EP1534825A2 - Analogues humains de genes de protease murine liberatrice d'ubiquitine - Google Patents

Analogues humains de genes de protease murine liberatrice d'ubiquitine

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Publication number
EP1534825A2
EP1534825A2 EP03711195A EP03711195A EP1534825A2 EP 1534825 A2 EP1534825 A2 EP 1534825A2 EP 03711195 A EP03711195 A EP 03711195A EP 03711195 A EP03711195 A EP 03711195A EP 1534825 A2 EP1534825 A2 EP 1534825A2
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Prior art keywords
hdub
dub
ubiquitin
polynucleotide
genes
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German (de)
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EP1534825A4 (fr
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Chang Hahn
Hong Liu
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Aventis Pharmaceuticals Inc
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Aventis Pharmaceuticals Inc
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Publication of EP1534825A4 publication Critical patent/EP1534825A4/fr
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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)

Definitions

  • 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 dose 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 2).
  • 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 Proteins ligated to polyubiquitin chains are usually degraded by the 26S proteasome complex that requires ATP hydrolysis for its action.
  • the 26S proteasome is formed by an ATP-dependent assembly of a 20S proteasome, a complex that contains the protease catalytic sites, with 19S "cap” or regulatory complexes.
  • the 19S complexes contain several ATPase subunits and other subunits that are presumably involved in the specific action of the 26S proteasome on ubiquitinylated proteins. The roles of ATP in the assembly of the 26S proteasome complex and in its proteolytic action are not understood.
  • the action of the 26S proteasome presumably generates several types of products: free peptides, short peptides still linked to ubiquitin via their Lys residues, and polyubiquitin chains (Step 4).
  • the latter two products are converted to free and reusable ubiquitin by the action of ubiquitin-C-terminal hydrolases or isopeptidases (Steps 5 and 6).
  • Some isopeptidases may also disassemble certain ubiquitin-protein conjugates (Step 7) and thus prevent their proteolysis by the 26S proteasome.
  • the latter type of isopeptidase action may have a correction function to salvage incorrectly 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 cell- cycle progression.
  • Cell growth and proliferation are further controlled by ubiquitin-mediated degradation of tumor suppressors, protooncogenes, and components of signal transduction systems.
  • the rapid degradation of numerous transcriptional regulators is involved in a variety 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.
  • 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 amino 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 1 , 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. J-n 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 and DUB-2 cytokines induced in T-cells specific deubiquitinating enzymes
  • DUB-1 is induced by stimulation of the cytokine receptors for IL-3, IL-5, and GM-CSF, suggesting a role in its induction for the ⁇ -common (betac) subunit of the interieukin 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.
  • DUB-1 arrests cells at Gi; therefore, the enzyme appears to regulate cellular growth via control of the G o -G ⁇ transition.
  • the catalytic conserved Cys residue of the enzyme is required for its activity.
  • DUB-2 is induced by IL-2 as an immediate early (IE) gene that is down-regulated shortly after the initiation of stimulation. The function of this enzyme is also obscure. It may stimulate or inhibit the degradation of a critical cell-cycle regulator.
  • 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 lympho tropic 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 STAT5 phosphorylation.
  • IL-2 interleukin-2
  • 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 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 of polymerase chain reaction amplification with degenerate primers for the DUB-2 complementary DNA, 3 murine bacterial artificial chromosome (BAC) clones that contain DUB gene sequences were isolated.
  • BAC 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.
  • DUB-2 A had functional deubiquitinating activity; mutation of its conserved amino acid residues abolished this activity.
  • the 5' flanking sequence of the DUB-2A gene has a hematopoietic-specific functional enhancer sequence.
  • Protein ubiquitination also serves regulatory functions in the cell that do not involve proteasome-mediated degradation.
  • 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.
  • 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.
  • UBPs deubiquitinating enzymes
  • ubiquitination like protein phosphorylation
  • 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 ⁇ l (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 interieukin- 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 hDUBs may achieve downregulation of specific cytokine receptor signaling, thus modulating specific immune responses.
  • DUB-1 cytokine-inducible, immediate-early gene
  • DUB-2 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.
  • the DUB-2 protein had deubiquitinating activity in vitro.
  • C60S serine
  • DUB-1 and DUB-2 proteins are highly related throughout their primary amino acid sequence except for a hypervariable region at their COOH terminus. Moreover, the DUB genes co-localize to a region of mouse chromosome 7, suggesting that they arose by a tandem duplication of an ancestral DUB gene. Additional DUB genes co-localize to this region, suggesting a larger family of cytokine-inducible DUB enzymes.
  • 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.
  • 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.
  • 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 transiently induced by IL-2. Interference of DUB enzymes with specific isopeptidase inhibitors may block specific cytokine signaling events.
  • Defensins constitute a major family of antimicrobial peptides in mammals. Depending on the distribution of the cysteines and the linkages of the disulfide bonds, human defensins can be divided into two categories: ⁇ -defensins, which can be found in granulocytes and in epithelial cells of the small intestine, and ⁇ -defensins, which are expressed by epithelial cells and leukocytes including macrophages. Some defensins are expressed constitutive manner in granulocytes and epithelial cells where as others are induces by either exposure to microbial pathogens or pro-inflammatory cytokines such as IL-1 ⁇ , TNF- ⁇ and interferon- ⁇ .
  • ⁇ -defensins may predate the a-defensin family during recent gene amplification since ⁇ -defensin cannot be detected even in many mammalians including cow.
  • Cow has at least 13 ⁇ -defensins but no ⁇ -defensin.
  • ⁇ -defensins contribute to early host defense against several bacterial and fungal pathogens, as an important mechanism of innate immune response.
  • the present invention is directed to analogs of murine DUBs, hematopoietic-specific, cytokine-inducible deubiquitinating proteases found as a cluster of genes on chromosomes 4 and 8 and respective regulatory regions.
  • novel human DUBs and four potential genes that express truncated form of DUBs not previously reported in public databases were identified by searching human genome database using murine DUB-1 and DUB-2 sequences. These genes share open reading frames (ORFs) that are 88 to 99% amino acid identity to each other, when gaps caused by deletion and N-terminal and/or C-terminal extension was not counted as mismatch, and exhibit approximately 50% identity to murine DUBs.
  • Eight of eleven ORFs generate a protein of 530 amino acids.
  • ORFs Two ORFs (hDUB8.3 and hDUB8.11) have internal in- frame deletions such that the genes are capable of generating 497 and 417 amino acid long polypeptides, respectively.
  • One ORF (hDUB4.5) exhibits extension at both 5' and 3' end of the ORF so that the gene is capable of expressing 574 amino acid long polypeptide.
  • this 5' extension results in specific pro-polypeptide sequence that can direct polypeptide targeting to the mitochondria.
  • the respective regulatory regions, putative promoters, of these genes also share close to 90% identity each other suggesting that their expression is coordinated.
  • two of these genes can be expressed under the control of separate promoters that can be controlled independently and expressing potentially distinctive protein products.
  • 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.
  • hDUB4.1 and hDUB4.2 Two of cluster genes (hDUB4.1 and hDUB4.2) possesses two distinctive promoter domains in front of their ORFs such that they can be regulated independently in their transcription potential.
  • the longer transcripts of these ORFs (called hDUB4.1a and hDUB4.2a) has 12 and 4 exons respectively and capable of generating 1016 and 1021 amino acid long polypeptides, respectively.
  • These polypeptides share C-terminal 530 amino acids with their shorter form that can be expressed separately from independent promoters (called hDUB4.1b and hDUB4.2b, respectively).
  • two other ORFs are capable of generating longer than 530 amino acid polypeptides (hDUB4.10 and hDUB4.11).
  • these two deduced polypeptides shares significant homology within portion of N-terminal portions (I added alignment file of these at the end of sequence file).
  • Three of the ORFs (hDUB4.5, 4.8, and 8.2) has N-terminal insertion that is typical for mitochondria targeting sequence. An alignment of these sequences is provide in the Tables.
  • the promoter sequences defined as upstream of initiation ATG of the ORF exhibit remarkable level of homology each other except that of hDUB4.1a.
  • the sequence identity among all promoter sequences except that of hDUB4.1a is approximately 90% in 2000 base pair span upstream of initiation ATG.
  • hDUB8.3 and 8.11 Two of the promoter sequences (hDUB8.3 and 8.11) have 334 nucleotides insertion at approximately 1000 base pair upstream of initiation ATG. Interestingly, hDUB8.3 and hDUB8.11 are the only ones with shorter ORFs due to the internal deletions. In addition to these ORFs, there are 5 ORFs that are capable of expressing polypeptides (hDUB4.4, hDUB4.9, hDUB8.2, hDUB8.9, and hDUB8.10) that share initiation codon with other 530 amino acid long polypeptides but terminate prematurely due to the in frame termination sequences.
  • hDUB8 genes are clustered with the defensin clusters within 2 Mb region in 8P23, implying that both acquisition and amplification are relatively recent event, perhaps during mammalian evolution. It is of interest that hDUB4 gene cluster is also in highly amplified cluster region of chromosome 4P16 that is yet to be assigned in chromosome location. These data suggest that hDUB4s and hDUB ⁇ s are within very dynamic region of the human chromosomes (both 4pl6 and 8p23) that are undergoing volatile amplifications. The data also suggest that expression of hDUB8 may also be coordinated in conjunction with defensins that are critical components of innate immune response and inflammation.
  • NM H0089 NM H0089
  • mDUB2A AF393637 DNA sequences were used to search against Ensembl entire "golden path" (as contigs) using Ensembl blast search engine (http://www.ensembl.org/perl/blastview). All three mDUBs have significant alignments with contig AC083981, AF252831, AF228730, AF252830, AC068974 on chromosome 8 with the high score above 2000 and the probability less than e-87.
  • exhaust search was performed using genomic aligned sequence to search against the "golden path" contigs. Two more contigs were found to have significant alignment that has probability less than e-100: one is AC074340 on chromosome 8 and the other is AC022770 on chromosome 4.
  • DNA sequences for contig AC083981, AF252831, AF228730, AF252830, AC068974, AC074340 and AC022770 were downloaded from Ensembl and gene annotation for each contig was performed using GenScan gene annotation program.
  • Genes having homolog with mDUBs were named in sequence based on their locations on chromosomes. For example, hDUB8.1 was derived from AF228730, 8.2, 8.3 were derived from AF252830, 8.5 were derived from AC074340, 8.6 were derived from AF252831, 8.7, 8.8 and 8.9 were derived from AC083981, and 8.10 and 8.11 were derived from AC068974.
  • hDUB4.1, 4.2, 4.3, 4.4, 4.5 were derived from AC022770 on chromosome 4.
  • ORFs Two ORFs (hDUB4.1 and hDUB4.2) are multi-exon ORFs that extend N-terminal part of polypeptides that shares minimal sequence identity. However, there is a conserved putative promoter sequences that encompass over 2,000 bases in the intron region proximal to the last exon that is conserved among all 5 genes. Three of the ORFs (hDUB4.5, 4.8, and 8.2) has N- terminal insertion that is typical for mitochondria targeting sequence. The hDUB genes cluster in 4P16 of the human chromosome, which is an unmapped part of the human chromosome.
  • hDUB gene clusters in chromosome 8 reveals that at least eleven ORFs in six different contigs (AC068974, AC074340, AC083981, AF228730, AF252830, and AF252831) were identified by nucleotide homology search with murine DUBl and 2. At least seven out of eleven ORFs share significant identities with similar length. There are conserved putative promoter sequences that encompass over 2,000 bases in all 11 genes.
  • the hDUB genes cluster in 8P23.1 of the human chromosome and clustered with defensin molecules (at lease 9 defensins are clustered with hDUB8s) and the whole domain belongs the olfactory GPCR cluster.
  • mDUB-2 The putative active site nucleophile of mDUB-2 is a cysteine residue (Cys "60 ) in the Cys domain.
  • Cys "60 The putative active site nucleophile of mDUB-2 is a cysteine residue (Cys "60 ) in the Cys domain.
  • mDUB-1 and mDUB-2 have a lysine rich region (Lys domain; amino acids 374-384 of mDUB-2) and a short hypervariable region (amino acids 385- 451 of mDUB-2), in which the mDUB-1 and mDUB-2 sequences diverge considerably.
  • the hypervariable (HV) region of mDUB-2 contains a duplication of the eight-amino acid sequence: PQEQNHQK.
  • 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 4.1 is unique for hDUB 4.1
  • Primer 4.2 covers hDUB 4.2, 4.3, 4.5 and 8.1
  • Primer 8.3 covers hDUB 8.3 and 8.11
  • Primer 8.5 is unique for hDUB 8.5
  • Primer 8.6 covers hDUB 8.6, 8.7 and 8.8 Table 1. Expression of hDUBs in PBMC stimulated with LPS (100 ng/ml) and PHA (5 ug/ml) for 7 hours.
  • T lymphocytes Donor 5
  • B lymphocytes Donor 6
  • Table 4 Expression of hDUB 4.2, 4.3, 4.5 and 8.1 examined by primer 4.2 in different human organ panel by TaqMan analysis.
  • CD19 (tonsillar - CD40L) 28.7 19.92 9.32 1.565 CD19 (tonsillar - LPS) 31.14 20.67 11.00 0.488
  • Monocyte INF-g (pool 1.5, 7, 34.62 17.27 17.88 0.004
  • Thl CD28/CD3 32.15 19.31 13.38 0.094
  • CD8 T cell a-CD3/CD28 4 hour 32.08 19.6 13.01 0.121
  • N-terminal primer 5'-atggaggacgactcactct-3' (19 mer)
  • C-terminal primer 5'-ctggcacacaagcaga-3' (19 mer)
  • Underlined triplet nucleotides in each primer represent translational initiation and termination codon.
  • This primer set can amplify most of hDUB4s and hDUB8s as well as potentially yet to be identified hDUBs that are similar enough to hDUB4s and hDUB8s due to the high homology in nucleotide sequences in this part of the ORF.
  • 1593 base pair fragment was successfully amplified from genomic DNA from two healthy human subjects and cloned into pCR2.1 vector and transformed into TOP10 strain of E coli. Over 300 independent clones with appropriate size insert were obtained and sequences are obtained by ABI automated DNA sequencers.
  • 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.
  • Ub-Met— gal is expressed from a pACYC184-based plasmid.
  • 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.
  • HDUB4s and hDUB8s are potential inflamatory cytokins specific Immediate-early Genes
  • 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.
  • inducibility as well as cell-type specific expression of these genes using multiple TaqMan analysis from human organ RNA samples and human immunocytes RNA samples.
  • Our data suggest that expression of hDUBs are not apparent in lymphocytes and granulocytes but high in fresh human PBMC from several donor. This strongly suggest that expression may be limited to the monocytes/macrophages and potentially NK cells.
  • hDUB4s and hDUB8s are upregulated in PBMC stimulated with stimuli (LPS and or PHA) that is known to upregulate various inflammatory cytokines such as TNF-alpha, IL-1 beta etc.
  • stimuli LPS and or PHA
  • TNF-alpha IL-1 beta
  • This increase of expression is almost completely disappeared 20 to 24 hours after stimulation suggesting this is an early gene.
  • the fact that there is only weak expression upregulation at 1.5 hours after stimulation suggests that stimuli by themselves may not upregulate hDUB4s and hDUB8s, but cytokines that are upregulated within couple of hours after stimulation may be responsible for upregulation of the hDUB4s and hDUB8s.
  • 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.
  • DUB 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 HV domain near their carboxyl terminus.
  • the regulatory regions, or promoter regions, of each of the DUBs was analyzed for putative transcription factor binding motifs using TRANSFACFind, a dynamic programming method, see Heinemeyer, T., et al., "Expanding the TRANSFAC database towards an expert system of regulatory molecular mechanisms” Nucleic Acids Res. 27, 318-322, (1999).
  • TRANSFACFind a dynamic programming method
  • Table 6 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 4.1a. The position is indicated by nucleotides.
  • Table 7 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 4.1b. The position is indicated by nucleotides.
  • Table 8 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 4.2a. The position is indicated by nucleotides.
  • Table 9 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 4.2b. The position is indicated by nucleotides.
  • Table 10 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 4.3. The position is indicated by nucleotides.
  • Table 11 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 4.4. The position is indicated by nucleotides.
  • Table 12 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 4.5. The position is indicated by nucleotides.
  • Table 13 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 8.1. The position is indicated by nucleotides.
  • Table 14 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 8.2. The position is indicated by nucleotides.
  • Table 15 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 8.3. The position is indicated by nucleotides.
  • Table 16 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 8.4. The position is indicated by nucleotides.
  • Table 17 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 8.5. The position is indicated by nucleotides.
  • Table 18 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 8.6. The position is indicated by nucleotides.
  • Table 19 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 8.7. The position is indicated by nucleotides.
  • Table 20 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 8.8. The position is indicated by nucleotides.
  • Table 22 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 8.10. The position is indicated by nucleotides.
  • Table 23 putative transcription factor binding motifs within the DUB regulatory or promoter, region of hDUB 8.11. The position is indicated by nucleotides.
  • DUB-2A a new member of the DUB subfamily of hematopoietic deubiquitinating enzymes, Blood 98, 636-42.
  • JAK2 is required for induction of the murine DUB-1 gene, Mol Cell Biol 17, 3364-72.
  • 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. Nucleotide sequence for hDUB4.
  • hDUB4.5 MRQRA HLKTLSEGIASFCNLRSQQKNLVILVPVDMEEDSLYLGGEWQFNHFSKLTSSRP 60 hDUB4.8 MRQRARHLKTLSEGIASCCKLRSQQKNLVILVPVDMEDDSLYLGGEWQFNHFSKLTSSRP 60 hDUB8.2 MRPESPSFED-SEEIASFCNLRSQPKNLVILVPGDMEDDSLYLGGEWQFNHFSKLTSSRP 59
  • CAGGGCTCCG TAGAACCACA GAATCTTGGG CGCAACCCTG CTCAAGCACC CAAATGTGCA TACGAACAGG GTCTCCGTGT GACGTGTGTGAAAA TCGGTTATGA GCATTCAAGC ACACCGATGC CCAGGTCCCG GCTGCAGGAA TAAGACCCTC CAGGGTCTTG TGTGAAGCCT CGGCATCTGC ATTGCTCATG CTTCTGGGGA TCATTCTCCT GAAAATGGTG GCTCCTTTCT CCCTGTGGAG CATCTTTCTA AGCAGTGCTC TTTTCTTCCC CCAGGACACT TTACATCCGG CACAGGAAGC CTTCTGATGG AGCACACCTG GCCCATGAAA AGACAAGGGA AACGGG GCCAAAGGTC ACAGTCCTCTCATCCCATCA TCCTCCTTAA AATCATCCTA ATTTCATGGG CCCTGAAG

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Abstract

L'invention concerne des analogues humains de protéases murines de libération de l'ubiquitine ('DUB'), inductibles par cytokine et spécifiques du système hématopoïétique, groupés sur les chromosomes 4 et 8 et leurs régions régulatrices respectives. Les nucléotides ou les protéines codées ainsi peuvent être utilisés dans des tests d'identification d'inhibiteurs des DUB humaines.
EP03711195A 2002-02-22 2003-02-20 Analogues humains de genes de protease murine liberatrice d'ubiquitine Ceased EP1534825A4 (fr)

Applications Claiming Priority (9)

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US35887502P 2002-02-22 2002-02-22
US35887302P 2002-02-22 2002-02-22
US358875P 2002-02-22
US358873P 2002-02-22
US36302002P 2002-03-08 2002-03-08
US363020P 2002-03-08
GBGB0208404.4A GB0208404D0 (en) 2002-03-08 2002-04-12 Human analogues of murine deubiquitinating protease genes
GB0208404 2002-04-12
PCT/US2003/005338 WO2003072724A2 (fr) 2002-02-22 2003-02-20 Analogues humains de genes de protease murine liberatrice d'ubiquitine

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WO2005049818A2 (fr) * 2003-11-14 2005-06-02 The Queen's University Of Belfast Gene
EP2049128A2 (fr) * 2006-05-12 2009-04-22 The Queen's University of Belfast Dub3 comme cible therapeutique du cancer

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
WO2000001817A2 (fr) * 1998-07-06 2000-01-13 Schering Corporation Genes mammiferes ; transporteur du type prostaglandine de cellules dendritiques (dc-pgt), hdtea84, hsljd37r et rankl, chimiokine hcc5, proteines de desubiquitination 11 et 12 (dub11, dub12), md-1, md-2 et cycline e2, reactifs apparentes et procedes associes
WO2005049818A2 (fr) * 2003-11-14 2005-06-02 The Queen's University Of Belfast Gene

Patent Citations (3)

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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
WO2000001817A2 (fr) * 1998-07-06 2000-01-13 Schering Corporation Genes mammiferes ; transporteur du type prostaglandine de cellules dendritiques (dc-pgt), hdtea84, hsljd37r et rankl, chimiokine hcc5, proteines de desubiquitination 11 et 12 (dub11, dub12), md-1, md-2 et cycline e2, reactifs apparentes et procedes associes
WO2005049818A2 (fr) * 2003-11-14 2005-06-02 The Queen's University Of Belfast Gene

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Title
BAEK K-H ET AL: "Essential regions of deubiquitinating enzyme activity and enhancer function for DUB-2A expressed in T-lymphocytes" ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, NEW YORK, US, US, vol. 430, no. 2, 15 October 2004 (2004-10-15), pages 191-197, XP004562711 ISSN: 0003-9861 *
BAEK KWANG-HYUN ET AL: "DUB-2A, a new member of the DUB subfamily of hematopoietic deubiquitinating enzymes" BLOOD, vol. 98, no. 3, 1 August 2001 (2001-08-01), pages 636-642, XP002356521 ISSN: 0006-4971 *
D'ANDREA A ET AL: "DEUBIQUITINATING ENZYMES A NEW CLASS OF BIOLOGICAL REGULATORS" CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, CRC PRESS, BOCA RATON, FL, US, vol. 33, no. 5, October 1998 (1998-10), pages 337-352, XP000943127 ISSN: 1040-9238 *
DATABASE EMBL [Online] 18 January 2004 (2004-01-18), "Homo sapiens deubiquitinating enzyme 3 (DUB3) mRNA, complete cds." XP002356525 retrieved from EBI accession no. EM_PRO:AY509884 Database accession no. AY509884 -& BURROWS JAMES F ET AL: "DUB-3, a cytokine-inducible deubiquitinating enzyme that blocks proliferation" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 279, no. 14, 2 April 2004 (2004-04-02), pages 13993-14000, XP002356519 ISSN: 0021-9258 *
LEE JIN-HIE ET AL: "Critical regions for deubiquitinating activity of DUB-2 expressed in T-lymphocytes" AMERICAN JOURNAL OF HEMATOLOGY, vol. 67, no. 4, August 2001 (2001-08), pages 270-272, XP009057991 ISSN: 0361-8609 *
MIGONE THI-SAU ET AL: "The deubiquitinating enzyme DUB-2 prolongs cytokine-induced signal transducers and activators of transcription activation and suppresses apoptosis following cytokine withdrawal" BLOOD, vol. 98, no. 6, 15 September 2001 (2001-09-15), pages 1935-1941, XP002356522 ISSN: 0006-4971 *
See also references of WO03072724A2 *
ZHU Y ET AL: "DUB-1, A DEUBIQUITINATING ENZYME WITH GROWTH-SUPPRESSING ACTIVITY" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 93, no. 8, April 1996 (1996-04), pages 3275-2379, XP002029887 ISSN: 0027-8424 *
ZHU YUAN ET AL: "DUB-2 is a member of a novel family of cytokine-inducible deubiquitinating enzymes" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 272, no. 1, 1997, pages 51-57, XP002169001 ISSN: 0021-9258 *
ZHU YUAN ET AL: "The murine DUB-1 gene is specifically induced by the beta-c subunit of the interleukin-3 receptor" MOLECULAR AND CELLULAR BIOLOGY, vol. 16, no. 9, 1996, pages 4808-4817, XP002356520 ISSN: 0270-7306 *

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AU2003215370A1 (en) 2003-09-09
EP1534825A4 (fr) 2006-02-01
AU2003215370B2 (en) 2008-06-12
CA2476770A1 (fr) 2003-09-04
BR0307895A (pt) 2006-01-17
IL163528A0 (en) 2005-12-18
WO2003072724A2 (fr) 2003-09-04
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