UBP43 (USP18) Specifically Removes ISG15 from Conjugated Proteinsстатья из журнала
Аннотация: UBP43 shows significant homology to well characterized ubiquitin-specific proteases and previously was shown to hydrolyze ubiquitin-β-galactosidase fusions in Escherichia coli. In our assays, the activity of UBP43 toward Ub fusions was undetectable in vitro directing us to investigate the possibility of Ub-like proteins such as SUMO, Nedd8, and ISG15 as probable substrates. We consequently demonstrate that UBP43 can efficiently cleave only ISG15 fusions including native ISG15 conjugates linked via isopeptide bonds. In addition to commonly used methods we introduce a new experimental design featuring ISG15-UBP43 fusion self-processing. Deletion of the UBP43 gene in mouse leads to a massive increase of ISG15 conjugates in tissues indicating that UBP43 is a major ISG15-specific protease. UBP43 is the first bona fide ISG15-specific protease reported. Both ISG15 andUBP43 genes are known to be strongly induced by interferon, genotoxic stress, and viral infection. We postulate that UBP43 is necessary to maintain a critical cellular balance of ISG15-conjugated proteins in both healthy and stressed organisms. UBP43 shows significant homology to well characterized ubiquitin-specific proteases and previously was shown to hydrolyze ubiquitin-β-galactosidase fusions in Escherichia coli. In our assays, the activity of UBP43 toward Ub fusions was undetectable in vitro directing us to investigate the possibility of Ub-like proteins such as SUMO, Nedd8, and ISG15 as probable substrates. We consequently demonstrate that UBP43 can efficiently cleave only ISG15 fusions including native ISG15 conjugates linked via isopeptide bonds. In addition to commonly used methods we introduce a new experimental design featuring ISG15-UBP43 fusion self-processing. Deletion of the UBP43 gene in mouse leads to a massive increase of ISG15 conjugates in tissues indicating that UBP43 is a major ISG15-specific protease. UBP43 is the first bona fide ISG15-specific protease reported. Both ISG15 andUBP43 genes are known to be strongly induced by interferon, genotoxic stress, and viral infection. We postulate that UBP43 is necessary to maintain a critical cellular balance of ISG15-conjugated proteins in both healthy and stressed organisms. interferon interferon-stimulated gene 15 kDa ubiquitin ubiquitin-like protein(s) ubiquitin-specific protease glutathione-S-transferase phosphate-buffered saline matrix-assisted laser desorption/ionization-time of flight N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine ISG15 is one of the most strongly induced genes after interferon (IFN)1 treatment (1Farrell P.J. Broeze R.J. Lengyel P. Nature. 1979; 279: 523-525Crossref PubMed Scopus (203) Google Scholar, 2Der S.D. Zhou A. Williams B.R. Silverman R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15623-15628Crossref PubMed Scopus (1544) Google Scholar) and is also significantly induced by influenza B virus (3Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (422) Google Scholar), lipopolysaccharide (4Li J. Peet G.W. Balzarano D. Li X. Massa P. Barton R.W. Marcu K.B. J. Biol. Chem. 2001; 276: 18579-18590Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), and genotoxic stress (5Memet S. Besancon F. Bourgeade M.F. Thang M.N. J. Interferon Res. 1991; 11: 131-141Crossref PubMed Scopus (45) Google Scholar).ISG15 was originally identified by Farrell et al.(1Farrell P.J. Broeze R.J. Lengyel P. Nature. 1979; 279: 523-525Crossref PubMed Scopus (203) Google Scholar) and later characterized by Knight and co-workers (6Blomstrom D.C. Fahey D. Kutny R. Korant B.D. Knight Jr., E. J. Biol. Chem. 1986; 261: 8811-8816Abstract Full Text PDF PubMed Google Scholar, 7Knight Jr., E. Fahey D. Cordova B. Hillman M. Kutny R. Reich N. Blomstrom D. J. Biol. Chem. 1988; 263: 4520-4522Abstract Full Text PDF PubMed Google Scholar). Subsequently, the sequence of ISG15 protein was noted to possess significant homology to a diubiquitin sequence, accounting for its cross-reactivity with affinity purified anti-ubiquitin antibodies (8Haas A.L. Ahrens P. Bright P.M. Ankel H. J. Biol. Chem. 1987; 262: 11315-11323Abstract Full Text PDF PubMed Google Scholar). Several reports demonstrate that ISG15 is released by various cell types and can act as cytokine leading to proliferation of NK cells (9Knight Jr., E. Cordova B. J. Immunol. 1991; 146: 2280-2284PubMed Google Scholar, 10D'Cunha J. Knight Jr., E. Haas A.L. Truitt R.L. Borden E.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 211-215Crossref PubMed Scopus (299) Google Scholar, 11D'Cunha J. Ramanujam S. Wagner R.J. Witt P.L. Knight Jr., E. Borden E.C. J. Immunol. 1996; 157: 4100-4108PubMed Google Scholar). Most remarkably, ISG15 was found to be conjugated to intracellular proteins via an isopeptide bond in a manner similar to ubiquitin (Ub), SUMO, and Nedd8 (12Loeb K.R. Haas A.L. J. Biol. Chem. 1992; 267: 7806-7813Abstract Full Text PDF PubMed Google Scholar). Conjugation of ubiquitin-like proteins (Ubls) involves a three-step mechanism whereby specific enzymes (or enzyme complexes) activate and covalently link Ubls to their substrates (13Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2944) Google Scholar, 14Ciechanover A. Orian A. Schwartz A.L. Bioessays. 2000; 22: 442-451Crossref PubMed Scopus (706) Google Scholar). Narasimhan et al. (15Narasimhan J. Potter J.L. Haas A.L. J. Biol. Chem. 1996; 271: 324-330Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) demonstrated that ISG15 conjugation occurs via a similar but distinct pathway compared with Ub conjugation. Yuan and Krug (3Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (422) Google Scholar) discovered that an activating enzyme for ISG15 is UBE1L. Although the role of Ub, Nedd8, and SUMO conjugation has been assessed in numerous studies (16Hochstrasser M. Science. 2000; 289: 563-564Crossref PubMed Scopus (102) Google Scholar, 17Hochstrasser M. Nat. Cell Biol. 2000; 2: E153-E157Crossref PubMed Scopus (370) Google Scholar, 18Yeh E.T. Gong L. Kamitani T. Gene (Amst.). 2000; 248: 1-14Crossref PubMed Scopus (420) Google Scholar, 19Jentsch S. Pyrowolakis G. Trends Cell Biol. 2000; 10: 335-342Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 20Callis J. Vierstra R.D. Curr. Opin. Plant Biol. 2000; 3: 381-386Crossref PubMed Scopus (170) Google Scholar), the biological significance of ISG15 modification remains unknown and the proteins that are targeted by ISG15 have not been identified. It is unknown whether ISG15 conjugates can be targeted to proteasomes in a way similar to Ub conjugates. Alternatively, ISG15 conjugation might antagonize binding of Ub and save proteins from degradation or modify biological activities of targeted proteins as is the case with Nedd8 and SUMO modification (16Hochstrasser M. Science. 2000; 289: 563-564Crossref PubMed Scopus (102) Google Scholar, 18Yeh E.T. Gong L. Kamitani T. Gene (Amst.). 2000; 248: 1-14Crossref PubMed Scopus (420) Google Scholar). Loeb and Haas (21Loeb K.R. Haas A.L. Mol. Cell. Biol. 1994; 14: 8408-8419Crossref PubMed Scopus (92) Google Scholar) demonstrated that a substantial amount of ISG15 conjugates are co-localized with intermediate filaments of the cytoskeleton. It is therefore possible that one of the physiological roles of ISG15 modification is re-organization of the cytoskeleton after IFN stimulation. ISG15 sequences have been reported in fish, chick, and mammals. Despite the presence of Nedd8 and SUMO analogs in yeast, ISG15 is absent from this unicellular eukaryote and, therefore, the ISG15 regulatory pathway is suggested to be a relatively recent functional divergence (22Potter J.L. Narasimhan J. Mende-Mueller L. Haas A.L. J. Biol. Chem. 1999; 274: 25061-25068Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Our search of fully sequenced genomes of a nematode (Caenorabditis), a plant (Arabidopsis), and an insect (Drosophila) also did not reveal a candidate for an ISG15 ortholog. It is therefore possible that the ISG15-conjugation system is restricted to higher animals with evolved IFN signaling. UBP43 (USP18 in standard nomenclature proposed by Baker et al. (23Baker R.T. Wang X.W. Woollatt E. White J.A. Sutherland G.R. Genomics. 1999; 59: 264-274Crossref PubMed Scopus (64) Google Scholar)) was initially cloned in our laboratory (24Liu L.Q. Ilaria Jr., R. Kingsley P.D. Iwama A. van Etten R.A. Palis J. Zhang D.E. Mol. Cell. Biol. 1999; 19: 3029-3038Crossref PubMed Scopus (132) Google Scholar, 25Schwer H. Liu L.Q. Zhou L. Little M.T. Pan Z. Hetherington C.J. Zhang D.E. Genomics. 2000; 65: 44-52Crossref PubMed Scopus (57) Google Scholar) and later, independently by three other groups; cDNA coding for porcine UBP43 was cloned in a differential screen from lung macrophages of virus-infected swine (26Zhang X. Shin J. Molitor T.W. Schook L.B. Rutherford M.S. Virology. 1999; 262: 152-162Crossref PubMed Scopus (73) Google Scholar), Li et al. (27Li X.L. Blackford J.A. Judge C.S. Liu M. Xiao W. Kalvakolanu D.V. Hassel B.A. J. Biol. Chem. 2000; 275: 8880-8888Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) identified mRNA for human UBP43 in a screen for RNase L substrates, and Kang et al. (28Kang D. Jiang H. Wu Q. Pestka S. Fisher P.B. Gene (Amst.). 2001; 267: 233-242Crossref PubMed Scopus (60) Google Scholar) cloned human UBP43 in a screen for genes induced by IFN in melanoma cell lines. Significant up-regulation of the UBP43 gene by viral infection (or double-stranded RNA), by IFN and lipopolysaccharide 2O. Malakhova, M. Malakhov, C. Hetherington, and D.-E. Zhang, submitted for publication. suggests that UBP43 might be involved in a number of processes including the control of cell proliferation, inflammation, stress, and immune response. Several regions in the UBP43 sequence exhibit homology to catalytic domains of Ub-specific proteases (USPs) which remove Ub from conjugated proteins. The USP family may include more than 100 proteins which differ in size and amino acid sequence, yet all share several highly homologous patches around the residues required for catalytic activity (29Wilkinson K.D. FASEB J. 1997; 11: 1245-1256Crossref PubMed Scopus (511) Google Scholar, 30Wilkinson K.D. Hochstrasser M. Peters J.M. Harris J.R. Finley D. Ubiquitin and the Biology of the Cell. Plenum Press, New York1998: 99-125Crossref Google Scholar, 31Chung C.H. Baek S.H. Biochem. Biophys. Res. Commun. 1999; 266: 633-640Crossref PubMed Scopus (157) Google Scholar, 32Yan N. Doelling J.H. Falbel T.G. Durski A.M. Vierstra R.D. Plant Physiol. 2000; 124: 1828-1843Crossref PubMed Scopus (110) Google Scholar). Using a conventional assay that involves co-transformation of two plasmids into Escherichia coli(first coding for USP enzyme and the second for Ub-β-galactosidase fusion as a substrate), UBP43 was demonstrated to have proteolytic activity against Ub fusion, and was thought to work on Ub conjugates (24Liu L.Q. Ilaria Jr., R. Kingsley P.D. Iwama A. van Etten R.A. Palis J. Zhang D.E. Mol. Cell. Biol. 1999; 19: 3029-3038Crossref PubMed Scopus (132) Google Scholar, 25Schwer H. Liu L.Q. Zhou L. Little M.T. Pan Z. Hetherington C.J. Zhang D.E. Genomics. 2000; 65: 44-52Crossref PubMed Scopus (57) Google Scholar). However, our subsequent experiments failed to detect activity of UBP43 toward Ub fusions in vitro. Similarity in the expression patterns of ISG15 and UBP43 genes lead us to hypothesize that ISG15 conjugates may be the preferred substrates for UBP43. We consequently investigated the ability of UBP43 to cleave ISG15 and other known Ub-like proteins (namely SUMO, Nedd8, and Ub) from artificial substrates. Here we demonstrate that UBP43, a member of the Ub-specific protease family, cleaves both artificial and native ISG15 conjugates. No other USPs were specific to ISG15. Therefore, UBP43 is the first ISG15-specific protease reported. Significantly, in vivomouse data also demonstrates that UBP43 is a major ISG15-specific protease and activity of this enzyme is crucial for proper cellular balance of ISG15-conjugated proteins. pcDNA6-UBP43 construct for expression UBP43-V5–6His fusion in mammalian cells has been previously described (25Schwer H. Liu L.Q. Zhou L. Little M.T. Pan Z. Hetherington C.J. Zhang D.E. Genomics. 2000; 65: 44-52Crossref PubMed Scopus (57) Google Scholar). Plasmids expressing the ubiquitin-specific proteases UBP41 (as GST fusion), UBP1 (33Tobias J.W. Varshavsky A. J. Biol. Chem. 1991; 266: 12021-12028Abstract Full Text PDF PubMed Google Scholar), Unp, and Unp(mut) (34Gilchrist C.A. Baker R.T. Biochim. Biophys. Acta. 2000; 1481: 297-309Crossref PubMed Scopus (22) Google Scholar) as well as pUb-GSTP1 (35Baker R.T. Smith S.A. Marano R. McKee J. Board P.G. J. Biol. Chem. 1994; 269: 25381-25386Abstract Full Text PDF PubMed Google Scholar) construct were provided by Dr. R. Baker (Australian National University, Canberra). pGEX-Nedd8-gsPESTc and pGEX-SUMO-gsPESTc plasmids were from Dr. K. Tanaka (The Tokyo Metropolitan Institute of Medical Science, Japan) (36Kawakami T. Suzuki T. Baek S.H. Chung C.H. Kawasaki H. Hirano H. Ichiyama A. Omata M. Tanaka K. J. Biochem. (Tokyo). 1999; 126: 612-623Crossref PubMed Scopus (16) Google Scholar). pGEX-ISG15-Rcap and pRSV-ISG17 plasmids were received from Dr. A. L. Haas (Medical Colledge of Wisconsin, Milwaukee, WI). To produce pGEX-ISG15-gsPESTc and pGEX-ISG17-gsPESTc expressing GST-ISG15-gsPESTc or GST-pro-ISG15-gsPESTc, respectively, the plasmid pGEX-Nedd8-gsPESTc was digested withBamHI and the excised Nedd8 was replaced withISG15 or ISG17 that had been PCR amplified from pGEX-ISG15-Rcap or pRSV-ISG17. To produce pET-Ub-gsPESTc plasmid expressing Ub-gsPESTc fusion the gsPESTc-coding sequence was excised from pGEX-Nedd8-gsPESTc using BamHI and EcoRI and cloned into pET-Ub-UBP43-H plasmid (see below) from which UBP43-H encoding sequence had been excised. Construction of pGEX-UBP43 (pGEX-4T-3-UBP43) has been previously described (24Liu L.Q. Ilaria Jr., R. Kingsley P.D. Iwama A. van Etten R.A. Palis J. Zhang D.E. Mol. Cell. Biol. 1999; 19: 3029-3038Crossref PubMed Scopus (132) Google Scholar). pET-S-UBP43-H was produced by cloning of the full-length UBP43 gene into pET29a vector (Novagen, Madison, WI). To generate a construct expressing GST-UBP43–6His the pET-S-UBP43-H was digested withSacI and SmaI restriction endonucleases to excise part of UBP43 together with the fused His6 tag. The resulting SacI-SmaI fragment was cloned into pGEX-UBP43(mut) digested with SacI andNotI (blunted). pET-Ub-UBP43-H and pET-ISG15-UBP43-H constructs expressing the fusions of Ub or ISG15 with UBP43 were produced by replacing the S-tag part in pET-S-UBP43-H (digested with NdeI andEcoR V) with Ub or ISG15 genes that had been PCR amplified from pUb-GSTP1 or pGEX-ISG15-Rcap plasmids. Inactive form of UBP43 was obtained by conversion of a critical cysteine residue (Cys61) into serine by site-directed mutagenesis of pBK/CMV-UBP43 plasmid (previously described (24Liu L.Q. Ilaria Jr., R. Kingsley P.D. Iwama A. van Etten R.A. Palis J. Zhang D.E. Mol. Cell. Biol. 1999; 19: 3029-3038Crossref PubMed Scopus (132) Google Scholar)) using the QuikChange kit (Stratagene, La Jolla, CA). To convert the constructs expressing the wild-type version of UBP43 into inactive forms, internal SacI-XbaI fragments of theUBP43 gene in the respective constructs were replaced withSacI-XbaI fragments of UBP43(mut) excised from pBK-CMV-UBP43(mut). All constructs generated in the course of this work were sequenced. The plasmids expressing Ubl-gsPESTc constructs were transformed into BL21(DE3) E. coli and the expression was induced with isopropyl-1-thio-β-d-galactopyranoside. GST-Nedd8-gsPESTc and GST-SUMO-gsPESTc fusions were purified on GSH-agarose (Sigma), digested with thrombin protease (Amersham Biosciences, Inc., Piscataway, NJ) while attached to GSH-agarose beads and the supernatants containing these Ubls-gsPESTc were directly used for labeling. GST-ISG15-gsPESTc fusion was purified on GSH-agarose and eluted with reduced glutathion. After dialysis GST-ISG15-gsPESTc was digested with thrombin protease and the solution containing both GST and ISG15-gsPESTc polypeptides used for labeling. Ub-gsPESTc fusion was purified by heat denaturation, (NH4)2SO4 precipitation and ion-exchange chromatography as described in Kawakami et al.(36Kawakami T. Suzuki T. Baek S.H. Chung C.H. Kawasaki H. Hirano H. Ichiyama A. Omata M. Tanaka K. J. Biochem. (Tokyo). 1999; 126: 612-623Crossref PubMed Scopus (16) Google Scholar). The Ubl-gsPESTc fusions were radiolabeled with Na125I (ICN, Costa Mesa, CA) using IODO-BEADS (Pierce, Rockford, IL) for 5 min in accordance with the manufacturer's instructions. The labeling conditions used resulted in almost exclusive labeling of gsPESTc extension. Expression of UBP43 inE. coli as either GST-UBP43 or UBP43–6His fusions produced less than 30% of full-length UBP43. Therefore the UBP43gene was expressed as GST-UBP43–6His fusion (both wild-type and mutant versions). One liter of BL21 E. coli culture transformed with pGEX-UBP43-H plasmid was grown in LB broth toA 600 = 0.8 and protein expression was induced with isopropyl-1-thio-β-d-galactopyranoside. After 3 h of induction at 21 °C, cells were harvested by centrifugation, washed with PBS, and lysed by sonication in PBS adjusted to 10 mm imidazole, 300 mm NaCl, 2 mmβ-mercaptoethanol, 2 mm phenylmethylsulfonyl fluoride. Lysate was cleared by 10 min centrifugation at 20,000 ×g and GST-UBP43–6His fusions were absorbed on 3 ml of Ni-NTA-agarose (Qiagen, Valencia, CA). After elution with PBS containing 300 mm NaCl, 250 mm imidazole, and 0.1% n-octyl β-d-glucopyranoside, the fusions were absorbed to 0.6 ml of GSH-agarose. The resin was washed with PBS, 0.1% n-octyl β-d-glucopyranoside, resuspended in 10 mm Tris, pH 7.5, 30% glycerol and stored at −20 °C until use. All purification procedures were performed at +4 °C. 2 × 106 of 293T cells were seeded on 60-mm cell culture plates and transfected with pcDNA6-UBP43 plasmids using Polyfect reagent (Qiagen) according to the manufacturer's instructions. Thirty-six hours after transfection cells were washed with PBS and harvested into 1 ml of lysis buffer (PBS adjusted to 10 mm imidazole, 0.1% n-octyl β-d-glucopyranoside, 300 mm NaCl, 2 mm β-mercaptoethanol, 2 mmphenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin) and then lysed by a 5-s pulse of sonication. Lysate was cleared by a 5-min centrifugation at 16,000 × g and UBP43-V5–6His fusions were absorbed to 40 μl of Ni-NTA-agarose for 1 h. Beads were washed once with 1 ml of PBS adjusted to 300 mm NaCl, 20 mm imidazole, and 0.1% n-octyl β-d-glucopyranoside, twice with the same buffer containing 4 m urea, and twice with the buffer with no urea. Beads were resuspended in 80 μl of 10 mm Tris, pH 7.5, 5 mm EDTA, 5 mm dithiothreitol, 20 mm imidazole, and 0.1% n-octyl β-d-glucopyranoside. The suspensions (15 μl per enzymatic reaction, 1 μg of total protein, and 0.1 μg of UBP43 fusion) were immediately used for enzymatic assay. Equal efficiency of expression, and purification between wild-type and mutant UBP43s was confirmed by Western blot with anti-V5 antibodies (Invitrogen). The peptide released from GST-proISG15-gsPESTc by UBP43 proteolytic activity was recovered in a trichloroacetic acid-soluble fraction as previously described (22Potter J.L. Narasimhan J. Mende-Mueller L. Haas A.L. J. Biol. Chem. 1999; 274: 25061-25068Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 37Woo S.K. Lee J.I. Park I.K. Yoo Y.J. Cho C.M. Kang M.S. Ha D.B. Tanaka K. Chung C.H. J. Biol. Chem. 1995; 270: 18766-18773Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). To remove buffer components, which interfere with mass spectroscopic analysis, the peptide was purified on C18-reverse phase matrix (Zip-Tip; Millipore, Bedford, MA). Matrix was equilibrated and washed with 1% (v/v) acetic acid in water and the peptide was eluted into 10 μl of 0.1% (v/v) acetic acid, 75% (v/v) acetonitrile in water. Linear matrix-assisted laser desorption/ionization (MALDI TOF)-mass spectrometry was performed by the Mass Spectrometry Core Facility of The Scripps Research Institute on Voyager-DE work station (PerSeptive Biosystems, Foster City, CA). To assess the ability of Ub-UBP43 and ISG15-UBP43 fusions to undergo self-hydrolysis,E. coli were transformed with pET-Ub-UBP43-H and pET-ISG15-UBP43-H carrying wild-type or inactive versions ofUBP43. Production of UBP43 in E. coli grown in rich media, such as LB, resulted in high yield but massive (more than 70%) degradation of UBP43. On the contrary, growth of E. coli in nutrient-poor medium M9 and induction of expression at low temperature, resulted in production of small quantities of full-length UBP43. Therefore, in this experiment, E. coli cultures were grown in 5 ml of M9 minimal medium to A 600 = 0.8 and induced with isopropyl-1-thio-β-d-galactopyranoside for 4 h at 21 °C. Cells were harvested, lysed by sonication, and after removal of insoluble material at 18,000 × gfor 5 min, supernatants were resolved on 10% SDS-PAGE, electroblotted, and then probed with antibodies against the NH2-terminal part of UBP43. Lungs were surgically removed from euthanized wild-type and UBP43−/−mice (3.5-week old) washed in cold PBS and kept on ice. For preparation of total protein extracts a pair of lungs was homogenized by sonication (four 8-s pulses) in 1 ml of 20 mm Tris, pH 7.5, 2 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma, product number P2714). Triton X-100 and β-mercaptoethanol were added to 1% and 2 mm, respectively, and suspensions were incubated on ice for 10 min. Particulate material was removed by centrifugation at 18,000 ×g for 5 min and supernatants were stored at −80 °C until use. For preparation of cytoskeleton-enriched fraction, lungs combined from three mice were homogenized using a tissue homogenizer in 5 ml of 20 mm Tris, pH 7.5, 2 mm EGTA, 100 mmNaCl, 2 mm MgCl2, 2 mmphenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin. Particulate was collected by centrifugation for 10 min at 16,000 ×g and thoroughly washed twice with 7 ml of the same buffer. The final pellet was then resuspended in 1 ml of 10 mmTris, pH 8.0, 5 mm EDTA, 100 mm NaCl, 5 mm β-mercaptoethanol, 2 mm CaCl2, and 0.5% n-octyl β-d-glucopyranoside. The suspension was then briefly sonicated and any insoluble material removed by 10 min centrifugation at 15,000 × g. Supernatant designated as “cytoskeleton-enriched fraction” was stored at −80 °C. Rabbit polyclonal IgGs against human ISG15 were kindly provided by Dr. E. Borden (Cleveland Clinic Foundation, OH) and were used at a final concentration of 0.5 μg/ml (11D'Cunha J. Ramanujam S. Wagner R.J. Witt P.L. Knight Jr., E. Borden E.C. J. Immunol. 1996; 157: 4100-4108PubMed Google Scholar). Rabbit anti-serum against Ub was purchased from Sigma and used at 1:300 dilution. For anti-UBP43 antibodies the NH2-terminal part (amino acids 1–120) of murine UBP43 was produced in E. coli as a GST fusion and purified on GSH-agarose. Immunization of rabbits with GST-UBP43(1–120) attached to GSH-agarose was performed at Charles River Pharmservices (Southbridge, MA). Total IgGs were purified on a protein A column (Amersham Biosciences, Inc.) and GST-specific antibodies were removed by passing the total IgG fraction through GST-agarose (Pierce). The resulting IgG fraction was subjected to purification of UBP43-specific antibodies on Affi-Gel resin (Bio-Rad, Hercules, CA) to which GST-UBP43 fusion protein was coupled. All chromatographic procedures were performed according to the instructions of the respective manufacturers. Proteins were electroblotted onto nitrocellulose membranes (Amersham Biosciences Inc.). Where indicated, membranes were stripped after the first blot in 50 mm Tris, pH 7.0, 2% SDS, and 50 mm dithiothreitol at 55 °C for 30 min. Chemiluminescence system was from PerkinElmer Life Sciences (Boston, MA). Despite significant homology of UBP43 to well characterized Ub-specific proteases and detection of activity in E. coli using Ub-β-galactosidase fusion protein (24Liu L.Q. Ilaria Jr., R. Kingsley P.D. Iwama A. van Etten R.A. Palis J. Zhang D.E. Mol. Cell. Biol. 1999; 19: 3029-3038Crossref PubMed Scopus (132) Google Scholar), we were not able to detect activity of UBP43 in vitro using a variety of Ub fusions. This fact prompted us to analyze activity of UBP43 toward other major Ubls, namely SUMO, Nedd8, and ISG15. A sensitive assay originally developed by Woo et al. (37Woo S.K. Lee J.I. Park I.K. Yoo Y.J. Cho C.M. Kang M.S. Ha D.B. Tanaka K. Chung C.H. J. Biol. Chem. 1995; 270: 18766-18773Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) was selected for this purpose. Each Ubl produced in E. coli had a cleavable carboxyl-terminal extension of the following sequence GSMHISPPEPESEEEEEHYC (referred to as gsPESTc) (36Kawakami T. Suzuki T. Baek S.H. Chung C.H. Kawasaki H. Hirano H. Ichiyama A. Omata M. Tanaka K. J. Biochem. (Tokyo). 1999; 126: 612-623Crossref PubMed Scopus (16) Google Scholar). To avoid covalent modifications to the backbones of Ubls caused by 125I attachment and possible artifacts in the assay we used conditions under which 125I exclusively labels the tyrosine in gsPESTc extension. As a consequence, Ubl-gsPESTc fusion (detectable when intact) after hydrolysis loses the labeled gsPESTc extension and appears as unlabeled and therefore undetectable on autoradiograms. gsPESTc due to its low molecular mass could be detected on Tris-Tricine (38Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar) but not on regular SDS-PAGE (39Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). As evidenced by disappearance of the ISG15-gsPESTc band and appearance of the gsPESTc band, UBP43 hydrolyzed only ISG15-gsPESTc and not the Ub-, Nedd8-, or SUMO-gsPESTc fusions (Fig. 1 A). The mutant version of UBP43, in which the cysteine residue critical for activity is converted to serine, was inactive toward any substrate. To confirm this observation with mammalian-expressed UBP43 we transfected 293T cells with either wild-type or mutant His6-tagged UBP43. The wild-type version of UBP43-H purified on Ni-agarose efficiently digested ISG15-gsPESTc fusion while mutated UBP43-H did not (Fig.1 B). Members of USP family including pro-ISG15 processing enzyme (22Potter J.L. Narasimhan J. Mende-Mueller L. Haas A.L. J. Biol. Chem. 1999; 274: 25061-25068Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) perform cleavage of substrates immediately after LRLRGG motif (identical in Ub and ISG15) yielding functional carboxyl-terminal diglycine available for conjugation. To confirm that hydrolysis catalyzed by UBP43 occurs at the same site, a larger processing reaction similar to that of Fig. 1 B was carried out on unlabeled GST-pro-ISG15-gsPESTc. As evidenced by band shift (Fig.1 C, inset), the incubation of GST-pro-ISG15-gsPESTc with wild-type UBP43-H resulted in removal of the carboxyl-terminal octapeptide extension with gsPESTc peptide (GTEPGGRSGSMHISPPEPESEEEEEHYC) leaving mature ISG15. The released peptide was recovered from the reaction by selective precipitation with trichloroacetic acid (22Potter J.L. Narasimhan J. Mende-Mueller L. Haas A.L. J. Biol. Chem. 1999; 274: 25061-25068Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 37Woo S.K. Lee J.I. Park I.K. Yoo Y.J. Cho C.M. Kang M.S. Ha D.B. Tanaka K. Chung C.H. J. Biol. Chem. 1995; 270: 18766-18773Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and its exact molecular mass was determined by MALDI analysis. The molecular mass obtained in this experiment (3059 Da; Fig. 1 C) corresponded with the predicted mass (3058.17 Da) of the released peptide suggesting that UBP43 cleaves ISG15 at the expected site following diglycine. The use of ISG15-gsPESTc fusion has never been reported before and UBP43 is the first enzyme to hydrolyze ISG15-gsPESTc. Therefore, to eliminate a possibility of experimental artifacts we tested a yeast (UBP1) and two mammalian USPs (UBP41 and Unp) in an identical assay. None of these three USPs could hydrolyze ISG15-gsPESTc (nor could they hydrolyze Nedd8- or SUMO-gsPESTc, not shown) while, as expected, all three efficiently hydrolyzed Ub-gsPESTc (Fig.2). The results presented in Figs.1 and 2 strongly suggest that UBP43 is an ISG15-specific protease that exhibits no proteolytic activity toward other Ubls. Detection of Ub specific activity in E. colico-transformed with Ub-β-galactosidase and UBP43 expressing constructs contradicted the ISG15-specific activity presented in Fig.1. In a standard co-transformation assay, Ub-β-galactosidase is expressed from low-copy number pACYC plasmid, whereas USPs are expressed from high-copy number plasmids. Such a difference in expression is likely to generate significant excess of enzyme over the substrate which may permit proteolytic activity on Ub fusions even though they are not preferred substrates. We therefore redesigned theE. coli assay to allow one copy of UBP43 to be synthesized per one copy of Ub or ISG15 by making Ub-UBP43 and ISG15-UBP43 fusions. Only ISG15-UBP43(wt) fusion could be efficiently hydrolyzed while expressed in E. coli (Fig. 3). No hydrolysis could be observed when ISG15-UBP43(mut) was expressed and, consistent with in vitro experiments, no hydrolysis was observed when either wild-type or mutated versions of UBP43 were fused to Ub. Use of artificial substrates such as Ub fusions (Ub-carboxyl-terminal extension proteins) in vitro is a commonly used technique, yet, even when an excess of enzyme is used and incubation times are extended, not all USPs that are able to hydrolyze Ub fusions are able to cleave an isopeptide bond, i.e. the bond between the Ub and the sid
Год издания: 2002
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: interferon and immune responses, Immunotherapy and Immune Responses, vaccines and immunoinformatics approaches
Другие ссылки: Journal of Biological Chemistry (PDF)
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PubMed (HTML)
Journal of Biological Chemistry (HTML)
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Открытый доступ: hybrid
Том: 277
Выпуск: 12
Страницы: 9976–9981