Modulation of COP9 signalsome isopeptidase activity

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates generally to the protein degradation pathways, and more specifically to compositions and methods for modulating the isopeptidase activity of the COP9 signalsome and, therefore, ubiquitin dependent protein degradation.

[0004] 2. Background Information

[0005] The function of a protein is regulated via various means in a cell. One way to regulate protein function is via conjugation and deconjugation of a modifier protein to a target protein, e.g., neddylation and deneddylation or ubiquitination and deubiquitination.

[0006] The major route for protein degradation in the nucleus and cytoplasm of eukaryotic cells is via the ubiquitin/26S proteasome pathway. The 26S proteasome comprises two major subparticles: the 20S proteasome and the 19S regulatory particle. The 20S proteasome is a cylindrical structure with an internal cavity that contains the peptidase active sites. Substrates of the proteasome are inserted into the cylinder, where they are susceptible to digestion by the peptidase active sites of the 20S proteasome. Entry into the 20S proteasome cylinder is governed by the 19S regulatory particle, which caps the ends of the 20S cylinder.

[0007] The 19S regulatory particle binds ubiquitinated substrates and translocates them into the inner cavity of the 20S cylinder, where they are degraded. The 19S regulatory particle can be further subdivided into two multiprotein complexes: the base and the lid. The base comprises a set of six ATPases that are thought to unfold substrates and translocate them into the 20S proteasome. The lid is comprised of a set of eight proteins of unknown function. Biochemical data indicate that the presence of the lid renders the proteasome selective for degrading ubiquitinated proteins, but the basis for this selectivity is not known.

[0008] The lid subcomplex of the 26S proteasome is evolutionarily related to the COP9-signalsome (CSN) complex, but the significance of this similarity has remained unknown. There are reports that 26S proteasome preparations contain a variety of associated ubiquitin isopeptidase activities (Eytan et al., J. Biol. Chem. 268:4668-74, 1993; Verma et al., Mol. Biol. Cell 11:3425, 2000), but it has not been demonstrated that an ubiquitinated substrate can be completely deubiquitinated by purified 26S proteasome to yield unmodified substrate. The failure to detect such a reaction product may be due to a tight coupling between the deubiquitination of a substrate and its subsequent degradation within the internal cavity of the 20S proteasome.

[0009] Proteins that are destined for degradation by the ubiquitin/26S pathway are marked by the attachment of a polyubiquitin chain to the side chains of lysine residues on the target protein. The ubiquitinated protein is recognized by the 26S proteasome by a mechanism that remains poorly understood. Subsequently the ubiquitinated protein is disengaged from any tightly bound partners, unfolded, and translocated into the central cavity of the 20S complex, where it is exhaustively degraded by the proteolytic active sites that are present in this inner cavity.

[0010] Despite many years of intensive study of this system, it remains unclear what happens to the substrate-bound polyubiquitin chains that target the substrate for degradation. It appears that ubiquitin is not degraded by the proteasome but, in fact, is recycled. However it remains unclear if the ubiquitin chains enter the inner cavity of the proteasome and emerge unscathed, or are cleaved from the substrate protein prior to or during its translocation into the inner cavity of the 20S. In prior work (Eytan et al., supra, 1993), it was demonstrated that there is an isopeptidase activity or activities associated with the intact 26S proteasome that is able to release free ubiquitin monomers from ubiquitinated substrates that are degraded by the 26S proteasome. It was demonstrated that this activity is sensitive to the metal ion chelator 1,10-phenanthroline, but the identity of a polypeptide that harbors this activity was not established, nor was it established that this activity is intrinsic to the 26S proteasome as opposed to being intrinsic to a protein that binds transiently to the 26S proteasome. This prior work also failed to disclose whether the ubiquitin isopeptidase activity is critical to the protein-degrading function of the 26S proteasome.

[0011] Nedd8 is an ubiquitin-like protein that is covalently linked via an isopeptide bond between its carboxy terminus and the epsilon amino group of lysine residues in target proteins (referred to as neddylation). The attachment of Nedd8 to target proteins requires the combined action of a Nedd8-activating enzyme composed of Ula1 and Uba3 subunits (analogous to ubiquitin-activating enzyme, E1), and Ubc12, which is homologous to the ubiquitin-conjugating enzymes (E2). There is no known requirement for an activity equivalent to the ubiquitin ligase (E3) component of ubiquitination pathways. The Nedd8 modification, like ubiquitination, is likely dynamic, although little is known about the nature of the enzymes that would cleave Nedd8 from its targets (i.e., deneddylate, also referred to as deneddylation or deneddylating activity).

[0012] A ubiquitin isopeptidase (USP21) can deneddylate Cul1-Nedd8 conjugates, and a second enzyme UCH-L3, can cleave Nedd8-containing fusion proteins at the C-terminus of Nedd8 to release mature Nedd8; Nedd8, like ubiquitin, is made as a precursor with additional C-terminal residues that must be removed before it can be conjugated to proteins. Despite the fact that both USP2 1 and UCH-L3 can metabolize Nedd8-based substrates, they also act on ubiquitin-based substrates, and it remains unclear whether their biochemical activity towards Nedd8 is relevant in the context of a cell.

[0013] In contrast to ubiquitin, the attachment of Nedd8 to proteins does not mark them for degradation. Rather, it appears that neddylation acts to modify protein function, much like phosphorylation. The cullins are the only proteins that have been found to be conjugated with Nedd8. The cullins are a family of six related proteins that bind a RING-H2 domain protein to form the catalytic core of multisubunit ubiquitin ligases. All cullins examined are modified by attachment of Nedd8, and neddylation of Cul1 potentiates the ubiquitin ligase of the SCF complex within which Cul1 resides. Thus, for Cul1-based ubiquitin ligases, neddylation serves as a positive regulator of activity. However, for other cullin-based ubiquitin ligases, the role, if any, of neddylation remains uncertain.

[0014] The COP9/signalsome (“CSN”) was originally identified as a regulator of photomorphogenetic development in plants. In seedlings grown in the dark, CSN enables a putative ubiquitin ligase, COP1, to mediate rapid turnover of the transcriptional regulatory protein HY5 in the nucleus. In seedlings that have been exposed to light or in CSN mutants, COP1 redistributes from the nucleus to the cytoplasm, thereby stabilizing HY5 and allowing it to accumulate in the nucleus. HY5, in turn, activates the transcription of a broad palette of genes that mediate photomorphogenetic development. Although the general physiological role of CSN in photomorphogenesis has been defined, little is known about other potential physiological functions for CSN, and the biochemical function of CSN remains elusive. Based on the observation that the eight subunits of CSN share homology to subunits of the lid subcomplex of the 19S regulator of the 26S proteasome, it has been suggested that CSN may play a role in ubiquitin-dependent proteolysis. However, a similar pattern of homology is shared with subunits of the eukaryotic initiation factor-3 (eIF3) complex. Besides plants, CSN complexes have been discovered in human cells, Drosophila, and the fission yeast Schizosaccharomyces pombe. Surprisingly, the budding yeast Saccharomyces cerevisiae does not contain an apparent CSN complex, although it contains a gene homologous to the CSN5/JAB1 subunit of CSN complex, thus implicating CSN5 as a critical component of CSN.

[0015] Given the role of protein degradation in maintaining normal cell function, and the disruption of this pathway in association with various disorders such as cancer, an understanding of the components involved in regulating ubiquitination and protein degradation would allow the development of drugs that can modify protein degradation. Thus, a need exists to identify the active domain and site of peptidase activity, particularly isopeptidase activity of a protein involved in deconjugation of a modifier protein from a target protein, e.g., deneddylation or deubiquitination such that screening assays can be developed to identify drugs that can selectively modulate protein modification and degradation. The present invention satisfies this need and provides additional advantages.

SUMMARY OF THE INVENTION

[0016] The present invention is based on a determination that a mutation affecting the isopeptidase activity of a COP9 signal some (CSN) synthetically enhances a mutation affecting the activity of an SCF ubiquitin ligase. Accordingly, the present invention relates to an isolated polypeptide comprising a mutant of a JAMM domain, which has an amino acid sequence of HXHXXXXXXXXXXD (SEQ ID NO: 1), wherein H is histidine, D is aspartate, and X is any amino acid, and wherein, in the mutant, at least one of the H residues is substituted with any amino acid residue other than H, the D residue is substituted with an amino acid residue other than D, or a combination thereof. Such a polypeptide comprising the mutant JAMM domain, or a protein comprising such a polypeptide, can have increased isopeptidase activity or can have reduced (or no) isopeptidase activity as compared to a polypeptide comprising the normal (i.e., unmutated) JAMM domain.

[0017] The JAMM domain can be a JAMM domain of any polypeptide, particularly a polypeptide of a CSN complex, for example, a JAMM domain generally represented by an amino acid sequence of GW(Y/I)H(S/T)HPXXXXXXSXXD (SEQ ID NO. 2), wherein G is glycine, W is tryptophan, Y is tyrosine, I is isoleucine, H is histidine, S is serine, T is threonine, P is proline, D is aspartate, X is any amino acid, Y/I is either Y or I, and S/T is either S or T. Such JAMM domains are exemplified by

[0018] VGRLENAIGWYHSHPGYGCWLSGIDVSTQ (SEQ ID NO: 5),

[0019] VGRMEHAVGWYHSHPGYGCWLSGIDVSTQ (SEQ ID NO: 6),

[0020] AGRLENVVGWYHSHPGYGCWLSGIDVSTQ (SEQ ID NO: 7),

[0021] EGRKEKVVGWYHSHPGYGCWLSGIDVSTQ (SEQ ID NO: 8), or

[0022] KGAKLNVVGWFHSHPGYDCWLSNIDIQTQ (SEQ ID NO: 9), and mutant JAMM domains are represented by KGAKLNVVGWFASHPGYDCWLSNIDIQTQ (SEQ ID NO: 10), KGAKLNVVGWFHSAPGYDCWLSNIDIQTQ (SEQ ID NO: 1), or

[0023] KGAKLNVVGWFHSHPGYACWLSNINIQTQ (SEQ ID NO: 12).

[0024] The present invention also relates to an isolated polypeptide comprising a mutant JAMM domain, which has an amino acid sequence of H(S/T)HXXXXXXXSXXD (SEQ ID NO: 65), wherein S is serine, (S/T) is serine or threonine, and X is any amino acid, and wherein, in the mutant JAMM domain, at least one of the H residues is substituted with an amino acid residue other than H; the D residue is substituted with an amino acid residue other than D; the S residue is substituted with an amino acid residue other than S; the T residue, when present, is substituted with an amino acid residue other than T, or a combination thereof. Such a polypeptide comprising the mutant JAMM domain can have reduced (or no) isopeptidase activity as compared to a polypeptide comprising the normal (i.e., not mutated) JAMM domain, or can have increased isopeptidase activity as compared to a polypeptide comprising the normal JAMM domain.

[0025] The present invention also relates to an isolated JAMM domain consisting essentially of a JAMM domain having an amino acid sequence of EXHyXHyHy(X)nHXHXXXXXXXXXXD (SEQ ID NO: 13), wherein E is glutamic acid, Hy is a hydrophobic amino acid residue, X is any amino acid, and (X)n indicates about 40 to 83 amino acid residues. Also provided is a fusion protein, comprising such an isolated JAMM domain (SEQ ID NO: 13) operatively linked to a heterologous polypeptide, and an isolated polynucleotide encoding such a JAMM domain. In addition, the present invention relates to an isolated polypeptide comprising a mutant JAMM domain, which has an amino acid sequence of EXHyXHyHy(X)nHXHXXXXXXXXXXD (SEQ ID NO: 13), wherein, in the mutant JAMM domain, at least one of the H residues is substituted with an amino acid residue other than H; the D residue is substituted with an amino acid residue other than D; the E residue is substituted with an amino acid residue other than E; or a combination thereof. The JAMM domain of such a polypeptide can have, for example, an amino acid sequence of EXHyXHyHy(X)nH(S/T)HXXXXXXXSXXD (SEQ ID NO: 14), wherein the S residue is substituted with an amino acid residue other than S; and/or the T residue, when present, is substituted with an amino acid residue other than T. Examples of polypeptide having such JAMM domains includes those having an amino acid sequence as set forth in any of SEQ ID NOS: 15 to 64.

[0026] The present invention also relates to a polynucleotide encoding a polypeptide comprising a mutant JAMM domain. The polynucleotide can be a deoxyribonucleic acid molecule (DNA), a ribonucleic acid molecule (RNA), or a hybrid thereof, and can be single stranded or double stranded. The polynucleotide can be contained in a vector, for example, an expression vector, and can be operatively linked to one or more expression control elements, including, for example, transcriptional or translational regulatory element. The polynucleotide, which can, but need not, be in a vector, can be exogenously introduced into a cell, which can be a prokaryotic or eukaryotic cell, and can be maintained in the cell transiently or stably, for example, integrated into the cell genome.

[0027] The present invention further relates to a method of identifying an inhibitor of an isopeptidase activity of a polypeptide by rational drug design, wherein the polypeptide comprises a JAMM domain. Such a method can be performed, for example, by designing a potential inhibitor for the polypeptide that will form a bond with the JAMM domain based upon the crystal structure co-ordinates of the polypeptide, synthesizing the potential inhibitor, and detecting inhibition of isopeptidase activity of the polypeptide by the potential inhibitor, thereby identifying the potential inhibitor as an inhibitor of the polypeptide. The isopeptidase activity can be, for example, deneddylation.

[0028] The polypeptide comprising the JAMM domain can be, for example, a component of a CSN, e.g., JAB1, or a component of a 26S proteasome, e.g., RPN11 (also known as POH1). As such, the JAMM domain can be a JAMM domain as exemplified herein (e.g., SEQ ID NOS: 5-9) or otherwise known in the art. A potential inhibitor can be designed based, for example, on a likelihood that it will form a bond with a metal ion bound by the JAMM domain, and can be, for example, a peptide, a peptide derivative, a peptoid, a peptidomimetic, a polynucleotide, a polynucleotide derivative, or a small organic molecule. In addition, a potential inhibitor can be designed based on the ability to form a bond or otherwise interact with one or more amino acid residues involved in binding to the metal ion, for example, with at least one of the two histidine residues or with the aspartate (D) residue as set forth in the HXHXXXXXXXXXXD (SEQ ID NO: 1) sequence of the JAMM domain; or with one or more amino acid residues involved in maintaining the structure of the metal ion coordinating site, for example, with the first serine residue (or the threonine residue, when present) as set forth in the EXHyXHyHy(X)nH(S/T)HXXXXXXXSXXD (SEQ ID NO: 66), which interacts via its hydroxyl group side with the main chain amide of the upstream glutamic acid residue, or with the second serine residue as set forth in SEQ ID NO: 66, which interacts with the main chain amide of the down stream aspartic acid residue; or with the glutamic acid residue as set forth in SEQ ID NO: 66, which, as disclosed herein, when mutated results in loss of the isopeptidase activity of Csn5.

[0029] The present invention also relates to a method of identifying an agent that modulates an isopeptidase activity of a polypeptide comprising a JAMM domain. Such a method can be performed, for example, by contacting the polypeptide comprising the JAMM domain and a test agent under conditions suitable for isopeptidase activity, wherein said isopeptidase activity cleaves a modifier protein from a target protein, and detecting a change in isopeptidase activity by the polypeptide in the presence of the test agent as compared to the absence of the test agent, thereby identifying an agent that modulates isopeptidase activity of the polypeptide comprising the JAMM domain. According to a screening assay of the invention, the test agent can be identified as one that decreases deconjugation of the modifier protein from the target protein, thereby identifying the test agent as an agent that reduces or inhibits the isopeptidase activity of the polypeptide; or can be identified as a test agent that increases deconjugation of the modifier protein from the target protein, thereby identifying the test agent as an agent that increases the isopeptidase activity of the polypeptide. The isopeptidase activity can be one that cleaves a peptide bond between a carboxy terminus of the modifier protein and an epsilon amino group of a lysine residue of the target protein. For example, the modifier protein is Nedd8, UBL1, SMT3H2, SMT3H1, APG12, FAT10, Fau, UCRP, URM1, or UBL5, and the target protein can be a protein that contains a cullin domain (e.g., Cul1, Cul2, Cul3, Cul4A, Cul4B, or Cul5). A screening assay of the invention can further include contacting the test agent and a 26S proteasome complex comprising the polypeptide comprising the JAMM domain (e.g. RPN11/POH1), wherein detecting isopeptidase activity can be performed by detecting degradation of a protein by the 26S proteasome complex.

[0030] In one embodiment of such a method, the target protein comprises a SCF ubiquitin ligase, and a change in isopeptidase activity of the polypeptide is detected by detecting a change in the SCF ubiquitin ligase activity. In another embodiment, the polypeptide comprising the JAMM domain is a polypeptide of a CSN. The target protein or the modifier protein or both can comprise a detectable label, for example, a peroxidase, alkaline phosphatase, luciferase, fluorescent small molecule, fluorescent protein, or a ligand or substrate thereof, such that detecting the isopeptidase activity of the polypeptide can be performed by detecting the detectable label. A fluorescent protein useful as a detectable label can be a green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, dsRed, or a derivative thereof. In one embodiment, each of the target protein and the modifier protein comprises a detectable label of a fluorescence resonance energy transfer (FRET) pair, such that detecting the isopeptidase activity of the polypeptide can be performed by detecting a change in FRET.

[0031] A test agent screened according to a method of the invention can be any type of compound including, for example, a peptide, a peptide derivative (e.g., a peptide hydroxamate or a phosphinic peptide), a peptoid, a peptidomimetic, a polynucleotide, a polynucleotide derivative, or a small organic molecule. Furthermore, the test agent can be one of a library of test agents, for example, a combinatorial library, which can be a combinatorial library of random test agents, biased test agents, or variegated test agents. Exemplary libraries of test agents include a hydroxamate compound library, reverse hydroxamate compound library, carboxylate compound library, thiol compound library, a phosphinic peptide library, or phosphonate compound library.

[0032] A screening assay of the invention can be performed in vitro, for example, using purified components such as a purified polypeptide comprising the JAMM domain, or using a biological sample, or an extract of a biological sample, containing such a polypeptide, or can be performed in vivo. The methods are particularly adaptable to a high or ultra-high throughput format, thus facilitating screening of hundreds, thousands, or hundreds of thousands of test agent in parallel. Such high throughput format assays can be performed, for example, by contacting each of a plurality of polypeptides, each polypeptide comprising a JAMM domain, with a test agent, wherein polypeptides in the plurality are different from other polypeptides of the plurality; or by contacting each of number of same or different polypeptides comprising a JAMM domain with one or more of a plurality of test agents, wherein at least one polypeptide of the plurality of polypeptides is contacted with at least one test agent of the plurality of test agents. Accordingly, the present invention provides an agent that modulates the isopeptidase activity of a polypeptide comprising a JAMM domain, wherein the agent is identified according to a method of the invention.

[0033] The present invention further relates to a method of treating a condition by modulating protein degradation in a cell associated with the condition. Such a method can be performed, for example, by administering to a subject having or susceptible to the condition at least a first agent that modulates an isopeptidase activity of a polypeptide comprising a JAMM domain. The condition can be a cell proliferative disorder such as a neoplastic growth (e.g., a benign or malignant tumor), or psoriasis; an inflammatory condition, which can be an acute inflammatory disorder due to an infection or a chronic inflammatory disorder; an autoimmune disorder such as multiple sclerosis or rheumatoid arthritis; a condition associated with undesirable angiogenesis as occurs during tumor growth; a condition such as asthma, or ischemia and reperfusion injury; or any other condition in which it is desirable to modulate protein degradation. For example, the condition can be a protein storage disorder or other condition characterized by undesirable protein accumulation, wherein the method is performed by administering to a subject in need an agent that increases isopeptidase activity of a polypeptide comprising the JAMM domain, thereby increasing protein degradation in the cells, and ameliorating the condition.

[0034] In one embodiment, a method of the invention utilizes administration of an agent that reduces or inhibits the isopeptidase activity of the polypeptide comprising the JAMM domain, for example, a JAB1 polypeptide or other polypeptide, which can comprise a CSN complex. In another embodiment, the method can further include administering to the subject at least a second agent, which can be any agent that can provide a therapeutic benefit to the subject being treated, for example, a chemotherapeutic agent for a cancer patient, or a corticosteroid for a subject having an autoimmune disorder, or a nutrient and/or vitamin composition, or the like. In one aspect, the second agent is an agent that modulates a component of a protein degradation pathway comprising the polypeptide comprising the JAMM domain. Generally, a second agent that modulates a component of a protein degradation pathway modulates the component similarly to the first agent that modulates that isopeptidase activity of the polypeptide comprising the JAMM domain; i.e., where the first agent reduces or inhibits the isopeptidase activity, the second agent is selected such that it has reducing or inhibiting activity with respect to the other component of the protein degradation pathway. Where a second agent is selected such that it modulates a component of the protein degradation pathway, the component can be, for example, an E2 ubiquitin conjugating enzyme such as Cdc34 or a Ubc4/5 family member, or an E3 ubiquitin ligase such as Skp1, Cul1/Cdc53, an F-box protein, or a combination thereof (e.g., an SCF ubiquitin ligase).

[0035] As further disclosed herein, the JAB subunit, more specifically the JAMM domain, is responsible for the peptidase activity of a protein, e.g., the protein's ability of cleaving a peptide bond between the carboxy terminus of a modifier protein and a free amino group of a target protein. The present invention provides polypeptides and crystalline polypeptides containing the JAMM domain and methods of using such domain to treat conditions associated with peptidase activity, especially isopeptidase activity and methods to screen for agents that are capable of modulating its peptidase activity. The present invention also provides methods of using such domain to identify or design compounds that are candidates for modulators of the peptidase activity.

[0036] Accordingly, the present invention provides an isolated polypeptide containing a JAMM domain consisting essentially of an amino acid sequence of HXHXXXXXXXXXXD (SEQ ID NO: 1), wherein H is histidine, D is aspartate, and X is any amino acid and wherein the JAMM domain is not adjacent to an amino acid sequence that is naturally adjacent to the domain. In another embodiment, the invention provides an isolated crystalline polypeptide containing a JAMM domain consisting essentially of an amino acid sequence of HXHXXXXXXXXXXD (SEQ ID NO: 1), wherein H is histidine, D is aspartate, and X is any amino acid. In yet another embodiment, the present invention provides an isolated monoclonal antibody that specifically binds to an epitope within a JAMM domain of a polypeptide, wherein the JAMM domain consists essentially of an amino acid sequence of HXHXXXXXXXXXXD (SEQ ID NO: 1), wherein H is histidine, D is aspartate, and X is any amino acid. In still another embodiment, the present invention provides an isolated JAMM domain consisting essentially of a JAMM domain having an amino acid sequence of EXHyXHyHy(X)nHXHXXXXXXXXXXD (SEQ ID NO: 13), wherein E is glutamic acid, Hy is a hydrophobic amino acid residue, X is any amino acid, and (X)n indicates about 40 to 83 amino acid residues, and wherein the JAMM domain is not adjacent to an amino acid sequence that can normally be found adjacent to the domain in a naturally occurring protein.

[0037] The present invention also provides a method of identifying an inhibitor of a polypeptide by rational drug design. The polypeptide comprises a JAMM domain consisting essentially of an amino acid sequence of HXHXXXXXXXXXXD (SEQ ID NO: 1), wherein H is histidine, D is aspartate, and X is any amino acid. The method comprises designing a potential inhibitor for the polypeptide that will form a bond with the JAMM domain based upon the crystal structure co-ordinates of the polypeptide, synthesizing the inhibitor, and determining whether the potential inhibitor inhibits the activity of the polypeptide. In one aspect of this method, the potential inhibitor is designed based on the potential ability to form a bond with at least one of the histidine residues or with the aspartate residue, or with any combination of those residues, as set forth in the HXHXXXXXXXXXXD (SEQ ID NO: 1) sequence of the JAMM domain. The JAMM domain of the polypeptide also can included an extended amino acid sequence, EXHyXHyHy(X)nH(S/T)HXXXXXXXSXXD (SEQ ID NO: 13), wherein S is serine, T is threonine, E is glutamic acid, Hy is a hydrophobic amino acid residue, X is any amino acid, and (X)n indicates about 40 to 83 amino acid residues, in which case the potential inhibitor also can be designed based on the potential ability to form a bond with one or more of the specified histidine, aspartic acid, glutamic acid, serine, and threonine (when present) residues.

[0038] In another embodiment, the potential inhibitor is designed based on an ability to form a bond with a metal ion bound by the JAMM domain. For example, the hydroxamate class of compounds act to inhibit matrix metalloproteases, which contain a zinc ion, by chelating the bound zinc on the enzyme. Zinc generally forms bonds to four ligands in zinc finger and RING finger proteins, whereas metalloproteases, including proteins having a JAMM domain, contain only three proteinaceous ligands (i.e., in a JAMM domain, the H, H, and D residues; see, e.g., SEQ ID NO: 1). The fourth coordination position binds the oxygen moiety of a water molecule, which, in a JAMM domain containing polypeptide, is activated by the glutamate residue (see SEQ ID NO: 13), which hydrogen bonds to the hydrogen atoms of the water molecule, to initiate hydrolytic attack of the target peptide bond. Classical hydroxamate, carboxylate, thiol, and phosphinic inhibitors of metalloproteases bind to the enzyme and supply a fourth ligand for the enzyme-bound zinc, thus displacing the water molecule and inhibiting catalysis. As such, a potential inhibitor useful for purposes of the present invention is distinguishable from molecules that generally bind zinc atoms, including, for example, from chelating agents such as EDTA, which bind the metal ion and extract or otherwise sequester it from a protein.

[0039] In another embodiment, the invention provides a method of deconjugating a modifier protein from a target protein. The modifier protein is conjugated to the target protein via a peptide bond between the carboxy terminus of the modifier protein and a free amino group of the target protein. The method comprises contacting the target protein to a polypeptide comprising a JAMM domain consisting essentially of an amino acid sequence of HXHXXXXXXXXXXD (SEQ ID NO: 1), wherein H is histidine, D is aspartate, and X is any amino acid. In yet another embodiment, the invention provides a method of screening for an agent that affects deconjugation of a modifier protein from a target protein. The modifier protein is conjugated to the target protein via a peptide bond between the carboxy terminus of the modifier protein and a free amino group of the target protein. The method comprises incubating in the presence and absence of a test agent, the target protein and a polypeptide comprising a JAMM domain consisting essentially of an amino acid sequence of HXHXXXXXXXXXXD (SEQ ID NO: 1), wherein H is histidine, D is aspartate, and X is any amino acid and determining the effect of the test agent. An increase or decrease in the amount of the target protein not conjugated to the modifier protein caused by the test agent is indicative of an agent affecting deconjugation of the modifier protein from the target protein. Accordingly, an agent identified according to a method of the invention also is provided.

[0040] The present invention further provides a method of increasing conjugation of a modifier protein to a target protein. The modifier protein is conjugated to the target protein via a peptide bond between the carboxy terminus of the modifier protein and a free amino group of the target protein in a cell comprising inhibiting the activity of a polypeptide comprising a JAMM domain consisting essentially of an amino acid sequence of HXHXXXXXXXXXXD (SEQ ID NO: 1), wherein H is histidine, D is aspartate, and X is any amino acid, thereby increasing the conjugation of the modifier protein to the target protein. In addition, the present invention provides a method of treating a condition of neoplastic growth, angiogenesis, infection, chronic inflammation, asthma, ischemia and reperfusion injury, multiple sclerosis, rheumatoid arthritis, or psoriasis. The method comprises administering an agent identified by the method of the present invention to a subject in need of such treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]
FIG. 1 shows the sequence alignment of human AMSH proteins (see FIG. 2; SEQ ID NOS: 66 to 68) with human JAB1 (SEQ ID NO: 3) and Rpn11 (SEQ ID NO: 4). Conserved active site residues are boxed.

[0042]
FIG. 2 shows the sequence alignment of AMSH (SEQ ID NO: 66), AMSH1 (SEQ ID NO: 67), and AMSH2 (SEQ ID NO: 68). The critical conserved active site residues are boxed.

[0043]
FIG. 3 shows that sequence alignment of COP9 subunit 5 (CSN5/JAB1) orthologs from different eukaryotic species (SEQ ID NOS: 69 to 79) reveals conserved histidine, serine, and aspartate residues that are also found in a set of prokaryotic and archaebacterial proteins.

[0044]
FIG. 4 shows the alignment of Rpn11 orthologs (Pad1; SEQ ID NOS: 80 to 83) and CSN5/JAB1 orthologs from different species (SEQ ID NOS: 69 to 72).

[0045]
FIG. 5 provides an alignment of predicted JAMM domains (SEQ ID NOS: 15 to 64). Selected sequences were aligned using T-Coffee (Notredame et al, J. Mol. Biol. 302:205, 2000, which is incorporated herein by reference) and modified manually to ensure correct superposition of the conserved motifs. Eukaryotic proteins are grouped to reflect orthologous relationships. Position of the aligned region in the sequence is shown by numbers; poorly conserved spacers are not shown and are designated by numbers. The consensus includes amino acid residues conserved in 80% of the aligned sequences; I indicates aliphatic residues (A, I, L, V; yellow shading), h indicates hydrophobic residues (F, Y, W, A, I, L, V, M; yellow shading), and s indicates small residues (G, A, C, S, D, N, V, P; blue letters). Predicted metal-chelating and catalytic residues are shown in yellow against a dark blue background. Secondary structure prediction was made using the PHD program with the multiple alignment submitted as the query (Rost et al., Comput. Appl. Biosci. 10:53, 1994, which is incorporated herein by reference); E indicates extended conformation (β-strand), and H indicates a-helix. Each protein is denoted by the GenBank identifier, followed by the gene name and abbreviated species name. Species abbreviations are as follows: Hsa, Homo sapiens; Dme, Drosophila melanogaster; Cel, Caenorhabditis elegans; Ath, Arabidopsis thaliana; S C, Saccharomyces cerevisiae; Afu, Archaeoglobus fulgidus; Pho, Pyrococcus horikoshii; Hsp, Halobacterium sp.; Aae, Aquifex aeolicus; Nsp, Nostoc sp.; Dra, Deinococcus radiodurans; Psa, Pseudomonas aeruginosa; Vch, Vibrio cholerae; Rso, Ralstonia solanacearum; Eco, Escherichia coli; Bsu, Bacillus subtilis; Mac, Methanosarcina acetivorans (see Cope et al., Science 298; 608611, 2002; Verma et al., Science 298:611-615, each of which is incorporated herein by reference).

DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention is based on a determination that inhibition of the COP9 Signalosome (CSN) isopeptidase activity inhibits ubiquitin ligase activity, thus providing a means to modulate protein degradation in a cell. The CSN has a biological role in cleaving the ubiquitin-like protein Nedd8 from the cullin subunit of SCF (Skp-1/Cullin/F-protein) ubiquitin ligase. In particular, a metalloenzyme motif (“JAMM domain”) in the JabI subunit of CSN underlies the Nedd8 conjugate cleavage activity of the CSN complex. As disclosed herein, specific inhibition of the isopeptidase activity of CSN reduces SCF ubiquitin ligase activity. As such, the present invention provides methods for identifying agents that can modulate the activity of the CSN isopeptidase, and further provides agents identified using such screening assays. Such agents, either alone or in combination with other agents (e.g., agents that modulate other components of ubiquitin dependent protein degradation pathways such as E2 ubiquitin conjugating enzyme activity or an F-box substrate binding activity), can be used to ameliorate cell proliferative disorders and autoimmune diseases by modulating SCF activity. Accordingly, medicaments are provided for treating cell proliferative disorders or autoimmune disease by modulating SCF activity in cells associated with the disorder, for example, cancer cells or plasma cells.

[0047] The present invention provides relates to an isolated polypeptide comprising a mutant of a JAMM domain, which has an amino acid sequence of HXHXXXXXXXXXXD (SEQ ID NO: 1), wherein H is histidine, D is aspartate, and X is any amino acid, and wherein, in the mutant, at least one of the H residues is substituted with any amino acid residue other than H, the D residue is substituted with an amino acid residue other than D, or a combination thereof. Also provided is an isolated polypeptide comprising a mutant JAMM domain, which has an amino acid sequence of H(S/T)HXXXXXXXSXXD (SEQ ID NO: 65), wherein S is serine, and (S/T) is serine or threonine, and wherein, in the mutant JAMM domain, at least one of the H residues is substituted with an amino acid residue other than H; the D residue is substituted with an amino acid residue other than D; the S residue is substituted with an amino acid residue other than S; the T residue, when present, is substituted with an amino acid residue other than T, or a combination thereof. In addition, an isolated JAMM domain consisting essentially of a JAMM domain having an amino acid sequence of EXHyXHyHy(X)nHXHXXXXXXXXXXD (SEQ ID NO: 13), wherein E is glutamic acid, Hy is a hydrophobic amino acid residue, X is any amino acid, and (X)n indicates about 40 to 83 amino acid residues, is provided, as is a mutant JAMM domain having an amino acid sequence of EXHyXHyHy(X)nHXHXXXXXXXXXXD (SEQ ID NO: 13), wherein, in the mutant JAMM domain, at least one of the H residues is substituted with an amino acid residue other than H; the D residue is substituted with an amino acid residue other than D; the E residue is substituted with an amino acid residue other than E; or a combination thereof.

[0048] The term “mutant”, when used herein in reference to a JAMM domain, means that at least one amino acid residue of the JAMM domain has been manipulated artificially, i.e., by the hand of man. As such, for purposes of the present invention, a mutant JAMM domain, as well as a polypeptide containing a mutant JAMM domain, is a non-naturally occurring molecule, which can, but need not, be based on a sequence of a naturally occurring polypeptide. Methods for making a mutant JAMM domain are disclosed herein or otherwise known in the art and include, for example, chemical synthesis of a peptide comprising a JAMM domain having an amino acid substitution with respect to the sequence of a JAMM domain in a naturally occurring polypeptide (e.g., a substitution of an alanine residue for a histidine residue such as an H residue as set forth in SEQ ID NO: 1), or expression of a recombinant nucleic acid molecule encoding the JAMM domain, such a recombinant nucleic acid molecule being, for example, an isolated otherwise naturally occurring nucleic acid molecule modified by a method such as site directed mutagenesis, such that a mutant JAMM domain is encoded. As such, it will be recognized that various naturally occurring proteins such as proteins that contain a JAB domain as well as an amino acid sequence that is reminiscent of a JAMM domain, but differs, for example, in one, two or three of the H, H and D residues as set forth in SEQ ID NO: 1, is not encompassed within the meaning of a “mutant” JAMM domain because such naturally occurring proteins are not manipulated by man to generate a mutant JAMM domain.

[0049] Protein degradation is regulated primarily by a pathway that involves ubiquitination of the target protein, thus ‘marking’ it for proteolysis. Ubiquitination occurs via a series of steps, wherein an ubiquitin activating enzyme (generally referred to a “E1”) activates ubiquitin, which is transferred to a thiol group of an ubiquitin conjugating enzyme (“E2”), then further transferred to a lysine group of a substrate that is presented to the E2 by a specificity factor (“E3”). SCF is a heterotetrameric E3 ubiquitin ligase that includes Skp1, Cul1/Cdc53, F-box, and Hrt1/Roc1/Rbx1 subunits. SCF recruits both an E2 ubiquitin conjugating enzyme (Ube) and a substrate, and catalyzes the transfer of ubiquitin from the former to the latter (Deshaies, Ann. Rev. Cell. Devel. Biol. 15:435-467, 1999, which is incorporated herein by reference). Depending upon the specific cell type, the SCF ubiquitin conjugating enzyme can be either Cdc34 or a member of the Ubc4/5 family. Once a substrate is ubiquitinated, it is typically targeted for degradation by the proteosome (Verma and Deshaies, Cell 101:341-344, 2000); however, SCF also can potentially modulate the function of a substrate (positively or negatively) by other mechanisms independent of degradation (Hicke, Cell 106:527-530, 2001).

[0050] The F-box protein subunit of SCF includes a substrate-binding domain, which recruits substrate to SCF, and an F-box motif, which links the substrate-binding domain to the remainder of the SCF complex. Many different F-box proteins have been discovered, with the human genome alone encoding at least 40 of them (Cenciarelli et al., Curr. Biol. 9:1177-1179, 1999, Winston et al., Curr. Biol. 9:1180-1182, 1999). As such, human cells can contain as many as 40 distinct SCF complexes, each with a distinctive substrate specificity. Owing in part to this diversity of F-box proteins, SCF ubiquitin ligases can control a broad range of cellular processes, including, for example, regulation of the cytoskeleton, gene transcription, cell cycle progression, and innate immune response (Deshaies, supra, 1999). Accordingly, drugs that target either the shared components of SCF complexes or individual F-box proteins have potential application for the treatment of many different diseases. Indeed, it was recently suggested that the anti-cancer effects of the proteasome inhibitor PS-341 are mediated by stabilization of IκB, which is a substrate of the SCF complex comprised of the F-box protein β-TRCP (Garber, Science 295:612-613, 2002).

[0051] The mechanism of action of SCF and the regulation of its activity have not yet been completely defined. However, the covalent attachment of the ubiquitin-like protein Nedd8 to the Cul1 subunit of SCF plays an important regulatory role. The gene encoding Nedd8 in fission yeast is essential for growth, and mutation in fission yeast of Cul1 lysine 713, which is the site of attachment of Nedd8, is lethal (Osaka et al., EMBO J. 19:3475-3484, 2000)). Thus, the execution of at least one essential function of SCF in fission yeast requires that lysine 713 of Cul1 be modified by Nedd8. Although all members of the cullin family (Cul1-Cul5) can be modified with Nedd8 (Hori et al., Oncogene 189:6829-6834, 1999), the role of this modification in cullin function has been described only for Cul1. In vitro experiments with purified SCF complexes indicate that the attachment of Nedd8 to Cul1 stimulates the biochemical activity of SCF by promoting recruitment of ubiquitin-conjugating enzyme to the complex (Kawakami et al., EMBO J. 20:4003-4012, 2001).

[0052] In addition to the attachment of Nedd8 to SCF being important for SCF ubiquitin ligase activity, the removal of Nedd8 conjugates from SCF also can be important for SCF function. The possibility that cyclical addition and removal of Nedd8 is important for SCF function was suggested by the observation that cosuppression of the Csn5 subunit of CSN in Arabidopsis thaliana exacerbated the effects of mutations in the AXR1 gene, which encodes a subunit of the Nedd8 attachment enzyme, on the responsiveness of plants to the hormone auxin (Schwechheimer et al., Science 292:1379-1382, 2001). However, the result is inconclusive for at least two reasons. First, it was not established that SCF is the major or only target of Csn5 and Axr1 in auxin signaling. As such, it is not clear why diminishing the activity of both Csn5 and Axr1 caused a synergistic defect in auxin responsiveness. Second, disruption of the Csn5 subunit compromises the integrity of the CSN complex. As CSN has been implicated in the regulation of inositol 1, 3, 4 triphosphate-5, 6 kinase (Wilson et al., J. Biol. Chem. 276:40998-41004, 2001) and an unknown protein kinase that phosphorylates p53 (Bech-Otschiretal., EMBO J. 20:1630-1639, 2001; Seegeretal., Curr. Biol. 11:R643-R646, 2001), in addition to its role in cleaving Nedd8 conjugates (Lyapina et al., Science 292:1382-1385, 2001, which is incorporated herein by reference), the observed genetic results may have arisen, for example, because altered activity of inositol 1, 3, 4 triphosphate-5, 6 kinase or the p53 kinase in Csn5-deficient plants compromised the activity of some protein that is involved in auxin signaling.

[0053] The present invention relates in general to active sites responsible for the peptidase activity, e.g., isopeptidase activity of a protein, and to screening assays for identifying agents that modulate the isopeptidase activity, such agents being useful for increasing or decreasing ubiquitin-mediated protein degradation. It is a discovery of the present invention that a polypeptide containing the JAB subunit, more specifically the JAMM domain has peptidase activity, e.g., cleavage of a peptide bond between the carboxy terminus of a modifier protein and a free amino group of a target protein. Polypeptides containing the JAB subunit, more specifically the JAMM domain can be used to treat conditions associated with the peptidase activity, screen for agents capable of modulating the peptidase activity, and identify or design compounds that are candidates for modulators of the peptidase activity.

[0054] According to the present invention, a modifier protein can be deconjugated, removed, or separated from a target protein by exposing or contacting the target protein to a polypeptide containing a subunit characterized as JAB subunit or a domain characterized as JAMM domain. The modifier protein is usually associated with a target protein via a peptide bond between the carboxy terminus of the modifier protein and a free amino group of the target protein. A free amino group generally includes, without limitation, an amino group of the amino terminus of a polypeptide or the epsilon amino group of lysine residues of a polypeptide.

[0055] According to the present invention, the JAB subunit may be the JAB1 subunit of COP9/signalsome (CSN) (see FIG. 1; SEQ ID NO: 3) or the Rpn11 subunit of 26S proteasome (FIG. 1; SEQ ID NO: 4). A polypeptide subunit can be characterized as JAB subunit if it has the sequence characteristics of the JAB1 or Rpn11 subunit. Normally a polypeptide subunit is deemed to have the sequence characteristics of the JAB1 or Rpn11 subunit if it contains the JAMM domain or is a homolog or ortholog of JAB1 or Rpn11 subunit. For example, FIG. 3 shows various orthologs of JAB1 from different species while FIG. 4 shows various orthologs of Rpn11 and JAB1 including an alignment of orthologs of Rpn11 and JAB1 from different species.

[0056] Polypeptides containing a subunit characterized as JAB subunit can be any known or to be discovered polypeptides, proteins, or complexes thereof. For example, the polypeptides may be a polypeptide complex of CSN containing JAB1 subunit, 26S proteasome containing Rpn11, AMSH, AMSH1, AMSH2, or C6-1A. CSN has previously been identified as a regulator of photomorphogenetic development in plants. 26S proteasome is involved in various activities, e.g., ubiquitin/26S proteasome pathway while AMSH, AMSH1, and AMSH2 are involved in cytokine signaling, TGF-β signaling, and survival of hippocampal neurons. C6-1A has been observed to be fused to the T cell receptor alpha chain gene by a chromosomal translocation in a patient with T-cell leukemia.

[0057] According to one embodiment of the invention, the deconjugation, removal, or separation of a modifier protein from a target protein is achieved by exposing or contacting the target protein to a polypeptide containing a domain characterized as the JAMM domain. The JAMM domain of the present invention includes any domain containing the amino acid sequence of HXHXXXXXXXXXXD (SEQ ID NO: 1), with H being histidine, D being aspartate, and X being any amino acid. In one embodiment, the JAMM domain is any domain having an amino acid sequence of GW(Y/I)H(S/T)HPXXXXXXSXXD (SEQ ID NO: 2), with G being glycine, W being tryptophan, Y being tyrosine, I being isoleucine, P being proline, S being serine, T being threonine, Y/I being either Y or I, and S/T being either S or T. In another embodiment, the JAMM domain is any domain having an amino acid sequence of EXHyXHyHy(X)nHXHXXXXXXXXXXD (SEQ ID NO: 13), wherein E is glutamic acid, Hy is a hydrophobic amino acid residue, and (X)nindicates about 40 to 83 amino acid residues.

[0058] In one aspect, the JAMM domain is from a human protein and has the amino acid sequence of SEQ ID NO: 1. In yet another embodiment, the JAMM domain includes any domain having the same peptidase activity, e.g., isopeptidase activity of a domain containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In still another embodiment, the JAMM domain contains a metalloenzyme, e.g., metalloprotease active site and provides peptidase activity, e.g., isopeptidase activity.

[0059] One feature of the present invention provides isolated polypeptides containing a domain characterized as the JAMM domain. For example, the present invention provides an isolated polypeptide containing the JAMM domain which is not surrounded by or adjacent to any amino acid sequences that are naturally adjacent to such JAMM domain. Another feature of the present invention provides isolated crystalline polypeptides containing the JAMM domain. The crystalline polypeptides can be made by any suitable means known to one skilled in the art. For example, polypeptides containing the JAMM domain can be crystallized by equilibrating saturated solutions of the polypeptides with salts, volatile organic compounds, and other organic compounds at various controlled temperatures. Yet another feature of the present invention provides isolated monoclonal antibodies that specifically bind to an epitope within the JAMM domain. Such monoclonal antibodies can be prepared by any means known to one skilled in the art. For example, whole or partial amino acid sequences of the JAMM domain can be used as an antigen to obtain monoclonal antibodies. In one embodiment, the amino acid sequence used as antigen contains the histidine or aspartate in SEQ ID NO: 1 or SEQ ID NO: 2.

[0060] Polypeptides containing the JAMM domain include all known or to be discovered polypeptides, proteins, or complexes thereof. For example, Table 1 shows various polypeptides, proteins, or fragments thereof from different species that contain the JAMM domain.

1TABLE 1JAMM-Domain-Containing Proteins From Model Organisms*BacteriaMost bacteria encode one and some 2-4 paralogs of the DNA repairprotein RadC, which is a distinct version of the JAMM domain.In addition:Aquifex aeolicus [aquificales] taxid 63363gi|15606783|ref|NP_214163.1| (NC_000918) hypothetical prot . . .Mycobacterium tuberculosis H37Rv [high GC Gram+]taxid 83332gi|15608474|ref|NP_215850.1| (NC_000962) hypothetical prot . . .Synechocystis sp. PCC 6803 [cyanobacteria] taxid 1148gi|16330214|ref|NP_440942.1| (NC_000911) unknown protein[ . . .Deinococcus radiodurans [eubacteria] taxid 1299gi|15805429|ref|NP_294125.1| (NC_001263) conserved hypothe . . .Pseudomonas aeruginosa [g-proteobacteria] taxid 287gi|15595836|ref|NP_249330.1| (NC_002516) conserved hypothe . . .gi|15597298|ref|NP_250792.1| (NC_002516) hypothetical prot . . .Coxiella burnetii [g-proteobacteria]taxid 777gi|10956045|ref|NP_052867.1| (NC_002131) hypothetical prot . . .gi|1070034|emb|CAA63684.1| (X93204) orf 112 [Coxiella burn . . .gi|10956011|ref|NP_052361.1| (NC_002118) orf 169; similari . . .ArclhaeaPyrococcus abyssi [euryarchaeotes] taxid 29292gi|14520886|ref|NP_126361.1| (NC_000868) hypothetical prot . . .gi|14521781|ref|NP_127257.1| (NC_000868) hypothetical prot . . .Methanothermobacter thermautotrophicus [euryarchaeotes] taxid 145262gi|15678989|ref|NP_276106.1| (NC_000916) unknown [Methanot . . .Halobacterium sp. NRC-1 [euryarchaeotes] taxid 64091gi|16554503|ref|NP_444227.1| (NC_002607) Uncharacterized c . . .gi|15789943|ref|NP_279767.1| (NC_002607) Vng0778c [Halobac . . .Archaeoglobus fulgidus [euryarchaeotes]taxid 2234gi|11499780|ref|NP_071023.1| (NC_000917) conserved hypothe . . .Aeropyrum pernix [crenarchaeotes] taxid 56636gi|14600889|ref|NP_147414.1| (NC_000854) hypothetical prot . . .Sulfolobus solfataricus [crenarchaeotes] taxid 2287gi|15897071|ref|NP_341676.1| (NC_002754) Hypothetical prot . . .EukaryotesSaccharomyces cerevisiae (baker's yeast) [fungi] taxid 4932gi|143l8526|ref|NP_116659.1| (NC_001138)Suppressor of mut . . . Rpnllpgi|63l9985|ref|NP_010065.1| (NC_001136) Hypothetical ORF; . . . RrilpSchizosaccharomyces pombe (fission yeast) [fungi] taxid 4896gi|3334476|sp|P41878|PAD1_SCHPO PROTEINPAD1/SKS1 >gi|7493 . . .gi|11281515|pir∥T44427 hypothetical protein - fission yea . . .gi|7492119|pir∥T37756 jun activation domain binding prote . . .gi|9588467|emb|CAC00558.1|(AL390814) similarity to human . . .Arabidopsis thaliana (thale cress) [eudicots] taxid 3702gi|15224003|ref|NP_177279.1| (NC_003070) c-Jun coactivator . . .gi|15219970|ref|NP_173705.1| (NC_003070) putative JUN kina . . .gi|15237785|ref|NP_197745.1| (NC_003076) 26S proteasome, n . . .gi|15229710|ref|NP_187736.1| (NC_003074) 26S proteasome re . . .gi|15239230|ref|NP_196197.1| (NC_003076) 26S proteasome re . . .gi|15218589|ref|NP_172530.1| (NC_003070) hypothetical prot . . .gi|15221964|ref|NP_175311.1| (NC_003070) hypothetical prot . . .gi|15231308|ref|NP_187338.l| (NC_003074) unknown protein [. . .gi|5902365|gb|AAD55467.1|AC009322_7 (AC009322) Putative sp . . .gi|5091556|gb|AAD39585.1|AC007067_25 (AC007067) T10O24.25 . . .gi|6573732|gb|AAF17652.1|AC009398_1 (AC009398) F20B24.2 [A . . .gi|15220090|ref|NP_178138.1| (NC_003070) hypothetical prot . . .gi|5902374|gb|AAD55476.1|AC009322_16 (AC009322) Hypothetic . . .Drosophila melanogaster (fruit fly) [flies] taxid 7227gi|17137694|ref|NP_477442.1|(NM_058094) CSNS-P1; Drosophi . . .gi|4732109|gb|AAD28608.1|AF129083_1 (AF129083) COP9 signal . . .gi|6434964|gb|AAF08394.1|AF145313_1 (AF145313) 26S proteas . . .gi|7291779|gb|AAF47199.1| (AE003464) Mov34 gene product [D . . .gi|7301945|gb|AAF57051.1| (AE003774) CG2224 gene product [. . .gi|7303518|gb|AAF58573.1| (AE003823) CG8877 gene product [. . .gi|7297828|gb|AAF53077.l| (AE003631) CG4751 gene product [. . .gi|6752672|gb|AAF27818.1|AF195189_1 (AF195189) yippee inte . . .Caenorhabditis elegans [nematodes] taxid 6239gi|17538322|ref|NP_500841.1| (NM_068440) B0547.1.p [Caenor . . .gi|17553290|ref|NP_498470.1| (NM_066069) F37A4.5.p [Caenor . . .gi|17535703|ref|NP_494712.1| (NM_062311) K07D4.3.p [Caenor . . .gi|17508685|ref|NP_491319.1| (NM_058918) R12E2.3.p [Caenor . . .Homo sapiens (human) [mammals] taxid 9606gi|12734403|ref|XP_011713.1| (XM_011713) COP9 (constitutiv . . .gi|5031981|ref|NP_005796.1| (NM_005805) 26S proteasome-ass . . .gi|7243127|dbj|BAA92611.1| (AB037794) KIAA1373 protein [Ho . . .gi|5453545|ref|NP_006454.1| (NM_006463) associated molecul . . .gi|16158201|ref|XP_055481.1| (XM_055481) KIAA1915 protein . . .gi|1168719|sp|P46736|C61A_HUMAN C6.lA PROTEIN >gi|2l35176| . . .gi|4581082|gb|AAD24592.1|AC007292_2 (AC007292) R31167_1, p . . .gi|7717235|gb|AAB30469.2| (S72931) T-cell receptor alpha c . . .gi|14249610|ref|NP_116257.1| (NM_032868) hypothetical prot . . .* Each protein is disclosed by accession numbers used in the protein and nucleic acid sequence databases (GenBank) maintained by the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov).

[0061] According to the present invention, a polypeptide containing the JAB subunit or JAMM domain can additionally include any other amino acid sequences. In one embodiment, a polypeptide containing the JAB subunit or JAMM domain has other amino acid sequences that do not interfere or inhibit the peptidase activity of the JAB subunit or JAMM domain. In another embodiment, a polypeptide containing the JAB subunit or JAMM domain has other amino acid sequences that enhance or facilitate the peptidase activity of the JAB subunit or JAMM domain.

[0062] In yet another embodiment, a polypeptide containing the JAB subunit or JAMM domain has other amino acid sequences that are associated with or determine the specificity of the peptidase activity of the JAB subunit or JAMM domain. In still another embodiment, a polypeptide containing the JAB subunit or JAMM domain has other amino acid sequences that inhibit or decrease the activity of the JAB subunit or JAMM domain, and such inhibition can be released by a signal, e.g., second messenger, covalent modification, calcium or phosphorylation.

[0063] The modifier protein of the present invention can be any protein that modifies the activity or function of a target protein. In one embodiment, the modifier protein modifies a target protein through conjugation and deconjugation to the target protein, e.g., formation and cleavage of a peptide bond between the carboxy terminus of the modifier protein and a free amino group of the target protein. For example, APG12 and URM1 modifies a target protein via forming an isopeptide bond between the carboxy terminus of APG12 or URM1 and a free amino group of the target protein. A free amino group of a target protein usually includes, without limitation, an amino group of the amino terminus or epsilon amino group of lysine residues of the target protein.

[0064] One major class of modifier proteins is ubiquitin. Proteins destined for degradation may be marked by the attachment of a multiubiquitin chain to the side chains of lysine residues of the protein. Another class of modifier proteins include ubiquitin-like proteins, e.g., NEDD8, UBL1/SUMO, SMT3H2, SMT3H1, FAT10, Fau, UCRP/ISG15, or UBL5. In one embodiment, the modifier protein of the present invention includes any protein containing two glycine amino acids at its carboxy terminus after being processed.

[0065] The target protein of the present invention can be any protein whose activity or function is modified by a modifier protein. In one embodiment, a target protein is a protein which forms a peptide bond with a modifier protein, e.g., a peptide bond between the carboxy terminus of the modifier protein and a free amino group of the target protein. In another embodiment, a target protein is specific to a class of modifier proteins.

[0066] For example, the target protein of the present invention may be cullin proteins such as Cul1, Cul2, Cul3, Cul4A, Cul4B, and Cul5, which are known to be the target proteins of Nedd8. In one embodiment, the target protein of the present invention includes any protein having ubiquitin ligase activity or is part of a protein complex having ubiquitin ligase activity. In another embodiment, the target protein of the present invention includes any protein to which ubiquitin conjugates for processing or degradation, e.g., p53, IκB, NF-κB, β-adrenergic receptor, cyclin E, p27Kipl, etc.

[0067] According to one embodiment of the present invention, the target protein used in the screening assays provided by the present invention can be any protein that produces a detectable signal upon deconjugation, removal, or separation from its modifier protein. Such detectable signal can be any assayable signal including, without limitation, enzymatic, spectroscopic, fluorescent, or functional signals, or a signal produced upon specific molecular interaction. For example, the target protein can be an enzyme such as peroxidase, alkaline phosphatase, and luciferase. The target protein can also be a fluorescent protein obtained via protein modification or a naturally fluorescent protein including, without limitation green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, dsRed, and the derivatives thereof.

[0068] In one embodiment, the target protein of the present invention is used in the screening assays of the present invention with a 26S proteasome and may be any protein that can structurally fit or refold within the inner chamber of a 20S proteasome. Normally the inner chamber of a 20S proteasome can structurally accommodate any protein that is 70 kDa or less.

[0069] According to the present invention, a target protein is deconjugated, removed, or separated from its modifier protein by being exposed or contacted to a polypeptide having the JAB subunit or JAMM domain. Such exposure or contact may be in vivo via administering the polypeptide to a subject in need of such treatment or in vitro via incubating the polypeptide with the target protein. In one embodiment, an inhibitor of a polypeptide having the JAB subunit or JAMM domain can be used to increase the association of a modifier protein to a target protein in vitro or in vivo.

[0070] According to another aspect of the invention, a polypeptide having the JAB subunit or JAMM domain, or a protein comprising such a polypeptide (e.g., CSN, or a proteasome comprising an Rpn11 polypeptide) can be used for screening assays for agents that affect the deconjugation, removal, or separation of a modifier protein from a target protein. The screening assays provided by the present invention can be carried out by incubating in the presence and absence of a test agent, a target protein, e.g., conjugated with a modifier protein and a polypeptide containing the JAB subunit or JAMM domain, and determining the effect of the test agent. In one embodiment, the screening assays provide a means to identify an agent that modulates (i.e., increases or decreases) the isopeptidase activity of a polypeptide comprising a JAMM domain (e.g., deneddylation activity), or of a protein comprising such a polypeptide.

[0071] Generally, an increase or decrease in the amount of the target protein not conjugated to the modifier protein caused by the test agent is indicative of an agent capable of affecting deconjugation, removal, or separation of the modifier protein from the target protein. For example, a test agent decreasing the amount of the target protein not conjugated to the modifier protein is indicative of an agent decreasing the deconjugation of the modifier protein from the target protein.

[0072] In one embodiment, the polypeptide (protein) used in the screening assays provided by the present invention is a polypeptide complex of 26S proteasome while the modifier protein is ubiquitin. The polypeptide complex of 26S proteasome can be obtained by any suitable means available in the art. For example, 26S proteasome can be purified from eukaryotic cells or tissues, e.g., S. cerevisiae or human.

[0073] In another embodiment, the incubation in the presence and absence of a test agent, a target protein, and 26S proteasome of the screening assays provided by the present invention is carried out in the presence of a 20S inhibitor or an inhibitor of the degradation process associated with the deubiquitination process of the 26S proteasome pathway. Any 20S inhibitor or inhibitor of the degradation process can be used for the purpose of the present invention. For example, a 20S inhibitor can be MG132, lactacystin, epoxomycin, PS-349, PS-519, LLnL, or the derivatives thereof. In general, such inhibitor prevents or decreases the degradation of a target protein that is not conjugated to a modifier protein, e.g., ubiquitin.

[0074] In yet another embodiment, the incubation is conducted further in the presence of an energy source, e.g., ATP. In still another embodiment, the incubation is conducted further in the presence of an inhibitor of deubiquitination by a conventional ubiquitin isopeptidase, e.g., an ubiquitin isopeptidase other than those that associated with a JAB subunit such as 26S proteasome. One example of such inhibitor is ubiquitin aldehyde, e.g., at 2-5 μM.

[0075] The test agent used for the screening methods of the present invention can be any agent from any library of compounds or molecules. In one embodiment, the test agent is selected or derived from compounds likely to inhibit the activity of metalloproteinase, e.g., compounds having zinc-binding functionality. For example, the test agent can be any compounds having a hydroxamate moiety or a member of a hydroxamate compound library, reverse hydroxamate compound library, thiol compound library, carboxylate compound library, or phosphinic acid compound library.

[0076] A screening method of the invention conveniently can be adapted to high throughput analysis and, therefore, can be used to screen combinatorial libraries of test agents, which can be a library of random test agents, biased test agents, or variegated test agents (see, for example, U.S. Pat. No. 5,571,698, which is incorporated herein by reference), in order to identify those agents that can modulate an isopeptidase activity of a polypeptide comprising a JAMM domain and, therefore, ubiquitin-mediated protein degradation in a cell. 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. Pat. 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. Pat. No. 5,264,563, which is incorporated herein by reference); a library of peptide derivative compounds such as a hydroxamate compound library, reverse hydroxamate compound library, a carboxylate compound library, thiol compound library, a phosphinic peptide library, or phosphonate compound library (see, for example, Dive et al., Biochem. Soc. Trans. 28:455-460, 2000; Ye and Marshall, Peptides: The Wave of the Future (Lebl and Houghten, ed.; American Peptide Society, 2001), each of which is incorporated herein by reference); a peptidomimetic library (Blondelle et al., Trends Anal. Chem. 14:83-92, 1995, which is incorporated herein by reference); incorporated herein by reference); 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. Expl. Med. Biol. 376:261-269, 1995; each of which is incorporated herein by reference); a lipoprotein library (de Kruif et al., FEBS Lett. 399:232-236, 1996, which is incorporated herein by reference); 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., J. Med. Chem. 37:1385-1401, 1994; Ecker and Crooke, Bio Technology 13:351-360, 1995; each of which is incorporated herein by reference).

[0077] Peptides and peptide derivatives can be useful as agents for modulating isopeptidase activity of a polypeptide comprising a JAMM domain, or as test agents to screen for such activity. Such peptides (or test agent peptides) 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, and, therefore, can be particularly useful in performing a method of modulating isopeptidase activity of a polypeptide comprising a JAMM domain as disclosed herein. Of course, the peptides 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.

[0078] Peptide derivatives comprising hydroxamate bonds or phosphinic bonds can be particularly useful for modulating isopeptidase activity of a polypeptide comprising a JAMM domain because the hydroxamate or phosphinic bonds of such peptide derivatives can coordinate with metal ions in polypeptides such as metalloproteases, thus inhibiting the activity of the metalloproteases (see, for example, Dive et al., supra, 2000; Ye and Marshall, supra, 2001). As such, hydroxamate and/or phosphinic peptide derivatives can be screened to identify those that similarly can reduce or inhibit isopeptidase activity of a polypeptide comprising a JAMM domain.

[0079] Test agents also can be antibodies that are raised against and specifically bind one or more epitopes of a polypeptide comprising a JAMM domain, particularly to an epitope associated with the isopeptidase activity of the polypeptide. 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. 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 1×10−6 M, generally at least about 1×10−7 M, usually at least about 1×10−8 M, and particularly at least about ×10−9 M or 1×10−10 M 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. In addition to specifically binding a particular epitope, an antibody agent can modulate the isopeptidase activity of a polypeptide comprising a JAMM domain, including increasing or decreasing such activity.

[0080] 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, 1988); 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).

[0081] A panel of test agent antibodies conveniently can be obtained by immunizing an animal using a peptide portion of a polypeptide that has isopeptidase activity and comprises a JAMM domain, for example, a JAB1 polypeptide. Where such a peptide is non-immunogenic, it can be made immunogenic by coupling the hapten to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressing the peptide portion as a fusion protein. Various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art (see, for example, by Harlow and Lane, supra, 1988). Methods for raising polyclonal antibodies, for example, in a rabbit, goat, mouse or other mammal, are well known in the art (see, for example, Green et al., “Production of Polyclonal Antisera,” in Immunochemical Protocols (Manson, ed., Humana Press 1992), pages 1-5; Coligan et al., “Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters,” in Curr. Protocols Immunol. (1992), section 2.4.1; each or which is incorporated herein by reference). In addition, monoclonal antibodies can be obtained using methods that are well known and routine in the art (see, for example, Kohler and Milstein, Nature 256:495, 1975, which is incorporated herein by reference; see, also, Harlow and Lane, supra, 1988). For example, spleen cells from a mouse immunized with a polypeptide comprising a JAMM domain, or an epitopic fragment thereof, can be fused to an appropriate myeloma cell line such as SP/02 myeloma cells to produce hybridoma cells. Cloned hybridoma cell lines can be screened using labeled antigen to identify clones that secrete monoclonal antibodies having the appropriate specificity, and hybridomas expressing antibodies having a desirable specificity and affinity can be isolated and utilized as a continuous source of the antibodies. A recombinant phage that expresses, for example, a single chain antibody that modulates the isopeptidase activity of a polypeptide comprising a JAMM domain also provides an antibody that can used for preparing standardized kits.

[0082] Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well established techniques, including, for example, affinity chromatography with Protein-A SEPHAROSE gel, size exclusion chromatography, and ion exchange chromatography (Coligan et al., supra, 1992, see sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; see, also, Barnes et al., “Purification of Immunoglobulin G (IgG),” in Meth. Molec. Biol. 10:79-104 (Humana Press 1992), which is incorporated herein by reference). Methods of in vitro and in vivo multiplication of monoclonal antibodies are well known in the art. Multiplication in vitro can be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture. Multiplication in vivo can be carried out by injecting cell clones into mammals histocompatible with the parent cells, for example, syngeneic mice, to cause growth of antibody producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.

[0083] As disclosed herein, therapeutic applications for agents, including antibody agents, identified according to a screening assay of the invention also are provided. Where the therapeutic procedure is for treating a human subject, the antibodies can be derived from a subhuman primate antibody (see, for example, Goldenberg et al., Intl. Publ. WO 91/11465, 1991; and Losman et al., Intl. J. Cancer 46:310, 1990, each of which is incorporated herein by reference). A therapeutically useful antibody for human treatment also can be derived from a “humanized” monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are known (see, for example, Orlandi et al., Proc. Natl. Acad. Sci., USA 86:3833, 1989, which is hereby incorporated in its entirety by reference). Techniques for producing humanized monoclonal antibodies also are known (see, for example, Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci., USA 89:4285, 1992; Sandhu, Crit. Rev. Biotechnol. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993; each of which is incorporated herein by reference). Alternatively, the antibodies can be derived from human antibody fragments isolated from a combinatorial immunoglobulin library (see, for example, Barbas et al., METHODS: A Companion to Methods in Immunology2:119, 1991; Winter et al., Ann. Rev. Immunol. 12:433, 1994; each of which is incorporated herein by reference).

[0084] The antibodies also can be derived from human monoclonal antibodies, which, for example, can be obtained from transgenic mice that have been genetically modified to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are well known (see, for example, by Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; and Taylor et al., Intl. Immunol. 6:579, 1994; each of which is incorporated herein by reference), and commercial sources of human antibodies are available (Abgenix, Inc.; Fremont Calif.).

[0085] Polynucleotides also can be useful as agents that can modulate isopeptidase activity of a polypeptide comprising a JAMM domain 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. Pat. No. 5,750,342, which is incorporated herein by reference). 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 gene or a portion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence, or the like, and can be single stranded or double stranded, as well as a DNA/RNA hybrid. A polynucleotide can be a naturally occurring nucleic acid molecule, which can be isolated from a cell, or a synthetic molecule, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR).

[0086] A polynucleotide agent (or 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).

[0087] 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, Bio Technology 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.

[0088] 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).

[0089] According to another aspect of the present invention, the JAMM domain can be used for rational drug design or as a guide for identifying agents that are capable of affecting the activity of the JAMM domain or any polypeptide containing the JAMM domain, e.g., identify inhibitors of the JAMM domain. In one embodiment, the structure coordinates or atomic coordinates of the JAMM domain or any polypeptide containing the JAMM domain are used to design a potential inhibitor that will form a covalent or non-covalent bond with one or more amino acids within the JAMM domain or metal ions bound by the JAMM domain. In another embodiment, the potential inhibitor is designed to form a covalent bond or non-covalent bond with histidine or aspartate of the JAMM domain. Such designed potential inhibitor can be synthesized by any suitable means and be tested for their ability to inhibit the activity, e.g., peptidase activity of the JAMM domain.

[0090] The structure or atomic coordinates of the JAMM domain or a polypeptide containing the JAMM domain refer to mathematical coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of the JAMM domain or the polypeptide containing the JAMM domain in crystal form. The diffraction data normally are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are generally used to establish the positions of the individual atoms within the unit cell of the crystal.

[0091] Various methods can be used to obtain the structure or atomic coordinates of the JAMM domain or a polypeptide containing the JAMM domain. For example, three dimensional diffraction data for a polypeptide containing the JAMM domain can be collected at temperatures ranging from 100-274 K using an area detector and radiation from a rotating-anode X-ray generator and from the Stanford synchrotron. These data, along with data collected from a heavy atom derivative of the polypeptide, can be processed and the structure can be solved by methods which make use of the isomorphous differences between a derivative and native polypeptide and/or make use of the anomalous X-ray scattering from the heavy atom in the derivative. In one embodiment, the structure or atomic coordinates of one JAMM containing polypeptide can be solved by using the phases of another JAMM containing polypeptide structure or sections thereof that has already been previously determined. High resolution data sets can be solved by direct methods.

[0092] Based on the three dimensional crystal structure of a JAMM domain polypeptide from Archaeglobulus fulgidis (GenBank Ace. No. AF21980; see, also, Cope et al., supra, 2002). The structure confirmed that the two histidine residues and the aspartic acid residue as set forth in the HXHXXXXXXXXXXD (SEQ ID NO: 1) sequence of the JAMM domain act to coordinate zinc metal ion binding to a polypeptide comprising the JAMM domain; the metal ion binding is required for isopeptidase activity. In addition, an extended structure having an amino acid sequence EXHyXHyHy(X)nH(S/T)HXXXXXXXSXXD (SEQ ID NO: 66), which comprises the JAMM domain (SEQ ID NO: 1), was determined to contribute to the structure of the metal ion coordinating site. As disclosed herein, mutation of the upstream glutamic acid residue resulted in a loss of isopeptidase activity of Csn5, thus demonstrating that this residue is essential to the isopeptidase activity. With respect to the three dimensional structure of the isopeptidase, the glutamate residue binds to the hydrogen atoms of a water molecule, whose oxygen atom is bound to the zinc ion immobilized within the JAMM domain (see SEQ ID NO: 66). The first serine residue (or the threonine residue, when present) as shown in SEQ ID NO: 66 appears to interact via its hydroxyl group side with the main chain amide of the upstream glutamic acid residue. In addition, the second serine residue as set forth in SEQ ID NO: 66 appears to interact with the main chain amide of the down stream aspartic acid residue; or with the glutamic acid residue as set forth in SEQ ID NO: 66. As such, these results provide targets for rational design of potential inhibitors of isopeptidase activity, including, for example, designing small molecules, peptides, peptide derivatives, or other molecules that interact with one or more of the above-described histidine, aspartic acid, serine, threonine (when present), and glutamic acid residues (see, for example, SEQ ID NO: 66).

[0093] According to another aspect of the present invention, any agent that is capable of inhibiting or decreasing the deconjugation, removal, or separation of a modifier protein from a target protein can be used therapeutically to treat various conditions associated with protein regulation, e.g., deubiquitination or deneddylation. For example, any agent identified by the screening methods of the present invention that is able to decrease the deconjugation of a modifier protein from a target protein, e.g., an inhibitor of the isopeptidase activity of 26S proteasome can be used therapeutically to treat a subject having a condition such as a neoplastic growth, infection, chronic inflammation, asthma, ischemia and reperfusion injury, multiple sclerosis, rheumatoid arthritis, or psoriasis; or to treat a subject in whom it is desired to modulate angiogenesis, including, for example, the angiogenesis associated with a condition such as tumor growth.

[0094] An agent useful for therapeutic treatment can be administered alone, in a composition with a suitable pharmaceutical carrier, or in combination with other therapeutic agents. A particularly effective combination of agents includes a first agent that modulates (i.e., increases or decreases) the isopeptidase activity of a polypeptide comprising a JAMM domain, e.g., a JAB domain of a CSN complex, and a second agent that similarly modulates (i.e., increases or decreases, respectively) a component of a protein degradation pathway that includes the polypeptide comprising the JAMM domain, particularly a component such as an E2 ubiquitin conjugating enzyme (e.g., Cdc34 or Ubc) or E3 ubiquitin ligase (e.g., SCF). Whereas previous data suggested that a CSN isopeptidase mutant likely would suppress the effect of an SCF mutant, as disclosed herein, the opposite effect occurs, i.e., inhibition of the CSN isopeptidase activity synthetically enhances an SCF mutant (see Example 4). As such, a particularly effective combination of agents for inhibiting protein degradation can include, for example, a first agent that reduces or inhibits the isopeptidase activity of a polypeptide comprising a JAMM domain and a second agent that reduces or inhibits the activity of an E3 ubiquitin ligase such as SCF. For example, a particularly effective combination of agents can include a first agent that reduces or inhibits the isopeptidase activity of a JAB domain of a CSN complex, and a second agent that reduces or inhibits F-box protein binding to the Skp1 domain of an E3 such as an SCF complex comprising the F-box protein and Skp1 domain, or a second agent that reduces or inhibits F-box protein binding to a substrate targeted for degradation, or a second agent that reduces or inhibits the interaction of Skp1 with Cul1; or a second agent that reduces or inhibits the charging of ubiquitin to a Ubc (E2) polypeptide that works together with SCF (e.g., UbcH5 or Cdc34), or otherwise inhibits the ability of the Ubc polypeptide to transfer ubiquitin to a substrate polypeptide bound to the F-box subunit of SCF.

[0095] An effective amount of an agent or combination of agents to be administered can be determined on a case-by-case basis. Factors to consider include, for example, age, body weight, stage of the condition, other disease conditions, duration of the treatment, and the response to the initial treatment. Typically, the agents are prepared as an injectable, either as a liquid solution or suspension. However, solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The agent can also be formulated into an enteric-coated tablet or gel capsule according to known methods in the art.

[0096] The agents of the present invention may be administered in any way that is medically acceptable which may depend on the disease condition or injury being treated. Possible administration routes include injections, by parenteral routes such as intravascular, intravenous, intraepidural or others, as well as oral, nasal, ophthalmic, rectal, topical, or pulmonary, e.g., by inhalation. The agents may also be directly applied to tissue surfaces, e.g., during surgery. Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants.

[0097] The following examples are intended to illustrate but not to limit the invention in any manner, shape, or form, either explicitly or implicitly. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLE 1

Deubiquitination by 26S Proteasome

[0098] To demonstrate that an isopeptidase activity associated with the 26S proteasome can deubiquitinate a target polypeptide in vitro, experiments were conducted to incubate multiubiquitinated Sic1 substrate (300 nM) for 0 or 5 min at 25° C. in the presence of purified 26S proteasome (100 nM), epoxomicin (100 μM), and ubiquitin aldehyde (5 μM).

[0099] Epoxomicin was included to uncouple the deubiquitination and degradation of a substrate by the 26S proteasome, since these two processes are normally tightly coupled. Epoxomicin forms a covalent adduct with the catalytically active N-terminal threonine residues of beta subunits of the 20S proteasome, thereby eliminating the proteolytic activity of this particle. At the end of the incubation, samples were supplemented with SDS-PAGE sample buffer, fractionated on an SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted with antibodies directed against Sic1. Filter-bound antibodies were decorated with goat-anti-rabbit antibody conjugated to horse radish peroxidase and visualized with ECL reagents.

[0100] This experiment demonstrated that inhibition of 20S peptidase activity is required to observe the production of deubiquitinated Sic1 by 26S proteasome. In the absence of epoxomicin, ubiquitinated Sic1 was completely degraded by the 26S proteasome. In the presence of epoxomicin, Sic1 was not degraded, but a majority of Sic1 was deubiquitinated such that the input substrate was converted from a heterogeneous smear of greater 220 kDa to a discrete species of about 50 kDa, which corresponds to Sic1 lacking any covalently attached ubiquitin molecules.

[0101] To test if this reaction is dependent upon ATP, multi-ubiquitinated Sic1 substrate (300 nM) and purified 26S proteasome (100 nM) were incubated in the presence or absence of epoxomicin (100 μM), glucose (30 mM) plus hexokinase (5 U/ml), apyrase (15 U/ml), and ubiquitin vinyl sulfone (Ub-PVS; 2.5 μM) as indicated.

[0102] This experiment revealed that ATP is required to observe the deubiquitination of target polypeptide by purified 26S proteasome. Specifically, in the presence of multi-ubiquitinated Sic1 substrate, 26S proteasome (which contains ATP), and epoxomicin, the input substrate was converted from a heterogeneous smear of >220 KD to a discrete species of ˜40 KD, which corresponds to Sic1 lacking any covalently attached ubiquitin molecules. However, in the additional presence of apyrase or glucose plus hexokinase (both of which consume ATP), no deubiquitination of the multi-ubiquitinated Sic1 substrate was observed. This reaction was not sensitive to ubiquitin vinyl sulfone, which is an inhibitor of conventional ubiquitin isopeptidases.

[0103] These results demonstrate that the 26S proteasome recognizes a ubiquitinated substrate, and begins to translocate it into the internal cavity of the 20S proteasome, where it is degraded. As the substrate is being translocated into the 20S proteasome, the ubiquitin chains are removed by a previously unknown isopeptidase in the 26S proteasome. Under normal circumstances this isopeptidase activity is not detected because substrate degradation and removal of the ubiquitin chains occurs contemporaneously. However, in the presence of an inhibitor of the 20S peptidases, the substrate is completely deubiquitinated and translocated into the central cavity of the 20S proteasome, but is not degraded. As such, the deubiquitinated product was readily detectable.

[0104] Mass spectrometric analysis of affinity-purified yeast 26S proteasome that is active in deubiquitinating Sic1 revealed only a single deubiquitinating enzyme, Ubp6 (Verma et al., supra, 2000). However, 26S proteasome purified from a ubp6Δ mutant yeast strain and assayed as described above was fully competent to deubiquitinate multi-ubiquitinated Sic1 to yield Sic1 lacking any covalently attached ubiquitin molecules.

[0105] The above demonstrations enable an assay in which the substrate is observed to be converted from a ubiquitinated molecule to a deubiquitinated molecule. Such conversion can be monitored, for example, by detecting the removal of a multi-ubiquitin chain from a protein based on a change in an enzymatic, spectroscopic, fluorescent, or molecular interaction properties of the ubiquitinated protein. For example, a protein that emits light e.g., luciferase, will be made inactive due to the attachment of multi-ubiquitin chains.

[0106] The multi-ubiquitin chains will be attached by enzymatic, chemical, molecular genetic, or a combination of these methods. The cleavage of ubiquitin chains from the inactive luciferase substrate as it is being translocated into the inner cavity of the 20S proteasome in the presence of a 20S peptidase inhibitor will enable the luciferase to fold and thereby acquire the ability to emit light upon ATP hydrolysis.

[0107] The inner chamber of the 20S proteasome can accommodate a folded protein of approximately 70 kD. Therefore, any protein domain less than 70 kD whose enzymatic, spectroscopic, fluorescent, or molecular interaction properties can be reversibly altered by attachment of ubiquitin or a multi-ubiquitin chain can be used as a substrate to monitor ubiquitin isopeptidase activity of the 26S proteasome based on the methods that are disclosed herein.

[0108] Inhibition of the ability of the 26S proteasome to degrade proteins by blockage of ubiquitin isopeptidase activity would be useful for treating cancer, ischemia and reperfusion injury, and diseases characterized by excessive inflammation or autoimmune responses, including, for example, asthma, rheumatoid arthritis, psoriasis, and multiple sclerosis.

[0109] To test whether the deconjugation of ubiquitin from a substrate protein is required for its degradation, the degradation of an ubiquitin-proteasome pathway substrate was examined in cells deficient in Rpn11-associated metalloprotease activity. A set of four congenic strains was generated, including 1) MPR (wild type), 2) mpr1-1, 3) mpr1-1 harboring an integrated copy of RPN11 in which the codons for histidine 109 and 111 have been mutated to alanine (mpr1-1 leu2::LEU2- rpn11 AxA), and 4) mpr1-1 harboring an integrated copy of wild type RPN11 (mpr1-1 leu2::LEU2-RPN11). mpr1-1 is a temperature-sensitive allele of RPN11, and complementation tests demonstrated that wild type RPN11 but not rpn11 AxA complemented the temperature-sensitive growth defect of mpr1-1.

[0110] Each of these strains was transformed with a plasmid that encodes UbV76Val-eΔK-βGal, which is an unstable protein that is degraded via the ubiquitin-proteasome pathway. All strains were pulse-radiolabeled with 35S-Translabel® probe for 5 min at 37° C., then at time 0 a chase was initiated by addition of 10 mM cold methionine and 500 μg/ml cycloheximide. At 7, 14, and 22 min following the initiation of chase, aliquots of the culture were removed and processed for immunoprecipitation with antibodies directed against β-galactosidase. Immunoprecipitated proteins were fractionated by SDS-PAGE and visualized by autoradiography.

[0111] The results demonstrated that the normally unstable UbV76Val-eΔK-βGal reporter was dramatically stabilized in mpr1-1 cells. Rapid turnover of the test protein in mpr1-1 cells was restored by an integrated copy of RPN11, but not rpn11 AxA. Thus, the metalloprotease active site of Rpn11 was required for rapid turnover of proteins by the ubiquitin-proteasome pathway in vivo.

[0112] This result was confirmed in vitro. Purified 26S proteasomes that lack critical metalloprotease active site residues of Rpn11 were unable to deubiquitinate and degrade ubiquitinated Sic1. 26S proteasomes were affinity-purified from Saccharomyces cerevisiae cells in which the PRE1 gene was modified to encode a Pre1 polypeptide tagged with a FLAG epitope (Verma et al., supra, 2000).

[0113] Wild type 26S proteasome was purified from a strain with the genotype pre1::PRE1-FLAG-HIS6 (URA3) his3-11 ade2-1 112 trp1-Δ2 can1-100, mpr1-1, leu2::LEU2-RPN11, and mutant 26S proteasome was purified from a strain with the genotype pre1::PRE1-FLAG-HIS6 (URA3) his3-11 ade2-1 112 trp1-Δ2 can1 -100 mpr1-1, leu2::LEU2-rpn11AXA.

[0114] Purified proteasomes were evaluated by immunoblotting with antibodies directed against Schizosaccharomyces pombe Pad1, which cross-reacted with Rpn11. Wild type and point mutant Rpn11 polypeptides were readily distinguished from Mpr1-1 polypeptide, because the latter is truncated due to a frameshift mutation. The immunoblot analysis revealed that both wild type and mutant proteasomes contained only full-length Rpn11 polypeptide, and none of the truncated Mpr1-1 polypeptide was detected. Thus, the results are directly attributable to the properties of the Rpn11 and Rpn11AxA proteins since no Mpr1-1 protein was present. Mass spectrometric analysis confirmed that the wild type and mutant proteasomes were of equal composition, and all subunits were present.

[0115] To evaluate their activity, wild type and Rpn11AxA mutant 26S proteasomes (100 nM) were incubated with ubiquitinated Sic1 (300 nM) plus ATP (2 mM) in the presence or absence of 00 μM epoxomicin for 0 or 5 min at 25° C. Reactions were terminated by the addition of SDS-PAGE sample buffer, fractionated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies directed against Sic1. Filter-bound antibodies were decorated with goat-anti-rabbit antibody conjugated to horse radish peroxidase and visualized with ECL reagents.

[0116] The results demonstrate that mutant proteasomes were unable to degrade multi-ubiquitinated Sic1 even in the absence of epoxomicin, and were unable to deubiquitinate multiubiquitinated Sic1 (i.e., to convert multiubiquitinated Sic1 from a heterogeneous smear of greater than about 220 kDa to a discrete species of about 40 kDa, which corresponds to Sic1 lacking any covalently attached ubiquitin molecules) in the presence of epoxomicin.

EXAMPLE 2

Deneddylation by COP9 Signalsome (CSN)

[0117] To identify a putative active site within the CSN complex that can mediate cleavage of Nedd8-cullin conjugates, all eight known subunits of the CSN were subjected to sequence analysis by computer. Detailed inspection revealed that the Jab1 subunit contains conserved histidine residues and a conserved aspartic acid that are reminiscent of zinc-coordinating residues in a metallo-β-lactamase. An alignment of these residues is presented in FIG. 3.

[0118] On the basis of this analysis, it was hypothesized that CSN represents the founding member of a novel class of metalloproteases. To test this hypothesis, the effect of a divalent cation chelator on the Nedd8 conjugate cleavage activity associated with CSN was examined. Mutant csn5Δ S. pombe cell extract that contained neddylated Pcu1 was incubated either alone (−) or following addition (+) of wild type lysate (which contained active CSN) supplemented with either methanol vehicle (MeOH), 1 mM 1,10-phenanthroline (O-PT), or 1 mM, 10 mM, or 20 mM EDTA.

[0119] Reactions were incubated 30 min at 30° C. and terminated by addition of SDS-PAGE sample buffer, fractionated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies directed against S. pombe Cul1. Filter-bound antibodies were decorated with goat-anti-rabbit antibody conjugated to horse radish peroxidase and visualized with ECL reagents (Lyapina et al., supra, 2001).

[0120] No inhibition was detected with 1 mM EDTA; less than 50% inhibition was observed with 10 mM EDTA; and almost complete inhibition occurred with 20 mM EDTA, wherein “inhibition” means that Nedd8 was not deconjugated from Cul1 upon addition of CSN and “no inhibition” means that Nedd8 was efficiently deconjugated from Cul1 upon addition of CSN. The status of Nedd8 conjugation to Cul1 was determined by the mobility of Cul1 on the SDS-polyacrylamide gel. Nedd8-conjugated Cul1 migrated more slowly than unmodified Cul.

[0121] Some metal-dependent enzymes are relatively insensitive to inhibition by EDTA, but are nevertheless potently inhibited by the chelator 1,10-phenanthroline, which, at 1 mM, completely inhibited the Nedd8 conjugate cleavage activity of CSN. These results indicate that CSN is a metalloprotease.

[0122] To further confirm the hypothesis that arose from the sequence analysis, the conserved histidine residues and aspartic acid in S. pombe csn5+ were individually mutated, and the ability of each point-mutated gene to complement the cullin deneddylation defect of a csn5Δ mutant was examined (Lyapina et al., supra, 2001; Zhou et al., BMC Biochemistry 2:7, 2001, which is incorporated herein by reference). Wild type S. pombe csn5+ and the indicated mutant derivatives were inserted into the S. pombe expression vector pREP41 and transfected into S. pombe csn5Δ cells.

[0123] Transformants were evaluated for modification state of Pcu1 by fractionating cell extracts on an SDS-polyacrylamide gel, transferring fractionated proteins to nitrocellulose, and immunoblotting with antibodies directed against S. pombe Cul1. Filter-bound antibodies were decorated with goat-anti-rabbit antibody conjugated to horse radish peroxidase and visualized with ECL reagents.

[0124] csn5Δ cells containing an empty vector accumulated Pcu1 exclusively in the Nedd8-modified form. This accumulation was reversed upon expression of wild type csn5+, but not the H118A, H120A, or D131 N mutants. Thus, the putative active site residues identified by computer analysis are absolutely required for CSN activity in deconjugating Nedd8 from Cul1 in vivo.

[0125] These results demonstrate that CSN is has metalloprotease activity. The active site of CSN resides, at least in part, in the Jab1/Csn5 subunit, and this active site is proposed to bind a metal ion which initiates hydrolytic attack of the isopeptide bonds cleaved by CSN. These findings enable the development of targeted screens for compounds that inhibit or enhance the catalytic activity of CSN, and also allow for the rationale design of compounds that modulate CSN activity based on knowledge of the mechanism of action of other metalloenzymes.

[0126] These results provide a basis for re-engineering the specificity of CSN, JabI, or related proteins to generate isopeptidases having desired specificities. Such modified isopeptidase enzymes can be useful in a variety of applications involving the cleavage of protein cross-links or conjugates. In addition, the results provide a basis for designing compounds that can inhibit the prokaryotic homologs of Csn5/Jab1 and for developing screening assays useful for isolating compounds having such inhibitory activity, such inhibitors being useful, for example, as antibiotics.

[0127] The role of Cys box (Cys 145) in association with the ability of CSN to deconjugate Nedd8 from Cul1 was examined using an in vitro deconjugation assay as described above. The results demonstrated that a Jab1 mutant, in which the conserved cysteine of the Cys box (Cys 145) was mutated to alanine, had wild-type levels of Nedd8 conjugate cleavage activity.

EXAMPLE 3

AMSH, AMSH1, and AMSH2

[0128] As disclosed above, the existence of a metalloprotease active site motif, the JAMM domain (:Jab1-associated metalloenzyme motif”), has been identified. The Rpn11 subunit of the 26S proteosome and the Csn5 subunit of the COP9-signalsome (CSN) both contain a JAMM domain. Thus, direct evidence is provided that the JAMM domain of Csn5 is essential for the Nedd8 isopeptidase activity of CSN, and the JAMM domain of Rpn11 is essential for the ubiquitin isopeptidase activity of the 26S proteosome.

[0129] These results demonstrate that eukaryotic JAMM domain proteins contain isopeptidase activity that deconjugate modifier proteins from target proteins, wherein the carboxy terminus of the modifier protein is attached via a peptide bond to a free amino group of the target protein, including, but not restricted to the amino terminus and the epsilon amino group of lysine residues in the target protein.

[0130] In addition to Csn5 and Rpn11, several other proteins expressed in human cells contain an intact JAMM domain (see Table 1), including, for example, the AMSH proteins, which have been implicated in cytokine signaling, TGF-β signaling, and survival of hippocampal neurons (Ishii et al., Mol. Cell Biol. 24:8626-8637, 2001; Itoh et al., EMBO J. 15:4132-4142, 2001; Tanaka et al., J. Biol. Chem. 27:19129-35, 1999).

[0131] The present results indicate that the histidine 335, histidine 337, and aspartate 348 residues of AMSH (and the equivalent residues in AMSH1 and AMSH2, see FIG. 2) specify a metalloprotease active site. Furthermore, compounds that inhibit the active site specified by these residues likely can inhibit the ability of AMSH, AMSH1, and AMSH2 to deconjugate a modifier protein from a target protein, for example, a modifier protein such as NEDD8, UBL1, SMT3H2, SMT3H1, APG12, FAT10, Fau, UCRP/ISG15, URM1, or UBL5.

EXAMPLE 4

CSN Isopeptidase Activity is Required for SCF Ubiquitin Ligase Activity

[0132] To confirm that diminished deneddylation activity exacerbates the effect of diminished SCF activity, it needs to be established that specifically disrupting the deneddylation activity of CSN exacerbates the effect of a reduction in SCF activity, and to further demonstrate that such an effect occurs for multiple SCF subunits.

[0133] To determine whether diminished CSN-associated deneddylation activity can exacerbate the effect of diminished SCF ubiquitin ligase activity, genetic interaction studies were performed in budding yeast. Although it is not clear whether budding yeast cells contain the entire CSN complex as found in plant and animal cells, they at least contain an ortholog of the Csn5 subunit, which is the most highly conserved member of the complex and is the subunit that contains the active site residues that catalyze deneddylation of substrate. Mutant rrilΔ cells accumulate the budding yeast Cul1 orthologue Cdc53 exclusively in a Nedd8-modified form (Lyapina et al., supra, 2001), indicating that RRI1 function is essential for Cdc53 deneddylation. Likewise, the Csn5 subunit of CSN is required to maintain the proper neddylation state of Cul1 in fission yeast (Lyapina et al., 2001) and in Arabidopsis thaliana (Schwechheimer et al., 2001).

[0134] It was previously disclosed that Csn5 orthologues in Drosophila melanogaster, Caenorhabditis elegans, S. pombe and humans contain two conserved histidine residues and a conserved aspartate residue that together coordinate a metal ion and form the active site that catalyzes cleavage of the isopeptide bond that links Nedd8 to substrate proteins. As shown in Table 2, S. cerevisiae Rri1 contains the same conserved residues. Whereas wild-type RRI1 on a plasmid complemented the Cdc53 deneddylation defect of an rrilΔ mutant, RRI1 plasmids in which the conserved histidine or aspartate residues have been altered were unable to restore the normal neddylation status of Cdc53 (not shown). This result demonstrates that the JAMM domain residues are required for deneddylation activity of Rr1.

2TABLE 2Alignment of Csn5 HomologsOrga-nismAlignmentAcc. No.HumanVGRLENAIGWYHSHPGYGCWLSGIDVSTQXP_034280(5)*Droso-VGRMEHAVGWYHSHPGYGCWLSGIDVSTQAAF55321phila(6)Arabi-AGRLENVVGWYHSHPGYGCWLSGIDVSTQAAC36343dopsis(7)C.EGRKEKVVGWYHSHPGYGCWLSGIDVSTQAAB37991elegans(8)S.KGAKLNVVGWFHSHPGYDCWLSNIDIQTQNP_010065cerevisiae(9)bold print indicates conserved JAMM domain consensus sequence (see, also, SEQ ID NO:1) *—SEQ ID NO: S. cerevisiae Rri1.

[0135] Plasmids encoding GST-Rri1 fusion proteins, including wild type Rri1 and Rri1 mutants having a point mutation in histidine 179 (H179A), histidine 181 (H 181 A), or aspartate 192 (D192A), were transformed into SKP1myc9 rrilΔ yeast cells. Logarithmically growing cultures of transformed cells were lysed with glass beads, and the lysate was subjected to immunoprecipitation with anti-myc monoclonal antibody 9E10. Washed immunoprecipitates were fractionated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-Cdc53 or anti-GST polyclonal antibodies. Essentially all of the Cdc53 in rrilΔ cells migrated as a Rub1-modified species (Rub1 is the S. cerevisiae ortholog of Nedd8). Re-introduction of wild type Rri1 expression promoted cleavage of Rub 1 from a fraction of the Cdc53 molecules. However, the H179A, H181A, and D192N mutants were unable to restore Rub1 cleavage activity. Screening with the anti-GST antibody confirmed that the wild type and mutant forms of GST-Rri1 were expressed.

[0136] The requirement of RRI1-associated Nedd8 isopeptidase activity to sustain SCF activity was examined in cells in which SCF function was compromised by mutation. The following nine strains were constructed: 1) rrilΔ; 2) cdc53-1; 3) cdc34-2; 4) cdc4-1; 5) skp1-12; 6) rrilΔ cdc53-1; 7) rrilΔ cdc34-2; 8) rrilΔ cdc4-1; and 9) rrilΔ skp1-12. The cdc53-1, cdc34-2, skp1-12, and cdc4-1 alleles are temperature-sensitive conditional lethal mutations. In S. cerevisiae, Cdc34 is the only Ubc (E2) reported to interact with SCF.

[0137] The strains were grown at an intermediate temperature (33° C.) that is normally permissive for the function of the temperature-sensitive gene products. Cells having the various genotypes were progressively diluted in 5-fold increments from left to right, and were spotted onto YPD plates and incubated at 25° C. (control; permissive temperature) or 33° C. for 1-2 days. All of the single mutant strains were able to grow at 33° C., whereas all of the double mutant strains exhibited a severe growth defect. Thus, the growth of all single mutants (cdc53-1, cdc34-2, cdc4-1, and skp1-12) was exacerbated by the rrilΔ mutation.

[0138] The above result indicates that Nedd8 isopeptidase activity is necessary in cells with compromised SCF pathway activity. However, the RRI1 gene (like the CSN complex in other organisms) also may have a second activity that promotes SCF pathway function. To address this possibility for the cdc34-2 and skp1-12 mutations, doubly-mutated strains (i.e., rrilΔ and SCF pathway deficient) were generated that contained a plasmid encoding GST fusion proteins comprising either the wild-type RRI1 gene or a mutant in which the conserved histidine 179, histidine 181, or aspartate 192 residue is substituted with an alanine residue. The GST-RRI1 fusion construct was expressed from the GAL1 promoter. Transformants were progressively diluted in 5-fold increments, spotted onto YPD (Glucose) or YPG (Galactose) plates, and incubated at 25° C., 33° C. or 34° C. for 1-2 days; the GST-RRI1 fusion is expressed only on galactose-containing medium. The wild-type, but not mutant, RRI1 plasmids restored growth at 33° C. to the rrilΔ cdc34-2 and rrilΔskp1-12 mutant strains. Together, these results demonstrate that inhibition of Nedd8 isopeptidase activity exacerbates the effect of treatments that diminish SCF activity.

[0139] To determine whether deneddylation is important for the role of the SCF pathway in promoting protein degradation, the degradation of the cell cycle regulatory protein Sic1 was examined in skp1-12 and skp1-12 rrilΔ cells. Sic1 is targeted for degradation in an SCF/Cdc34-dependent manner (Schwob et al., Cell 79:233-244, 1994). Immunoblot analysis of these strains at various time points after SIC1 transcription was extinguished revealed that the Sic1 polypeptide level declined more rapidly in skp1-12 cells than in skp1-12 rril cells. This result demonstrates that deneddylation by CSN is required to maintain optimal activity of the SCF pathway.

[0140] In summary, this Example demonstrates that inhibition of the isopeptidase activity of CSN, for example, by introducing mutations that alter the conserved JAMM domain residues of the isopeptidase, synthetically enhance the effect of mutations that diminish SCF activity. This result is in contrast with the result predicted from prior biochemical studies, i.e., that a CNS mutant would suppress an SCF mutant. As such, these results indicate that SCF activity can be reduced or inhibited using an agent that decreases CNS isopeptidase activity, and that such an agent can be used in combination, for example, with a second agent that more directly inhibits SCF activity, thus providing a means to enhance the efficacy of agents that inhibit SCF ubiquitin ligase.

[0141] Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Number	Date	Country
60355334	Feb 2002	US
60261314	Jan 2001	US
60322322	Sep 2001	US
60322030	Sep 2001	US

	Number	Date	Country
Parent	10047253	Jan 2002	US
Child	10340578	Jan 2003	US
Parent	10046961	Jan 2002	US
Child	10340578	Jan 2003	US

Modulation of COP9 signalsome isopeptidase activity

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