu Lys Pro Tyr Phe
Leu Glu Ala Tyr Arg 130 135 140Pro Ile Arg Lys Gly Asp Ile Phe Leu
Val Arg Gly Gly Met Arg Ala145 150 155 160Val Glu Phe Lys Val Val
Glu Thr Asp Pro Ser Pro Tyr Cys Ile Val 165 170 175Ala Pro Asp
Thr
Val Ile His Cys Glu Gly Glu Pro Ile Lys Arg Glu 180 185 190Asp Glu
Glu Glu Ser Leu Asn Glu Val Gly Tyr Asp Asp Ile Gly Gly 195 200
205Cys Arg Lys Gln Leu Ala Gln Ile Lys Glu Met Val Glu Leu Pro Leu
210 215 220Arg His Pro Ala Leu Phe Lys Ala Ile Gly Val Lys Pro Pro
Arg Gly225 230 235 240Ile Leu Leu Tyr Gly Pro Pro Gly Thr Gly Lys
Thr Leu Ile Ala Arg 245 250 255Ala Val Ala Asn Glu Thr Gly Ala Phe
Phe Phe Leu Ile Asn Gly Pro 260 265 270Glu Ile Met Ser Lys Leu Ala
Gly Glu Ser Glu Ser Asn Leu Arg Lys 275 280 285Ala Phe Glu Glu Ala
Glu Lys Asn Ala Pro Ala Ile Ile Phe Ile Asp 290 295 300Glu Leu Asp
Ala Ile Ala Pro Lys Arg Glu Lys Thr His Gly Glu Val305 310 315
320Glu Arg Arg Ile Val Ser Gln Leu Leu Thr Leu Met Asp Gly Leu Lys
325 330 335Gln Arg Ala His Val Ile Val Met Ala Ala Thr Asn Arg Pro
Asn Ser 340 345 350Ile Asp Pro Ala Leu Arg Arg Phe Gly Arg Phe Asp
Arg Glu Val Asp 355 360 365Ile Gly Ile Pro Asp Ala Thr Gly Arg Leu
Glu Ile Leu Gln Ile His 370 375 380Thr Lys Asn Met Lys Leu Ala Asp
Asp Val Asp Leu Glu Gln Val Ala385 390 395 400Asn Glu Thr His Gly
His Val Gly Ala Asp Leu Ala Ala Leu Cys Ser 405 410 415Glu Ala Ala
Leu Gln Ala Ile Arg Lys Lys Met Asp Leu Ile Asp Leu 420 425 430Glu
Asp Glu Thr Ile Asp Ala Glu Val Met Asn Ser Leu Ala Val Thr 435 440
445Met Asp Asp Phe Arg Trp Ala Leu Ser Gln Ser Asn Pro Ser Ala Leu
450 455 460Arg Glu Thr Val Val Glu Val Pro Gln Val Thr Trp Glu Asp
Ile Gly465 470 475 480Gly Leu Glu Asp Val Lys Arg Glu Leu Gln Glu
Leu Val Gln Tyr Pro 485 490 495Val Glu His Pro Asp Lys Phe Leu Lys
Phe Gly Met Thr Pro Ser Lys 500 505 510Gly Val Leu Phe Tyr Gly Pro
Pro Gly Cys Gly Lys Thr Leu Leu Ala 515 520 525Lys Ala Ile Ala Asn
Glu Cys Gln Ala Asn Phe Ile Ser Ile Lys Gly 530 535 540Pro Glu Leu
Leu Thr Met Trp Phe Gly Glu Ser Glu Ala Asn Val Arg545 550 555
560Glu Ile Phe Asp Lys Ala Arg Gln Ala Ala Pro Cys Val Leu Phe Phe
565 570 575Asp Glu Leu Asp Ser Ile Ala Lys Ala Arg Gly Gly Asn Ile
Gly Asp 580 585 590Gly Gly Gly Ala Ala Asp Arg Val Ile Asn Gln Ile
Leu Thr Glu Met 595 600 605Asp Gly Met Ser Thr Lys Lys Asn Val Phe
Ile Ile Gly Ala Thr Asn 610 615 620Arg Pro Asp Ile Ile Asp Pro Ala
Ile Leu Arg Pro Gly Arg Leu Asp625 630 635 640Gln Leu Ile Tyr Ile
Pro Leu Pro Asp Glu Lys Ser Arg Val Ala Ile 645 650 655Leu Lys Ala
Asn Leu Arg Lys Ser Pro Val Ala Lys Asp Val Asp Leu 660 665 670Glu
Phe Leu Ala Lys Met Thr Asn Gly Phe Ser Gly Ala Asp Leu Thr 675 680
685Glu Ile Cys Gln Arg Ala Cys Lys Leu Ala Ile Arg Glu Ser Ile Glu
690 695 700Ser Glu Ile Arg Arg Glu Arg Glu Arg Gln Thr Asn Pro Ser
Ala Met705 710 715 720Glu Val Glu Glu Asp Asp Pro Val Pro Glu Ile
Arg Arg Asp His Phe 725 730 735Glu Glu Ala Met Arg Phe Ala Arg Arg
Ser Val Ser Asp Asn Asp Ile 740 745 750Arg Lys Tyr Glu Met Phe Ala
Gln Thr Leu Gln Gln Ser Arg Gly Phe 755 760 765Gly Ser Phe Arg Phe
Pro Ser Gly Asn Gln Gly Gly Ala Gly Pro Ser 770 775 780Gln Gly Ser
Gly Gly Gly Thr Gly Gly Ser Val Tyr Thr Glu Asp Asn785 790 795
800Asp Asp Asp Leu Tyr Gly 805
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