AU2003230701C1 - Human deubiquitinating protease gene on chromosome 7 and its murine ortholog - Google Patents

Human deubiquitinating protease gene on chromosome 7 and its murine ortholog Download PDF

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AU2003230701C1
AU2003230701C1 AU2003230701A AU2003230701A AU2003230701C1 AU 2003230701 C1 AU2003230701 C1 AU 2003230701C1 AU 2003230701 A AU2003230701 A AU 2003230701A AU 2003230701 A AU2003230701 A AU 2003230701A AU 2003230701 C1 AU2003230701 C1 AU 2003230701C1
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Chang Hahn
Hong Liu
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Description

WO 03/083050 PCT/US03/08590 HUMAN DEUBIQUITINATING PROTEASE GENE ON CHROMOSOME 7 AND ITS MURINE ORTHOLOG 5 Background of the Invention The role of ubiquitin in protein degradation was discovered and the main enzymatic reactions of this system elucidated in biochemical studies in a cell-free system from reticulocytes. In this system, proteins are targeted for degradation by covalent ligation to o ubiquitin, a 76-amino-acid-residue protein. Briefly, ubiquitin-protein ligation requires the sequential action of three enzymes. The C-terminal Gly residue of ubiquitin is activated in an ATP-requiring step by a specific activating enzyme, El (Step 1). This step consists of an intermediate formation of ubiquitin adenylate, with the release of PPi, followed by the binding of ubiquitin to a Cys residue of El in a thiolester linkage, with the release of AMP. Activated 5 ubiquitin is next transferred to an active site Cys residue of a ubiquitin-carrier protein, E2 (Step 2). In the third step catalyzed by a ubiquitin-protein ligase or E3 enzyme, ubiquitin is linked by its C-terminus in an amide isopeptide linkage to an -amino group of the substrate protein's Lys residues (Step 3). ! Proteins ligated to polyabiquitin chains are usually degraded by the 26S proteasome complex that requires ATP hydrolysis for its action. The 26S proteasome is formed by an ATP-dependent assembly of a 20S proteasome, a complex that contains the protease catalytic sites, with 19S "cap" or regulatory complexes. The 19S complexes contain several ATPase subunits and other subunits that are presumably involved in the specific action of the 26S Z5 proteasome on ubiquitinylated proteins. The roles of ATP in the assembly of the 26S proteasome complex and in its proteolytic action are not understood. The action of the 26S proteasome presumably generates several types of products: free peptides, short peptides still linked to ubiquitin via their Lys residues, and polyubiquitin chains (Step 4). The latter two products are converted to free and reusable ubiquitin by the action of ubiquitin-C-terminal 30 hydrolases or isopeptidases (Steps 5 and 6). Some isopeptidases may also disassemble certain ubiquitin-protein conjugates (Step 7) and thus prevent their proteolysis by the 26S proteasome. The latter type of isopeptidase action may have a correction function to salvage incorrectly WO 03/083050 PCT/US03/08590 2 ubiquitinylated proteins or may have a regulatory role. Short peptides formed by the above processes can be further degraded to free amino acids by cytosolic peptidases (Step 8). Ubiquitin-mediated degradation of protein is involved in various biological processes. 5 The selective and programmed degradation of cell-cycle regulatory proteins, such as cyclins, inhibitors of cyclin-dependent kinases, and anaphase inhibitors are essential events in cell cycle progression. Cell growth and proliferation are further controlled by ubiquitin-mediated degradation of tumor suppressors, protooncogenes, and components of signal transduction systems. The rapid degradation of numerous transcriptional regulators is involved in a variety .0 of signal transduction processes and responses to environmental cues. The ubiquitin system is clearly involved in endocytosis and down-regulation of receptors and transporters, as well as in the degradation of resident or abnormal proteins in the endoplasmic reticulum. There are strong indications for roles of the ubiquitin system in development and apoptosis, although the target proteins involved in these cases have not been identified. Dysfunction in several [5 ubiquitin-mediated processes causes pathological conditions, including malignant transformation. Our knowledge of different signals in proteins that mark them for ubiquitinylation is also limited. Recent reports indicate that many proteins are targeted for degradation by 2o phosphorylation. It was observed previously that many rapidly degraded proteins contain PEST elements, regions enriched in Pro, Glu, Ser, and Thr residues. More recently, it was pointed out that PEST elements are rich in S/TP sequences, which are minimum consensus phosphorylation sites for Cdks and some other protein kinases. Indeed, it now appears that in several (though certainly not all) instances, PEST elements contain phosphorylation sites 25 necessary for degradation. Thus multiple phosphorylations within PEST elements are required for the ubiquitinylation and degradation of the yeast G1 cyclins Cln3 and Cln2, as well as the Gcn4 transcriptional activator. Other proteins, such as the mammalian G1 regulators cyclin E and cyclin D1, are targeted for ubiquitinylation by phosphorylation at specific, single sites. In the case of the IkBa inhibitor of the NF-kB transcriptional regulator, phosphorylation at two 30 specific sites, Ser-32 and Ser-36, is required for ubiquitin ligation. P-cateinin, which is targeted for ubiquitin-mediated degradation by phosphorylation, has a sequence motif similar to that of IkBa around these phosphorylation sites. However, the homology in phosphorylation patterns of these two proteins is not complete, because phosphorylation of WO 03/083050 PCT/US03/08590 3 other sites of B-catenin is also required for its degradation. Other proteins targeted for degradation by phosphorylation include the Cdk inhibitor Sicip and the STAT1 transcription factor. Though different patterns of phosphorylation target different proteins for degradation, a common feature appears to be that the initial regulatory event is carried out by a protein 5 kinase, while the role of a ubiquitin ligase would be to recognize the phosphorylated form of the protein substrate. It further appears that different ubiquitin ligases recognize different phosphorylation patterns as well as additional motifs in the various protein substrates. However, the identity of such E3s is unknown, except for some PULC-type ubiquitin ligases that act on some phosphorylated cell-cycle regulators in the budding yeast. The multiplicity of 10 signals that target proteins for ubiquitin-mediated degradation (and of ligases that have to recognize such signals) is underscored by observations that the phosphorylation of some proteins actually prevents their degradation. Thus the phosphorylation of the c-Mos protooncogene on Ser3 and the multiple phosphorylations of c-Fos and c-Jun protooncogenes at multiple sites by MAP kinases suppress their ubiquitinylation and degradation. 15 In addition to the families of enzymes involved in conjugation of ubiquitin, a very large family of deubiquitinating enzymes has recently been identified from various organisms. These enzymes have several possible functions. First, they may have peptidase activity and cleave the products of ubiquitin genes. Ubiquitin is encoded by two distinct classes of genes. 20 One is a polyubiquitin gene, which encodes a linear polymer of ubiquitins linked through peptide bonds between the C-terminal Gly and N-terminal Met of contiguous ubiquitin molecules. Each copy of ubiquitin must be released by precise cleavage of the peptide bond between Gly-76-Met-1 of successive ubiquitin moieties. The other class of ubiquitin genes encodes ubiquitin C-terminal extension proteins, which are peptide bond fusions between the 25 C-terminal Gly of ubiquitin and N-terminal Met of the extension protein. To date, the extensions described are ribosomal proteins consisting of 52 or 76-80 amino acids. These ubiquitin fusion proteins are processed to yield ubiquitin and the corresponding C-terminal extension proteins. Second, deubiquitinating enzymes may have isopeptidase activities. When a target protein is degraded, deubiquitinating enzymes can cleave the polyubiquitin chain from 30 the target protein or its remnants. The polyubiquitin chain must also be disassembled by deubiquitinating enzymes during or after proteolysis by the 26 S proteasome, regenerating free monomeric ubiquitin. In this way, deubiquitinating enzymes can facilitate the ability of the 26 S proteasome to degrade ubiquitinated proteins. Third, deubiquitinating enzymes may WO 03/083050 PCT/US03/08590 4 hydrolyze ester, thiolester, and amide linkages to the carboxyl group of Gly-76 of ubiquitin. Such nonfunctional linkages may arise from reactions between small intracellular compounds such as glutathione and the El-, E2-, or E3 -ubiquitin thiolester intermediates. Fourth, deubiquitinating enzymes may compete with the conjugating system by removing ubiquitin 5 from protein substrates, thereby rescuing them from degradation or any other function mediated by ubiquitination. Thus generation of ubiquitin by deubiquitinating enzymes from the linear polyubiquitin and ubiquitin fusion proteins and from the branched polyabiquitin ligated to proteins should be essential for maintaining a sufficient pool of free ubiquitin. Many deubiquitinating enzymes exist, suggesting that these deubiquitinating enzymes recognize .0 distinct substrates and are therefore involved in specific cellular processes. Although there is recent evidence to support such specificity of these deubiquitinating enzymes, the structure function relationships of these enzymes remain poorly studied. Deubiquitinating enzymes can be divided broadly on the basis of sequence homology [5 into two classes, the ubiquitin-specific processing protease (UBP or USP, also known as type 2 ubiquitin C-terminal hydrolase (type 2 UCH)) and the UCH, also known as type 1 UCH). UCH (type 1 UCH) enzymes hydrolyze primarily C-terminal esters and amides of ubiquitin but may also cleave ubiquitin gene products and disassemble polyubiquitin chains. They have in common a 210-amino acid catalytic domain, with four highly conserved blocks of 20 sequences that identify these enzymes. They contain two very conserved motifs, the CYS and HIS boxes. Mutagenesis studies revealed that the two boxes play important roles in catalysis. Some UCH enzymes have significant C-terminal extensions. The functions of the C-terminal extensions are still unknown but appear to be involved in proper localization of the enzyme. The active site of these UCH enzymes contains a catalytic triad consisting of cysteine, 25 histidine, and aspartate and utilizes a chemical mechanism similar to that of papain. The crystal structure of one of these, UCH-L3, has been solved at 1.8 A resolution. The enzyme comprises a central antiparallel B-sheet flanked on both sides by helices. The B-sheet and one of the helices are similar to those observed in the thiol protease cathepsin B. The similarity includes the three amino acid residues that comprise the active site, Cys 95 , His 16 9 , and Asp 184 . 30 The active site appears to fit the binding of ubiquitin that may anchor also at an additional site. The catalytic site in the free enzyme is masked by two different segments of the molecule that limit nonspecific hydrolysis and must undergo conformational rearrangement after substrate binding.
WO 03/083050 PCT/US03/08590 5 UBP (type 2 UCH) enzymes are capable of cleaving the ubiquitin gene products and disassembling polyubiquitin chains after hydrolysis. It appears that there is a core region of about 450 amino acids delimited by CYS and HIS boxes. Many of these isoforms have N 5 terminal extensions and a few have C-terminal extensions. In addition, there are variable sequences in the core region of many of the isoforms. The functions of these divergent sequences remain poorly characterized. Another interesting function of specific UBPs is the regulation of cell proliferation. It was observed that cytokines induced in T-cells specific de ubiquitinating enzymes (DUBs), termed DUB-1 and DUB-2. DUB-1 is induced by stimulation to of the cytokine receptors for IL-3, IL-5, and GM-CSF, suggesting a role in its induction for the B-common (betac) subunit of the interleukin receptors. Overexpression of a dominant negative mutant of JAK2 inhibits cytokine induction of DUB-1, suggesting that the regulation of the enzyme is part of the cell response to the JAK/STAT signal transduction pathway. Continued expression of DUB-1 arrests cells at G 1 ; therefore, the enzyme appears to regulate cellular 15 growth via control of the Go-G 1 transition. The catalytic conserved Cys residue of the enzyme is required for its activity. DUB-2 is induced by IL-2 as an immediate early (IE) gene that is down-regulated shortly after the initiation of stimulation. The function of this enzyme is also obscure. It may stimulate or inhibit the degradation of a critical cell-cycle regulator. 20 Cytokines, such as interleukin-2 (IL-2), activate intracellular signaling pathways via rapid tyrosine phosphorylation of their receptors, resulting in the activation of many genes involved in cell growth and survival. The deubiquitinating enzyme DUB-2 is induced in response to IL-2 and is expressed in human T-cell lymphotropic virus-I (HTLV-1) transformed T cells that exhibit constitutive activation of the IL-2 JAK/STAT (signal 25 transducers and activators of transcription) pathway, and when expressed in Ba/F3 cells DUB 2 markedly prolonged IL-2-induced STAT5 phosphorylation. Although DUB-2 does not enhance IL-2-mediated proliferation, when withdrawn from growth factor, cells expressing DUB-2 had sustained STAT5 phosphorylation and enhanced expression of IL-2-induced genes cis and c-myc. DUB-2 expression markedly inhibited apoptosis induced by cytokine 30 withdrawal allowing cells to survive. Therefore, DUB-2 has a role in enhancing signaling through the JAK/STAT pathway, prolonging lymphocyte survival, and, when constitutively expressed, may contribute to the activation of the JAK/STAT pathway observed in some transformed cells. (Migone, T.-S., et al., Blood. 2001;98:1935-1941).
WO 03/083050 PCT/US03/08590 6 Protein ubiquitination is an important regulator of cytokine-activated signal transduction pathways and hematopoietic cell growth. Protein ubiquitination is controlled by the coordinate action of ubiquitin-conjugating enzymes and deubiquitinating enzymes. 5 Recently a novel family of genes encoding growth-regulatory deubiquitinating enzymes (DUB-1 and DUB-2) has been identified. DUBs are immediate-early genes and are induced rapidly and transiently in response to cytokine stimuli. By means of polymerase chain reaction amplification with degenerate primers for the DUB-2 complementary DNA, 3 murine bacterial artificial chromosome (BAC) clones that contain DUB gene sequences were isolated. One [0 BAC contained a novel DUB gene (DUB-2A) with extensive homology to D UB-2. Like DUB 1 and DUB-2, the D UB-2A gene consists of 2 exons. The predicted DUB-2A protein is highly related to other DUBs throughout the primary amino acid sequence, with a hypervariable region at its C-terminus. In vitro, D UB-2A had functional deubiquitinating activity; mutation of its conserved amino acid residues abolished this activity. The 5' flanking sequence of the 15 DUB-2A gene has a hematopoietic-specific functional enhancer sequence. It is proposed that there are at least 3 members ofthe D UB subfamily (DUB-1, DUB-2, and DUB-2A) and that different hematopoietic cytokines induce specific DUB genes, thereby initiating a cytokine specific growthresponse. (Baek, K.-H., et al, Blood. 2001;98:636-642). 20 Protein ubiquitination also serves regulatory functions in the cell that do not involve proteasome-mediated degradation. For example, Hicke and Riezman have recently demonstrated ligand-inducible ubiquitination of the Ste2 receptor in yeast. Ubiquitination of the Ste2 receptor triggers receptor endocytosis and receptor targeting to vacuoles, not proteasomes. Also, Chen et al. have demonstrated that activation of the IB kinase requires a 25 rapid, inducible ubiquitination event. This ubiquitination event is a prerequisite for the specific phosphorylation of IB and does not result in subsequent proteolysis of the kinase complex. The ubiquitination of Ste2 and IB kinase appears reversible, perhaps resulting from the action of a specific deubiquitinating enzyme. 30 A large superfamily of genes encoding deubiquitinating enzymes, or UBPs, has recently been identified. UBPs are ubiquitin-specific thiol-proteases that cleave either linear ubiquitin precursor proteins or post-translationally modified proteins containing isopeptide WO 03/083050 PCT/US03/08590 7 ubiquitin conjugates. The large number of UBPs suggests that protein ubiquitination, like protein phosphorylation, is a highly reversible process that is regulated in the cell. Interestingly, UBPs vary greatly in length and structural complexity, suggesting 5 functional diversity. While there is little amino acid sequence similarity throughout their coding region, sequence comparison reveals two conserved domains. The Cys domain contains a cysteine residue that serves as the active enzymatic nucleophile. The His domain contains a histidine residue that contributes to the enzyme's active site. More recent evidence demonstrates six homology domains contained by all members of the ubp superfamily. 10 Mutagenesis of conserved residues in the Cys and His domains has identified several residues that are essential for UBP activity. Recently, a growth regulatory deubiquitinating enzyme, DUB-1, that is rapidly induced in response to cytokine receptor stimulation was identified. DUB-1 is specifically induced by 15 the receptors for IL-3, granulocyte macrophage-colony-stimulating factor, and IL-5, suggesting a specific role for the c subunit shared by these receptors. In the process of cloning the D UB-1 gene, a family of related, cross-hybridizing DUB genes was identified. From this, other DUB genes might be induced by different growth factors. Using this approach, an IL-2 inducible DUB enzyme, DUB-2 and closely related DUB-2a were identified. DUB-i and 20 DUB-2 are more related to each other than to other members of the ubp superfamily and thereby define a novel subfamily of deubiquitinating enzymes. Hematopoietic-specific, cytokine induced DUBs in murine system have shown to prolong cytokine receptor, see Migone, T. S., et al. (2001). The deubiquitinating enzyme 25 DUB-2 prolongs cytokine-induced signal transducers and activators of transcription activation and suppresses apoptosis following cytokine withdrawal, Blood 98, 1935-41; Zhu, Y., et al., (1997). DUB-2 is a member of a novel family of cytokine-inducible deubiquitinating enzymes, J Biol Chem 272, 51-7 and Zhu, Y., et al., (1996). The murine DUB-1 gene is specifically induced by the betac subunit of interleukin-3 receptor, Mol Cell Biol 16, 4808 30 17.). These effects are likely due to the deubiquitination of receptors or other signaling intermediates by DUB-1 or DUB-2, murine analogs of hDUBs. Inhibition of hDUBs may achieve downregulation of specific cytokine receptor signaling, thus modulating specific immune responses.
WO 03/083050 PCT/US03/08590 8 Cytokines regulate cell growth by inducing the expression of specific target genes. A recently identified a cytokine-inducible, immediate-early gene, DUB-1, encodes a deubiquitinating enzyme with growth regulatory activity. In addition, a highly related gene, 5 DUB-2, that is induced by interleukin-2 was identified. The DUB-2 mRNA was induced in T cells as an immediate-early gene and was rapidly down-regulated. Like DUB-1, the DUB-2 protein had deubiquitinating activity in vitro. When a conserved cysteine residue of D UB-2, required for ubiquitin-specific thiol protease activity, was mutated to serine (C60S), deubiquitinating activity was abolished. DUB-1 and DUB-2 proteins are highly related [0 throughout their primary amino acid sequence except for a hypervariable region at their COOH terminus. Moreover, the DUB genes co-localize to a region of mouse chromosome 7, suggesting that they arose by a tandem duplication of an ancestral DUB gene. Additional DUB genes co-localize to this region, suggesting a larger family of cytokine-inducible DUB enzymes. We propose that different cytokines induce specific DUB genes. Each induced DUB 15 enzyme thereby regulates the degradation or the ubiquitination state of an unknown growth regulatory factor, resulting in a cytokine-specific growth response. On the basis of these structural criteria, additional members of the DUB subfamily can be identified in the GenBanki". The highest degree of homology is in the Cys and His domains. Additionally, this putative humanDUB protein contains a Lys domain (amino acids 400-410) 20 and ahypervariable region (amino acids 413-442). Murine DUB (mDUB) subfamily members differ from other UBPs by functional criteria as well. mDUB subfamily members are cytokine-inducible, immediate-early genes and may therefore play regulatory roles in cellular growth or differentiation. Also, DUB proteins 25 are unstable and are rapidly degraded by ubiquitin-mediated proteolysis shortly after their induction. mDUB reports demonstrate that specific cytokines, such as IL-2 and IL-3, induce specific deubiquitinating enzymes (DUBs). The DUB proteins may modify the ubiquitin 30 proteolytic pathway and thereby mediate specific cell growth or differentiation signals. These modifications are temporally regulated. The DUB-2 protein, for instance, is rapidly but transiently induced by IL-2. Interference of DUB enzymes with specific isopeptidase inhibitors may block specific cytokine signaling events.
WO 03/083050 PCT/US03/08590 9 The prior art teaches some partial sequences with homology to DUBs; specifically Human cDNA sequence SEQ ID NO:17168 in EP1074617-A2; a human protease and protease inhibitor PPIM-4 encoding cDNA; in W0200110903-A2 and human ubiquitin 5 protease 23431 coding sequence in W0200123589-A2. References 1. Baek, K. H., Mondoux, M. A., Jaster, R., Fire-Levin, E., and D'Andrea, A. D. (2001). DUB-2A, a new member of the DUB subfamily of hematopoietic deubiquitinating enzymes, Blood 98, 63 6-42. 10 2. Jaster, R., Baek, K. H., and D'Andrea, A. D. (1999). Analysis of cis-acting sequences and trans acting factors regulating the interleukin-3 response element of the DUB-1 gene, Biochim Biophys Acta 1446, 308-16. 3. Jaster, R., Zhu, Y., Pless, M., Bhattacharya, S., Mathey-Prevot, B., and D'Andrea, A. D. (1997). JAK2 is required for induction of the murine DUB-1 gene, Mol Cell Biol 17, 3364-72. 15 4. Migone, T. S., Humbert, M., Rascle, A., Sanden, D., D'Andrea, A., Johnston, J. A., Baek, K. H., Mondoux, M. A., Jaster, R., Fire-Levin, E., et al. (2001). The deubiquitinating enzyme DUB-2 prolongs cytokine-induced signal transducers and activators of transcription activation and suppresses apoptosis following cytokine withdrawal, Blood 98, 1935-41. 5. Zhu, Y., Carroll, M., Papa, F. R., Hochstrasser, M., and D'Andrea, A. D. (1996a). DUB-1, a 20 deubiquitinating enzyme with growth-suppressing activity, Proc Natl Acad Sci U S A 93, 3275-9. 6. Zhu, Y., Lambert, K., Corless, C., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and D'Andrea, A. D. (1997). DUB-2 is a member of a novel family of cytokine-inducible deubiquitinating enzymes, J Biol Chem 272, 51-7. 7. Zhu, Y., Pless, M., Inhom, R., Mathey-Prevot, B., and D'Andrea, A. D. (1996b). The murine DUB-1 25 gene is specifically induced by the betac subunit of interleukin-3 receptor, Mol Cell Biol 16, 4808-17. Scott Emr described a role for monoubiquitination in protein sorting in the late endosome, which has a role in determining which proteins, both newly synthesized and endocytosed, will be delivered to the lumen of the vacuole and which to its limiting membrane. Proteins 30 destined for lumen are sorted into internal vesicles at the multivesicular body (MVB) stage of endosome maturation, whereas proteins destined for the vacuolar membrane, or for recycling to the plasma membrane, remain in the endosome's limiting membrane. Emr showed that the sorting of a vacuolar hydrolase into MVB vesicles requires the monoubiqutination of this cargo molecule at a specific lysine residue (Katzmann et al., 2001). Thus, monoubiquitination 35 is a green light for traffic to proceed from this important intracellular intersection to the lumen WO 03/083050 PCT/US03/08590 10 of the vacuole. The policeman directing the traffic is an endosome-localized protein complex called ESCRT-I, one of whose components, Vps23, plays a key role in recognizing the cargo's ubiquitin signal (Katzmann et al., 2001). Vps23 is one of a small family of UTEV proteins (ubiquitin E2 variants) that resemble E2s but cannot perform canonical E2 functions. The ESCRT-I complex binds ubiquitin, and a mutation in Vps23 that cripples ubiquitin-dependent sorting in the MVB pathway abolishes ubiquitin binding to ESCRT-I. A model in which Vsp23 binds ubiquitin directly, while still inferential, received support from structural studies of a different UEV protein. Intriguingly, the mammalian homolog of Vps23, known as tsg1O1, is a tumor suppressor (Li and Cohen, 1996) The current results suggest that mutations in tsglO could cause persistent signaling by growth factor receptors because of inappropriate receptor recycling to the plasma membrane, thus leading to tumorigenesis. A role for monoubiquitination in triggering the first step of endocytosis-the internalization of plasma membrane proteins-is well established (Hicke, 2001), but how this signal is 5 recognized has been unclear. Linda Hicke reported that yeast Ent1 is vital for the ubiquitin dependent endocytosis of yeast factor receptor (see also Wendland et al., 1999). Ent1 carries a proposed ubiquitin binding motif called the UIM domain (Hofnann and Falquet, 2001 ), and Hicke showed that Entl indeed binds ubiquitin directly. Ent1 also binds clathrin (Wendland et al., 1999) and so is poised to link monoubiquitinated cargo molecules to the endocytic 0 machinery. Hicke's and Emr's results suggest that the ability of monoubiquitin to signal two different trafficking outcomes relies in part on distinct localizations of the relevant signal recognizing components-Ent1 resides at the plasma membrane, while ESCRT-I is associated with late endosomes. !5 Fanconi Anemia (FA) is a rare cancer susceptibility disorder associated with cellular sensitivity to DNA damage that can be caused by mutations in at least seven genes. Alan D'Andrea shed new light on the molecular basis of FA: monoubiquitination of a specific lysine residue in one FA protein, known as D2, requires the activities of four upstream FA genes and leads to the relocalization of D2 within the nucleus (Garcia-Higuera et al., 2001). 30 in normal cells, monoubiquitination of D2 is strongly augmented following DNA damage and is strictly required for damage-associated targeting of D2 and BRCA1 to subnuclear foci. Thus, D2 monoubiquitination links an FA protein complex to the BRCA1 repair machinery. Although the downstream events in this pathway are still unclear, localization of the signal- WO 03/083050 PCT/US03/08590 11 recognizing factor(s) will likely be critical. This new function of ubiquitin carries a strong flavor of certain roles of Sumo-1, a UbL that has been implicated in protein targeting to specific subnuclear structures (Hochstrasser, 2000). 5 Polyubiquitin chains are well known as a signal for substrate destruction by 26S proteasomes. But there are several kinds of chains, linked through different lysines of ubiquitin, suggesting that different chains might be distinct signals (Pickart, 2000). James Chen provided rigorous proof of this hypothesis by showing that noncanonical polyubiuqitination can activate phosphorylation-in contrast to numerous examples of the converse regulation (Hershko and 10 Ciechanover, 1998). Postreplicative DNA repair and the activation of IkBa kinase (IKK) require chains linked through Lys63, rather than the Lys48-chains that usually signal proteasomal proteolysis. Chen found that Tak1 kinase is a downstream target of Lys63-chain signaling in the IKK activation pathway. The assembly of these chains depends on an unusual UEV/E2 complex and a RING finger protein, Traf6 (Deng et al., 2000). (The RING finger 15 defines a large E3 family.) Modification of Traf6 with a Lys63 -chain leads to the activation of Takl, which in turn phosphorylates IKK (Wang et al., 2001). Activated IKK then phosphorylates IkBc and triggers its tagging with Lys48-chains. Only then do proteasomes enter the picture-they degrade IkBc and thereby free its partner, NFkB, to translocate to the nucleus and activate the expression of inflammatory response genes. Chen's results suggest 20 that Traf6 is the target of the Lys63-chain, as well as a catalyst of its assembly. Indeed, many other RING E3s also self-modify-although the consequence is more apt to be suicide (cf. tagging with Lys48-chains) than the kind of personality change seen with Traf6 (Joazeiro and Weissman, 2000). It remains to be seen if a similar mechanism applies in DNA repair, where a different RING protein, the Rad5 helicase, binds to a related UEV/E2 complex (Ulrich and 25 Jentsch, 2000). New genetic data reported by Helle Ulrich confirmed the central importance of Rad5 in Lys-63 chain signaling in DNA repair (Ulrich, 2001). These reports suggest a variety of new functions of protein ubiquitination and its potential involvement of subcellular trafficking including nucleus and the lumen of the intracellular 30 vesicles. Thus regulation of ubiquitination by deubiquitinating proteases in various subcellular localization is become a critical issue.
WO 03/083050 PCT/US03/08590 12 Recently, a number of proteins have been identified as capable of transducing, that is, moving across cellular and nuclear membranes in an energy-independent manner. Transducing sequences have been identified in proteins involved in circadian rhythm, such as human Period proteins. It is thought that these proteins move more freely through cellular and 5 nuclear membranes, and that this movement permits concerted control. No other enzymes involved in the deubiquitination activities have been identified as being capable of transducing or having NLS until now. The presence of an NLS at the C-terminal suggests that the hDUB7 and its urine ortholog, [0 mDUB7, are capable of translocating to the nucleus, possibly by importin-dependent manner and that these DUBs have a role in deubiquitinating ubiquitinated nuclear proteins and/or ubiquitinated proteins that are translocated to the nucleus. This has never been identified before. Protein ubiquitination targets selectively to proteasome degradation and/or provides facilitating protein localization. Thus, nuclear protein deubiquitination may have a role in 15 unique function in regulation of nuclearprotein degradation as well as nuclear protein localization. The same logic can be applied to the vesicular targeting of DUB7 by targeting sequence, regulating vesicular protein degradation as well as invloved in traficking of vesicular proteins. 20 References Katzmann D.J., Babst M. and Emr S.D. (2001) Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell, 106:145-155. Li L. and Cohen S.N. (1996) tsg101: a novel tumor susceptibility gene isolated by controlled 25 homozygous functional knockout of allelic loci in mammalian cells. Cell, 85:319-329. Hicke L. (2001) A new ticket for entry into budding vesicles-ubiquitin. Cell, 106:527-530. Wendland B., Steece K.E. and Emr S.D. (1999) Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin, and are required for endocytosis. EMBO J., 18:4383-4393. Hofiann K. and Falquet L. (2001) A ubiquitin-interacting motif conserved in components of 30 the proteasomal and lysosomal protein degradation systems. Trends Biochen. Sci., 26:347 350.
13 Garcia-Higuera I., Taniguchi T., Ganesan S., Meyn M.S., Timmers C., Hejna J., Grompe M. and D'Andrea A.D. (2001) Interaction of the Fanconi Anemia proteins and BRCA1 in a common pathway. Mol. Cell, 7:249-262. Hochstrasser M. (2000) Evolution and function of ubiquitin-like protein 5 conjugation systems. Nat. Cell Biol., 2:E153-E157.Pickart C.M. (2000) Ubiquitin in chains. Trends Biochem. Sci., 25:544-548. Pickart C.M. (2000) Ubiquitin in chains. Trends Biochem. Sci., 25:544-548. Hershko A. and Ciechanover A. (1998) The ubiquitin system. Annu. Rev. Biochem., 67:425-479. 10 Deng L., Wang C., Spencer E., Yang L., Braun A., You J., Slaughter C., Pickart C. and Chen Z.J. (2000) Activation of the IkB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell, 103:351-361. Wang C., Deng L., Hong M., Akkaraju G.R., Inoue J.-l. and Chen Z.J. 15 (2001) TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature, 412:346 351. Joazeiro C.A.P. and Weissman A.M. (2000) RING finger proteins: mediators of ubiquitin ligase activity. Cell, 102:549-552. Ulrich H. (2001) The srs2 suppressor of UV sensitivity acts specifically on 20 the RAD5- and MMS2-dependent branch of the RAD6 pathway. Nucleic Acids Res., 29:3487-3494. Ulrich H.D. and Jentsch S. (2000) Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J., 19:3388-3397. 25 It should be understood that comprises/comprising and grammatical variations thereof, when used in this specification, are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but are not limited so as to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. 30 SUMMARY OF THE INVENTION The present invention is directed to identification of human homolog of murine DUBs, hematopoietic-specific, cytokine-inducible deubiquitinating proteases found on chromosome 7, respective regulatory region and its murine 14 ortholog, named as hDUB7 and mDUB7, respectively. Therefore, according to the present invention, there is provided an isolated polynucleotide and a polypeptide encoding a deubiquitating protease selected from the group consisting of hDUB7 and mDUB7. 5 Both hDUB7 and its murine ortholog mDUB7 were identified by searching human and mouse genome databases using murine DUB-1 and DUB-2 sequences. These genes (hDUB7 and mDUB7) share open reading frames (ORFs) that are 67% amino acid identity to each other, when gaps caused by deletion was not counted as mismatch, and exhibit 75% identity in nucleotide 10 sequence. Furthermore, both hDUB7 and mDUB7 share 48% identity to murine DUBs, DUB1 and DUB2 within 297 amino acids core DUB sequences. In addition, hDUB7 and mDUB7 genes share open reading frames that are greater than 92% amino acid identity within 540 amino acids N-terminal ubiquitin protease domain (with 98.4% identity within 313 amino acid core). These genes 15 also exhibit 74% identity within 138 amino acids C-terminal conserved domain containing several putative nuclear localization sequences (NLSs) and stretchs of amino acid sequences that is known to possess transducing capacity (KAKKHKKSKKKKKSKDKHR and HRHKKKKKKKKRHSRK). Therefore, the present invention is also directed to a transducing peptide 20 comprising an NLS or transducing sequence of hDUB7 or mDUB7 linked to a cargo molecule. The invention also includes a transducing peptide comprising an NLS or transducing sequence is selected from the group consisting of a peptidyl fragment comprising KAKKHKKSKKKKKSKDKHR, HRHKKKKKKKKRHSRK, KKHKKSKKKKKSKDKHR, and HRHRKKKKKKKRHSRK. 25 The invention also comprises a transducing peptide wherein the cargo molecule is a biologically active protein, therapeutically effective compound, antisense nucleotide, or test compound. Preferably, the invention comprises a transducing peptide comprising an NLS or transducing sequence of hDUB7 or mDUB7 linked to a cargo molecule. 30 The invention also includes a method of delivering a biologically active protein, therapeutically effective compound, antisense nucleotide, or test compound to a cell wherein a transducing peptide is added exogenously to a cell.
14a The invention also includes methods of reducing inflammation and methods of modulating an autoimmune disease or immune reaction by administering a compound capable of inhibiting a polypeptide encoding a deubiquitating protease selected from the group consisting of hDUB7 and 5 mDUB7. Manipulation of these gene products by small molecular compounds can (1) reduce inflammation by regulating proinflammatory cytokine signaling, (2) modulate autoimmune diseases by regulating cytokine receptor signaling that are critical for lymphocytes proliferation, and (3) immune over-reaction during 10 infection using above mechanisms. SEARCH METHODS FOR IDENTIFYING hDUB7 AND mDUB7 mDUB1 (U41636), mDUB2 (U70368), and mDUB2A (AF393637) DNA sequences were used to search against nr (All non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF) in GenBank for potential homologs. 15 Homology was found to a cDNA (AK022759) whose C terminal was incomplete (3660 nucleotides capable of expressing 1197 amino acids run-off translation). In order to in silico clone the full length. Both EST extending and genomic sequence annotation methods were used. Sequence of AK022759 was searched against human ESTs and genomic sequences. AK022759 was extended 20 manually based on matching ESTs and mapped genomic sequence on contig NT_007844.8 from chromosome 7. From these full-length sequence for open reading frame for hDUB7 was generated (3951 nucleotides long DNA segment capable of generating 1316 amino acids long polypeptide). 25 WO 03/083050 PCT/US03/08590 15 For in silico cloning of the putative full length of mDUB7, hDUB7 amino acid sequence was used to search against nr by blastp. The highest match to Mouse proteins is a protein similar to mDUB2. The accession number for this protein is BAB27190 and for nucleotide sequence is AK010801 (1485 nucleotide long capable of translating 487 amino acids run-on 5 translation). Based on Genbank annotation, the gene has partial sequence with C terminal incomplete. In order to get the full length of mDUB7, nucleotide sequence of AK010801 was used to search against Mouse Genomic sequence. There was no match to Mouse curated NT contigs database and match was found on contig_70795 from Mouse ArachneNov30 database (preliminary assembly of the mouse WGS reads based on an Nov 9th freeze of the D WGS data) in Genbank. Putative genes from contig_70795 were annotated by GENSCAN prediction. There is one putative protein with extended/finished C terminal aligned perfectly with BAB27190 except having 33 amino acids missing in the middle of sequence. The nucleotide sequence of the 33 amino acids segment from BAB27190 was searched against the Mouse genomic sequence and found it matched to the genomic sequence region that generates 5 the putative full length mDUB7 and has potential splice sites on the borders. It implies that exon was missed by GENSCAN annotation. A full length mouse DUB7 was constructed by adding 33 amino acids to the putative protein according to the genomic sequence alignment (3981 nucleotides long open reading frame capable of generating 1326 amino acids long polypeptide). The final niDUB7 sequence was aligned with hDUB7 and showed 67% D homology in amino acid level and 75% homology in nucleotide level. TaqMan real time PCR analysis of expression of hDUB7 in human immunocytes upon various stimulation 15 Protocol of reverse transcription (RT) from total cellular RNA using random hexamer as primer (using TaqMan Reverse Transcription Reagents Cat# N808-0234) 1 ug of total RNA preparation in 100 ul of lxTaqMan RT Buffer Mix, 5.5mM MgCl 2 , 0.5 mM dNTPs, 2.5 uM Random Hexamers, 40 U RNAse inhibitor, 125U Multiscribe Reverse 30 Transcriptase. Mix by pipeting up and down. Incubate 25'C for 10 minutes (annealing step), 48 0 C for 30 minutes (reverse transcription), and 95'C for 5 minutes (heat killing of the enzyme). The samples can be left at the machine at 4'C, or alternatively, can be stored at 20'C. Yield of cDNA synthesis can be measured by incorporation of small portion of WO 03/083050 PCT/US03/08590 16 radioactive dATP (or dCTP). Average efficiency for this protocol is between 60-80% of conversion of RNA to cDNA. Protocol of TaqMan real-time quantitative PCR 5 1 ul of TaqMan RT product in 12.5 ul of lx master Mix (Applied Biosystems Cat# 4304437)containing all necessary reaction components except primers and probes, 0.9 uM forward primer, 0.9 uM reverse primer, 0.2 uM probe. Mix by pipetting up and down. Samples containing GADPH primer pair and probe were also prepared as control. Thermal 10 cycling and detection of the real-time amplification were performed using the ABI PRISM 7900HT Sequuence Detection System. The quantity of target gene is given relative to the GADPH control based on Ct values determined during the exponential phase of PCR. Primer-probe set used is as follow: 15 Forward Primer 5'-CCACGACAGAACTGCACTTGTAG-3' Reverse Primer 5'- CCGGGACTTTCCATTTTCG-3' Probe sequence 5'- CAACTGTAACCTCTCTGATCGGTTTCACGAA-3' 20 Table 1. Expression of hDUB7 in PBMC stimulated with LPS (100 ng/ml) for 1.5, 7and 24 hours by TaqMan (Donor 1). LPS Stimulation/Time 1.5 hours 7 hours 24 hours Fold Upregulation upon 0.9 1.5 1.0 stimulation Table 2. Expression of DUB7 in PBMC stimulated with LPS (100 ng/ml) and/or PHA (5 25 ug/ml) for 1.5, 7, 24 hours by TaqMan (donor 2, donor 3) Donor 2 Fold Upregulation upon stimulation Stimuli/time LPS PHA LPS + PHA 1.5 hours 1.1 1.2 1.1 7 hours 3.3 9.2 9.2 24 hours 0.2 0.3 0.3 Donor 3 Fold Upregulation upon stimulation Stimuli/time LPS PHA LPS + PHA 1.5 hours 1.2 1.0 1.2 7 hours 3.5 8.2 9.3 24 hours 0.5 0.5 0.6 WO 03/083050 PCT/US03/08590 17 Table 3. Expression of hDUB7 in enriched B cells stimulated with LPS (100 ng/ml) or IL-4 and anti-CD40 mAb for 4 and 20 hours by TaqMan (Donor 4). Donor 4 Fold Upregulation upon stimulation Stimuli/time LPS IL-4, anti-CD40 mAb 4 hours 1.11 2.44 20 hours 0.70 1.0 5 Table 4. Expression of hDUB7 in entiched CD4* T cells stimulated with anti-CD3 and anti CD28 mAbs for 3,6 and 18 hours by TaqMan (Donor 5). mAbs Stimulation/Time 3 hours 6 hours 18 hours Fold Upregulation upon 1.36 1.74 0.37 stimulation 10 Table 5. Expression of hDUB7 in differentiated ThO, Thl and Th2 CD4* T cells (Day 4 after differentiation) stimulated with anti-CD3 and anti-CD28 mAbs for 8 hours by TaqMan (Donor 6). mAbs Stimulation ThO Th1 Th2 Fold Upregulation upon stimulation 2.60 0.36 1.72 15 Table 6. Expression of hDUB7 in differentiated ThO, Th1 and Th2 CD4* T cells (Day 7 after differentiation) stimulated with anti-CD3 and anti-CD28 mAbs for 8 and 18 hours by TaqMan (Donor 6). mAbs Stimulation ThO Th1 Th2 Fold Upregulation in 8 hours 1.38 1.11 1.71 Fold Upregulation in 18 hours 0.94 0.81 1.47 20 Table 7. Expression of hDUB7 in various tissue examined by Affymatrix chip analysis Tissue Relative Intensity Con_Adipose_1 2287 Con_Adipose _2 4190 CV Heart 1 2545 CV Heart 2 3907 CVHeart_3 5367 CVPericardia 1 3682 Dig_Colon_1 2387 DigColon_2 2894 DigEsophagusj 5004 DigEsophagus_2 1658 WO 03/083050 PCT/US03/08590 18 DigFetalLiver_1 1288 DigFetalLiver_2 4676 DigFetalLiver_3 829 DigFetalLiver_4 3161 Dig_Liver_1 3094 Dig_Liver_2 1527 Dig_Liver_3 3410 DigPancreas_1 3731 DigPancreas_2 4837 DigRectum_1 2329 DigRectum_2 1851 DigSalivaryGland_1 2337 DigSalivaryGland_2 2110 DigSmallIntestine_1 2838 DigSmallIntestine_2 2662 DigStomach_1 2187 End AdrenalGland_1 591 EndAdrenalGland_2 2199 End_Thyroid_1 2564 End_Thyroid_2 2392 End_Thyroid_3 3522 ExoBreast_1 3673 ExoBreast_2 6173 Exo_MammaryGland 3741 1 ImmBoneMarrow_1 1090 InmSpleen1 2429 1mm_Thymus_1 3666 Imm_Thymus_2 1759 RepCervix_1 4482 RepCervix_2 3362 RepPlacenta_1 1248 RepPlacenta_2 2378 RepPlacenta_3 1622 RepProstate_1 5128 RepProstate_2 2762 Rep_Testis_1 2252 Rep_Testis_2 3196 RepUterus_1 4720 RepUterus_2 3789 ResLung_1 2313 ResLung_2 3177 ResLung_3 4409 ResLung_4 2366 ResTrachea_1 2152 ResTrachea_2 2358 ResTrachea_3 812 Res Trachea 4 812 Sk SkeletalMuscle_1 2838 Sk SkeletalMuscle_2 6106 WO 03/083050 PCT/US03/08590 19 SkinSkin_1 5500 UriKidneyl 3593 UriKidney_2 1311 Ui_Kidney_3 2747 UriKidney_4 1530 NS Brain 1 3214 NS Brain 2 2173 NS Brain 3 1332 NS Brain 4 2604 NSBrain_5 1663 NSCerebellum_1 3175 NSCerebellum_2 1766 NSFetalBrain_1 4299 NS FetalBrain 2 2549 NSFetalBrain_3 4027 NSSpinalCord_1 2976 NSSpinalCord_2 3999 NSSpinalCord_3 4614 Table 8. Expression of mDUB7 in various tissue examined by Affymatrix chip analysis Mouse # Organ Relative Intentisty A stomach 56 A stomach 11 B stomach 175 B stomach 97 C stomach 178 C stomach 126 A lymph 516 A lymph 365 B lymph 494 B lymph 335 C lymph 475 C lymph 509 A thymus 913 A thymus 1015 B thymus 881 B thymus 927 C thymus 834 C thymus 975 A prostate 327 A prostate 350 B prostate 75 B prostate 423 C prostate 405 C prostate 267 A uterus 549 A uterus 372 B uterus 225 B uterus 418 WO 03/083050 PCT/US03/08590 20 C uterus 335 C uterus 401 Deubiquitination Assay Confirmation that the DUB is a deubiquitinating enzyme may be shown using 5 previously identified deubiquitination assay of ubiquitin--galactosidase fusion proteins, as described previously in the literature. Briefly, a fragment of the DUB, of approximately 1,500 nucleotides, based on the wild-type DUB cDNA (corresponding to amino acids 1 to about 500) and a cDNA containing a missense mutation are generated by PCR and inserted, in frame, into pGEX (Pharmacia), downstream of the glutathione S-transferase (GST) coding 10 element. Ub-Met--gal is expressed from a pACYC 184-based plasmid. Plasmids are co transformed as indicated into MC 1061 Escherichia coli. Plasmid-bearing E. coli MC 1061 cells are lysed and analyzed by immunoblotting with a rabbit anti--gal antiserum (Cappel), a rabbit anti-GST antiserum (Santa Cruz), and the ECL system (Amersham Corp.). in vitro deubiquitinating enzyme activity may be shown from purified hDUB fusion protein using 15 commercial polyubiquitinated protein as substrate. HDUB7 and mDUB7 are potential inflamatory cytokins specific Inmediate-early Genes mDUB-1 was originally cloned as an IL-3-inducible immediate-early gene. Similarly, mDUB-2 was cloned as an IL-2-inducible immediate-early gene. We examined inducibility as Zo well as cell-type specific expression of these genes using Affymatrix-Chip analysis and multiple TaqMan analysis from human organ RNA samples and human immunocytes RNA samples. Our data suggest that expression of hDUB7 are not apparent in monoocytes and other myoloid cell types but high in fresh human PBMC from several donor. Furthermore, enriched cell populations of several lymphocytes, including B cells, CD4+ T cells of Th-1 and Z5 Th-2 differentiation conditions as well as bulk CD4+ T cells showed significant upregulation upon appropriate stimulations. Currently, we can not rule out the possibility of upregulation upon stimulation in CD8+ T cells and potentially NK/NK-T cells. The DUB Subfamily of the ubp Superfamily 3o From these data we propose that hDUB4s and hDUB8s are members of a discrete subfamily of deubiquitinating enzymes that shows the strongest similarity to mDUB subfamily including mDUB1, mDUB2, and mDUB2A, called the DUB subfamily.DUB subfamily members contain distinct structural features that distinguish them from other ubps.
WO 03/083050 PCT/US03/08590 21 First, DUB subfamily members are comparatively small enzymes of approximately 500-550 amino acids. Second, DUB subfamily members share amino acid similarity not only in the Cys and His domains but also throughout their primary amino acid sequence. For instance, DUB proteins contain a lysine-rich region (Lys domain) and a HV domain near their carboxyl 5 terminus. The regulatory regions, or promoter regions, of each of the hDUB7 was analyzed for putative transcription factor binding motifs using TRANSFACFind, a dynamic programming method, see Heinemeyer, T., et al., "Expanding the TRANSFAC database towards an expert 10 system of regulatory molecular mechanisms" Nucleic Acids Res. 27, 318-322, (1999). The Transfac database provides eukaryotic cis- and trans-acting regulatory elements. The data is shown as table X. Table 9, putative transcription factor binding motifs within the hDUB7 regulatory or promoter 15 region. The position is indicated by nucleotides used in the table 9. Transfac Position(Score) Name Description M00148 1960..1966(100) SRY sex-determining region Y gene product 876..870(100) 1357..1351(92) 1881..1875(92) 1749..1755(90) 118..124(90) 267..261(90) 275..269(90) 1663..1669(90) 1313..1319(90) 1860..1854(90) 108..114(90) M00240 491..497(100) Nkx-2.5 homeo domain factor Nkx-2.5/Csx, tinman homolog 1512..1506(90) 1894..1888(90) M00028 1844..1848(100) HSF heat shock factor (Drosophila) 1835..1839(100) 251..247(100) 265..261(100) 273..269(100) 1429..1433(100) 1315..1319(100) WO 03/083050 PCT/US03/08590 22 1264..1268(100) 1060..1064(100) 1014..1010(100) 1540..1536(100) 1559..1555(100) 1619..1615(100) 110..114(100) 66..70(100) 1950..1946(100) 1737..1741(95) 1635..1639(95) 651..647(95) 1103..1107(95) 1082..1078(95) 16..20(95) 1674..1678(94) 1189..1185(94) 880..876(91) M00029 247..243(100) HSF heat shock factor (yeast) 1667..1671(100) 1210..1206(100) 1745..1741(100) 71..75(100) 1844..1848(96) 1835..1839(96) 265..261(96) 273..269(96) 1429..1433(96) 1315..1319(96) 1264..1268(96) 1060..1064(96) 1014..1010(96) 1540..1536(96) 1559..1555(96) 1619..1615(96) 110..114(96) 1950..1946(96) 1674..1678(95) 1189..1185(95) 1737..1741(93) 1635..1639(93) 651..647(93) 1103..1107(93) 1082..1078(93) 16..20(93) 1120..1124(90) 139..143(90) M00101 1418..1412(100) CdxA CdxA 1689..1695(98) WO 03/083050 PCT/US03/08590 23 1566..1572(98) 1460..1466(98) 1319..1325(98) 969..975(98) 1463..1457(98) 1614..1608(98) 1065..1059(94) 1599..1605(93) 1375..1369(93) 1840..1834(93) 1859..1865(92) 1168..1174(92) 1218..1212(92) 1478..1484(90) M00048 447..452(100) ADR1 alcohol dehydrogenase gene regulator 1 535..540(95) 1716..1721(93) 459..454(93) 558..553(93) 1180..1185(93) 305..310(93) 38..43(92) M00354 1951..1941(99) Dof3 Dof3 - single zinc finger transcription factor 1560..1550(95) 104..114(93) 65..75(91) M00227 1920..1928(98) v-Myb v-Myb M00141 521..513(98) Lyf-1 LyF-1 828..820(98) M00344 806..795(98) RAV1 3'-part of bipartite RAV1 binding site, interacting with AP2 domain 806..817(92) 1949.. 1960(92) M00253 1139..1146(98) cap cap signal for transcription initiation 681..688(96) 374..381(96) 299..306(95) 1674..1667(94) 1737..1730(91) 31..24(91) 16..9(91) 1701..1694(91) 1909..1902(90) 619..626(90) 1368..1375(90) M00286 577..564(97) GKLF gut-enriched Krueppel-like factor 271..258(96) M00199 684..676(96) AP-1 AP-1 binding site WO 03/083050 PCT/US03/08590 24 676..684(95) M00183 227..218(96) c-Myb c-Myb 28..37(95) 1247..1238(90) M00154 1714..1721(96) STRE stress-response element M00140 1824..1831(96) Bcd Bicoid 834..841(93) 527..534(93) M00100 1418..1412(96) CdxA CdxA 1209..1215(92) 1348..1354(91) M00291 1652..1667(95) Freac-3 Fork head RElated ACtivator-3 M00073 1948..1958(95) deltaEF1 deltaEF1 807..797(95) 1452..1442(92) 805..815(90) M00216 1176..1167(95) TATA Retroviral TATA box M00120 1952..1942(95) dl dorsal 1561..1551(93) M00042 1861..1852(95) Sox-5 Sox-5 1790..1781(91) M00174 675..685(95) AP-1 activator protein 1 M00230 1797..1808(95) Skn-1 maternal gene product M00272 1024..1033(94) p53 tumor suppressor p53 1033..1024(94) M00160 1862..1851(94) SRY sex-determining region Y gene product M00022 111..120(94) Hb Hunchback 436..427(91) 584..575(91) M00053 447..456(94) c-Rel c-Rel M00249 1244..1256(93) CHOP- heterodimers of CHOP and C/EBPalpha C/EBPalpha M00142 1367..1362(93) NIT2 activator of nitrogen-regulated genes 1348..1343(91) M00289 1670..1658(93) HFH-3 HNF-3/Fkh Homolog 3 (= Freac-6) M00019 1381..1366(93) Dfd Deformed 1593..1608(91) M00147 1903..1912(92) HSF2 heat shock factor 2 M00184 806..815(92) MyoD myoblast determining factor M00345 225..218(92) GAmyb GA-regulated myb gene from barley M00094 1658..1670(92) BR-C Broad-Complex Z4 1398..1386(90) M00349 1200..1191(92) GATA-2 GATA-binding factor 2 M00077 443..451(92) GATA-3 GATA-binding factor 3 M00087 388..399(91) Ik-2 Ikaros 2 M00099 1268..1283(91) S8 S8 M00285 1399..1411(91) TCF11 TCF11/KCR-F1/Nrfl homodimers M00241 1224..1217(91) Nkx-2.5 homeo domain factor Nkx-2.5/Csx, tinman WO 03/083050 PCT/US03/08590 25 homolog 1526..1519(91) M00283 1863..1878(90) Zeste Zeste transvection gene product M00046 1113..1105(90) GCR1 GCR1 M00353 1069..1079(90) Dof2 Dof2 - single zinc finger transcription factor 1951..1941(90) M00263 985..994(90) StuAp Aspergillus Stunted protein M00051 448..457(90) NF-kappaB NF-kappaB(p50) M00350 1200..1191(90) GATA-3 GATA-binding factor 3 M00276 1851..1860(90) Matl-Mc M-box interacting with Matl-Mc M00075 1936..1945(90) GATA-1 GATA-binding factor 1 442..451(90) M00355 279. .269(90) PBF PBF (M\PBF) 1897..1887(90) ______ M00352 1775.. 1785(90) Dofi Dof2 / MNB1a - single zinc finger transcription factor M00294 1670..1658(90) BFH-8 HNF-3/Fkh Homolog-8 M00131 1762..1748(90) HNF-3beta Hepatocyte Nuclear Factor 3beta M00137 1320..1332(90) Oct-1 octamer factor 1 M00054 448..457(90) NF-kappaB NF-kappaB WO 03/083050 PCT/US03/08590 26 Table 10. Nucleotide sequence of coding region of human DU1B7 (hDUB7) ATGACCATAGTTGACAAAGCTTCTGAATCTTCAGACCCATCAGCCTATCAGAATC AGCCTGGCAGCTCCGAGGCAGTCTCACCTGGAGACATGGATGCAGGTTCTGCCAG 5 CTGGGGTGCTGTGTCTTCATTGAATGATGTGTCAAATCACACACTTTCTTTAGGAC CAGTACCTGGTGCTGTAGTTTATTCGAGTTCATCTGTACCTGATAAATCAAAACCA TCACCACAAAAGGATCAAGCCCTAGGTGATGGCATCGCTCCTCCACAGAAAGTTC TTTTCCCATCTGAGAAGATTTGTCTTAAGTGGCAACAAACTCATAGAGTTGGAGCT GGGCTCCAGAATTTGGGCAATACCTGTTTTGCCAATGCAGCACTGCAGTGTTTAA [o CCTACACACCACCTCTTGCCAATTACATGCTATCACATGAACACTCCAAAACATGT CATGCAGAAGGCTTTTGTATGATGTGTACAATGCAAGCACATATTACCCAGGCAC TCAGTAATCCTGGGGACGTTATTAAACCAATGTTTGTCATCAATGAGATGCGGCG TATAGCTAGGCACTTCCGTTTTGGAAACCAAGAAGATGCCCATGAATTCCTTCAA TACACTGTTGATGCTATGCAGAAAGCATGCTTGAATGGCAGCAATAAATTAGACA 15 GACACACCCAGGCCACCACTCTTGTTTGTCAGATATTTGGAGGATACCTAAGATC TAGAGTCAAATGTTTAAATTGCAAGGGCGTTTCAGATACTTTTGATCCATATCTTG ATATAACATTGGAGATAAAGGCTGCTCAGAGTGTCAACAAGGCATTGGAGCAGTT TGTGAAGCCGGAACAGCTTGATGGAGAAAACTCGTACAAGTGCAGCAAGTGTAA AAAGATGGTTCCAGCTTCAAAGAGGTTCACTATCCATAGATCCTCTAATGTTCTTA 20 CACTTTCTCTGAAACGTTTTGCAAATTTTACCGGTGGAAAAATTGCTAAGGATGTG AAATACCCTGAGTATCTTGATATTCGGCCATATATGTCTCAACCCAACGGAGAGC CAATTGTCTACGTCTTGTATGCAGTGCTGGTCCACACTGGTTTTAATTGCCATGCT GGCCATTACTTCTGCTACATAAAAGCTAGCAATGGCCTCTGGTATCAAATGAATG ACTCCATTGTATCTACCAGTGATATTAGATCGGTACTCAGCCAACAAGCCTATGTG 25 CTCTTTTATATCAGGTCCCATGATGTGAAAAATGGAGGTGAACTTACTCATCCCAC CCATAGCCCCGGCCAGTCCTCTCCCCGCCCCGTCATCAGTCAGCGGGTTGTCACCA ACAAACAGGCTGCGCCAGGCTTTATCGGACCACAGCTTCCCTCTCACATGATAAA GAATCCACCTCACTTAAATGGGACTGGACCATTGAAAGACACGCCAAGCAGTTCC ATGTCGAGTCCTAACGGGAATTCCAGTGTCAACAGGGCTAGTCCTGTTAATGCTT 30 CAGCTTCTGTCCAAAACTGGTCAGTTAATAGGTCCTCAGTGATCCCAGAACATCCT AAGAAACAAAAAATTACAATCAGTATTCACAACAAGTTGCCTGTTCGCCAGTGTC AGTCTCAACCTAACCTTCATAGTAATTCTTTGGAGAACCCTACCAAGCCCGTTCCC TCTTCTACCATTACCAATTCTGCAGTACAGTCTACCTCGAACGCATCTACGATGTC AGTTTCTAGTAAAGTAACAAAACCGATCCCCCGCAGTGAATCCTGCTCCCAGCCC 35 GTGATGAATGGCAAATCCAAGCTGAACTCCAGCGTGCTGGTGCCCTATGGCGCCG AGTCCTCTGAGGACTCTGACGAGGAGTCAAAGGGGCTGGGCAAGGAGAATGGGA TTGGTACGATTGTGAGCTCCCACTCTCCCGGCCAAGATGCCGAAGATGAGGAGGC CACTCCGCACGAGCTTCAAGAACCCATGACCCTAAACGGTGCTAATAGTGCAGAC AGCGACAGTGACCCGAAAGAAAACGGCCTAGCGCCTGATGGTGCCAGCTGCCAA 40 GGCCAGCCTGCCCTGCACTCAGAAAATCCCTTTGCTAAGGCAAACGGTCTTCCTG GAAAGTTGATGCCTGCTCCTTTGCTGTCTCTCCCAGAAGACAAAATCTTAGAGAC CTTCAGGCTTAGCAACAAACTGAAAGGCTCGACGGATGAAATGAGTGCACCTGG AGCAGAGAGGGGCCCTCCCGAGGACCGCGACGCCGAGCCTCAGCCTGGCAGCCC CGCCGCCGAATCCCTGGAGGAGCCAGATGCGGCCGCCGGCCTCAGCAGCACCAA 45 GAAGGCTCCGCCGCCCCGCGATCCCGGCACCCCCGCTACCAAAGAAGGCGCCTGG GAGGCCATGGCCGTCGCCCCCGAGGAGCCTCCGCCCAGCGCCGGCGAGGACATC GTGGGGGACACAGCACCCCCTGACCTGTGTGATCCCGGGAGCTTAACAGGCGATG CGAGCCCGTTGTCCCAGGACGCAAAGGGGATGATCGCGGAGGGCCCGCGGGACT CGGCGTTGGCGGAAGCCCCGGAAGGGTTGAGTCCGGCTCCGCCTGCGCGGTCGGA 50 GGAGCCCTGCGAGCAGCCACTCCTTGTTCACCCCAGCGGGGACCACGCCCGGGAC WO 03/083050 PCT/US03/08590 27 GCTCAGGACCCATCCCAGAGCTTGGGCGCACCCGAGGCCGCAGAGCGGCCGCCA GCTCCTGTGCTGGACATGGCCCCGGCCGGTCACCCGGAAGGGGACGCTGAGCCTA GCCCCGGCGAGAGGGTCGAGGACGCCGCGGCGCCGAAAGCCCCAGGCCCTTCCC CAGCGAAGGAGAAAATCGGCAGCCTCAGAAAGGTGGACCGAGGCCACTACCGCA 5 GCCGGAGAGAGCGCTCGTCCAGCGGGGAGCCCGCCAGAGAGAGCAGGAGCAAG ACTGAGGGCCACCGTCACCGGCGGCGCCGCACCTGCCCCCGGGAGCGCGACCGC CAGGACCGCCACGCCCCGGAGCACCACCCCGGCCACGGCGACAGGCTCAGCCCT GGCGAGCGCCGCTCTCTGGGCAGGTGCAGTCACCACCACTCCCGACACCGGAGCG GGGTGGAGCTGGACTGGGTCAGACACCACTACACCGAGGGCGAGCGTGGCTGGG .0 GCCGGGAGAAGTTCTACCCCGACAGGCCGCGCTGGGACAGGTGCCGGTACTACC ATGACAGGTACGCCCTGTACGCTGCCCGGGACTGGAAGCCCTTCCACGGCGGCCG CGAGCACGAGCGGGCCGGGCTGCACGAGCGGCCGCACAAGGACCACAACCGGGG CCGTAGGGGCTGCGAGCCGGCCCGGGAGAGGGAGCGGCACCGCCCCAGCAGCCC CCGCGCAGGCGCGCCCCACGCCCTCGCCCCGCACCCCGACCGCTTCTCCCACGAC [5 AGAACTGCACTTGTAGCCGGAGACAACTGTAACCTCTCTGATCGGTTTCACGAAC ACGAAAATGGAAAGTCCCGGAAACGGAGACACGACAGTGTGGAGAACAGTGACA GTCATGTTGAAAAGAAAGCCCGGAGGAGCGAACAGAAGGATCCTCTAGAAGAGC CTAAAGCAAAGAAGCACAAAAAATCAAAGAAGAAAAAGAAATCCAAAGACAAA CACCGAGACCGCGACTCCAGGCATCAGCAGGACTCAGACCTCTCAGCAGCGTGCT !o CTGACGCTGACCTCCACAGACACAAAAAAAAGAAGAAGAAAAAGAAGAGACATT CAAGAAAATCAGAGGACTTTGTTAAAGATTCAGAACTGCACTTACCCAGGGTCAC CAGCTTGGAGACTGTCGCCCAGTTCCGGAGAGCCCAGGGTGGCTTTCCTCTCTCTG GTGGCCCGCCTCTGGAAGGCGTCGGACCTTTCCGTGAGAAAACGAAACACTTACG GATGGAAAGCAGGGATGACAGGTGTCGTCTCTTTGAGTATGGCCAGGGTGATTGA 25 Table 11. Deduced amino acid sequence of coding region of hDUB7 C-terminal potential nuclear localization (as well as targeting) sequences are underlined. 30 MTIVDKASESSDPSAYQNQPGSSEAVSPGDMDAGSASWGAVSSLNDVSNHTLSLGPV PGAVVYSSSSVPDKSKPSPQKDQALGDGIAPPQKVLFPSEKICLKWQQTHRVGAGLQ NLGNTCFANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQAHITQALSNP 35 GDVIKPMFVINEMRRIARHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQA TTLVCQIFGGYLRSRVKCLNCKGVSDTFDPYLDITLEIKAAQSVNKALEQFVKPEQLD GENSYKCSKCKKMVPASKRFTIHRSSNVLTLSLKRFANFTGGKIAKDVKYPEYLDIRP YMSQPNGEPIVYVLYAVLVHTGFNCHAGHYFCYIKASNGLWYQMNDSIVSTSDIRSV LSQQAYVLFYIRSHDVKNGGELTHPTHSPGQSSPRPVISQRVVTNKQAAPGFIGPQLPS 40 HI4KNPPHLNGTGPLKDTPSSSMSSPNGNSSVNRASPVNASASVQNWSVNRSSVIPEH PKKQKITISIHNKLPVRQCQSQPNLHSNSLENPTKPVPSSTITNSAVQSTSNASTMSVSS KVTKPIPRSESCSQPVMNGKSKLNSSVLVPYGAESSEDSDEESKGLGKENGIGTIVSSH SPGQDAEDEEATPHELQEPMTLNGANSADSDSDPKENGLAPDGASCQGQPALHSENP FAKANGLPGKLMPAPLLSLPEDKILETFRLSNKLKGSTDEMSAPGAERGPPEDRDAEP 45 QPGSPAAESLEEPDAAAGLSSTKKAPPPRDPGTPATKEGAWEAMAVAPEEPPPSAGE DIVGDTAPPDLCDPGSLTGDASPLSQDAKGMIAEGPRDSALAEAPEGLSPAPPARSEEP CEQPLLVHPSGDHARDAQDPSQSLGAPEAAERPPAPVLDMAPAGHPEGDAEPSPGER VEDAAAPKAPGPSPAKEKIGSLRKVDRGHYRSRRERSSSGEPARESRSKTEGHRHRR RTCPRERDRQDRHAPEHHPGHGDRLSPGERRSLGRCSHHHSRHRSGVELDWVRHHY 50 TEGERGWGREKFYPDRPRWDRCRYYHDRYALYAARDWKPFHGGREHERAGLHERP WO 03/083050 PCT/US03/08590 28 HKDHNRGRRGCEPARERERHRPSSPRAGAPHALAPHPDRFSHDRTALVAGDNCNLSD RFHEHENGKSRKRRHDSVENSDSHVEKKARRSEQKDPLEEPKAKKHKKSKKKKKSK DKHRDRDSRHQQDSDLSAACSDADLHRHKKKKKKKKRHSRKSEDFVKDSELHLPRV TSLETVAQFRRAQGGFPLSGGPPLEGVGPFREKTKHLRMESRDDRCRLFEYGQGD 5 Table 12. Putative promoter sequence of hDUB7 (2 Kb sequence upstream of initiation AUG) GTAAAGTCTAAACTGAGAAGTGGAAGTGTGAACTGGCTGGAGGTGGAAGGTTGG AAAAGAGTCGGAGAAAAGAACAGCATGTGCAGAGCCCAGAGACAGCAGGGACA [0 AAAGAAAAAAAAACAAGACTTCAGCATGGTGGGAACGTGACGGAGAGGGTGTTT GGCGAGGTTATTAGGTCAGACAATGTGAAGTCCAGACATTAAGATGTTGTGCTGT GGGCAGTTGGGCCACTCCTGAAAGGTGTTCTTTCTTCCTTTCCTTTTCTTTCTTTCT TTTCTTGAGGCAGAGTCTCTCTATGTCAGTCTGGAGTGCAGTGGCATGATCTCGGC TCACTGCAATCTCTGCCTTCCAGGTTCAAGCAATTTTCCTTGCCTCAGCCTCCCAA [5 GTAGCTGGGAATACAGGCGTGCGCCACCATGCCTGGTTAATTTTTTTATTTTTAGT AGAGATGGGGTTTCCCCATGTTGGCCAGGCTGGTCTCGAACTCCTGGACTCAAGT GATCCACCCACTTTGGCCTCCCAAAGTGCTGGGATTACAGGGGTGTGAGCCACTG CGCCCCGCCCGGCCTTTTTTTTTTTTTTTTTTGAGACTTAATCTTGCTCTGTCACCA AGGCTGGATATCAGTGGCACGGTTTTGGCTCTCTGCAACTTCTGTCTCCCAGGTTC 20 AAGCGATTTTCCTGACTCAGCCTCCCAAGTAGTTGAGATTACAGGTACGTGCCAC CACGCCCGGCTAATTTTTGTATTTTTAGTAGAGATGAGGTTTCACTATGTTGGCCA GACTGGTCTCAAACGCCTGACCTCAGGTGATTCACCTGCCTCGGCCTCCCAAAAT GCTGGGATTACAGGTGTGCACCACCATGCCTGGGTAATTTTTGTTTTTCGTAGAGA CAGGGTCTCACCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAAGCGATCT 25 GCCCACCTTGGCCTCCCAAGGTGCTGCAATTATAGGCATGAGCCACCGCGCCCGG CCTCCTGAAAGGTTTTCTACATAGGAGTGGCATGTCTAGATGTGGCTACTGTTGGG CGATTTTAGAAATATCCCTAAAAGCCTTCTGTTGACAGGGTGGCATAACCAGAAG GAAGCCTGGCTGGGAACGCTGGACCTGGCTCTCAGTCCCAGTTGCTGACTGGTTG CTTCATTTTATAGGCCCTGGGGATTCTGTCTGATCTCTCATACGTTCTTTATAAAA 30 ATTAAGTTAATGTATGTCCAGCAGTTGATGCAATGCCCAGTACATAGAAAATGCT CAATTAGTGGTAGCCCTAATATTTTAAAATAGGACTCAGAAAGAAAATTATAATC AAGTCCTTTCATAACAGATATTTGTGTTTGAGTTTGATATCAGTAATGGCTTACGG GTTTTATTTAAAAAGTCATACATTCCATATAAATGAGCCTCTTCAGAAAAATGGTT TTAAAGGTGAGATCTCTATAATTATAATTTTAAAAAATATAATGTATTTCACTTGG 35 TGCCATTTGCACTTTAAGCACAAAATTAAGTCTAGATTTTTTCTGTGTAGTTGATG CTTTTCTCTGAGGAATTATACTCAAATTGAAGATGTAGTCAAATGTATTACTGTGT ATAATTTTTCTAGTTTTAAGCAGTATAGAAGGAAAATATAGGTACTTAGTAAATA AACAGAACTGAGAATTGAAATGTCCAATTATAAACTGAAATGCCAGACTTTTAGG GGGCATGAAATGAAAATGAGAAGTTCTTTTAATCAAATACTTCACTGAAGATTTT 40 AAAATAAAGATTGTTGACATTCAGATTATCATGATGCTAAATGTCCCAAGGGGAT TATTACAGAAATGTTAGAAAGTACTATTGTTTTTATATTTGAGTGATGTGTTTGAA AATCACTTTAAAATGGCTGGAATGATCTTCCAAGATCTAACGGTAGGGTAAGGAG ATTGCTTTTCTCACCTGATGAAACAAATACATACTTTTCATCTTTTGCAGAGTTGA ACAATG 45 Table 13. Nucleotide sequence of coding region of murine DUB7 (mDUB7) ATGACCATAGTTGACAAAACTGAACCTTCAGACCCATCAACCTGTCAGAACCAGC 50 CTGGCAGTTGTGAGGCGGTCTCACCTGAAGACATGGACACAGGCTCTGCCAGCTG WO 03/083050 PCT/US03/08590 29 GGGCGCTGTGTCTTCAATAAGTGATGTCTCAAGTCACACACTTCCATTAGGGCCA GTGCCTGGTGCTGTAGTTTATTCTAACTCGTCTGTACCTGAAAAATCAAAGCCATC ACCACCAAAGGATCAAGTCCTAGGTGATGGCATTGCTCCTCCTCAAAAGGTCCTG TTTCCATCTGAAAAGATTTGTCTTAAGTGGCAACAAAGTCATCGAGTTGGCGCTG 5 GGCTCCAGAATTTGGGCAACACCTGTTTTGCCAATGCCGCATTGCAGTGTCTGACT TACACGCCACCCCTCGCCAATTACATGTTATCCCATGAACACTCCAAGACATGCC ACGCAGAAGGATTTTGTATGATGTGCACGATGCAGACACACATTACCCAGGCACT TAGCAACCCTGGGGATGTTATCAAGCCGATGTTCGTCATCAATGAAATGCGGCGT ATAGCTAGACACTTCCGTTTTGGAAACCAAGAAGATGCCCATGAATTTCTTCAGT to ACACGGTCGATGCCATGCAGAAAGCATGTTTAAATGGCAGCAATAAATTAGACA GACACACCCAGGCCACCACCCTGGTCTGCCAGATATTTGGAGGCTACCTAAGATC CCGAGTTAAATGTTTAAATTGCAAGGGTGTTTCAGATACCTTTGATCCATATCTGG ACATAACGTTGGAGATTAAGGCTGCACAGAGTGTTACCAAGGCGTTAGAGCAGTT TGTGAAGCCAGAACAACTGGATGGAGAAAACTCCTACAAGTGCAGCAAGTGCAA 15 AAAAATGGTTCCAGCTTCAAAGAGATTCACAATCCATAGGTCCTCTAATGTTCTTA CCATCTCACTGAAGCGCTTTGCCAACTTCACCGGTGGAAAGATTGCTAAGGATGT GAAATATCCTGAGTACCTTGATATCCGGCCCTATATGTCTCAGCCCAATGGAGAG CCAATTATTTATGTTTTGTATGCTGTGCTGGTGCACACTGGTTTTAATTGTCATGCT GGCCACTACTTTTGCTACATCAAGGCTAGCAATGGCCTCTGGTATCAGATGAATG 20 ACTCCATCGTGTCCACCAGTGATATCAGAGCAGTGCTTAACCAGCAAGCTTACGT GCTCTTTTATATCAGGTCCCATGATGTGAAAAATGGAGGGGAGTCTGCTCATCCT GCCCATAGCCCCGGCCAATCCTCTCCCCGCCCAGGAGTCAGTCAGCGGGTAGTCA ACAACAAGCAGGTGGCTCCAGGGTTTATTGGACCCCAGCTGCCTTCCCATGTGAT GAAGAACACGCCACACTTGAATGGCACCACGCCAGTGAAAGACACACCAAGTAG 25 TTCTGTGTCAAGCCCTAACGGAAACACCAGCGTCAATAGGGCCAGTCCTGCTACT GCTTCGACTTCTGTGCAGAACTGGTCTGTTACCAGACCCTCAGTTATTCCAGATCA CCCCAAGAAACAAAAAATCACCATCAGTATTCACAACAAGTTGCCTGCTCGCCAG GGTCAGGCACCACTGAATAACAGCCTCCATGGCCCTTGTCTGGAGGCTCCTAGTA AGGCGGCACCCTCCTCCACCATCACTAACCCTTCTGCAATACAGTCTACCTCGAAC 30 GTACCCACAACGTCGACTTCCCCCAGTGAGGCCTGTCCCAAGCCCATGGTGAACG GCAAGGCTAAAGTGGGCGCCAGTGTGCTTGTCCCCTATGGGGCCGAGTCCTCAGA AGAGTCTGATGAGGAGTCGAAGGGCCTGGCCAAGGAGAACGGTGTGGACATGAT GGCCGGCACTCACTCCGATAGGCCAGAAGCTGCTGCAGATGACGGTGCTGAGGCT TCCTCCCATGAGCTTCAAGAACCCGTCCTGCTAAATGGTGCTAATAGCGCAGACA 35 GTGACTCACAAGAGAACAGCCTGGCATTTGACAGTGCCAGCTGCCAGGTCCAGCC CGAGCTACACACAGAAAACCTCTTTTCCAAACTTAATGGTCTTCCTGGAAAGGTG ACGCCTGCTCCTTTGCAGTCTGTTCCTGAAGACAGAATCCTTGAGACCTTCAAGCT TACCAACCAGGCAAAGGGTCCAGCGGGTGAAGAGAGTTGGACTACGACAGGGGG AAGCTCTCCAAAGGACCCTGTTTCACAGCTGGAGCCCATCAGTGATGAGCCCAGT 40 CCCCTTGAGATACCGGAGGCTGTCACCAATGGGAGCACACAGACCCCTTCCACCA CATCACCCCTGGAGCCCACCATCAGCTGTACCAAAGAAGACTCGTCCGTTGTTGT CTCAGCTGAACCTGTGGAGGGTTTGCCTTCCGTCCCTGCTCTTTGTAACAGCACTG GTACTATCTTGGGGGATACCCCAGTGCCCGAATTGTGTGACCCTGGAGACTTGAC TGCCAACCCGAGCCAGCCAACCGAAGCAGTGAAAGGTGATACAGCTGAGAAGGC 45 TCAGGACTCTGCCATGGCTGAAGTGGTGGAGAGGCTGAGCCCTGCTCCCTCAGTA CTCACAGGTGACGGGTGTGAGCAGAAACTCTTACTTTACCTCAGCGCAGAGGGGT CAGAGGAGACAGAAGACTCTTCCAGAAGCTCGGCGGTCTCTGCTGACACGATGCC CCCTAAGCCTGACAGGACCACCACCAGCTCCTGTGAAGGGGCTGCCGAGCAGGCT GCTGGGGACAGAGGCGATGGAGGCCATGTGGGACCCAAAGCTCAGGAGCCTTCC 50 CCAGCCAAGGAAAAGATGAGCAGCCTCCGGAAAGTGGACCGAGGACACTATCGG WO 03/083050 PCT/US03/08590 30 AGCCGGAGAGAGCGCTCCTCCAGTGGGGAGCACGTGAGGGACAGCAGGCCCCGG CCGGAGGACCATCACCATAAGAAGCGGCACTGCTACAGCCGAGAGCGGCCCAAG CAGGACCGACACCCTACTAATTCATACTGCAATGGGGGCCAGCACTTGGGCCACG GGGACAGAGCCAGCCCTGAGCGCCGCTCCCTGAGCAGGTATAGTCACCACCACTC 5 ACGGATTAGGAGTGGCCTGGAGCAGGACTGGAGCCGGTACCACCATTTGGAAAA TGAGCATGCTTGGGTCAGGGAGAGATTCTACCAGGACAAGCTGCGGTGGGACAA GTGCAGGTATTACCACGACAGGTACACGCCCCTATACACGGCCCGGGACGCCCGA GAATGGCGGCCTCTGCATGGTCGTGAGCATGACCGCCTTGTCCAGTCTGGACGGC CATACAAGGACAGCTACTGGGGCCGCAAGGGCTGGGAGCTGCAATCCCGGGGGA o AGGAACGGCCCCACTTCAACAGCCCCCGAGAGGCCCCTAGCCTTGCTGTGCCCCT CGAGAGACATCTCCAAGAGAAGGCTGCGCTGAGTGTGCAGGACAGCAGCCACAG TCTCCCTGAGCGCTTTCATGAACACAAAAGTGTCAAGTCGAGGAAGCGGAGGTAT GAGACTCTAGAAAATAATGATGGCCGTCTAGAAAAGAAAGTCCACAAAAGCCTG GAGAAGGACACGCTAGAGGAGCCAAGGGTGAAGAAGCACAAAAAGTCTAAAAA .5 GAAAAAGAAGTCCAAAGATAAACACCGGGATCGAGAAAGCAGGCACCAGCAGG AGTCTGATTTTTCAGGAGCATACTCTGATGCTGACCTCCATAGACACCGGAAGAA AAAGAAGAAAAAGAAAAGGCATTCCAGGAAGTCGGAGGACTTTATAAAGGATGT TGAGATGCGTTTACCGAAGCTCTCCAGCTACGAGGCCGGCGGCCATTTCCGGAGA ACAGAGGGCAGCTTTCTCCTGGCTGATGGTCTGCCTGTGGAAGACAGCGGCCCTT !o TCCGGGAGAAAACGAAGCATTTAAGGATGGAAAGCCGGCCTGACAGATGCCGTC TGTCGGAGTATGGCCAGGATTCAACATTTTGA Table 14. Deduced amino acid sequence of coding region of nDUB7 5 C-terminal potential nuclear localization (as well as targeting) sequences are underlined. MTIVDKTEPSDPSTCQNQPGSCEAVSPEDMDTGSASWGAVSSISDVSSHTLPLGPVPG AVVYSNSSVPEKSKPSPPKDQVLGDGIAPPQKVLFPSEKICLKWQQSHRVGAGLQNL o GNTCFANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQTHITQALSNPGD VIKPMFVINEMRRIARHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQATT LVCQIFGGYLRSRVKCLNCKGVSDTFDPYLDITLEIKCAAQSVTKALEQFVKPEQLDGE NSYKCSKCKKMVPASKRFTIHRSSNVLTISLKRFANFTGGKIAKDVKYPEYLDIRPYM SQPNGEPIIYVLYAVLVHTGFNCHAGHYFCYIKASNGLWYQMNDSIVSTSDIRAVLNQ 35 QAYVLFYIRSHDVKNGGESAHPAHSPGQSSPRPGVSQRVVNNKQVAPGFIGPQLPSH VMKNTPHLNGTTPVKDTPSSSVSSPNGNTSVNRASPATASTSVQNWSVTRPSVIDHP KKQKITISIHNKLPARQGQAPLNNSLHGPCLEAPSKAAPSSTITNPSAIQSTSNVPTTSTS PSEACPKPMVNGKAKVGASVLVPYGAESSEESDEESKGLAKENGVDMMAGTHSDRP EAAADDGAEASSHELQEPVLLNGANSADSDSQENSLAFDSASCQVQPELHTENLFSK to LNGLPGKVTPAPLQSVPEDRILETFKLTNQAKGPAGEESWTTTGGSSPKDPVSQLEPIS DEPSPLEIPEAVTNGSTQTPSTTSPLEPTISCTKEDSSVVVSAEPVEGLPSVPALCNSTGT ILGDTPVPELCDPGDLTANPSQPTEAVKGDTAEKAQDSAMAEVVERLSPAPSVLTGD GCEQKLLLYLSAEGSEETEDSSRSSAVSADTMPPKPDRTTTSSCEGAAEQAAGDRGD GGHVGPKAQEPSPAKEKMSSLRKVDRGHYRSRRERSSSGEHVRDSRPRPEDHHHKK 45 RHCYSRERPKQDRHPTNSYCNGGQHLGHGDRASPERRSLSRYSHHHSRIRSGLEQDW SRYHHLENEHAWVRERFYQDKLRWDKCRYYHDRYTPLYTARDAREWRPLHGREHD RLVQSGRPYKDSYWGRKGWELQSRGKERPHFNSPREAPSLAVPLERHLQEKAALSV
QDSSHSLPERFHEHKSVKSRKRRYETLENNDGRLEKKVHKSLEKDTLEEPRVKKHKK
WO 03/083050 PCT/US03/08590 31 SKKKKKSKDKHRDRESRHQQESDFSGAYSDADLHRHRKKKKKKKRHSRKSEDFIKD VEMRLPKLSSYEAGGHFRRTEGSFLLADGLPVEDSGPFREKTKHLRMESRPDRCRLSE YGQDSTF 5 Table 15. Nucleotide sequence aligmnent of hDUB7 and mDUB7 HDUB7 ATGACCATAGTTGACAAAGCTTCTGAATCTTCAGACCCATCAGCCTATCAGAATCAGCCT 60 MDUB7 ATGACCATAGTTGACAAAA--- CTGAACCTTCAGACCCATCAACCTGTCAGAACCAGCCT 57 10 ****************** ***** ************** *** ****** ****** HDUB7 GGCAGCTCCGAGGCAGTCTCACCTGGAGACATGGATGCAGGTTCTGCCAGCTGGGGTGCT 120 MDUB 7 GGCAGTTGTGAGGCGGTCTCACCTGAAGACATGGACACAGGCTCTGCCAGCTGGGGCGCT 117 ***** * ***** ********** ********* **** ************** *** 15 HDUB 7 GTGTCTTCATTGAATGATGTGTCAAATCACACACTTTCTTTAGGACCAGTACCTGGTGCT 180 MDUB 7 GTGTCTTCAATAAGTGATGTCTCAAGTCACACACTTCCATTAGGGCCAGTGCCTGGTGCT 177 ********* * * ****** **** ********** * ***** ***** ********* 20 HDUB7 GTAGTTTATTCGAGTTCATCTGTACCTGATAAATCAAAACCATCACCACAAAAGGATCAA 240 MDUB7 GTAGTTTATTCTAACTCGTCTGTACCTGAAAAATCAAAGCCATCACCACCAAAGGATCAA 237 *********** * ** *********** ******** ********** ********** HDUB7 GCCCTAGGTGATGGCATCGCTCCTCCACAGAAAGTTCTTTTCCCATCTGAGAAGATTTGT 300 25 MDUB7 GTCCTAGGTGATGGCATTGCTCCTCCTCAAAAGGTCCTGTTTCCATCTGAAAAGATTTGT 297 * *************** ******** ** ** ** ** ** ******** ********* HDUB7 CTTAAGTGGCAACAAACTCATAGAGTTGGAGCTGGGCTCCAGAATTTGGGCAATACCTGT 360 MDUB7 CTTAAGTGGCAACAAAGTCATCGAGTTGGCGCTGGGCTCCAGAATTTGGGCAACACCTGT 357 30 **************** **** ******* *********************** ****** HDUB7 TTTGCCAATGCAGCACTGCAGTGTTTAACCTACACACCACCTCTTGCCAATTACATGCTA 420 MDUB7 TTTGCCAATGCCGCATTGCAGTGTCTGACTTACACGCCACCCCTCGCCAATTACATGTTA 417 *********** *** ******** * ** ***** ***** ** ************ ** 35 HDUB7 TCACATGAACACTCCAAAACATGTCATGCAGAAGGCTTTTGTATGATGTGTACAATGCAA 480 MDUB7 TCCCATGAACACTCCAAGACATGCCACGCAGAAGGATTTTGTATGATGTGCACGATGCAG 477 ** ************** ***** ** ******** ************** ** ***** 40 HDUB7 GCACATATTACCCAGGCACTCAGTAATCCTGGGGACGTTATTAAACCAATGTTTGTCATC 540 MDUB7 ACACACATTACCCAGGCACTTAGCAACCCTGGGGATGTTATCAAGCCGATGTTCGTCATC 537 **** ************** ** ** ******** ***** ** ** ***** ****** 5 DUB7 AATGAGATGCGGCGTATAGCTAGGCACTTCCGTTTTGGAAACCAAGAAGATGCCCATGAA 600 45 MDUB7 AATGAAATGCGGCGTATAGCTAGACACTTCCGTTTTGGAAACCAAGAAGATGCCCATGAA 597 ***** ***************** ************************************ HDUB7 TTCCTTCAATACACTGTTGATGCTATGCAGAAAGCATGCTTGAATGGCAGCAATAAATTA 660 MDUB7 TTTCTTCAGTACACGGTCGATGCCATGCAGAAAGCATGTTTAAATGGCAGCAATAAATTA 657 50 ** ***** ***** ** ***** ************** ** ****************** HDUB7 GACAGACACACCCAGGCCACCACTCTTGTTTGTCAGATATTTGGAGGATACCTAAGATCT 720 MDUB7 GACAGACACACCCAGGCCACCACCCTGGTCTGCCAGATATTTGGAGGCTACCTAAGATCC 717 *********************** ** ** ** ************** *********** 55 HDUB7 AGAGTCAAATGTTTAAATTGCAAGGGCGTTTCAGATACTTTTGATCCATATCTTGATATA 780 MDUB7 CGAGTTAAATGTTTAAATTGCAAGGGTGTTTCAGATACCTTTGATCCATATCTGGACATA 777 **** ******************** *********** ************** ** *** WO 03/083050 PCT/US03/08590 32 H-DUB7 ACATTGGAGATAAAGGCTGCTCAGAGTGTCAACAAGGCATTGGAGCAGTTTGTGAAGCCG 840 MDUB7 ACGTTGGAGATTAAGGCTGCACAGAGTGTTACCAAGGCGTTAGAGCAGTTTGTGAAGCCA 837 ** ******** ******** ******** * ****** ** ***************** 5 HDUB7 GAACAGCTTGATGGAGAAAACTCGTACAAGTGCAGCAAGTGTAAGAAAATGGTTCCAGCT 900 MDUB7 GAACAACTGGATGGAGAAAACTCCTACAAGTGCAGCAAGTGCAAAAAAATGGTTCCAGCT 897 ***** ** ************** ***************** ***** ************ HDUB7 TCAAAGAGGTTCACTATCCATAGATCCTCTAATGTTCTTACACTTTCTCTGAAACGTTT 960 10 MDUB 7 TCAAAGAGATTCACAATCCATAGGTCCTCTAATGTTCTTACCATCTCACTGAAGCGCTTT 957 ******** ***** ******** ***************** * ** ***** ** *** HDUB7 GCAAATTTTACCGGTGGAAAAATTGCTAAGGATGTGAAATACCCTGAGTATCTTGATATT 1020 MDUB 7 GCCAACTTCACCGGTGGAAAGATTGCTAAGGATGTGAAATATCCTGAGTACCTTGATATC 1017 15 ** ** ** *********** ******************** ******** ******** HDUB7 CGGCCATATATGTCTCAACCCAACGGAGAGCCAATTGTCTACGTCTTGTATGCAGTGCTG 1080 MDUB 7 CGGCCCTATATGTCTCAGCCCAATGGAGAGCCAATTATTTATGTTTTGTATGCTGTGCTG 1077 ***** *********** ***** ************ * ** ** ******** ****** 20 HDUB7 GTCCACACTGGTTTTAATTGCCATGCTGGCCATTACTTCTGCTACATAAAAGCTAGCAAT 1140 MDUB7 GTGCACACTGGTTTTAATTGTCATGCTGGCCACTACTTTTGCTACATCAAGGCTAGCAAT 1137 ** ***************** *********** ***** ******** ** ********* 25 HDUB7 GGCCTCTGGTATCAAATGAATGACTCCATTGTATCTACCAGTGATATTAGATCGGTACTC 1200 MDUB7 GGCCTCTGGTATCAGATGAATGACTCCATCGTGTCCACCAGTGATATCAGAGCAGTGCTT 1197 ************** ************** ** ** *********** *** * ** ** HDUB7 AGCCAACAAGCCTATGTGCTCTTTTATATCAGGTCCCATGATGTGAAAAATGGAGGTGAA 1260 30 MDUB 7 AACCAGCAAGCTTACGTGCTCTTTTATATCAGGTCCCATGATGTGAAAAATGGAGGGGAG 1257 * *** ***** ** ***************************************** ** HDUB7 CTTACTCATCCCACCCATAGCCCCGGCCAGTCCTCTCCCCGCCCCGTCATCAGTCAGCGG 1320 MDUB7 TCTGCTCATCCTGCCCATAGCCCCGGCCAATCCTCTCCCCGCCCAGGAGTCAGTCAGCGG 1317 35 * ******* **************** ************** * *********** HDUB7 GTTGTCACCAACAAACAGGCTGCGCCAGGCTTTATCGGACCACAGCTTCCCTCTCACATG 1380 MDUB7 GTAGTCAACAACAAGCAGGTGGCTCCAGGGTTTATTGGACCCCAGCTGCCTTCCCATGTG 1377 ** **** ****** **** ** ***** ***** ***** ***** ** ** ** ** 40 HDUB7 ATAAAGAATCCACCTCACTTAAATGGGACTGGACCATTGAAAGACACGCCAAGCAGTTCC 1440 MDUB7 ATGAAGAACACGCCACACTTGAATGGCACCACGCCAGTGAAAGACACACCAAGTAGTTCT 1437 ** ***** * ** ***** ***** ** *** ********** ***** ***** 45 HDUB7 ATGTCGAGTCCTAACGGGAATTCCAGTGTCAACAGGGCTAGTCCTGTTAATGCTTCAGCT 1500 MDUB7 GTGTCAAGCCCTAACGGAAACACCAGCGTCAATAGGGCCAGTCCTGCTACTGCTTCGACT 1497 **** ** ******** ** **** ***** ***** ******* ** ****** ** HDUB7 TCTGTCCAAAACTGGTCAGTTAATAGGTCCTCAGTGATCCCAGAACATCCTAAGAAACAA 1560 50 MDUB7 TCTGTGCAGAACTGGTCTGTTACCAGACCCTCAGTTATTCCAGATCACCCCAAGAAACAA 1557 ***** ** ******** **** ** ******* ** ***** ** ** ********* HDUB7 AAAATTACAATCAGTATTCACAACAAGTTGCCTGTTCGCCAGTGTCAGTCTCAACCTAA- 1619 MDUB7 AAAATCACCATCAGTATTCACAACAAGTTGCCTGCTCGCCAGGGTCAGGCACCACTGAAT 1617 55 ***** ** ************************* ******* ***** * * ** ** HDUB7 ----- CCTTCATAGTAATTCTTTGGAGAACCCTACCAAGCCCGTTCCCTCTTCTACCATT 1674 MDUB7 AACAGCCTCCATGGCCCTTGTCTGGAGGCTCCTAGTAAGGCGGCACCCTCCTCCACCATC 1677 *** *** * ** * ***** **** *** * * ***** ** ***** 60 WO 03/083050 PCT/US03/08590 33 HDUB7 ACCAA- - -TTCTGCAGTACAGTCTACCTCGAACGCATCTACGATGTCAGTTTCTAGTAAA 1731 MDUB7 ACTAACCCTTCTGCAATACAGTCTACCTCGAACGTACCCACAACGTCGACTTC------- 1730 ** ** ******* ****************** * * ** * *** *** 5 HDUB7 GTAACAAAACCGATCCCCCGCAGTGAATCCTGCTCCCAGCCCGTGATGAATGGCAAATCC 1791 MDUB7 ------------------ CCCCAGTGAGGCCTGTCCCAAGCCCATGGTGAACGGCAAGGCT 1773 ** ****** **** ** ***** ** **** ***** * HDUB7 AAGCTGAACTCCAGCGTGCTGGTGCCCTATGGCGCCGAGTCCTCTGAGGACTCTGACGAG 1851 .0 MDUB7 AAAGTGGGCGCCAGTGTGCTTGTCCCCTATGGGGCCGAGTCCTCAGAAGAGTCTGATGAG 1833 ** ** * **** ***** ** ******** *********** ** ** ***** *** HDUB7 GAGTCAAAGGGGCTGGGCAAGGAGAATGGGATTGGTACGATTGTGAGCTCCCACTCTCCC 1911 MDUB7 GAGTCGAAGGGCCTGGCCAAGGAGAACGGTGTGGACATGATGGCCGGCACTCACTCCGAT 1893 [5 ***** ***** **** ********* ** * * * *** * ** * ***** HDUB7 GGCCAAGA--- TGCCGAAGATGAGG------AGGCCACTCCGCACGAGCTTCAAGAACCC 1962 MDUB7 AGGCCAGAAGCTGCTGCAGATGACGGTGCTGAGGCTTCCTCCCATGAGCTTCAAGAACCC 1953 * * *** *** * ****** * **** * * ** *************** HDUB7 ATGACCCTAAACGGTGCTAATAGTGCAGACAGCGACAGTGACCCGAAAGAAAACGGCCTA 2022 MDUB7 GTCCTGCTAAATGGTGCTAATAGCGCAGA------CAGTGACTCACAAGAGAACAGCCTG 2007 * **** ********** ***** ******* * **** *** **** Z5 HDUB7 GCGCCTGATGGTGCCAGCTGCCAAGGCCAGCCTGCCCTGCACTCAGAAAATCCCTTTGCT 2082 MDUB7 GCATTTGACAGTGCCAGCTGCCAGGTCCAGCCCGAGCTACACACAGAAAACCTCTTTTCC 2067 ** *** ************* * ****** * ** *** ******* * **** * HDUB7 AAGGCAAACGGTCTTCCTGGAAAGTTGATGCCTGCTCCTTTGCTGTCTCTCCCAGAAGAC 2142 30 MDUB7 AAACTTAATGGTCTTCCTGGAAAGGTGACGCCTGCTCCTTTGCAGTCTGTTCCTGAAGAC 2127 ** ** *************** *** ************** **** * ** ****** HDUB7 AAAATCTTAGAGACCTTCAGGCTTAGCAACAAACTGAAAGGCTCGACGGATGAAATGAGT 2202 MDUB7 AGAATCCTTGAGACCTTCAAGCTTACCAACCAGGCAAAGGGTCCAGCGGGTGAAGAGAGT 2187 35 * **** * ********** ***** **** * ** ** * *** **** **** HDUB7 GCACCTGGAGCAGAGAGGGGCCCTCCCGAGGACCGCGACGCCGAGCCTCAGCCTGGCAGC 2262 MDUB7 TGGACTACGACAGGGGGAAGCTCTCCAAAGGACCCTGTTTCACAGCTGGAGCCCATCAGT 2247 ** *** * * ** **** ****** * * *** **** *** 40 HDUB7 CCCGCCGCCGAATCCCTGGAGGAGCCAGATGCGGCCGCCGGCCTCAGCA---GCACCAAG 2319 MDUB7 GATGAGCCCAGTCCCCTTGAGATACCGGAGGCTGTCACCAATGGGAGCACACAGACCCCT 2307 * * *** ** * ** ** * * ** **** ** 45 HDUB7 AAGGCTCCGCCGCCCCGCGATCCCGGCACCCCCGCTACCAAAGAAGGCGCCTGGGAGGCC 2379 MDUB7 TCCACCACATCACCCCTGGAGCCCACCATCAGCTGTACCAAAGAAGACTCGTCCGTTGTT 2367 * * ******* *** ** * * *********** * * * * * HDUB7 ATGGCCGTCGCCCCCGAGGAG-------CCTCCGCCC------------AGCGCCGGCGAG 2421 50 MDUB7 GTCTCAGCTGAACCTGTGGAGGGTTTGCCTTCCGTCCCTGCTCTTTGTAACAGCACTGGT 2427 * ** * *** * **** ** * * * * HDUB7 GACATCGTGGGGGACACAGCACCCCCTGACCTGTGTGATCCCGGGAGCTTAACAGGCGAT 2481 MDUB7 ACTATCTTGGGGGATACCCCAGTGCCCGAATTGTGTGACCCTGGAGACTTGACTGCCAAC 2487 55 *** ******* ** ** ** ** ******* ** ** *** ** * * * HDUB7 GCGAGCCCGTTGTCCCAGGACGCAAAGGGGATGATCGCGGAGGGCCCGCGGGACTCGGCG 2541 MDUB7 CCGAGCCAGCCAACCGAAGCAGTGAAAGGTGATACAGCTGAGAAGGCTCAGGACTCTGCC 2547 0* ** ** * ** *** * * ****** ** 60 WO 03/083050 PCT/US03/08590 34 HDUB7 TTGGCGGAAGCCCCGGAAGGGTTGAGTCCGGCTCCGCCTGCGCGGTCGGAGGAGCCCTGC 2601 MDUB7 ATGGCTGAAGTGGTGGAGAGGCTGAGCCCTGCTCCCTCAGTACTCACAGGTGACGGGTGT 2607 **** **** *** ** **** ** ***** * * * * * ** ** 5 HDUB7 GAGCAGCCACTCCTTGTTCACCCCAGCGGGGACCACGCCCGGGACGCTCAGGACCCATCC 2661 MDUB7 GAGCAGAAACTCTTACTTTACCTCAGCGCAGAGGGGTCAGAGGAGACAGAAGACTCTTCC 2667 ****** **** * ** *** ***** ** * *** * * *** * *** HDUB7 CAGAGCTTGGGCGCACCCGAGGCCGCAGAGCGGCCGCCAGCTCCTGTGCTGGACATGGCC 2721 10 MDUB7 AGAAGCTCGGCGGTCTCTGCTGACACGATGC--------- -CCCCTAAGCCTGACAGGACC 2718 **** ** * * * * * * ** * *** ** **** * ** HDUB7 CCGGCCGGTCACCCGGAAGGGGACGCTGAGCCTAGCCCCGGCGAGAGGGTCGA-GGACGC 2780 MDUB7 ACCACCAGCTCCTGTGAAGGGGCTGCCGAGCAGGCTGCTGGGGACAGAGGCGATGGAGGC 2778 15 * ** * * ******* ** **** * ** ** ** * *** *** ** HDUB7 C--GCGGCGCCGAAAGCCCCAGGCCCTTCCCCAGCGAAGGAGAAAATCGGCAGCCTCAGA 2838 MDUB7 CATGTGGGACCCAAAGCTCAGGAGCCTTCCCCAGCCAAGGAAAAGATGAGCAGCCTCCGG 2838 * * ** ** ***** * * *********** ***** ** ** ******** * 20 HDUB7 AAGGTGGACCGAGGCCACTACCGCAGCCGGAGAGAGCGCTCGTCCAGCGGGGAGCCCGCC 2898 MDUB7 AAAGTGGACCGAGGACACTATCGGAGCCGGAGAGAGCGCTCCTCCAGTGGGGAGCACGTG 2898 ** *********** ***** ** ***************** ***** ******* ** 25 HDUB7 AGAGAGAGCAGGAGCAAGACTGAGGGCCACCGTCACCGGCGGCGCCGCACCTGCCCCCGG 2958 MDUB7 AGGGACAGCAGGCCCCGGCCGGAGGACCATCACCATAAGAAGCGGCACTGCTACAGCCGA 2958 ** ** ****** * * * **** *** * ** * *** * * ** * *** HDUB7 GAGCGCGACCGCCAGGACCGCCACGCCCC- ------------------- GGAGCACCACCCC 3000 30 MDUB7 GAGCGGCCCAAGCAGGACCGACACCCTACTAATTCATACTGCAATGGGGGCCAGCACTTG 3018 ***** * ******** *** * * ** ** *** HDUB7 GGCCACGGCGACAGGCTCAGCCCTGGCGAGCGCCGCTCTCTGGGCAGGTGCAGTCACCAC 3060 MDUB7 GGCCACGGGGACAGAGCCAGCCCT -- GAGCGCCGCTCCCTGAGCAGGTATAGTCACCAC 3075 35 ******** ***** ******* *********** *** ****** ********* HDUB7 CACTCCCGACACCGGAGCGGGGTGGAGCTGGACTGGGTCAGACACCACTACACCGAGGGC 3120 MDUB7 CACTCACGGATTAGGAGTGGCCTGGAGCAGGACTGGAGCCGGTACCACCATTTGGAAAAT 3135 ***** ** **** ** ****** ******* * * ***** * ** 40 HDUB7 GAGCGTGGCTGGGGCCGGGAGAAGTTCTACCCCGACAGGCCGCGCTGGGACAGGTGCCGG 3180 MDUB7 GAGCATGCTTGGGTCAGGGAGAGATTCTACCAGGACAAGCTGCGGTGGGACAAGTGCAGG 3195 **** ** **** * ****** ******* **** ** *** ******* **** ** 45 HDUB7 TACTACCATGACAGGTACGC- --CCTGTACGCTGCCCGGGACT - --GGAAGCCCTTCCA 3233 MDUB7 TATTACCACGACAGGTACACGCCCCTATACACGGCCCGGGACGCCCGAGAATGGCGGCCT 3255 ** ***** ********* * *** *** * ********* *** * ** HDUB7 CGGC- -GGCCGCGAGCACGAGCGGGCCGGGCTGCACGAGCGGCCGCACAAGGACCACAAC 3291 50 MDUB7 CTGCATGGTCGTGAGCATGACCGCCTTGTCCAGTCTGGACGGCCATACAAGGACAGCTAC 3315 * ** ** ** ***** ** ** * * * * ***** ******** * ** HDUB7 CGGGGCCGTAGGGGCTGCGAGCCGG--- CCCGGGAGAGGGAGCGGCACCGCCCCAGCAGC 3348 MDUB7 TGGGGCCGCAAGGGCTGGGAGCTGCAATCCCGGGGGAAGGAACGGCCCCACTTCAACAGC 3375 55 ******* * ****** **** * ****** ** *** **** ** * ** HDUB7 CCCCGCGCAGGCGCGCCCCACGCCCTCGCCCCGCACCCCGACCGCTTCTCCCACGACAGA 3408 MDUB7 CCCCGAGAGG------CCCCTAGCCTTGCTGTGCCCCTCGAGAGACATCTCCAAGAGAAG 3429 ***** * * *** *** ** ** ** *** * *** ** * 60 WO 03/083050 PCT/US03/08590 35 HDUB7 ACTGCACT --- TGTAGCCGGAGACAACTGTAACCTCTCTGATCGGTTTCACGAACACGAA 3465 MDUB7 GCTGCGCTGAGTGTGCAGGACAGCAGCCACAGTCTCCCTGAGCGCTTTCATGAACACAAA 3489 **** ** *** * ** * * *** **** ** ***** ****** ** 5 HDUB7 AATGGAAAGTCCCGGAAACGGAGACACGACAGTGTGGAGAACAGTGACAGTCATGTTGAA 3525 MDUB7 AGTGTCAAGTCGAGGAAGCGGAGGTATGAGACTCTAGAAAATAATGATGGCCGTCTAGAA 3549 * ** ***** **** ***** * ** * * * ** ** * *** * * * * *** HDUB7 AAGAAAGCCCGGAGGAGCGAACAGAAGGATCCTCTAGAAGAGCCTAAAGCAAAGAAGCAC 3585 10 MDUB7 AAGAAAGTCCACAAAAGCCTGGAGAAGGACACGCTAGAGGAGCCAAGGGTGAAGAAGCAC 3609 ******* ** * *** ******* * ***** ***** * * ********* HDUB7 AAAAAATCAAAGAAGAAAAAGAAATCCAAAGACAAACACCGAGACCGCGACTCCAGGCAT 3645 MDUB7 AAAAAGTCTAAAAAGAAAAAGAAGTCCAAAGATAAACACCGGGATCGAGAAAGCAGGCAC 3669 15 ***** ** ** *********** ******** ******** ** ** ** ****** HDUB7 CAGCAGGACTCAGACCTCTCAGCAGCGTGCTCTGACGCTGACCTCCACAGACACAAAAAA 3705 MDUB7 CAGCAGGAGTCTGATTTTTCAGGAGCATACTCTGATGCTGACCTCCATAGACACCGGAAG 3729 ******** ** ** * **** *** * ****** *********** ****** ** 20 HDUB7 AAGAAGAAGAAAAAGAAGAGACATTCAAGAAAATCAGAGGACTTTGTTAAAGATTCAGAA 3765 MDUB7 AAAAAGAAGAAAAAGAAAAGGCATTCCAGGAAGTCGGAGGACTTTATAAAGGATGTTGAG 3789 ** ************** ** ***** ** ** ** ********* * ** *** ** 25 HDUB7 CTGCACTTACCCAGGGTCACCAGCTTGGAGACTGTCGCCCAGTTCCGGAGAGCCCAGGGT 3825 MDUB7 ATGCGTTTACCGAAGCTCTCCAGCTACGAGGCCGGCGGCCATTTCCGGAGAACAGAGGGC 3849 *** ***** * * ** ****** *** * * ** *** ********* * **** HDUB7 GGCTTTCCTCTCTCTGGTGGCCCGCCTCTGGAAGGCGTCGGACCTTTCCGTGAGAAAACG 3885 30 MDUB7 AGCTTTCTCCTGGCTGATGGTCTGCCTGTGGAAGACAGCGGCCCTTTCCGGGAGAAAACG 3909 ****** ** *** *** * **** ****** * *** ******** ********* HDUB7 AAACACTTACGGATGGAAAGCAGGGATGACAGGTGTCGTCTCTTTGAGTATGGCCAGGGT 3945 MDUB7 AAGCATTTAAGGATGGAAAGCCGGCCTGACAGATGCCGTCTGTCGGAGTATGGCCAGGAT 3969 35 ** ** *** *********** ** ****** ** ***** * ************* * HDUB7 GATTGA------ 3951 MDUB7 TCAACATTTTGA 3981 * 40 Table 16. Deduced amino acid sequence alignment of hDUB7 and mDUB7 HDUB7 MTIVDKASESSDPSAYQNQPGSSEAVSPGDMDAGSASWGAVSSLNDVSNHTLSLGPVPGA 60 MD*UB7 MTIVDKT-EPSDPSTCQNQPGSCEAVSPEDMDTGSASWGAVSSISDVSSHTLPLGPVPGA 59 45 ******: *.****: *********** ***:*********************** HDUB7 VVY$SSSVPDKSKPSPQKDQALGDGIAPPQKVLFPSEKICLKWQQTHRVGAGLQNLGNTC 120 MDUB7 VVYSNSSVPEKSKPSPPKDQVLGDGIAPPQKVLFPSEKICLKWQQSHRVGAGLQNLGNTC 119 *******:****** *** *************************************** 50 HDUB7 FANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQAHITQALSNPGDVIKPMFVI 180 MDUB7 FANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQTHITQALSNPGDVIKPMFVI 179 55 HDUB*7 NEMRRIARHFRFGNQEDAHEFLQYTVDAQKACLNGSNKLDRHTQATTLVCQIFGGYLRS 240 MDUB7 NEMRRIARHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQATTLVCQIFGGYLRS 239 HDUB7 RVKCLNCKGVSDTFDPYLDITLEIKAAQSVNKALEQFVKPEQLDGENSYKCSKCKKMVPA 300 60 MDUB7 RVKCLNCKGVSDTFDPYLDITLEIKAAQSVTKALEQFVKPEQLDGENSYKCSKCKKMVPA 299 WO 03/083050 PCT/US03/08590 36 HDUB7 SKRFTIHRSSNVLTLSLKRFA FTGGKIAKDVKYPEYLDIRPYMSQPNGEPIVYVLYAVL 360 MDUB7 SKRFTIHRSSNVLTISLKRFANFTGGKIAKDVKYPEYLDIRPYMSQPNGEPIIYVLYAVL 359 5 HDUB7 VHTGFNCHAGHYFCYIKASNGLWYQMNDSIVSTSDIRSVLSQQAYVLFYIRSHDVKNGGE 420 MDUB7 VHTGFNCHAGHYFCYIKASNGLWYQMNDSIVSTSDIRAVLNQQAYVLFYIRSHDVKNGGE 419 *************************** **** ******************* 10 HDUB7 LTHPTHSPGQSSPRPVISQRVVTNKQAAPGFIGPQLPSHMIKNPPHLNGTGPLKDTPSSS 480 MDUB7 SAHPAHSPGQSSPRPGVSQRVVNNKQVAPGFIGPQLPSHVMKNTPHLNGTTPVKDTPSSS 479 .**.********** .*****.***.************::.**.* ***** *:******* 15 HDUB7 MSSPNGNSSVNRASPVNASASVQNWSVNRSSVIPEHPKKQKITISIHNKLPVRQCQSQPN 540 MDUB7 VSSPNGNTSVNRASPATASTSVQNWSVTRPSVIPDHPKKQKITISIHNKLPARQGQAPLN 539 .******.*******.**. *******.*.****.****************.** *: * HDUB7 --LHSNSLENPTKPVPSSTITN-SAVQSTSNASTMSVSSKVTKPIPRSESCSQPVMNGKS 597 20 MDUB7 NSLHGPCLEAPSKAAPSSTITNPSAIQSTSNVPTTSTS--------PSEACPKPMVNGKA 591 **. .** *.*..******* **:*****..* *.**:.*:** HDUB7 KLNSSVLVPYGAESSEDSDEESKGLGKENGIGTIVSSHS--PGQDAED-EEATPHELQEP 654 MDUB7 KVGASVLVPYGAESSEESDEESKGLAKENGVDMMAGTHSDRPEAAADDGAEASSHELQEP 651 25 :.:************:************: . .:** * *- ** ****** HDUB7 MTLNGANSADSDSDPKENGLAPDGASCQGQPALHSENPFAKANGLPGKLMPAPLLSLPED 714 MDUB7 VLLNGANSADSDS--QENSLAFDSASCQVQPELHTENLFSKLNGLPGKVTPAPLQSVPED 709 . *********** :**. ** *.**** ** **:** *:* ******: **** *:*** 30 HDUB7 KILETFRLSNKLKGSTDEMSAPGAERGPPEDRDAEPQPGSPAAESLEEPDAAA-GLSSTK 773 MDUB7 RILETFKLTNQAKGPAGEESWTTTGGSSPKDPVSQLEPISDEPSPLEIPEAVTNGSTQTP 769 .* ** .* *.**. .* * .;.:* : * * ... ** *:*.: * :. 35 HDUB7 KAPPPRDPGTPATKEGAWEAMAVAPEEPPP------SAGEDIVGDTAPPDLCDPGSLTGD 827 MDUB7 STTSPLEPTISCTKEDSSVVVSAEPVEGLPSVPALCNSTGTILGDTPVPELCDPGDLTAN 829 .:.* : .. ** : . :. * * .: *:***. *:*****.**. HDUB7 ASPLSQDAKGMIAEGPRDSALAEAPEGLSPAPPARSEEPCEQPLLVHPSGDHARDAQDPS 887 40 MDUB7 PSQPTEAVKGDTAEKAQDSAMAEVVERLSPAPSVLTGDGCEQKLLLYLSAEGSEETEDSS 889 * . .* * .. ** ** * ** * : :*** **:: *.: :.: *. HDUB7 QSLGAPEAAERPPAPVLDMAPAGHPEGDAEPSPGERVED-AAAPKAPGPSPAKEKIGSLR 946 MDUB7 RSS -AVSADTMPPKP- -DRTTTSSCEGAAEQAAGDRGDGGHVGPKAQEPSPAKEKMSSLR 946 45 :* * .* ** * * :.:. ** ** :.*:* :. ** *******:*** HDUB7 KVDRGHYRSRRERSSSGEPARESRSKTEGHRHRRRRTCPRERDRQDRHAP------EHHP 1000 MDUB7 KVDRGHYRSRRERSSSGEHVRDSRPRPEDHHHKKRHCYSRERPKQDRHPTNSYCNGGQHL 1006 **** *** ******* .*:*.: *.* *:* .*** :** .. : 50 HDUB7 GHGDRLSPGERRSLGRCSHHHSRHRSGVELDWVRHHYTEGERGWGREKFYPDRPRWDRCR 1060 MDUB7 GHGDRASP-ERRSLSRYSHHHSRIRSGLEQDWSRYHHLENEHAWVRERFYQDKLRWDKCR 1065 ***** ** *****.* ****** ***:* ** *:*: *.*:.* **:** *: ***:** 55 HDUB7 YYHDRYA-LYAAR-- -DWKPFHGGREHERAGLHERPHKDHNRGRRGCEP-ARERERHRPS 1115 MDUB7 YYHDRYTPLYTARDAREWRPLHG-REHDRLVQSGRPYKDSYWGRKGWELQSRGKERPHFN 1124 ******. **.** .*.*:** ***:* **:** **: * * :* :**:. HDUB7 SPRAGAPHALAPHPDRFSHDRTALVAGDNCN-LSDRFHEHENGKSRKRRHDSVENSDSHV 1174 60 MDUB7 SPREAP--SLAVPLERHLQEKAALSVQDSSHSLPERFHEHKSVKSRKRRYETLENNDGRL 1182 WO 03/083050 PCT/US03/08590 37 HDUB7 EKKARRSEQKDPLEEPKAKKHKKSKKKKKSKDKHRDRDSRHQQDSDLSAACSDADLHRHK 1234 MDUB7 EKKVHKSLEKDTLEEPRVKKHKKSKKKKKSKDKHRDRESRHQQESDFSGAYSDADLHRHR 1242 5***.::* :**.**: **********:**:*** ********: 5 HDUB7 KKKKKKKRHSRKSEDFVKDSELHLPRVTSLETVAQFRRAQGGFPLSGGPPLEGVGPFREK 1294 MDUB7 KKKKKKKRHSRKSEDFIKDVEMRLPKLSSYEAGGHFRRTEGSFLLADGLPVEDSGPFREK 1302 ****************:** *::**:::* *: .:***::*.* *:.* *:*. ****** 10 HDUB7 TKHLRMESRDDRCRLFEYGQGD-- 1316 MDUB7 TKHLRMESRPDRCRLSEYGQDSTF 1326 ********* ***** ****.. 15 Table 17. Amino acid sequence alignment of catalytic domain among murine DUB1, DUB2, hDUB7 and mDUB7. Amino acids that are involved in catalysis in DUB1 (Cys-60, Asp-133, and His-307) are underlined. mDUB1 MVVALSFPEADPALSSPDAPELHQDEAQVVEELTVNGKHSLSWESPQGPGCGLQNTGNSC 60 20 mDUB2 MVVSLSFPEADPALSSPGAQQLHQDEAQVVVELTANDKPSLSWECPQGPGCGLQNTGNSC 60 hDUB7 VVYSSSSVPDKSKPSPQKDQALGDGIAPPQKVLFPSEKICLKWQQTHRVGAGLQNLGNTC 120 mDUB7 VVYSNSSVPEKSKPSPPKDQVLGDGIAPPQKVLFPSEKICLKWQQSHRVGAGLQNLGNTC 119 .* .* .. *. * : * * * .*.*: .: * * * **:* 25 mDUBI YLNAALQCLTHTPPLADYMLSQEHSQTCCSPEGCKLCAMEALVTQSLLHSHSGDVMKPSH 120 mDUB2 YLNAALQCLTHTPPLADYMLSQEYSQTCCSPEGCKMCAMEAHVTQSLLHSHSGDVMKPSQ 120 hDUB7 FANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQAHITQALSN--PGDVIKPMF 178 mDUB7 FANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQTHITQALSN--PGDVIKPMF 177 . ********.*****.****.**** : * :*:*:: : **:* :.*:* 30 mDUB1 ILTSA ------ -FHKHQQEDAHEFLMFTLETMHESCLQVHRQSKPTSEDSSPIHDIFGGWW 174 mDUB2 ILTSA ------ FHKHQQEDAHEFLMFTLETMHESCLQVHRQSEPTSEDSSPIHDIFGGLW 174 hDUB7 VINEMRRIARHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQATTLVCQIFGGYL 238 mDUB7 VINEMRRIARHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQATTLVCQIFGGYL 237 35 : .. *- -******** * * : :: : * mDUB1 RSQIKCLLCQGTSDTYDRFLDIPLDISSAQSVKQAL.WDTEKSEELCGDNAYYCGKCRQKM 234 mDUB2 RSQIKCLHCQGTSDTYDRFLDVPLDISSAQSVNQALWDTEKSEELRGENAYYCGRCRQKM 234 hDUB7 RSRVKCLNCKGVSDTFDPYLDITLEIKAAQSVNKALEQFVKPEQLDGENSYKCSKCKKMV 298 40 mDUB7 RSRVKCLNCKGVSDTFDPYLDITLEIKAAQSVTKALEQFVKPEQLDGENSYKCSKCKKMV 297 **..*** *.* ***.* .*.* .**** .** : *.* *:*:* * .:*:: mDUB1 PASKTLHVHIAPKVLMVVLNRFSAFTGNKLDRKVSYPEFLDLKPYLSEPTGGPLPYALYA 294 mDUB2 PASKTLHIHSAPKVLLLVLKRFSAFMGNKLDRKVSYPEFLDLKPYLSQPTGGPLPYALYA 294 45 hDUB7 PASKRFTIHRSSNVLTLSLKRFANFTGGKIAKDVKYPEYLDIRPYMSQPNGEPIVYVLYA 358 mDUB7 PASKRFTIHRSSNVLTISLKRFANFTGGKIAKDVKYPEYLDIRPYMSQPNGEPIIYVLYA 357 .*.**. * * *. . *.***:**::**:*:*.* *- * *** mDUB1 VLVHDGATSHSGHYFCCVKAGHGKWYKMDDTKVTRCDVTSVLNENAYVLFYVQQANLKQ 352 50 mDUB2 VLVHEGATCHSGHYFSYVKARHGAWYKMDDTKVTSCDVTSVLNENAYVLFYVQQTDLKQ 352 hDUB7 VLVHTGFNCHAGHYFCYIKASNGLWYQMNDSIVSTSDIRSVLSQQAYVLFYIRSHDVKN 417 mDUB7 VLVHTGFNCHAGHYFCYIKASNGLWYQMNDSIVSTSDIAVLNQQAYVLFYIRSHDVKN 416 **** * .. . . *.*. :******::. : 55

Claims (15)

1. An isolated polynucleotide encoding a deubiquitinating protease selected from the group consisting of hDUB7 and mDUB7.
2. An isolated deubiquitinating protease polypeptide selected from the group consisting of hDUB7 and mDUB7.
3. A method of using a polynucleotide according to claim 1, wherein the polynucleotide is used in an assay to identify an inhibitor of a hDUB or mDUB7 of claim 1.
4. A method of using a polypeptide according to claim 2, wherein the polypeptide is used in an assay to identify an inhibitor of a hDUB or mDUB7 of claim 2.
5. A method of reducing inflammation by regulating proinflammatory cytokine signaling, by administering a compound capable of specifically inhibiting a polypeptide according to claim 2.
6. A method of modulating an autoimmune disease by altering cytokine receptor signaling involved in lymphocytes proliferation, by administering a compound specifically capable of inhibiting a polypeptide according to claim 2.
7. A method of modulating an immune reaction during infection, by administering a compound capable of specifically inhibiting a polypeptide according to claim 2.
8. A method of reducing inflammation by regulating proinflammatory cytokine signaling, by administering a compound capable of specifically altering regulation of transcription of a polynucleotide of claim 1.
9. A method of modulating an autoimmune disease by altering cytokine receptor signaling involved in lymphocytes proliferation, by administering a 39 compound capable of specifically altering regulation of transcription of a polynucleotide of claim 1.
10. A method of modulating an immune reaction during infection, by administering a compound capable of specifically altering regulation of 5 transcription of a polynucleotide of claim 1.
11. A method of identifying a modulator of a deubiquitinating protease, wherein a compound is added to the reporter assay comprising a polynucleotide immediately 5' to a human deubiquitinating protease selected from the group consisting of hDUB7 and mDUB7 operatively linked to a reporter gene, and the 10 effect of the compound is determined.
12. A transducing peptide comprising an NLS or transducing sequence of hDUB7 or mDUB7 linked to a cargo molecule.
13. The transducing peptide of claim 12, wherein the NLS or transducing sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, 15 SEQ ID NO:3, and SEQ ID NO:4.
14. The transducing peptide of claim 12, wherein the cargo molecule is a biologically active protein, therapeutically effective compound, antisense nucleotide, or test compound.
15. A method of delivering a biologically active protein, therapeutically effective 20 compound, antisense nucleotide, or test compound to a cell wherein a transducing peptide of claim 12 is added exogenously to a cell. AVENTIS PHARMACEUTICALS INC WATERMARK PATENT & TRADE MARK ATTORNEYS P24403AU00
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