Kinetic Analysis of the Conjugation of Ubiquitin to Picornavirus 3C Proteases Catalyzed by the Mammalian Ubiquitin-protein Ligase E3α
2001; Elsevier BV; Volume: 276; Issue: 43 Linguagem: Inglês
10.1074/jbc.m102659200
ISSN1083-351X
AutoresT. Glen Lawson, Molly E. Sweep, Peter E. Schlax, Richard N. Bohnsack, Arthur L. Haas,
Tópico(s)Viral-associated cancers and disorders
ResumoThe 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3α. The conjugation of ubiquitin to these proteins requires internal ten-amino acid-long protein destruction signal sequences. To evaluate how these destruction signals modulate interactions that must occur between E3α and the 3C proteases, we have kinetically analyzed the formation of ubiquitin-3C protease conjugates in a reconstituted system of purified E1, HsUbc2b/E214Kb, and human E3α. Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with K m = 42 ± 11 µm and V max = 0.051 ± 0.01 pmol/min whereas the parameters for the ubiquitination of the hepatitis A virus 3C protease are K m = 20 ± 5 µm and V max = 0.018 ± 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3α conjugates ubiquitin to the altered 3C protease proteins. The K m andV max values for these reactions change proportionally in the same direction. These results suggest differences in rates of conjugation of ubiquitin to 3C proteases are primarily ak cat effect. Replacing specific encephalomyocarditis virus 3C protease lysine residues with arginine residues was found to increase, rather than decrease, the rate of ubiquitin conjugation, and the K m andV max values for these reactions are both higher than for the wild type protein. The ability of E3α to catalyze the conjugation of ubiquitin to both 3C proteases was found to be inhibited by lysylalanine and phenylalanylalanine, demonstrating that the same sites on E3α that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases. The 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3α. The conjugation of ubiquitin to these proteins requires internal ten-amino acid-long protein destruction signal sequences. To evaluate how these destruction signals modulate interactions that must occur between E3α and the 3C proteases, we have kinetically analyzed the formation of ubiquitin-3C protease conjugates in a reconstituted system of purified E1, HsUbc2b/E214Kb, and human E3α. Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with K m = 42 ± 11 µm and V max = 0.051 ± 0.01 pmol/min whereas the parameters for the ubiquitination of the hepatitis A virus 3C protease are K m = 20 ± 5 µm and V max = 0.018 ± 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3α conjugates ubiquitin to the altered 3C protease proteins. The K m andV max values for these reactions change proportionally in the same direction. These results suggest differences in rates of conjugation of ubiquitin to 3C proteases are primarily ak cat effect. Replacing specific encephalomyocarditis virus 3C protease lysine residues with arginine residues was found to increase, rather than decrease, the rate of ubiquitin conjugation, and the K m andV max values for these reactions are both higher than for the wild type protein. The ability of E3α to catalyze the conjugation of ubiquitin to both 3C proteases was found to be inhibited by lysylalanine and phenylalanylalanine, demonstrating that the same sites on E3α that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases. ubiquitin-activating enzyme ubiquitin carrier protein ubiquitin-protein ligase encephalomyocarditis virus hepatitis A virus polyacrylamide gel electrophoresis The selection of proteins for destruction by the ubiquitin 26 S/proteasome pathway depends upon specific interactions that occur between the targeted substrates and enzymes involved in the formation of the ubiquitin-target protein conjugates. A hierarchical family of pathways, each composed of at least three enzymes, accomplishes the attachment of ubiquitin to proteins destined to be degraded (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-497Crossref PubMed Scopus (6817) Google Scholar, 2Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2894) Google Scholar, 3Ciechanover A. Orian A. Schwartz A.L. Bioessays. 1999; 22: 442-451Crossref Scopus (694) Google Scholar). Common to all of these pathways is the ubiquitin-activating enzyme, E1,1 which recruits free ubiquitin through the ATP-dependent formation of a thiolester bond between a cysteine in the E1 and the C-terminal glycine of the ubiquitin molecule. This ubiquitin is then transferred to one of several members of the E2 family of proteins that are referred to as ubiquitin carrier proteins or ubiquitin-conjugating enzymes. Finally, the ubiquitin is transferred from the E2 to the target substrate protein through the action of an ubiquitin-protein ligase, or E3. Although each E2 protein appears to function with several specific ubiquitin-protein ligases, each E3 can specifically interact with only a limited number of substrate proteins. Regardless of the E3 involved in the ubiquitination process, following the conjugation of the first ubiquitin molecule to a primary amine on the substrate protein, the E3, or the E3 plus E2 proteins, can catalyze additional conjugating reactions that result in the synthesis of a chain of ubiquitin molecules attached to the substrate (4Finley D. Sadis S. Monia B.P. Boucher P. Ecker D.J. Crooke S.T. Chau V. Mol. Cell. Biol. 1994; 14: 5501-5509Crossref PubMed Scopus (301) Google Scholar). Important unanswered questions remain as to precisely how the E3 ubiquitin-protein ligases recognize and interact with their substrate proteins. It appears that proteins degraded by the ubiquitin/26 S proteasome system contain structural features, often short primary sequence elements (2Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2894) Google Scholar, 5Laney J.D. Hochstrasser M. Cell. 1999; 97: 427-430Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar), that act as protein destruction signals, and presumably it is these structural features that serve as sites for interaction with specific E3 proteins. Very few precisely mapped protein destruction signal structures have been matched with their cognate ubiquitin-protein ligase, however (2Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2894) Google Scholar). Among the most well studied E3 proteins are mammalian E3α, which functions in conjunction with the ubiquitin carrier protein HsUbc2 (E214K; see Refs.6Reiss Y. Heller H. Hershko A. J. Biol. Chem. 1989; 264: 10378-10383Abstract Full Text PDF PubMed Google Scholar and 7Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar), and the yeast homologue of E3α, Ubr1p, which requires the presence of yeast Ubc2p/Rad6 ubiquitin carrier protein (8Dohman R.J. Madura K. Bartel B. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7351-7355Crossref PubMed Scopus (203) Google Scholar, 9Xie Y. Varshavsky A. EMBO J. 1999; 18: 6832-6844Crossref PubMed Scopus (142) Google Scholar). E3α and Ubr1p were first shown to recognize proteins with N-terminal basic (type I) or bulky hydrophobic (type II) amino acids as substrates (10Bachmair A. Finley D. Varshavsky A. Science. 1986; 234: 179-186Crossref PubMed Scopus (1364) Google Scholar, 11Reiss Y. Kaim D. Hershko A. J. Biol. Chem. 1988; 263: 2693-2698Abstract Full Text PDF PubMed Google Scholar, 12Gonda D.K. Bachmair A. Wünning I. Tobias J.W. Lane W.S. Varshavsky A. J. Biol. Chem. 1989; 264: 16700-16712Abstract Full Text PDF PubMed Google Scholar, 13Reiss Y. Hershko A. J. Biol. Chem. 1990; 265: 3685-3690Abstract Full Text PDF PubMed Google Scholar, 14Baker R.T. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1090-1094Crossref PubMed Scopus (70) Google Scholar, 15Baker R.T. Varshavsky A. J. Biol. Chem. 1995; 270: 12065-12074Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (715) Google Scholar). Based on the affinity resin binding behavior of E3α (13Reiss Y. Hershko A. J. Biol. Chem. 1990; 265: 3685-3690Abstract Full Text PDF PubMed Google Scholar), measurements of the degradation rates of artificial substrates in reticulocyte lysate and in intact yeast cells (7Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar, 12Gonda D.K. Bachmair A. Wünning I. Tobias J.W. Lane W.S. Varshavsky A. J. Biol. Chem. 1989; 264: 16700-16712Abstract Full Text PDF PubMed Google Scholar), and in vitro and in vivo dipeptide competition studies (11Reiss Y. Kaim D. Hershko A. J. Biol. Chem. 1988; 263: 2693-2698Abstract Full Text PDF PubMed Google Scholar,13Reiss Y. Hershko A. J. Biol. Chem. 1990; 265: 3685-3690Abstract Full Text PDF PubMed Google Scholar, 14Baker R.T. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1090-1094Crossref PubMed Scopus (70) Google Scholar), it was proposed that these enzymes contain both type I and type II N-terminal amino acid binding sites (13Reiss Y. Hershko A. J. Biol. Chem. 1990; 265: 3685-3690Abstract Full Text PDF PubMed Google Scholar, 15Baker R.T. Varshavsky A. J. Biol. Chem. 1995; 270: 12065-12074Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (715) Google Scholar). These binding sites were assumed to provide the means by which substrate proteins are recognized by E3α and Ubr1p. In recent years it has been discovered that E3α and Ubr1p can catalyze the ubiquitination of proteins lacking destabilizing N-terminal amino acids (type III substrates). The short-lived yeast proteins Gpap and Cup9p, neither of which contains a destabilizing N-terminal amino acid, have been reported to be substrates for Ubr1p (17Madura K. Varshavsky A. Science. 1994; 265: 1454-1458Crossref PubMed Scopus (133) Google Scholar, 18Byrd C. Turner G.C. Varshavsky A. EMBO J. 1998; 17: 269-277Crossref PubMed Scopus (104) Google Scholar, 19Schauber C. Chen L. Tongaonkar P. Vega I. Madura K. Genes Cells. 1998; 3: 307-319Crossref PubMed Scopus (19) Google Scholar). Ribonuclease S, the subtilisn-derived fragment of ribonuclease A, has a stabilizing serine N terminus, but it is known to be a substrate for mammalian E3α-dependent ubiquitin-protein conjugate synthesis (20Baboshina O.V. Crinelli R. Siepmann T.J. Haas A.L. J. Biol. Chem. 2001; 276: 39428-39437Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). This indicates these E3 enzymes can recognize substrate proteins through associations with other types of structural elements. E3α has recently been found to catalyze the conjugation of ubiquitin to two additional proteins that, based upon their N-terminal amino acids, would not be predicted to be N-end rule substrates for degradation. The 3C proteases produced by the encephalomyocarditis virus (EMCV) and the hepatitis A virus (HAV), both members of the picornavirus family, have been shown to serve as substrates for E3α-dependent ubiquitination (21Gladding R.L. Haas A.L. Gronros D.L. Lawson T.G. Biochem. Biophys. Res. Commun. 1997; 238: 119-125Crossref PubMed Scopus (16) Google Scholar, 22Lawson T.G. Gronros D.L. Werner J.A. Wey A.C. DiGeorge A.M. Lockhart J.L. Wilson J.W. Wintrode P.L. J. Biol. Chem. 1994; 269: 28429-28435Abstract Full Text PDF PubMed Google Scholar, 23Lawson T.G. Gronros D.L. Evans P.E. Bastien M.C. Michalewich K.M. Clark J.K. Edmonds J.H. Graber K.H. Werner J.A. Lurvey B.A. Cate J.M. J. Biol. Chem. 1999; 274: 9871-9980Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 2M. E. Sweep, T. G. Lawson, and A. L. Haas, unpublished results.2M. E. Sweep, T. G. Lawson, and A. L. Haas, unpublished results. The ten-amino acid sequence 34LLVRGRTLVV43, located in what is probably a strand-turn-strand structure, has been discovered to function as a protein destruction signal in the EMCV 3C protease (22Lawson T.G. Gronros D.L. Werner J.A. Wey A.C. DiGeorge A.M. Lockhart J.L. Wilson J.W. Wintrode P.L. J. Biol. Chem. 1994; 269: 28429-28435Abstract Full Text PDF PubMed Google Scholar). The HAV 3C protease contains the sequence32LGVKDDWLLV41 in a location homologous to that of the EMCV protein destruction signal sequence, and this sequence has been shown to be required for the ubiquitination and degradation of the HAV 3C protein (21Gladding R.L. Haas A.L. Gronros D.L. Lawson T.G. Biochem. Biophys. Res. Commun. 1997; 238: 119-125Crossref PubMed Scopus (16) Google Scholar, 23Lawson T.G. Gronros D.L. Evans P.E. Bastien M.C. Michalewich K.M. Clark J.K. Edmonds J.H. Graber K.H. Werner J.A. Lurvey B.A. Cate J.M. J. Biol. Chem. 1999; 274: 9871-9980Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 3V. P. Losick, P. E. Schlax, R. E. Emmons, and T. G. Lawson, unpublished results.3V. P. Losick, P. E. Schlax, R. E. Emmons, and T. G. Lawson, unpublished results. The identification of two substrate proteins recognized by E3α, both of which contain precisely mapped, internal sequences known to be required for E3α-dependent ubiquitin conjugation, provides excellent model systems for detailed studies of the interactions that take place between E3α and substrate proteins lacking a destabilizing N-terminal amino acid. The recent development of an affinity chromatography purification method, based upon the specific binding of mammalian E3α to HsUbc2b, 4The HsUbc2b is identical in sequence to its rabbit ortholog (27Wing S.S. Dumas F. Banville D. J. Biol. Chem. 1992; 267: 6494-6501Abstract Full Text PDF Google Scholar) and is functionally indistinguishable from the “a” isoform (26Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar).4The HsUbc2b is identical in sequence to its rabbit ortholog (27Wing S.S. Dumas F. Banville D. J. Biol. Chem. 1992; 267: 6494-6501Abstract Full Text PDF Google Scholar) and is functionally indistinguishable from the “a” isoform (26Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). has made it possible to obtain sufficient quantities of pure E3α to allow biochemically defined kinetic studies of the E3α-catalyzed conjugation of ubiquitin to different types of substrate proteins (20Baboshina O.V. Crinelli R. Siepmann T.J. Haas A.L. J. Biol. Chem. 2001; 276: 39428-39437Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). We have used a reconstituted system of purified E1, HsUbc2b, and affinity-purified human E3α to evaluate the kinetics of the conjugation of ubiquitin to the EMCV and HAV 3C proteases. We have determined the K m and V maxvalues for the E3α-dependent conjugation of ubiquitin to the wild type 3C proteases and to 3C protease proteins containing mutations in their defined, internally located protein destruction signal regions. The kinetics with which EMCV 3C proteases containing selected lysine to arginine substitutions are ubiquitinated were also evaluated. Our results indicate that differences in the ability of the 3C proteases to serve as substrates for E3α are most likely the result of differences in the k cat values with which the catalysis of ubiquitin conjugation occurs. We also obtained data demonstrating that the interaction of E3α with the 3C protease proteins involves the same site, or sites, with which it associates with both basic and hydrophobic destabilizing N-terminal amino acids. Our findings explicate the ability of E3α to target non-N-end rule substrates for degradation and reveal that the mechanism by which the ubiquitin-protein ligase E3α selects substrate proteins for ubiquitination is considerably more complicated than suggested by earlier studies. The construction of the expression plasmids pETE3B′CD′*, pETHAV3C, and pETP3C have already been described (21Gladding R.L. Haas A.L. Gronros D.L. Lawson T.G. Biochem. Biophys. Res. Commun. 1997; 238: 119-125Crossref PubMed Scopus (16) Google Scholar, 22Lawson T.G. Gronros D.L. Werner J.A. Wey A.C. DiGeorge A.M. Lockhart J.L. Wilson J.W. Wintrode P.L. J. Biol. Chem. 1994; 269: 28429-28435Abstract Full Text PDF PubMed Google Scholar, 23Lawson T.G. Gronros D.L. Evans P.E. Bastien M.C. Michalewich K.M. Clark J.K. Edmonds J.H. Graber K.H. Werner J.A. Lurvey B.A. Cate J.M. J. Biol. Chem. 1999; 274: 9871-9980Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). pETE3CA38+, which contains the sequences coding for the EMCV 3C protease with an alanine inserted between amino acid positions 38 and 39, pETE3CL34A, pETE3CR39D, pETE3CK10,14R, pETE3CK74,77R, and pETE3CK98,101R were prepared using polymerase chain reaction-based oligonucleotide-directed mutagenesis. pE3C (23Lawson T.G. Gronros D.L. Evans P.E. Bastien M.C. Michalewich K.M. Clark J.K. Edmonds J.H. Graber K.H. Werner J.A. Lurvey B.A. Cate J.M. J. Biol. Chem. 1999; 274: 9871-9980Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) was employed as a template, and DNA insert fragments containing the mutated EMCV 3C protease coding sequences were synthesized using the appropriate mutagenic primer (CTTGCCTCCTTGTGAGAGGCGCCCGCACCTTGGTAGTTAATAG for pETE3CA38+, GAGGCCGCACCGCGGTAGTAAATAGACACATG for pETE3CL34A, and CTTCTTGTGAGAGGCGACACCTTGGTAGTAAATAG for pETE3CR39D). The inserts were ligated into pET3d at theNcoI and BamHI sites (24Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.E. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5991) Google Scholar). Likewise, the expression plasmids pETHAV3CA37+, which contains the sequences coding for the HAV 3C protease with an alanine inserted between amino acid positions 37 and 38, and pETHAV3CD37R were prepared using polymerase chain reaction. pHAV3C (21Gladding R.L. Haas A.L. Gronros D.L. Lawson T.G. Biochem. Biophys. Res. Commun. 1997; 238: 119-125Crossref PubMed Scopus (16) Google Scholar) was employed as a template, and DNA insert fragments containing the mutated HAV 3C protease coding sequences were synthesized using the appropriate mutagenic primer (CTTGGGAGTGAAAGATGATGGCTGGCTGCTTGTGCCTTC for pETHAV3CA37+, and CACAAGCAGCCAACGATCTTTCACTCCCAAG for pETE3CD37R). The inserts were ligated into pET3d. pETP3CN33R, which contains the sequences coding for a mutated poliovirus 3C protease, was constructed using pETP3C as a template. A DNA insert fragment containing the mutated poliovirus 3C protease coding sequence was synthesized using the mutagenic primer GGAGTCCACGACCGCGTGGCTATTTTACCAACC. The insert was ligated into pET3d at the NcoI and BamHI sites. Human erythrocyte fraction II was prepared using procedures described previously (25Hershko A. Heller H. Elias S. Ciechanover A. J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar). Human ubiquitin-activating enzyme E1 was purified from this material using a ubiquitin affinity column and fast protein liquid chromatography methods described previously (26Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Human recombinant HsUbc2b (27Wing S.S. Dumas F. Banville D. J. Biol. Chem. 1992; 267: 6494-6501Abstract Full Text PDF Google Scholar) was expressed inEscherichia coli and purified using methods reported previously (20Baboshina O.V. Crinelli R. Siepmann T.J. Haas A.L. J. Biol. Chem. 2001; 276: 39428-39437Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Some of this protein was employed in the preparation of an affinity column, which was then used to purify ubiquitin-protein ligase E3α from the fraction II preparation as reported recently (20Baboshina O.V. Crinelli R. Siepmann T.J. Haas A.L. J. Biol. Chem. 2001; 276: 39428-39437Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The wild type EMCV 3C and mutated (3C(+A38), 3C(L34A), 3C(R39D), 3C(K10,14R), 3C(K74,77R), and 3C(K98,101R)) protease proteins were expressed in E. coli from pETE3B′CD′*, pETE3C+A38, pETE3CL34A, and pETE3CR39D and were purified from refolded inclusion body material using procedures reported previously (22Lawson T.G. Gronros D.L. Werner J.A. Wey A.C. DiGeorge A.M. Lockhart J.L. Wilson J.W. Wintrode P.L. J. Biol. Chem. 1994; 269: 28429-28435Abstract Full Text PDF PubMed Google Scholar). Wild type HAV 3C protease was expressed in E. coli from pETHAV3C and purified as described previously (21Gladding R.L. Haas A.L. Gronros D.L. Lawson T.G. Biochem. Biophys. Res. Commun. 1997; 238: 119-125Crossref PubMed Scopus (16) Google Scholar). The mutated HAV 3C(+A37) and 3C(D37R) proteins were expressed from pETHAV3CA37+ and pETE3CD37R, but the presence of the mutations necessitated an altered purification scheme. Cleared lysates from induced cells were passed through a Q-Sepharose column (Amersham Pharmacia Biotech) equilibrated in TDE buffer (50 mm Tris-HCl, pH 7.6, 1 mm dithiothreitol, and 0.1 mm EDTA) containing 0.1 mm phenylmethylsulfonyl fluoride. The bound 3C protease proteins were eluted with a gradient of 300 to 650 mm NaCl in TDE buffer. The column fractions containing the 3C protease proteins were fractionated further and concentrated by precipitation with 30 to 50% (NH4)2SO4. The precipitates were resuspended in 10 mmKH2PO4-K2HPO4, pH 6.9, and applied to a column of Bio-Gel HTP hydroxyapatite (Bio-Rad) equilibrated in the same buffer. Bound material was eluted with a step gradient of 100 to 300 mmKH2PO4-K2HPO4, pH 7.2. The eluted proteins were dialyzed against TDE buffer containing 10% glycerol. Stock solutions of the 3C protease preparations were prepared to be ≤ 2 mg/ml. We have observed that at least some of the 3C proteins form aggregates, detectable by size exclusion chromatography, during long term storage at greater concentrations than this. All of the purified 3C protease proteins, with the exception of the wild type EMCV 3C protease, have an N-terminal methionine instead of the naturally occurring glycine or serine residues found in the mature proteins. The non-mutated EMCV 3C protease expressed in E. coli from pETE3B'CD'* undergoes self-processing to produce a protein with a glycine residue at the N terminus (22Lawson T.G. Gronros D.L. Werner J.A. Wey A.C. DiGeorge A.M. Lockhart J.L. Wilson J.W. Wintrode P.L. J. Biol. Chem. 1994; 269: 28429-28435Abstract Full Text PDF PubMed Google Scholar). For convenience, both non-mutated proteins are referred to here as wild type proteins. Bovine ubiquitin (Sigma) was purified further to apparent homogeneity (28Anchordoguy T.J. Hand S.C. J. Exp. Biol. 1995; 198: 1299-1305PubMed Google Scholar) and radioiodinated by the Chloramine T procedure (14Baker R.T. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1090-1094Crossref PubMed Scopus (70) Google Scholar). Some of this material was subjected to reductive methylation (29Hershko A. Heller H. Biochem. Biophys. Res. Commun. 1985; 128: 1079-1086Crossref PubMed Scopus (193) Google Scholar). The initial rates of 125I-ubiquitin conjugation were measured using an adaptation of methods employed previously (20Baboshina O.V. Crinelli R. Siepmann T.J. Haas A.L. J. Biol. Chem. 2001; 276: 39428-39437Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The reaction mixtures typically contained, in a final volume of 25 µl, 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 10 mm creatine phosphate, 2 mm ATP, 1 mm dithiothreitol, 1 international unit/ml creatine phosphokinase (Sigma), 1 international unit/ml high protein liquid chromatography-purified yeast inorganic pyrophosphate (Sigma), 50 nm purified E1, 500 nm purified HsUbc2b, 0.2 µg of affinity-purified E3α preparation, 0 to 20 µm exogenous protein substrates, and 4 µm125I-ubiquitin or 125I-methylated ubiquitin (about 12,000 cpm/pmol). The mixtures were incubated for 15 min at 37 ° and then boiled for 4 min in the presence of 25 µl of added sample buffer. This incubation time was selected to yield a linear initial rate of monoubiquitination. The samples were analyzed by 12% SDS-PAGE and autoradiography. The amounts of monoubiquitin-substrate protein conjugates formed were determined by cutting slices from the dried gels and subjecting them to γ-counting (30Haas A.L. Rose I.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6845-6848Crossref PubMed Scopus (106) Google Scholar). Control experiments confirmed that the initial rate was linear with [E3α]0 and independent of [E1]0 or [HsUbc2b]0 (20Baboshina O.V. Crinelli R. Siepmann T.J. Haas A.L. J. Biol. Chem. 2001; 276: 39428-39437Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Reaction rate data sets were generated by measuring the initial rates of monoubiquitinated conjugates produced in several simultaneously incubated reaction mixtures containing varying concentrations of 3C protease substrate. Two to four data sets were generated for each substrate protein using the same preparations of E1, HsUbc2b, and E3α. The rate versus substrate concentration data sets were simultaneously fit for each substrate to the K mvalue using a non-linear least squares regression analysis program (Sigma Plot 5.0). The V max values for each substrate were calculated by averaging the values derived from the fits for each data set. A coupled in vitro transcription-translation rabbit reticulocyte system (Promega) was employed to prepare35S-labeled poliovirus 3C and 3C(N33R) proteins encoded within pETP3C and pETP3CN33R. The ability of these proteins to serve as substrates for ubiquitination was evaluated by incubating 7 µl of transcription-translation reaction mixtures in a final volume of 20 µl containing 20 mm HEPES-KOH, pH 7.5, 1 mm dithiothreitol, 0.1 mm methylated ubiquitin, 0.1 mg/ml cycloheximide, and 60% by volume reticulocyte lysate containing an energy-generating system at 30 ° for 40 min (21Gladding R.L. Haas A.L. Gronros D.L. Lawson T.G. Biochem. Biophys. Res. Commun. 1997; 238: 119-125Crossref PubMed Scopus (16) Google Scholar, 22Lawson T.G. Gronros D.L. Werner J.A. Wey A.C. DiGeorge A.M. Lockhart J.L. Wilson J.W. Wintrode P.L. J. Biol. Chem. 1994; 269: 28429-28435Abstract Full Text PDF PubMed Google Scholar, 23Lawson T.G. Gronros D.L. Evans P.E. Bastien M.C. Michalewich K.M. Clark J.K. Edmonds J.H. Graber K.H. Werner J.A. Lurvey B.A. Cate J.M. J. Biol. Chem. 1999; 274: 9871-9980Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Aliquots of the reaction mixtures were analyzed by 12% SDS-PAGE and fluorography. Under appropriate conditions, kinetic analysis provides a sensitive and accurate means of quantifying enzyme-substrate interactions and the catalytic competence of the resulting Michaelis complex. We employed a biochemically defined, reconstituted N-end rule ubiquitin ligation system, comprised of affinity-purified human E1 and E3α and recombinant human HsUbc2b, to quantitatively evaluate the targeting of the EMCV and HAV 3C proteases for ubiquitin attachment and to assess the effect disrupting their respective destruction signal sequences has on their selection as N-end rule pathway substrates. A comparison of the labeled products generated by the reconstituted system with either α-lactalbumin or wild type EMCV 3C protease as the substrate is shown in the autoradiogram in Fig.1A, lanes 1–3. In the absence of a substrate protein, the otherwise complete assay mixture containing E1, HsUbc2b, and E3α catalyzes the synthesis of hyperconjugates to trace protein contaminants in the enzyme preparations, seen at the top of lane 1 (see Fig.1 A and Ref. 20Baboshina O.V. Crinelli R. Siepmann T.J. Haas A.L. J. Biol. Chem. 2001; 276: 39428-39437Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Monoubiquitin-HsUbc2b conjugates are also generated under these conditions, at the concentration of this enzyme employed to assure E3α-limiting conditions. In reaction mixtures in which α-lactalbumin was the substrate most of the products synthesized during the incubation period were high molecular mass polyubiquitin-α-lactalbumin conjugates, and the characteristic ladder of sequential ubiquitin adducts is apparent (Fig. 1 A,lane 2). The conjugation of ubiquitin to the EMCV 3C protease occurred at a markedly lower rate, and the majority of these products consisted of monoubiquitinated 3C protease (Fig.1 A, lane 3). The rate of monoubiquitin-3C protease synthesis was found to be linear for up to 15 min. An increasingly large fraction of the products consisted of polyubiquitinated 3C protease at longer times (data not shown). This suggests the attachment of the first ubiquitin molecule to the 3C protease occurs more slowly than subsequent polyubiquitinated conjugate synthesis. It should be noted that the synthesis of polyubiquitinated α-lactalbumin conjugates has been shown to also be linear with respect to time and E3α concentration in a reaction system very similar to the one used here (20Baboshina O.V. Crinelli R. Siepmann T.J. Haas A.L. J. Biol. Chem. 2001; 276: 39428-39437Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Because our goal in this study was to attempt to detect potentially subtle differences in the kinetics with which related substrate proteins are ubiquitinated by the same ubiquitin-protein ligase, we preferred to avoid artifacts that might result from the use of ubiquitin mutants or derivatives that do not support polyubiquitin chain synthesis. In addition, the rate of the first ubiquitin attachment is more likely than subsequent steps to reflect substrate recognition events mediated by E3α. To confirm that the formation of polyubiquitinated 3C protease was not a major event during the incubation time, reactions were carried out in which125I-methylated ubiquitin was used in place of125I-ubiquitin. Measurements of the
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