U.S. patent application number 10/105992 was filed with the patent office on 2002-10-17 for 23413, a novel human ubiquitin protease.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Hunter, John Joseph, Kapeller-Libermann, Rosana.
Application Number | 20020152481 10/105992 |
Document ID | / |
Family ID | 23606333 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020152481 |
Kind Code |
A1 |
Kapeller-Libermann, Rosana ;
et al. |
October 17, 2002 |
23413, a novel human ubiquitin protease
Abstract
The present invention relates to a newly identified human
ubiquitin protease belonging to the family of mammalian
deubiquitinating enzymes. The invention also relates to
polynucleotides encoding the ubiquitin protease. The invention
further relates to methods using the ubiquitin protease
polypeptides and polynucleotides as a target for diagnosis and
treatment in ubiquitin-mediated or -related disorders. The
invention further relates to drug-screening methods using the
ubiquitin protease polypeptides and polynucleotides to identify
agonists and antagonists for diagnosis and treatment. The invention
further encompasses agonists and antagonists based on the ubiquitin
protease polypeptides and polynucleotides. The invention further
relates to procedures for producing the ubiquitin protease
polypeptides and polynucleotides.
Inventors: |
Kapeller-Libermann, Rosana;
(Chestnut Hill, MA) ; Hunter, John Joseph;
(Somerville, MA) |
Correspondence
Address: |
ALSTON & BIRD LLP
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
23606333 |
Appl. No.: |
10/105992 |
Filed: |
March 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10105992 |
Mar 25, 2002 |
|
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09406045 |
Sep 27, 1999 |
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Current U.S.
Class: |
800/8 ; 435/226;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61P 25/08 20180101;
A61P 43/00 20180101; A61P 21/00 20180101; A01K 2217/05 20130101;
C12N 9/6472 20130101; A61P 35/00 20180101; A61P 31/04 20180101;
A61K 48/00 20130101; A61P 3/00 20180101; A61P 5/14 20180101; A61P
25/14 20180101; A61P 21/04 20180101; A61K 38/00 20130101; A61P 3/10
20180101; A61P 25/18 20180101; A61P 17/02 20180101; A61P 25/28
20180101; A61P 25/16 20180101; A61P 13/00 20180101; A61P 37/02
20180101 |
Class at
Publication: |
800/8 ; 435/226;
435/69.1; 435/325; 435/320.1; 536/23.2 |
International
Class: |
A01K 067/00; C07H
021/04; C12N 009/64; C12P 021/02; C12N 005/06 |
Claims
That which is claimed:
1. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence having at least 65% sequence identity to the nucleotide
sequence set forth in SEQ ID NO:2, wherein said sequence identity
is calculated using the GAP algorithm with a gap weight of 40 and a
length weight of 4 and said nucleotide sequence encodes a
polypeptide having ubiquitin protease activity; and (b) a
nucleotide sequence complementary the nucleotide sequence of
(a).
2. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule comprises a nucleotide sequence selected from the group
consisting of: (a) a nucleotide sequence having at least 70%
sequence identity to the nucleotide sequence set forth in SEQ ID
NO:2, wherein said sequence identity is calculated using the GAP
algorithm with a gap weight of 40 and a length weight of 4 and said
nucleotide sequence encodes a polypeptide having ubiquitin protease
activity; (b) a nucleotide sequence complementary to the nucleotide
sequence of (a).
3. The nucleic acid molecule of claim 2, wherein said nucleic acid
molecule comprises a nucleotide sequence selected from the group
consisting of: (a) a nucleotide sequence having at least 80%
sequence identity to the nucleotide sequence set forth in SEQ ID
NO:2, wherein said sequence identity is calculated using the GAP
algorithm with a gap weight of 40 and a length weight of 4 and said
nucleotide sequence encodes a polypeptide having ubiquitin protease
activity; (b) a nucleotide sequence complementary to the nucleotide
sequence of (a).
4. The nucleic acid molecule of claim 3, wherein said nucleic acid
molecule comprises a nucleotide sequence selected from the group
consisting of: (a) a nucleotide sequence having at least 90%
sequence identity to the nucleotide sequence set forth in SEQ ID
NO:2, wherein said sequence identity is calculated using the GAP
algorithm with a gap weight of 40 and a length weight of 4 and said
nucleotide sequence encodes a polypeptide having ubiquitin protease
activity; (b) a nucleotide sequence complementary to the nucleotide
sequence of (a).
5. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence encoding a polypeptide having ubiquitin protease activity,
wherein said nucleotide sequence hybridizes to the complement of
the nucleotide sequence shown in SEQ ID NO:2 under stringent
conditions, said stringent conditions comprising hybridization in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by one or more washes in 0.2 .times. SSC, 0.1% SDS at
65.degree. C.; (b) a nucleotide sequence encoding a polypeptide
having ubiquitin protease activity, wherein said nucleotide
sequence hybridizes to the cDNA insert contained in ATCC Patent
Deposit No. PTA-1652 under stringent conditions, said stringent
conditions comprising hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more washes in 0.2.times. SSC, 0.1% SDS at 65.degree. C.;
and (c) a nucleotide sequence complementary to the nucleotide
sequence of (a) or (b).
6. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence encoding a fragment of the amino acid sequence set forth
in SEQ ID NO:1, wherein said fragment has ubiquitin protease
activity and consists of at least 15 contiguous amino acids of the
amino acid sequence set forth in SEQ ID NO:1; (b) a nucleotide
sequence encoding a fragment of the amino acid sequence encoded by
the cDNA insert of the plasmid deposited with ATCC as Patent
Deposit No. PTA-1652, wherein said fragment has ubiquitin protease
activity and consists of at least 15 contiguous amino acids of the
amino acid sequence encoded by the cDNA insert of the plasmid
deposited with ATCC as Patent Deposit No. PTA-1652; (c) a
nucleotide sequence encoding a fragment of the amino acid sequence
set forth in SEQ ID NO:1, wherein said fragment has ubiquitin
protease activity and consists of at least 25 contiguous amino
acids of the amino acid sequence set forth in SEQ ID NO:1; (d) a
nucleotide sequence encoding a fragment of the amino acid sequence
encoded by the cDNA insert of the plasmid deposited with ATCC as
Patent Deposit No. PTA-1652, wherein said fragment has ubiquitin
protease activity and consists of at least 25 contiguous amino
acids of the amino acid sequence encoded by the cDNA insert of the
plasmid deposited with ATCC as Patent Deposit No. PTA-1652; (e) a
nucleotide sequence encoding a fragment of the amino acid sequence
set forth in SEQ ID NO:1, wherein said fragment has ubiquitin
protease activity and consists of at least 35 contiguous amino
acids of the amino acid sequence set forth in SEQ ID NO:1; and (f)
a nucleotide sequence encoding a fragment of the amino acid
sequence encoded by the cDNA insert of the plasmid deposited with
ATCC as Patent Deposit No. PTA-1652, wherein said fragment has
ubiquitin protease activity and consists of at least 35 contiguous
amino acids of the amino acid sequence encoded by the cDNA insert
of the plasmid deposited with ATCC as Patent Deposit No. PTA-1652;
(g) a nucleotide sequence encoding a fragment of the amino acid
sequence set forth in SEQ ID NO:1, wherein said fragment has
ubiquitin protease activity and consists of at least 45 contiguous
amino acids of the amino acid sequence set forth in SEQ ID NO:1;
and (h) a nucleotide sequence encoding a fragment of the amino acid
sequence encoded by the cDNA insert of the plasmid deposited with
ATCC as Patent Deposit No. PTA-1652, wherein said fragment has
ubiquitin protease activity and consists of at least 45 contiguous
amino acids of the amino acid sequence encoded by the cDNA insert
of the plasmid deposited with ATCC as Patent Deposit No.
PTA-1652.
7. A method for producing a polypeptide comprising an amino acid
sequence selected from the group consisting of: (a) an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence set forth in SEQ ID NO:1 wherein said polypeptide has
ubiquitin protease activity and said sequence identity is
calculated by the GAP algorithm using a Blossum 62 scoring matrix
with a gap weight of 12 and a length weight of 4; (b) an amino acid
sequence having at least 80% sequence identity to the amino acid
sequence set forth in SEQ ID NO:1 wherein said polypeptide has
ubiquitin protease activity and said sequence identity is
calculated by the GAP algorithm using a Blossum 62 scoring matrix
with a gap weight of 12 and a length weight of 4; (c) an amino acid
sequence having at least 90% sequence identity to the amino acid
sequence set forth in SEQ ID NO:1 wherein said polypeptide has
ubiquitin protease activity and said sequence identity is
calculated by the GAP algorithm using a Blossum 62 scoring matrix
with a gap weight of 12 and a length weight of 4; said method
comprising the steps of introducing a nucleic acid molecule
encoding the polypeptide into a host cell and culturing the host
cell under conditions in which the polypeptide is expressed from
the nucleic acid molecule.
8. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) the amino acid sequence
set forth in SEQ ID NO:1; (b) the amino acid sequence encoded by
the cDNA insert of the plasmid deposited with the ATCC as Patent
Deposit Number PTA-1652; (c) the amino acid sequence of a sequence
variant of the amino acid sequence shown in SEQ ID NO:1, wherein
said sequence variant has ubiquitin protease activity and is
encoded by a nucleotide sequence having at least about 70% sequence
identity to the nucleotide sequence set forth in SEQ ID NO:2; (d)
the amino acid sequence of an sequence variant of the amino acid
sequence encoded by the cDNA insert of the plasmid deposited with
the ATCC as Patent Deposit Number PTA-1652, wherein said sequence
variant has ubiquitin protease activity and is encoded by a
nucleotide sequence having at least about 70% sequence identity to
the nucleotide sequence of the cDNA insert of the plasmid deposited
with the ATCC as Patent Deposit Number PTA-1652; (e) the amino acid
sequence of a sequence variant of the amino acid sequence shown in
SEQ ID NO:1, wherein the sequence variant has ubiquitin protease
activity and is encoded by a nucleic acid molecule that hybridizes
to the complement of the nucleotide sequence set forth in SEQ ID
NO:2 under stringent conditions; (f) the amino acid sequence of a
sequence variant of the amino acid sequence encoded by the cDNA
insert of the plasmid deposited with the ATCC as Patent Deposit
Number PTA-1652, wherein the sequence variant has ubiquitin
protease activity and is encoded by a nucleic acid molecule that
hybridizes under stringent conditions the cDNA insert of the
plasmid deposited with the ATCC as Patent Deposit Number PTA-1652;
(g) the amino acid sequence of the mature polypeptide from about
amino acid 6 to the last amino acid set forth in SEQ ID NO:1; (h)
the amino acid sequence of a fragment of the amino acid sequence
set forth in SEQ ID NO:1, wherein said fragment has ubiquitin
protease activity and consists of at least 15 contiguous amino
acids of the amino acid sequence set forth in SEQ ID NO:1; and (i)
the amino acid sequence of a fragment of the amino acid sequence
encoded by the cDNA insert of the plasmid deposited with the ATCC
as Patent Deposit Number PTA-1652, wherein the fragment has
ubiquitin protease activity and consists of at least 15 contiguous
amino acids of the amino acid sequence encoded by the cDNA insert
of the plasmid deposited with the ATCC as Patent Deposit Number
PTA-1652.
9. An isolated antibody that selectively binds to a polypeptide of
claim 8.
10. A method for detecting the presence of at least one
polypeptides of claim 8 in a sample, said method comprising
contacting said sample with an agent that specifically allows
detection of the presence of the polypeptide in the sample and then
detecting the presence of the polypeptide.
11. The method of claim 10, wherein said agent is an antibody.
12. The method of claim 10, wherein said agent is ubiquitinated
protein or polyubiquitin.
13. A kit comprising reagents used for the method of claim 8,
wherein the reagents comprise an agent that specifically binds to
said polypeptide.
14. A method for identifying an agent that binds to any of the
polypeptides in claim 8, said method comprising contacting the
polypeptide with an agent that binds to the polypeptide and
assaying the complex formed with the agent bound to the
polypeptide.
15. A method for identifying an agent that modulates the level or
activity of any of the polypeptides in claim 1 in a cell, the
method comprising contacting the agent with a cell capable of
expressing said polypeptide such that said polypeptide level or
activity can be modulated in said cell by said agent and measuring
said polypeptide level or activity.
16. A method for identifying an agent that interacts with any of
the polypeptides in claim 8 in a cell, the method comprising
contacting said agent with a cell expressing a polypeptide of claim
8 under conditions such that said polypeptide can interact with
said agent and measuring the interaction between said polypeptide
and said agent.
17. A method of screening a cell to identify an agent that
modulates the level or activity of any of the polypeptides in claim
8 in said cell, said method comprising contacting said agent with a
cell capable of expressing said polypeptide such that said
polypeptide level or activity can be modulated in said cell by said
agent and measuring said polypeptide level or activity.
18. A method of screening a cell to identify an agent that
interacts with any of the polypeptides in claim 8, said method
comprising contacting said agent with a cell capable of allowing an
interaction between said polypeptide and said agent such that said
polypeptide can interact with said agent and measuring the
interaction.
19. A method for modulating the activity of any of at least one
polypeptide of claim 8, the method comprising contacting a
polypeptide of claim 8 with an agent under conditions that allow
the agent to modulate the activity of the polypeptide.
20. The method of claim 19 wherein said modulation is in cells
derived from tissues selected from the group consisting of breast,
testes, brain, lung, kidney, liver, and colonic liver
metastases.
21. The method of claim 20 wherein said cells are malignant breast,
lung, and colonic liver metastatic cells.
22. The method of claim 20 wherein said modulation is in a patient
having breast cancer.
23. A composition containing any of the polypeptides in claim 8 in
a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Application Ser.
No. 09/406,045, filed Sep. 27, 1999, which is hereby incorporated
in its entirety by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to a newly identified human
ubiquitin protease belonging to the family of mammalian
deubiquitinating enzymes. The invention also relates to
polynucleotides encoding the ubiquitin protease. The invention
further relates to methods using the ubiquitin protease
polypeptides and polynucleotides as a target for diagnosis and
treatment in ubiquitin-mediated or -related disorders. The
invention further relates to drug-screening methods using the
ubiquitin protease polypeptides and polynucleotides to identify
agonists and antagonists for diagnosis and treatment. The invention
further encompasses agonists and antagonists based on the ubiquitin
protease polypeptides and polynucleotides. The invention further
relates to procedures for producing the ubiquitin protease
polypeptides and polynucleotides.
BACKGROUND OF THE INVENTION
[0003] The Ubiquitin System
[0004] Several biological processes are controlled by the
ubiquitination of cellular protein. Cellular processes that are
affected by ubiquitin modification include the regulation of gene
expression, regulation of the cell cycle and cell division,
cellular housekeeping, cell-specific metabolic pathways, disposal
of mutated or post-translationally damaged proteins, the cellular
stress response, modification of cell surface receptors, DNA
repair, import of proteins into mitochondria, uptake of precursors
into neurons, biogenesis of mitochondria, ribosomes, and
peroxisomes, apoptosis, and growth factor-mediated signal
transduction.
[0005] For some protein substrates ubiquitination leads to protein
degradation by the 26S proteasomal complex. A wide variety of
protein substrates is degraded by the 26S proteasomal complex
following ubiquitination of the substrate. Degradation of a protein
by the ubiquitin system involves two steps. The first involves the
covalent attachment of multiple ubiquitin molecules to the
substrate protein. The second involves degradation of the
ubiquitinated protein by the 26S proteasome. In some cases,
degradation of the ubiquitinated protein can occur by means of the
lysosomal pathway.
[0006] The 26S proteasome comprises a 20S core catalytic complex
which is flanked by two 19S regulatory complexes. The 26S complex
recognizes ubiquitinated proteins. Substrate recognition by the 26S
proteasome, however, may be mediated by the interaction of specific
subunits of the 19S complex with the ubiquitin chain. The
ubiquitinated protein is degraded by specific and energy-dependent
proteases into free amino acids and free and reutilizable
ubiquitin.
[0007] The 19S regulatory complex consists of many subunits that
can be classified into ATPases and non-ATPases. This complex is
thought to act in recognition, unfolding, and translocation of the
substrates into the 20S proteasome for proteolysis. The regulatory
complex contains isopeptidases capable of deubiquitinating
substrates (Spataro et al. (1998) British Journal of Cancer
77:448-455).
[0008] The ubiquitin proteasome pathway functions to degrade
abnormal proteins, short-lived normal proteins, long-lived normal
proteins, and proteins of the endoplasmic recticulum. Important
regulatory proteins rapidly inactivated by proteolysis include
c-JUN, c-FOS, and p53 (Lecker et al. (1999) Journal of Nutrition
129:227S-237S). Conditions that stimulate protein degradation by
the ubiquitin proteasome pathway include eating disorders, renal
tubular defects, diabetes, uremia, neuromuscular disease,
immobilization, burn injuries, sepsis, cancer, cachexia,
hyperadrenocortisolism and hyperthyroidism.
[0009] Cellular proteins degraded by the ubiquitin system include
cell cycle regulators, including mitotic cyclins, G1 cyclins, CDK
inhibitors, anaphase inhibitors, transcription factors, tumor
suppressors, and oncoproteins such as NF-.eta.B and I.eta.B.alpha.,
p53, JUN, .beta.-catenin, E2F-1, and membrane proteins such as
Ste2p, GH receptor, T-cell receptor, platelet-derived growth
factor, lymphocyte homing receptor, MET tyrosine kinase receptor,
hepatocyte growth factor-scatter factor, connexin 43, the high
affinity IgE receptor, the prolactin receptor, and the EGF receptor
(Hershko et al. (1998) Annual Review of Biochemistry
67:425-479).
[0010] Ubiquitination does not only result in proteolytic
degradation. For some protein substrates, ubiquitination is a
reversible post-translational modification that can regulate
cellular targeting and enzymatic activity. This includes targeting
to the vacuole, activation of enzyme activity, such as Ik.beta.
kinase activation, and activation of cytokine receptor-mediated
signal transduction (D'Andrea et al. (1998) Critical Reviews In
Biochemistry and Molecular Biology 33:337-352). The T-cell receptor
undergoes ubiquitination in response to receptor engagement.
Platelet derived growth factor undergoes multiple ubiquitination
following ligand binding. Soluble steel factor has been shown to
stimulate rapid polyubiquitination of the c-KIT receptor.
[0011] It has been shown that protein degradation accounts for
regulation of proteins such as cyclins, cyclin-dependent kinase
inhibitors, p53, c-JUN and c-FOS (Spitaro et al. above). The
ubiquitin system has also shown to be involved in antigen
presentation. The 26S proteasome is responsible for processing
MHC-restricted class I antigens (Spitaro et al. above).
[0012] The ubiquitin system has been implicated in various
diseases. One group includes pathology that results from loss of
function, a mutation in an enzyme or substrate that leads to
stabilization of the protein and consequent build up of a protein
to abnormally high levels. The second involves pathologies that
result from a gain of function that produces increased protein
degradation.
[0013] The ubiquitin system has been implicated in various
malignancies. In cervical carcinoma, low levels of p53 have been
found. This protein is targeted for degradation by HPV
E6-associated protein. Removal of the suppressor by this
oncoprotein may be a mechanism utilized by the virus to transform
cells. Other results have shown that c-JUN, but not the
transforming counterpart, v-JUN, is ubiquitinated and subsequently
degraded. Other studies show that low levels of p27, a cell
division kinase inhibitor whose degradation is necessary for proper
cell cycle progression, is correlated with colorectal, and breast
carcinomas. The low level of this enzyme is due to activation of
the ubiquitin system.
[0014] Human genetic diseases involving aberrant proteolysis have
been reviewed (Kato (1999) Human Mutation 13:87-98). Cystic
fibrosis has been correlated with the ubiquitin system. The cystic
fibrosis transmembrane regulator in cystic fibrosis patients is
almost completely degraded by the ubiquitin system so that an
abnormally low amount of the wild type protein is found on the cell
surface. In Angelman's syndrome, one of the enzymes involved in
ubiquitination (E3) is affected. In Liddle syndrome, the E3 enzyme
is also affected.
[0015] The ubiquitin system can also affect the immune and
inflammatory response. The persistence of EBNA-1 contributes to
some virus related pathologies. A sequence on this protein was
found to inhibit degradation by the ubiquitin system. This
inhibited processing and subsequent presentation of viral epitopes
by MHC protein.
[0016] The ubiquitin system has also been implicated in
neurodegenerative diseases. Ubiquitin immunohistochemistry has
shown enrichment of ubiquitin conjugates in senile plaques,
lysosomes, endosomes, and a variety of inclusion bodies and
degenerative fibers in many neurodegenerative diseases, such as
Alzheimer's, Parkinson's and Lewy body diseases, amyotrophic
lateral sclerosis, and Creutzfeld-Jakob disease. Further, in
Huntington disease and spinocerebellar ataxias, the proteins
encoded by the affected genes aggregate in ubiquitin- and
proteasome-positive intranuclear inclusion bodies.
[0017] The ubiquitin system has been associated with muscle wasting
(Mitch et al. (1999) American Journal of Physiology 276:C1132-C1138
and Lecker et al. above) and muscle-wasting diseases and in such
pathological states as fasting, starvation, sepsis, and
denervation, all of which result from accelerated
ubiquitin-mediated proteolysis (see Ciechanover, EMBO Journal
17:7151-7160 (1998)).
[0018] The ubiquitin system is also involved in development. The
involvement in human brain development is indicated by the fact
that a mutation in an E3 enzyme is implicated as the cause of
Angelman's syndrome, a disorder characterized by mental
retardation, seizures, and abnormal gait (Hershko et al.
above).
[0019] The ubiquitin system is also associated with apoptosis.
Ubiquitin-proteasome-mediated proteolysis is reported to play an
important role in apoptosis of nerve growth factor-deprived neurons
(Sadoul et al. (1996) EMBO Journal 15:3845-3852). One of the first
genes shown to be involved in programmed cell death is the
polyubiquitin gene that is regulated during metamorphosis of
Manduca sexta. Radiation-induced apoptosis in human lymphocytes has
been shown to be accompanied by increased ubiquitin mRNA and
ubiquitinylated nuclear proteins. Further, drugs that interfere
with proteasome function, such as lactacystin, prevent
radiation-induced cell death of thymocytes (Hershko et al.
above).
[0020] Deubiquitinating Enzymes
[0021] Deubiquitinating enzymes are cysteine proteases that
specifically cleave ubiquitin conjugates at the ubiquitin carboxy
terminus. These enzymes are responsible for processing linear
polyubiquitin chains to generate free ubiquitin from precursor
fusion proteins. They also affect pools of free ubiquitin by
recycling branched chain ubiquitin. These enzymes also remove
ubiquitin from ubiquitin- and polyubiquitin-conjugate- d target
protein, thereby regulating localization or activity of the target.
Further, these enzymes can remove ubiquitin from a ubiquitinated
tagged protein and thereby rescue the protein from degradation by
the 26S proteasome. The end result of each of these activities, is
to affect the level of free intracellular ubiquitin (D'Andrea et
al., above) and the level of specific proteins.
[0022] Ubiquitin is synthesized in a variety of
functionally-distinct forms. One of these is a linear head-to-tail
polyubiquitin precursor. Release of the free molecules involves
specific enzymatic cleavage between the fused residues. The last
ubiquitin moiety in many of these precursors is encoded with an
extra C-terminal residue that must be removed to expose the active
C-terminal Gly. In general, the recycling enzymes are thiol
proteases that recognize the C-terminal domain/residue of
ubiquitin. These are divided into two classes. The first is
designated ubiquitin C-terminal hydrolase (UCH) and the second is
designated ubiquitin-specific protease (UBP; isopeptidases)
(Ciechanover, above). These enzymes have been reviewed in detail in
D'Andrea, above.
[0023] UBPs contain six conserved regions. One surrounds the
conserved cysteine, one surrounds the aspartic acid, one surrounds
the histidine, and three additional regions of unknown function
have been identified. These six domains provide a molecular
signature for the UBP family. Short sequences surrounding the
cysteine residue and histidine residue are highly conserved among
all UBPs. Sequence comparison of several UBP family members reveals
that there are various subfamilies. One subfamily, designated DUB,
contains enzymes that are transcriptionally induced in response to
cytokines. The UBP family contains enzymes whose members have
multiple ubiquitin binding sites. Identified members of this family
include DUB1, isoT, UBP3, Doa4, Tre2, and FAF (D'Andrea et al.
above).
[0024] The UCH family is distinct from the UBP family. These
enzymes are cysteine proteases but do not contain the six homology
domains characteristic of the UBP family. Further, there is only
one binding site for ubiquitin. With respect to substrate
specificity, the UCH family preferentially cleaves ubiquitin from
small molecules, such as peptides and amino acids. Further, the two
families share little sequence homology with each other, although
the UCH signature can be found in some UBPs.
[0025] The deubiquitinating enzymes can promote either degradation
or stabilization of a given substrate. One of the best
characterized deubiquitinating enzymes is the yeast UBP14p enzyme
which has a human homolog designated isopeptidase-T. Isopeptidase-T
hydrolyzes free polyubiquitin chains and stimulates degradation of
polyubiquitinated protein substrates by the 26S proteasome. In
vitro data suggest that the cellular role of isopeptidase-T is to
dissemble unanchored polyubiquitin chains. The isopeptidase-T then
sequentially degrades these polyubiquitin chains into ubiquitin
monomers.
[0026] The yeast Doa4 promotes ubiquitin-mediated proteolysis of
cellular substrates. The primary function appears to be the
hydrolysis of isopeptide-linked ubiquitin chains from peptides that
are the by-products of proteasome degradation. The function appears
to be the clipping of polymeric ubiquitin from peptide degradation
products. In summary, with respect to a degradation function,
isopeptidases can produce free ubiquitin monomers from straight
chain polyubiquitin, branched chain polyubiquitin, ubiquitin or
polyubiquitin attached to substrate proteins, and ubiquitin or
polyubiquitin attached to substrate remnants, such as peptides or
amino acids.
[0027] Deubiquitinating enzymes that promote stabilization of
substrates include the FAF protein. Results show that the FAF
protein deubiquitinates and rescues a ubiquitin-conjugated target,
preventing its degradation by the proteasome. Another
deubiquitinating enzyme, designated PA700 isospeptidase, also
prevents proteasome degradation. This enzyme has been isolated from
the 19S regulatory complex. This enzyme appears to remove one
ubiquitin at a time starting from the distal end of a polyubiquitin
chain.
[0028] The enzymes have been associated with growth control. The
mammalian oncoprotein Tre-2 is a member of the UBP superfamily. The
transforming isoform of the Tre-2 oncoprotein is a truncated UPB
lacking the histidine domain and lacking deubiquitinating activity.
The full length Tre-2 protein has deubiquitinating activity but no
transforming activity. Accordingly, it has been suggested that this
protein acts as a growth suppressor within the cell.
[0029] Another UBP that regulates cellular function is designated
DUB. DUB-1 was originally shown to be induced by interleukin-3
stimulation. It has been postulated that the DUB protein family is
generally responsive to cytokines. It has also been shown that
another family member, DUB-2, is induced by interleukin-2. Zhu et
al. (1997) Journal of Biological Chemistry 272:51-57.
[0030] The enzymes may deubiquitinate cell surface growth factor
receptors thereby prolonging receptor half life and amplifying
growth signals. They may also deubiquitinate proteins involved in
signal transduction and deubiquitinate cell cycle regulators such
as cyclins or cyclin-CDK inhibitors. See D'Andrea above.
[0031] UBPs have also been linked to the chromatin regulatory
process, transcriptional silencing. UBP-3 has been reported to
complex with SIR-4, a trans-acting factor that is required for
establishment and maintenance of silencing. Accordingly, UBP-3 may
act as an inhibitor of silencing by either stabilizing an inhibitor
or by removing a positive regulator.
[0032] The murine UNP protooncogene has been shown to encode a
nuclear ubiquitin protease whose overexpression leads to oncogenic
transformation in NIH3T3 cells. A cDNA was cloned corresponding to
the human homolog of this gene. It was shown to map to a region
frequently rearranged in human tumor cells. Further, it was shown
that levels of this gene are elevated in small cell tumors and
adenocarcinomas of the lung, suggesting a causative role of the
gene in the neoplastic process (Gray et al. (1995) Oncogene
10:2179-2183).
[0033] A novel ubiquitin-specific protease, designated UBP-43, was
cloned from a leukemia fusion protein in AML1-ETO Knockin mice.
This protease was shown to function in hematopoitic cell
differentiation. The overexpression of this gene was shown to block
cytokine-induced terminal differentiation of monocytic cells (Liu
et al. (1999) Molecular and Cellular Biology 19:3029-3038).
[0034] In summary, deubiquitinating enzymes are potentially
powerful targets for modulating ubiquitination. Modulation of
ubiquitination can increase or decrease the proteolysis of specific
proteins, particularly key proteins in cellular processes, can
increase or decrease levels of general proteolysis, thus affecting
the basic metabolic state, and may increase or decrease the pool of
free ubiquitin monomers available for ubiquitination.
[0035] Accordingly, ubiquitin proteases are a major target for drug
action and development. Thus, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown ubiquitin proteases. The present invention advances the
state of the art by providing a previously unidentified human
deubiquitinating enzyme.
SUMMARY OF THE INVENTION
[0036] It is an object of the invention to identify novel ubiquitin
proteases.
[0037] It is a further object of the invention to provide novel
ubiquitin protease polypeptides that are useful as reagents or
targets in assays applicable to treatment and diagnosis of
ubiquitin-mediated or -related disorders, especially disorders
mediated by or related to deubiquitinating enzymes.
[0038] It is a further object of the invention to provide
polynucleotides corresponding to the novel ubiquitin protease
polypeptides that are useful as targets and reagents in assays
applicable to treatment and diagnosis of ubiquitin or ubiquitin
protease-mediated or -related disorders and useful for producing
novel ubiquitin protease polypeptides by recombinant methods.
[0039] A specific object of the invention is to identify compounds
that act as agonists and antagonists and modulate the expression of
the novel ubiquitin protease.
[0040] A further specific object of the invention is to provide
compounds that modulate expression of the ubiquitin protease for
treatment and diagnosis of ubiquitin and ubiquitin protease-related
disorders.
[0041] The invention is thus based on the identification of a novel
human ubiquitin protease. The amino acid sequence is shown in SEQ
ID NO:1. The nucleotide sequence is shown in SEQ ID NO:2.
[0042] The invention provides isolated ubiquitin protease
polypeptides, including a polypeptide having the amino acid
sequence shown in SEQ ID NO:1 or the amino acid sequence encoded by
the cDNA deposited with the Patent Depository of the American Type
Culture Collection (ATCC), 10801 University Boulevard, Manassas,
Va., on Apr. 6, 2000, and assigned Patent Deposit No. PTA-1652
("the deposited cDNA"). This deposit will be maintained under the
terms of the Budapest Treaty on the International Recognition of
the Deposit of Microorganisms for the Purposes of Patent Procedure.
This deposit was made merely as a convenience for those of skill in
the art and is not an admission that a deposit is required under 35
U.S.C. .sctn.112. The deposited sequences, as well as the
polypeptides encoded by the sequences, are incorporated herein by
reference and controls in the event of any conflict, such as a
sequencing error, with description in this application.
[0043] The invention also provides isolated ubiquitin protease
nucleic acid molecules having the sequence shown in SEQ ID NO:2 or
in the deposited cDNA.
[0044] The invention also provides variant polypeptides having an
amino acid sequence that is substantially homologous to the amino
acid sequence shown in SEQ ID NO:1 or encoded by the deposited
cDNA.
[0045] The invention also provides variant nucleic acid sequences
that are substantially homologous to the nucleotide sequence shown
in SEQ ID NO:2 or in the deposited cDNA.
[0046] The invention also provides fragments of the polypeptide
shown in SEQ ID NO:1 and nucleotide sequence shown in SEQ ID NO:2,
as well as substantially homologous fragments of the polypeptide or
nucleic acid.
[0047] The invention further provides nucleic acid constructs
comprising the nucleic acid molecules described herein. In a
preferred embodiment, the nucleic acid molecules of the invention
are operatively linked to a regulatory sequence.
[0048] The invention also provides vectors and host cells for
expressing the ubiquitin protease nucleic acid molecules and
polypeptides, and particularly recombinant vectors and host
cells.
[0049] The invention also provides methods of making the vectors
and host cells and methods for using them to produce the ubiquitin
protease nucleic acid molecules and polypeptides.
[0050] The invention also provides antibodies or antigen-binding
fragments thereof that selectively bind the ubiquitin protease
polypeptides and fragments.
[0051] The invention also provides methods of screening for
compounds that modulate expression or activity of the ubiquitin
protease polypeptides or nucleic acid (RNA or DNA).
[0052] The invention also provides a process for modulating
ubiquitin protease polypeptide or nucleic acid expression or
activity, especially using the screened compounds. Modulation may
be used to treat conditions related to aberrant activity or
expression of the ubiquitin protease polypeptides or nucleic acids
or of the ubiquitin system.
[0053] The invention also provides assays for determining the
activity of or the presence or absence of the ubiquitin protease
polypeptides or nucleic acid molecules in a biological sample,
including for disease diagnosis.
[0054] The invention also provides assays for determining the
presence of a mutation in the polypeptides or nucleic acid
molecules, including for disease diagnosis.
[0055] In still a further embodiment, the invention provides a
computer readable means containing the nucleotide and/or amino acid
sequences of the nucleic acids and polypeptides of the invention,
respectively.
DESCRIPTION OF THE DRAWINGS
[0056] FIG. 1 shows the nucleotide sequence (SEQ ID NO:2) and the
deduced amino acid sequence (SEQ ID NO:1) of the novel ubiquitin
protease. The underlined amino acids designate the conserved
cysteine region and conserved histidine region. These regions are
conserved among members of the UBP protein family.
[0057] FIG. 2 shows a comparison of the ubiquitin protease against
the ProDom database of protein patterns (SEQ ID NOS:3, 4, 5, 6, 7,
8, 9, 10, 11 and 12), specifically showing some homology to the UCH
family.
[0058] FIG. 3 shows an analysis of the ubiquitin protease amino
acid sequence: .alpha..beta.turn and coil regions; hydrophilicity;
amphipathic regions; flexible regions; antigenic index; and surface
probability plot.
[0059] FIG. 4 shows a hydrophobicity plot of the ubiquitin protease
of SEQ ID NO:1.
[0060] FIG. 5 shows an analysis of the ubiquitin protease open
reading frame for amino acids corresponding to specific functional
sites of SEQ ID NO:1. Glycosylation sites are found from about
amino acid 188 to about amino acid 191 and from about amino acid
335 to about amino acid 338, with the actual modified residue being
the first amino acid. Cyclic AMP and cyclic GMP-dependent protein
kinase phosphorylation sites are found from about amino acid 84 to
about amino acid 87 and from about amino acid 288 to about amino
acid 291, with the actual modified residue being the last amino
acid. Protein kinase C phosphorylation sites are found from about
amino acid 169 to about amino acid 171, from about amino acid 185
to about amino acid 187, from about amino acid 223 to about amino
acid 225, from about amino acid 260 to about amino acid 262, and
from about amino acid 266 to about amino acid 268, with the actual
modified residue being the first amino acid. Casein kinase II
phophorylation sites are found from about amino acid 22 to about
amino acid 25, from about amino 197 to about amino acid 200, from
about amino acid 208 to about amino acid 211, and from about amino
acid 343 to about amino acid 346, with the actual modified residue
being the first amino acid. A tyrosine kinase phosphorylation site
is found from about amino acid 119 to about amino acid 125, with
the actual modified residue being the last amino acid.
N-myristoylation sites are found from about amino acid 61 to about
amino acid 66, and from about amino acid 312 to about amino acid
317, with the actual modified residue being the first amino acid.
An amidation site is found from about amino acid 233 to about amino
acid 236. In addition, amino acids corresponding to the UCH
signature are found at amino acids 302-319.
[0061] FIG. 6 shows expression of the protease in normal breast,
lung, liver, and colon, and shows enhanced expression in malignant
breast, lung, liver, and colon metastases. The liver metastases are
derived from malignant colonic tissue.
[0062] FIG. 7 shows expression of the protease in cDNA libraries
from various tissues and cell types in culture. These data were
derived from RT-PCR of various cDNA libraries and hence, are not
relatively quantitative.
[0063] FIG. 8 shows expression of the protease in various normal
human tissues. Expression was obtained by Taqman analysis and hence
are relatively quantitative.
DETAILED DESCRIPTION OF THE INVENTION
[0064] Polypeptides
[0065] The invention is based on the identification of a novel
human ubiquitin protease. Specifically, an expressed sequence tag
(EST) was selected based on homology to ubiquitin protease
sequences. This EST was used to design primers based on sequences
that it contains and used to identify a cDNA from an endothelial
cell cDNA library. Positive clones were sequenced and the
overlapping fragments were assembled. Analysis of the assembled
sequence revealed that the cloned cDNA molecule encodes a ubiquitin
protease containing the conserved HIS and CYS boxes of the UBP
family of deubiquitinating enzymes.
[0066] The invention thus relates to a novel ubiquitin protease
having the deduced amino acid sequence shown in FIG. 1 (SEQ ID
NO:1) or having the amino acid sequence encoded by the deposited
cDNA, ATCC Patent Deposit No. PTA-1652. "Ubiquitin protease
polypeptide" or "ubiquitin protease protein" refers to the
polypeptide in SEQ ID NO:1 or encoded by the deposited cDNA. The
term "ubiquitin protease protein" or "ubiquitin protease
polypeptide", however, further includes the numerous variants
described herein, as well as fragments derived from the full-length
ubiquitin proteases and variants.
[0067] Tissues and/or cells in which the ubiquitin protease nucleic
acid is found include, but are not limited to, those shown in FIGS.
6, 7, and 8. Tissues in which the gene is highly expressed include
breast, testes, liver, and fetal liver. The gene is also
significantly expressed in thymus, brain, skeletal muscle,
prostate, thyroid, fetal kidney, fetal heart, and ovary. The
ubiquitin protease is particularly expressed in tissues involved in
breast and lung cancer. The gene is also particularly expressed in
liver metastases. These liver metastases are derived from malignant
colonic tissue. Expression has been confirmed by Northern blot
analysis.
[0068] The present invention thus provides an isolated or purified
ubiquitin protease polypeptide and variants and fragments
thereof.
[0069] Based on a BLAST search, highest homology was shown to
murine UBP43 (Liu, above).
[0070] As used herein, a polypeptide is said to be "isolated" or
"purified" when it is substantially free of cellular material when
it is isolated from recombinant and non-recombinant cells, or free
of chemical precursors or other chemicals when it is chemically
synthesized. A polypeptide, however, can be joined to another
polypeptide with which it is not normally associated in a cell and
still be considered "isolated" or "purified."
[0071] The ubiquitin protease polypeptides can be purified to
homogeneity. It is understood, however, that preparations in which
the polypeptide is not purified to homogeneity are useful and
considered to contain an isolated form of the polypeptide. The
critical feature is that the preparation allows for the desired
function of the polypeptide, even in the presence of considerable
amounts of other components. Thus, the invention encompasses
various degrees of purity.
[0072] In one embodiment, the language "substantially free of
cellular material" includes preparations of the ubiquitin protease
having less than about 30% (by dry weight) other proteins (i.e.,
contaminating protein), less than about 20% other proteins, less
than about 10% other proteins, or less than about 5% other
proteins. When the polypeptide is recombinantly produced, it can
also be substantially free of culture medium, i.e., culture medium
represents less than about 20%, less than about 10%, or less than
about 5% of the volume of the protein preparation.
[0073] A ubiquitin protease polypeptide is also considered to be
isolated when it is part of a membrane preparation or is purified
and then reconstituted with membrane vesicles or liposomes.
[0074] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the ubiquitin protease
polypeptide in which it is separated from chemical precursors or
other chemicals that are involved in its synthesis. In one
embodiment, the language "substantially free of chemical precursors
or other chemicals" includes preparations of the polypeptide having
less than about 30% (by dry weight) chemical precursors or other
chemicals, less than about 20% chemical precursors or other
chemicals, less than about 10% chemical precursors or other
chemicals, or less than about 5% chemical precursors or other
chemicals.
[0075] In one embodiment, the ubiquitin protease polypeptide
comprises the amino acid sequence shown in SEQ ID NO:1. However,
the invention also encompasses sequence variants. Variants include
a substantially homologous protein encoded by the same genetic
locus in an organism, i.e., an allelic variant.
[0076] Variants also encompass proteins derived from other genetic
loci in an organism, but having substantial homology to the
ubiquitin protease of SEQ ID NO:1. Variants also include proteins
substantially homologous to the ubiquitin protease but derived from
another organism, i.e., an ortholog. Variants also include proteins
that are substantially homologous to the ubiquitin protease that
are produced by chemical synthesis. Variants also include proteins
that are substantially homologous to the ubiquitin protease that
are produced by recombinant methods. It is understood, however,
that variants exclude any amino acid sequences disclosed prior to
the invention.
[0077] As used herein, two proteins (or a region of the proteins)
are substantially homologous when the amino acid sequences are at
least about 70-75%, typically at least about 80-85%, and most
typically at least about 90-95% or more homologous. A substantially
homologous amino acid sequence, according to the present invention,
will be encoded by a nucleic acid sequence hybridizing to the
nucleic acid sequence, or portion thereof, of the sequence shown in
SEQ ID NO:2 under stringent conditions as more fully described
below.
[0078] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% of the length of
the reference sequence (e.g., when aligning a second sequence to
the amino acid sequence herein having 372 amino acid residues, at
least 111, preferably at least 149, more preferably at least 186,
even more preferably at least 223, and even more preferably at
least 260, 297, 335, and 372 amino acid residues are aligned). The
amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position (as
used herein amino acid or nucleic acid "identity" is equivalent to
amino acid or nucleic acid "homology"). The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[0079] The invention also encompasses polypeptides having a lower
degree of identity but having sufficient similarity so as to
perform one or more of the same functions performed by the
ubiquitin protease. Similarity is determined by conserved amino
acid substitution. Such substitutions are those that substitute a
given amino acid in a polypeptide by another amino acid of like
characteristics. Conservative substitutions are likely to be
phenotypically silent. Typically seen as conservative substitutions
are the replacements, one for another, among the aliphatic amino
acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues
Ser and Thr, exchange of the acidic residues Asp and Glu,
substitution between the amide residues Asn and Gln, exchange of
the basic residues Lys and Arg and replacements among the aromatic
residues Phe, Tyr. Guidance concerning which amino acid changes are
likely to be phenotypically silent are found in Bowie et al.,
Science 247:1306-1310 (1990).
1TABLE 1 Conservative Amino Acid Substitutions. Aromatic
Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine
Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine
Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine
Methionine Glycine
[0080] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991).
[0081] A preferred, non-limiting example of such a mathematical
algorithm is described in Karlin et al. (1993) Proc. Natl. Acad.
Sci. USA 90:5873-5877. Such an algorithm is incorporated into the
NBLAST and XBLAST programs (version 2.0) as described in Altschul
et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., NBLAST) can be used. In one embodiment, parameters
for sequence comparison can be set at score=100, wordlength=12, or
can be varied (e.g., W=5 or W=20).
[0082] In a preferred embodiment, the percent identity between two
amino acid sequences is determined using the Needleman et al.
(1970) (J. Mol. Biol. 48:444-453) algorithm which has been
incorporated into the GAP program in the GCG software package using
either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6. In yet another preferred embodiment, the percent identity
between two nucleotide sequences is determined using the GAP
program in the GCG software package (Devereux et al. (1984) Nucleic
Acids Res. 12(1):387) using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6.
[0083] Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the CGC sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art and include ADVANCE and ADAM as described in Torellis et
al. (1994) Comput. Appl. Biosci. 10:3-5; and FASTA described in
Pearson et al. (1988) PNAS 85:2444-8.
[0084] A variant polypeptide can differ in amino acid sequence by
one or more substitutions, deletions, insertions, inversions,
fusions, and truncations or a combination of any of these.
[0085] Variant polypeptides can be fully ftmctional or can lack
function in one or more activities. Thus, in the present case,
variations can affect the function, for example, of ubiquitin
binding, ubiquitin recognition, interaction with ubiquitinated
substrate protein, such as binding or proteolysis, subunit
interaction, particularly within the proteasome, activation or
binding by ATP, developmental expression, temporal expression,
tissue-specific expression, interacting with cellular components,
such as transcriptional regulatory factors, and particularly
trans-acting transcriptional regulatory factors, proteolytic
cleavage of peptide bonds in polyubiquitin and peptide bonds
between ubiquitin or polyubiquitin and substrate protein, and
proteolytic cleavage of peptide bonds between ubiquitin or
polyubiquitin and a peptide or amino acid.
[0086] Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. Functional variants can also contain
substitution of similar amino acids, which results in no change or
an insignificant change in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree.
[0087] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0088] As indicated, variants can be naturally-occurring or can be
made by recombinant means or chemical synthesis to provide useful
and novel characteristics for the ubiquitin protease polypeptide.
This includes preventing immunogenicity from pharmaceutical
formulations by preventing protein aggregation.
[0089] Useful variations further include alteration of catalytic
activity. For example, one embodiment involves a variation at the
binding site that results in binding but not hydrolysis, or slower
hydrolysis, of the peptide bond. A further useful variation results
in an increased rate of hydrolysis of the peptide bond. A further
useful variation at the same site can result in higher or lower
affinity for substrate. Useful variations also include changes that
provide for affinity for a different ubiquitinated substrate
protein than that normally recognized. Other useful variations
involving altered recognition affect recognition of the type of
substrate normally recognized. For example, one variation could
result in recognition of ubiquitinated intact substrate but not of
substrate remnants, such as ubiquitinated amino acid or peptide
that are proteolysis products that result from the hydrolysis of
the intact ubiquitinated substrate. Alternatively, the protease
could be varied so that one or more of the remnant products is
recognized but not the intact protein substrate. Another variation
would affect the ability of the protease to rescue a ubiquitinated
protein. Thus, protein substrates that are normally rescued from
proteolysis would be subject to degradation. Further useful
variations affect the ability of the protease to be induced by
activators, such as cytokines, including but not limited to, those
disclosed herein. Another useful variation would affect the
recognition of ubiquitin substrate so that the enzyme could not
recognize one or more of a linear polyubiquitin, branched chain
polyubiquitin, linear polyubiquitinated substrate, or branched
chain polyubiquitin substrate. Specific variations include
truncation in which, for example, a HIS domain is deleted, the
variation resulting in decrease or loss of deubiquitination
activity. Another useful variation includes one that prevents
activation by ATP. Another useful variation provides a fusion
protein in which one or more domains or subregions are
operationally fused to one or more domains or subregions from
another UBP or from a UCH. Specifically, a domain or subregion can
be introduced that provides a rescue function to an enzyme not
normally having this function or for recognition of a specific
substrate wherein recognition is not available to the original
enzyme. Other variations include those that affect ubiquitin
recognition or recognition of a ubiquitinated substrate protein.
Further variations could affect specific subunit interaction,
particularly in the proteasome. Other variations would affect
developmental, temporal, or tissue-specific expression. Other
variations would affect the interaction with cellular components,
such as transcriptional regulatory factors.
[0090] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al
(1985) Science 244:1081-1085). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity,
such as peptide hydrolysis in vitro or ubiquitin-dependent in vitro
activity, such as proliferative activity, receptor-mediated signal
transduction, and other cellular processes including, but not
limited, those disclosed herein that are a function of the
ubiquitin system. Sites that are critical for binding or
recognition can also be determined by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith et al. (1992) J. Mol. Biol. 224:899-904; de Vos et
al. (1992) Science 255:306-312).
[0091] The assays for deubiquitinating enzyme activity are well
known in the art and can be found, for example, in Zhu et al.
(1997) Journal of Biological Chemistry 272:51-57, Mitch et al.
(1999)
[0092] American Journal of Physiology 276:C1132-C1138, Liu et al.
(1999) Molecular and Cell Biology 19:3029-3038, and such as those
cited in various reviews, for example, Ciechanover et al. (1994)
The FASEB Journal 8:182-192, Chiechanover (1994) Biol. Chem.
Hoppe-Seyler 375:565-581, Hershko et al. (1998) Annual Review of
Biochemistry 67:425-479, Swartz (1999) Annual Review of Medicine
50:57-74, Ciechanover (1998) EMBO Journal 17:7151-7160, and
D'Andrea et al. (1998) Critical Reviews in Biochemistry and
Molecular Biology 33:337-352. These assays include, but are not
limited to, the disappearance of substrate, including decrease in
the amount of polyubiquitin or ubiquitinated substrate protein or
protein remnant, appearance of intermediate and end products, such
as appearance of free ubiquitin monomers, general protein turnover,
specific protein turnover, ubiquitin binding, binding to
ubiquitinated substrate protein, subunit interaction, interaction
with ATP, interaction with cellular components such as trans-acting
regulatory factors, stabilization of specific proteins, and the
like.
[0093] Substantial homology can be to the entire nucleic acid or
amino acid sequence or to fragments of these sequences.
[0094] The invention thus also includes polypeptide fragments of
the ubiquitin protease. Fragments can be derived from the amino
acid sequence shown in SEQ ID NO:1. However, the invention also
encompasses fragments of the variants of the ubiquitin proteases as
described herein.
[0095] The fragments to which the invention pertains, however, are
not to be construed as encompassing fragments that may be disclosed
prior to the present invention.
[0096] Accordingly, a fragment can comprise at least about 10, 15,
20, 25, 30, 35, 40, 45, 50 or more contiguous amino acids.
Fragments can retain one or more of the biological activities of
the protein, for example the ability to bind to ubiquitin or
hydrolyze peptide bonds, as well as fragments that can be used as
an immunogen to generate ubiquitin protease antibodies.
[0097] Biologically active fragments (peptides which are, for
example, 5, 7, 10, 12, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100
or more amino acids in length) can comprise a domain or motif,
e.g., catalytic site, UBP or UCH signature, membrane-associated
regions and sites for glycosylation, cAMP and cGMP-dependent
protein kinase phosphorylation, protein kinase C phosphorylation,
casein kinase II phosphorylation, tyrosine kinase phosphorylation,
N-myristoylation, and amidation. Further possible fragments include
the catalytic site or domain including the cysteine or histidine
boxes as shown in FIG. 1, ubiquitin recognition sites, ubiquitin
binding sites, sites important for subunit interaction, and sites
important for carrying out the other functions of the protease as
described herein.
[0098] Such domains or motifs can be identified by means of routine
computerized homology searching procedures.
[0099] Fragments, for example, can extend in one or both directions
from the functional site to encompass 5, 10, 15, 20, 30, 40, 50, or
up to 100 amino acids. Further, fragments can include sub-fragments
of the specific domains mentioned above, which sub-fragments retain
the function of the domain from which they are derived.
[0100] These regions can be identified by well-known methods
involving computerized homology analysis.
[0101] The invention also provides fragments with immunogenic
properties. These contain an epitope-bearing portion of the
ubiquitin protease and variants. These epitope-bearing peptides are
useful to raise antibodies that bind specifically to a ubiquitin
protease polypeptide or region or fragment. These peptides can
contain at least 10, 12, at least 14, or between at least about 15
to about 30 amino acids.
[0102] Non-limiting examples of antigenic polypeptides that can be
used to generate antibodies include but are not limited to peptides
derived from an extracellular site. Regions having a high
antigenicity index are shown in FIG. 3. However,
intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular peptide
regions.
[0103] The epitope-bearing ubiquitin protease polypeptides may be
produced by any conventional means (Houghten, R. A. (1985) Proc.
Natl. Acad. Sci. USA 82:5131-5135). Simultaneous multiple peptide
synthesis is described in U.S. Pat. No. 4,631,211.
[0104] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the ubiquitin protease fragment and
an additional region fused to the carboxyl terminus of the
fragment.
[0105] The invention thus provides chimeric or fusion proteins.
These comprise a ubiquitin protease peptide sequence operatively
linked to a heterologous peptide having an amino acid sequence not
substantially homologous to the ubiquitin protease. "Operatively
linked" indicates that the ubiquitin protease peptide and the
heterologous peptide are fused in-frame. The heterologous peptide
can be fused to the N-terminus or C-terminus of the ubiquitin
protease or can be internally located.
[0106] In one embodiment the fusion protein does not affect
ubiquitin protease function per se. For example, the fusion protein
can be a GST-fusion protein in which the ubiquitin protease
sequences are fused to the C-terminus of the GST sequences. Other
types of fusion proteins include, but are not limited to, enzymatic
fusion proteins, for example beta-galactosidase fusions, yeast
two-hybrid GAL-4 fusions, poly-His fusions and Ig fusions. Such
fusion proteins, particularly poly-His fusions, can facilitate the
purification of recombinant ubiquitin protease. In certain host
cells (e.g., mammalian host cells), expression and/or secretion of
a protein can be increased by using a heterologous signal sequence.
Therefore, in another embodiment, the fusion protein contains a
heterologous signal sequence at its N-terminus.
[0107] EP-A-O 464 533 discloses fusion proteins comprising various
portions of immunoglobulin constant regions. The Fc is useful in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). In drug discovery, for
example, human proteins have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
(Bennett et al. (1995) J Mol. Recog. 8:52-58 (1995) and Johanson et
al. J Biol. Chem. 270:9459-9471). Thus, this invention also
encompasses soluble fusion proteins containing a ubiquitin protease
polypeptide and various portions of the constant regions of heavy
or light chains of immunoglobulins of various subclass (IgG, IgM,
IgA, IgE). Preferred as immunoglobulin is the constant part of the
heavy chain of human IgG, particularly IgG1, where fusion takes
place at the hinge region. For some uses it is desirable to remove
the Fc after the fusion protein has been used for its intended
purpose, for example when the fusion protein is to be used as
antigen for immunizations. In a particular embodiment, the Fc part
can be removed in a simple way by a cleavage sequence, which is
also incorporated and can be cleaved with factor Xa.
[0108] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see Ausubel et al. (1992)
Current Protocols in Molecular Biology). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A ubiquitin protease-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the ubiquitin protease.
[0109] Another form of fusion protein is one that directly affects
ubiquitin protease functions. Accordingly, a ubiquitin protease
polypeptide is encompassed by the present invention in which one or
more of the ubiquitin protease domains (or parts thereof) has been
replaced by homologous domains (or parts thereof) from another UBP
or UCH species. Accordingly, various permutations are possible. One
or more functional sites as disclosed herein from the specifically
disclosed protease can be replaced by one or more functional sites
from a corresponding UBP family member or from a UCH family member.
Thus, chimeric ubiquitin proteases can be formed in which one or
more of the native domains or subregions has been replaced by
another.
[0110] Additionally, chimeric ubiquitin protease proteins can be
produced in which one or more functional sites is derived from a
different ubiquitin protease family. It is understood however that
sites could be derived from ubiquitin protease families that occur
in the mammalian genome but which have not yet been discovered or
characterized. Such sites include but are not limited to any of the
functional sites disclosed herein.
[0111] The isolated ubiquitin proteases can be purified from cells
that naturally express it, such as from thymus, testes, brain,
breast, skeletal muscle, liver, prostate, thyroid, ovary, fetal
kidney, fetal heart, fetal liver, liver metastases derived from
colon, and malignant lung and breast tissue, especially purified
from cells that have been altered to express it (recombinant), or
synthesized using known protein synthesis methods.
[0112] In one embodiment, the protein is produced by recombinant
DNA techniques. For example, a nucleic acid molecule encoding the
ubiquitin protease polypeptide is cloned into an expression vector,
the expression vector introduced into a host cell and the protein
expressed in the host cell. The protein can then be isolated from
the cells by an appropriate purification scheme using standard
protein purification techniques. Polypeptides often contain amino
acids other than the 20 amino acids commonly referred to as the 20
naturally-occurring amino acids. Further, many amino acids,
including the terminal amino acids, may be modified by natural
processes, such as processing and other post-translational
modifications, or by chemical modification techniques well known in
the art. Common modifications that occur naturally in polypeptides
are described in basic texts, detailed monographs, and the research
literature, and they are well known to those of skill in the
art.
[0113] Accordingly, the polypeptides also encompass derivatives or
analogs in which a substituted amino acid residue is not one
encoded by the genetic code, in which a substituent group is
included, in which the mature polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence for purification of
the mature polypeptide or a pro-protein sequence.
[0114] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphatidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0115] Such modifications are well-known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as by Wold,
F., Posttranslational Covalent Modification of Proteins, B. C.
Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.
(1990) Meth. Enzymol. 182: 626-646) and Rattan et al. (1992) Ann.
N.Y. Acad. Sci. 663:48-62).
[0116] As is also well known, polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
ubiquitination, and they may be circular, with or without
branching, generally as a result of post-translation events,
including natural processing events and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translational natural processes and by synthetic methods.
[0117] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. Blockage of the amino or carboxyl group in a
polypeptide, or both, by a covalent modification, is common in
naturally-occurring and synthetic polypeptides. For instance, the
aminoterminal residue of polypeptides made in E. coli, prior to
proteolytic processing, almost invariably will be
N-formylmethionine.
[0118] The modifications can be a function of how the protein is
made. For recombinant polypeptides, for example, the modifications
will be determined by the host cell posttranslational modification
capacity and the modification signals in the polypeptide amino acid
sequence. Accordingly, when glycosylation is desired, a polypeptide
should be expressed in a glycosylating host, generally a eukaryotic
cell. Insect cells often carry out the same posttranslational
glycosylations as mammalian cells and, for this reason, insect cell
expression systems have been developed to efficiently express
mammalian proteins having native patterns of glycosylation. Similar
considerations apply to other modifications.
[0119] The same type of modification may be present in the same or
varying degree at several sites in a given polypeptide. Also, a
given polypeptide may contain more than one type of
modification.
[0120] Polypeptide Uses
[0121] The protein sequences of the present invention can be used
as a "query sequence" to perform a search against public databases
to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score 100, wordlength=12 to obtain nucleotide
sequences homologous to the nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the proteins of the invention. To obtain gapped
alignments for comparison purposes, Gapped BLAST can be utilized as
described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used.
[0122] The ubiquitin protease polypeptides are useful for producing
antibodies specific for the ubiquitin protease, regions, or
fragments. Regions having a high antigenicity index score are shown
in FIG. 3.
[0123] The ubiquitin protease polypeptides are useful for
biological assays related to ubiquitin protease function. Such
assays involve any of the known functions or activities or
properties useful for diagnosis and treatment of ubiquitin- or
ubiquitin protease-related conditions. Potential assays have been
disclosed herein and generically include disappearance of
substrate, appearance of end product, and general or specific
protein turnover.
[0124] The ubiquitin protease polypeptides are also useful in drug
screening assays, in cell-based or cell-free systems. Cell-based
systems can be native, i.e., cells that normally express the
ubiquitin protease, as a biopsy or expanded in cell culture. In one
embodiment, however, cell-based assays involve recombinant host
cells expressing the ubiquitin protease.
[0125] Determining the ability of the test compound to interact
with the ubiquitin protease can also comprise determining the
ability of the test compound to preferentially bind to the
polypeptide as compared to the ability of a known binding molecule
(e.g., ubiquitin) to bind to the polypeptide.
[0126] The polypeptides can be used to identify compounds that
modulate ubiquitin protease activity. Such compounds, for example,
can increase or decrease affinity for polyubiquitin, either linear
or branched chain, ubiquitinated protein substrate, or
ubiquitinated protein substrate remnants. Such compounds could
also, for example, increase or decrease the rate of binding to
these components. Such compounds could also compete with these
components for binding to the ubiquitin protease or displace these
components bound to the ubiquitin protease. Such compounds could
also affect interaction with other components, such as ATP, other
subunits, for example, in the 19S complex, and transcriptional
regulatory factors. It is understood, therefore, that such
compounds can be identified not only by means of ubiquitin, but by
means of any of the components that functionally interact with the
disclosed protease. This includes, but is not limited to, any of
those components disclosed herein.
[0127] Both ubiquitin protease and appropriate variants and
fragments can be used in high-throughput screens to assay candidate
compounds for the ability to bind to the ubiquitin protease. These
compounds can be further screened against a functional ubiquitin
protease to determine the effect of the compound on the ubiquitin
protease activity. Compounds can be identified that activate
(agonist) or inactivate (antagonist) the ubiquitin protease to a
desired degree. Modulatory methods can be performed in vitro (e.g.,
by culturing the cell with the agent) or, alternatively, in vivo
(e.g., by administering the agent to a subject.
[0128] The ubiquitin protease polypeptides can be used to screen a
compound for the ability to stimulate or inhibit interaction
between the ubiquitin protease protein and a target molecule that
normally interacts with the ubiquitin protease protein. The target
can be ubiquitin, ubiquitinated substrate, or polyubiquitin or
another component of the pathway with which the ubiquitin protease
protein normally interacts (for example, ATP). The assay includes
the steps of combining the ubiquitin protease protein with a
candidate compound under conditions that allow the ubiquitin
protease protein or fragment to interact with the target molecule,
and to detect the formation of a complex between the ubiquitin
protease protein and the target or to detect the biochemical
consequence of the interaction with the ubiquitin protease and the
target. Any of the associated effects of protease function can be
assayed. This includes the production of hydrolysis products, such
as free terminal peptide substrate, free terminal amino acid from
the hydrolyzed substrate, free ubiquitin, lower molecular weight
species of hydrolyzed polyubiquitin, released intact substrate
protein resulting from rescue from proteolysis, free polyubiquitin
formed from hydrolysis of the polyubiquitin from intact substrate,
and substrate remnants, such as amino acids and peptides produced
from proteolysis of the substrate protein, and biological endpoints
of the pathway.
[0129] Determining the ability of the ubiquitin protease to bind to
a target molecule can also be accomplished using a technology such
as real-time Bimolecular Interaction Analysis (BIA). Sjolander et
al. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr.
Opin. Struct. Biol. 5:699-705. As used herein, "BIA" is a
technology for studying biospecific interactions in real time,
without labeling any of the interactants (e.g., BIAcore.TM.).
Changes in the optical phenomenon surface plasmon resonance (SPR)
can be used as an indication of real-time reactions between
biological molecules.
[0130] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to polypeptide libraries, while the
other four approaches are applicable to polypeptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, K. S.
(1997) Anticancer Drug Des. 12:145).
[0131] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in DeWitt et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678;
Cho et al. (1993) Science 261:1303; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem.
37:1233. Libraries of compounds maybe presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S.
patent '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci.
USA 89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 97:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra).
[0132] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al. (1991)
Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al. (1993) Cell 72:767-778); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0133] One candidate compound is a soluble full-length ubiquitin
protease or fragment that competes for substrate binding. Other
candidate compounds include mutant ubiquitin proteases or
appropriate fragments containing mutations that affect ubiquitin
protease function and compete for substrate. Accordingly, a
fragment that competes for substrate, for example with a higher
affinity, or a fragment that binds substrate but does not hydrolyze
the peptide bond, is encompassed by the invention.
[0134] Other candidate compounds include ubiquitinated protein or
protein analog that binds to the protease but is not released or
released slowly. Other candidate compounds include analogs of the
other natural substrates, such as substrate remnants that bind to
but are not released or released more slowly. Further candidate
compounds include activators of the proteases such as cytokines,
including but not limited to, those disclosed herein.
[0135] The invention provides other end points to identify
compounds that modulate (stimulate or inhibit) ubiquitin protease
activity. The assays typically involve an assay of events in the
pathway that indicate ubiquitin protease activity. This can include
cellular events that result from deubiquitination, such as cell
cycle progression, programmed cell death, growth factor-mediated
signal transduction, or any of the cellular processes including,
but not limited to, those disclosed herein as resulting from
deubiquitination. Specific phenotypes include changes in stress
response, DNA replication, receptor internalization, cellular
transformation or reversal of transformation, and transcriptional
silencing.
[0136] Assays are based on the multiple cellular functions of
deubiquitinating enzymes. These enzymes act at various different
levels in the regulation of protein ubiquitination. A
deubiquitinating enzyme can degrade a linear polyubiquitin chain
into monomeric ubiquitin molecules. Deubiquitinating enzymes, such
as isopeptidase-T, can degrade a branched multiubiquitin chain into
monomeric ubiquitin molecules. Deubiquitinating enzymes can remove
ubiquitin from a ubiquitin-conjugated target protein. The
deubiquitinating enzyme, such as FAF or PA700 isopeptidase, can
remove polyubiquitin from a ubiquitinated target protein, and
thereby rescue the target from degradation by the 26S proteasome.
Deubiquitinating enzymes such as Doa-4 can remove polyubiquitin
from proteasome degradation products. The result of all of these is
to regulate the cellular pool of free monomeric ubiquitin.
Accordingly, assays can be based on detection of any of the
products produced by hydrolysis/deubiquitination.
[0137] Further, the expression of genes that are up- or
down-regulated by action of the ubiquitin protease can be assayed.
In one embodiment, the regulatory region of such genes can be
operably linked to a marker that is easily detectable, such as
luciferase.
[0138] Accordingly, any of the biological or biochemical functions
mediated by the ubiquitin protease can be used as an endpoint
assay. These include all of the biochemical or
biochemical/biological events described herein, in the references
cited herein, incorporated by reference for these endpoint assay
targets, and other functions known to those of ordinary skill in
the art.
[0139] Binding and/or activating compounds can also be screened by
using chimeric ubiquitin protease proteins in which one or more
domains, sites, and the like, as disclosed herein, or parts
thereof, can be replaced by their heterologous counterparts derived
from other ubiquitin proteases. For example, a recognition or
binding region can be used that interacts with different substrate
specificity and/or affinity than the native ubiquitin protease.
Accordingly, a different set of pathway components is available as
an end-point assay for activation. Further, sites that are
responsible for developmental, temporal, or tissue specificity can
be replaced by heterologous sites such that the protease can be
detected under conditions of specific developmental, temporal, or
tissue-specific expression.
[0140] The ubiquitin protease polypeptides are also useful in
competition binding assays in methods designed to discover
compounds that interact with the ubiquitin protease. Thus, a
compound is exposed to a ubiquitin protease polypeptide under
conditions that allow the compound to bind to or to otherwise
interact with the polypeptide. Soluble ubiquitin protease
polypeptide is also added to the mixture. If the test compound
interacts with the soluble ubiquitin protease polypeptide, it
decreases the amount of complex formed or activity from the
ubiquitin protease target. This type of assay is particularly
useful in cases in which compounds are sought that interact with
specific regions of the ubiquitin protease. Thus, the soluble
polypeptide that competes with the target ubiquitin protease region
is designed to contain peptide sequences corresponding to the
region of interest.
[0141] Another type of competition-binding assay can be used to
discover compounds that interact with specific functional sites. As
an example, ubiquitin and a candidate compound can be added to a
sample of the ubiquitin protease. Compounds that interact with the
ubiquitin protease at the same site as ubiquitin will reduce the
amount of complex formed between the ubiquitin protease and
ubiquitin. Accordingly, it is possible to discover a compound that
specifically prevents interaction between the ubiquitin protease
and ubiquitin. Another example involves adding a candidate compound
to a sample of ubiquitin protease and polyubiquitin. A compound
that competes with polyubiquitin will reduce the amount of
hydrolysis or binding of the polyubiquitin to the ubiquitin
protease. Accordingly, compounds can be discovered that directly
interact with the ubiquitin protease and compete with
polyubiquitin. Such assays can involve any other component that
interacts with the ubiquitin protease, such as ubiquitinated
substrate protein, ubiquitinated substrate remnants, and cellular
components with which the protease interacts such as
transcriptional regulatory factors.
[0142] To perform cell free drug screening assays, it is desirable
to immobilize either the ubiquitin protease, or fragment, or its
target molecule to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
[0143] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase/ubiquitin
protease fusion proteins can be adsorbed onto glutathione sepharose
beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized
microtitre plates, which are then combined with the cell lysates
(e.g., .sup.35S-labeled) and the candidate compound, and the
mixture incubated under conditions conducive to complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads are washed to remove any unbound label, and
the matrix immobilized and radiolabel determined directly, or in
the supernatant after the complexes is dissociated. Alternatively,
the complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of ubiquitin protease-binding protein found
in the bead fraction quantitated from the gel using standard
electrophoretic techniques. For example, either the polypeptide or
its target molecule can be immobilized utilizing conjugation of
biotin and streptavidin using techniques well known in the art.
Alternatively, antibodies reactive with the protein but which do
not interfere with binding of the protein to its target molecule
can be derivatized to the wells of the plate, and the protein
trapped in the wells by antibody conjugation. Preparations of a
ubiquitin protease-binding target component, such as ubiquitin,
polyubiquitin, ubiquitinated substrate protein, ubiquitinated
substrate protein remnant, or ubiquitinated remnant amino acid, and
a candidate compound are incubated in the ubiquitin
protease-presenting wells and the amount of complex trapped in the
well can be quantitated. Methods for detecting such complexes, in
addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies
reactive with the ubiquitin protease target molecule, or which are
reactive with ubiquitin protease and compete with the target
molecule; as well as enzyme-linked assays which rely on detecting
an enzymatic activity associated with the target molecule.
[0144] Modulators of ubiquitin protease activity identified
according to these drug screening assays can be used to treat a
subject with a disorder mediated by the ubiquitin protease pathway,
by treating cells that express the ubiquitin protease, including
but not limited to, those shown in FIGS. 6, 7, and 8 and especially
liver, breast, brain, and testes. In one embodiment, the cells
treated are lung or breast cancer cells. In another embodiment of
the invention the cells that are treated are colon metastases to
the liver. These methods of treatment include the steps of
administering the modulators of ubiquitin protease activity in a
pharmaceutical composition as described herein, to a subject in
need of such treatment.
[0145] Disorders involving the liver include, but are not limited
to, hepatic injury; jaundice and cholestasis, such as bilirubin and
bile formation; hepatic failure and cirrhosis, such as cirrhosis,
portal hypertension, including ascites, portosystemic shunts, and
splenomegaly; infectious disorders, such as viral hepatitis,
including hepatitis A-E infection and infection by other hepatitis
viruses, clinicopathologic syndromes, such as the carrier state,
asymptomatic infection, acute viral hepatitis, chronic viral
hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and
toxin-induced liver disease, such as alcoholic liver disease;
inborn errors of metabolism and pediatric liver disease, such as
hemochromatosis, Wilson disease, a.sub.1-antitrypsin deficiency,
and neonatal hepatitis; intrahepatic biliary tract disease, such as
secondary biliary cirrhosis, primary biliary cirrhosis, primary
sclerosing cholangitis, and anomalies of the biliary tree;
circulatory disorders, such as impaired blood flow into the liver,
including hepatic artery compromise and portal vein obstruction and
thrombosis, impaired blood flow through the liver, including
passive congestion and centrilobular necrosis and peliosis hepatis,
hepatic vein outflow obstruction, including hepatic vein thrombosis
(Budd-Chiari syndrome) and veno-occlusive disease; hepatic disease
associated with pregnancy, such as preeclampsia and eclampsia,
acute fatty liver of pregnancy, and intrehepatic cholestasis of
pregnancy; hepatic complications of organ or bone marrow
transplantation, such as drug toxicity after bone marrow
transplantation, graft-versus-host disease and liver rejection, and
nonimmunologic damage to liver allografts; tumors and tumorous
conditions, such as nodular hyperplasias, adenomas, and malignant
tumors, including primary carcinoma of the liver and metastatic
tumors.
[0146] Disorders involving the brain include, but are limited to,
disorders involving neurons, and disorders involving glia, such as
astrocytes, oligodendrocytes, ependymal cells, and microglia;
cerebral edema, raised intracranial pressure and herniation, and
hydrocephalus; malformations and developmental diseases, such as
neural tube defects, forebrain anomalies, posterior fossa
anomalies, and syringomyelia and hydromyelia; perinatal brain
injury; cerebrovascular diseases, such as those related to hypoxia,
ischemia, and infarction, including hypotension, hypoperfusion, and
low-flow states--global cerebral ischemia and focal cerebral
ischemia--infarction from obstruction of local blood supply,
intracranial hemorrhage, including intracerebral (intraparenchymal)
hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms,
and vascular malformations, hypertensive cerebrovascular disease,
including lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-bome
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicalla-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyelitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer disease and Pick
disease, degenerative diseases of basal ganglia and brain stem,
including Parkinsonism, idiopathic Parkinson disease (paralysis
agitans), progressive supranuclear palsy, corticobasal
degeneration, multiple system atrophy, including striatonigral
degeneration, Shy-Drager syndrome, and olivopontocerebellar
atrophy, and Huntington disease; spinocerebellar degenerations,
including spinocerebellar ataxias, including Friedreich ataxia, and
ataxia-telanglectasia, degenerative diseases affecting motor
neurons, including amyotrophic lateral sclerosis (motor neuron
disease), bulbospinal atrophy (Kennedy syndrome), and spinal
muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B.sub.1) deficiency and vitamin B.sub.12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease.
[0147] Disorders involving the heart, include but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left-to-right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
[0148] Disorders involving the thymus include developmental
disorders, such as DiGeorge syndrome with thymic hypoplasia or
aplasia; thymic cysts; thymic hypoplasia, which involves the
appearance of lymphoid follicles within the thymus, creating thymic
follicular hyperplasia; and thymomas, including germ cell tumors,
lynphomas, Hodgkin disease, and carcinoids. Thymomas can include
benign or encapsulated thymoma, and malignant thymoma Type I
(invasive thymoma) or Type II, designated thymic carcinoma.
[0149] Disorders involving the kidney include, but are not limited
to, congenital anomalies including, but not limited to, cystic
diseases of the kidney, that include but are not limited to, cystic
renal dysplasia, autosomal dominant (adult) polycystic kidney
disease, autosomal recessive (childhood) polycystic kidney disease,
and cystic diseases of renal medulla, which include, but are not
limited to, medullary sponge kidney and nephronophthisis-uremic
medullary cystic disease complex, acquired (dialysis-associated)
cystic disease and simple cysts; glomerular diseases including
pathologies of glomerular injury that include, but are not limited
to, in situ immune complex deposition, that includes, but is not
limited to, anti-GBM nephritis, Heymann nephritis, and antibodies
against planted antigens, circulating immune complex nephritis,
antibodies to glomerular cells, cell-mediated immunity in
glomerulonephritis, activation of alternative complement pathway,
epithelial cell injury, and pathologies involving mediators of
glomerular injury including cellular and soluble mediators, acute
glomerulonephritis, such as acute proliferative (poststreptococcal,
postinfectious) glomerulonephritis, including but not limited to,
poststreptococcal glomerulonephritis and nonstreptococcal acute
glomerulonephritis, rapidly progressive (crescentic)
glomerulonephritis, nephrotic syndrome, membranous
glomerulonephritis (membranous nephropathy), minimal change disease
(lipoid nephrosis), focal segmental glomerulosclerosis,
membranoproliferative glomerulonephritis, IgA nephropathy (Berger
disease), focal proliferative and necrotizing glomerulonephritis
(focal glomerulonephritis), hereditary nephritis, including but not
limited to, Alport syndrome and thin membrane disease (benign
familial hematuria), chronic glomerulonephritis, glomerular lesions
associated with systemic disease, including but not limited to,
systemic lupus erythematosus, Henoch-Schonlein purpura, bacterial
endocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary
and immunotactoid glomerulonephritis, and other systemic disorders;
diseases affecting tubules and interstitium, including, but not
limited to, acute tubular necrosis and tubulointerstitial
nephritis, including but not limited to, pyelonephritis and urinary
tract infection, acute pyelonephritis, chronic pyelonephritis and
reflux nephropathy, tubulointerstitial nephritis induced by drugs
and toxins, including but not limited to, acute drug-induced
interstitial nephritis, analgesic abuse nephropathy, and
nephropathy associated with nonsteroidal anti-inflammatory drugs,
and other tubulointerstitial diseases including, but not limited
to, urate nephropathy, hypercalcemia and nephrocalcinosis, and
multiple myeloma; diseases of blood vessels including, including
but not limited to, benign nephrosclerosis, malignant hypertension
and accelerated nephrosclerosis, renal artery stenosis, and
thrombotic microangiopathies, including, but not limited to,
classic (childhood) hemolytic-uremic syndrome, adult
hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura,
idiopathic HUS/TTP, and other vascular disorders including, but not
limited to, atherosclerotic ischemic renal disease, atheroembolic
renal disease, sickle cell disease nephropathy, diffuse cortical
necrosis, and renal infarcts; urinary tract obstruction
(obstructive uropathy); urolithiasis (renal calculi, stones); and
tumors of the kidney including, but not limited to, benign tumors,
such as renal papillary adenoma, renal fibroma or hamartoma
(renomedullary interstitial cell tumor), angiomyolipoma, and
oncocytoma, and malignant tumors, including renal cell carcinoma
(hypemephroma, adenocarcinoma of kidney), which includes urothelial
carcinomas of renal pelvis.
[0150] Disorders of the breast include, but are not limited to,
disorders of development; inflammations, including but not limited
to, acute mastitis, periductal mastitis (recurrent subareolar
abscess, squamous metaplasia of lactiferous ducts), mammary duct
ectasia, fat necrosis, granulomatous mastitis, and pathologies
associated with silicone breast implants; fibrocystic changes;
proliferative breast disease including, but not limited to,
epithelial hyperplasia, sclerosing adenosis, and small duct
papillomas; tumors including, but not limited to, stromal tumors
such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial
tumors, such as large duct papilloma; carcinoma of the breast
including in situ (noninvasive) carcinoma that includes ductal
carcinoma in situ (including Paget's disease) and lobular carcinoma
in situ, and invasive (infiltrating) carcinoma including, but not
limited to, invasive ductal carcinoma, no special type, invasive
lobular carcinoma, medullary carcinoma, colloid (mucinous)
carcinoma, tubular carcinoma, and invasive papillary carcinoma, and
miscellaneous malignant neoplasms. Disorders in the male breast
include, but are not limited to, gynecomastia and carcinoma.
[0151] Disorders involving the testis and epididymis include, but
are not limited to, congenital anomalies such as cryptorchidism,
regressive changes such as atrophy, inflammations such as
nonspecific epididymitis and orchitis, granulomatous (autoimmune)
orchitis, and specific inflammations including, but not limited to,
gonorrhea, mumps, tuberculosis, and syphilis, vascular disturbances
including torsion, testicular tumors including germ cell tumors
that include, but are not limited to, seminoma, spermatocytic
seminoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma,
teratoma, and mixed tumors, tumors of sex cord-gonadal stroma
including, but not limited to, Leydig (interstitial) cell tumors
and Sertoli cell tumors (androblastoma), and testicular lymphoma,
and miscellaneous lesions of tunica vaginalis.
[0152] Disorders involving the prostate include, but are not
limited to, inflammations, benign enlargement, for example, nodular
hyperplasia (benign prostatic hypertrophy or hyperplasia), and
tumors such as carcinoma.
[0153] Disorders involving the thyroid include, but are not limited
to, hyperthyroidism; hypothyroidism including, but not limited to,
cretinism and myxedema; thyroiditis including, but not limited to,
hashimoto thyroiditis, subacute (granulomatous) thyroiditis, and
subacute lymphocytic (painless) thyroiditis; Graves disease;
diffuse and multinodular goiter including, but not limited to,
diffuse nontoxic (simple) goiter and multinodular goiter; neoplasms
of the thyroid including, but not limited to, adenomas, other
benign tumors, and carcinomas, which include, but are not limited
to, papillary carcinoma, follicular carcinoma, medullary carcinoma,
and anaplastic carcinoma; and cogenital anomalies.
[0154] Disorders involving the skeletal muscle include tumors, such
as rhabdomyosarcoma.
[0155] The ubiquitin protease polypeptides are thus useful for
treating a ubiquitin protease-associated disorder characterized by
aberrant expression or activity of a ubiquitin protease. The
polypeptides can also be useful for treating a disorder
characterized by excessive amounts of polyubiquitin or
ubiquitinated substrate/remnant/amino acid. In one embodiment, the
method involves administering an agent (e.g., an agent identified
by a screening assay described herein), or combination of agents
that modulates (e.g., upregulates or downregulates) expression or
activity of the protein. In another embodiment, the method involves
administering the ubiquitin protease as therapy to compensate for
reduced or aberrant expression or activity of the protein.
[0156] Methods for treatment include but are not limited to the use
of soluble ubiquitin protease or fragments of the ubiquitin
protease protein that compete for substrates including those
disclosed herein. These ubiquitin proteases or fragments can have a
higher affinity for the target so as to provide effective
competition.
[0157] Stimulation of activity is desirable in situations in which
the protein is abnormally downregulated and/or in which increased
activity is likely to have a beneficial effect. Likewise,
inhibition of activity is desirable in situations in which the
protein is abnormally upregulated and/or in which decreased
activity is likely to have a beneficial effect. In one example of
such a situation, a subject has a disorder characterized by
aberrant development or cellular differentiation. In another
example, the subject has a proliferative disease (e.g., cancer) or
a disorder characterized by an aberrant hematopoietic response. In
another example, it is desirable to achieve tissue regeneration in
a subject (e.g., where a subject has undergone brain or spinal cord
injury and it is desirable to regenerate neuronal tissue in a
regulated manner).
[0158] In yet another aspect of the invention, the proteins of the
invention can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO
94/10300), to identify other proteins (captured proteins) which
bind to or interact with the proteins of the invention and modulate
their activity.
[0159] The ubiquitin protease polypeptides also are useful to
provide a target for diagnosing a disease or predisposition to
disease mediated by the ubiquitin protease, including, but not
limited to, diseases involving tissues in which the ubiquitin
proteases are expressed as disclosed herein, such as in breast
cancer. Accordingly, methods are provided for detecting the
presence, or levels of, the ubiquitin protease in a cell, tissue,
or organism. The method involves contacting a biological sample
with a compound capable of interacting with the ubiquitin protease
such that the interaction can be detected.
[0160] The polypeptides are also useful for treating a disorder
characterized by reduced amounts of these components. Thus,
increasing or decreasing the activity of the protease is beneficial
to treatment. The polypeptides are also useful to provide a target
for diagnosing a disease characterized by excessive substrate or
reduced levels of substrate. Accordingly, where substrate is
excessive, use of the protease polypeptides can provide a
diagnostic assay. Furthermore, for example, proteases having
reduced activity can be used to diagnose conditions in which
reduced substrate is responsible for the disorder.
[0161] One agent for detecting ubiquitin protease is an antibody
capable of selectively binding to ubiquitin protease. A biological
sample includes tissues, cells and biological fluids isolated from
a subject, as well as tissues, cells and fluids present within a
subject.
[0162] The ubiquitin protease also provides a target for diagnosing
active disease, or predisposition to disease, in a patient having a
variant ubiquitin protease. Thus, ubiquitin protease can be
isolated from a biological sample and assayed for the presence of a
genetic mutation that results in an aberrant protein. This includes
amino acid substitution, deletion, insertion, rearrangement, (as
the result of aberrant splicing events), and inappropriate
post-translational modification. Analytic methods include altered
electrophoretic mobility, altered tryptic peptide digest, altered
ubiquitin protease activity in cell-based or cell-free assay,
alteration in binding to or hydrolysis of polyubiquitin, binding to
ubiquitinated substrate protein or hydrolysis of the ubiquitin from
the protein, binding to ubiquitinated protein remnant, including
peptide or amino acid, and hydrolysis of the ubiquitin from the
remnant, general protein turnover, specific protein turnover,
antibody-binding pattern, altered isoelectric point, direct amino
acid sequencing, and any other of the known assay techniques useful
for detecting mutations in a protein in general or in a ubiquitin
protease specifically, including assays discussed herein.
[0163] In vitro techniques for detection of ubiquitin protease
include enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. Alternatively, the
protein can be detected in vivo in a subject by introducing into
the subject a labeled anti-ubiquitin protease antibody. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques. Particularly useful are methods, which
detect the allelic variant of the ubiquitin protease expressed in a
subject, and methods, which detect fragments of the ubiquitin
protease in a sample.
[0164] The ubiquitin protease polypeptides are also useful in
pharmacogenomic analysis. Pharmacogenomics deal with clinically
significant hereditary variations in the response to drugs due to
altered drug disposition and abnormal action in affected persons.
See, e.g., Eichelbaum, M. (1996) Clin. Exp. Pharmacol. Physiol.
23(10-11):983-985, and Linder, M. W. (1997) Clin. Chem.
43(2):254-266. The clinical outcomes of these variations result in
severe toxicity of therapeutic drugs in certain individuals or
therapeutic failure of drugs in certain individuals as a result of
individual variation in metabolism. Thus, the genotype of the
individual can determine the way a therapeutic compound acts on the
body or the way the body metabolizes the compound. Further, the
activity of drug metabolizing enzymes affects both the intensity
and duration of drug action. Thus, the pharmacogenomics of the
individual permit the selection of effective compounds and
effective dosages of such compounds for prophylactic or therapeutic
treatment based on the individual's genotype. The discovery of
genetic polymorphisms in some drug metabolizing enzymes has
explained why some patients do not obtain the expected drug
effects, show an exaggerated drug effect, or experience serious
toxicity from standard drug dosages. Polymorphisms can be expressed
in the phenotype of the extensive metabolizer and the phenotype of
the poor metabolizer. Accordingly, genetic polymorphism may lead to
allelic protein variants of the ubiquitin protease in which one or
more of the ubiquitin protease functions in one population is
different from those in another population. The polypeptides thus
allow a target to ascertain a genetic predisposition that can
affect treatment modality. Thus, in a ubiquitin-based treatment,
polymorphism may give rise to catalytic regions that are more or
less active. Accordingly, dosage would necessarily be modified to
maximize the therapeutic effect within a given population
containing the polymorphism. As an alternative to genotyping,
specific polymorphic polypeptides could be identified.
[0165] The ubiquitin protease polypeptides are also useful for
monitoring therapeutic effects during clinical trials and other
treatment. Thus, the therapeutic effectiveness of an agent that is
designed to increase or decrease gene expression, protein levels or
ubiquitin protease activity can be monitored over the course of
treatment using the ubiquitin protease polypeptides as an end-point
target. The monitoring can be, for example, as follows: (i)
obtaining a pre-administration sample from a subject prior to
administration of the agent; (ii) detecting the level of expression
or activity of the protein in the pre-administration sample; (iii)
obtaining one or more post-administration samples from the subject;
(iv) detecting the level of expression or activity of the protein
in the post-administration samples; (v) comparing the level of
expression or activity of the protein in the pre-administration
sample with the protein in the post-administration sample or
samples; and (vi) increasing or decreasing the administration of
the agent to the subject accordingly.
[0166] Antibodies
[0167] The invention also provides antibodies that selectively bind
to the ubiquitin protease and its variants and fragments. An
antibody is considered to selectively bind, even if it also binds
to other proteins that are not substantially homologous with the
ubiquitin protease. These other proteins share homology with a
fragment or domain of the ubiquitin protease. This conservation in
specific regions gives rise to antibodies that bind to both
proteins by virtue of the homologous sequence. In this case, it
would be understood that antibody binding to the ubiquitin protease
is still selective.
[0168] To generate antibodies, an isolated ubiquitin protease
polypeptide is used as an immunogen to generate antibodies using
standard techniques for polyclonal and monoclonal antibody
preparation. Either the full-length protein or antigenic peptide
fragment can be used. Regions having a high antigenicity index are
shown in FIG. 3.
[0169] Antibodies are preferably prepared from these regions or
from discrete fragments in these regions. However, antibodies can
be prepared from any region of the peptide as described herein. A
preferred fragment produces an antibody that diminishes or
completely prevents substrate hydrolysis or binding. Antibodies can
be developed against the entire ubiquitin protease or domains of
the ubiquitin protease as described herein. Antibodies can also be
developed against specific functional sites as disclosed
herein.
[0170] The antigenic peptide can comprise a contiguous sequence of
at least 12, 14, 15, or 30 amino acid residues. In one embodiment,
fragments correspond to regions that are located on the surface of
the protein, e.g., hydrophilic regions. These fragments are not to
be construed, however, as encompassing any fragments, which may be
disclosed prior to the invention.
[0171] Antibodies can be polyclonal or monoclonal. An intact
antibody, or a fragment thereof (e.g. Fab or F(ab').sub.2) can be
used.
[0172] Detection can be facilitated by coupling (i.e., physically
linking) the antibody to a detectable substance. Examples of
detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent
materials, and radioactive materials. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0173] An appropriate immunogenic preparation can be derived from
native, recombinantly expressed, or chemically synthesized
peptides.
[0174] Antibody Uses
[0175] The antibodies can be used to isolate a ubiquitin protease
by standard techniques, such as affinity chromatography or
immunoprecipitation. The antibodies can facilitate the purification
of the natural ubiquitin protease from cells and recombinantly
produced ubiquitin protease expressed in host cells.
[0176] The antibodies are useful to detect the presence of
ubiquitin protease in cells or tissues to determine the pattern of
expression of the ubiquitin protease among various tissues in an
organism and over the course of normal development.
[0177] The antibodies can be used to detect ubiquitin protease in
situ, in vitro, or in a cell lysate or supernatant in order to
evaluate the abundance and pattern of expression.
[0178] The antibodies can be used to assess abnormal tissue
distribution or abnormal expression during development.
[0179] Antibody detection of circulating fragments of the full
length ubiquitin protease can be used to identify ubiquitin
protease turnover.
[0180] Further, the antibodies can be used to assess ubiquitin
protease expression in disease states such as in active stages of
the disease or in an individual with a predisposition toward
disease related to ubiquitin or ubiquitin protease function. When a
disorder is caused by an inappropriate tissue distribution,
developmental expression, or level of expression of the ubiquitin
protease protein, the antibody can be prepared against the normal
ubiquitin protease protein. If a disorder is characterized by a
specific mutation in the ubiquitin protease, antibodies specific
for this mutant protein can be used to assay for the presence of
the specific mutant ubiquitin protease. However,
intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular ubiquitin protease
peptide regions.
[0181] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Antibodies can be developed against the whole
ubiquitin protease or portions of the ubiquitin protease.
[0182] The diagnostic uses can be applied, not only in genetic
testing, but also in monitoring a treatment modality. Accordingly,
where treatment is ultimately aimed at correcting ubiquitin
protease expression level or the presence of aberrant ubiquitin
proteases and aberrant tissue distribution or developmental
expression, antibodies directed against the ubiquitin protease or
relevant fragments can be used to monitor therapeutic efficacy.
[0183] Antibodies accordingly can be used diagnostically to monitor
protein levels in tissue as part of a clinical testing procedure,
e.g., to, for example, determine the efficacy of a given treatment
regimen.
[0184] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic ubiquitin
protease can be used to identify individuals that require modified
treatment modalities.
[0185] The antibodies are also useful as diagnostic tools as an
immunological marker for aberrant ubiquitin protease analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0186] The antibodies are also useful for tissue typing. Thus,
where a specific ubiquitin protease has been correlated with
expression in a specific tissue, antibodies that are specific for
this ubiquitin protease can be used to identify a tissue type.
[0187] The antibodies are also useful in forensic identification.
Accordingly, where an individual has been correlated with a
specific genetic polymorphism resulting in a specific polymorphic
protein, an antibody specific for the polymorphic protein can be
used as an aid in identification.
[0188] The antibodies are also useful for inhibiting ubiquitin
protease function, for example, blocking ubiquitin or polyubiquitin
binding, or binding to ubiquitinated substrate or substrate
remnants.
[0189] These uses can also be applied in a therapeutic context in
which treatment involves inhibiting ubiquitin protease function. An
antibody can be used, for example, to block ubiquitin binding.
Antibodies can be prepared against specific fragments containing
sites required for function or against intact ubiquitin protease
associated with a cell.
[0190] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. For an overview of this
technology for producing human antibodies, see Lonberg et al.
(1995) Int. Rev. Immunol. 13:65-93. For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, e.g., U.S.
Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No.
5,545,806.
[0191] The invention also encompasses kits for using antibodies to
detect the presence of a ubiquitin protease protein in a biological
sample. The kit can comprise antibodies such as a labeled or
labelable antibody and a compound or agent for detecting ubiquitin
protease in a biological sample; means for determining the amount
of ubiquitin protease in the sample; and means for comparing the
amount of ubiquitin protease in the sample with a standard.
[0192] The compound or agent can be packaged in a suitable
container. The kit can further comprise instructions for using the
kit to detect ubiquitin protease.
[0193] Polynucleotides
[0194] The nucleotide sequence in SEQ ID NO:2 was obtained by
sequencing the deposited human cDNA. Accordingly, the sequence of
the deposited clone is controlling as to any discrepancies between
the two and any reference to the sequence of SEQ ID NO:2 includes
reference to the sequence of the deposited cDNA.
[0195] The specifically disclosed cDNA comprises the coding region
and 5' and 3' untranslated sequences in SEQ ID NO:2.
[0196] The invention provides isolated polynucleotides encoding the
novel ubiquitin protease. The term "ubiquitin protease
polynucleotide" or "ubiquitin protease nucleic acid" refers to the
sequence shown in SEQ ID NO:2 or in the deposited cDNA. The term
"ubiquitin protease polynucleotide" or "ubiquitin protease nucleic
acid" further includes variants and fragments of the ubiquitin
protease polynucleotide.
[0197] An "isolated" ubiquitin protease nucleic acid is one that is
separated from other nucleic acid present in the natural source of
the ubiquitin protease nucleic acid. Preferably, an "isolated"
nucleic acid is free of sequences which naturally flank the
ubiquitin protease nucleic acid (i.e., sequences located at the 5'
and 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. However, there can be some
flanking nucleotide sequences, for example up to about 5 KB. The
important point is that the ubiquitin protease nucleic acid is
isolated from flanking sequences such that it can be subjected to
the specific manipulations described herein, such as recombinant
expression, preparation of probes and primers, and other uses
specific to the ubiquitin protease nucleic acid sequences.
[0198] Moreover, an "isolated" nucleic acid molecule, such as a
cDNA or RNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0199] In some instances, the isolated material will form part of a
composition (for example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0200] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0201] In some instances, the isolated material will form part of a
composition (or example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0202] The ubiquitin protease polynucleotides can encode the mature
protein plus additional amino or carboxyterminal amino acids, or
amino acids interior to the mature polypeptide (when the mature
form has more than one polypeptide chain, for instance). Such
sequences may play a role in processing of a protein from precursor
to a mature form, facilitate protein trafficking, prolong or
shorten protein half-life or facilitate manipulation of a protein
for assay or production, among other things. As generally is the
case in situ, the additional amino acids may be processed away from
the mature protein by cellular enzymes.
[0203] The ubiquitin protease polynucleotides include, but are not
limited to, the sequence encoding the mature polypeptide alone, the
sequence encoding the mature polypeptide and additional coding
sequences, such as a leader or secretory sequence (e.g., a pre-pro
or pro-protein sequence), the sequence encoding the mature
polypeptide, with or without the additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but non-translated
sequences that play a role in transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding
and stability of mRNA. In addition, the polynucleotide may be fused
to a marker sequence encoding, for example, a peptide that
facilitates purification.
[0204] Ubiquitin protease polynucleotides can be in the form of
RNA, such as mRNA, or in the form DNA, including cDNA and genomic
DNA obtained by cloning or produced by chemical synthetic
techniques or by a combination thereof. The nucleic acid,
especially DNA, can be double-stranded or single-stranded.
Single-stranded nucleic acid can be the coding strand (sense
strand) or the non-coding strand (anti-sense strand).
[0205] Ubiquitin protease nucleic acid can comprise the nucleotide
sequence shown in SEQ ID NO:2, corresponding to human cDNA.
[0206] In one embodiment, the ubiquitin protease nucleic acid
comprises only the coding region.
[0207] The invention further provides variant ubiquitin protease
polynucleotides, and fragments thereof, that differ from the
nucleotide sequence shown in SEQ ID NO:2 due to degeneracy of the
genetic code and thus encode the same protein as that encoded by
the nucleotide sequence shown in SEQ ID NO:2.
[0208] The invention also provides ubiquitin protease nucleic acid
molecules encoding the variant polypeptides described herein. Such
polynucleotides may be naturally occurring, such as allelic
variants (same locus), homologs (different locus), and orthologs
(different organism), or may be constructed by recombinant DNA
methods or by chemical synthesis. Such non-naturally occurring
variants may be made by mutagenesis techniques, including those
applied to polynucleotides, cells, or organisms. Accordingly, as
discussed above, the variants can contain nucleotide substitutions,
deletions, inversions and insertions.
[0209] Typically, variants have a substantial identity with a
nucleic acid molecule of SEQ ID NO:2 and the complements thereof.
Variation can occur in either or both the coding and non-coding
regions. The variations can produce both conservative and
non-conservative amino acid substitutions.
[0210] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. These variants comprise a
nucleotide sequence encoding a ubiquitin protease that is at least
about 60-65%, 65-70%, typically at least about 70-75%, more
typically at least about 80-85%, and most typically at least about
90-95% or more homologous to the nucleotide sequence shown in SEQ
ID NO:2. Such nucleic acid molecules can readily be identified as
being able to hybridize under stringent conditions, to the
nucleotide sequence shown in SEQ ID NO:2 or a fragment of the
sequence. It is understood that stringent hybridization does not
indicate substantial homology where it is due to general homology,
such as poly A sequences, or sequences common to all or most
proteins or all deubiquitinating enzymes. Moreover, it is
understood that variants do not include any of the nucleic acid
sequences that may have been disclosed prior to the invention.
[0211] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a polypeptide
at least about 60-65% homologous to each other typically remain
hybridized to each other. The conditions can be such that sequences
at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 90%, at least about 95% or more
identical to each other remain hybridized to one another. Such
stringent conditions are known to those skilled in the art and can
be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated by reference.
One example of stringent hybridization conditions are hybridization
in 6.times. sodium chloride/sodium citrate (SSC) at about
45.degree. C., followed by one or more washes in 0.2.times. SSC,
0.1% SDS at 50-65.degree. C. In another non-limiting example,
nucleic acid molecules are allowed to hybridize in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more low stringency washes in 0.2.times. SSC/0.1% SDS at
room temperature, or by one or more moderate stringency washes in
0.2.times. SSC/0.1% SDS at 42.degree. C., or washed in 0.2.times.
SSC/0.1% SDS at 65.degree. C. for high stringency. In one
embodiment, an isolated nucleic acid molecule that hybridizes under
stringent conditions to the sequence of SEQ ID NO:1 corresponds to
a naturally-occurring nucleic acid molecule. As used herein, a
"naturally-occurring" nucleic acid molecule refers to an RNA or DNA
molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes a natural protein).
[0212] As understood by those of ordinary skill, the exact
conditions can be determined empirically and depend on ionic
strength, temperature and the concentration of destabilizing agents
such as formamide or denaturing agents such as SDS. Other factors
considered in determining the desired hybridization conditions
include the length of the nucleic acid sequences, base composition,
percent mismatch between the hybridizing sequences and the
frequency of occurrence of subsets of the sequences within other
non-identical sequences. Thus, equivalent conditions can be
determined by varying one or more of these parameters while
maintaining a similar degree of identity or similarity between the
two nucleic acid molecules.
[0213] The present invention also provides isolated nucleic acids
that contain a single or double stranded fragment or portion that
hybridizes under stringent conditions to the nucleotide sequence of
SEQ ID NO:2 or the complement of SEQ ID NO:2. In one embodiment,
the nucleic acid consists of a portion of the nucleotide sequence
of SEQ ID NO:2 or the complement of SEQ ID NO:2. The nucleic acid
fragments of the invention are at least about 15, preferably at
least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50,
100, 200, 500 or more nucleotides in length. Longer fragments, for
example, 30 or more nucleotides in length, which encode antigenic
proteins or polypeptides described herein are useful.
[0214] Furthermore, the invention provides polynucleotides that
comprise a fragment of the full-length ubiquitin protease
polynucleotides. The fragment can be single or double-stranded and
can comprise DNA or RNA. The fragment can be derived from either
the coding or the non-coding sequence.
[0215] In another embodiment an isolated ubiquitin protease nucleic
acid encodes the entire coding region. In another embodiment the
isolated ubiquitin protease nucleic acid encodes a sequence
corresponding to the mature protein that may be from about amino
acid 6 to the last amino acid. Other fragments include nucleotide
sequences encoding the amino acid fragments described herein.
[0216] Thus, ubiquitin protease nucleic acid fragments further
include sequences corresponding to the domains described herein,
subregions also described, and specific functional sites. Ubiquitin
protease nucleic acid fragments also include combinations of the
domains, segments, and other functional sites described above. A
person of ordinary skill in the art would be aware of the many
permutations that are possible.
[0217] Where the location of the domains or sites have been
predicted by computer analysis, one of ordinary sill would
appreciate that the amino acid residues constituting these domains
can vary depending on the criteria used to define the domains.
[0218] However, it is understood that a ubiquitin protease fragment
includes any nucleic acid sequence that does not include the entire
gene.
[0219] The invention also provides ubiquitin protease nucleic acid
fragments that encode epitope bearing regions of the ubiquitin
protease proteins described herein.
[0220] Nucleic acid fragments, according to the present invention,
are not to be construed as encompassing those fragments that may
have been disclosed prior to the invention.
[0221] Polynucleotide Uses
[0222] The nucleotide sequences of the present invention can be
used as a "query sequence" to perform a search against public
databases, for example, to identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol.
Biol. 215:403-10. BLAST protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to the proteins of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al. (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used.
[0223] The nucleic acid fragments of the invention provide probes
or primers in assays such as those described below. "Probes" are
oligonucleotides that hybridize in a base-specific manner to a
complementary strand of nucleic acid. Such probes include
polypeptide nucleic acids, as described in Nielsen et al. (1991)
Science 254:1497-1500. Typically, a probe comprises a region of
nucleotide sequence that hybridizes under highly stringent
conditions to at least about 15, typically about 20-25, and more
typically about 40, 50 or 75 consecutive nucleotides of the nucleic
acid sequence shown in SEQ ID NO:2 and the complements thereof.
More typically, the probe further comprises a label, e.g.,
radioisotope, fluorescent compound, enzyme, or enzyme
co-factor.
[0224] As used herein, the term "primer" refers to a
single-stranded oligonucleotide which acts as a point of initiation
of template-directed DNA synthesis using well-known methods (e.g.,
PCR, LCR) including, but not limited to those described herein. The
appropriate length of the primer depends on the particular use, but
typically ranges from about 15 to 30 nucleotides. The term "primer
site" refers to the area of the target DNA to which a primer
hybridizes. The term "primer pair" refers to a set of primers
including a 5' (upstream) primer that hybridizes with the 5' end of
the nucleic acid sequence to be amplified and a 3' (downstream)
primer that hybridizes with the complement of the sequence to be
amplified.
[0225] The ubiquitin protease polynucleotides are thus useful for
probes, primers, and in biological assays.
[0226] Where the polynucleotides are used to assess ubiquitin
protease properties or functions, such as in the assays described
herein, all or less than all of the entire cDNA can be useful.
Assays specifically directed to ubiquitin protease functions, such
as assessing agonist or antagonist activity, encompass the use of
known fragments. Further, diagnostic methods for assessing
ubiquitin protease function can also be practiced with any
fragment, including those fragments that may have been known prior
to the invention. Similarly, in methods involving treatment of
ubiquitin protease dysfunction, all fragments are encompassed
including those, which may have been known in the art.
[0227] The ubiquitin protease polynucleotides are useful as a
hybridization probe for cDNA and genomic DNA to isolate a
full-length cDNA and genomic clones encoding the polypeptide
described in SEQ ID NO:1 and to isolate cDNA and genomic clones
that correspond to variants producing the same polypeptide shown in
SEQ ID NO:1 or the other variants described herein. Variants can be
isolated from the same tissue and organism from which the
polypeptides shown in SEQ ID NO:1 were isolated, different tissues
from the same organism, or from different organisms. This method is
useful for isolating genes and cDNA that are
developmentally-controlled and therefore may be expressed in the
same tissue or different tissues at different points in the
development of an organism.
[0228] The probe can correspond to any sequence along the entire
length of the gene encoding the ubiquitin protease. Accordingly, it
could be derived from 5' noncoding regions, the coding region, and
3' noncoding regions.
[0229] The nucleic acid probe can be, for example, the full-length
cDNA of SEQ ID NO:2 or a fragment thereof, such as an
oligonucleotide of at least 12, 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to mRNA or DNA.
[0230] Fragments of the polynucleotides described herein are also
useful to synthesize larger fragments or full-length
polynucleotides described herein. For example, a fragment can be
hybridized to any portion of an mRNA and a larger or full-length
cDNA can be produced.
[0231] The fragments are also useful to synthesize antisense
molecules of desired length and sequence.
[0232] Antisense nucleic acids of the invention can be designed
using the nucleotide sequence of SEQ ID NO:2, and constructed using
chemical synthesis and enzymatic ligation reactions using
procedures known in the art. For example, an antisense nucleic acid
(e.g., an antisense oligonucleotide) can be chemically synthesized
using naturally occurring nucleotides or variously modified
nucleotides designed to increase the biological stability of the
molecules or to increase the physical stability of the duplex
formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate derivatives and acridine substituted nucleotides
can be used. Examples of modified nucleotides which can be used to
generate the antisense nucleic acid include 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluraci- l, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest).
[0233] Additionally, the nucleic acid molecules of the invention
can be modified at the base moiety, sugar moiety or phosphate
backbone to improve, e.g., the stability, hybridization, or
solubility of the molecule. For example, the deoxyribose phosphate
backbone of the nucleic acids can be modified to generate peptide
nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4:5). As used herein, the terms "peptide nucleic acids"
or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which
the deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of PNAs has been shown to allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93:14670. PNAs can be further modified, e.g., to
enhance their stability, specificity or cellular uptake, by
attaching lipophilic or other helper groups to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other
techniques of drug delivery known in the art. The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996),
supra, Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63, Mag et
al. (1989) Nucleic Acids Res. 17:5973, and Peterser et al. (1975)
Bioorganic Med. Chem. Lett. 5:1119.
[0234] The nucleic acid molecules and fragments of the invention
can also include other appended groups such as peptides (e.g., for
targeting host cell ubiquitin proteases in vivo), or agents
facilitating transport across the cell membrane (see, e.g.,
Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;
Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT
Publication No. WO 88/0918) or the blood brain barrier (see, e.g.,
PCT Publication No. WO 89/10134). In addition, oligonucleotides can
be modified with hybridization-triggered cleavage agents (see,
e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating
agents (see, e.g., Zon (1988) Pharm Res. 5:539-549).
[0235] The ubiquitin protease polynucleotides are also useful as
primers for PCR to amplify any given region of a ubiquitin protease
polynucleotide.
[0236] The ubiquitin protease polynucleotides are also useful for
constructing recombinant vectors. Such vectors include expression
vectors that express a portion of, or all of, the ubiquitin
protease polypeptides. Vectors also include insertion vectors, used
to integrate into another polynucleotide sequence, such as into the
cellular genome, to alter in situ expression of ubiquitin protease
genes and gene products. For example, an endogenous ubiquitin
protease coding sequence can be replaced via homologous
recombination with all or part of the coding region containing one
or more specifically introduced mutations.
[0237] The ubiquitin protease polynucleotides are also useful for
expressing antigenic portions of the ubiquitin protease
proteins.
[0238] The ubiquitin protease polynucleotides are also useful as
probes for determining the chromosomal positions of the ubiquitin
protease polynucleotides by means of in situ hybridization methods,
such as FISH. (For a review of this technique, see Verma et al.
(1988) Human Chromosomes: A Manual of Basic Techniques (Pergamon
Press, New York), and PCR mapping of somatic cell hybrids. The
mapping of the sequences to chromosomes is an important first step
in correlating these sequences with genes associated with
disease.
[0239] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0240] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland et al. ((1987) Nature 325:783-787).
[0241] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
a specified gene, can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or
translocations, that are visible from chromosome spreads, or
detectable using PCR based on that DNA sequence. Ultimately,
complete sequencing of genes from several individuals can be
performed to confirm the presence of a mutation and to distinguish
mutations from polymorphisms.
[0242] The ubiquitin protease polynucleotide probes are also useful
to determine patterns of the presence of the gene encoding the
ubiquitin proteases and their variants with respect to tissue
distribution, for example, whether gene duplication has occurred
and whether the duplication occurs in all or only a subset of
tissues. The genes can be naturally occurring or can have been
introduced into a cell, tissue, or organism exogenously.
[0243] The ubiquitin protease polynucleotides are also usefid for
designing ribozymes corresponding to all, or a part, of the mRNA
produced from genes encoding the polynucleotides described
herein.
[0244] The ubiquitin protease polynucleotides are also useful for
constructing host cells expressing a part, or all, of the ubiquitin
protease polynucleotides and polypeptides.
[0245] The ubiquitin protease polynucleotides are also useful for
constructing transgenic animals expressing all, or a part, of the
ubiquitin protease polynucleotides and polypeptides.
[0246] The ubiquitin protease polynucleotides are also useful for
making vectors that express part, or all, of the ubiquitin protease
polypeptides.
[0247] The ubiquitin protease polynucleotides are also useful as
hybridization probes for determining the level of ubiquitin
protease nucleic acid expression. Accordingly, the probes can be
used to detect the presence of, or to determine levels of,
ubiquitin protease nucleic acid in cells, tissues, and in
organisms. The nucleic acid whose level is determined can be DNA or
RNA. Accordingly, probes corresponding to the polypeptides
described herein can be used to assess gene copy number in a given
cell, tissue, or organism. This is particularly relevant in cases
in which there has been an amplification of the ubiquitin protease
genes.
[0248] Alternatively, the probe can be used in an in situ
hybridization context to assess the position of extra copies of the
ubiquitin protease genes, as on extrachromosomal elements or as
integrated into chromosomes in which the ubiquitin protease gene is
not normally found, for example as a homogeneously staining
region.
[0249] These uses are relevant for diagnosis of disorders involving
an increase or decrease in ubiquitin protease expression relative
to normal, such as a proliferative disorder, a differentiative or
developmental disorder, or a hematopoietic disorder.
[0250] The ubiquitin protease is expressed in tissues including,
but not limited to normal human thymus, testes, brain, breast,
ovary, skeletal muscle, liver, prostate, and thyroid. As such, the
gene is particularly relevant for the treatment of disorders
involving these tissues. The gene is also expressed in fetal
kidney, fetal heart, and fetal liver. The gene is also expressed in
liver metastases derived from colon, and malignant lung and breast
and therefore, treatment is relevant to these disorders.
[0251] Disorders involving the above tissues are discussed herein
above.
[0252] Thus, the present invention provides a method for
identifying a disease or disorder associated with aberrant
expression or activity of ubiquitin protease nucleic acid, in which
a test sample is obtained from a subject and nucleic acid (e.g.,
mRNA, genomic DNA) is detected, wherein the presence of the nucleic
acid is diagnostic for a subject having or at risk of developing a
disease or disorder associated with aberrant expression or activity
of the nucleic acid.
[0253] One aspect of the invention relates to diagnostic assays for
determining nucleic acid expression as well as activity in the
context of a biological sample (e.g., blood, serum, cells, tissue)
to determine whether an individual has a disease or disorder, or is
at risk of developing a disease or disorder, associated with
aberrant nucleic acid expression or activity. Such assays can be
used for prognostic or predictive purpose to thereby
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with expression or activity
of the nucleic acid molecules.
[0254] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[0255] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express the ubiquitin protease,
such as by measuring the level of a ubiquitin protease-encoding
nucleic acid in a sample of cells from a subject e.g., mRNA or
genomic DNA, or determining if the ubiquitin protease gene has been
mutated.
[0256] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate ubiquitin protease nucleic acid
expression (e.g., antisense, polypeptides, peptidomimetics, small
molecules or other drugs). A cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of the mRNA in the presence of the candidate compound is
compared to the level of expression of the mRNA in the absence of
the candidate compound. The candidate compound can then be
identified as a modulator of nucleic acid expression based on this
comparison and be used, for example to treat a disorder
characterized by aberrant nucleic acid expression. The modulator
can bind to the nucleic acid or indirectly modulate expression,
such as by interacting with other cellular components that affect
nucleic acid expression.
[0257] Modulatory methods can be performed in vitro (e.g., by
culturing the cell with the agent) or, alternatively, in vivo
(e.g., by administering the gent to a subject) in patients or in
transgenic animals.
[0258] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the ubiquitin protease gene. The method
typically includes assaying the ability of the compound to modulate
the expression of the ubiquitin protease nucleic acid and thus
identifying a compound that can be used to treat a disorder
characterized by undesired ubiquitin protease nucleic acid
expression.
[0259] The assays can be performed in cell-based and cell-free
systems. Cell-based assays include cells naturally expressing the
ubiquitin protease nucleic acid or recombinant cells genetically
engineered to express specific nucleic acid sequences.
[0260] Alternatively, candidate compounds can be assayed in vivo in
patients or in transgenic animals.
[0261] The assay for ubiquitin protease nucleic acid expression can
involve direct assay of nucleic acid levels, such as mRNA levels,
or on collateral compounds involved in the pathway (such as free
ubiquitin pool or protein turnover). Further, the expression of
genes that are up- or down-regulated in response to the ubiquitin
protease activity can also be assayed. In this embodiment the
regulatory regions of these genes can be operably linked to a
reporter gene such as luciferase.
[0262] Thus, modulators of ubiquitin protease gene expression can
be identified in a method wherein a cell is contacted with a
candidate compound and the expression of mRNA determined. The level
of expression of ubiquitin protease mRNA in the presence of the
candidate compound is compared to the level of expression of
ubiquitin protease mRNA in the absence of the candidate compound.
The candidate compound can then be identified as a modulator of
nucleic acid expression based on this comparison and be used, for
example to treat a disorder characterized by aberrant nucleic acid
expression. When expression of mRNA is statistically significantly
greater in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of
nucleic acid expression. When nucleic acid expression is
statistically significantly less in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of nucleic acid expression.
[0263] Accordingly, the invention provides methods of treatment,
with the nucleic acid as a target, using a compound identified
through drug screening as a gene modulator to modulate ubiquitin
protease nucleic acid expression. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or effects on nucleic acid activity
(e.g. when nucleic acid is mutated or improperly modified).
Treatment is of disorders characterized by aberrant expression or
activity of the nucleic acid, including the disorders described
herein.
[0264] Alternatively, a modulator for ubiquitin protease nucleic
acid expression can be a small molecule or drug identified using
the screening assays described herein as long as the drug or small
molecule inhibits the ubiquitin protease nucleic acid
expression.
[0265] The ubiquitin protease polynucleotides are also useful for
monitoring the effectiveness of modulating compounds on the
expression or activity of the ubiquitin protease gene in clinical
trials or in a treatment regimen. Thus, the gene expression pattern
can serve as a barometer for the continuing effectiveness of
treatment with the compound, particularly with compounds to which a
patient can develop resistance. The gene expression pattern can
also serve as a marker indicative of a physiological response of
the affected cells to the compound. Accordingly, such monitoring
would allow either increased administration of the compound or the
administration of alternative compounds to which the patient has
not become resistant. Similarly, if the level of nucleic acid
expression falls below a desirable level, administration of the
compound could be commensurately decreased.
[0266] Monitoring can be, for example, as follows: (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of a specified
mRNA or genomic DNA of the invention in the pre-administration
sample; (iii) obtaining one or more post-administration samples
from the subject; (iv) detecting the level of expression or
activity of the mRNA or genomic DNA in the post-administration
samples; (v) comparing the level of expression or activity of the
mRNA or genomic DNA in the pre-administration sample with the mRNA
or genomic DNA in the post-administration sample or samples; and
(vi) increasing or decreasing the administration of the agent to
the subject accordingly.
[0267] The ubiquitin protease polynucleotides are also useful in
diagnostic assays for qualitative changes in ubiquitin protease
nucleic acid, and particularly in qualitative changes that lead to
pathology. The polynucleotides can be used to detect mutations in
ubiquitin protease genes and gene expression products such as mRNA.
The polynucleotides can be used as hybridization probes to detect
naturally-occurring genetic mutations in the ubiquitin protease
gene and thereby to determine whether a subject with the mutation
is at risk for a disorder caused by the mutation. Mutations include
deletion, addition, or substitution of one or more nucleotides in
the gene, chromosomal rearrangement, such as inversion or
transposition, modification of genomic DNA, such as aberrant
methylation patterns or changes in gene copy number, such as
amplification. Detection of a mutated form of the ubiquitin
protease gene associated with a dysfunction provides a diagnostic
tool for an active disease or susceptibility to disease when the
disease results from overexpression, underexpression, or altered
expression of a ubiquitin protease.
[0268] Mutations in the ubiquitin protease gene can be detected at
the nucleic acid level by a variety of techniques. Genomic DNA can
be analyzed directly or can be amplified by using PCR prior to
analysis. RNA or cDNA can be used in the same way.
[0269] In certain embodiments, detection of the mutation involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080;
and Nakazawa et al. (1994) PNAS 91:360-364), the latter of which
can be particularly useful for detecting point mutations in the
gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682).
This method can include the steps of collecting a sample of cells
from a patient, isolating nucleic acid (e.g., genomic, mRNA or
both) from the cells of the sample, contacting the nucleic acid
sample with one or more primers which specifically hybridize to a
gene under conditions such that hybridization and amplification of
the gene (if present) occurs, and detecting the presence or absence
of an amplification product, or detecting the size of the
amplification product and comparing the length to a control sample.
Deletions and insertions can be detected by a change in size of the
amplified product compared to the normal genotype. Point mutations
can be identified by hybridizing amplified DNA to normal RNA or
antisense DNA sequences.
[0270] It is anticipated that PCR and/or LCR may be desirable to
use as a preliminary amplification step in conjunction with any of
the techniques used for detecting mutations described herein.
[0271] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), or any
other nucleic acid amplification method, followed by the detection
of the amplified molecules using techniques well-known to those of
skill in the art. These detection schemes are especially useful for
the detection of nucleic acid molecules if such molecules are
present in very low numbers.
[0272] Alternatively, mutations in a ubiquitin protease gene can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[0273] Further, sequence-specific ribozymes (U.S. Pat. No.
5,498,531) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
[0274] Perfectly matched sequences can be distinguished from
mismatched sequences by nuclease cleavage digestion assays or by
differences in melting temperature.
[0275] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method.
[0276] Furthermore, sequence differences between a mutant ubiquitin
protease gene and a wild-type gene can be determined by direct DNA
sequencing. A variety of automated sequencing procedures can be
utilized when performing the diagnostic assays ((1995)
Biotechniques 19:448), including sequencing by mass spectrometry
(see, e.g., PCT International Publication No. WO 94/16101; Cohen et
al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993)
Appl. Biochem. Biotechnol. 38:147-159).
[0277] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.
(1985) Science 230:1242); Cotton et al. (1988) PNAS 85:4397;
Saleeba et al. (1992) Meth. Enzymol. 217:286-295), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al. (1989) PNAS 86:2766; Cotton et al. (1993) Mutat. Res.
285:125-144; and Hayashi et al. (1992) Genet. Anal. Tech. Appl.
9:73-79), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al. (1985)
Nature 313:495). The sensitivity of the assay may be enhanced by
using RNA (rather than DNA), in which the secondary structure is
more sensitive to a change in sequence. In one embodiment, the
subject method utilizes heteroduplex analysis to separate double
stranded heteroduplex molecules on the basis of changes in
electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
Examples of other techniques for detecting point mutations include,
selective oligonucleotide hybridization, selective amplification,
and selective primer extension.
[0278] In other embodiments, genetic mutations can be identified by
hybridizing a sample and control nucleic acids, e.g., DNA or RNA,
to high density arrays containing hundreds or thousands of
oligonucleotide probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified in two dimensional
arrays containing light-generated DNA probes as described in Cronin
et al. supra. Briefly, a first hybridization array of probes can be
used to scan through long stretches of DNA in a sample and control
to identify base changes between the sequences by making linear
arrays of sequential overlapping probes. This step allows the
identification of point mutations. This step is followed by a
second hybridization array that allows the characterization of
specific mutations by using smaller, specialized probe arrays
complementary to all variants or mutations detected. Each mutation
array is composed of parallel probe sets, one complementary to the
wild-type gene and the other complementary to the mutant gene.
[0279] The ubiquitin protease polynucleotides are also useful for
testing an individual for a genotype that while not necessarily
causing the disease, nevertheless affects the treatment modality.
Thus, the polynucleotides can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship). In the
present case, for example, a mutation in the ubiquitin protease
gene that results in altered affinity for ubiquitin could result in
an excessive or decreased drug effect with standard concentrations
of ubiquitin or analog. Accordingly, the ubiquitin protease
polynucleotides described herein can be used to assess the mutation
content of the gene in an individual in order to select an
appropriate compound or dosage regimen for treatment.
[0280] Thus polynucleotides displaying genetic variations that
affect treatment provide a diagnostic target that can be used to
tailor treatment in an individual. Accordingly, the production of
recombinant cells and animals containing these polymorphisms allow
effective clinical design of treatment compounds and dosage
regimens.
[0281] The methods can involve obtaining a control biological
sample from a control subject, contacting the control sample with a
compound or agent capable of detecting mRNA, or genomic DNA, such
that the presence of mRNA or genomic DNA is detected in the
biological sample, and comparing the presence of mRNA or genomic
DNA in the control sample with the presence of mRNA or genomic DNA
in the test sample.
[0282] The ubiquitin protease polynucleotides are also useful for
chromosome identification when the sequence is identified with an
individual chromosome and to a particular location on the
chromosome. First, the DNA sequence is matched to the chromosome by
in situ or other chromosome-specific hybridization. Sequences can
also be correlated to specific chromosomes by preparing PCR primers
that can be used for PCR screening of somatic cell hybrids
containing individual chromosomes from the desired species. Only
hybrids containing the chromosome containing the gene homologous to
the primer will yield an amplified fragment. Sublocalization can be
achieved using chromosomal fragments. Other strategies include
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to chromosome-specific libraries. Further mapping
strategies include fluorescence in situ hybridization, which allows
hybridization with probes shorter than those traditionally used.
Reagents for chromosome mapping can be used individually to mark a
single chromosome or a single site on the chromosome, or panels of
reagents can be used for marking multiple sites and/or multiple
chromosomes. Reagents corresponding to noncoding regions of the
genes actually are preferred for mapping purposes. Coding sequences
are more likely to be conserved within gene families, thus
increasing the chance of cross hybridizations during chromosomal
mapping.
[0283] The ubiquitin protease polynucleotides can also be used to
identify individuals from small biological samples. This can be
done for example using restriction fragment-length polymorphism
(RFLP) to identify an individual. Thus, the polynucleotides
described herein are useful as DNA markers for RFLP (See U.S. Pat.
No. 5,272,057).
[0284] Furthermore, the ubiquitin protease sequence can be used to
provide an alternative technique, which determines the actual DNA
sequence of selected fragments in the genome of an individual.
Thus, the ubiquitin protease sequences described herein can be used
to prepare two PCR primers from the 5' and 3' ends of the
sequences. These primers can then be used to amplify DNA from an
individual for subsequent sequencing.
[0285] Panels of corresponding DNA sequences from individuals
prepared in this manner can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences. It is estimated that allelic variation in humans
occurs with a frequency of about once per each 500 bases. Allelic
variation occurs to some degree in the coding regions of these
sequences, and to a greater degree in the noncoding regions. The
ubiquitin protease sequences can be used to obtain such
identification sequences from individuals and from tissue. The
sequences represent unique fragments of the human genome. Each of
the sequences described herein can, to some degree, be used as a
standard against which DNA from an individual can be compared for
identification purposes.
[0286] If a panel of reagents from the sequences is used to
generate a unique identification database for an individual, those
same reagents can later be used to identify tissue from that
individual. Using the unique identification database, positive
identification of the individual, living or dead, can be made from
extremely small tissue samples.
[0287] The ubiquitin protease polynucleotides can also be used in
forensic identification procedures. PCR technology can be used to
amplify DNA sequences taken from very small biological samples,
such as a single hair follicle, body fluids (e.g. blood, saliva, or
semen). The amplified sequence can then be compared to a standard
allowing identification of the origin of the sample.
[0288] The ubiquitin protease polynucleotides can thus be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As described
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to the
noncoding region are particularly useful since greater polymorphism
occurs in the noncoding regions, making it easier to differentiate
individuals using this technique.
[0289] The ubiquitin protease polynucleotides can further be used
to provide polynucleotide reagents, e.g., labeled or labelable
probes which can be used in, for example, an in situ hybridization
technique, to identify a specific tissue. This is useful in cases
in which a forensic pathologist is presented with a tissue of
unknown origin. Panels of ubiquitin protease probes can be used to
identify tissue by species and/or by organ type.
[0290] In a similar fashion, these primers and probes can be used
to screen tissue culture for contamination (i.e. screen for the
presence of a mixture of different types of cells in a
culture).
[0291] Alternatively, the ubiquitin protease polynucleotides can be
used directly to block transcription or translation of ubiquitin
protease gene sequences by means of antisense or ribozyme
constructs. Thus, in a disorder characterized by abnormally high or
undesirable ubiquitin protease gene expression, nucleic acids can
be directly used for treatment.
[0292] The ubiquitin protease polynucleotides are thus useful as
antisense constructs to control ubiquitin protease gene expression
in cells, tissues, and organisms. A DNA antisense polynucleotide is
designed to be complementary to a region of the gene involved in
transcription, preventing transcription and hence production of
ubiquitin protease protein. An antisense RNA or DNA polynucleotide
would hybridize to the mRNA and thus block translation of mRNA into
ubiquitin protease protein.
[0293] Examples of antisense molecules useful to inhibit nucleic
acid expression include antisense molecules complementary to a
fragment of the 5' untranslated region of SEQ ID NO:2 which also
includes the start codon and antisense molecules which are
complementary to a fragment of the 3' untranslated region of SEQ ID
NO:2.
[0294] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of ubiquitin
protease nucleic acid. Accordingly, these molecules can treat a
disorder characterized by abnormal or undesired ubiquitin protease
nucleic acid expression. This technique involves cleavage by means
of ribozymes containing nucleotide sequences complementary to one
or more regions in the mRNA that attenuate the ability of the mRNA
to be translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the ubiquitin protease protein.
[0295] The ubiquitin protease polynucleotides also provide vectors
for gene therapy in patients containing cells that are aberrant in
ubiquitin protease gene expression. Thus, recombinant cells, which
include the patient's cells that have been engineered ex vivo and
returned to the patient, are introduced into an individual where
the cells produce the desired ubiquitin protease protein to treat
the individual.
[0296] The invention also encompasses kits for detecting the
presence of a ubiquitin protease nucleic acid in a biological
sample. For example, the kit can comprise reagents such as a
labeled or labelable nucleic acid or agent capable of detecting
ubiquitin protease nucleic acid in a biological sample; means for
determining the amount of ubiquitin protease nucleic acid in the
sample; and means for comparing the amount of ubiquitin protease
nucleic acid in the sample with a standard. The compound or agent
can be packaged in a suitable container. The kit can further
comprise instructions for using the kit to detect ubiquitin
protease mRNA or DNA.
[0297] Computer Readable Means
[0298] The nucleotide or amino acid sequences of the invention are
also provided in a variety of mediums to facilitate use thereof. As
used herein, "provided" refers to a manufacture, other than an
isolated nucleic acid or amino acid molecule, which contains a
nucleotide or amino acid sequence of the present invention. Such a
manufacture provides the nucleotide or amino acid sequences, or a
subset thereof (e.g., a subset of open reading frames (ORFs)) in a
form which allows a skilled artisan to examine the manufacture
using means not directly applicable to examining the nucleotide or
amino acid sequences, or a subset thereof, as they exists in nature
or in purified form.
[0299] In one application of this embodiment, a nucleotide or amino
acid sequence of the present invention can be recorded on computer
readable media. As used herein, "computer readable media" refers to
any medium that can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media,
such as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. The skilled artisan will readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide or amino acid sequence
of the present invention.
[0300] As used herein, "recorded" refers to a process for storing
information on computer readable medium. The skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide or amino acid sequence information of the
present invention.
[0301] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of dataprocessor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0302] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[0303] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. The most preferred
sequence length of a target sequence is from about 10 to 100 amino
acids or from about 30 to 300 nucleotide residues. However, it is
well recognized that commercially important fragments, such as
sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[0304] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[0305] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software includes, but is not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[0306] For example, software which implements the BLAST (Altschul
et al. (1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al.
(1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system
can be used to identify open reading frames (ORFs) of the sequences
of the invention which contain homology to ORFs or proteins from
other libraries. Such ORFs are protein encoding fragments and are
useful in producing commercially important proteins such as enzymes
used in various reactions and in the production of commercially
useful metabolites.
[0307] Vectors/Host Cells
[0308] The invention also provides vectors containing the ubiquitin
protease polynucleotides. The term "vector" refers to a vehicle,
preferably a nucleic acid molecule that can transport the ubiquitin
protease polynucleotides. When the vector is a nucleic acid
molecule, the ubiquitin protease polynucleotides are covalently
linked to the vector nucleic acid. With this aspect of the
invention, the vector includes a plasmid, single or double stranded
phage, a single or double stranded RNA or DNA viral vector, or
artificial chromosome, such as a BAC, PAC, YAC, OR MAC.
[0309] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the ubiquitin protease polynucleotides.
Alternatively, the vector may integrate into the host cell genome
and produce additional copies of the ubiquitin protease
polynucleotides when the host cell replicates.
[0310] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
ubiquitin protease polynucleotides. The vectors can function in
procaryotic or eukaryotic cells or in both (shuttle vectors).
[0311] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the ubiquitin protease
polynucleotides such that transcription of the polynucleotides is
allowed in a host cell. The polynucleotides can be introduced into
the host cell with a separate polynucleotide capable of affecting
transcription. Thus, the second polynucleotide may provide a
trans-acting factor interacting with the cis-regulatory control
region to allow transcription of the ubiquitin protease
polynucleotides from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself.
[0312] It is understood, however, that in some embodiments,
transcription and/or translation of the ubiquitin protease
polynucleotides can occur in a cell-free system.
[0313] The regulatory sequence to which the polynucleotides
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lambda., the lac, TRP,
and TAC promoters from E. coli, the early and late promoters from
SV40, the CMV immediate early promoter, the adenovirus early and
late promoters, and retrovirus long-terminal repeats.
[0314] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0315] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
[0316] A variety of expression vectors can be used to express a
ubiquitin protease polynucleotide. Such vectors include
chromosomal, episomal, and virus-derived vectors, for example
vectors derived from bacterial plasmids, from bacteriophage, from
yeast episomes, from yeast chromosomal elements, including yeast
artificial chromosomes, from viruses such as baculoviruses,
papovaviruses such as SV40, Vaccinia viruses, adenoviruses,
poxviruses, pseudorabies viruses, and retroviruses. Vectors may
also be derived from combinations of these sources such as those
derived from plasmid and bacteriophage genetic elements, e.g.
cosmids and phagemids. Appropriate cloning and expression vectors
for prokaryotic and eukaryotic hosts are described in Sambrook et
al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0317] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e. tissue specific) or may provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0318] The ubiquitin protease polynucleotides can be inserted into
the vector nucleic acid by well-known methodology. Generally, the
DNA sequence that will ultimately be expressed is joined to an
expression vector by cleaving the DNA sequence and the expression
vector with one or more restriction enzymes and then ligating the
fragments together. Procedures for restriction enzyme digestion and
ligation are well known to those of ordinary skill in the art.
[0319] The vector containing the appropriate polynucleotide can be
introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0320] As described herein, it may be desirable to express the
polypeptide as a fusion protein. Accordingly, the invention
provides fusion vectors that allow for the production of the
ubiquitin protease polypeptides. Fusion vectors can increase the
expression of a recombinant protein, increase the solubility of the
recombinant protein, and aid in the purification of the protein by
acting for example as a ligand for affinity purification. A
proteolytic cleavage site may be introduced at the junction of the
fusion moiety so that the desired polypeptide can ultimately be
separated from the fusion moiety. Proteolytic enzymes include, but
are not limited to, factor Xa, thrombin, and enterokinase. Typical
fusion expression vectors include pGEX (Smith et al. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein. Examples of suitable inducible
non-fusion E. coli expression vectors include pTrc (Amann et al.
(1988) Gene 69:301-315) and pET 11d (Studier et al. (1990) Gene
Expression Technology: Methods in Enzymology 185:60-89).
[0321] Recombinant protein expression can be maximized in a host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S. (1990) Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif. 119-128).
Alternatively, the sequence of the polynucleotide of interest can
be altered to provide preferential codon usage for a specific host
cell, for example E. coli. (Wada et al. (1992) Nucleic Acids Res.
20:2111 -2118).
[0322] The ubiquitin protease polynucleotides can also be expressed
by expression vectors that are operative in yeast. Examples of
vectors for expression in yeast e.g., S. cerevisiae include
pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234 ), pMFa (Kurjan
et al. (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene
54:113-123), and pYES2 (Invitrogen Corporation, San Diego,
Calif.).
[0323] The ubiquitin protease polynucleotides can also be expressed
in insect cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL
series (Lucklow et al. (1989) Virology 170:31-39).
[0324] In certain embodiments of the invention, the polynucleotides
described herein are expressed in mammalian cells using mammalian
expression vectors. Examples of mammalian expression vectors
include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman
et al. (1987) EMBO J. 6:187-195).
[0325] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
ubiquitin protease polynucleotides. The person of ordinary skill in
the art would be aware of other vectors suitable for maintenance
propagation or expression of the polynucleotides described herein.
These are found for example in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[0326] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the
polynucleotide sequences described herein, including both coding
and non-coding regions. Expression of this antisense RNA is subject
to each of the parameters described above in relation to expression
of the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0327] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0328] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y).
[0329] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the ubiquitin protease polynucleotides can be
introduced either alone or with other polynucleotides that are not
related to the ubiquitin protease polynucleotides such as those
providing trans-acting factors for expression vectors. When more
than one vector is introduced into a cell, the vectors can be
introduced independently, co-introduced or joined to the ubiquitin
protease polynucleotide vector.
[0330] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0331] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the polynucleotides described herein or
may be on a separate vector. Markers include tetracycline or
ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0332] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0333] Where secretion of the polypeptide is desired, appropriate
secretion signals are incorporated into the vector. The signal
sequence can be endogenous to the ubiquitin protease polypeptides
or heterologous to these polypeptides.
[0334] Where the polypeptide is not secreted into the medium, the
protein can be isolated from the host cell by standard disruption
procedures, including freeze thaw, sonication, mechanical
disruption, use of lysing agents and the like. The polypeptide can
then be recovered and purified by well-known purification methods
including ammonium sulfate precipitation, acid extraction, anion or
cationic exchange chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0335] It is also understood that depending upon the host cell in
recombinant production of the polypeptides described herein, the
polypeptides can have various glycosylation patterns, depending
upon the cell, or maybe non-glycosylated as when produced in
bacteria. In addition, the polypeptides may include an initial
modified methionine in some cases as a result of a host-mediated
process.
[0336] Uses of Vectors and Host Cells
[0337] It is understood that "host cells" and "recombinant host
cells" refer not only to the particular subject cell but also to
the progeny or potential progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term as used herein.
[0338] The host cells expressing the polypeptides described herein,
and particularly recombinant host cells, have a variety of uses.
First, the cells are useful for producing ubiquitin protease
proteins or polypeptides that can be further purified to produce
desired amounts of ubiquitin protease protein or fragments. Thus,
host cells containing expression vectors are useful for polypeptide
production.
[0339] Host cells are also useful for conducting cell-based assays
involving the ubiquitin protease or ubiquitin protease fragments.
Thus, a recombinant host cell expressing a native ubiquitin
protease is useful to assay for compounds that stimulate or inhibit
ubiquitin protease function. This includes disappearance of
substrate (polyubiquitin, ubiquitinated substrate protein,
ubiquitinated substrate remnants), appearance of end product
(ubiquitin monomers, polyubiquitin hydrolyzed from substrate or
substrate remnant, free substrate that has been rescued by
hydrolysis of ubiquitin), general or specific protein turnover, and
the various other molecular functions described herein that
include, but are not limited to, substrate recognition, substrate
binding, subunit association, and interaction with other cellular
components. Modulation of gene expression can occur at the level of
transcription or translation.
[0340] Host cells are also useful for identifying ubiquitin
protease mutants in which these functions are affected. If the
mutants naturally occur and give rise to a pathology, host cells
containing the mutations are useful to assay compounds that have a
desired effect on the mutant ubiquitin protease (for example,
stimulating or inhibiting function) which may not be indicated by
their effect on the native ubiquitin protease.
[0341] Recombinant host cells are also useful for expressing the
chimeric polypeptides described herein to assess compounds that
activate or suppress activation or alter specific function by means
of a heterologous domain, segment, site, and the like, as disclosed
herein.
[0342] Further, mutant ubiquitin proteases can be designed in which
one or more of the various functions is engineered to be increased
or decreased (e.g., binding to ubiquitin, polyubiquitin, or
ubiquitinated protein substrate) and used to augment or replace
ubiquitin protease proteins in an individual. Thus, host cells can
provide a therapeutic benefit by replacing an aberrant ubiquitin
protease or providing an aberrant ubiquitin protease that provides
a therapeutic result. In one embodiment, the cells provide
ubiquitin proteases that are abnormally active.
[0343] In another embodiment, the cells provide ubiquitin proteases
that are abnormally inactive. These ubiquitin proteases can compete
with endogenous ubiquitin proteases in the individual.
[0344] In another embodiment, cells expressing ubiquitin proteases
that cannot be activated, are introduced into an individual in
order to compete with endogenous ubiquitin proteases for ubiquitin
substrates. For example, in the case in which excessive ubiquitin
substrate or analog is part of a treatment modality, it may be
necessary to inactivate this molecule at a specific point in
treatment. Providing cells that compete for the molecule , but
which cannot be affected by ubiquitin protease activation would be
beneficial.
[0345] Homologously recombinant host cells can also be produced
that allow the in situ alteration of endogenous 23413
polynucleotide sequences in a host cell genome. The host cell
includes, but is not limited to, a stable cell line, cell in vivo,
or cloned microorganism. This technology is more fully described in
WO 93/09222, WO 91/12650, WO 91/06667, U.S. Pat. No. 5,272,071, and
U.S. Pat. No. 5,641,670. Briefly, specific polynucleotide sequences
corresponding to the 23413 polynucleotides or sequences proximal or
distal to a 23413 gene are allowed to integrate into a host cell
genome by homologous recombination where expression of the gene can
be affected. In one embodiment, regulatory sequences are introduced
that either increase or decrease expression of an endogenous
sequence. Accordingly, a 23413 protein can be produced in a cell
not normally producing it. Alternatively, increased expression of
23413 protein can be effected in a cell normally producing the
protein at a specific level. Further, expression can be decreased
or eliminated by introducing a specific regulatory sequence. The
regulatory sequence can be heterologous to the 23413 protein
sequence or can be a homologous sequence with a desired mutation
that affects expression. Alternatively, the entire gene can be
deleted. The regulatory sequence can be specific to the host cell
or capable of functioning in more than one cell type. Still
further, specific mutations can be introduced into any desired
region of the gene to produce mutant 23413 proteins. Such mutations
could be introduced, for example, into the specific functional
regions such as the ligand-binding site.
[0346] In one embodiment, the host cell can be a fertilized oocyte
or embryonic stem cell that can be used to produce a transgenic
animal containing the altered ubiquitin protease gene.
Alternatively, the host cell can be a stem cell or other early
tissue precursor that gives rise to a specific subset of cells and
can be used to produce transgenic tissues in an animal. See also
Thomas et al., Cell 51:503 (1987) for a description of homologous
recombination vectors. The vector is introduced into an embryonic
stem cell line (e.g., by electroporation) and cells in which the
introduced gene has homologously recombined with the endogenous
ubiquitin protease gene is selected (see e.g., Li, E. et al. (1992)
Cell 69:915). The selected cells are then injected into a
blastocyst of an animal (e.g., a mouse) to form aggregation
chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,
Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted
into a suitable pseudopregnant female foster animal and the embryo
brought to term. Progeny harboring the homologously recombined DNA
in their germ cells can be used to breed animals in which all cells
of the animal contain the homologously recombined DNA by germline
transmission of the transgene. Methods for constructing homologous
recombination vectors and homologous recombinant animals are
described further in Bradley, A. (1991) Current Opinion in
Biotechnology 2:823-829 and in PCT International Publication Nos.
WO 90/11354; WO 91/01140; and WO 93/04169.
[0347] The genetically engineered host cells can be used to produce
non-human transgenic animals. A transgenic animal is preferably a
mammal, for example a rodent, such as a rat or mouse, in which one
or more of the cells of the animal include a transgene. A transgene
is exogenous DNA which is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal in one or more cell types or tissues of the
transgenic animal. These animals are useful for studying the
function of a ubiquitin protease protein and identifying and
evaluating modulators of ubiquitin protease protein activity.
[0348] Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[0349] In one embodiment, a host cell is a fertilized oocyte or an
embryonic stem cell into which ubiquitin protease polynucleotide
sequences have been introduced.
[0350] A transgenic animal can be produced by introducing nucleic
acid into the male pronuclei of a fertilized oocyte, e.g., by
microinjection, retroviral infection, and allowing the oocyte to
develop in a pseudopregnant female foster animal. Any of the
ubiquitin protease nucleotide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[0351] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the
ubiquitin protease protein to particular cells.
[0352] Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the transgene
in its genome and/or expression of transgenic mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0353] In another embodiment, transgenic non-human animals can be
produced which contain selected systems, which allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
PNAS 89:6232-6236. Another example of a recombinase system is the
FLP recombinase system of S. cerevisiae (O'Gorman et al. (1991)
Science 251:1351-1355. If a cre/loxP recombinase system is used to
regulate expression of the transgene, animals containing transgenes
encoding both the Cre recombinase and a selected protein is
required. Such animals can be provided through the construction of
"double" transgenic animals, e.g., by mating two transgenic
animals, one containing a transgene encoding a selected protein and
the other containing a transgene encoding a recombinase.
[0354] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wihnut
et al. (1997) Nature 385:810-813 and PCT International Publication
Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic
cell, from the transgenic animal can be isolated and induced to
exit the growth cycle and enter G.sub.o phase. The quiescent cell
can then be fused, e.g., through the use of electrical pulses, to
an enucleated oocyte from an animal of the same species from which
the quiescent cell is isolated. The reconstructed oocyte is then
cultured such that it develops to morula or blastocyst and then
transferred to a pseudopregnant female foster animal. The offspring
born of this female foster animal will be a clone of the animal
from which the cell, e.g., the somatic cell, is isolated.
[0355] Transgenic animals containing recombinant cells that express
the polypeptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
affect, for example, binding, activation, and protein turnover, may
not be evident from in vitro cell-free or cell-based assays.
Accordingly, it is useful to provide non-human transgenic animals
to assay in vivo ubiquitin protease function, including substrate
interaction, the effect of specific mutant ubiquitin proteases on
ubiquitin protease function and substrate interaction, and the
effect of chimeric ubiquitin proteases. It is also possible to
assess the effect of null mutations, that is mutations that
substantially or completely eliminate one or more ubiquitin
protease functions.
[0356] In general, methods for producing transgenic animals include
introducing a nucleic acid sequence according to the present
invention, the nucleic acid sequence capable of expressing the
receptor protein in a transgenic animal, into a cell in culture or
in vivo. When introduced in vivo, the nucleic acid is introduced
into an intact organism such that one or more cell types and,
accordingly, one or more tissue types, express the nucleic acid
encoding the receptor protein. Alternatively, the nucleic acid can
be introduced into virtually all cells in an organism by
transfecting a cell in culture, such as an embryonic stem cell, as
described herein for the production of transgenic animals, and this
cell can be used to produce an entire transgenic organism. As
described, in a further embodiment, the host cell can be a
fertilized oocyte. Such cells are then allowed to develop in a
female foster animal to produce the transgenic organism.
[0357] Pharmaceutical Compositions
[0358] The ubiquitin protease nucleic acid molecules, protein
modulators of the protein, and antibodies (also referred to herein
as "active compounds") can be incorporated into pharmaceutical
compositions suitable for administration to a subject, e.g., a
human. Such compositions typically comprise the nucleic acid
molecule, protein, modulator, or antibody and a pharmaceutically
acceptable carrier.
[0359] The term "administer" is used in its broadest sense and
includes any method of introducing the compositions of the present
invention into a subject. This includes producing polypeptides or
polynucleotides in vivo as by transcription or translation, in
vivo, of polynucleotides that have been exogenously introduced into
a subject. Thus, polypeptides or nucleic acids produced in the
subject from the exogenous compositions are encompassed in the term
"administer."
[0360] As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, such media can be used in the compositions of the
invention. Supplementary active compounds can also be incorporated
into the compositions. A pharmaceutical composition of the
invention is formulated to be compatible with its intended route of
administration. Examples of routes of administration include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral
(e.g., inhalation), transdermal (topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampules, disposable
syringes or multiple dose vials made of glass or plastic.
[0361] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0362] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a ubiquitin protease
protein or anti-ubiquitin protease antibody) in the required amount
in an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0363] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For oral administration, the agent can be
contained in enteric forms to survive the stomach or further coated
or mixed to be released in a particular region of the GI tract by
known methods. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules. Oral compositions can
also be prepared using a fluid carrier for use as a mouthwash,
wherein the compound in the fluid carrier is applied orally and
swished and expectorated or swallowed. Pharmaceutically compatible
binding agents, and/or adjuvant materials can be included as part
of the composition. The tablets, pills, capsules, troches and the
like can contain any of the following ingredients, or compounds of
a similar nature: a binder such as microcrystalline cellulose, gum
tragacanth or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0364] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser, which contains a suitable propellant, e.g., a gas
such as carbon dioxide, or a nebulizer.
[0365] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0366] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0367] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0368] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. "Dosage unit form" as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0369] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) PNAS
91:3054-3057). The pharmaceutical preparation of the gene therapy
vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded. Alternatively, where the complete
gene delivery vector can be produced intact from recombinant cells,
e.g. retroviral vectors, the pharmaceutical preparation can include
one or more cells which produce the gene delivery system.
[0370] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0371] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
[0372] The skilled artisan will appreciate that certain factors may
influence the dosage required to effectively treat a subject,
including but not limited to the severity of the disease or
disorder, previous treatments, the general health and/or age of the
subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a protein,
polypeptide, or antibody can include a single treatment or,
preferably, can include a series of treatments. In a preferred
example, a subject is treated with antibody, protein, or
polypeptide in the range of between about 0.1 to 20 mg/kg body
weight, one time per week for between about 1 to 10 weeks,
preferably between 2 to 8 weeks, more preferably between about 3 to
7 weeks, and even more preferably for about 4, 5, or 6 weeks. It
will also be appreciated that the effective dosage of antibody,
protein, or polypeptide used for treatment may increase or decrease
over the course of a particular treatment. Changes in dosage may
result and become apparent from the results of diagnostic assays as
described herein.
[0373] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds.
[0374] It is understood that appropriate doses of small molecule
agents depends upon a number of factors within the ken of the
ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention. Exemplary doses
include milligram or microgram amounts of the small molecule per
kilogram of subject or sample weight (e.g., about 1 microgram per
kilogram to about 500 milligrams per kilogram, about 100 micrograms
per kilogram to about 5 milligrams per kilogram, or about 1
microgram per kilogram to about 50 micrograms per kilogram. It is
furthermore understood that appropriate doses of a small molecule
depend upon the potency of the small molecule with respect to the
expression or activity to be modulated. Such appropriate doses may
be determined using the assays described herein. When one or more
of these small molecules is to be administered to an animal (e.g.,
a human) in order to modulate expression or activity of a
polypeptide or nucleic acid of the invention, a physician,
veterinarian, or researcher may, for example, prescribe a
relatively low dose at first, subsequently increasing the dose
until an appropriate response is obtained. In addition, it is
understood that the specific dose level for any particular animal
subject will depend upon a variety of factors including the
activity of the specific compound employed, the age, body weight,
general health, gender, and diet of the subject, the time of
administration, the route of administration, the rate of excretion,
any drug combination, and the degree of expression or activity to
be modulated.
[0375] This invention may be embodied in many different forms and
should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will fully convey the invention to those skilled in the
art. Many modifications and other embodiments of the invention will
come to mind in one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Although specific terms are employed, they
are used as in the art unless otherwise indicated.
Sequence CWU 1
1
12 1 372 PRT Homosapiens 1 Met Ser Lys Ala Phe Gly Leu Leu Arg Gln
Ile Cys Gln Ser Ile Leu 1 5 10 15 Ala Glu Ser Ser Gln Ser Pro Ala
Asp Leu Glu Glu Lys Lys Glu Glu 20 25 30 Asp Ser Asn Met Lys Arg
Glu Gln Pro Arg Glu Arg Pro Arg Ala Trp 35 40 45 Asp Tyr Pro His
Gly Leu Val Gly Leu His Asn Ile Gly Gln Thr Cys 50 55 60 Cys Leu
Asn Ser Leu Ile Gln Val Phe Val Met Asn Val Asp Phe Thr 65 70 75 80
Arg Ile Leu Lys Arg Ile Thr Val Pro Arg Gly Ala Asp Glu Gln Arg 85
90 95 Arg Ser Val Pro Phe Gln Met Leu Leu Leu Leu Glu Lys Met Gln
Asp 100 105 110 Ser Arg Gln Lys Ala Val Arg Pro Leu Glu Leu Ala Tyr
Cys Leu Gln 115 120 125 Lys Cys Asn Val Pro Leu Phe Val Gln His Asp
Ala Ala Gln Leu Tyr 130 135 140 Leu Lys Leu Trp Asn Leu Ile Lys Asp
Gln Ile Thr Asp Val His Leu 145 150 155 160 Val Glu Arg Leu Gln Ala
Leu Tyr Thr Ile Arg Val Lys Asp Ser Leu 165 170 175 Ile Cys Val Asp
Cys Ala Met Glu Ser Ser Arg Asn Ser Ser Met Leu 180 185 190 Thr Leu
Pro Leu Ser Leu Phe Asp Val Asp Ser Lys Pro Leu Lys Thr 195 200 205
Leu Glu Asp Ala Leu His Cys Phe Phe Gln Pro Arg Glu Leu Ser Ser 210
215 220 Lys Ser Lys Cys Phe Cys Glu Asn Cys Gly Lys Lys Thr Arg Gly
Lys 225 230 235 240 Gln Val Leu Lys Leu Thr His Leu Pro Gln Thr Leu
Thr Ile His Leu 245 250 255 Met Arg Phe Ser Ile Arg Asn Ser Gln Thr
Arg Lys Ile Cys His Ser 260 265 270 Leu Tyr Phe Pro Gln Ser Leu Asp
Phe Ser Gln Ile Leu Pro Met Lys 275 280 285 Arg Glu Ser Cys Asp Ala
Glu Glu Gln Ser Gly Gly Gln Tyr Glu Leu 290 295 300 Phe Ala Val Ile
Ala His Val Gly Met Ala Asp Ser Gly His Tyr Cys 305 310 315 320 Val
Tyr Ile Arg Asn Ala Val Asp Gly Lys Trp Phe Cys Phe Asn Asp 325 330
335 Ser Asn Ile Cys Leu Val Ser Trp Glu Asp Ile Gln Cys Thr Tyr Gly
340 345 350 Asn Pro Asn Tyr His Trp Gln Glu Thr Ala Tyr Leu Leu Val
Tyr Met 355 360 365 Lys Met Glu Cys 370 2 1869 DNA Homo sapiens
misc_feature (0)...(0) 23413 Ubiquitin 2 caccccgcgt ccgcagcagc
ggaggctgga cgcttgcatg gcgcttgaga gattccatcg 60 tgcctggctc
acataagcgc ttcctggaag tgaagtcgtg ctgtcctgaa cgcgggccag 120
gcagctgcgg cctgggggtt ttggagtgat cacga atg agc aag gcg ttt ggg 173
Met Ser Lys Ala Phe Gly 1 5 ctc ctg agg caa atc tgt cag tcc atc ctg
gct gag tcc tcg cag tcc 221 Leu Leu Arg Gln Ile Cys Gln Ser Ile Leu
Ala Glu Ser Ser Gln Ser 10 15 20 ccg gca gat ctt gaa gaa aag aag
gaa gaa gac agc aac atg aag aga 269 Pro Ala Asp Leu Glu Glu Lys Lys
Glu Glu Asp Ser Asn Met Lys Arg 25 30 35 gag cag ccc aga gag cgt
ccc agg gcc tgg gac tac cct cat ggc ctg 317 Glu Gln Pro Arg Glu Arg
Pro Arg Ala Trp Asp Tyr Pro His Gly Leu 40 45 50 gtt ggt tta cac
aac att gga cag acc tgc tgc ctt aac tcc ttg att 365 Val Gly Leu His
Asn Ile Gly Gln Thr Cys Cys Leu Asn Ser Leu Ile 55 60 65 70 cag gtg
ttc gta atg aat gtg gac ttc acc agg ata ttg aag agg atc 413 Gln Val
Phe Val Met Asn Val Asp Phe Thr Arg Ile Leu Lys Arg Ile 75 80 85
acg gtg ccc agg gga gct gac gag cag agg aga agc gtc cct ttc cag 461
Thr Val Pro Arg Gly Ala Asp Glu Gln Arg Arg Ser Val Pro Phe Gln 90
95 100 atg ctt ctg ctg ctg gag aag atg cag gac agc cgg cag aaa gca
gtg 509 Met Leu Leu Leu Leu Glu Lys Met Gln Asp Ser Arg Gln Lys Ala
Val 105 110 115 cgg ccc ctg gag ctg gcc tac tgc ctg cag aag tgc aac
gtg ccc ttg 557 Arg Pro Leu Glu Leu Ala Tyr Cys Leu Gln Lys Cys Asn
Val Pro Leu 120 125 130 ttt gtc caa cat gat gct gcc caa ctg tac ctc
aaa ctc tgg aac ctg 605 Phe Val Gln His Asp Ala Ala Gln Leu Tyr Leu
Lys Leu Trp Asn Leu 135 140 145 150 att aag gac cag atc act gat gtg
cac ttg gtg gag aga ctg cag gcc 653 Ile Lys Asp Gln Ile Thr Asp Val
His Leu Val Glu Arg Leu Gln Ala 155 160 165 ctg tat acg atc cgg gtg
aag gac tcc ttg att tgc gtt gac tgt gcc 701 Leu Tyr Thr Ile Arg Val
Lys Asp Ser Leu Ile Cys Val Asp Cys Ala 170 175 180 atg gag agt agc
aga aac agc agc atg ctc acc ctc cca ctt tct ctt 749 Met Glu Ser Ser
Arg Asn Ser Ser Met Leu Thr Leu Pro Leu Ser Leu 185 190 195 ttt gat
gtg gac tca aag ccc ctg aag aca ctg gag gac gcc ctg cac 797 Phe Asp
Val Asp Ser Lys Pro Leu Lys Thr Leu Glu Asp Ala Leu His 200 205 210
tgc ttc ttc cag ccc agg gag tta tca agc aaa agc aag tgc ttc tgt 845
Cys Phe Phe Gln Pro Arg Glu Leu Ser Ser Lys Ser Lys Cys Phe Cys 215
220 225 230 gag aac tgt ggg aag aag acc cgt ggg aaa cag gtc ttg aag
ctg acc 893 Glu Asn Cys Gly Lys Lys Thr Arg Gly Lys Gln Val Leu Lys
Leu Thr 235 240 245 cat ttg ccc cag acc ctg aca atc cac ctc atg cga
ttc tcc atc agg 941 His Leu Pro Gln Thr Leu Thr Ile His Leu Met Arg
Phe Ser Ile Arg 250 255 260 aat tca cag acg aga aag atc tgc cac tcc
ctg tac ttc ccc cag agc 989 Asn Ser Gln Thr Arg Lys Ile Cys His Ser
Leu Tyr Phe Pro Gln Ser 265 270 275 ttg gat ttc agc cag atc ctt cca
atg aag cga gag tct tgt gat gct 1037 Leu Asp Phe Ser Gln Ile Leu
Pro Met Lys Arg Glu Ser Cys Asp Ala 280 285 290 gag gag cag tct gga
ggg cag tat gag ctt ttt gct gtg att gcg cac 1085 Glu Glu Gln Ser
Gly Gly Gln Tyr Glu Leu Phe Ala Val Ile Ala His 295 300 305 310 gtg
gga atg gca gac tcc ggt cat tac tgt gtc tac atc cgg aat gct 1133
Val Gly Met Ala Asp Ser Gly His Tyr Cys Val Tyr Ile Arg Asn Ala 315
320 325 gtg gat gga aaa tgg ttc tgc ttc aat gac tcc aat att tgc ttg
gtg 1181 Val Asp Gly Lys Trp Phe Cys Phe Asn Asp Ser Asn Ile Cys
Leu Val 330 335 340 tcc tgg gaa gac atc cag tgt acc tac gga aat cct
aac tac cac tgg 1229 Ser Trp Glu Asp Ile Gln Cys Thr Tyr Gly Asn
Pro Asn Tyr His Trp 345 350 355 cag gaa act gca tat ctt ctg gtt tac
atg aag atg gag tgc 1271 Gln Glu Thr Ala Tyr Leu Leu Val Tyr Met
Lys Met Glu Cys 360 365 370 taatggaaat gcccaaaacc ttcagagatt
gacacgctgt cattttccat ttccgttcct 1331 ggatctacgg agtcttctaa
gagattttgc aatgaggaga agcattgttt tcaaactata 1391 taactgagcc
ttatttataa ttagggatat tatcaaaata tgtaaccatg aggcccctca 1451
ggtcctgatc agtcagaatg gatgctttca ccagcagacc cggccatgtg gctgctcggt
1511 cctgggtgct cgctgctgtg caagacatta gccctttagt tatgagcctg
tgggaacttc 1571 aggggttccc agtggggaga gcagtggcag tgggaggcat
ctgggggcca aaggtcagtg 1631 gcagggggta tttcagtatt atacaactgc
tgtgaccaga cttgtatact ggctgaatat 1691 cagtgctgtt tgtaattttt
cactttgaga accaacatta attccatatg aatcaagtgt 1751 tttgtaactg
ctattcattt attcagcaaa tatttattga tcatctcttc tccataagat 1811
agtgtgataa acacagtcat gaataaagtt attttccaca aaaaaaaaaa aaaaaagg
1869 3 18 PRT Artificial Sequence UCH-1 consensus domain 3 Gly Leu
Glu Asn Leu Gly Asn Thr Cys Tyr Met Asn Ser Val Leu Gln 1 5 10 15
Cys Leu 4 44 PRT Artificial Sequence UCH-2 consensus domain 4 Tyr
Asp Leu Tyr Gly Val Val Cys His Tyr Gly Ala Thr Leu Ser Gly 1 5 10
15 Gly His Tyr Thr Ala Tyr Val Lys Lys Glu Leu Glu His Glu Val Leu
20 25 30 Lys Asn Lys Trp Tyr Leu Phe Asp Asp Glu Thr Val 35 40 5
292 PRT Artificial Sequence ProDom consensus sequence 5 Ser Trp Asp
Ser Lys Arg Gly Pro Gly Tyr Thr Gly Leu Lys Asn Leu 1 5 10 15 Gly
Asn Thr Cys Tyr Met Asn Ser Val Leu Gln Cys Leu Tyr His Val 20 25
30 Pro Pro Leu Arg Glu Tyr Phe Leu Glu Asp Glu Tyr Glu Ser Glu Met
35 40 45 Val Asn Asn Glu Ser Asn Pro Leu Gly Met Lys Gly Glu Leu
Ala Thr 50 55 60 Ala Tyr Ala Lys Leu Val His Gln Met Trp Ser Asn
Ser Ser Asn Lys 65 70 75 80 Ser Val Ala Pro Thr Gln Phe Leu Thr Thr
Val Gly Lys Phe Ser Pro 85 90 95 Gln Phe Ser Glu Gly Tyr Gln Gln
Gln Asp Ser Gln Glu Phe Leu Lys 100 105 110 Phe Leu Gln Asp Asp Ala
His Glu Asp Phe Asn Ser Leu Met Glu Lys 115 120 125 Pro Tyr Val Glu
Glu Gln Val Lys Asp Ser Asn Glu Lys Ser Thr Ala 130 135 140 Leu Val
Asn Val Ser Glu Glu Ala Trp Glu Asn His Lys Lys Arg Asn 145 150 155
160 Asp Ser Ile Ile Thr Asp Ile Phe Gln Gly Gln Phe Lys Ser Thr Ile
165 170 175 Lys Cys Pro Ser Cys Glu His Thr Ser Glu Thr Thr Phe Glu
Pro Phe 180 185 190 Met Asp Leu Ser Leu Pro Ile Pro Ser Asp Ser Ala
Asp Asn His Gln 195 200 205 Asn Leu Gln Asp Cys Leu Glu Ser Phe Thr
Lys Lys Glu Thr Leu Glu 210 215 220 Gly Asp Asn Lys Trp Tyr Cys Pro
Lys Cys Lys Lys Lys Gln Glu Ala 225 230 235 240 Thr Lys Lys Leu Asp
Ile Trp Lys Leu Pro Pro Val Leu Val Ile His 245 250 255 Leu Lys Arg
Phe Ser Tyr Asp Arg Gln Trp Gly Arg Arg Asp Lys Leu 260 265 270 Asn
Thr Thr Val Glu Phe Pro Leu Glu Asp Leu Asp Met Ser Pro Tyr 275 280
285 Val Asp Lys Lys 290 6 84 PRT Artificial Sequence ProDom
consensus sequence 6 Ile Met Ser Glu Ser Thr Ser Ser Asn Glu Thr
Lys Ser Asn Asn Pro 1 5 10 15 Tyr Lys Tyr Glu Leu Tyr Gly Val Ile
Val His Ser Gly Ser Ser Met 20 25 30 Asn Gly Gly His Tyr Val Ala
Tyr Val Lys Asn Arg Ser Lys Asn Asn 35 40 45 Gly Lys Trp Tyr Lys
Phe Asp Asp Glu Lys Val Thr Glu Val Ser Glu 50 55 60 Glu Asp Val
Ile Lys Thr Ser Gly Asp Ser Ser Ala Tyr Ile Leu Phe 65 70 75 80 Tyr
Glu Arg Val 7 79 PRT Artificial Sequence ProDom consensus sequence
7 Ser Ile Glu Lys Ser Ile Lys Asp Phe Phe Asn Pro Glu Leu Ile Lys 1
5 10 15 Val Asp Lys Glu Gln Lys Gly Tyr Val Cys Glu Lys Cys His Lys
Thr 20 25 30 Thr Asn Ala Val Lys His Ser Ser Ile Leu Arg Ala Pro
Glu Thr Leu 35 40 45 Leu Val His Leu Lys Lys Phe Arg Phe Asn Gly
Thr Ser Ser Ser Lys 50 55 60 Met Lys Gln Ala Val Ser Tyr Pro Met
Phe Leu Asp Leu Thr Glu 65 70 75 8 87 PRT Artificial Sequence
ProDom consensus sequence 8 Lys Gly Lys Val Ile Lys Lys Asp Val Val
Val Gln Leu Pro Asp Ile 1 5 10 15 Leu Ile Val His Leu Ser Arg Ser
Thr Phe Asn Gly Ile Thr Tyr Ser 20 25 30 Arg Asn Pro Cys Asn Val
Lys Phe Gly Glu Arg Ile Thr Leu Ser Glu 35 40 45 Tyr Thr Leu Ala
Glu Ser Gly Thr Ile Thr Glu Asn Arg Gln Val Lys 50 55 60 Tyr Asn
Leu Lys Ser Val Val Lys His Thr Gly Ser His Ser Ser Gly 65 70 75 80
His Tyr Met Cys Tyr Arg Arg 85 9 142 PRT Artificial Sequence ProDom
consensus sequence 9 Met His Ala His Pro Pro Ile Arg Ser Tyr Phe
Glu Ile Glu Met Phe 1 5 10 15 Ile Ala Tyr Glu Cys Lys Ser Cys Lys
His Val Ser Asn Ala Pro Asp 20 25 30 Lys Ala Ile Tyr Val Ser Ile
Asp Leu Ser Ser Lys Thr Lys Gly Thr 35 40 45 Met Gln Ser Met Val
Asp Lys Met Ala Asn Pro Ile Pro Val Val Gly 50 55 60 Met Asn Cys
Lys Ser Cys Gly Gln Glu Thr Leu Cys Ser Thr Thr Arg 65 70 75 80 Phe
Thr Lys Leu Pro Glu Val Leu Leu Tyr Phe Val Pro Arg Val Lys 85 90
95 Asp Gln Gln Arg Gly Lys Asp Met Thr Val Leu Asn Val Gln Arg Gln
100 105 110 Leu Ile Leu Lys Asp Asp Asn Asn Ala His Asn Tyr Glu Leu
Cys Ser 115 120 125 Phe Ile Ala His Cys Gly Lys Asn Gly Asp Asn Gly
His Tyr 130 135 140 10 48 PRT Artificial Sequence ProDom consensus
sequence 10 Thr Leu His Leu Thr Leu Tyr Asn Pro Ser Asn Arg Pro Leu
Thr Ile 1 5 10 15 Arg Arg Gly Asp Leu Val Ala Val Ala Val Pro Cys
Tyr Cys His Pro 20 25 30 Ala Lys Ala Pro Ser Gln Asp Val Cys Phe
Cys Glu Glu Arg Gly Arg 35 40 45 11 40 PRT Artificial Sequence
ProDom consensus sequence 11 Cys Arg His Ile Gln Asp His Cys Glu
Gln Gln Ile Gln Asp Leu Glu 1 5 10 15 Arg Arg His Arg Gln Gln Gln
Gly His Leu Arg Asp Gln His Gln Glu 20 25 30 Glu Arg Arg Asp Trp
Glu Phe Pro 35 40 12 81 PRT Artificial Sequence ProDom consensus
sequence 12 Asp Ala Ser Lys Gln Gly Tyr Gln His Phe Phe Ala Leu Leu
Gly Ala 1 5 10 15 Ala Ser Ala Val Thr Thr Gly His Pro Glu Ala Arg
Lys Leu Leu Asp 20 25 30 Tyr Thr Ile Glu Ile Ile Glu Lys Tyr Phe
Trp Ser Glu Glu Glu Gln 35 40 45 Met Cys Leu Glu Ser Trp Asp Glu
Ala Phe Ser Lys Thr Glu Glu Tyr 50 55 60 Arg Gly Gly Asn Ala Asn
Met His Ala Val Glu Ala Phe Leu Ile Val 65 70 75 80 Tyr
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