AU2003230701B2 - Human deubiquitinating protease gene on chromosome 7 and its murine ortholog - Google Patents

Description

WO 03/083050 PCT/US03/08590 HUMAN DEUBIOUITINATING PROTEASE GENE ON CHROMOSOME 7 AND ITS MURINE ORTHOLOG 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 0 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 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 ubiquitin is next transferred to an active site Cys residue of a ubiquitin-carrier protein, E2 (Step 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).

!0 Proteins ligated to polyubiquitin chains are usually degraded by the 26S proteasome complex that requires ATP hydrolysis for its action. The 26S proteasome is formedby 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 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 The latter two products are converted to free and reusable ubiquitin by the action of ubiquitin-C-terminal hydrolases or isopeptidases (Steps 5 and Some isopeptidases may also disassemble certain ubiquitin-protein conjugates (Step 7) and thus prevent their proteolysis by the 26S proteasome.

The latter type ofisopeptidase 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.

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 cellcycle 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 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 0 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 proteinkinases. Indeed, it now appears that in several (though certainly not all) instances, PEST elements contain phosphorylation sites 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 Dl, are targeted for ubiquitinylation by phosphorylation at specific, single sites. In the case of the IkBca inhibitor of the NF-kB transcriptional regulator, phosphorylation at two 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 Siclp 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 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 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.

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.

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-l of successive ubiquitinmoieties. The other class of ubiquitin genes encodes ubiquitin C-terminal extension proteins, which are peptide bond fusions between the 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 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 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 polyubiquitin 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 structurefunction relationships of these enzymes remain poorly studied.

Deubiquitinating enzymes can be divided broadly on the basis of sequence homology 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 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, 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 169 and Asp 84 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 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. Manyof these isoforms have Nterminal 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 deubiquitinating 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 growth via control of the Go-GI transition. The catalytic conserved Cys residue of the enzyme is required for its activity. DUB-2 is inducedby 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.

Cytokines, such as interleukin-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 transducers and activators of transcription) pathway, and when expressed in Ba/F3 cells DUB- 2 markedly prolonged IL-2-induced STATS phosphorylation. Although DUB-2 does not enhance IL-2-mediatedproliferation, when withdrawn from growth factor, cells expressing DUB-2 had sustained STATS phosphorylation and enhanced expression of IL-2-induced genes cis and c-myc. DUB-2 expression markedly inhibited apoptosis induced by cytokine 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, 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.

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 ofpolymerase 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 BAC contained a novel DUB gene (DUB-2A) with extensive homology to DUB-2. Like DUB- 1 and DUB-2, the DUB-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 D UB-2A gene has a hematopoietic-specific functional enhancer sequence. It is proposed that there are at least 3 members of the DUB subfamily (DUB-1, DUB-2, and DUB-2A) and that different hematopoietic cytokines induce specific DUB genes, thereby initiating a cytokinespecific growthresponse. (Back, et al, Blood. 2001;98:636-642).

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 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.

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 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.

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 the receptors for IL-3, granulocyte macrophage-colony-stimulating factor, and suggesting a specific role for the c subunit shared by these receptors. In the process of cloning the DUB-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-2inducible DUB enzyme, DUB-2 and closely related DUB-2a were identified. DUB-1 and 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. 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; Zhu, 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, et al., (1996). The murine DUB-1 gene is specifically induced by the betac subunit ofinterleukin-3 receptor, Mol Cell Biol 16, 4808- These effects are likely due to the deubiquitination of receptors or other signaling intermediates by DUB-1 or DUB-2, murine analogs ofhDUBs. Inhibition ofhDUBs 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-i, encodes a deubiquitinating enzyme with growth regulatory activity. In addition, a highly related gene, 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-I, the DUB-2 proteinhad deubiquitinating activity in vitro. When a conserved cysteine residue of DUB-2, required for ubiquitin-specific thiol protease activity, was mutated to serine deubiquitinating activity was abolished. DUB-1 and DUB-2 proteins are highly related to 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 ofcytokine-inducible DUB enzymes. We propose that different cytokines induce specific DUB genes. Each induced DUB 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 GenBankTl. 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) 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 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). TheDUB proteins may modify the ubiquitinproteolytic 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 ofDUB 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 protease 23431 coding sequence in W0200123589-A2.

References 1. Baek, K. Mondoux, M. Jaster, Fire-Levin, and D'Andrea, A. D. (2001). DUB-2A, a new member of the DUB subfamily of hematopoietic deubiquitinating enzymes, Blood 98, 636-42.

2. Jaster, Baek, K. and D'Andrea, A. D. (1999). Analysis of cis-acting sequences and transacting factors regulating the interleukin-3 response element of the DUB-1 gene, Biochim Biophys Acta 1446, 308-16.

3. Jaster, Zhu, Pless, Bhattacharya, Mathey-Prevot, and D'Andrea, A. D. (1997).

JAK2 is required for induction of the murine DUB-1 gene, Mol Cell Biol 17, 3364-72.

4. Migone, T. Humbert, Rascle, Sanden, D'Andrea, Johnston, J. Back, K. H., Mondoux, M. Jaster, Fire-Levin, 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.

Zhu, Carroll, Papa, F. Hochstrasser, and D'Andrea, A. D. (1996a). DUB-1, a deubiquitinating enzyme with growth-suppressing activity, Proc Natl Acad Sci U S A 93, 3275-9.

6. Zhu, Lambert, Corless, Copeland, N. Gilbert, D. Jenkins, N. 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, Pless, Inhor, Mathey-Prevot, and D'Andrea, A. D. (1996b). The murine DUB-1 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 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 is a green light for traffic to proceed from this important intracellular intersection to the lumen WO 03/083050 PCT/US03/08590 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 UEV 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 tsgl0l, is a tumor suppressor (Li and Cohen, 1996) The current results suggest that mutations in tsglOl 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 recognized has been unclear. Linda Hicke reported that yeast Entl is vital for the ubiquitindependent endocytosis of yeast factor receptor (see also Wendland et al., 1999). Entl carries a proposed ubiquitin binding motif called the UIM domain (Hofinann and Falquet, 2001 and Hicke showed that Entl indeed binds ubiquitin directly. Entl 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 signalrecognizing components-Entl resides at the plasma membrane, while ESCRT-I is associated with late endosomes.

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).

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).

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 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 Takl kinase is a downstream target of Lys63-chain signaling in the 1KK 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 defines a large E3 family.) Modification of Traf6 with a Lys63-chain leads to the activation of Taki, which in turn phosphorylates IKK (Wang et al., 2001). Activated IKK then phosphorylates IkBa and triggers its tagging with Lys48-chains. Only then do proteasomes enter the picture-they degrade IkBa and thereby free its partner, NFkB, to translocate to the nucleus and activate the expression of inflammatory response genes. Chen's results suggest 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 Jentsch, 2000). New genetic data reported by Helle Ulrich confirmed the central importance of 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 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 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 murine ortholog, 0t 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 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.

References Katzmann 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) tsglOl: a novel tumor susceptibility gene isolated by controlled 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 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 18:4383-4393.

Hofmann K. and Falquet L. (2001) A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems. Trends Biochem. Sci., 26:347- 350.

00 SGarcia-Higuera Taniguchi Ganesan Meyn Timmers C., c Hejna 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 proteinconjugation 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.

C Biochem., 67:425-479.

Deng Wang Spencer Yang Braun You Slaughter C., N 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 Deng Hong Akkaraju Inoue and Chen Z.J.

(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 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.

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.

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 00 O ortholog, named as hDUB7 and mDUB7, respectively. Therefore, according to the C present invention, there is provided an isolated polynucleotide and a polypeptide encoding a human deubiquitating protease selected from the group consisting of hDUB7 and mDUB7.

\D

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 0(ORFs) that are 67% amino acid identity to each other, when gaps caused by c- deletion was not counted as mismatch, and exhibit 75% identity in nucleotide 0 10 sequence. Furthermore, both hDUB7 and mDUB7 share 48% identity to murine N 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 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 comprising an NLS or transducing sequence of hDUB7 or mDUB7 linked to a cargo molecule. The invention also includes a transducing peptide comprsing an NLS or transducing sequence is selected from the group consisting of a peptidyl fragment comprising KAKKHKKSKKKKKSKDKHR, HRHKKKKKKKKRHSRK, KKHKKSKKKKKSKDKHR, and HRHRKKKKKKKRHSRK.

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.

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 00 O The invention also includes methods of reducing inflammation and c methods of modulating an autoimmune disease or immune reaction by administering a compound capable of inhibiting a polypeptide encoding a human deubiquitating protease selected from the group consisting of hDUB7 and \0 mDUB7.

Manipulation of these gene products by small molecular compounds can reduce inflammation by regulating proinflammatory cytokine signaling, (2) Smodulate autoimmune diseases by regulating cytokine receptor signaling that are C- critical for lymphocytes proliferation, and immune over-reaction during 0 10 infection using above mechanisms.

C 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.

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 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).

WO 03/083050 PCT/US03/08590 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 rn-on 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 database (preliminary assembly of the mouse WGS reads based on an Nov 9th freeze of the 0 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 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 mDUB7 sequence was aligned with hDUB7 and showed 67% 0 homology in amino acid level and 75% homology in nucleotide level.

TaqMan real time PCR analysis of expression ofhDUB7 in human immunocytes upon various stimulation 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 IxTaqMan RT Buffer Mix, 5.5mM MgCl 2 mM dNTPs, 2.5 uM Random Hexamers, 40 U RNAse inhibitor, 125U Multiscribe Reverse Transcriptase. Mix by pipeting up and down. Incubate 25 0 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 0 C, or alternatively, can be stored at 0 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 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 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 Ctvalues determined during the exponential phase of PCR.

Primer-probe set used is as follow: Forward Primer Reverse Primer Probe sequence 5'-CCACGACAGAACTGCACTTGTAG-3' CCGGGACTTTCCATTTTCG-3' CAACTGTAACCTCTCTGATCGGTTTCACGAA-3' Table 1. Expression ofhDUB7 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 Uprcgulation upon 0.9 1.5 stimulation Table 2. Expression of DUB7 in PBMC stimulated with LPS (100 ng/ml) and/or PHA 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 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 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 Stimuli/time 4 hours hours

I

Fold Upregulation upon stimulation LPS IL-4, anti-CD40 mAb 1.11 2.44 0.70 Table 4. Expression ofhDUB7 in entiched CD4 T cells stimulated with anti-CD3 and anti- CD28 mAbs for 3,6 and 18 hours by TaqMan (Donor mAbs Stimulation/Time 3 hours 6 hours 18 hours Fold Upregulation upon 1.36 1.74 0.37 stimulation Table 5. Expression ofhDUB7 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 Thl Th2 Fold Upregulation upon stimulation 2.60 0.36 1.72 Table 6. Expression ofhDUB7 in differentiated ThO, Thl 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 Thl Th2 Fold Upregulation in 8 hours 1.38 1.11 1.71 Fold Upregulation in 18 hours 0.94 0.81 1.47 Table 7. Expression of hDUB7 in various tissue examined by Affymatrix chip analysis Tissue Con_Adipose_l Con_Adipose_2 CV Heart 1 CV Heart 2 CV Heart 3 CVPericardia_1 Dig_Colon_l Dig_Colon_2 Dig_Esophagus_l Dig_Esophagus_2 Relative Intensity 2287 4190 2545 3907 5367 3682 2387 2894 5004 1658 WO 03/083050 18 Dig FetalLiver_1 1288 Dig FetalLiver2 4676 Dig FetalLiver_3 829 DigFetalLiver_-4 3161 Dig Liver_1 3094 Dig Liver2 1527 Dig Liver3 3410 DigPancreas_1 3731 DigPancreas_2 4837 Dig Rectum1 2329 DigRectum2 1851 DigSalivaryGland 1 2337 DigSalivaryGland 2 2110 DigSmallIntestine_1 2838 DigSmallIntestine_2 2662 DigStomach_1 2187 EndAdrenaiGland_1 591 EndAdrenalGiand_2 2199 End_Thyroidl 2564 End_Thyroid 2 2392 End_Thyroid_3 3522 ExoBreast_1 3673 ExoBreast_2 6173 ExoMammaryGland 3741 1 1mmBoneMarrow_1 1090 1mm Spleen_ 2429 hmmThymusi1 3666 ImmThymus 2 1759 RepCervix_1 4482 RepCervix_2 3362 RepPlacenta_1 1248 RepPlacenta2 2378 Rep Placenta3 1622 RepProstate_1 5128 Rep Prostate_2 2762 RepTestis_1 2252 Rep Testis_2 3196 Rep Uterus1 4720 Rep Uterus2 3789 ResLung-1 2313 ResLung-2 3177 Res_Lung_3 4409 Res_Lung_4 2366 Res Trachea 1 2152 ResTrachea_2 2358 ResTrachea_3 812 ResTrachea_4 812 SkSkeletailuscle_1 2838 SkSkeletalMuscle_2 6106 PCT/USO3/08590 WO 03/083050 PCT/US03/08590 19 Skin Skin 1 5500 Uri_Kidney_l 3593 UriKidney_2 1311 UriKidney_3 2747 Uri_Kidney_4 1530 NS Brain 1 3214 NS Brain 2 2173 NS Brain 3 1332 NS Brain 4 2604 NS Brain 5 1663 NS Cerebellum 1 3175 NS Cerebellum 2 1766 NSFetalBrain 1 4299 NS FetalBrain_2 2549 NS FetalBrain 3 4027 NS_SpinalCord_l 2976 NS SpinalCord_2 3999 NS_SpinalCord_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 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 C uterus 335 C uterus 401 Deubiquitination Assay Confirmation that the DUB is a deubiquitinating enzyme may be shown using 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 element. Ub-Met--gal is expressed from a pACYC 84-based plasmid. Plasmids are cotransformed as indicated into MC 1061 Escherichia coli. Plasmid-bearing E. coli MC1061 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 commercial polyubiquitinated protein as substrate.

HDUB7 and mDUB7 are potential inflamatory cytokins specific Immediate-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 Z0 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 ofhDUB7 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 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 NIKNK-T cells.

The DUB Subfamily of the ubp Superfamily o0 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 theirprimary amino acid sequence. For instance, DUB proteins contain a lysine-rich region (Lys domain) and a HV domain near their carboxyl 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, et al., "Expanding the TRANSFAC database towards an expert 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 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) 1835..1839(100) 251..247(100) 265..261(100) 273..269(100) 1429..1433(100) 1315..1319(100) HSF heat shock factor (Drosophila) WO 03/083050 WO 03/83050PCT/USO3/08590 1264.1268 100) 1060.. 1064(100) 1014.. 1010(100) 1540.. 1536(100) 1559.. 1555(1 00) 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) 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) HF heat shock factor kyeast) I i MO001 1418..1412(100) 16-89..1695(98) CdxA CdxA WO 03/083050 WO 03/83050PCT/USO3/08590 1566..1572(98) 1460.. 1466(98) 1319..1325(98) 969..975(98) 1463.. 1457(98) 16 14..1608(98) 1065.. 1059(94) 1599.. 1605(93) 1375.. 1369(93) 1840..1834(93) 1859..1865(92) 1168..1174(92) 1218.. 1212(92) 1484(90) M00048 447..452(100) ADRi alcohol dehydrogenase gene regulator 1 535..540(95) 1716..1721(93) 459..454(93) 558..553(93) 1180..1185(93) 305..310(93) M00354 1951l..1941(99) Do±3 Dof3- single zinc finger transcription factor 1560..1550(95) 104..114(93) M00227 1920..1928(98) v-Myb v-Myb M00141 521..513(98j Lyf-1 LyF-1 820(98) M00344 806..795(98) RAVi 3'-part of bipartite RAVi binding site, interacting with AP2 domain 806..817(92) M00253 1139..1 146(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) M0O0286 577. .564(97) GKLF gut-enriched Krueppel-like factor M00199 684..676(96) AP-I AP-1 binding site WO 03/083050 WO 03/83050PCT/USO3/08590 .684(95) M00183 227..218(96) c-Myb c-Myb 28..37(95) 1238(90) M00154 1714..1721(96) STRE stress-response element M00140 1824..1831(96) Bed Bicoid 834..841(93) .534(93) M00100 1418..1412(96) CdxA CdxA 1209..1215(92) M00291 1652..1667(95) Freac-3 Fork head R-Elated ACtivator-3 M00073 1948.. 1958(95) deltaEFl deltaF1 807. .797(95) 1452.. 1442(92) 805..815(90) M00216 1176..1 167(95) TATA Retroviral TATA box M00120 1952.. 1942(95) dI dorsal 1561 155 1(93) M00042 1861..1852(95) Sox-5 1790.. 178 1(9 1) M00174 675..685(95) AlP-I activator protein 1 M00230 1797..1808(95) Skn-1 maternal gene product M00272 1024.. 1033(94) p53 tumor suppressor p53 1033..1024(94) M001 60 1862..185 1(94) SRY sex-detenmining region Y gene produact M00022 111..120(94) Hb Hunchback 436..427(91) M00053 447. .456(94) c-Rel c-Rel M00249 1244.. 1256(93) CHOP- heterodimers of CHOP and C/EBPalpha, M00142 1367.. 1362(93) NJT2 activator of nitrogen-regulated genes 1348.. 1343(9 1) M00289 1670.. 165 8(93) H.FH-3 HNF-3/Fldl Homolog 3 Freac-6) M00019 1381..1366(93) D fd Deformned 1593..1608(91) M00147 1903.. 1912(92) HSF2 heat shock factor 2 M001984 806..815(92) MyoD myoblast determining factor M00345 225. .218(92) GAmyb GA-regulated mnyb gene from barley M00094 1658.. 1670(92) BR-C Broad-Complex Z4 S1398.. 1386(90) M00349 1200..1 19 1(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) TCF11I TCF11/KCR-FI/Nrfl homodimers M00241 1224..1217(91) Nkx-2.5 homeo doinain factor Nkx-2.5/Csx, finnian WO 03/083050 WO 03/83050PCT/USO3/08590 homolog M00283 1863..1878(90) Zeste Zeste transvection gene product M00046 1 113..1105(90) GCR1 GCR1 M00353 1069..1079(90) Dof2 Dof2 single zinc finger transcription factor 195 1..1941 M00263 985 994(90) StuAp Aspergillus Stunted protein M00051 448..457(90) NF-kappaB N-F-kappaB M00350 1200..1 191(90) GATA-3 GATA-binding factor 3 M00276 185 1..1860(90) Matl-Mc M-box interacting with Matl-Mc M00075 1936..1945(90) GATA-1 GATA-bindrng factor 1 ____442..451(90) M00355 279..269(90) PBF PBF (MPBF) M00352 1775..1785(90) Dofi Dofi MINB1 a single zinc finger transcription factor M00294 1670..1658(90) HFH-8 KNF-3/Fkh Homolog-8 M00131 1762..1748(90) HNF-3beta Hepatocyte Nuclear Factor 3beta M00137 1320..1332(90) Oct-i octamner factor 1 M00054 1448. .457(90) 1NE-kappaB NE-kappaB WO 03/083050 PCT/US03/08590 26 Table 10. Nucleotide sequence of coding region of human DUB7 (hDUB7)

ATGACCATAGTTGACAAAGCTTCTGAATCTTCAGACCCATCAGCCTATCAGAATC

AGCCTGGCAGCTCCGAGGCAGTCTCACCTGGAGACATGGATGCAGGTTCTGCCAG

CTGGGGTGCTGTGTCTTCATTGAATGATGTGTCAAATCACACACTTTCTTTAGGAC

CAGTACCTGGTGCTGTAGTTTATTCGAGTTCATCTGTACCTGATAAAkTCAAAACCA

TCACCACAAAAGGATCAAGCCCTAGGTGATGGCATCGCTCCTCCACAGAAAGTTC

TTTTCCCATCTGAGAAGATTTGTCTTAAGTGGCAACAAACTCATAGAGTTGGAGCT

GGGCTCCAGAATTTGGGCAATACCTGTTTTGCCAATGCAGCACTGGAGTGTTTAA

GCTACACACCACCTCTTGCCAATTACATGCTATCACATGAACACTCCAAAACATGT

CATGCAGAAGGCTTTTGTATGATGTGTAGAATGCAAGCACATATTACCCAGGCAC

TCAGTAATCCTGGGGACGTTATTAAACCAATGTTTGTCATCAATGAGATGCGGCG

TATAGCTAGGCACTTCCGTTTTGGAAACCAAGAAGATGCCCATGAATTCCTTCAA

TACACTGTTGATGCTATGCAGAAAGCATGCTTGAATGGCAGCAATAAATTAGACA

GACACACCCAGGCCACCACTCTTGTTTGTCAGATATTTGGAGGATACCTAAGATC

TAGAGTCAAATGTTTAAATTGCAAGGGCGTTTCAGATACTTTTGATCCATATCTTG

ATATAACATfTGGAGATAAAGGCTGCTCAGAGTGTCAACAAGGCATTGGAGCAGTT

TGTGAAGCCGGAACAGCTTGATGGAGAAAACTCGTACAAGTGCAGCAAGTGTAA

AAAGATGGiTTCCAGCTTCAAAGAGGTTCACTATCCATAGATCCTCTAATGTTCTTA zo CACTTTCTCTGAA-ACGTTTTGCAAATTTTACCGGTGGAAAAATTGCTAAGGATGTG

AAATACCCTGAGTATCTTGATATTCGGCCATATATGTCTCAACGCAACGGAGAGC

CAATTGTCTACGTCTTGTATGCAGTGCTGGTCCACACTGGTTTTAATTGCCATGCT

GGCCATTACTTCTGCTACATAAAAGCTAGCAATGGCCTCTGGTATCAAATGAATG

ACTCCATTGTATCTACCAGTGATATTAGATCGGTACTCAGCCAACAAGCCTATGTG

CTCTTTTATATGAGGTCCCATGATGTGAAAAATGGAGGTGAACTTACTCATCCCAC

CCATAGCCCCGGCCAGTCCTGTCCCCGCCGCGTCATCAGTCAGCGGGTTGTCACCA

ACAAACAGGCTGCGCCAGGCTTTATCGGACCACAGCTTCCCTCTCACATGATAAA

GAATCCACCTCACTTAAATGGGACTGGACCATTGAAAGACACGCCAAGCAGTTCC

ATGTCGAGTCCTAACGGGAATTCCAGTGTCAACAGGGCTAGTCGTGTTAATGCTT

GAGCTTCTGTCCAAAACTGGTCAGTTAATAGGTCCTCAGTGATCCCAGAACATCCT

AAGAAACAAAAAATTACAATCAGTATTCACA-ACAAGTTGCCTGTTCGCCAGTGTC

AGTCTCAACCTAACCTTCATAGTAATTCTTTGGAGAACCCTACCAAGCCCGTTCCC

TCTTCTACCATTACCAATTCTGCAGTACAGTCTACCTCGAACGCATCTACGATGTC

AGTTTCTAGTAAAGTAACAAAACGGATCCCCCGCAGTGAATCCTGCTCCCAGCCC

GTGATGAATGGCAAATCCAAGCTGAACTCCAGCGTGCTGGTGCCCTATGGCGCCG

AGTCCTCTGAGGACTCTGACGAGGAGTCAAAGGGGCTGGGCAAGGAGAATGGGA

TTGGTACGATTGTGAGCTCCCACTCTCCCGGCCAAGATGCCGAAGATGAGGAGGC

CACTCCGCACGAGCTTCAAGAACCCATGACCCTAAACGGTGCTAATAGTGCAGAC

AGCGACAGTGACCCGAAAGAAAACGGCCTAGCGCCTGjATGGTGCCAGCTGCCAA

GGCCAGCCTGCCCTGCACTCAGAAAATCCCTTTGCTAAGGCAACGGTCTTCCTG

GAAAGTTGATGCCTGCTCCTTTGCTGTCTCTCCCAGAAGACAAAATCTTAGAGAC

CTTCAGGCTTAGCAACAAACTGAAAGGCTCGACGGATGAAATGAGTGCACCTGG

AGCAGAGAGGGGCCCTCCCGAGGACCGCGACGCCGAGCCTCAGCCTGGCAGCCC

CGCCGCCGAATCCCTGGAGGAGCCAGATGCGGCCCCGGCCTCAGCAGCACCA

GAAGGCTCCGCCGCCCCGCGATCCCGGCACCCCCGCTACCAAAGAAGGCGCCTGG

GAGGCCATGGCCGTGGCCCCCGAGGAGCCTCCGCCCAGCGCCGGCGAGGACATC

GTGGGGGACACAGCACCCCCTGACCTGTGTGATCCCGGGAGCTTAACAGGCGATG

CGAGCCCGTTGTCCCAGGACGCAAAGGGGATGATCGCGGAGGGCCCGCGGGACT

CGGCGTTGGCGGAAGCCCCGGAAGGGTTGAGTCCGGCTGCGCCTGCGCGGTCGGA

GGAGCCCTGCGAGCAGCCACTCCTTGTTCACCCCAGCGGGGACCACGCCCGGGAC

WO 03/083050 PCT/US03/08590 27

GCTCAGGACCCATCCCAGAGCTTGGGCGCACCCGAGGCCGCAGAGCGGCCGCCA

GCTCCTGTGCTGGACATGGCCCCGGCCGGTCACCCGGAAGGGGACGCTGAGCCTA

GCCCCGGCGAGAGGGTCGAGGACGCCGCGGCGCCGAAAGCCCCAGGCCCTTCCC

CAGCGAAGGAGAAAATCGGCAGCCTCAGAAAGGTGGACCGAGGCCACTACCGCA

GCCGGAGAGAGCGCTCGTCCAGCGGGGAGCCCGCCAGAGAGAGCAGGAGCAAG

ACTGAGGGCCACCGTCACCGGCGGCGCCGCACCTGCCCCCGGGAGCGCGACCGC

CAGGACCGCCACGCCCCGGAGCACCACCCCGGCCACGGCGACAGGCTCAGCCCT

GGCGAGCGCCGCTGTCTGGGCAGGTGCAGTCACCACCACTCCCGACACCGGAGCG

GGGTGGAGCTGGACTGGGTCAGACACCACTACACCGAGGGCGAGCGTGGCTGGG

.0 GCCGGGAGAAGTTCTACCCCGACAGGCCGCGCTGGGACAGGTGCCGGTACTACC

ATGACAGGYTACGCCCTGTACGCTGCCCGGGACTGGAAGCCCTTCCACGGCGGCCG

CGAGCACGAGCGGGCCGGGCTGCACGAGCGGCCGCAGAAGGACCACAACCGGGG

CCGTAGGGGCTGCGAGCCGGCCCGGGAGAGGGAGCGGCACCGCCCCAGCAGCCC

CCGCGCAGGCGCGCCCCACGCCCTCGCCCCGCACCCCGACCGCTTCTCCCACGAC

'AGAACTGCACTTGTAGCCGGAGACAACTGTAACCTCTCTGATCGGTTTCACGAC

ACGAAAATGGAAAGTCCCGGAAACGGAGACACGACAGTGTGGAGAACAGTGACA

GTCATGTTGAAAAGAAAGCCCGGAGGAGCGAACAGAAGGATCCTCTAGAAGAGC

CTAAAGCAAAGAAGCACAAAAATCAAAGAAGAAAAAGAAATCCAAGACAA

CACCGAGACCGCGACTCCAGGCATCAGCAGGACTCAGACCTCTCAGCAGCGTGCT

O CTGACGCTGACCTCCACAGACACAAAAAAAAGAAGAAGAAAAAGAAGAGACATT

CAAGAAAATCAGAGGACTTTGTTAAAGATTCAGAACTGCACTTACCCAGGGTCAC

CAGCTTGGAGACTGTCGCCCAGTTCCGGAGAGCCCAGGGTGGCTTTCCTCTCTCTG

GTGGCCCGCCTCTGGAAGGCGTCGGACCTTTCCGTGAGAAAACGAACACTTACG

GATGGAAAGCAGGGATGACAGGTGTCGTCTCTTTGAGTATGGCCAGGGTGATTGA

Table 1 1. Deduced amino acid sequence of coding region of hDUB7 C-terminal potential nuclear localization (as well as targeting) sequences are underlined.

MTWVKASESSDPSAYQNQPGSSEAVSPGDMDAGSASWGAVSSLNDVSNHTLSLGPV

PGAVVYSSSSVPDKSKPSPQKDQALG-DGIAPPQKVTLFPSEKICLKWQQTHRVGAGLQ

NLGNTCFANAALQCLTYTPPLAYMLSHHSKTCHAEGFCMMCTMQITQLS

GDVIIKPMFVNEMRIA1TFRPGNQEDAHEFLQYTVDAMQKACLNGSKIDRTQA TTLVCQIFGGYLRSRVKCLNCKGVSDTFDPYLDITLErAAQSVNLEQFVKPEQLD GENSYKCSKCKI(.'MVPASKRFTII1RSSNVLTLSLKRFAINFTGGKARKDVKYPYLDR

YMSQPNGEPIVVLYAVLVITGFNCHAGIIYFCYIKASNGLWYQMNDSIVSTSDIRSV

LSQQAYVLFYIRSHDVKNGGELTKPTHSPGQSSPRPVISQRVVTNKQAAPGFIGPQLPS

ITM1KINPPHLNGTGPLKDTPSSSMSSPNGNSSVNRASPVNASASVQNWSVNRSSVIPFH PKKQKITISIIMLPVRQCQSQPN1HSNSLENPTKPVPSSTITNSAVQSTSNASTMSVSS

KVTKPIPRSESCSQPVMNGKSKLNSSYLVPYGAESSEDSDEESKGLGKENGIGTWVSSH

SPQADETHLEMLGNASSPEGADACGPLSN

FAKANGLPGKLMIPAPLLSLPEDKILETFRLSNKLKGSTDEMSAPGAERGPPEDRDAEP

QPGSPAAESLEEPDAAAGLSSTKKAPPPRDPGTPATKEGAWEAMAVAPEEPPPSAGE

DIVGDTAPPDLCDPGSLTGDASPLSQDAKGMEGPRSALEAEGLSPAPASEEP

CEQPLLVHIPSGDHARDAQDPSQSLGAPAARPPAPVLDMPAGHPEGDAEPSPGER

VEDAAAPKAPGPSPAKKIGSLRKVDRGHYRSRRERSSSGEPSRSKTEGRR

RTCPRERDRQDRAEHPGHGDRLSPGERRSLGRCSH mSRH SGVELDHH TEGERGWGREKFYPDRPRWDRCRYYTDRYALYAvKFHGGEHEGLHERP WO 03/083050 PCT/US03/08590 28

HKDHNRGRRGCEPARERERIIRPSSPRAGAPHALAPHIPDRFSHDRTALVAGDNCNLSD

RFHEI-ENGKSRKRRHDSVENSDSHVEKKARRSEQKDPLEEPIkAKKIKKSKKKKKSK

DKHRDRDSRHQQDSDLSAACSDADLHRIIKKKKKKKRHSRKSEDFVKDSELIILPRV

TSLETVAQFRRAQGGFPLSGGPPLEGVGPFREKTKHLRMESRDDRCRLFEYGQGD

Table 12. Putative promoter sequence of hDUTB7 (2 Kb sequence upstream of initiation AUG)

GTAAAGTCTAAACTGAGAAGTGGAAGTGTGAACTGGCTGGAGGTGGAAGGTTGG

AAAAGAGTCGGAGAAAAGAACAGCATGTGCAGAGCCCAGAGACAGCAGGGACA

[0 AAAGAA AA AA A AACAAGACTTCAGCATGGTGGGAACGTGACGGAGAGGGTGTTT

GGCGAGGTTATTAGGTCAGACAATGTGAAGTCCAGACATTAAGATGTTGTGCTGT

GG GCAGTTGGGCCACTCCTGAAAGGTGTTCTTTCTTCCTTTCCTTTTCTTTCTTTCT TTTCTTGAGGCAGAGiTCTGTCTATGTCAGTCTGGAGTGCAGTGGCATGATCTCGGC

TCACTGCAATCTCTGCCTTCCAGGTTCAAGCAATTTTCCTTGCCTCAGCCTCCCAA

GTAGCTGGGAATACAGGCGTGCGCCACCATGCGTGGTTAATTTTTTTATTTTTAGT

AGAGATGGGGTTTCCCCATGTTGGCCAGGCTGGTCTCGAACTCCTGGACTCAAGT

GATCCACCCACTTTGGCCTCCCAAAGTGCTGGGATTACAGGGGTGTGAGCCACTG

CGCCCCGCCCGGCCTTTTTTTTTTTTTTTTTTGAGACTTAATCTTGCTCTGTCACCA

AGGCTGGATATCAGTGGCACGGT'JTTGGCTCTCTGCAACTTCTGTCTCCCAGGTTC

AAGCGATTTTCCTGACTCAGCCTCCCAAGTAGTTGAGATTACAGGTACGTGCCAC

CACGCCCGGCTAATTTTTGTATTTTTAGTAGAGATGAGGTTTGACTATGTTGGCCA

GACTGGTCTCAAACGCCTGACCTCAGGTGATTCACCTGCCTCGGCCTCCCAAAAT

GCTC3GGATTACAGGTGTGCACCACCATGCCTGGGTAATTTTTGTTTTTCGTAGAGA

CAGGGTCTCACCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAAGCGATCT

GCCCACCTTGGCCTCCCAAGGTGCTGCAATTATAGGCATGAGCCACCGCGCCCGG

CCTCCTGAAAGGTTTTCTACATAGGAGTGGCATGTCTAGATGTGGCTACTGTTGGG

CGATTTTAGAAATATCCCTAAAAGCCTTCTGTTGACAGGGTGGCATAACCAGAAG

GAAGCCTGGCTGGGAACGCTGGACCTGGCTCTCAGTCCCAGTTGCTGACTGGTTG

CTTCATTTTATAGGCCCTGGGGATTCTGTCTGATCTCTCATACGTTCTTTATAAAA

ATTAAGTTAATGTATGTCCAGCAGTTGATGCAATGCCCAGTACATAGAAAATGCT

CAATTAGTGGTAGCCCTAATATTTTAAAATAGGACTCAGAAAGAAAATTATAATC

AAGTCCTTTCATAACAGATATTTGTGTTTGAGTTTGATATCAGTAATGGCTTACGG

GTTTTATTTAAAAAGTCATACATTCCATATAAATGAGCCTCTTCAGAAAAATGGTT

TTAAAGGTGAGATCTCTATAATTATAATTTTAAANAAATATAATGTATTTCACTTGG

TGCCATTTGCACTTTAAGCACAAAATTAAGTCTAGATTTTTTCTGTGTAGTTGATG

CTTTTCTCTGAGGAATTATACTCAAATTGAAGATGTAGTCAAkATGTATTACTGTGT

ATAATTTTTCTAGTTTTAAGCAGTATAGAAGGAAAATATAGGTACTTAGTATA

AACAGAACTGAGAATTGAAATGTCCAATTATAAACTGAAATGCCAGACTTTTAGG

GGGCATGAAATGAAAATGAGAAGiTTCTTTTAATCAAATACTTCACTGAAGATTTT

AAAATAAAGATTGTTGAGATTCAGATTATCATGATGCTAAATGTCCCAAGGGGAT

TATTACAGAAATGTTAGAAAGTACTATTGTTTTTATATTTGAGTGATGTGTTTGAA

AATCACTTTAAAATGGCTGGAATGATCTTCCAAGATCTAACGGTAGGGTAAGGAG

ATTGCTTTTCTCACCTGATGAAACAAATACATACTTTTCATCTTTTGCAGAGTTGA

ACAATG

Table 13. Nucleotidle seqnence of coding region of murine DUB7 (mDIJB7)

ATGACCATAGTTGACAAAACTGAACCTTCAGACCCATCAACCTGTCAGAACCAGC

CTGGCAGTTGTGAGGCGGTCTCACCTGAAGACATGGACACAGGCTCTGCCAGCTG

WO 03/083050 PCT/US03/08590 29

GGGCGCTGTGTCTTCAATAAGTGATGTCTCAAGTCACACACTTCCATTAGGGCCA

GTGCCTGGTGCTGTAGTTTATTCTAACTCGTCTGTACCTGAAAAATCAA-AGCCATC

ACCACCAAAGGATCAAGTCCTAGGTGATGGCATTGCTCCTCCTCAAAAGGTCCTG

TTTCCATCTGAAAAGATTTGTCTTAAGTGGCAACAAAGTCATCGAGTTGGCGCTG

GGCTCCAGAATTTGGGCAACACCTGTTTTGCCAATGCCGCATTGCAGTGTCTGACT

TACACGCCACCCCTCGCCAATTACATGTTATCCCATGAACACTCCAAGACATGCC

ACGCAGAAGGATTTTGTATGATGTGCACGATGCAGACACACATTACCCAGGCACT

TAGCAACCCTGGGGATGTTATCAAGCCGATGTTCGTCATCAATGAAATGCGGCGT

ATAGCTAGACACTTCCGTTTTGGAAACCAAGAAGATGCCGATGAATTTCTTCAGT

ACACGGTCGATGCCATGCAGAAAGCATGTTTAAATGGCAGCAATAAATTAGACA

GAGACACCCAGGCCACCACGCTGGTCTGCCAGATATTTGGAGGCTACCTAAGATC

CCGAGTTAAATGTTTAAATTGCAAGGGTGTTTCAGATACCTTTGATCCATATCTGG

ACATAACGTTGGAGATTAAGGCTGCACAGAGTGTTACCAAGGCGTTAGAGCAGTT

TGTGAAGCCAGAACAACTGGATGGAGAAAACTCCTACAAGTGCAGCAAGTGCAA

AAAAATGG-TTCCAGCTTCAAAGAGATTCACAATCCATAGGTCCTGTAATGTTCTTA

CCATCTCACTGAAGCGCTTTGCCAACTTCACCGGTGGAAAGATTGCTAAWGATGT

GAAA'1'ATCCTGAGTACCTTGATATCCGGCCCTATATGTCTCAGCCCAATGGAGAG

CCAATTATTTATGTTTTGTATGCTGTGCTGGTGCACACTGGTTTTAATTGTCATGCT

GGCCACTACTTTTGCTACATCAAGGCTAGCAATGGCCTCTGGTATCAGATGATG

ACTCCATCGTGTCCACCAGTGATATCAGAGCAGTGCTTAACCAGCAAGCTTACGT

GCTCTTTTATATCAGGTCCCATGATGTGAAAAATGGAGGGGAGTCTGCTCATCCT

GCGCATAGCCCCGGCCAATCCTCTCCCCGCCCAGGAGTCAGTCAGCGGGTAGTCA

ACAACAAGCAGGTGGCTCCAGGGTTTATTGGACGCCAGCTGCCTTCCCATGTGAT

GAAGAACACGCCACACTTGAATGGCACCACGCCAGTGAAAGACACACCAAGTAG

TTCTGTGTCAAGCCCTAACGGAAACACCAGCGTCAATAGGGCCAGTCCTGCTACT

GCTTCGACTTCTGTGCAGAACTGGTCTGTTACCAGACCCTCAGTTATTCCAGATCA

CCGCAAGAAACAAAAAATCACCATCAGTATTCACAACAAGTTGCCTGCTCGCCAG

GGTCAGGCACCACTGAATAACAGCCTCCATGGCCCTTGTCTGGAGGCTCCTAGTA

AGGCGGCACCCTCCTCCACCATCACTAACCGTTCTGCAATACAGTCTACCTCGAAC

GTACCCACAACGTCGACTTCCCCCAGTGAGGCCTGTCCCAAGCGCATGGTGAACG

GCAAGGCTAAAGTGGGCGCCAGTGTGCTTGTCCCCTATGGGGCCGAGTCCTCAGA

AGAGTCTGATGAGGAGTCGAAGGGCCTGGCCAAGGAGAACGGTGTGGACATGAT

GGCCGGCACTCACTCCGATAGGCCAGAAGCTGCTGCAGATGACGGTGCTGAGGCT

TCCTCCCATGAGCTTCAAGAACCCGTCCTGCTAAATGGTGCTAATAGCGCAGACA

GTGACTCACAAGAGAACAGCCTGGCATTTGACAGTGCCAGCTGCCAGGTCCAGCC

CGAGCTACACACAGAAAACCTCTTTTCCAAACTTAATGGTCTTCCTGGAAAGGTG

ACGCCTGCTCCTTTGCAGTCTGTTCCTGAAGACAGAATCCTTGAGACCTTCAGCT

TACCAACCAGGCAAAGGGTCCAGCGGGTGAAGAGAGTTGGACTACGACAGGGGG

AAGCTCTCCAAAGGACCCTGTTTCACAGCTGGAGCCCATCAGTGATGAGCCCAGT

CCCCTTGAGATACCGGAGGCTGTCACCAATGGGAGCACACAGACCCCTTCCACCA

CATCACCCCTGGYAGCCCACCATCAGCTGTACCAAAGAAGACTCGTCCGTTGTTGT

CTCAGCTGAACCTGTGGAGGGTTTGCCTTCCGTCCCTGCTCTTTGTAACAGCACTG

GTACTATCTTGGGGGATACCCCAGTGCCCGAA'JTGTGTGACCCTGGAGACTTGAC

TGCCAACCCGAGCCAGCCAACCGAAGCAGTGAAAGGTGATACAGCTGAGAAGGC

TCAGGACTCTGCCATGGCTGAAGTGGTGGAGAGGCTGAGCCCTGCTCCCTCAGTA

CTCACAGGTTGACGGGTGTGAGCAGAAAGTCTTACTTTACGTCAGCGCAGAGGGGT

CAGAGGAGACAG:AAGAkCTCTTCCAGiAAGiCTCGGCGGTCTCTGCTGACACGATGCG

CCCTAAGCCTGACAGGACCACCACCAGCTCCTGTGAAGGGGCTGCCGAGCAGGCT

GCTGGGGACAGANGGCGATGGAGGCCATGTGGGACCCAAAGCTCAGGAGCCTTCC

CCAGCCAAGGAAAAGATGAGCAGCCTCCGGAAAGTGGACCGAGGACACTATCGG

WO 03/083050 PCT/US03/08590

AGCCGGAGAGAGCGCTCCTCCAGTGGGGAGCACGTGAGGGAGAGCAGGCCCCGG

CCGGAGGACCATCACCATAAGAAGCGGCACTGCTACAGCCGAGAGCGGCCCAAG

CAGGACCGACACCCTACTAATTCATACTGCAATGGGGGCCAGCACTTGGGCCACG

GGGACAGAGCCAGCCCTGAGCGCCGCTCCCTGAGCAGGTATAGTCACCACCACTC

ACGGATTAGGAGTGGCCTGGAGCAGGACTGGAGCCGGTACCACCATTTGGAAAA

TGAGCATGCTTGGGTCAGGGAGAGATTCTACCAGGACAAGCTGCGGTGGGACAA

GTGCAGGTATTACCACGACAGGTACACGCCCCTAkTACACGGCCCGGGACGCCCGA

GAATGGCGGCCTCTGCATGGTCGTGAGCATGACCGCCTTGTCCAGTCTGGACGGC

CATACAAGGACAGCTACTGGGGCCGCAAGGGCTGGGAGCTGCAATCCCGGG

GGA

o0 AGGAACGGCCCCACTTCAACAGCCCCCGAGAGGCCCCTAGCCTTGCTGTGCCCCT

CGAGAGACATCTCCAAGAGAAGGCTGCGCTGAGTGTGCAGGACAGCAGCCACAG

TCTCCCTGAGCGCTTTCATGAACACAAAAGTGTCAAGTCGAGGAAGCGGAGGTAT

GAGACTCTAGAAAATAATGATGGCCGTCTAGAAAAGAAAGTCCACAAAAGCCTG

GAGAAGGACACGCTAGAGGAGCCAAGGGTGAAGAAGCACAAAAAGTCTAAA

GAAAAAGAAGTCCAAAGATAAACACCGGGATCGAGAAAGCAGGCACCAGCAGG

AGTCTGATTTTTCAGGAGCATACTCTGATGCTGACCTCCATAGACACCGGAAGAA

AAAGAAGAAAAAGAAAAGGCATTCCAGGAAGTCGGAGGACTTTATAAAGGATGT

TGAGATGCGTTTACCGAAGCTCTCCAGCTACGAGGCCGGCGGCCATTTCCGGAGA

ACAGAGGGCAGCTTTCTCCTGGCTGATGGTCTGCCTGTGGAAGACAGCGGCCCTT

O TCCGGGAGAAAACGAAGCATTTAAGGATGGAAAGCCGGCCTGACAGATGCCGTC

TGTCGGAGTATGGCCAGGATTCAACATTTTGA

Table 14. Deduced amino acid sequence of coding region of mLJB37 C-terminal. potential nuclear localization (as well as targeting) sequences are underlined.

MTIVDKTEPSDPSTCQNQPGSCEAVSPEDMDTGSASWGAVSSISDVSSTLPLGPVPG

AVVYSNSSVPEKSKPSPPKDQVLGDGIAPPQKVLFPSEKTCLKWQQSHRVGAGLQNL

GNTCFANAALQCLTYTPPLMNYMLSHEHSKTCHAEGFCMMCTMQTHITQALSNPGD

VIKPMFVINEMRR{FRFGNQEDAEFLQYTVDAMQKACLNGSNKLDRITQATT

LVCQTFGGYLRSRVK<CL-NCKGVSDTFDPYLDITLELK-AAQSVTKALEQFVIPEQLDGE

NSYKCSKCKKMVPASKRETI{RSSNVLTISLKRFANTGGKAkKDVKYPEYLDRPYM SQPNGEPIIYVLYAkVLVHTGFNCHAGHYFCYKASNGLWYQMNDSVSTSDRVLNQ

QAYVLFYIRSHDVKNGGESAIIIAHSPGQSSPRPGVSQRVVNNKQVAPGFIGPQLPSH

VMKNTPHLNGTTPVKiDTPSSSVSSPNGNTSVNRASPATASTSVQNWSVTRPSVIPDBP 1KKQKITISIINKLPARQGQAPLNNSLHGPCLEAPSKAAPSSTITNPSAIQSTSNVPTTSTS

PSEACPKPMVNGKAKVGASVLVPYGAESSEESDEESKGLAKENGVDMMAGTHSDRP

EAAADDGAEASSHELQEPVLLNGANSADSDSQENSLAFDSASCQVQPELHTENLFSK

LNGLPGKVTPAPLQSVPEDRILETFKLTNQAKGPAGEESWTTTGGSSPKDPVSQLEPIS

DEPSPLEIPEAVTNGSTQTPSTTSPLEPTISCTK-EDSSVVVSAEPVEGLPSVPALCNSTGT

1LGDTPVPELCDPGDLTANPSQPTEAVKGDTAEKAQDSAMAEVVERLSPAPSVLTGD

GCEQKLLLYLSAEGSEETEDSSRSSAVSADTMPPKPDRTTTSSCEGAAEQAAGDRGD

GGHVGPKAQEPSPAKEKMSSLRKVDRGHYRSRRERSSSGEHVRDSRPRPEDHHI1KK

PIICYSRERPKQDRIIPTNSYCNGGQHLGHGDRASPERRSLSRYSHHHSRIRSGLEQDW

SRYHHLENE1AWYFJERFYQDKLRWDKCRYHDRYTPLYTARDAREWRPLHGRH

RLVQSGRPYKDSYWGRKGWIELQSRGRERPJJFNSPREAPSLAVPLERI{LQEKAALSV

QDSSHSLPER1FHEHKSVKSRKRRYETLEN1NDGRLEKKVHKSLEKDTL-EEPRVIKKHKK WO 03/083050 PCT/US03/08590 31

SKKX<KKS}CDKITRDRESRHQQESDFSGAYSDADLIHHKKKKKKKRHSRKSEDFTKD

VEMRLPKLSSYEAGGK:FRRTEGSFLLADGLPVLDSGPFREKTKIILRMESRPDRCRLSE

YGQDSTF

Table 15. Nucleotide sequence alignment of hDUIB7 and miDUB7 H-DUB 7 ATGACCATAGTTGACAAAGCTTCTGAATCTTCAGACCCATCAGCCTATCAGAATCAGCCT MDUB7 ATGACCATAGTTGACAAAA- CTGAACCTTCAGACCCATCAACCTGTCAGAACCAGCCT 57 HDUB7 GGCAGCTCCGAGGCAGTCTCACCTGGAGACATGGATGCAGGTTCTGCCAGCTGGGGTGCT 120 MDUB 7 GGCAGTTGTGAGGCGGTCTCACCTGAAGACATGGACACAGGCTCTGCCAGCTGGGGCGCT 117 HDUB 7 GTGTCTTCATTGAATGATGTGTCAAATCACACACTTTCTTTAGGACCAGTACCTGGT-CT 180 MDUB 7 GTGTCTTCAATAAGTGATGTCTCAAGTCACACACTTCCATTAGGGCCAGTGCCTGGTGCT 177 *DB TCA 24 DUB7 GTAGTTTATTCGAGTTCATCTGTACCTGATkAAATCAAAACCATCACCACAAAAGGATCAA 240 HDUB7 GCCCTh-GGTGATGGCATCGCTCCTCCACA-GAAAGTTCTTTTCCCATCTGAGAAGATTTGT 300 MDUB 7 GTCCTAGGTGATGGCATTGCTCCTCCTCAAZAAGGTCCTGTTTCCATCTGAAAAGATTTGT 297 *D B 36 HDUB7 CTTAAC-TGGCAACAAACTCATAGAGTTGGCGCTGGGCTCCAGAATTTGGGCAATACCTGT 350 HDU3B7 TTTGCCAATGCAGCACTGCAGTGTTTAACCTACACACCACCTCTTGCCAATTACATGCTA 420 MOUB 7 TTTGCCAATGCCGCATTGCAGTGTCTGACTTACACGCCACCCCTCGCCAATTACATGTTA 417 HDUB 7 TCACATGAACACTCCAAAACATGTCATGCAGAAGGCTTTTGTATGATGTGThCAATGCAA 480 MDUB 7 TCCCATGAACACTCCAALGACATGCCACGCAGAAGGATTTTGTATGATGTGCACGATGCAG 477 EDUB 7 GCACATATTACCCACGCACTCAGTAALTCCTGGGGACGTTATTAAALCCAATGTTTGTCATC 540 MDU'B7 ACACACATTACCCAGGCACTTAZGCAACCCTGGGGATGTTATCALAGCCGATG3TTCGTCATC 537 MUB7 AATGAALTGCCGCGTATAGCTAGGCACTTCCGTTTTGGAALACCAAGAALGATGCCCATGAA 600 UDUB 7 TTCCTTcAATACAcTGTTGATGCTATGcAGaAQA(CATGCTTCAATGGCACCAATAAATTA 660 MDUB7 TTTCTTCAGTACACGGTCGATGCCATGCAGAAAGCATGTTTAAATGGCAGCAATAAALTTA 657 BDUB7 3ACAGACACACCCkGGCCACCACTCTTGTTTGTCAGATATTTDGAGGATACCTAAGATCT 720 MDUB7 GACAGACACACCCAGGCCACCACCCTGGTCTGCCAGATATTTGGAGGCTACCTAAGATCC 717 BIDUB7 AGAGTCAAkTGTTTAAATTGCAAGGGCGTTTCAGATACTTTTGATCCATATCTTGATATA 780 NVDUTB7 CGAGTTAAATGTTTAAATTGCAAGGGTGTTTCAGATACCTTTGATCCATATCTGGACATA 777 WO 03/083050 PCT/US03/08590 32 HDUB7 ACATTGGAGATAAGGCTGCTCAGATGTCACA-GCATTGAGCAGTTTGTGAGCCG 840 M~DUB7 ACGTTGGAGATT1AAGGCTGCACAGAGTGTTACCAAGGCGTTAGAGCAGTTTGTGAACCCA 837 HDUB7 GACGTGTGGAATGAAGTCGAGGAAAAGTCAC 900 M'DUB 7 GAACAACTGGATGGAGAACTCCTACAAGTGCAGCAAGTGCAAAAATGGTTCCAGCT 897 IiDUB7 TCAAAGAGGTTCACTATCCATAGATCCTCTATGTTCTTCCTTTCTCTAACGTTTT 960 MDIJE7 TCAAAGAGATTCACAATCCATAGGTCCTCTAATGTTCTTACCATCTCACTGAAGCGCTTT 957 HDUD7 OCATTACGGAAATCAGGTTAAACTATTTGTT 1020 MDUB7 GCCAACTTCACCGGTGGAAAGATTGCTAAGGATGTGAAATATCCTGAGTACCTTGATATC 1017 UDUD 7 CGCAAAGCC2,CACGGGCATGCAGCTTTCGGT 1080 MDUB37 CGGCCCTATATGTCTCAGCCCAATGGAGAGCCAATTATTTATGTTTTGTATGCTGTGCTG 1077 I{DUB 7 GTCCACACTGGTTTTAATTGCCATGCTQGCCATTACTTCTGCTACATAAGCTAGCAAT 1140 MDUB7 GTGCACACTGGTTTTAATTGTCATGCTGGCCACTACTTTTGCTACATCAAGGCTAGCAAT 1137 IIDUB7 GGCCTCTGGTATCAAX1TGAATGACTCCATGTATCTACCAGTGATATTAGATCGGTACTC 1200 MDUB 7 GGCCTCTGGTATCAGATGAATGACTCCATCGTGTCCACCAGTGATATCAGAGCAGTGCTT 1197 IDTB 7 ACCAAGCAGGTTTAACGTCAGTTAALTGGTA 1260 MDUB7 AACCAGCAAGCTTACGTGCTCTTTTATATCAGTCCCATGATGTGAAATGGAGGDGAG 12S7 HDUB7 132 MDUB7 CTACTCATCCCACCCATAGCCCCGGCCAGLTCCTCTCCCCGCCCCGATCAGTCAGCGG 1320 HDTUh7 GTTGTCACCAACAAACAGGCTGCGCCAGGCTTTATCGGACCACAGCTTCCCTCTCACATG 1380 MDUB7 GTAGTCAACACAACCAGGTGGCTCCAGGGTTTATTGGACCCCAGCTGCCTTCCCATGTG 1377 HDTI87 ATAAGAATCCACCTCACTTAALTGGGACTGGACCATTGAAAGACACGCCAGCAGTTCC 1440 MDUB7 ATGAAGACCGCCACACTTGAPTGGCACCACGCCAGTGAAAGACACACCAAGTAGTTCT 1437 HDUB7 ATGTCGAGTCCTAACGGGAATTCCAGTGTCACAGGGCTAGTCCTGTTATGCTTCAGCT 1500 MDUB7 GTGTCAACCCTACGGACACCAGCTCAATAGGGCCAGTCCTGCTACTGCTTCGACT 1497 HDUD7 TCTGTCCAACTGGTCAGTTAATAGGTCCTCAGTGATCCCAGACATCCTAGAACAA 1560 MDUB37 TCrTCGATGCGTCAACTCGTTCAACCCAGACA 1557 HDUB7 AAATTACAATCAGTATTCACAACAAGTTGCCTGTTCGCCAGTGTCAGTCTCACCTA 1619 MDUB7 AAAATCACCATCAGTATTCACAACAAGTTGCCTGCTCGCCAGGGTCAGGCACCACTGAAT 1617 *4 *4 IIDU37---------CCTTCATAGTAATTCTTTGGAGAACCCTACCAAGCCCGTTCCCTCTTCTACCATT 1674 MDLT37 AACAGCCTCCATGGCCCTTGTCTGGAGGCTCCTAGTLGGCGGCACCCTCCTCCACCATC 1677 WO 03/083050 PCT/US03/08590 33 HDTJB7 ACCAA- TTCTGCAGTACAGTCTACCTCG AC3CATCTACGATGTCAGTTTCTAGTAAA 1731 MDUJB7 ACTAACCCTTCTGCAATACAGTCTACCTCGAACGTACCCACALCGTCGACTTC--------- 1730 HDUB7 GTAAAACACCCCGGATCTCCCGCGGTATGAAC 1791 CCCCAGTGAGGCCTGTCCCAAGCCCATGQTGAACGGCAAQGCT 1773 HDUB7 AAGCTGAACTCCAGCGTGCTGGTGCCCTATGGCGCCGAGTCCTCTGAGGACTCTGACGAG 1851 .0 MVDUB7 AAAGTGGGCGCCAGTGTGCTTGTCCCCTATGGGGCCGAGTCCTCAGAAGAGTCTGATGAG 1833 I-DUB37 CAGTCA. GGGGCTGGGCAGGAGATGGATTOTACGATTGTGAGCTCCCACTCTCCC 1911 MDUB7 GAGTCGAAGGGCCTGGCCAAGGAGAACGGTGTGGACATGATGGCCGGCACTCACTCCGAT 1893 HDUB7 GGCCAAGA--- -TGCCGAZAGATGAGG GCCACTCCGCACGAGCTTCAAGAACCC 1962 MDUB7 AGGCCAGAAGCTGCTGCAGATGACGGTGCTGAGGCTTCCTCCCATGAGCTTCACACCC 1953 HDUB7 ATGACCCTAAACGGTGCTAATAGTGCAGACAGCGACATGACCCGAGAAACGGCCTA 2022 MDUR7 GTCCTGCTAAATGGTGCTAATAGCGCAGA CAGTGACTCACAAGAGAALCAGCCTG 2007 IHDUB7 GCGCCTGATQGTGCCAGCTGCCAAGGCCAGCCTGCCCTCACTCAGAATCCCTTTGCT 2082 MDUB7 GCATTTGACAGTGCCAGCTGCCAGGTCCAGCCCGAGCTACACACAGAAAACCTCTTTTCC 2067 HDUB37 AAGWCAAACGGTCTTCCTGGAA-AGTTGATGCCTGCTCCTTTGCTGTCTCTCCCAGAAGAC 2142 MDUB7 AACTTAATGaTCTTCCTGGAA-AGGTGACGCCTGCTCCTTTGCAQTCTQTTCCTGAAGAC 2127 HDUE37 AAACTGGCTCGCTGAAAATAAGTGCGTAAGG 2202 MDUB7 AGATCCTTGAGACCTTCAACTTCCAACCAGGCAAGGGTCCAGCGGGTGAGAGAGT 2187 IIDUB7 GCACCTGGAGCAGAGAGGGGCCCTCCCGAGGACCGCGACGCCGAGCCTCAGCCTGGCAGC 2262 MDTB 7 TGGACTACGAWAGGGGGAAGCTCTCCAALAGGACCCTGTTTCACAGCTGGAGCCCATCAGT 2247 IIDUB7 CCCGCCGCCGAATCCCTGGAGGAGCCAGATGCGGCCGCCGGCCTCAGCA -GCACCAAG 2319 MDUB7 GATGAGCCCAGTCCCCTTGAGATACCGGAGGCTGTCACCAATGGCAGCACACAGACCCCT 2307 HDUB7 AGGCTCCGCCGCCCCGCGATCCCGGCACCCCCGCTACCAGAGGCGCCTGGGAGGCC 2379 MDU"B7 TCCACCACATCACCCCTGACCCACCATCAGCTGTACCAGAGACTCGTCCGTTGTT 2367 IHDUB7 ATGGCCGTCGCCCCCGAGGAG-------- AGCGCCGGCGAG 2421 MDEJB7 GTCTCAGCTGAIACCTGTGGAGGGTTTGCCTTCCGTCCCTGCTCTTTGTALCAGCACTGGT 2427 HDUB7 GACATCGTGGGGGACACAGCACCCCCTGACCTGTGTGATCCCGGGAGCTTAACAGGCGAT 2481 MDTJB7 ACTATCTTGGGGGATACCCCAGTGCCCGA.ATTGTGTGACCCTGGAGACTTGACTGCCAAC 2487 HDUB7 GCGAGCCCGTTGTCCCAGGACGCAAAGGGGATGATCGCGGAGGGCCCGCGGGACTCGGCG 2541 MDUB37 CCGAGCCAGCCAACCGAAGCAGTGAALAGGTGATACAGCTGAGAAGGCTCAGGACTCTGCC 2547 WO 03/083050 PCT/US03/08590 34 HDUB7 TTGOAACCGAGGTATCGTCCTCCGCGGACCG 2601 MflUB7 ATGGCTGAAGTGGTGGAGAGGCTGAGCCCTGCTCCCTCAGTACTCACAGGTGACGGGTGT 2607 HDU1B7 CACGCCCTGTACCCGGGCAGCGGCCCGACA 2661 MLTTB7 GAGCAGAAACTCTTACTTTACCTC-AGCGCAGAGGGGTCAGAGGAGACAGAAGACTCTTCC 2667 HDU137 CAGAGCTTGGGCGCACCCGAGGCCGCAGAGCGGCCaCCAGCTCCTGTGCTGACATG3GCC 2721 MDUB7 AGAAGCTCGGCGGTCTCTGCTGACACGATGC---------- CCCCTAAGCCTGACAGGACC 2718 HDUB7 CCGCGCCCGAGGCCGGCTGCCGGrrGCAGAG 2780 MDUB 7 ACCACCAGCTCCTGTGAAGGGGCTGCCGAGCAGGCTGCTGGGGACAGAGGCGATGGAGGC 2778 EDTJB7 C- -GCGGCGCCGAA AGCCCCAGGCCCTTCCCCAG3CGAAGGAGAAAATCGGCAGCCTCAGA 2838 MDUJB7 CATGTGGGACCCAA-AGCTCAGGAGCCTTCCCCAGCCAAGGAAAAGATGAGCAGCCTCCGG 2838 HJUB7 AAGGTGGACCGZAGGCCACTACCGCAGCCGGAGAGAGCGCTCGTCCAGCGGGGAGCCCGCC 2898 MDUB7 AAAGTGGACCGAGGACACTATCGGAGCCGGAGAGAGCGCTCCTCCAGTGGGGAGCACGTG 2898 1DUB7 AGAGAGAGCAGGAGCAAGACTGAGGGCCACCGTCACCGGCGGCGCCGCACCTGCCCCCGG 2958 MDLTB7 AGGGACAGCAGGCCCCGCCGGAGGACCATCACCATA-AGAAGCGGCACTGCTACAGCCGA 2958 I1IDlUB7 G2AGCGCGACCcCCAGGACCGCCACGCCCC-------------------- GGAGCACCACCCC 3000 MDUB7 GAGCGGCCCA7\GCAGGACCGACACCCTACTAATTCATACTGCAATGGGGGCCAGCACTTG 3018 HDUB7 GGCCACGGCGACAGGCTCAGCCCTGGCGAGCGCCGCTCTCTGGGCAGGTGCAGTCACCAC 3060 MDUB7 GGCCOGCAACACC -GAGCGCCGCTCCCTGAGCAGGTATAGTCACCAC 3075 HIJUB7 CACTCCCGACACCGGAGCGGGGTGGAGCTGGACTGGGTCAGACACCACTACACCGAGGGC 3120 MDLUB7 CACTCACGGATTAGGAGTGGCCTGGAGCAGGACTGGAGCCGGTACCACCATTTGGAAAAT 3135 HJJUB7 GAGCGTGGCTGGGGCCGGGAGAAGTTCTACCCCGACAGGCCGCGCTGGGACAGGTGCCGG 3180 MDUB7 GAGCATGCTTGGGTCAGGGAGAGATTCTACCAGGACAAGCTGCGGTGGGACAAGTGCAGG 3195 HDUB7 TACTACCATGACAGGTACGC--- -CCTGTACGCTGCCCGGGACT GGAAGCCCTTCCA 3233 MDUB7 TATTACCACGACAGGTACACGCCCCTATACACGGCCCGGGACGCCCGAGAATGGCGGCCT 3255 HDUB7 CGGC- -GGCCGCGAGCACGAGCGGGCCGGGCTGCACGAGCGGCCGCACAAGGACCACAAC 3291 MDLU37 CTGCATGGTCGTGAGCATGACCGCCTTGTCCAGTCTGGACGGCCATACAAGGACAGCTAC 3315 NJUUB7 CGGGGCCGTAGGGGCTGCGAGCCGG- -CCCGGGAGAGGGAGCGGCACCGCCCCAGCAGC 3348 MDUB7 TGGGGCCGCAAGGGCTGGGAGCTGCAATCCCGGGGGAAGGAACGGCCCCACTTCAACAGC 3375 FIDUB7 CCCCGCGCAGGCGCGCCCCACGCCCTCGCCCCGCACCCCGACCGCTTCTCCCACGACAGA 3408 MDUB7 CCCCGAGAGG CCCCTAGCCTTGCTGTGCCCCTCGAGAGACATCTCCAAGAGAAG 3429 WO 03/083050 PCT/US03/08590 HDUB7 ACTGCACT--- TGTAGCCGGAGAC7\ACTGTAACCTCTCTGATCGGTTTCACGAACACGAA 3465 MDUB7 GCTGCGCTGAGIGTGCAGGACAGCAGCCACAGTCTCCCTGAGCGCTTTCATGAACACAAA 3489 IIDUB7 AATGGPAAGTCCCGGAAACGGAGACACGACAGTGTGGAGAACAGTGACAGTCATGTTGAA 3525 NDUB7 AGTGTCAAGTCGAGGAAGCGGAGGTATGAGACTCTAGAAAATAATGATGGCCGTCTAGAA 3549 HDUB7 AAGAAAGCCCGGAGGAGCGAACAGAAGGATCCTCTAGAAGAGCCTAAAGCAAAGAAGCAC! 3585 MDU-B7 AAGPAAGTCCACA4AGCCTGGAGAAGGACACGCTAGAGGAGCCAAGGGTGAAGAAGCAC 3609 U-DUB7 2XAAAsTCAAAGAAGAAAAAGAAATCCA4AGACAflACACCCAOACCGCGACTCCAGGC!AT 3645 MDUB 7 AAAACTCTAAAAAGAAAAAGAAGTCCAAAGATAAACACCGGGATCGAGAAA0CAGGCAC 3669 N4DUIB7 CAGCAC-GACTCAGACCTCTCAGCAGCGTGCTCTQACGCTGACCTCCACACACACAAA-AAA 3705 MDUB7 CAGCAGGAGTCTGATTTTCAGGAGCATACTCTGATGCTGACCTCCATAGACACCGGAAG 3729 HDUB 7 AAGAAGAAGAAAAAGAAGAGACATTCAAGAAAATCAGAGGACTTTGTTAAAGATTCAGAA 3765 ?ADUB 7 AAAAAGAAGAAAAAGAAAASGCATTCCAOGAACTCGGAGGACTTTATAAAGGATGTTGAG 3789 UDUB7 CTGCACTTACCCAGGQTCACCAGCTTGGAGACTGTCGCCCAGTTCCGGAGAGCCCAGGGT 3825 MDU-B7 ATGCGTTTACCGAAGCPCTCCAGCTACGAGGCCCGCGGCCATTTCCGGAGAACAGAGGGC 3849 HDUB7 GGCTTTCCTCTCTCTGGTGGCCCGCCTCTGGAAGGCGTCGGACCTTTCCGTGAGAAAACG 3885 MDUB7 AGCTTTCTCCTGGCTGATGGTCTOCCTGTGGAAGACAGCGGCCCTTTCCGGGAGAAAACG 3909 B4DUB7 AALACACTTACGGATGGAAAGCAGGGATGACAGGTGTCGTCTCTTTGAGTATGGCCAGGGT 3945 MDU-B7 AAGCATTTAAOGATGGAAAGCCGGCCTGACAGATGCCGTCTGTCGGAGTATOGCCAGGAT 3969 BDUB7 GATTGA 3951 MDUB7 TCAACATTTTGA 3981 Table 16. Deduced amino acid sequence alignment of hDUB7 and mDUB7 H-DUB7 MTIVDKASESSDPSAYQNQPGSSEAVSFGDMDAGSASWGAVSSLNDVSNHTLSLGPVPGA MDIJB7 MTIVDKT- EPSDPSTCQNQPGSCEAVSFEDMDTGSASWGAVSS TSDVSSHTLPLGPVPGA 59 HDIJB7 WYSSSSVPDKSKPSPQKDQALGDGIAPQKVLFPSEKICLCWQQTHRVGAGLQNLGNTC 12(C IADIB7 VVYSNSSVPEKSKPSPPKDQVLGDGIAFPQKVIJFPSEKICLKWQQSHRVGAGLQNLGNTC 119 HDUB7 FANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCD4MCTMQ1ITQLSNPGDVIKPMFVI 180 MDUB7 FANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQTHITQAIJSNPGDVIKPMFVI 179 HDUB7 NENRRIARHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQATTLVCQIFGGYLRS 240 MDUJB7 NEMRRIARHFRFGNQEDAHEFLQYTVDAMQKACLJ3GSNKLDRHTQATTLVCQIFGGYLRS 239 HDUB MDU37 RVKCLNCKGVSDTFDPYLDITLEIKAAQSVNKALEQFVKPEQLDGENSYKCSKCKKMVPA 300 WO 03/083050 PCT/US03/08590 36 HDUB7 ****************GIADKYEYDRPMSPGEIVVYAL 6 HDUB7 SKRFTIHRSSNVLTLSLKRFANFTGGKIAKDVKYFEYLDIRPYSQPNEPflTYVLYAVL 360 HDUB7 VHTGFNCHAGEYFCYTKASNGLWYQMNDSIVSTSDIRSVLSQQAYVLFYIRSEDVKNGGE 420 MDUB7 vHTGFNCHAGEYFCYIKASNGLWYQ4NDS IVSTSDIRAVLNQQAYVLFYIRSHDVKWGGE 419 HDUB7 ITHPTHSPGQSSPRPVTSQRVVTNKQAAPGFIGPQLPSHMIKNPPHLNGTGPLKDTPSSS 480 NDUB7 SAHPAESPGQSSPRPGVSQRVVNNKQVAPGFIGPQLPSHVMKNTPHLNGTTPVKDTPSSS 479 BDUB7 MSSPNGNSSVNRASPVNASASVQNWSVNRSSVIPEHPKKQKITISIHNKLPVRQCQSQPN 540 MDUB7 VSSPNONTSVNRASPATASTSVQNWSVTRPSVIPD-PKKQKITISIHNKLDARQGQAPLN 539 BDUB7 LHSNSLENPTKPVPSSTITN-SAVQSTSNASTMSVSSKVTKPIPRSESCSQPVMNGKS 597 DDUB7 NSLHGPCLEAPSKAAFSSTITNPSAIQSTSNVPTTSTS---------FPSEACPKPNVNGKA, 592 HDUB7 KLNSSVLVPYGAESSEDSDBESKGLGKENGIGTIVSSHS- -PGQDAED-EEATPHELQEF 654 NDUB7 KVGASVJTVPYGAESSEESDEESIGLAKENGVDMMAGTHSDRPEAAADDGAEASSHELQEP 652L HDUB7 MTLNGANSADSDSDPKENGLAPDGASCQGQFALHSENPFAKANGLPGKLMFAFLLSLPED 714 MDUB7 VLLNGANSADSDS- -QENSLAFDSASCQVQFELHTENLFSKLNOLPGKVTPAFLQSVPED 709 BDUB7 KTLETFRLSNKLKGSTDEMSAFGIAERGPPEDRDAEPQPGSFAAESLEEPDAA.A-GLSSTK 773 MDUB7 RILETFKLTNQAKGPAGEESWTTTGGSSPKDPVSQLEPISDEPSPLEIPEAVTNGSTQTP 76S9 HDUB7 KAPPPRDPGTPATKEGAWEANAVAPEEPPP SAGEDIVGDTAPFDLCDPGSLTGD 827 MDIJB7 STTSFLEPTISCTKEDSSVVVSAEPVEGLFSVPALCNSTGTILGDTPVPELCDPGDLTAN 829 HDUTS7 ASPLSQDAKGMIAEGPRDSAT 4 AEAPBGLSPAPPARSEEPCEQPLLVHPSGDIARDAQDPS 887 MDTJB 7 PSQPTEAVKGDTAEKAQDSAMAEWERLSPAPSVJTGDGOEQKLLLYLSAEGSEBTEDSS 889 HDITB7 QSLGAPEAAERPPAPVLDMAPAGEPEGDAEPSPGERVED -AAAPKAFGFSPAKEKIGSLR 946 MDIJB7 RSS -AVSADTMPPKP- -ERTTTSSCEGAAEQAAGDRGDGGHVGPKAQEPSFAKEKMSSLR 946 EDU'B7 KVDRGEYRSRRERSSSGEPARESRSKTEGHRHRRRRTCPRERDRQDRIAP--- EHHlP 1000 MDUB7 KVDRGHYRSRRERSSSGEHVRDSRPRPEDHEHKKRECYSRERPKQDRHFTNSYCNGGQHL 1006 EDUEB7 GHGDRLSPGERRSLGRCSHEHSRHRSGVELDWVRHHYTEGERGWGREKFYPDRPRWDRCR 1060 MDUB7 GHGDRASP-ERRSLSRYSIIEHSRIRSGLEQDWSRYHHLENEHAWVRERFYQDKLRWDKCR 1065 HIDUE7 YYEDRYA- LYAAR- -DWKPFEGGREIIERAGLHERFHIODUNRGRRGCEF -ARERERHRPS 1115 MDUB7 YYHDRYTPLYTARDAREWRFLHG-REHDRLVQSGRPYKDSYWGRKGWELQSRGKERPHEN 1124 E4DUB37 SPPAQAPHALAPHPDRFSHDRTALVAGDNCbT-LSDRFHEHENGKSRKRRHDSVENSDSHV 1174 MDUB7 SPREAP- -SLAVPLERELQEKAALSVQDSSHSLPERFHEKSVKSRKRRYETLENlDGRL 1182 WO 03/083050 PCT/US03/08590 37 HIDJB 7 EKKARRSEQKDPLEEPKAKKHKKSK1(KKKSITKHRDRDSRHQQDSDLSAACSDADLHRHK 1234 MDUB7 EKKVHKSLEKDTLEHPRVKKHKKSKKKI{KSKDKHRIDRESRHQQESDFSGAYSDADLHRHR 1242 HDUB7 KKKKKTSKRHSRKSEDFVKDSELHLPRVTSLETVAQFRRAQGGFPLSGGPFLEGVGPFREK 1294 MDUB 7 KKKKKKECRHSRKSEDFTKDVEHRLPKLSSYEAGGHFRRTEGSFLLJADGLPVEDSGPFREK 1302 HDTJR7 TKHLRMESRDDRCRLFEYGQGD-- 1316 MDUE7 TKHTRMESRPDRCRLSEYGQDSTF 1326 Table 17. Amino acid sequence aligniment of catalytic domain among murine DUJBi, DUB2, KDUB7 and nYDUB7. Amino acids that are involved in catalysis in DUBi (Cys-60, Asp-133, and His-3 07) are underlined.

InDUB 1 mnDUB2 hDU137 MDUB 7 mDUB1 MDUB2 hDUB 7 n-LDUB 7 inDUB 1 mDUB2 bflUB7 mDUB7 mDfUBi mDUB2 hDUE 7 mDUB7 mDUB 1 mnfUB2 hDUE7 niDUB 7 rnfUB 1 inDUB32 hDUB 7 niDUB 7 MVVALSFPEADPALSSPDAPELHQDEAQVVEEJTVNGKHSL9WES

PQGPGCGLQNTGNSC

MVVSLSPPEADPALSSPGAQQLHQDEAQVVVELTANDKPSLSWECPQGPGCGLQNTGNSC

VVYSN\SSVPEKSKPSPPKDQVLGDGIAPPQKVLFPSEKICLKWQQSHRVGAGLQNLGNTC

**.*VTQS rH T4ST)V KPS

YLNAAI

4

QCLTHTPPLANYMLSQEYSQTCCEGCKMCTMEAHVTQSLLSHEGDVIKPSQ

FANAALQCLTYTPPLANYMLSBEHSKTCHAEGFCMMCTMQAITQALSN-

-PGDVIKPMF

ILTSA FHKHQQEDAHEFLMFTLETMHESCLQVHRQSKPTSEDSPI{DIFGGWW ILTSA FHKHQQEDAHEFLMFTLETMKESCLQVHRQSEPTSEDSSPIHDIFGGLW VINEMRRIJ4RHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQATTLVCQIFGGYL

VINBMRRTARHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQATTLVCQIFGGYL

ELGDNA YCGKRQK RSQIKCLLCQGT5DTYDRFLDTPLDI SSAQSVKQAIJWDTEKSEELCGDNAYYCGKCRQKM

RSQIKCLBCQGTSDTDFLDTLDTSAQSVNQAIJNDTEKSEELRGENAYYCGRCRQKMV

RSRVKCLNCKGVBDTFDPYLDITLEI KAAQSVNKAIJEQFVKPEQLDGENSYCCSKCKKMV

PABKTLHVHIAFI(VLMVVLNRFSAFTGNKLDRKVSYEFLDLKPYLSEPTOGPLPYALYA

PASKTLHIHSAFKVLLLVLKRFSAFMGNKLDRKVSYEFLD)LKPYLSQPTGGPLPYALYA

PASKRPTIHR5 SNVLTLSLKRFANFTGGKIAKDVKYFEYLDIRPYMSQPNGEPIVYVLYA PASKRFTIHRS SNVTTTSLKRFANFTGGKIAKDVKYPEYLDIRPYMSQFNGEPI

IYVLYA

*:QANLK

VLVHDGATSHSGHYFCCVKAGHGKWYKMDDTKVTRCDVTSVLNENAYVLFYVQQTDLKQ

VTVHEGATCHSGHYFSYVKARHGAWYKMDKVTCDVTSVIJNENAYVLFYVQQTDLKQ

VLVI{TGFbCHAGHYFCYIKASNOLWYQMNDS IVSTSDIRS-VLSQQAYVLFYIRSHDVKN

Claims (11)

1. An isolated polynucleotide encoding a human deubiquitinating protease selected from the group consisting of hDUB7 and mDUB7. \N

2. A polypeptide encoding a human deubiquitinating protease selected from the group consisting of hDUB7 and mDUB7. m 3. A method of using a polynucleotide according to claim 1, wherein the Spolynucleotide is used in an assay to identify an inhibitor of a hDUB or mDUB7 of Sclaim 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. 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 00 O compound capable of specifically altering regulation of transcription of a c polynucleotide of claim 1. A method of modulating an immune reaction during infection, by \D administering a compound capable of specifically altering regulation of transcription of a polynucleotide of claim 1.

11. A method of identifying a modulator of a human 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 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, 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. A method of delivering a biologically active protein, therapeutically effective 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 WO 03/083050 WO 03/83050PCT/USO3/08590 usav2002-0022 .ST25 .txt SEQU ENCE LISTING <-110> Aventis Pharmaceuticals, Inc. Hahn, Chang S Liu, Hong S <120> HUMAN DEUBIQUITINATING PROTEASE GENE ON CHROMOSOME 7 AND ITS MURINE ORTHOLOG <130> usAV2002-0022 WO PCT <150> GB 0218518.9 <151> 2002-09-08 <150> us 60/366,601 <151> 2002-03-22 <160> 12 <170> Patentln version 3.2 <210> 1 <211> 19 <212> PRT <213> Homo sapiens <400> 1 Lys Ala Lys Lys His Lys Lys Ser Lys Lys Lys Lys Lys Ser Lys Asp 1 5 10 Lys His Arg <210> 2 <211> 16 <212> PRT <213> Homo sapiens <400> 2 His Afl9 His Lys Lys Lys Lys Lys Lys Lys Lys Arg His ser Arg Lys 1 5 10 <210> 3 <211> 17 <212> PRT <213> Homlo sapiens <400> 3 Lys Lys His Lys Lys Ser Lys Lys Lys Lys Lys Ser Lys Asp Lys His 1 5 10 Arg <210> 4 <211> 16 <212> PRT <213> Homo sapiens <400> 4 Page 1 WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022.ST25.txt HiS Arg His Arg Lys LYS LYS Lys Lys LYS LYS Arg His Ser Arg LYS 1 5 10 <210> <211> 23 <212> DNA <213> HOMO sapiens <400> ccacgacaga actgcacttg tag <210> 6 <211> 19 <212> DNA <213> HOMO Sapiens <400> 6 ccgggacttt ccattttcg <210> 7 <211> 31 <212> DNA <213> HOMO sapiens <400> 7 caactgtaac ctctctgatc ggtttcacga a <210> 8 -<211> 3951 <212> DNA <213> HOMO <400> 8 atgaccatag ggcagctccg gtgtcttcat gtagtttatt gccctaggtg cttaagtggc tttgccaatg tcacatgaac gcacatatta aatgagatgc ttccttcaat gacagacaca agagtcaaat acattggaga sapiens ttgacaaagc aggcagtctc tgaatgatgt cgagttcatc atggcatcgc aacaaactca cagcactgca actccaaaac cccaggcact ggcgtatagc acactgttga cccaggccac gtttaaattg taaaggctgc ttctgaatct acctggagac gtcaaatcac tgtacctgat tcctccacag tagagttgga gtgtttaacc atgtcatgca cagtaatcct taggcacttc tgctatgcag cactcttgtt caagggcgtt tcagagtgtc tcagacccat atggatgcag acactttctt aaatcaaaac aaagttcttt gctgggCtcc tacacaccac gaaggctttt ggggacgtta cgttttggaa aaagcatgct tgtcagatat tcagatactt aacaaggcat cagcctat ca gttctgccag taggaccagt catcaccaca tcccatctga agaatttggg ctcttgccaa gtatgatgtg ttaaaccaat accaagaaga tgaatggcag ttggaggata ttgatccata tggagcagtt gtaaaaagat gaatcagcct ctggggtgct acctggtgct aaaggatcaa gaagatttgt caatacctgt ttacatgcta tacaatgcaa gtttgtcatc tgcccatgaa caataaatta cctaagatct tcttgatata tgtgaagccg ggttccagct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 gaacagcttg atggagaaaa ctcgtacaag tgcagcaagt Page 2 WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022.ST25 .txt tagatcctct aatgttctta cactttctct gaaacgtttt tcaaagaggt gcaaatttta cggccatata gtccacactg ggcCtctggt agccaacaag cttactcatc gttgtcacca ataaagaatc atgtcgagtc tctgtccaaa aaaattacaa cttcatagta tctgcagtac ccgatccccc tccagcgtgc gggctgggca gccgaagatg aatagtgcag tgccaaggcc ggaaagttga aggcttagca ggccctcccg gaggagccag cccggcaccc cctccgccca cccgggagct gagggcccgc gcgcggtcgg cgggacgctc gctcctgtgc ggcgagaggg aaaatcggca tcactatcca ccggtggaaa tgtctcaacc gttttaattg atcaaatgaa cctatgtgct ccacccatag acaaacaggc cacctcactt ctaacgggaa actggtcagt tcagtattca attctttgga agtctacctc gcagtgaatc tggtgcccta aggagaatgg aggaggccac acagcgacag agcctgccct tgcctgctcc acaaactgaa aggaccgcga atgcggccgc ccgctaccaa gcgccggcga taacaggcga gggactcggc aggagccctg aggacccatc tggacatggc tcgaggacgc gcctcagaaa aattgctaag caacggagag ccatgctggc tgact ccatt cttttatatc ccccggccag tgcgccaggc aaatgggact ttccagtgtc taataggtcc caacaagttg gaaccctacc gaacgcatct ctgctcccag tggcgccgag gattggtacg tccgcacgag tgacccgaaa gcactcagaa tttgctgtct aggctcgacg cgccgagcct cggcctcagc agaaggcgcc ggacatcgtg tgcgagcccg gttggcggaa cgagcagcca ccagagcttg cccggccggt cgcggcgccg ggtggaccga gatgtgaaat ccaattgtct cat-tacttct gtatctacca aggtcccatg tcctctcccc tttatcggac ggaccattga aacagggcta tcagtgatcc cctgttcgcc aagcccgttc acgatgtcag cccgtgatga tcctctgagg attgtgagct cttcaagaac gaaaacggcc aatccctttg ctcccagaag gatgaaatga cagcctggca agcaccaaga tgggaggcca ggggacacag ttgtcccagg gccccggaag ctccttgttc ggcgcacccg cacccggaag aaagccccag ggccactacc accctgagta tcttgatatt acgtcttgta gctacataaa gtgatattag atgtgaaaaa gccccgtcat cacagcttcc aagacacgcc gtcctgttaa cagaacatcc agtgtcagtc cctcttctac tttctagtaa atggcaaatc actctgacga cccactctcc ccatgaccct tagcgcctga ctaaggcaaa acaaaatctt gtgcacctgg gccccgccgc aggctccgcc tggccgtcgc caccccctga acgcaaaggg ggttgagtcc accccagcgg aggccgcaga gggacgctga gcccttcccc gcagccggag tgcagtgctg agctagcaat atcggtactc tggaggtgaa cagtcagcgg ctctcacatg aagcagttcc tgcttcagct taagaaacaa tcaacctaac cattaccaat agtaacaaaa caagctgaac ggagtcaaag cggccaagat aaacggtgct tggtgccagc cggtcttcct agagaccttc agcagagagg cgaatccctg gccccgcgat ccccgaggag cctgtgtgat gatgatcgcg ggctccgcct ggaccacgcc gcggccgcca gcctagcccc agcgaaggag agagcgctcg 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 tccagcgggg agcccgccag agagagcagg agcaagactg agggccaccg tcaccggcgg Page 3 WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022 .ST25 .txt gcgcgaccgc caggaccgcc acgccccgga cgccgcacct ggccacggcg cactcccgac gagcgtggct tactaccatg cgcgagcacg aggggctgcg gcgccccacg gccggagaca aaacggagac agcgaacaga aaaaagaaat ctctcagcag aagagacatt gtcaccagct ggtggcccgc gaaagcaggg gcccccggga acaggctcag accggagcgg ggggCCggga acaggtacgc agcgggccgg agccggcccg ccctcgcccc actgtaacct acgacagtgt aggatcctct ccaaagacaa cgtgctctga caagaaaatc tggagactgt ctctggaagg atgacaggtg ccctggcgag ggtggagctg gaagttctac cctgtacgct gctgcacgag ggagagggag gcaccccgac ctctgatcgg ggagaacagt agaagagcct acaccgagac cg ctgacctc agaggacttt cgcccagttc cgtcggacct tcgtctcttt cgccgctctc gactgggtca cccgacaggc gcccgggact cggccgcaca cggcaccgcc cgcttctccc tttcacgaac gacag-tcatg aaagcaaaga cgcgactcca cacagacaca gttaaagatt cggagagccc ttccgtgaga gagtatggcc tgggcaggtg gacaccacta cgcgctggga ggaagccctt aggaccacaa ccagcagccc acgacagaac acgaaaatgg ttgaaaagaa ag cacaaaaa ggcatcagca aaaaaaagaa cagaactgca agggtggctt aaacgaaaca agggtgattg gcaccacccc cagtcaccac caccgagggc caggtgccgg ccacggcggc ccggggccgt ccgcgcaggc tgcacttgta aaagtcccgg agcccggagg atcaaagaag ggactcagac gaagaaaaag cttacccagg tcctctctct cttacggatg a 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3951 <210> 9 <211> 1316 <212> PRT <213> Homo sapiens <400> 9 Met Thr le Val Asp 1 5 LYS Ala ser GIUl ser 10 sen ASP Pro Sen Al a Ty GI n ASfl Gi n Ala Gly Sen Gly Sen Sen Glu val sen Pro Gly ASP Val Sen Ala Sen Tnp Gly Val Sen sen Leu Asn His1Thr Leu Sen Leu Pro Vai Pro GIy val Val Tyr Sen Ser Sen val Pro ASP LYSsSen Lys Pro Sen Pro Gin Lys ASP Gin 75 Ala LeU Gly ASP ie Ala Pro Pro LYS Vai Leu Phe Pro Sen GlU Lys lie LeU LYS TnP Gin Thn HiS Arg Val Page 4 Gly Ala Gly 110 WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022.ST25 .txt Leu Leu ser 145 Al a Met Gi y Met Gin 225 Arg Ty r Al a Ty r Th r 305 Al a Ty r val Gin Th r 130 Lys His Phe As n Gi n 210 Al a Val Leu Leu LYS 290 Ile As n LeU Tyr Asn 115 Ty r Th r lie Val Gin 195 Lys Th r Lys Asp Gi u 275 CYS H-is Ph e Asp Vai 355 Leu Th r cys Th r Ile 180 Giu Al a Th r Cys Ile 260 Gin ser Arg Th r Ile 340 Leu Gly Pro His Gin 165 Asn ASP Cys Leu Leu 245 Th r Phe Lys Se r Gi y 325 Arg Ty r ASfl ihr Pro Leu 135 Ala Gl u 150 Ala Leu GlLJ met Ala His Leu Asn 215 Val cys 230 Asn Cys Leu Gbu Val Lys Cys Lys 295 Ser Asn 310 Gly Lys Pro Tyr Ala Val cys 120 Al a Gi y Se r Arg Glu 200 Gb y Gin Lys Ile Pro 280 Lys Val Ile Met Leu 360 Phe Ala Asn Ala Met met 155 Gi y Ala Gin Lys Gi y 235 Ser Al a Leu Pro Leu 315 Asp Pro Th r Al a 125 Se r cys Val His Th r 205 Asp Ty r Th r Ser Gly 285 Ser Leu LYS Gi y rhe 365 His Th r le Phe 190 Val Arg Leu Phe Val 270 GlU Lys Lys Tyr GiU 350 Asn Glu HIS met Gin 160 Lys Pro 175 Arg Phe ASP Al a HIS Thr Arg Ser 240 Asp Pro 255 Asn LYS Asn Ser Arg Phe Arg Phe 320 Pro Giu 335 Pro ie Cys His Leu Gin Cys Ala Gl y 370 His Tyr Phe Cys lie LYS Ala Ser Page Gly LeU Trp Tyr WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022 .ST25 .txt Gi n Met 385 ser Gi n Asfl Giy Pro Arg Pro Gly 450 Pro His 465 Met Ser Asn Ala lie Pro Lys Leu 530 Ser Leu 545 Ser Ala Lys Val Met Asn Al a Giu 610 GiU ASfl 625 ASfl ASP Gin Al a Gly Giu 420 Pro Val 435 Phe :lie Leu Asn Ser Pro Ser Aia 500 Giu His 515 Pro Val Giu ASfl Val Gin Thr Lys 580 Gly Lys 595 ser Ser Gly Ilie ser Ty r 405 Leu Ilie Gil y Gi y As n 485 Se r Pro Arg Pro Se r 565 Pro Se r Glu Gi y GiUl 645 Ile 390 Vai Th r Ser Pro Th r 470 Gi y Val Lys Gin Th r 550 Th r Ilie Lys ASP Th r 630 Val ser Thr Ser Leu Hi s Gi n Gin 455 Gi y Asn Gin Lys Cys 535 LYS ser Pro Leu Se r 615 Ile Ph e Pro Arg 440 Leu Pro Ser Asn Gin 520 Gin Pro As n Arg Asnf 600 Asp Val Tyr Th r 425 Val Pro Leu Ser irp 505 Lys Se r val Al a Se r 585 Se r Gi u Se r Ilie 410 His Val ser Lys Vai 490 Se r Ilie Gin Pro Se r 570 Giu Se r Gi u Se r Gi U 650 Asp 395 Arg Ser Th r HIS ASP 475 Asn Val Th r Pro Se r 555 Th r Se r Val Se r His 635 ie Arg Ser Val Ley 400 Ser His Pro Gly Asn Lys 445 Met Ilie 460 Thr Pro Arg Aia Asn Arg Ie Ser 525 Asfl Leu 540 ser Thr Met Ser Cys ser Leu x'ai 605 Lys Gly 620 ser Pro ASP Gin 430 Gin Lys Se r Ser Ser 510 Ilie His lie Vial Gin 590 Pro LeU Gi y Val LYS 415 ser ser Ala Ala Asn Pro Ser Ser 480 Pro Val 495 Ser Val His A~n ser Asn Thr Asn 560 ser Ser 575 Pro \'al Tyr Gly Gly Lys Gin ASP 640 Met Thr 655 Ala GiU ASP GIU Ala Thr Pro His LeU Gin Giu Pro Page 6 WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022 .ST25 .txt Leu Asn Gly Leu Ser Glu 690 Pro Al a 705 Arg Leu Gly Ala Gly Ser Leu Ser 770 Ala Thr 785 Pro Pro ASP Leu Gin ASP Ala G1U 850 Glu Pro 865 Arg ASP Glu Arg Gi y Al a 675 Asfl Pro se r GlU Pro 755 Se r Lys Pro Cys Al a 835 Al a cys Al a Pro Asn Asp Phe Leu LYS 725 Gl y Al a LYS Gl y Al a 805 Pro Gl y Glu Gi n ASP 885 Al a ser Ala ASP Gl y Al a Ser 710 Leu Pro GlU LYS Ala 790 Gi y Gi y Met Gly Pro 870 Pro Pro Ala LYS 695 Leu Lys Pro Se r Ala 775 Trp Glu se r Ile Leu 855 Leu Se r Val Ser 680 Al a Pro Gl y Gl U Leu 760 Pro Gl U ASP Leu Al a 840 Se r Leu Glnf Leu Se r 665 cys Asfl GlU ser ASP 745 Gl U Pro Al a Il e Th r 825 Glu Pro Val Se r ASP 905 ASP Ser Asp Pro Gln Gly Gly Leu ASP LYS 715 Thr ASP 730 Arg ASP GlU Pro Pro Ang Met Ala 795 Val Gly 810 Gly ASP G~ly Pro Ala Pro His Pro 875 Leu Gly 890 Met Ala Gln Pro 700 Ile Gl U Al a ASP ASP 780 Val Asp Al a Arg Pro 860 Se r Al a Pro Pro 685 Gl y Leu Met Glu Al a 765 Pro Al a Th r Se r ASP 845 Al a Gl y Pro Al a Lys Glu Asn 670 Ala LeU His[ Lys Leu Met Glu Thr Phe 720 Ser Ala Pro 735 Pro Oln Pro 750 Ala Ala Gly Gly Thr Pro Pro Glu GlU S00 Ala Pro Pro 815 Pro Leu Ser 830 Ser Ala Leu Arg Ser Glu Asp His Ala 880 Glu Ala Ala 895 Gly His Pro 910 GlU Gly ASP 915 Ala GlU Pro Ser Pro Gly GlU Arg Val GlU ASP Ala Ala 920 925 Page 7 WO 03/083050 WO 03/83050PCT/US03/08590 Ala Pro LYS 930 LeU Arg LYS 945 ser Ser Gly Ang His Ang Arg HiS Ala 995 usav2002-0022 .ST25 .txt Ser Pro Ala Lys GlU Lys Tie Gly Ser 940 His Tyr Arg Sen Arg Arg Glu Arg Sen 955 960 Glu Ser Arg sen Lys Thr Giu Gly His 970 975 Cys Pro Arg Glu Arg ASP Arg Gin ASP 985 990 Pro Gly His Gly Asp Arg Leu ser Pro 1000 1005 Gly His Glu Pro Ty r Gi U Gi y Pro Pro Asn Sen Gi U 1010 Ang 1025 Gi y 1040 Arg 1055 Al a 1070 Ang 1085 Ang 1100 Sen 1115 Asp 1130 cys 1145 Arg 1160 Arg Gi y Ang Asp Arg Gi y Gi y Pro Ph e Leu Afl9 Leu G1 U irp Cys Trp His GlU Al a His ASP His Gi y 1015 LeU 1030 GIlY 1045 Arg 1060 Lys 1075 GlLJ 1090 Pro 1105 Gi y 1120 Asp 1135 Any 1150 Asp 1165 Any 1180 Ang Asp A rg Tyr Pro A ng Ala Al a Any Ph e Sen His His 1020 His His 1035 Tyr Pro 1050 Arg Tyr 1065 Gly Any 1080 ASP Hi S 1095 Gl u Arg 1110 Leu Ala 1125 Val Al a 1140 GI u Asn 1155 Sen Asp 1170 Sen Ty r ASP Al a Gl U As n His Pro Gly Gly sen Val GIlU 1175 Lys LYS Ala Arg Sen Glu Gin LYS ASP 1185 Pro Leu GIU Page 8 WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022 .ST25 .txt GlU Pro 1190 Ser Lys 1205 Ser Asp 1220 Lys Lys 1235 Asp Phe 1250 Leu Glu 1265 Lys Ala LyS Lys Asp Lys His Arg Leu Ser Ala Ala Lys LYS Lys LYS 'ial Lys Asp Sen Thr Val Ala Gin His 1195 Lys Lys Sen Lys Lys 1200 Lys Lys Lys Asp Arg Asp Sen Arg His Gin Gin ASP 1210 1215 cys 1225 LYS 1240 Gi U 1255 Ph e 1270 Sen Asp Ala ASP LYS Arg His Sen Leu His Leu Pro Ang Arg Ala Gin Leu 1230 Ang 1245 Ang 1260 Gi y 1275 His Arg His LySsSer Glu Val Thr Sen Gly Phe Pro Leu Sen Gly Gly Pro Pro Leu GlU Gly \'al Gly Pro Phe Arg Glu 1280 1285 1290 LYS Thr 1295 Leu Phe 1310 LYS HIS LeU Arg GlU Tyr Gly Gin Met GiU Sen Ang Asp Asp Ang Cys Arg 1300 1305 Gly ASP 1315 <210> <211> 2001 <212> DNA <213> HOMO sapiens <400> gtaaagtcta aactgagaag gtcggagaaa agaacagcat caagacttca gcatggtggg acaatgtgaa gtccagacat gtgttctttc ttcctttcct agtctggagt gcagtggcat aattttcctt gcctcagcct gttaattttt ttatttttag actcctggac tcaagtgatc gtgagccact gcgccccgcc tgtcaccaag gctggatatc tggaagtgtg aactggctgg aggtggaagg ttggaaaaga gtgcagagcc aacgtgacgg taagatgttg tttctttctt gatctcggct cccaagtagc tagagatggg cacccacttt cggccttttt agtggcacgg cagagacagc agagggtgtt tgctgtgggc tcttttcttg cactgcaatc tgggaataca gtttccccat ggcctcccaa tttttttttt ttttggctct agggacaaaa tggcgaggtt agttgggcca aggcagagtc tctgccttcc ggcgtgcgcc gttggccagg agtgctggga tttgagactt ctg caactt C gattacaggt gaaaaaaaaa attaggtcag ctcctgaaag tctctatgtc aggttcaagc accatgcctg ctggtctcga ttacaggggt aatcttgctc tgtctcccag acgtgccacc gttcaagcga ttttcctgac tcagcctcc aagtagttga Page 9 WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022.ST25 .txt tttttagtag agatgaggtt tcactatgtt acgcccggct gtctcaaacg caggtgtgca gttggccagg ggtgctgcaa gagtggcatg ctgttgacag agt cccagtt catacgttct catagaaaat ttataat caa acgggtttta ttaaaggtga atttgcactt tgaggaatta gttttaagca aaatgtccaa gttcttttaa atcatgatgc ttatatttga ctaacggtag cttttgcaga aatttttgta cctgacctca ccaccatgcc ctggtctcaa ttataggcat tctagatgtg ggtggcataa gctgactggt ttataaaaat gctcaattag gtcctttcat tttaaaaagt gatctctata taaqcacaaa tactcaaatt gtatagaagg ttataaactg tcaaatactt taaatgtccc gtgatgtgtt ggtaaggaga gttgaacaat ggtgattcac tgggtaattt actcctgacc gagccaccgc gctactgttg ccagaaggaa tgcttcattt taagttaatg tggtagccct aacagatatt catacattcc attataattt attaagt cta gaagatgtag aaaatatagg aaatgccaga cactgaagat aaggggatta tgaaaatcac ttgcttttct ctgcctcggc ttgtttttcg tcaagcgatc gcccggcctc ggcgatttta gcctggctgg tataggccct tatgtccagc aatattttaa tgtgtttgag atataaatga taaaaaatat gatttttt ct tcaaatgtat tacttagtaa cttttagggg tttaaaataa ttacagaaat tttaaaatgg cacctgatga ctcccaaaat tagagacagg tgcccacctt ctgaaaggtt gaaatatccc gaacgctgga ggggattctg agttgatgca aataggactc tttgatatca gcctcttcag aatgtatttc gtgtagttga tactgtgtat ataaacagaa gcatgaaatg agattgttga gttagaaagt ctggaatgat aacaaataca ggccagactg gctgggatta gtctcaccat ggcctcccaa ttctacatag taaaagcctt cctggctctc tctgatctct atgcccagta agaaagaaaa gtaatggctt aaaaatggtt acttggtgcc tgcttttctc aatttttcta ctgagaattg aaaatgagaa cattcagatt actattgttt cttccaagat tacttttcat 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2001 <210> 11 <211> 3981 <212> DNA <213> Mus musculus <400> 11 atgaccatag ttgacaaaac agttgtgagg cggtctcacc tcttcaataa gtgatgtctc gtttattcta actcgtctgt ctaggtgatg gcattgctcc aagtggcaac aaagtcatcg gccaatgccg cattgcagtg catgaacact ccaagacatg cacattaccc aggcacttag tgaaccttca tgaagacatg aagtcacaca acctgaaaaa tcctcaaaag agttggcgct tctgacttac ccacgcagaa caaccctggg gacccatcaa cctgtcagaa gacacaggct ctgccagctg cttccattag ggccagtgcc tcaaagccat caccaccaaa gtcctgtttc catctgaaaa gggctccaga atttgggcaa acgccacccc tcgccaatta ggattttgta tgatgtgcac gatgttatca agccgatgtt Page ccagcctggc gggcgctgtg tggtgctgta ggatcaagtc gatttgtctt cacctgtttt catgttatCC gatgcagaca cgtcatcaat WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022 .ST25 .txt gaaatgcggc cttcagtaca agacacaccc gttaaatgtt ttggagatta caactggatg aagagattca aacttcaccg ccctatatgt cacactggtt ctctggtatc cagcaagctt gctcatcctg gtcaacaaca aagaacacgc tcaagcccta gtgcagaact at ca ccat ca agcctccatg aacccttctg gcctgtccca tatggggccg ggtgtggaca gctgaggctt gacagtgact gagctacaca gctcctttgc gcaaagggtc cctgtttcac gtcaccaatg tgtaccaaag gtccCtgCtC tgtgaccctg gtatagctag cggtcgatgc aggccaccac taaattgcaa aggctgcaca gagaaaactc caatccatag gtggaaagat ctcagcccaa ttaattgtca agatgaatga acgtgctctt cccatagccc agcaggtggc cacacttgaa acggaaacac ggtctgttac gtattcacaa gcccttgtct caatacagtc agcccatggt agtcctcaga tgatggccgg cctcccatga cacaagagaa cagaaaacct agtctgttcc cagcgggtga agctggagcc ggagcacaca aagactcgtc tttgtaacag gagacttgac acacttccgt catgcagaaa cctggtctgc gggtgtttca gagtgttacc ctacaagtgc gtcctctaat tgctaaggat tggagagcca tgctggccac ctccatcgtg ttatatcagg cggccaatcc tccagggttt tggcaccacg cagcgtcaat cagaccctca caagttgcct ggaggctcct tacctcgaac gaacggcaag agagtctgat cactcactcc gcttcaagaa cagcctggca cttttccaaa tgaagacaga agagagttgg catcagtgat gaccccttcc cgttgttgtC cactggtact tgccaacccg tttggaaacc gcatgtttaa cagatatttg gatacctttg aaggcgttag agcaagtgca gttcttacca gtgaaatatc attatttatg tacttttgct tccaccagtg tcccatgatg tctcCCCgc attggacccc ccagtgaaag agggccagtc gttattccag gctcgccagg agtaaggcgg gtacccacaa gctaaagtgg gaggagtcga gataggccag cccgtcctgc tttgacagtg cttaatggtc atccttgaga actacgacag gagcccagtc accacatcac tcagctgaac atcttggggg agccagccaa aagaagatgc atggcagcaa gaggctacct atccatatct agcagtttgt aaaaaatggt tctcactgaa ctgagtacct ttttgtatgc acatcaaggc atatcagagc tgaaaaatgg caggagtcag agctgccttc acacaccaag ctgctactgc atcaccccaa gtcaggcacc caccctcctc cgtcgacttc gcgccagtgt agggcctggc aagctgctgc taaatggtgc ccagctgcca ttcctggaaa ccttcaagct ggggaagctc cccttgagat ccctggagcc ctgtggaggg ataccccagt ccgaagcagt ccatgaattt taaattagac aagatcccga ggacataacg gaagccagaa tccagcttca gcgctttgcc tgatatccgg tgtgctggtg tagcaatggc agtgcttaac aggggagtct tcagcgggta ccatgtgatg tagttctgtg ttcgacttct gaaacaaaaa actgaataac caccatcact ccccagtgag gcttgtcCC caaggagaac agatgacggt taatagcgca ggtccagccc ggtgacgcct taccaaccag tccaaaggac accggaggct caccatcagc tttgccttcc gcccgaattg gaaaggtgat 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 acagctgaga aggctcagga ctctgccatg acagtgaa agctagg cttgcatggctgaagtgg tggagaggct gagccctgct Page 11 WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022. ST25 .txt ccctcagtac gggtcagagg cctaagcctg gacagaggcg aagatgagca tccagtgggg cggcactgct aatgggggcc aggtatagtc caccatttgg tgggacaagt cgagaatggc tacaaggaca. ccccacttca caagagaagg gaacacaaaa. cgtctagaaa aagaagcaca agcaggcacc caccggaaga gatgttgaga acagagggca, gagaaaacga ggccaggatt tcacaggtga agacagaaga acaggaccac atggaggcca gcctccggaa agcacgtgag acagccgaga agcacttggg accaccactc aaaatgagca gcaggtatta ggcctctgca gctactgggg acagcccccg ctgcgctgag gtgtcaagtc agaaagtcca aaaagtctaa agcaggagtc aaaagaagaa tgcgtttacc gctttctcct ag catttaag caacattttg cgggtgtgag ctcttccaga caccagctcc tgtgggaccc agtggaccga ggacagcagg gcggcccaag ccacggggac acggattagg tgcttgggtc ccacgacagg tggtcgtgag ccgcaagggc agaggcccct tgtgcaggac gaggaagcgg caaaagcctg aaagaaaaag tgatttttca aaagaaaagg gaagctctcc ggctgatggt gatggaaagc cagaaactct agctcggcgg tgtgaagggg aaagctcagg ggacactatc ccccggccgg caggaccgac agagccagcc agtggcctgg agggagagat tacacgcccc c at gac cgc c tgggagctgc agccttgctg agcagccaca aggtatgaga gagaaggaca aagtccaaag ggagcatact cattccagga agctacgagg ctgcctgtgg cggcctgaca tactttacct tctctgctga ctgccgagca agccttcccc ggagccggag aggaccatca accctactaa ctgagcgccg agcaggactg tctaccagga tatacacggc ttgtccagtc aatcccgggg tgcccctcga gtctccctga ctctagaaaa cgctagagga ataaacaccg ctgatgctga agtcggagga ccggcggcca aagacagcgg gatgccgtct cagcgcagag cacgatgccc ggctgctggg agccaaggaa agagcgctcc ccataagaag ttcatactgc ctccctgagc gagccggtac caagctgcgg ccgggacgcc tggacggcca. gaaggaacgg gagacatctc gcgctttcat taatgatggc gccaagggtg ggatcgagaa cctccataga. ctttataaag tttccggaga ccctttccgg gtcggagtat 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 3981 <210> 12 <211> 1326 <212> PRT <213> Mus musculus <400> 12 met Thr Ile Val ASP LYS Thr 1 5 Glu Pro Ser ASP Pro 10 Asfl Gin Pro Gly ser Cys GlU Ala Val Ser Pro Glu 25 Gly ser Ala ser Trp Gly Ala V'al ser Ser Ile Ser 40 Page 12 Ser Thr CYS Gin Asp met ASP Thr Asp Val Ser Ser WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022. ST25 .txt Th r Se r Gi y le As n Tyr 130 Th r Ile Val Gln LYS 210 Th r LYS ASP Gu CYS 290 Pro Pro ci y Leu 100 ci y Pro His Gin As n 180 ASP cys Leu Leu Th r 260 Phe Lys Leu Giy Pro 55 GiU LyS Ser lie Ala Pro Lys Trp Gin Asn Thr Cys Pro Leu Ala 135 Aia Glu Gly 150 Aia Leu Ser 165 Giu Met Arg Ala His Glu Leu Asn Gly 215 Val Cys Gin 230 Asn Cys Lys 245 Leu Glu Ilie Vai Lys Pro Cys Lys Lys. 295 Val Pro Gdy Ala Val Val Tyr Ser Asfl Pro Gin Se r 105 Al a Ty r Cys Pro Ilie 185 Leu As n Ph e Val Al a 265 GI n Val Pro 75 Vlal Arg Ala Leu Met 155 ASP Arg Ty r Leu Gi y 235 ASP Gin Asp Al a Se r 315 \'al Gi U Leu Leu Ser Th r 160 Met Gi y Met Gin Arg 240 Ty r Al a Ty r Th r Al a 320 His Arg Ser Ser Val Leu Thr Ilie Leu Lys Ang Phe Page 13 WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022. Asfl Phe Leu Asp Tyr Val Gly His 370 Met Asn 385 Gin Gin Gly Gly Arg Pro Gly Phe 450 His Leu 465 Sen Sen Ala Sen Pro Asp Leu pro 530 Pro CYS 545 Asn Pro Ser pro Th r Ilie Leu 355 Ty r Asp Al a GlU Gi Y 435 Ile Asnf Pro Th r His 515 Al a Leu sen sen Gly Gly 325 Ang Pro 340 Tyr Ala Phe Cys Ser Ilie Tyr Val 405 sen Ala 420 Val Ser Gly Pro Gly Thnr Asn Gly 485 Sen Val 500 Pro Lys Arg Gin GiU Ala Ala Ile 565 Giu Ala 580 Lys Ilie Ala Lys Met Leu Iie 375 Ser Phe Pro Ang Leu 455 Pro Th r ASfl Gin Gin 535 Sen Sen Pro Gin 345 His Al a Ser le His 425 Val Sen Lys Val Sen 505 le Pro Al a Sen Pro 585 Asp 330 Pro Th r Sen Asp Arg 410 Sen As n His Asp As n 490 Val Th r Leu Al a Asfl 570 Val LYS Tyr Pro Giu Tyr 335 Gly Phe Giy 380 Ang His Gi y Lys Met 460 Pro Al a Ang Sen AS n 540 Sen Pro Pro Ile 350 Cys Hi S Tnp Tyr Val Leu Val Lys 415 Sen Sen 430 Val Ala Asn Thr Sen Sen Pro Al a 495 Sen Val 510 His Asn Leu His Thr Ile Thr Sen 575 le Al a Gln Asn 400 Asfl Prno Prno Pro Val 480 Th r le Lys ci y Th n 560 Th r met X/al Asn Gly LYS Ala LYS 590 Page 14 WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022 .ST25 .txt V/al sen Met 625 Al a Al a Se r sen Sen 705 Ala sen Se r Pro ASP 785 Val Val Pro Al a 595 Glu Gl y Ala Se r Sen 675 Leu Pro Gi y LYS Leu 755 rh r sen Al a GlU GlU 835 Sen Val Glu Ser Thn Hi S Sen Sen 645 Ala ASP 660 CYS Gin ASfl Gly Glu ASP Pro Ala 725 ASP Pro 740 G1u lie Thn Ser Val Val Leu Cys 805 Leu Cys 820 Ala Val Leu Val Pro Tyr Gly 600 Ala Glu Sen Sen Glu GlU 605 Gi y 615 ASP Gl u ASP Gin Pro 695 le Gi U Sen Glu Leu 775 Se r Sen Prno Gi y Al a Prno Gin Gin 665 GlU Lys Glu Sen Leu 745 Val Prno Gi U Gl y ASP 825 Th n GIly Val Ala ASP LeU Leu LeU Ala 670 GlU Asfl 685 Ala Pro Leu Thn Thn Gly Sen Asp 750 Sen Thn 765 Cys Thn Gly LeU Gly Asp Asfl Pro 830 Ala Gln 845 Met dl y 640 Gi y Asp Phe Gin Gin 720 Sen Pro Th r Gi U Sen 800 Pro Gi n Sen Al a Met 850 Ala Clu Val Val Ang Leu Sen Pno Pro Sen Val Leu Page WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022. ST25 .txt Th r G- 865 Gdy S1 ASp TI Gly A' Gly P1 9: LeU A] 945 Ser Si His H Arg H' Gin LYS LeU Leu Tyr Leu Ser Ala Arg 890 Th r Arg Ala Arg Arg 970 Arg cys 1000 ASfl Gly Gly Gin His LeU Gly His 1005 Asp Arg 1010 HIS His 1025 Tyr Hi S 1040 Tyr Gi n 1055 Ang Tyr 1070 Pro Leu 1085 Pro Tyr 1100 ser Arg 1115 Glu 1015 Arg 1030 As n 1045 Arg 1060 Tyr 1075 Gi u 1090 Tyr 1105 A rg 1120 Leu GlU Trp cys Asp Leu Lys Asn Se r 1020 Gi n 1035 Val 1050 Arg 1065 Al a 1080 Val 1095 Gly 1110 Se r 1125 Tyr Trp Gi U Tyr Glu Se r Glu Arg Page 16 WO 03/083050 WO 03/83050PCT/US03/08590 usav2002-0022. ST2 5. txt Pro 1130 Al a 1145 Hi s 1160 Leu 1175 Leu 1190 LYS 1205 Glu 1220 ASP 1235 Arg 1250 A ng 1265 Arg 1280 ASP 1295 Ser 1310 Th r 1325 Leu Se r His Asn Lys LYS Ang ASP Se r Pro GiU Gi y Pro Al a Val Pro 1135 Val Gin ASP 1150 Lys Ser Val 1165 Asn ASP Gly 1180 Asp Thr Leu 1195 LYS Lys LYS 1210 His Gin Gin 1225 Leu His Arg 1240 Ang Lys Ser 1255 Lys Leu Sen 1270 Gly Ser Phe 1285 Pro Phe Ang 1300 Asp Arg Cys 1315 Leu Gl U Ang Hi S Leu Gi n G] U Lys 1140 Sen Sen His ser Leu Pro Giu Arg 1155 Lys Sen Arg Lys Ang Arg Tyr GlU 1170 Ang Leu Giu Lys Lys Val His Lys 1185 Giu Glu Pro Ang Val LYS Lys His 1200 Lys Sen Lys Asp Lys His Ang Asp 1215 Giu Sen Asp Phe Sen Gly Ala Tyr 1230 His Ang Lys Lys Lys Lys Lys Lys 1245 GlU Asp Phe Ilie Lys Asp Val Glu 1260 Sen Tyrl Glu Ala Gly Gly His Phe 1275 Leu Leu Ala Asp Gly Leu Pro Val 1290 Glu Lys Tr Lys His Leu Arg met 1305 Ang Leu Sen Glu Tyr Gly Gin Asp 1320 Page 17