EP1495117A2 - Gene humain de la protease de desubiquitination positionnee sur le chromosome 7 et son orthologue murin - Google Patents

Gene humain de la protease de desubiquitination positionnee sur le chromosome 7 et son orthologue murin

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Publication number
EP1495117A2
EP1495117A2 EP03723791A EP03723791A EP1495117A2 EP 1495117 A2 EP1495117 A2 EP 1495117A2 EP 03723791 A EP03723791 A EP 03723791A EP 03723791 A EP03723791 A EP 03723791A EP 1495117 A2 EP1495117 A2 EP 1495117A2
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European Patent Office
Prior art keywords
mdub7
hdub7
dub
ubiquitin
compound
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German (de)
English (en)
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EP1495117A4 (fr
Inventor
Chang Hahn
Hong Liu
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Aventis Pharmaceuticals Inc
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Aventis Pharmaceuticals Inc
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Application filed by Aventis Pharmaceuticals Inc filed Critical Aventis Pharmaceuticals Inc
Publication of EP1495117A2 publication Critical patent/EP1495117A2/fr
Publication of EP1495117A4 publication Critical patent/EP1495117A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • ubiquitin a 76-amino-acid-residue protein.
  • ubiquitin-protein ligation requires the sequential action of three enzymes.
  • the C-terminal Gly residue of ubiquitin is activated in an ATP -requiring step by a specific activating enzyme, El (Step 1).
  • This step consists of .an intermediate formation of ubiquitin adenylate, with the release of PP;, followed by the binding of ubiquitin to a Cys residue of El in a thiolester linkage, with the release of AMP.
  • ubiquitin is next transferred to an active site Cys residue of a ubiquitin-carrier protein, E2 (Step 2).
  • E2 a ubiquitin-carrier protein
  • 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).
  • the 26S proteasome complex that requires ATP hydrolysis for its action.
  • the 26S proteasome is formed by an ATP-dependent assembly of a 20S proteasome, a complex that contains the protease catalytic sites, with 19S "cap” or regulatory complexes.
  • the 19S complexes contain several ATPase subunits and other subunits that are presumably involved in the specific action of the 26S
  • Step 4 The action of the 26S proteasome presumably generates several types of products: free peptides, short peptides still linked to ubiquitin via their Lys residues, and polyubiquitin chains (Step 4). The latter two products .are converted to free and reusable ubiquitin by the action of ubiquitin-C-terminal 0 hydrolases or isopeptidases (Steps 5 and 6). Some isopeptidases may also disassemble certain ubiquitin-protein conjugates (Step 7) and thus prevent their proteolysis by the 26S proteasome.
  • 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 cell- cycle progression.
  • Cell growth and proliferation are further controlled by ubiquitin-mediated degradation of tumor suppressors, protoonco genes, and components of signal transduction systems.
  • the rapid degradation of numerous transcriptional regulators is involved in a variety 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 reticumm.
  • Dysfunction in several ubiquitin-mediated processes causes pathological conditions, including malignant transformation.
  • proteins such as the mammalian Gl regulators cyclin E and cyclin Dl, are targeted for ubiquitinylation by phosphorylation at specific, single sites.
  • phosphorylation at two specific sites, Ser-32 and Ser-36 is required for ubiquitin ligation.
  • ⁇ -cateinin which is targeted for ubiquitin-mediated degradation by phosphorylation, has a sequence motif similar to that of IkB ⁇ around these phosphorylation sites.
  • the homology in phosphorylation patterns of these two proteins is not complete, because phosphorylation of other sites of ⁇ -catenin is also required for its degradation.
  • 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 ubiquitin moieties.
  • 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.
  • the extensions described are ribosomal proteins consisting of 52 or 76-80 a ino acids. These ubiquitin fusion proteins are processed to yield ubiquitin and the corresponding C-terminal extension proteins.
  • 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.
  • deubiquitinating enzymes may 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 E1-, E2-, or E3 -ubiquitin thiolester intermediates.
  • 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.
  • 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 distinct substrates and are therefore involved in specific cellular processes. Although there is recent evidence to support such specificity of these deubiquitinating enzymes, the structure- function relationships of these enzymes remain poorly studied.
  • Deubiquitinating enzymes can be divided broadly on the basis of sequence homology 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).
  • UBP ubiquitin-specific processing protease
  • 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.
  • 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 ⁇ -sheet flanked on both sides by helices.
  • the ⁇ -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 184 .
  • 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.
  • UBP type 2 UCH enzymes are capable of cleaving the ubiquitin gene products and disassembling polyubiquitin chains after hydrolysis. It appears that there is a core region of about 450 amino acids delimited by CYS and HIS boxes. Many of these isoforms have N- terminal extensions and a few have C-terminal extensions.
  • DUB-1 cytokines induced in T-cells specific deubiquitinating enzymes
  • DUB-2 cytokine receptors for IL-3, IL-5, and GM-CSF
  • DUB-1 is induced by IL-2 as an immediate early (IE) gene that is down-regulated shortly after the initiation of stimulation. The function of this enzyme is also obscure. It may stimulate or inhibit the degradation of a critical cell-cycle regulator.
  • IE immediate early
  • Cytokines such as interleukin-2 (IL-2), activate intracellular signaling pathways via rapid tyrosine phosphorylation of their receptors, resulting in the activation of many genes involved in cell growth and survival.
  • the deubiquitinating enzyme DUB-2 is induced in response to IL-2 .and is expressed in human T-cell lymphotropic virus-I (HTLV-1)- transformed T cells that exhibit constitutive activation of the IL-2 JAK/STAT (signal transducers and activators of transcription) pathway, and when expressed in BaF3 cells DUB- 2 markedly prolonged IL-2-induced STAT5 phosphorylation.
  • HTLV-1 human T-cell lymphotropic virus-I
  • DUB-2 does not enhance IL-2-mediated proliferation, when withdrawn from growth factor, cells expressing DUB-2 had sustained STAT5 phosphorylation and enhanced expression of IL-2-induced genes cis .and c-myc. DUB-2 expression markedly inhibited apoptosis induced by cytokine withdrawal allowing cells to survive. Therefore, DUB-2 has a role in enhancing signaling through the JAK/STAT pathway, prolonging lymphocyte survival, and, when constitutively expressed, may contribute to the activation of the JAK/STAT pathway observed in some transformed cells. (Migone, T.-S., et al., Blood. 2001;98:1935-1941).
  • Protein ubiquitmation is an important regulator of cytokine-activated signal transduction pathways and hematopoietic cell growth. Protein ubiquitmation 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 of polymerase chain reaction amplification with degenerate primers for the DUB-2 complementary DNA, 3 murine bacterial artificial chromosome (BAG) clones that contain D UB gene sequences were isolated.
  • BAG murine bacterial artificial chromosome
  • DUB-2 A DUB gene with extensive homology to DUB-2.
  • DUB-2 A gene consists of 2 exons.
  • the predicted DUB-2 A protein is highly related to other DUBs throughout the primary amino acid sequence, with a hypervariable region at its C-terminus. h vitro, DUB-2 A had functional deubiquitinating activity; mutation of its conserved amino acid residues abolished this activity.
  • the 5' flanking sequence of the DUB-2 A gene has a hematopoietic-specific functional enhancer sequence.
  • Protein ubiquitmation also serves regulatory functions in the cell that do not involve proteasome-mediated degradation.
  • Hicke and Riezman have recently demonstrated ligand-inducible ubiquitmation of the Ste2 receptor in yeast. Ubiquitmation of the Ste2 receptor triggers receptor endocytosis and receptor targeting to vacuoles, not proteasomes.
  • Chenet al. have demonstrated that activation of the IB kinase requires a rapid, inducible ubiquitmation event. This ubiquitmation event is a prerequisite for the specific phosphorylation of LB and does not result in subsequent proteolysis of the kinase complex. The ubiquitmation of Ste2 and IB kinase appears reversible, perhaps resulting from the action of a specific deubiquitinating enzyme.
  • UBPs deubiquitinating enzymes
  • thiol-proteases that cleave either linear ubiquitin precursor proteins or post-translationally modified proteins containing isopeptide ubiquitin conjugates.
  • the large number of UBPs suggests that protein ubiquitmation, like protein phosphorylation, is a highly reversible process that is regulated in the cell.
  • 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.
  • DUB-1 a growth regulatory deubiquitinating enzyme, 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 IL-5, suggesting a specific role for the c subunit shared by these receptors.
  • IL-3 granulocyte macrophage-colony-stimulating factor
  • IL-5 a growth regulatory deubiquitinating enzyme
  • 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.
  • DUB-2 Hematopoietic-specific, cytokine induced DUBs in murine system have shown to prolong cytokine receptor, see Migone, T. S., 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, Y., et al., (1997).
  • DUB-2 is a member of a novel family of cytokine-inducible deubiquitinating enzymes, J Biol Chem 272, 51-7 and Zhu, Y., et al., (1996).
  • the murine DUB-1 gene is specifically induced by the betac subunit of interleukin-3 receptor, Mol Cell Biol 16, 4808- 17.). These effects are likely due to the deubiquitination of receptors or other signaling intermediates by DUB-1 or DUB-2, murine analogs of hDUBs. Inhibition of lDUBs may achieve downregulation of specific cytokine receptor signaling, thus modulating specific immune responses. Cytokines regulate cell growth by inducing the expression of specific target genes. A recently identified a cytokine-inducible, immediate-early gene, DUB-1, encodes a deubiquitinating enzyme with growth regulatory activity. In addition, a highly related gene, DUB-2, that is induced by interleukin-2 was identified.
  • the DUB-2 mRNA was induced in T cells as an immediate-early gene and was rapidly down-regulated. Like DUB-1, the DUB-2 proteinhad deubiquitinating activity in vitro. When a conserved cysteine residue of DUB-2, required for ubiquitin-specific thiol protease activity, was mutated to serine (C60S), deubiquitinating activity was abolished. DUB-1 and DUB-2 proteins are highly related throughout their primary amino acid sequence except for a hypervariable region at their
  • DUB genes co-localize to a region of mouse chromosome 7, suggesting that they arose by a tandem duplication of an ancestral DUB gene. Additional DUB genes co-localize to this region, suggesting a larger family of cytokine-inducible DUB enzymes.
  • cytokines induce specific DUB genes. Each induced DUB enzyme thereby regulates the degradation or the ubiquitmation state of an unknown growth regulatory factor, resulting in a cytokine-specific growth response.
  • additional members of the DUB subfamily can be identified in the GenBankTM. The highest degree of homology is in the Cys and His domains. Additionally, this putative human DUB protein contains a Lys domain (amino acids 400-410) and a hypervariable 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).
  • the DUB proteins may modify the ubiquitin- proteolytic pathway and thereby mediate specific cell growth or differentiation signals. These modifications are temporally regulated.
  • the DUB-2 protein for instance, is rapidly but tr-ansiently induced by IL-2. Interference of DUB enzymes with specific isopeptidase inhibitors may block specific cytokine signaling events.
  • the prior art teaches some partial sequences with homology to DUBs; specifically Human cDNA sequence SEQ ID NO: 17168 in EP 1074617- A2; a human protease and protease inhibitor PP -4 encoding cDNA; in WO200110903-A2 and human ubiquitin protease 23431 coding sequence in WO200123589-A2.
  • DUB-1 a deubiquitinating enzyme with growth-suppressing activity, Proc Natl Acad Sci U S A 93, 3275-9.
  • DUB-2 is a member of a novel family of cytokine-inducible deubiquitinating enzymes, J Biol Chem 272, 51-7.
  • 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.
  • MNB multivesicular body
  • Emr showed that the sorting of a vacuolar hydrolase into MNB vesicles requires the monoubiqutination of this cargo molecule at a specific lysine residue (Katzmann et al, 2001).
  • monoubiquitination is a green light for traffic to proceed from this important intracellular intersection to the lumen of the vacuole.
  • the policeman directing the traffic is an endosome-localized protein complex called ESCRT-I, one of whose components, Nps23, plays a key role in recognizing the cargo's ubiquitin signal (Katzmann et al., 2001).
  • Nps23 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 Nps23 that cripples ubiquitin-dependent sorting in the MNB pathway abolishes ubiquitin binding to ESCRT-I.
  • a model in which Nsp23 binds ubiquitin directly, while still inferential, received support from structural studies of a different UEN protein.
  • tsglOl the mammalian homolog of Nps23, known as tsglOl, is a tumor suppressor (Li and Cohen, 1996)
  • tsglOl the mammalian homolog of Nps23
  • FA Fanconi Anemia
  • 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- 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 IkB ⁇ kinase (IKK) require chains linked through Lys63, rather than the Lys48-chains that usually signal proteasomal proteolysis.
  • IKK IkB ⁇ kinase
  • Takl kinase is a downstream target of Lys63-chain signaling in the IKK activation pathway.
  • the assembly of these chains depends on an unusual UEV/E2 complex and a RING finger protein, Traf6 (Deng et al., 2000). (The RING finger defines a large E3 family.) Modification of Traf6 with a Lys63 -chain leads to the activation of Takl, which in turn phosphorylates IKK (Wang et al., 2001). Activated IKK then phosphorylates IkB ⁇ and triggers its tagging with Lys48-chains.
  • hDUB7 .and its murine ortholog, 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.
  • Protein ubiquitination targets selectively to proteasome degradation and/or provides facilitating protein localization.
  • 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 gettingking of vesicular proteins.
  • 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.
  • ENTH domain bind clathrin, and are required for endocytosis. EMBO J., 18:4383-4393.
  • 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.
  • 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 ortholog, n-amed as hDUB7 and mDUB7, respectively.
  • Both hDUB7 and its murine ortholog mDUB7 were identified by searching human and mouse genome databases using murine DUB-1 and DUB-2 sequences.
  • These genes (hDUB7 and mDUB7) share open reading frames (ORFs) that are 67% amino acid identity to each other, when gaps caused by deletion was not counted as mismatch, and exhibit 15% identity in nucleotide sequence.
  • hDUB7 and mDUB7 share 48% identity to murine DUBs, DUB1 and DUB2 within 297 amino acids core DUB sequences.
  • hDUB7 and mDUB7 genes share open reading frames that are greater than 92%o amino acid identity within 540 amino acids N-terminal ubiquitin protease domain (with 98.4%o identity within 313 amino acid core). These genes also exhibit 74% identity within 138 amino acids C-teraiinal conserved domain containing several putative nuclear localization sequences (NLSs) and stretchs of amino acid sequences that is known to possess transducing capacity (K-AKKm XSKKKKKSKDKHR and HRHKKKKKKKKRHSRK).
  • NLSs putative nuclear localization sequences
  • the present invention is also directed to a transducing peptide comprising an NLS or transducing sequence of l DUB7 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 KLAKKHKKSKKK-KKSKDKHR, HRHKKKKKKKKRHSRK, KKHKKSKKKKKSKDKHR, and H RKKKKKKKRHSRK.
  • the invention also comprsises a transducing peptide wherein the cargo molecule is a biologically active protein, therapeutically effective compound, antisense nucleotide, or test compound.
  • 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.
  • Manipulation of these gene products by small molecular compounds can (1) reduce inflammation by regulating proinflammatory cytokine signaling, (2) modulate autoimmune diseases by regulating cytokine receptor signaling that are critical for lymphocytes proliferation, and (3) immune over-reaction during infection using above mechanisms.
  • mDUBl U41636
  • mDUB2 U70368
  • mDUB2A AF393637
  • 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 cliromosome 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). 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.
  • accession number for this protein is BAB27190 and for nucleotide sequence is AK010801 (1485 nucleotide long capable of translating 487 amino acids run-on translation).
  • the gene has partial sequence with C terminal incomplete, hi 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 Arachne_Nov30 database (preliminary assembly of the mouse WGS reads based on an Nov 9th freeze of the WGS data) in Genbank. Putative genes from contig_70795 were annotated by GENSCAN prediction.
  • RNA preparation 1 ug of total RNA preparation in 100 ul of lxTaqMan RT Buffer Mix, 5.5mM MgCl 2 , 0.5 mM dNTPs, 2.5 uM Random Hexamers, 40 U RNAse inhibitor, 125U Multiscribe Reverse Transcriptase. Mix by pipeting up and down. Incubate 25°C for 10 minutes (annealing step), 48°C for 30 minutes (reverse transcription), and 95°C for 5 minutes (heat killing of the enzyme). The samples can be left at the machine at 4°C, or alternatively, can be stored at - 20°C Yield of cDNA synthesis can be measured by incorporation of small portion of radioactive dATP (or dCTP). Average efficiency for this protocol is between 60-80% of conversion of RNA to cDNA.
  • Primer-probe set used is as follow:
  • a lymph 516 A lymph 516
  • a prostate 350 A prostate 350
  • DUB is a deubiquitinating enzyme
  • 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.
  • GST glutathione S-transferase
  • Plasmids are co- transformed as indicated into MCI 061 Escherichia coli. Plasmid-bearing E. coli MCI 061 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.
  • mDUB-1 was originally cloned as an IL-3 -inducible immediate-early gene.
  • mDUB-2 was cloned as an IL-2-inducible immediate-early gene.
  • 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.
  • B cells 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.
  • CD8+ T cells including B cells, CD4+ T cells of Th-1 and Th-2 differentiation conditions as well as bulk CD4+ T cells.
  • hDUB4s and hDUB8s are members of a discrete subfamily of deubiquitinating enzymes that shows the strongest similarity to mDUB subfamily including mDUBl, mDUB2, and mDUB2A, called the DUB subfamily.
  • D UB subfamily members contain distinct structural features that distinguish them from other ubps. First, DUB subfamily members are comparatively small enzymes of approximately 500-550 amino acids. Second, DUB subfamily members share amino acid similarity not only in the Cys and His domains but also throughout their primary amino acid sequence. For instance, DUB proteins contain a lysine-rich region (Lys domain) and a HN 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 TRA ⁇ SFACFind, a dynamic programming method, see Heinemeyer, T., et al., "Expanding the TRA ⁇ SFAC database towards an expert system of regulatory molecular mechamsms" 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.

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Abstract

Selon l'invention, on a identifié des analogues humains et murins d'enzymes de désubiquitination, des protéases de désubiquitination à spécificité hématopoïétique, inductibles par cytokine, groupées sur le chromosome 7 et leurs régions régulatrices respectives. Le nucléotide ou les protéines codées par celui-ci peuvent être utilisés dans des analyses afin d'identifier les inhibiteurs de hDUB7 ou de mDUB7. L'invention concerne également des peptides de transduction comprenant NLS ou une séquence de transduction de hDUB7 ou mDUB7 liés à une molécule cargo ainsi que des méthodes permettant d'administrer une protéine bioactive, un composé efficace sur le plan thérapeutique, un nucléotide antisens ou un composé de test à une cellule en ajoutant, par voie exogène, un peptide de transduction à une cellule.
EP03723791A 2002-03-22 2003-03-21 Gene humain de la protease de desubiquitination positionnee sur le chromosome 7 et son orthologue murin Withdrawn EP1495117A4 (fr)

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GBGB0218518.9A GB0218518D0 (en) 2002-03-22 2002-08-09 Human deubiquitinating protease gene on chromosome 7 and its murine ortholog
PCT/US2003/008590 WO2003083050A2 (fr) 2002-03-22 2003-03-21 Gene humain de la protease de desubiquitination positionnee sur le chromosome 7 et son orthologue murin

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997006247A2 (fr) * 1995-08-09 1997-02-20 Dana Farber Cancer Institute Enzymes de desubiquitination regulant la croissance des cellules
WO2001010903A2 (fr) * 1999-08-09 2001-02-15 Incyte Genomics, Inc. Proteases et inhibiteurs de proteases
WO2001023589A2 (fr) * 1999-09-29 2001-04-05 Millennium Pharmaceuticals, Inc. Nouvelle ubiquitine protease humaine nommee 23431

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997006247A2 (fr) * 1995-08-09 1997-02-20 Dana Farber Cancer Institute Enzymes de desubiquitination regulant la croissance des cellules
WO2001010903A2 (fr) * 1999-08-09 2001-02-15 Incyte Genomics, Inc. Proteases et inhibiteurs de proteases
WO2001023589A2 (fr) * 1999-09-29 2001-04-05 Millennium Pharmaceuticals, Inc. Nouvelle ubiquitine protease humaine nommee 23431

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BAKER R T ET AL: "Identification, Functional Characterization, and Chromosomal Localization of USP15, a Novel Human Ubiquitin-Specific Protease Related to the UNP Oncoprotein, and a Systematic Nomenclature for Human Ubiquitin-Specific Proteases" GENOMICS, ACADEMIC PRESS, SAN DIEGO, US, vol. 59, no. 3, 1 August 1999 (1999-08-01), pages 264-274, XP004444843 ISSN: 0888-7543 *
JANS D A ET AL: "SIGNALS MEDIATING NUCLEAR TARGETING AND THEIR REGULATION: APPLICATION IN DRUG DELIVERY" MEDICINAL RESEARCH REVIEWS, NEW YORK, NY, US, vol. 18, no. 4, July 1998 (1998-07), pages 189-223, XP002922553 ISSN: 0198-6325 *
See also references of WO03083050A2 *

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AU2003230701A1 (en) 2003-10-13
AU2003230701B2 (en) 2008-06-05
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