WO2009009773A1

WO2009009773A1 - Use of otubain enzyme to cleave lysine-48-linked polyubiquitin

Info

Publication number: WO2009009773A1
Application number: PCT/US2008/069887
Authority: WO
Inventors: Cynthia Wolberger; Tao Wang; Robert E. Cohen
Original assignee: The Johns Hopkins University
Priority date: 2007-07-11
Filing date: 2008-07-11
Publication date: 2009-01-15

Abstract

The invention relates generally to agents and methods for treating an Otubain-mediated disorder, methods for detecting ubiquitinated proteins, methods of diagnosing an Otubain-mediated disorder in a subject, and methods for identifying an agent that modulates Otubain-mediated cleavage of lysine-48-linked polyubiquitin peptide bonds.

Description

USE OF OTUBAIN ENZYME TO CLEAVE LYSINE-48-LINKED POLYUBIQUITIN

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0001] The invention relates generally to agents and methods for treating an Otubain- mediated disorder, and more specifically to methods for identifying an agent that modulates Otubain-mediated cleavage of polyubiquitin peptide bonds.

BACKGROUND INFORMATION

[0002] Covalent modification of proteins by ubiquitin (Ub) is used to signal protein degradation by the 26S proteasome and is essential as well as for many proteasome independent aspects of cellular regulation. Conjugation of ubiquitin to a protein substrate requires the Ub-activating enzyme, El, a Ub-conjugating enzyme, E2, and a Ub ligase, E3, to form an isopeptide bond between the C-terminal α-carboxylate of Ub and an ε-amine of a lysine within the protein substrate. Ubiquitin itself can be modified in this way at one of its seven surface lysine residues, giving rise to a polyubiquitin chain. All seven types of polyubiquitin chains, which are distinguished by isopeptide linkages with Ub at residues lysine(K)48, K63, K33, K29, K27, K6, or Kl 1, have been identified in vivo. The fate of ubiquitinated substrates depends, in part, on the length and linkage type of the polyubiquitin chain. Substrates modified with K48 -linked polyubiquitin of n > 4 usually are targeted for degradation by the 26S proteasome, whereas K63 -linked polyubiquitin plays a non- degradative role in DNA damage tolerance and NF-κB activation pathways.

[0003] Ubiquitination is a reversible modification. Deubiquitination is catalyzed by deubiquitinating enzymes (DUBs) that disassemble polyubiquitin chains or cleave Ub (or polyubiquitin) from conjugated proteins, including other Ub molecules and non-protein adducts. Deubiquitination plays important roles in regulating Ub-dependent pathways, including processing of Ub precursors, editing of polyubiquitin chains, release of proteins from Ub conjugates, and recycling of Ub. Current evidence suggests that many, and possibly most, DUBs regulate a limited number of proteins and pathways, which suggests that they target specific substrates. [0004] Six different classes of deubiquitinating enzymes have been identified. These include ubiquitin C-terminal hydrolases (UCHs), ubiquitin specific proteases (UBPs), ovarian tumor proteases (OTUs), Machado-Joseph disease protein domain proteases (MJDs), MPN+/JAMM motif proteases, and viral DUBs. With the exception of the MPN+/JAMM class DUBs which are Zn -metalloproteases, all other deubiquitinating enzymes are cysteine proteases.

[0005] Otubains are known to associate with the ubiquitin E3 ligase GRAIL and to control GRAIL expression. Expression of GRAIL has been associated with production of T cell anergy, immune suppression, auto-immunity, and other regulatory T cell functions. In autoimmune disease, T cells are inappropriately activated against particular tissues and proliferate, e.g. causing the inflammation associated with rheumatoid arthritis. Regulation of T cells is beneficial in the modulation of disease.

SUMMARY OF THE INVENTION

[0006] The present invention is based, in part, on the discovery that Otubain mediates cleavage of lysine-48 -linked polyubiquitin. As such, the invention provides methods to identify agents that can modulate Otubain-mediated activity, including high throughput screening methods, and further provides a means to identify agents that are useful for treating patients having Otubain-mediated disorders, methods for detecting ubiquitinated proteins, and methods of diagnosing an Otubain-mediated disorder in a subject.

[0007] The present invention relates to a method for treating an Otubain-mediated disorder by administering an agent that modulates Otubain-mediated cleavage of lysine-48- linked polyubiquitin to a subject in need thereof. An Otubain-mediated disorder can be, for example, a cellular proliferative disorder such as cancer, T-cell anergy, an autoimmune disease, immunosuppression, or Parkinson's Disease. The autoimmune disease can be acute disseminated encephalomyelitis, Addison's Disease, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, Bullous Pemphigoid, Celiac Disease, Chagas Disease, chronic obstructive pulmonary disease, dermatomyositis, diabetes mellitus type 1, endometriosis, Goodpasture's Syndrome, Graves' Disease, Guillain-Barre Syndrome, Hashimoto's Disease, Hidradenitis Suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, Lupus erythematosus, morphea, multiple sclerosis, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, pernicious anemia, polymyositis, primary biliary cirrhosis, rheumatoid arthritis, schizophrenia, Sjogren's syndrome, temporal arteritis, ulcerative colitis, vasculitis, vitiligo, or Wegener's granulomatosis. An agent that modulates Otubain-mediated activity could be, for example, lysine-48-linked polyubiquitin, lysine-63 -linked polyubiquitin, lysine-29/lysine-6- linked polyubiquitin, or monoubiquitin.

[0008J The present invention also relates to a method for monitoring a therapeutic regimen for treating a subject having a Otubain-mediated disorder, by determining a change in the Otubain-mediated cleavage of lysine-48-linked polyubiquitin during therapy.

[0009] The present invention also relates to a method for detecting ubiquitinated proteins by contacting a sample comprising a protein with detectably-labeled lysine-48-linked polyubiquitin and Otubain; and detecting Otubain-mediated cleavage of detectably-labeled lysine-48-linked polyubiquitin. In one embodiment, the protein is a cellular protein, which can be, for example, a disease marker. The detectably-labeled lysine-48-linked polyubiquitin can be either linear or cyclic lysine-48-linked polyubiquitin. In another embodiment, the method can be performed in a high throughput format. The sample can be a cell sample obtained from a subject, or it can be a cell free sample.

[0010] The present invention further relates to a method of diagnosing an Otubain- mediated disorder in a subject by comparing the Otubain-mediated activity in a test sample from the subject with the Otubain-mediated activity in a corresponding normal sample, where a difference in Otubain-mediated activity in the test sample as compared to the Otubain- mediated activity in the normal sample is diagnostic of an Otubain-mediated disorder. In one embodiment, the method involves contacting the test sample and a corresponding normal sample with an antibody that specifically binds Otubain and comparing the Otubain-mediated activity in the test sample and the Otubain-mediated activity in the normal sample. A greater decrease in Otubain-mediated activity detected in the test sample as compared to the Otubain- mediated activity in the normal sample is indicative of an Otubain-mediated disorder. The Otubain can be a mammalian Otubain such as, for example, human Otubain. In another embodiment the method of diagnosing an Otubain-mediated disorder in a subject can be performed in a high throughput format. [0011] The present invention also relates to a method of modulating protein stability by contacting lysine-48-linked polyubiquitin peptide with an agent that cleaves lysine-48-linked polyubiquitin peptide bonds. In one embodiment, the method includes reducing or inhibiting lysine-48-linked polyubiquitin peptide cleavage. The agent can be Otubain, and can be a mammalian Otubain such as, for example, human Otubain. The agent can be a functional fragment of Otubain, or Otubain with a deletion of 41 amino-terminal amino acids. In one embodiment, the method of modulating protein stability includes administering the agent to a subject having a disorder associated with lysine-48-linked polyubiquitin peptide cleavage. The disorder can be, for example, a cellular proliferative disorder. The lysine-48-linked polyubiquitin can be free, or it can be linked to a substrate. The substrate linked to the lysine- 48-linked polyubiquitin can be a cellular protein, for example, a disease marker. In one embodiment, the lysine-48-linked polyubiquitin can be linear or cyclic. In another embodiment, the agent can be lysine-48-linked polyubiquitin, lysine-63 -linked polyubiquitin, lysine-29/lysine-6-linked polyubiquitin, or monoubiquitin.

[0012] The present invention further relates to a method of identifying an agent that modulates Otubain-mediated cleavage of lysine-48-linked polyubiquitin peptide bonds by contacting a sample containing lysine-48-linked polyubiquitin and Otubain with a test agent under conditions sufficient for Otubain-mediated cleavage of lysine-48-linked polyubiquitin peptide bonds, and detecting a change in Otubain-mediated cleavage of lysine-48-linked polyubiquitin peptide bonds in the presence of the test agent as compared to the activity in the absence of the test agent. The lysine-48-linked polyubiquitin can be free, or it can be linked to a substrate. The substrate linked to the lysine-48-linked polyubiquitin can be a cellular protein, for example, a disease marker. In one embodiment, the lysine-48-linked polyubiquitin can be linear or cyclic. In another embodiment, the lysine-48-linked polyubiquitin can be detectibly labeled. In one embodiment, the lysine-48-linked polyubiquitin peptide bonds is cleaved at the proximal lysine-48-linked polyubiquitin bond and/or at the distal lysine-48-linked polyubiquitin bond. In one embodiment, the Otubain can be a mammalian Otubain such as, for example, human Otubain. The agent that modulates Otubain-mediated cleavage of lysine-48-linked polyubiquitin peptide bonds can either inhibit or stimulate Otubain-mediated activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure IA shows the sequence alignment of otubain proteins from different species. The proteins include: Homo sapiens otubain 1 (h Otul, encoded by Genbank accession number NMJ 17670) (SEQ ID NO:1) and otubain 2 (h_Otu2, encoded by NM_023112) (SEQ ID NO:2), Mus musculus otubain 1 (m_Otul, encoded by NM_134150) (SEQ ID NO:3), Caenorhabdits elegans otubain (ce Otu, encoded by Q9XVR6) (SEQ ID NO:4), Saccharomyces cerevisiae Otul (Sc_Otul, encoded by nucleotides 44655 to 45560 of NC OOl 138) (SEQ ID NO:5), Drosophila melanogaster otubain (d_Otu, encoded by Q9 VLOO) (SEQ ID NO: 6), and Arabidopsis thaliana otubain (a_Otu, encoded by NM 102577) (SEQ ID NO:7). Figure IB shows human otubain 1 aligned with other OTU domain ubiquitinating enzymes. The numbers indicate amino acid residues.

[0014] Figure 2A shows Coomassie Blue staining of an SDS-PAGE gel after hOtul was incubated with K48-Ub₂, K63-Ub₂, or K29/K6-Ub₂ for the times indicated. Figure 2B shows silver staining of proteins on an SDS-PAGE gel after hOtul or hCezcat was incubated with Kl 1-Ub₂ for the times indicated. Figure 2C shows detection of proteins on an SDS-PAGE gel after hOtul was incubated with hOtul mixed-linkage Ub₄ chains Ub-K48-Ub-K63-Ub- K48-Ub and Ub-K63-Ub-K48-Ub-K63-Ub for the times indicated. Figure 2D shows detection of proteins on an SDS-PAGE gel after hOtul was incubated with E2-25 kDa-(K48- linked) Ub₄ or Ubcl3-(K63-linked)Ub₄ for the times indicated. Figure 2E shows detection of proteins on an SDS-PAGE gel after hOtul was incubated with E2-25 kDa-(K48-linked) Ub₄ for the times indicated.

[0015] Figure 3 shows an SDS-PAGE gel of the degradation of a fluorescent tetraubiquitin substrate with the fluorophore Lucifer Yellow attached to the distal end of the tetraubiquitin after incubation with the hOtul or isopeptidase T (isoT) ubiquitinating enzymes.

[0016] Figure 4 shows detection of ⁵I-labeled proteins after hOtul was incubated with K48-Ub2, K63-Ub₂, K29/K6-Ub₂, or diubiquitin in the presence of no competitor or equal, 5- fold, or 10-fold molar excess of unlabeled monoubiquitin or diubiquitin of different linkage types. [0017] Figure 5A shows Coomassie Blue staining of polyubiquitin protein cleaved by hCezcat. Figure 5B shows Coomassie Blue staining of polyubiquitin protein cleaved by recombinant human otubain 1 fragment hOtulΔN41 and recombinant putative C. elegans otubain (ceOtu).

[0018] Figure 6 shows silver stained SDS-Page gels of Ub₅ proteins incubated with hOtul, hCezcat, ceOtu, or hOtulΔN41 for the times indicated.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention is based on the discovery that otubain 1 deubiquitinating activity is specific for K48 linkages. The invention relates generally to agents and methods for treating an Otubain-mediated disorder, methods for detecting ubiquitinated proteins, methods of diagnosing an Otubain-mediated disorder in a subject, and methods for identifying an agent that modulates Otubain-mediated cleavage of lysine-48-linked polyubiquitin peptide bonds.

[0020] The present invention is not limited to the particular methodology, protocols, cell lines, vectors, reagents, described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof (e.g., antigen binding antibody fragments) known to those skilled in the art, and so forth.

[0021] Otubain (OTU) domain deubiquitinating enzymes are ovarian-tumor-domain containing proteins identified initially through a bioinformatics analysis. Using novel DUB- specific probes generated by a chemical ligation method, an OTU-domain containing protein was isolated. Of the more than 100 OTU-domain proteins to date, the otubains are among only a few that have been shown to have deubiquitinating (DUB) activities in vitro or in vivo. Otubain proteins from different species share a high degree of sequence homology (Figure IA). Human otubain 1 has been reported to cleave isopeptide-linked tetraubiquitin, but not a peptide substrate that mimics a derivatized Ub C-terminus (Leu-Arg-Gly-Gly-7-amido-4- methycoumarin; LRGG-AMC) (SEQ ID NO: 8). Human otubain 2, on the other hand, is inactive in vitro against peptide and isopeptide-linked substrates, but cleaves Ub-AMC.

[0022] Two isoforms of human otubain 1 were found to regulate T cell anergy by forming a complex with GRAIL 1, an E3 ligase. Otubain 1 effects both GRAIL 1 expression and GRAIL-mediated ubiquitination. The yeast homolog of otubain 1, Otul, recently was reported to bind to Cdc48, a chaperone-like AAA ATPase. The binding of Otu 1 to Cdc48 counteracts the association of Cdc48 with Ufd2, a U-box containing E4 Ub ligase that extends polyubiquitin chains on ubiquitin conjugates. In addition, the transcription factor Spt23 was identified as an Otul substrate in vivo. In vitro, yeast Otul can efficiently cleave ubiquitin from Ub-AMC and disassemble K48-linked polyubiquitin chains, but not K63- linked polyubiquitin chains.

[0023] One of the salient features of the normal immune system is its ability to mount responses against foreign antigens while not attacking self-antigens. This is known as "tolerance." One mechanism for inducing tolerance, termed "peripheral tolerance," can be induced by activation of T cell receptors (TCR) without costimulation. Costimulation is necessary for a productive response to antigen in T cells, a predominant costimulatory receptor is CD28, which binds the costimulatory ligands B7-1 (CD80) and B7-2 (CD86) expressed on the surface of antigen-presenting cells (APC). Combined engagement of TCR and CD28 results in full activation of a number of signaling pathways that ultimately lead to IL-2 production and cell proliferation.

[0024] TCR engagement in the absence of costimulation results in a partial response. The incompletely stimulated T cells enter a long-lived unresponsive state, known as tolerance or anergy. Critically, once tolerance is induced, the anergic T cell is blocked from the response evoked by exposure to an antigen presented by an APC. In such cells, the combined engagement of the TCR and CD28 does not trigger the level of IL-2 production and the extent of proliferation that occurs in fully activated T cells. There is considerable evidence that tumors can induce immune tolerance in order to functionally inactivate T cells that may mount a tumor-specific response.

[0025] Other OTU proteins known to have DUB activity are Cezanne (cellular zinc finger anti-NF-KB), TRABID, A20, and VCIPL35 (Figure IB). A20 can cleave both K48 and K63 linked polyubiquitin chains in vitro, and the N-terminal, OTU domain-containing portion of A20 cleaves K63 -linked polyubiquitin. The Cezanne catalytic domain has been reported to cleave Ub-AMC and linear or branched (either K48 or K63-linked) polyubiquitin. The deubiquitinating activity of VCIP 135, which is required for p97-p47-mediated cisternal regrowth, cleaves K48-linked polyubiquitin in vitro.

[0026] Accordingly, the invention provides a method for detecting ubiquitinated proteins. In one embodiment, the method includes contacting a sample that contains Otubain and a protein with detectably-labeled lysine-48-linked polyubiquitin and detecting Otubain- mediated cleavage of detectably-labeled lysine-48-linked polyubiquitin. The detection can be performed in a cell free format or is performed in a cell based assay.

[0027] The invention also provides a method of modulating protein stability, comprising contacting lysine-48-linked polyubiquitin peptide with an agent that cleaves lysine-48-linked polyubiquitin peptide bonds. Protein stability can be modulated by, for example, reducing or inhibiting cleavage of lysine-48-linked polyubiquitin. An agent such as Otubain can be used in the present invention. The agent can be mammalian Otubain including human Otubain. Furthermore, the agent can be a functional fragment of Otubain.

[0028] A sample that is examined according to a method of the invention can be any sample that contains, or to which can be added, Otubain and a protein with detectably-labeled lysine-48-linked polyubiquitin such that Otubain activity can be detected. In one aspect, the sample is a biological sample, including, for example, a bodily fluid, an extract from a cell, which can be a crude extract or a fractionated extract, a chromosome, an organelle, or a cell membrane; a cell; genomic DNA, RNA, or cDNA, which can be in solution or bound to a solid support; a tissue; or a sample of an organ. A biological sample, for example, from a human subject, can be obtained using well known and routine clinical methods (e.g., a biopsy procedure).

[0029] Otubain 1 is a cysteine protease, and is known to associate with the ubiquitin E3 ligase GRAIL and to control GRAIL expression. Expression of GRAIL has been associated with production of T cell anergy, immune suppression, autoimmunity, and other regulatory T cell functions. [0030] Otubain 1 is specific, cleaving only lysine 48 (K48)-linked polyubiquitin chains but not K63-, K29/K6-, or Kl 1-linked polyubiquitin, or linear α-linked polyubiquitin. This specificity for K48 linkages is not limited to the distal or proximal end of a polyubiquitin chain, and both free and substrate-linked polyubiquitin are disassembled. Cleavage of K48- Ub₂ by otubain 1 is inhibited by addition of K48-Ub₂, K63-Ub₂, K29/K6-Ub₂, or monoubiquitin.

[0031] The substrate specificities of the OTU class of deubiquitinating enzymes appear to be quite diverse. As with many other DUBs, very little was known about OTU domain deubiquitination substrates in vivo. Accordingly, this invention is based on the observation that the cleavage activity of human otubain 1 (hOtul) is specific for K48 Ub isopeptide linkages. This specificity holds true even with polyubiquitin substrates containing mixtures of K48 and K63 linkages within the same chain. hOtul is the first clear-cut example of a deubiquitinating enzyme that cleaves only K48 linkages. Previous studies of UbpY (USP8), USP 14, and yeast Otul had shown that K63 -linked polyubiquitin is a very poor substrate as compared to K48-linked polyubiquitin. However, UbpY also can disassemble linear polyubiquitin and Ub-protein fusions. While USP14 does prefer K48 over K63 linkages, the activity was low in both cases and reflected the enzyme in an inactivated state. Similarly, yeast Otul prefers K48 over K63 linkages, but overnight enzyme cleavage results in disappearance of longer K48-linked polyubiquitin chains yet not K48-Ub₂.

[0032] The discrimination by hOtul among substrates with different linkage types cannot be attributed simply to preferential binding to K48-linked polyubiquitin substrates. Deubiquitins of different linkage types could inhibit K48-Ub₂ cleavage by hOtul equally well, and even monoubiquitin could serve as an inhibitor. These results suggest that some aspect of the enzyme mechanism other than initial substrate binding must contribute to the strict selectivity for K48 linkages.

[0033] T cells play an important role in the immune response. However, in auto-immune disease T cells are inappropriately activated against particular tissues and proliferate, e.g. causing the inflammation associated with rheumatoid arthritis. Inhibition of the proliferation of T cells is beneficial in the modulation of autoimmune disease. [0034] An immunosuppressed individual is one whose activation or efficacy of the immune system is reduced. The immune system can be suppressed by infection, such as infection with Human Immunodeficiency Virus, or immunosuppression may occur as an adverse reaction to treatment of other conditions. Deliberately induced immunosuppression is generally done to prevent the body from rejecting an organ transplant, treating graft- versus- host disease after a bone marrow transplant, or for the treatment of auto-immune diseases such as rheumatoid arthritis or Crohn's disease.

[0035] The specificity of hOtul for K48-linked chains is important for its function in regulating T cell anergy. T cell anergy, or tolerance, is a result of incompletely stimulated T cells which enter a long-lived unresponsive state. There is considerable evidence that tumors can induce immune tolerance in order to functionally inactivate T cells that may mount a tumor-specific response.

[0036] The term "anergy," refers to downregulation of at least one response of an immune cell, e.g., a B cell or a T cell. Such downregulated responses may include, e.g., decreased proliferation in response to antigen stimulation, or decreased cytokine (e.g., IL-2) production.

[0037] Once disease is established and a treatment protocol is initiated, screening assays of the invention may be repeated on a regular basis to evaluate whether the level of Otubain- mediated activity in the patient begins to approximate that which is observed in the normal patient. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months. Accordingly, the invention is also directed to methods for monitoring a therapeutic regimen for treating a subject having a Otubain-mediated disorder. A comparison of the Otubain-mediated activity prior to and during therapy indicates the efficacy of the therapy. Therefore, one skilled in the art will be able to recognize and adjust the therapeutic approach as needed.

[0038] The present invention also provides methods for diagnosing Otubain-mediated disorders in a subject. In one embodiment, agents identified as modulating Otubain-mediated activity may be used for the diagnosis of conditions or diseases characterized by Otubain- mediated disorders, or in assays to monitor patients being treated for Otubain-mediated disorders. The agents useful for diagnostic purposes may be prepared in the same manner as those described herein for therapeutics. Diagnostic assays for Otubain-mediated disorders can be performed in samples such as human body fluids or extracts of cells or tissues. The agents may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules which are known in the art may be used.

[0039] The term "disorder" or "disease" as used herein refers to a condition associated with Otubain-mediated cleavage of lysine-48-polyubiquitin. The term "Otubain-mediated disorder" is used herein to refer specifically to a condition in which the activity of Otubain is increased or decreased above the level of Otubain activity in a corresponding normal cell. Otubain-mediated disorders include, but are not limited to, a cellular proliferative disorder such as cancer, T-cell anergy, an autoimmune disease, immunosuppression, or Parkinson's Disease. The autoimmune disease can be acute disseminated encephalomyelitis, Addison's Disease, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, Bullous Pemphigoid, Celiac Disease, Chagas Disease, chronic obstructive pulmonary disease, dermatomyositis, diabetes mellitus type 1, endometriosis, Goodpasture's Syndrome, Graves' Disease, Guillain-Barre Syndrome, Hashimoto's Disease, Hidradenitis Suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, Lupus erythematosus, morphea, multiple sclerosis, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, pernicious anemia, polymyositis, primary biliary cirrhosis, rheumatoid arthritis, schizophrenia, Sjogren's syndrome, temporal arteritis, vasculitis, vitiligo, or Wegener's granulomatosis.

[0040] The terms "cell proliferative disorder" or "cellular proliferative disorder" refer to any disorder in which the proliferative capabilities of the affected cells are different from the normal proliferative capabilities of unaffected cells. An example of a cell proliferative disorder is neoplasia. Malignant cells (i.e., cancer) develop as a result of a multistep process. The term "malignant" refers to a tumor that is metastastic or no longer under normal cellular growth control.

[0041] The term "cancer" as used herein, includes any malignant tumor including, but not limited to, carcinoma and sarcoma. Cancer arises from the uncontrolled and/or abnormal division of cells that then invade and destroy the surrounding tissues. As used herein, "proliferating" and "proliferation" refer to cells undergoing mitosis. As used herein, "metastasis" refers to the distant spread of a malignant tumor from its sight of origin. Cancer cells may metastasize through the bloodstream, through the lymphatic system, across body cavities, or any combination thereof. The term "cancerous cell" as provided herein, includes a cell afflicted by any one of the cancerous conditions provided herein. The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate surrounding tissues, and to give rise to metastases.

[0042] A cell proliferative disorder as described herein may be a neoplasm. Such neoplasms are either benign or malignant. The term "neoplasm" refers to a new, abnormal growth of cells or a growth of abnormal cells that reproduce faster than normal. A neoplasm creates an unstructured mass (a tumor) which can be either benign or malignant. The term "benign" refers to a tumor that is noncancerous, e.g. its cells do not proliferate or invade surrounding tissues.

[0043] The terms "activate," "modulate," "induce," "inhibit," "elevate," "increase," "decrease," "reduce," or the like, denote quantitative differences between two states, e.g., a statistically significant difference, between the two states.

[0044] "Inhibitors," "activators," and "modulators" of Otubain-mediated activity are used to refer to activating, inhibitory, or modulating molecules identified using in vitro and in vivo assays of Otubain-mediated cleavage of lysine-48-linked polyubiquitin. "Inhibitors" are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of Otubain, e.g., antagonists. "Activators" are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate Otubain activity, e.g., agonists. Inhibitors, activators, or modulators also include genetically modified versions of Otubain, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, substrates, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, small chemical molecules and the like. Such assays for inhibitors and activators include, e.g., expressing Otubain in vitro, in cells, or cell membranes, applying putative modulator compounds, and then determining the functional effects on activity, as described above.

[0045] As used herein, the terms "sample" and "biological sample" refer to any sample suitable for the methods provided by the present invention. In one embodiment, the biological sample of the present invention is a tissue sample, e.g., a biopsy specimen such as samples from needle biopsy. In other embodiments, the biological sample of the present invention is a sample of bodily fluid, e.g., blood, serum, plasma, urine, and ejaculate.

[0046] Samples or assays comprising Otubain that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%. Inhibition of Otubain is achieved when the activity value relative to the control is about 80%, at least about 50%, or at least about 25-0%. Activation of Otubain is achieved when the activity value relative to the control (untreated with activators) is 110%, as much as about 150%, as much as 200-500% (i.e., two to five fold higher relative to the control), or as much as 1000-3000% higher.

[0047] "Ubiquitin," or "Ub," is a highly conserved 76 amino acid protein expressed in all eukaryotic cells and is best known for its role in targeting proteins for degradation by the 26S proteasome. All seven conserved lysines of Ub (K6, 11, 27, 29, 33, 48 and 63) may be used as branching sites for the generation of Ub polymers.

[0048] A "deubiquitinating enzyme" denotes a protease which hydrolyses a peptide bond at the C-terminus of ubiquitin, and thereby mediates the removal and processing of ubiquitin from its conjugates (e.g., polyubiquitin chains or chimeric ubiquitin fusion polypeptides).

[0049] As used herein, the term "agent" means any compound that is being examined for the ability to modulate Otubain-mediated cleavage of lysine-48-linked polyubiquitin. An agent can be any type of molecule, including, for example a peptide, a polynucleotide, an antibody, a glycoprotein, a carbohydrate, a small organic molecule, or a peptidomimetic.

[0050] Polynucleotides are known to specifically interact with proteins and, therefore, can be useful as test agents to be screened for the ability to modulate Otubain-mediated activity. The term "polynucleotide" is used broadly herein to mean a sequence of two or more deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond. As such, the term "polynucleotide" includes RNA and DNA, which can be a synthetic RNA or DNA sequence, and can be single stranded or double stranded, as well as a DNA/RNA hybrid. Furthermore, the term "polynucleotide" as used herein includes naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as synthetic molecules, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR). In various embodiments, a polynucleotide useful as a test agent can contain nucleoside or nucleotide analogs, or a backbone bond other than a phosphodiester bond. In general, the nucleotides comprising a polynucleotide are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2'-deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose. However, a polynucleotide also can contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides. Such nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220-5234, 1994; Jellinek et al., Biochemistry 34:11363-11372, 1995; Pagratis et al., Nature Biotechnol. 15:68-73, 1997, each of which is incorporated herein by reference).

[0051] The covalent bond linking the nucleotides of a polynucleotide generally is a phosphodiester bond. However, the covalent bond also can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides (see, for example, Tam et al., Nucl. Acids Res. 22:977-986, 1994; Ecker and Crooke, BioTechnology 13:351360, 1995, each of which is incorporated herein by reference). The incorporation of non-naturally occurring nucleotide analogs or bonds linking the nucleotides or analogs can be particularly useful where the polynucleotide is to be exposed to an environment that can contain a nucleolytic activity, including, for example, a tissue culture medium or upon administration to a living subject, since the modified polynucleotides can be less susceptible to degradation.

[0052] A polynucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template. In comparison, a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template (Jellinek et al., supra, 1995). [0053] Peptides also can be useful as test agents. The term "peptide" is used broadly herein to refer to a molecule containing two or more amino acids or amino acid analogs (or modified forms thereof) linked by peptide bonds. As such, peptide agents can contain one or more D-amino acids and/or L-amino acids; and/or one or more amino acid analogs, for example, an amino acid that has been derivatized or otherwise modified at its reactive side chain. In addition, one or more peptide bonds in the peptide can be modified, and a reactive group at the amino terminus or the carboxy terminus or both can be modified. Peptides containing D-amino acids, or L-amino acid analogs, or the like, can have improved stability to a protease, an oxidizing agent or other reactive material the peptide may encounter in a biological environment. Further, the stability of a peptide agent (or test agent) can be improved by generating (or linking) a fusion protein comprising the peptide and a second polypeptide (e.g., an Fc domain of an antibody) that increases the half-life of the peptide agent in vivo. Peptides also can be modified to have decreased stability in a biological environment, if desired, such that the period of time the peptide is active in the environment is reduced.

[0054] Antibodies provide an example a molecule useful as test agents in a screening assay of the invention. As used herein, the term "antibody" is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies. Antibodies are characterized, in part, in that they specifically bind to an antigen, particularly to one or more epitopes of an antigen. The term "binds specifically" or "specific binding activity" or the like, when used in reference to an antibody, means that an interaction of the antibody and a particular epitope has a dissociation constant of at least about 1 x ICT⁶ M, generally at least about 1 x 10^" M, usually at least about 1 x 10^" M, and particularly at least about 1 x 10^"9 M or 1 x 10^"10M or less. As such, Fab, F(ab')₂, Fd and Fv fragments of an antibody that retain specific binding activity are included within the definition of an antibody.

[0055] The term "antibody" as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains (see Huse et al., Science 246:1275-1281, 1989, which is incorporated herein by reference). These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known (Winter and Harris, Immunol. Today 14:243-246, 1993; Ward et al., Nature 341 :544-546, 1989; Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1999); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference). In addition, modified or derivatizcd antibodies, or antigen binding fragments of antibodies, such as pegylated (polyethylene glycol modified) antibodies, can be useful for the present methods.

[0056] Antibodies can be tested for anti-target polypeptide activity using a variety of methods well-known in the art. Various techniques may be used for screening to identify antibodies having the desired specificity, including various immunoassays, such as enzyme- linked immunosorbent assays (ELISAs), including direct and ligand-capture ELISAs, radioimmunoassays (RIAs), immunoblotting, and fluorescent activated cell sorting (FACS). Numerous protocols for competitive binding or immunoradiometric assays, using either polyclonal or monoclonal antibodies with established specificities, are well known in the art. See, e.g., Harlow and Lane. Such immunoassays typically involve the measurement of complex formation between the target polypeptide and a specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non- interfering epitopes on the target polypeptide is preferred, but other assays, such as a competitive binding assay, may also be employed. See, e.g., Maddox et al, 1983, J. Exp. Med. 158:1211.

[0057] An antibody useful in the methods of the invention can be an intact antibody or antigen binding fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding the epitopic determinant. The antibodies used in the method can be polyclonal or, more preferably, monoclonal antibodies. Monoclonal antibodies with different epitopic specificities are made from antigen containing fragments of the protein by methods well known in the art.

[0058] Where an agent is identified as having Otubain-mediated lysine-48-linked polyubiquitin activity, a method of identifying an agent can further include a step of determining an amount by which the agent increases or decreases Otubain-mediated activity. For example, where an agent is identified that increases Otubain-mediated activity, a method of the invention can further include determining an amount by which the agent increases Otubain-mediated activity above a basal level in a corresponding normal sample. Such an agent can be identified by measuring the amount of Otubain-mediated activity in a single sample both before adding the test agent and after adding the test agent, or can be identified for example, using two samples, wherein one sample serves as a control (no test agent added) and the other sample includes the test agent. As such, a method of the invention provides a means to obtain agents or panels of agents that variously modulate Otubain-mediated activity.

[0059] As used herein, a "corresponding normal sample" is any sample taken from a subject of similar species that is considered healthy or otherwise not suffering from a Otubain-mediated associated disorder. As such, a normal/standard level of Otubain-mediated activity denotes the level of Otubain-mediated activity present in a sample from the normal sample. A normal level of Otubain-mediated activity can be established by combining body fluids or cell extracts taken from normal healthy subjects, preferably human, with an antibody to Otubain under conditions suitable for Otubain-mediated activity. Levels of Otubain- mediated activity in subject, control, and disease samples from biopsied tissues can be compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease. A normal level of Otubain-mediated activity also can be determined as an average value taken from a population of subjects that is considered to be healthy, or is at least free of a Otubain-mediated associated disorder. A variety of protocols including ELISA, RJA, and FACS are useful for measuring levels of Otubain-mediated activity, and provide a basis for diagnosing altered or abnormal levels of Otubain-mediated activity.

[0060] Delivery of an agent can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia or preferably an RNA virus such as a retrovirus. A number of the known retroviruses can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting a polynucleotide sequence of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is target specific. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the antisense polynucleotide.

[0061] Another targeted delivery system for an agent is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery systems in vivo and in vitro. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form. In order for a liposome to be an effective gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information.

[0062] In order to express a biologically active Otubain protein or fragment thereof, the nucleotide sequence coding for the full length protein, or a functional Otubain fragment is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.

[0063] More specifically, methods which are well known to those skilled in the art can be used to construct expression vectors containing the Otubain or Otubain fragment sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See e.g., the techniques described in Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N. Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N. Y.

[0064] As disclosed herein, the methods of the invention provide the advantage that they can be adapted to high throughput analysis and, therefore, can be used to screen combinatorial libraries of test agents in order to identify those agents that can modulate Otubain-mediated activity. Methods for preparing a combinatorial library of molecules that can be tested for a desired activity are well known in the art and include, for example, methods of making a phage display library of peptides, which can be constrained peptides (see, for example, U.S. Patent No. 5,622,699; U.S. Pat. No. 5,206,347; Scott and Smith, Science 249:386-390, 1992; Markland et al., Gene 109:13 19, 1991 ; each of which is incorporated herein by reference); a peptide library (U.S. Patent No. 5,264,563, which is incorporated herein by reference); a peptidomimetic library (Blondelle et al., Trends Anal. Chem. 14:83 92, 1995; a nucleic acid library (O'Connell et al., Proc. Natl. Acad. ScI, USA 93:5883-5887, 1996; Tuerk and Gold, Science 249:505-510, 1990; Gold et al., Ann. Rev. Biochem. 64:763-797, 1995; each of which is incorporated herein by reference); an oligosaccharide library (York et al., Carb. Res., 285:99 128, 1996; Liang et al., Science, 274:1520 1522, 1996; Ding et al., Adv. Expt. Med. Biol. 376:261 269, 1995; each of which is incorporated herein by reference); a lipoprotein library (de Kruif et al., FEBS Lett. 1>99:21>2 236, 1996); a glycoprotein or glycolipid library (Karaoglu et al., J Cell Biol. 130:567 577, 1995, which is incorporated herein by reference); or a chemical library containing, for example, drugs or other pharmaceutical agents (Gordon et al., X Med. Chem. 37:1385-1401, 1994; Ecker and Crooke, BioTechnology 13:351-360, 1995; each of which is incorporated herein by reference). Polynucleotides can be particularly useful as agents that can modulate a specific interaction of molecules because nucleic acid molecules having binding specificity for cellular targets, including cellular polypeptides, exist naturally, and because synthetic molecules having such specificity can be readily prepared and identified (see, for example, U.S. Patent No. 5,750,342).

[0065] An additional advantage of arranging the samples in an array, particularly an addressable array, is that an automated system can be used for adding or removing reagents from one or more of the samples at various times, or for adding different reagents to particular samples. In addition to the convenience of examining multiple test agents and/or samples at the same time, such high throughput assays provide a means for examining duplicate, triplicate, or more aliquots of a single sample, thus increasing the validity of the results obtained, and for examining control samples under the same conditions as the test samples, thus providing an internal standard for comparing results from different assays.

[0066] Various protocols may be employed for screening a library of chemical compounds. To some degree, the selection of the appropriate protocol will depend upon the nature of the preparation of the compounds. For example, the compounds may be bound to individual particles, pins, membranes, or the like, where each of the compounds is segregatable. In addition, the amount of compound available will vary, depending upon the method employed for creating the library. Furthermore, depending upon the nature of the attachment of the compound to the support, one may be able to release aliquots of a compound, so as to carry out a series of assays. In addition, the manner in which the compounds are assayed will be affected by the ability to identify the compound which is shown to have activity.

[0067] Where the agents are individually located on a surface in a grid, so that at each site of the grid one knows the identification of each agent, one can provide a cellular lawn which is similarly organized as a grid and may be placed in registry with the agents bound to the solid surface. Once the lawn and solid substrate are in registry, one may release the agents from the surface in accordance with the manner in which the agents are attached. After sufficient time for the agents to bind to the proteins on the cellular surface, one may wash the cellular lawn to remove non-specifically bound agents. One or more washings may be involved, where the washings may provide for varying degrees of stringency, depending upon the desired degree of affinity. Since the preparative process can be repeated, a plurality of solid substrates can be prepared, where the same compounds are prepared at the comparable sites, so that the screening could be repeated with the same or different cells to determine the activity of the individual compounds.

[0068] In some instances, the identity of the agent can be determined by a nucleic acid tag, using the polymerase chain reaction for amplification of the tag. See, for example, WO93/20242, incorporated herein by reference. In this instance, the agents which are active may be determined by taking the lysate and introducing the lysate into a polymerase chain reaction medium comprising primers specific for the nucleic acid tag. Upon expansion, one can sequence the nucleic acid tag or determine its sequence by other means, which will indicate the synthetic procedure used to prepare the agent.

[0069] Otubain-mediated activity represents a specific target for the development of anti- Otubain activity. Accordingly, the invention provides methods of using an agent that can modulate Otubain-mediated activity to treat a Otubain-mediated associated disorder. As such, the methods provide for the administration of a therapeutically effective amount of an agent that modulates Otubain-mediated activity.

[0070] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.

[0071] For administration to a subject, an agent that modulates Otubain-mediated activity is administered by a route and under conditions that facilitate contact of the agent with the target cell and, if appropriate, entry into the cell. Thus, the agent can be administered to the site of the cells to be treated, or can be administered by any method that provides the target cells with the agent. Furthermore, the agent generally is formulated in a composition (e.g., a pharmaceutical composition) suitable for administration to the subject. Thus, the invention provides pharmaceutical compositions containing an agent that modulates Otubain-mediated activity in a pharmaceutically acceptable carrier. As such, the agents are useful as medicaments for treating a subject suffering from a Otubain-mediated associated disorder. Further, such a composition can include one or more other compounds that, alone or in combination with the agent modulates Otubain-mediated activity, provides a therapeutic advantage to the subject, for example, an antibiotic if the subject is susceptible to a bacterial infection, one or more additional antiviral agents known to be useful for treating the particular disease or disorder, a nutrient or vitamin or the like, a diagnostic reagent, toxin, a therapeutic agent such as a cancer chemotherapeutic agent, or any other compound as desired, provided the additional compound(s) does not adversely affect the activity of the agent that modulates Otubain-mediated activity or, if the compound does affect the activity of the agent, does so in a manner that is predictable and can be accounted for in formulating the agent.

[0072] Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of the agent. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the physico-chemical characteristics of the agent that alters protein-protein interactions that affect hearing and on the route of administration of the composition, which can be, for example, orally or parenterally such as intravenously, and by injection, intubation, or other such method known in the art.

[0073] An agent that modulates Otubain-mediated activity can be incorporated within an encapsulating material such as into an oil-in-water emulsion, a microemulsion, micelle, mixed micelle, liposome, microsphere or other polymer matrix (see, for example, Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, FL 1984); Fraley et al., Trends Biochem. Sci. 6:77, 1981, each of which is incorporated herein by reference). Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. "Stealth" liposomes (see, for example, U.S. Patent Nos. 5,882,679; 5,395,619; and 5,225,212, each of which is incorporated herein by reference) are an example of such encapsulating materials particularly useful for preparing a composition useful for practicing a method of the invention, and other "masked" liposomes similarly can be used, such liposomes extending the time that the therapeutic agent remain in the circulation. Cationic liposomes, for example, also can be modified with specific receptors or ligands (Morishita et al., J. CHn. Invest. 91:2580-2585, 1993, which is incorporated herein by reference). In addition, a polynucleotide agent can be introduced into a cell using, for example, adenovirus- polylysine DNA complexes (see, for example, Michael et al., J Biol. Chem. 268:6866-6869, 1993, which is incorporated herein by reference).

[0074] The route of administration of a pharmaceutical composition containing an agent that modulates Otubain-mediated activity as discussed herein will depend, in part, on the chemical structure of the molecule. Polypeptides and polynucleotides, for example, are not particularly useful when administered orally because they can be degraded in the digestive tract. However, methods for chemically modifying polypeptides, for example, to render them less susceptible to degradation by endogenous proteases or more absorbable through the alimentary tract are well known (see, for example, Blondelle et al., supra, 1995; Ecker and Crook, supra , 1995). In addition, a peptide agent can be prepared using D-amino acids, or can contain one or more domains based on peptidomimetics, which are organic molecules that mimic the structure of peptide domain; or based on a peptoid such as a vinylogous peptoid.

[0075] A pharmaceutical composition as disclosed herein can be administered to an individual by various routes including, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intrarectally, intracisternally or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively. Furthermore, the pharmaceutical composition can be administered by injection, intubation, orally or topically, the latter of which can be passive, for example, by direct application of an ointment, or active, for example, using a nasal spray or inhalant, in which case one component of the composition is an appropriate propellant. A pharmaceutical composition also can be administered to the site of a pathologic condition, for example, intravenously or intra-arterially into a blood vessel supplying a tissue or organ comprising retrovirus infected cells.

[0076] The pharmaceutical composition also can be formulated for oral formulation, such as a tablet, or a solution or suspension form; or can comprise an admixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications, and can be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, or other form suitable for use. The carriers, in addition to those disclosed above, can include glucose, lactose, mannose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form. In addition auxiliary, stabilizing, thickening or coloring agents and perfumes can be used, for example a stabilizing dry agent such as triulose (see, for example, U.S. Patent No. 5,314,695, incorporated herein by reference).

[0077] The total amount of an agent that modulates Otubain-mediated activity to be administered in practicing a method of the invention can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a prolonged period of time. An advantage of using a fractionated method is that, upon normal division of a retrovirus infected cell, replication of the retrovirus can be reduced or inhibited due to the presence of the agent. One skilled in the art would know that the amount of the composition to treat a retrovirus infection in a subject depends on many factors including the age and general health of the subject as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan would adjust the particular dose as necessary. In general, the formulation of the pharmaceutical composition and the routes and frequency of administration for treatment of human subjects are determined, initially, using Phase I and Phase II clinical trials.

[0078] The following examples are intended to illustrate but not limit the invention.

EXAMPLE 1 EXPERIMENTAL PROCEDURES

[0079] Plasmid construction- DNAs encoding full length human otubain 1 and Cezanne were cloned by PCR from a human fetus cDNA library (Clontech). DNA encoding full- length putative otubain from Caenorhabditis elegans (ceOtu) was cloned from a cDNA library (Invitrogen). Dynazme (MJ Research) was used as the DNA polymerase in PCR. DNAs encoding full-length human otubain 1, human otubain 1 with an N-terminal 41 -residue truncation, and a full length ceOtu were subcloned respectively into a pProEx-c vector (Life Technologies) through Ncol (5') and BamHI (3') sites. DNA encoding the catalytic domain of human Cezanne (amino acids 125-455) was subcloned into pProEx-c using Sfol (5') and Notl (3') sites.

[0080] Protein expression and purification- For expression of human otubainl (hOtul) and the human Cezanne catalytic domain 125-455 aa (hCezcat), plasmids pProEx-hOtul and pProEx-hCezcat were transformed into Rosetta E. coli cells (Novagen). The transformed cells were grown overnight at 37°C in 10 ml LB medium containing 100 μg/ml ampicillin and 33 μg/ml chloramphenicol. The overnight culture was diluted 100-fold into LB containing 100 μg/ml ampicillin and 33 μg/ml chloramphenicol, and the cultures were grown at 37°C to an OD_όoo_nm of 1 -0. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was then added to a final concentration of 1.0 niM to induce protein expression and the temperature was reduced to 15⁰C; after overnight growth, the cells were harvested by centrifugation. The cell pellet was resuspended in 20 mM Tris pH 7.8, 4 mM MgCl₂, 60 μg/ml DNAse I, 500 mM NaCl, 5 mM immidazole, 5% glycerol, 1 mM Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), and protease inhibitor cocktail (COMPLETE™ without EDTA; Roche), and lysed using a microfluidizer. The lysate was clarified by centrifugation at 12000 rpm in a GSA rotor (Beckman) and applied to a Talon column (Clontech), which retained the His₆-tagged hOtul or His₆-tagged hCezcat. The column was washed with 20 mM Tris pH 7.8, 0.5 M NaCl, 10 mM (for His₆-h0tul) or 20 mM imidazole, 1 mM TCEP, and 5% glycerol (for His₆- hCezcat). His₆-tagged hOtul was eluted with 150 mM imidazole; His₆-tagged hCezcat was eluted with 300 mM imidazole. After cleavage of the His₆ tag with HiS₆-TEV protease, the protein solutions were dialyzed against 20 mM Tris, pH 7.8, 100 mM NaCl, and 1 mM TCEP. The His₆ tag and HiS₆-TEV were removed by applying the protein solutions to a Talon column. The flowthrough was applied to a Hiload 26/60 Superdex 75 gel filtration column eluted with buffer 50 mM Tris-HCl (pH 7.8), 200 mM NaCl, 1 mM TCEP, and 1 mM EDTA. Peak fractions were combined, concentrated to 10 mg/ml and stored in small aliquots at -8O⁰C. As estimated by SDS-PAGE and Coomassie blue staining, purity of the hOtul and hCezcat were 99% and 95%, respectively.

[0081] Human otubainl with an N-terminal 41-residue truncation (hOtulΔN41) and C. elegans full-length putative otubain (ceOtu) were expressed and purified using the protocol described above to 99% homogeneity. [0082] Synthesis of KIl -linked Ub₂- The enzyme E2-EPF specifically generates KI l- linked polyubiquitin chains. GST-E2EPF was induced in BL21 (DE3) E. coli cells (Novagen) for 4 h at 37°C with 0.1 niM IPTG. Cells were pelleted, frozen, and resuspended in a buffer containing TDE (20 niM Tris, pH 7.6, 1 mM DTT, and 0.1 mM EDTA), 0.02% NP-40, and 1 mM PMSF. Cells were then lysed with 0.4 mg/ml lysozyme and DNA was digested by adding 10 mM MgCl₂ and 20 μg/ml DNAse I. The soluble extract was applied to glutathione Sepharose resin equilibrated with TDE and 150 mM NaCl and bound in batch for 1 h at 4°C. The beads were put onto a column and washed extensively with the same buffer. The GST- E2EPF was then eluted with the same buffer containing 20 mM reduced glutathione. The protein was further dialyzed into TDE and applied to a 1 ml Mono Q column equilibrated with TDE. GST-E2EPF was eluted with an 80 ml linear gradient from 0-1 M NaCl. Peak fractions were pooled and exchanged into TDE by repeated concentration and dilution.

[0083] For synthesis of Kl 1 -linked Ub₂, GST-E2EPF (10 μM) was incubated with 1 mg/ml of Kl IR mutant Ub, 1 mg/ml C-terminally blocked Ub (D77), and 0.1 μM El in conjugation buffer (50 mM Tris, pH 8, 5 mM MgCl₂, 10 mM creatine phosphate, 0.6 units/ml creatine phosphokinase, 0.6 units/ml pyrophosphatase, 2 mM ATP and 0.5 mM DTT) for 30 min before the reaction was stopped with 5 mM EDTA. GST-E2EPF was removed from the reaction mixture by a glutathione Sepharose column. The flow-through fraction was collected, dialyzed into 50 mM ammonium acetate, pH 4.5, and applied to a Mono S column equilibrated in the same buffer. Kl 1- linked Ub₂ was eluted with a salt gradient at around 0.33 M NaCl.

[0084] Syntheses of^l25I-labeled E2-25K-(K48-linked) Ub₄ and ^mI-labeled Ubcl3-(K63- linked) Ub₄- K48 and K63-linked polyubiquitin tetramers, prepared as described previously (Piotrowski, J., et al, (1997) The Journal of Biological Chemistry, 272(38), 23712-23721; Hofmann, R. M., and Pickart, C. M., (2001) The Journal of Biological Chemistry, 276(30), 27936-27943), were iodinated and conjugated to E2-25K or Ubcl3, respectively, by autoubiquitination as follows. 5 μM ¹²⁵I-labeled K48-Ub₄ was incubated with 15 μM E2-25K and 0.1 μM El for 90 min at 37°C in conjugation buffer (50 mM Tris, pH 8, 5 mM MgCl₂, 10 mM creatine phosphate, 0.6 units/ml creatine phosphokinase, 0.6 units/ml pyrophosphatase, 2 mM ATP and 0.5 mM DTT). The reaction was stopped by adding N-ethyl-maleimide (NEM) to a final concentration of 4 mM and incubated for 5 more minutes. The NEM was then consumed by adding DTT to a final concentration of 5 niM. The proteins were exchanged into HDE buffer (20 mM Hepes, pH 7.3, 1 mM DTT and 0.1 niM EDTA), supplemented with 0.5 mg/ml BSA, and bound to a Q-Sepharose column equilibrated with HDE. E2-25K-(K48- linked) Ub₄ conjugates bind to the column under these conditions, but free, unconjugated K48-Ub₄ chains do not. The Q-Sepharose resin was then washed with HDE containing 20 mM NaCl, and the E2-25K-(K48-linked) Ub₄ conjugates were eluted with HDE containing 30O mM NaCl.

[0085] To generate ¹²⁵I-K63Ub₄-Ubcl 3, 5 μM ¹²⁵I-labeled K63-Ub₄ was incubated with 15 μM Ubcl3/Mms2-His₆ heterodimer and 0.1 μM El for 90 min at 37°C in conjugation buffer. The reaction was stopped as above with 4 mM NEM followed by 5 mM DTT. After this reaction, the bulk (~95%) of the conjugated ¹²⁵I-K63-Ub₄ was in the form of ¹²⁵I- K63Ub₄-Ubcl3 rather than ¹²⁵I-K63Ub₄-Mms2-His₆. To separate the small amount of ¹²⁵I- K63Ub₄-Mms2-His₆ conjugate, the mixture was diluted ten-fold into Ni -NTA binding buffer (0.1 % NP40, 10 mM imidazole, 10 mM Tris, pH 8.0, 300 mM NaCl and 0.1 mg/ml BSA) and incubated for 60 min with Ni²⁺-NTA resin equilbrated in the same buffer. The flow through fraction was collected, which specifically contained the ^I25I-K63Ub₄-Ubcl3 conjugates. These were exchanged into HDE and purified from the free, unconjugated ^l I- K63Ub₄ using a Q-Sepharose column as described for ⁵I-K48Ub₄-E2-25K above.

[0086] Deubiquitination assays- Deubiquitin substrates K48-Ub₂ (proximal UbD77) (Chen, Z., and Pickart, C. M. (1990) The Journal of Biological Chemistry, 265(35), 21835- 21842), K63-Ub₂ (proximal UbD77) (Hofmann, R. M., and Pickar, C. M. (1999) Cell, 96(5), 645-653), a mixture of K29-Ub₂ and K6-Ub₂ (You, J., and Pickar, C. M. (2001) The Journal of Biological Chemistry 276(23), 19871-19878), linear Ub₅ (Jonnalagadda, S., et al., (1987) The Journal of Biological Chemistry 262(36), 17750-17756), and distal-end Lucifer Yellow- labeled (LY) K48-Ub₄ (UbC48LY-Ub3) were prepared as described (Yao, T., and Cohen, R. E. (2002) Nature, 419(6905), 403-407). Two mixed-linkage tetraubiquitin chains, K48-K63- K48 Ub₄ (catalog #UCM310) and K63-K48-K63 Ub₄ (catalog #UCM21O), were from Boston Biochem.

[0087] DUB assays were performed at 37°C. Specified amounts of substrate were incubated in a 10 μl volume with hOtul, hOtulΔN41, ceOtu, or hCezcat in 20 mM Tris pH 7.5 (at 37°C), 50 mM NaCl, and 5 mM DTT; 0.05% BSA was included as a carrier protein in some reactions. The reactions were quenched with SDS sample buffer and applied to a 4- 12% acrylamide gel for SDS-PAGE. Fluorescent LY-tagged substrates and ¹²⁵I-labeled substrates were detected by a phosphorimager (Typhoon 4001, Molecular Dynamics). Otherwise, protein bands were detected by silver staining or Coomassie Brilliant blue staining.

[0088] Competition assays- I-radioiodinated K48-Ub₂ (7 μM with specific activity of 1.6xlO⁴ cpm/reaction) was incubated with 0.16 μM hOtul in 50 Mm Tris, pH 7.5, 5 mM DTT, and 0.05% BSA at 25°C. Unlabeled K48-Ub₂, K63-Ub₂, (K29+K6)-Ub₂, or Ub was added at molar concentrations 1, 5, and 10-fold above the amount of ¹²⁵I-labeled substrate. The reactions were quenched with SDS sample buffer and applied to 4-12% gels for SDS-PAGE. The gels were dried and the ¹²⁵I-labeled Ub and Ub₂ band intensities were quantified using a phosphorimager.

EXAMPLE 2 OTUBAIN 1 CLEAVES K48-LINKED POLYUBIQUITIN

[0089] This example illustrates that Otubain 1 specifically cleaves K48-linked polyubiquitin. Human otubain 1 has been reported to cleave tetraubiquitin but not Leu-Arg- Gly-Gly-7-amido-4-methylcoumarin (LRGG-AMC) (SEQ ID NO: 8) or ubiquitin fused to Green Fluorescent Protein (GFP). One of the yeast otubain proteins, Otul, cleaves Ub-AMC and K48-linked polyubiquitin chains but not K63-linked chains. The possibility that Human otubain 1 may be a highly selective isopeptidase was further investigated. Human otubain 1 was cloned from a cDNA library, and the recombinant human protein (hOtul) was expressed in E. coli and purified for biochemical studies. The ability of hOtul to cleave forms of diubiquitin (Ub₂) that differ in their isopeptide linkages was examined. Among the five different Ub₂ substrates tested, only K48-Ub₂ was processed significantly. As shown in Figure 2A, hOtul readily cleaved K48-Ub₂, but negligible K63- Ub₂ and no K29/K6- Ub₂ were hydrolyzed. The same results were obtained when 10-fold more hOtul was used, or if the incubation times were extended to 20 h. It was also found that hOtul could not cleave Kl 1-Ub₂ (Figure 2B).

[0090] To determine if otubain 1 could selectively cut K48-Ub-Ub linkages in the context of polyubiquitin chains with mixed linkages, tetraubiquitin chains having either K48-K63- K48 or K63-K48-K63 isopeptide linkages were incubated with hOtul (Figure 2C). In each case, the products obtained were consistent with cleavage at the K48 linkage(s) only; processing of K63-K48-K63 tetraubiquitin yielded only diubiquitin, whereas K48-K63-K48 tetraubiquitin was cleaved into triubiquitin, diubiquitin, and ubiquitin monomers. The K48- K63-K48 tetraubiquitin substrate contained an additional and unexpected faster-migrating band (asterisk in Figure 2C). This species is a cyclized side-product of the K48-K63-K48 tetraubiquitin synthesis that, analogous to cyclic K48-linked chains, can form in vitro. hOtul was able to cleave both the linear and cyclic forms ofK48-K63-K48 tetraubiquitin.

[0091] The deubiquitination of polyubiquitin-protein conjugates was also investigated. The activity of hOtul against E2-25K-(K48-linked) Ub₄ and Ubcl 3 -(K63 -linked) Ub₄ was determined. Using a strategy similar to that of Liu et al. ((2006) Molecular Cell, 24(1), 39- 50), E2-25K-(K48-linked) Ub₄ was synthesized in which a 25 kDa ubiquitin conjugating (E2) enzyme was self-ubiquitinated with a preformed K48-Ub₄ chain; similarly, Ubcl3-(K63- linked) Ub₄ was made by self ubiquitination of Ubcl3 with K63-linked Ub₄. As shown in Figure 2D₃ hOtul cleaved E2-25K-(K48-linked) Ub₄ but not Ubcl3-(K63-linked) Ub₄. In the case of E2-25K-(K48-linked) Ub4, there was cleavage at each Ub-Ub bond, with Ub₃, Ub₂ and Ub produced. In addition, a protein band at ~34 kDa was generated that corresponds to the size of either free Ub₄ or monoUb-E2-25K. Even with prolonged incubation, this species persisted longer than Ub₃, which suggests that at least some and perhaps all of it was monoUb-E2-25K. The existence of monoUb-E2-25K was confirmed by applying the product mixture to a Q-Sepharose column. The -34 kDa protein bound and eluted from the column, indicating that it must be monoUb-E2-25K. With short incubations (i.e., 0.5 - 10 min; Figure 2E), the Ub₄/monoUb-E2-25K band was much weaker than the Ub₃ band, which indicates that cleavage at the proximal Ub to release Ub₄ from the E2-25K is much slower than cleavage at Ub-Ub linkages. Notably, hOtul may not cleave as efficiently between the second and third ubiquitins of E2-25K-(K48-linked) Ub₄. This is because that isopeptide bond is not to lysine but is instead to the side chain of aminoethylcysteine, which was introduced to make the polyubiquitin chains with defined lengths (Evans, P. C, et al., (2003) The Journal of Biological Chemistry, 278(25), 23180-23186). EXAMPLE 3

OTUBAIN 1 CLEAVES K48-LINKED POLYUBIQUITIN AT BOTH THE DISTAL

AND PROXIMAL ENDS

[0092] This example illustrates that cleavage of K48-linked polyubiquitin by otubain 1 is not limited to either end of the chain. Some DUBs are known to release Ub specifically from distal ends of free or protein conjugated polyubiquitin chains. Other DUBs reverse Ub conjugation by cleaving the isopeptide bond that links to proximal Ub of polyubiquitin to a protein or disassemble free chains by releasing monomers from the proximal end. Experiments were performed to determine if otubain 1 cleavage of K48-linked polyubiquitin proceeds from the distal or proximal end. The substrate used was K48-linked Ub₄ labeled with a Lucifer Yellow fluorophore on the distal Ub (UbC48^LY-Ub₃). When hOtul was incubated with UbC48 -Ub₃ at 37°C from 0 to 32 min, fluorescently-labeled products UbC48^LY-Ub₂, UbC48^LY-Ub, and UbC48^LY appeared simultaneously (Figure 3). In contrast, when isopeptidase T was used to cleave the same substrate, mostly UbC48 -Ub₂ was generated first; UbC48^LY-Ub and UbC48^LY appeared only as the reaction progressed further. This indicates that, unlike isopeptidase T, which is a proximal-end specific isopeptidase, otubain 1 is not specific for either end of a polyubiquitin chain. Otubain 1 recognizes and cleaves the isopeptide bond wherever there is a K48 linkage between two ubiquitin molecules.

EXAMPLE 4

OTUBAIN 1 SPECIFICITY IS NOT DUE TO LINKAGE-SPECIFIC BINDING TO

POLYUBIQUITIN SUBSTRATES

[0093] This example illustrates that the specificity of otubain 1 is not due to linkage- specific binding to polyubiquitin substrates. There was a possibility that the strong preference by hOtul for cleaving within K48-linked polyubiquitin is due to specific binding of ubiquitin joined by this linkage type. To address this, possible inhibition of hOtul by K48- Ub2, K63-Ub2, K29/K6-Ub2, or monoubiquitin was examined in assays with radioiodinated K48-linked Ub2 as the substrate (Figure 4). Surprisingly, monoubiquitin and all of the Ub2 species inhibited the reaction; moreover, all inhibited with approximately equal potency. These results indicate that the binding of hOtul to substrate is not specific for a particular type of Ub-Ub isopeptide linkage. Therefore, the cleavage specificity of hOtul is not determined simply by selective binding of K48-linked Ub-Ub, but appears instead to involve a subsequent catalytic step.

EXAMPLE 5 OTU DOMAIN LINKAGE SPECIFICITY

[0094] This example illustrates the linkage specificity of other OTU domain proteins. The cleavage specificity of hOtul was compared to that of another human OTU class deubiquitinating enzyme, Cezanne. Initially identified as a protein that contains a TRAF binding domain and negatively regulates NF-κB, Cezanne contains an N-terminal OTU domain and has been reported to cleave ubiquitin monomers from both α-amine linked (linear) and branched polyubiquitin chains. In contrast with hOtul, the catalytic domain of human Cezanne (hCezcat, 125-455 aa) cleaves both K48-and K63-linked Ub₂ (Figure 5A), but barely any K29/K6-Ub₂. In addition, hCezcat also cleaves Kl 1-Ub₂ (Figure 2B). The catalytic domain of Cezanne therefore exhibits far less linkage specificity than otubain 1. To explore whether residues outside of the catalytic domain contributes to the greater specificity observed for hOtul, human otubain 1 lacking the N-terminal 41 residues, hOtulΔN41, as well as a putative full-length otubain from C. elegans (ceOtu), was expressed and purified (see Figure IB). The results indicate that ceOtu and hOtulΔN41 share the same specificity for K48-linked polyubiquitin as hOtul, although the full-length proteins hOtul and ceOtu appear to cleave more efficiently than the truncated hOtulΔN41 (Figure 5B).

EXAMPLE 6 OTU DOMAIN PROTEINS DO NOT CLEAVE α-LINKED PEPTIDE BONDS

[0095] This example illustrates that OTU domain proteins/fragments do not cleave α- linked peptide bonds. There have been conflicting reports on whether OTU domain proteins cleave α-linked peptide bonds. Otubain 1 has been reported to cleave Ub-AMC but not LRGG-AMC (SEQ ID NO: 8). The activity of OTU domain proteins against penta-ubiquitin connected by α-amine-linkages between the Ub C-termini and the N-termini of each succeeding Ub {i.e., linear Ub₅) was examined. When incubated with hOtul, hCezcat, ceOtu, or hOtulΔN41, the linear Ub₅ was not cleaved (Figure 6). In contrast, linear Ub₅ was cleaved, albeit slowly, by isopeptidase T, which was included as a positive control in the assay (Figure 6).

[0096] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention.

Claims

What is claimed is:

1. A method for treating an Otubain-mediated disorder comprising administering to a subject in need thereof an agent that modulates Otubain-mediated cleavage of lysine-48- linked polyubiquitin, thereby treating an Otubain-mediated disorder.

2. The method of claim I₅ wherein the disorder is a cellular proliferative disorder, T-cell anergy, an autoimmune disease, immunosuppression, or Parkinson's Disease.

3. The method of claim 2, wherein the cellular proliferative disorder is cancer.

4. The method of claim 2, wherein the autoimmune disease is acute disseminated encephalomyelitis, Addison's Disease, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, Bullous Pemphigoid, Celiac Disease, Chagas Disease, chronic obstructive pulmonary disease, dermatomyositis, diabetes mellitus type 1, endometriosis, Goodpasture's Syndrome, Graves' Disease, Guillain-Barre Syndrome, Hashimoto's Disease, Hidradenitis Suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, Lupus erythematosus, morphea, multiple sclerosis, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, pernicious anemia, polymyositis, primary biliary cirrhosis, ulcerative colitis, rheumatoid arthritis, schizophrenia, Sjogren's syndrome, temporal arteritis, vasculitis, vitiligo, or Wegener's granulomatosis.

5. The method of claim 2, wherein the immunosuppression is due to Human Immunodeficiency Virus infection.

6. The method of claim 1 , wherein the agent is lysine-48-linked polyubiquitin, lysine-63 -linked polyubiquitin, lysine-29/lysine-6-linked polyubiquitin, or monoubiquitin.

7. A method for monitoring a therapeutic regimen for treating a subject having a Otubain-mediated disorder, comprising determining a change in the Otubain-mediated cleavage of lysine-48-linked polyubiquitin during therapy.

8. A method for detecting ubiquitinated proteins, comprising:

a) contacting a sample comprising a protein with detectably-labeled lysine-48-linkcd polyubiquitin and Otubain; and

b) detecting Otubain-mediated cleavage of detectably-labeled lysine-48 -linked polyubiquitin.

9. The method of claim 8, wherein the protein comprises a cellular protein.

10. The method of claim 9 wherein the cellular protein comprises a disease marker.

1 1. The method of claim 8, wherein the lysine-48-linked polyubiquitin comprises linear lysine-48-linked polyubiquitin.

12. The method of claim 8, wherein the lysine-48-linked polyubiquitin comprises cyclic lysine-48-linked polyubiquitin.

13. The method of claim 8, which is performed in a high throughput format.

14. The method of claim 8, wherein the sample comprises a cell sample.

15. The method of claim 14, wherein the cell sample is obtained from a subject.

16. The method of claim 8, wherein the sample comprises a cell free sample.

17. A method of diagnosing an Otubain-mediated disorder in a subject, comprising comparing the Otubain-mediated activity in a test sample from the subject with the Otubain-mediated activity in a corresponding normal sample, wherein a difference in Otubain-mediated activity in the test sample as compared to the Otubain-mediated activity in the normal sample is diagnostic of a Otubain-mediated disorder in the subject.

18. The method of claim 17, further comprising contacting the test sample and a corresponding normal sample with an antibody that specifically binds Otubain; thereafter comparing the Otubain-mediated activity in the test sample and the Otubain-mediated activity in the normal sample; and detecting a greater decrease in Otubain-mediated activity in the test sample as compared to the Otubain-mediated activity in the normal sample.

19. The method of claim 17, wherein the Otubain comprises a mammalian Otubain.

20. The method of claim 19, wherein the mammalian Otubain is human Otubain.

21. The method of claim 17, which is performed in a high throughput format.

22. A method of modulating protein stability, comprising contacting lysine-48- linked polyubiquitin peptide with an agent that cleaves lysine-48 -linked polyubiquitin peptide bonds, thereby modulating protein stability.

23. The method of claim 22, which comprises reducing or inhibiting lysine-48- linked polyubiquitin peptide cleavage.

24. The method of claim 22, wherein the agent comprises Otubain.

25. The method of claim 24, wherein the agent comprises a mammalian Otubain.

26. The method of claim 24, wherein the agent comprises a human Otubain.

27. The method of claim 23, wherein the agent comprises a functional fragment of Otubain.

28. The method of claim 27, wherein the agent comprises Otubain with a deletion of 41 amino-terminal amino acids.

29. The method of claim 22, which comprises administering the agent to a subject having a disorder associated with lysine-48-linked polyubiquitin peptide cleavage.

30. The method of claim 29, wherein the disorder comprises a cellular proliferative disorder.

31. The method of claim 22, wherein the lysine-48-linked polyubiquitin comprises free lysine-48-linked polyubiquitin.

32. The method of claim 22, wherein the lysine-48-linked polyubiquitin comprises substrate-linked lysine-48-linked polyubiquitin.

33. The method of claim 32, wherein the substrate-linked lysine-48-linked polyubiquitin comprises a cellular protein.

34. The method of claim 33, wherein the cellular protein comprises a disease marker.

35. The method of claim 22, wherein the lysine-48-linked polyubiquitin comprises linear lysine-48-linked polyubiquitin.

36. The method of claim 22, wherein the lysine-48-linked polyubiquitin comprises cyclic lysine-48-linked polyubiquitin.

37. The method of claim 22, wherein the agent is lysine-48-linked polyubiquitin, lysine-63 -linked polyubiquitin, lysine-29/lysine-6-linked polyubiquitin, or monoubiquitin.

38. A method of identifying an agent that modulates Otubain-mediated cleavage of lysine-48-linked polyubiquitin peptide bonds, comprising:

a) contacting a sample comprising lysine-48-linked polyubiquitin and Otubain with a test agent under conditions sufficient for Otubain-mediated cleavage of lysine-48-linked polyubiquitin peptide bonds; and

b) detecting a change in Otubain-mediated cleavage of lysine-48-linked polyubiquitin peptide bonds in the presence of the test agent as compared to the activity in the absence of the test agent;

wherein a change in Otubain-mediated cleavage of lysine-48-linked polyubiquitin peptide bonds, identifies the test agent as an agent that modulates Otubain-mediated cleavage of lysine-48-linked polyubiquitin peptide bonds.

39. The method of claim 38, wherein the lysine-48-linked polyubiquitin comprises free lysine-48-linked polyubiquitin.

40. The method of claim 38, wherein the lysine-48-linked polyubiquitin comprises substrate-linked lysine-48-linked polyubiquitin.

41. The method of claim 39, wherein the substrate-linked lysine-48-linked polyubiquitin comprises a cellular protein.

42. The method of claim 41 , wherein the cellular protein comprises a disease marker.

43. The method of claim 38, wherein the lysine-48-linked polyubiquitin comprises linear lysine-48-linked polyubiquitin.

44. The method of claim 38, wherein the lysine-48-linked polyubiquitin comprises cyclic lysine-48-linked polyubiquitin.

45. The method of claim 38, wherein the lysine-48-linked polyubiquitin peptide bonds is cleaved at the proximal lysine-48-linked polyubiquitin bond and at the distal lysine- 48-linked polyubiquitin bond.

46. The method of claim 38, wherein the Otubain comprises a mammalian Otubain.

47. The method of claim 46, wherein the mammalian Otubain is human Otubain.

48. The method of claim 38, wherein the lysine-48-linked polyubiquitin is detectably labeled.

49. The method of claim 38, wherein the agent inhibits Otubain-mediated activity.

50. The method of claim 38, wherein the agent stimulates Otubain-mediated activity.