The SANT2 Domain of the Murine Tumor Cell DnaJ-like Protein 1 Human Homologue Interacts with α1-Antichymotrypsin and Kinetically Interferes with Its Serpin Inhibitory Activity
2004; Elsevier BV; Volume: 279; Issue: 12 Linguagem: Inglês
10.1074/jbc.m310903200
ISSN1083-351X
AutoresBarbara Kroczyńska, Christina M. Evangelista, Shalaka Samant, Ebrahim C. Elguindi, Sylvie Y. Blond,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoThe murine tumor cell DnaJ-like protein 1 or MTJ1/ERdj1 is a membrane J-domain protein enriched in microsomal and nuclear fractions. We previously showed that its lumenal J-domain stimulates the ATPase activity of the molecular chaperone BiP/GRP78 (Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) J. Biol. Chem. 275, 19620–19627). MTJ1/ERdj1 also contains a large carboxyl-terminal cytosolic extension composed of two tryptophan-mediated repeats or SANT domains for which the function(s) is unknown. Here we describe the cloning of the human homologue HTJ1 and its interaction with α1-antichymotrypsin (ACT), a member of the serine proteinase inhibitor (serpin) family. The interaction was initially identified in a two-hybrid screening and further confirmed in vitro by dot blots, native electrophoresis, and fluorescence studies. The second SANT domain of HTJ1 (SANT2) was found to be sufficient for binding to ACT, both in yeast and in vitro. Single tryptophan-alanine substitutions at two strictly conserved residues significantly (Trp-497) or totally (Trp-520) abolished the interaction with ACT. SANT2 binds to human ACT with an intrinsic affinity equal to 0.5 nm. Preincubation of ACT with nearly stoichiometric concentrations of SANT2 wild-type but not SANT2: W520A results in an apparent loss of ACT inhibitory activity toward chymotrypsin. Kinetic analysis indicates that the formation of the covalent inhibitory complex ACT-chymotrypsin is significantly delayed in the presence of SANT2 with no change on the catalytic efficiency of the enzyme. This work demonstrates for the first time that the SANT2 domain of MTJ1/HTJ1/ERdj1 mediates stable and high affinity protein-protein interactions. The murine tumor cell DnaJ-like protein 1 or MTJ1/ERdj1 is a membrane J-domain protein enriched in microsomal and nuclear fractions. We previously showed that its lumenal J-domain stimulates the ATPase activity of the molecular chaperone BiP/GRP78 (Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) J. Biol. Chem. 275, 19620–19627). MTJ1/ERdj1 also contains a large carboxyl-terminal cytosolic extension composed of two tryptophan-mediated repeats or SANT domains for which the function(s) is unknown. Here we describe the cloning of the human homologue HTJ1 and its interaction with α1-antichymotrypsin (ACT), a member of the serine proteinase inhibitor (serpin) family. The interaction was initially identified in a two-hybrid screening and further confirmed in vitro by dot blots, native electrophoresis, and fluorescence studies. The second SANT domain of HTJ1 (SANT2) was found to be sufficient for binding to ACT, both in yeast and in vitro. Single tryptophan-alanine substitutions at two strictly conserved residues significantly (Trp-497) or totally (Trp-520) abolished the interaction with ACT. SANT2 binds to human ACT with an intrinsic affinity equal to 0.5 nm. Preincubation of ACT with nearly stoichiometric concentrations of SANT2 wild-type but not SANT2: W520A results in an apparent loss of ACT inhibitory activity toward chymotrypsin. Kinetic analysis indicates that the formation of the covalent inhibitory complex ACT-chymotrypsin is significantly delayed in the presence of SANT2 with no change on the catalytic efficiency of the enzyme. This work demonstrates for the first time that the SANT2 domain of MTJ1/HTJ1/ERdj1 mediates stable and high affinity protein-protein interactions. The rough endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; ACT, α1-antichymotrypsin; BiP, immunoglobulin heavy chain-binding protein; HA, hemagglutinin; RSL, reactive site loop; SD, minimal synthetic dropout medium; Succ-AAPF-pNA, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; X-α-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. 1The abbreviations used are: ER, endoplasmic reticulum; ACT, α1-antichymotrypsin; BiP, immunoglobulin heavy chain-binding protein; HA, hemagglutinin; RSL, reactive site loop; SD, minimal synthetic dropout medium; Succ-AAPF-pNA, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; X-α-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. is the primary site for the synthesis and maturation of secreted and membrane proteins. At this site molecular chaperones and their associated enzymes promote the folding and assembly of newly synthesized polypeptides (1Lamande S.R. Bateman J.F. Semin. Cell Dev. 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In the yeast Saccharomyces cerevisiae BiP/Kar2p is assisted by at least three J-domain proteins: Sec63p, Jem1p, and Scj1p (12Nishikawa S.I. Fewell S.W. Kato Y. Brodsky J.L. Endo T. J. Cell Biol. 2001; 153: 1061-1070Crossref PubMed Scopus (255) Google Scholar, 14Scidmore M.A. Okamura H.H. Rose M.D. Mol. Biol. Cell. 1993; 4: 1145-1159Crossref PubMed Scopus (115) Google Scholar, 15Corsi A.K. Schekman R. J. Cell Biol. 1997; 137: 1483-1493Crossref PubMed Scopus (124) Google Scholar, 16Nishikawa S. Endo T. J. Biol. Chem. 1997; 272: 12889-12892Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 17Silberstein S. Schlenstedt G. Silver P.A. Gilmore R. J. Cell Biol. 1998; 143: 921-933Crossref PubMed Scopus (76) Google Scholar). The integral membrane protein Sec63p is an essential component of the ER membrane translocon (18Johnson A.E. van Waes M.A. Annu. Rev. Cell Dev. Biol. 1999; 15: 799-842Crossref PubMed Scopus (495) Google Scholar). Jem1p together with BiP/Kar2p are part of a nuclear fusion complex (19Nishikawa S. Endo T. Biochem. Biophys. Res. Commun. 1998; 244: 785-789Crossref PubMed Scopus (10) Google Scholar, 20Brizzio V. Khalfan W. Huddler D. Beh C.T. Andersen S.S. Latterich M. Rose M.D. Mol. Biol. Cell. 1999; 10: 609-626Crossref PubMed Scopus (37) Google Scholar), and Scj1p is likely to be involved in protein folding and assembly in the ER lumen (17Silberstein S. Schlenstedt G. Silver P.A. Gilmore R. J. Cell Biol. 1998; 143: 921-933Crossref PubMed Scopus (76) Google Scholar, 21Bies C. Guth S. Janoschek K. Nastainczyk W. Volkmer J. Zimmermann R. Biol. Chem. 1999; 380: 1175-1182Crossref PubMed Scopus (41) Google Scholar). Both Jem1p and Scj1p may also facilitate the retrotranslocation of lumenal ER-associated protein degradation substrates to the cytosol by preventing aggregation of misfolded polypeptides in the ER (12Nishikawa S.I. Fewell S.W. Kato Y. Brodsky J.L. Endo T. J. Cell Biol. 2001; 153: 1061-1070Crossref PubMed Scopus (255) Google Scholar). In mammals, five ER J-domain proteins have been identified so far, all of which stimulate BiP ATPase activity: MTJ1/ERdj1 (22Brightman S.E. Blatch G.L. Zetter B.R. Gene (Amst.). 1995; 153: 249-254Crossref PubMed Scopus (65) Google Scholar, 23Chevalier M. Rhee H. Elguindi E.C. Blond S.Y. J. Biol. Chem. 2000; 275: 19620-19627Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 24Dudek J. Volkmer J. Bies C. Guth S. Muller A. Lerner M. Feick P. Schafer K.H. Morgenstern E. Hennessy F. Blatch G.L. Janoscheck K. Heim N. Scholtes P. Frien M. Nastainczyk W. Zimmermann R. EMBO J. 2002; 21: 2958-2967Crossref PubMed Scopus (57) Google Scholar), the human Sec63 homologue/ERdj2 (25Skowronek M.H. Rotter M. Haas I.G. Biol. Chem. 1999; 380: 1133-1138Crossref PubMed Scopus (52) Google Scholar, 26Tyedmers J. Lerner M. Bies C. Dudek J. Skowronek M.H. Haas I.G. Heim N. Nastainczyk W. Volkmer J. Zimmerman R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7214-7219Crossref PubMed Scopus (124) Google Scholar), the Scj1 homologue or HEDJ/ERdj3 (21Bies C. Guth S. Janoschek K. Nastainczyk W. Volkmer J. Zimmermann R. Biol. Chem. 1999; 380: 1175-1182Crossref PubMed Scopus (41) Google Scholar, 27Yu M. Haslam R.H. Haslam D.B. J. Biol. Chem. 2000; 275: 24984-24992Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), ERdj4 (28Shen Y. Meunier L. Hendershot L.M. J. Biol. Chem. 2002; 277: 15947-15956Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), and ERdj5 (29Cunnea P.M. Miranda-Vizuete A. Bertoli G. Simmen T. Damdimopoulos A.E. Herman S. Leinonen S. Pelto Huikko M. Gustafsson J.A. Sitia R. Spyrou G. J. Biol. Chem. 2003; 278: 1059-1066Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). We previously showed that the lumenal J-domain of murine MTJ1 forms a stable complex with BiP and stimulates its ATPase activity at stoichiometric concentrations (23Chevalier M. Rhee H. Elguindi E.C. Blond S.Y. J. Biol. Chem. 2000; 275: 19620-19627Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). More recently, Zimmermann and colleagues (24Dudek J. Volkmer J. Bies C. Guth S. Muller A. Lerner M. Feick P. Schafer K.H. Morgenstern E. Hennessy F. Blatch G.L. Janoscheck K. Heim N. Scholtes P. Frien M. Nastainczyk W. Zimmermann R. EMBO J. 2002; 21: 2958-2967Crossref PubMed Scopus (57) Google Scholar) identified MTJ1 as having higher affinity for BiP than the more abundant Sec63 homologue in dog pancreas. The NH2-terminal of MTJ1 that carries the J-domain is followed by one transmembrane helix and a cytosolic carboxyl-terminal domain composed of a spacer region that possesses homology with Sec63p flanked by two tryptophan-mediated repeats or SANT domains for which no function has been attributed (23Chevalier M. Rhee H. Elguindi E.C. Blond S.Y. J. Biol. Chem. 2000; 275: 19620-19627Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 24Dudek J. Volkmer J. Bies C. Guth S. Muller A. Lerner M. Feick P. Schafer K.H. Morgenstern E. Hennessy F. Blatch G.L. Janoscheck K. Heim N. Scholtes P. Frien M. Nastainczyk W. Zimmermann R. EMBO J. 2002; 21: 2958-2967Crossref PubMed Scopus (57) Google Scholar). SANT domains are structurally related to the Myb DNA-binding domain (30Aasland R. Stewart A.F. Gibson T. Trends Biochem. Sci. 1996; 21: 87-88Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 31Hanaoka S. Nagadoi A. Yoshimura S. Aimoto S. Li B. de Lange T. Nishimura Y. J. Mol. Biol. 2001; 312: 167-175Crossref PubMed Scopus (47) Google Scholar) and are present at one to five copies in a variety of transcription factors or regulators including Swi3, Ada2, N-CoR, TFIIIB (30Aasland R. Stewart A.F. Gibson T. Trends Biochem. Sci. 1996; 21: 87-88Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 32Andres M.E. Burger C. Peral-Rubio M.J. Battaglioli E. Anderson M.E. Grimes J. Dallman J. Ballas N. Mandel G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9873-9878Crossref PubMed Scopus (358) Google Scholar, 33Sterner D.E. Wang X. Bloom M.H. Simon G.M. Berger S.L. J. Biol. Chem. 2002; 277: 8178-8186Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 34Humphrey G.W. Wang Y. Russanova V.R. Hirai T. Qin J. Nakatani Y. Howard B.H. J. Biol. Chem. 2001; 276: 6817-6824Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) or components involved in chromatin remodeling (35Boyer L.A. Langer M.R. Crowley K.A. Tan S. Denu J.M. Peterson C.L. Mol. Cell. 2002; 10: 935-942Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 36Yu J. Li Y. Ishizuka T. Guenther M.G. Lazar M.A. EMBO J. 2003; 22: 3403-3410Crossref PubMed Scopus (139) Google Scholar). Besides these reports, very little is known about the function of SANT-containing proteins. In the present study, we have cloned the human homologue of MTJ1, which we call HTJ1, and used its COOH-terminal domain as bait in a two-hybrid screening of a human liver cDNA library. We have identified α1-antichymotrypsin (ACT), a member of the serpin family, as a potential interacting candidate. Targeted mutagenesis indicates that the second SANT domain of HTJ1 (SANT2) mediates the interaction with ACT and that this interaction can be totally disrupted by a tryptophan to alanine single mutation (W520A). SANT2-ACT interaction was confirmed in vitro using purified proteins. ACT once bound to SANT2 was found to have no inhibitory activity toward chymotrypsin. SANT2 significantly slows down the kinetic of formation of the ACT-chymotrypsin acyl complex with no significant effect on the catalytic efficiency of the proteinase. This report documents for the first time that the human homologue of MTJ1/ERdj1 can mediate high affinity protein-protein interactions through its cytosolic domain. In another report, we describe that HTJ1 also interacts with a member of the inter-α-trypsin inhibitor and protects it from being processed by its natural protease. 2B. Kroczynska, L. Yang, H. Lu, and S. Y. Blond, submitted for publication. 2B. Kroczynska, L. Yang, H. Lu, and S. Y. Blond, submitted for publication. Our data suggests that HTJ1/ERdj1 may play a role in the secretion of protease inhibitors in mammalian cells. Materials, Antibodies, Proteins, and Strains—All components of the two-hybrid system, including cloning vectors pGBKT7 and pACT2, human liver cDNA library, S. cerevisiae strain AH109, anti-c-Myc monoclonal antibody, anti-HA tag polyclonal antibody, X-α-gal, dropout supplements, and cobalt-chelating resin Talon were obtained from Clontech. Bacterial vector pUC19 was from New England BioLabs, Beverly, MA. pET15b plasmid and the BL21(DE3) codon+ strain were from Novagen, Madison, WI. Pfu polymerase was from Stratagene, La Jolla, CA. All restriction enzymes and T4 DNA ligase were from MBI Fermentas, Amherst, NY. The ECL kit was from Amersham Biosciences. Human plasma ACT was from Calbiochem-Novabiochem Corp., San Diego, CA. Bovine pancreas chymotrypsin, and its substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Succ-AAPF-pNA) were from Sigma. Monoclonal anti-α1-antichymotrypsin antibody was obtained from Fitzgerald Industries International, Inc. Concord, MA. Cloning of HTJ1 cDNA—A nucleotidic sequence from a 10-week-old human embryo (AK027263, GI: 14041830) coding for a putative MTJ1 homologue was used as a template to design the set of primers for the amplification of a human liver cDNA library. The forward primer (5′-ATGACGGCTCCTTGCTCCCAG-3′) and reverse primer (5′-TCAGCTTTTAGCTTGTTTTTTCTTTTGGACC-3′) were used in PCR with Pfu polymerase. The PCR product (1700 bp) was then subcloned into the SmaI site of pUC19. The construct (pUCHTJ1) was sequenced on both strands and sequence analysis was carried out using BLAST sequence analysis software. Yeast and Bacterial Plasmids—cDNA coding for HTJ1, full-length ACT, and fragments were amplified by PCR using the forward and reverse primers listed in Table I. PCR products were subcloned in the yeast pGBKT7 plasmid, in-frame with the NH2-terminal GAL4 DNA-binding domain and the c-Myc epitope tag (pGBKT7 constructs, Table I). A number of PCR products containing varying lengths of HTJ1 and full-length ACT cDNA were cloned in pET15b Escherichia coli expression vector in-frame with a NH2-terminal hexahistidine tag (p(His)6 constructs, Table I). Finally two PCR products of ACT coding the ACT344–400 and ACT35–343 fragments were fused to the NH2-terminal GAL4 activation domain and the HA epitope tag of the yeast pACT2 plasmid (pACT2 constructs, Table I). The clone pGBKT7/HTJ1242–493 was generated by digestion of pGBKT7/HTJ1242–554 with PstI and religation. The HTJ1493–554:W497A mutant was engineered using PCR and a forward mutagenic primer (Table I). The HTJ1493–554:W520A mutant was created using the QuikChange site-directed mutagenesis kit (Stratagene) and two overlapping mutagenic primers (Table I). The construct pGBKT7/HTJ1493–554:W497A was used as a template to generate the double mutant pGBKT7/HTJ1493–554:W497A,W520A. The constructs pGBKT7/HTJ1493–554, pGBKT7/HTJ1493–554:W497A, pGBKT7/HTJ1493–554:W520A, and pGBKT7/HTJ1493–554:W497A,W520A were digested with NdeI and XhoI and the inserts were ligated into pET15b to generate p(His)6HTJ1493–554, p(His)6HTJ1493–554:W497A, p(His)6HTJ1493–554:W520A, and p(His)6HTJ1493–554:W497A,W520A, respectively. All constructs were sequenced.Table IPrimer sequences and templates for PCR, and restriction sites for cloning Restriction sites are underlined; positions mutated are in boldface.Forward primerReverse primerRestriction sitesConstruct nameTemplate5′-CATGCCATGGATCCAGGATGCT GGGCAGTTTTATGC-3′5′-TCAGCTTTTAGCTTGTTTTTTCTTTTGGACC-3′Ncol-SmalpGBKT7/HTJ1242-554pUCHTJ15′-CATGCCATGGATCCAGGATGCTGGGCAGTTTTATGC-3′5′-CGGGATCCGATCATGTCATCGGGCAAGGTG-3′Ncol-BamHIpGBKT7/HTJI242-411pUCHTJI5′-GGAATTCCATATGGCAGAGGAGCCGTGGACTCAAAATC-3′5′-CCGCTCGAGTCAGCTTTTAGCTTGTTTTTTCTTTTGGACCAG-3′Ndel-SmaIpGBKT7/HTJI493-554pUCHTJI5′-GGAATTCCATATGGCAGAGGAGCCGGCGACTCAAAATC-3′5′-CCGCTCGAGTCAGCTTTTAGCTTGTTTTTTCTTTTGGACCAG-3′Ndel-SmaIpGBKT7/HTJI493-554:W497ApGBKT7/HTJI493-5545′-CTCTGACCGCGCGGACAAAATAG-3′5′-CTATTTTGTCCGCGCGGTCAGAG-3′NAaNA, not applicablepGBKT7/HTJI493-554:W520ApGBKT7/HTJI493-5545′-CTCTGACCGCGCGGACAAAATAG-3′5′-CTATTTTGTCCGCGCGGTCAGAG-3′NApGBKT7/HTJI493-554W497A:W520ApGBKT7/HTJI493-554:W497A5′-GGAATTCCATATGGCGCGCGGCTGGGAGAGCGGAGAC-3′5′-TTTTCTCACCCGCCTGTAGTAGAATAC-3′Ndel-SmaIpGBKT7/J-HTJI45-148pUCHTJ15′-GGAATTCCATATGATCCAGGATGCTGGGCAGTTTTATGC-3′5′-CCGCTCGAGTCAGCTTTTAGCTTGTTTTTTCTTTTGGACCAG-3′NdeI-XhoIp(His)6-HTJI242-554pUCHTJI5′-GGAATTCCATATGGTGACAGATGTGACAACCAAAGC-3′5′-CCGCTCGAGAGACCGAGCTCTCTCTTTTCTC-3′NdeI-XhoIp(His)6-HTJI378-492pUCHTJI5′-GGAATTCCATATGATCCAGGATGCTGGGCAGTTTTATGC-3′5′-CCGCTCGAGAGATCGACCCAATTCGTGGGCAATC-3′NdeI-XhoIp(His)6-HTJI242-177pUCHTJI5′-GGAATTCCATATGCACCCTTAACAGCCCACTTGACGAG-3′5′-CGGGATCCCTAGGCTTGCTTGGGATTGGTG-3′NdeI-BamH1p(His)6-ACT1-400cDNA human liver library5′-CGGGATCCGAGAGGAGGGCACAGAAGCATC-3′5′-CGGAATTCTTACTAGGCTTGCTTGGATTGGTG-3′BamH1-EcoR1pACT2/ACT144-400p(His)6-ACT1-1005′-CCCCCCGGGGTTCAGCCTGTACAAGCAGTTAGTC-3′5′-CGGAATTCAATTAAAATACATCAAGCACAGCC-3′Xma1-EcoR1pACT2/ACT35-343p(His)6-ACT1-400a NA, not applicable Open table in a new tab Yeast Two-hybrid Library Screening—The construct pGBKT7/HTJ1242–554 (TRP1) was used as bait to screen a human liver cDNA library fused to the pACT2 (LEU2) GAL4-activation domain and the HA epitope tag. The bait pGBKT7/HTJ1242–554 and the library-containing target pACT2 vector were sequentially transformed into the two-hybrid yeast strain AH109 as described (38Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2000) Google Scholar). As strain AH109 possesses three reporter genes (ADE2, HIS3, and MEL1) that are under the control of the Gal4 upstream activating sequences and TATA boxes, transformants were grown on minimal synthetic dropout four medium lacking tryptophan (Trp), leucine (Leu), histidine (His), and adenine (Ade) (SD/-Trp/-Leu/-His/-Ade) to select for potential interactors. Approximately 2 × 106 clones, representing 1/3 library equivalents, were screened against the pGBKT7/HTJ1242–554 bait. Transformants with the ability to grow on SD/-Trp/-Leu/-His/-Ade that turned blue in the presence of X-α-gal were analyzed further as described under "Results." Western Blot Analysis of Protein Expression in Yeast Protein Extracts—The yeast strain AH109 was transformed separately with pGBKT7 or pACT2-derived constructs (Table I) and grown overnight on selective SD medium at 30 °C. The yeast protein extracts were prepared according to the manufacturer's procedure (Clontech, protocol number PT3024-1, version PR91200). Following electrophoresis on a 10% SDS-PAGE gel, proteins were transferred onto nitrocellulose membrane in 50 mm Tris (pH 8.0), 380 mm glycine, 0.1% SDS, and 20% methanol (transfer buffer). The recombinant proteins expressed from pGBKT7-derived constructs were detected using an anti-c-Myc monoclonal primary antibody and an anti-mouse conjugated to horseradish peroxidase secondary antibody. The expression of the pACT2-derived fusion proteins was detected by using a rabbit polyclonal antibody to HA. Immunoreactivity was visualized by enhanced chemiluminescence using the ECL kit. Expression and Purification of Recombinant Proteins—The recombinant histidine-tagged proteins were expressed from pET15-derived constructs (Table I). The E. coli BL21(DE3) codon+ cells were grown at 37 °C in Luria broth (LB) supplemented with carbenicillin at 100 μg/ml final concentration. Synthesis of recombinant proteins was induced by addition of isopropyl-1-thio-β-d-galactopyranoside at 0.4 mm final concentration at mig-log exponential phase (A600 ∼ 0.5–0.6). Cells were harvested after a 16-h induction, lysed using a French press at 10,000 p.s.i., and the histidine-tagged protein was purified on cobalt-chelating resin as described by the manufacturer (Talon resin, Clontech). All recombinant proteins were expressed in the soluble fraction except for His6-ACT1–400, which accumulates in inclusion bodies and was purified from lysate pellets resolubilized in 20 mm Tris (pH 8.0), 8 m urea, 100 mm NaCl and purified in denaturing conditions. Fractions eluted from the resin were dialyzed overnight at 4 °C in 20 mm Tris (pH 8.0), 100 mm NaCl to promote renaturation. The renatured soluble recombinant ACT was further purified by fast pressure liquid chromatography using gel filtration on Superdex 75 (Amersham Biosciences) equilibrated in the same buffer. Protein concentrations were estimated by the method of Bradford (39Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211571) Google Scholar). Protein purity and homogeneity were analyzed by PAGE performed in both denaturing and native conditions (40Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205416) Google Scholar). Dot Blot Protein Analysis—Dot blots were performed as described (41Blacque O.E. Worrall D.M. J. Biol. Chem. 2002; 272: 9950-9955Google Scholar) with the following modifications. Various amounts of recombinant His6-HTJ1 proteins were applied onto a nitrocellulose membrane, dried, then incubated overnight with a solution of soluble renatured His6-ACT1–400 (5 μg/ml) in 20 mm Tris-Cl (pH 8.0), 150 mm NaCl. After washing in phosphate-buffered saline, bound ACT was detected with an anti-human ACT monoclonal antibody (1:20,000). In control experiments, purified His6-J-MTJ1 and His6-J-MTJ1:H89Q prepared as described (23Chevalier M. Rhee H. Elguindi E.C. Blond S.Y. J. Biol. Chem. 2000; 275: 19620-19627Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) were applied to the nitrocellulose membrane, and incubated with a solution of recombinant His6-BiP (5 μg/ml) in Tris-Cl (pH 8.0), 150 mm NaCl. Bound BiP was detected by chemiluminescence using a commercially available anti-BiP antibody (PAI-014, Affinity Bioreagents Inc., Golden, CO). In all experiments, immunoreactivity was visualized by ECL. Native PAGE and Immunodetection of ACT-HTJ1 Interaction—Human plasma ACT (0.64 μm) was incubated at 25 °C with increasing concentrations of purified His6-HTJ1493–554 wild-type or mutant (His6-HTJ1493–554:W497A) in 20 mm Tris (pH 8.0), 100 mm NaCl prior to electrophoresis in non-denaturing conditions (40Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205416) Google Scholar). ACT was detected by Western blotting using a commercially available monoclonal antibody. Tryptophan Fluorescence Quenching Measurements—All measurements were carried out on a Cary Eclipse fluorescence spectrophotometer at a temperature of 25 °C regulated by a thermostatted cell holder and a Cary PCB15 Water peltier system (Varian Instruments, Walnut Creek, CA). All data were analyzed using the Cary Eclipse software (Varian Instruments). The emission spectra were recorded between 300 and 450 nm at an excitation wavelength of 280 nm. The excitation and emission slits were set to 10 nm. The emission wavelength increment was 4 nm and the acquisition time 3 s. Tryptophan fluorescence intensities were corrected for the contribution of ACT alone analyzed in the same conditions of temperature, buffer (20 mm Tris (pH 8.0), 100 mm NaCl), and concentrations. In titration experiments, the fraction of ACT bound to SANT2 (4 nm) at every serpin concentration (0–16 nm) was calculated relative to the fluorescence intensity (excitation wavelength = 280 nm; emission wavelength = 350 nm) at saturating concentrations of ACT (100% bound) minus the intensity of SANT2 alone (0% bound). The intrinsic dissociation equilibrium constant, Kdiss, was determined using the Scatchard representation corresponding to the equation, [ACT]b/[ACT]f=-1/Kdiss[ACT]b+n[SANT2]Tot/Kdiss(Eq. 1) where [ACT]b represents the concentration of bound ACT; [ACT]f is the concentration of free ACT; n is the number of SANT2 binding site(s) per ACT molecule. Plot of [ACT]b/[ACT]f = f([ACT]b) gives a straight line with a negative slope that equals -1/Kdiss[ACT]b and extrapolation on the x axis gives the maximum concentration of bound ACT and is used to deduce the stoichiometry of the complex. As SANT2 and ACT contain two and three tryptophans, respectively, we used Lehrer representation to determine the fraction of tryptophan residues quenched upon SANT2-ACT interactions as described (42Zargarian L. Le Tilly V. Jamin N. Chaffotte A. Gabrielsen O.S. Toma F. Alpert B. Biochemistry. 1999; 38: 1921-1929Crossref PubMed Scopus (25) Google Scholar), using the equation, F0/F0-F=1/faK[ACT]+1/fa(Eq. 2) where F0 is the tryptophan fluorescence of SANT2 alone; F is the tryptophan fluorescence of the SANT2-ACT complex at a given ACT concentration [ACT]; fa is the maximum fraction of accessible fluorophore; K is the Lehrer quenching constant of an individual tryptophan. The Lehrer representation F0/F0 - F = f(1/[ACT]) gives a straight line whose extrapolation on the y axis ([ACT] = 0) corresponds to the value of 1/fa (42Zargarian L. Le Tilly V. Jamin N. Chaffotte A. Gabrielsen O.S. Toma F. Alpert B. Biochemistry. 1999; 38: 1921-1929Crossref PubMed Scopus (25) Google Scholar). Chymotrypsin Activity—All steady state experiments were carried out at 25 °C in 20 mm Tris (pH 8.0), 100 mm NaCl with the chromogenic substrate Succ-AAPF-pNA. Absorbance was recorded spectrophotometrically at 405 nm. Km and Vmax were determined using the Lineweaver-Burk representation and the Michaelis-Menten equation at 4 nm chymotrypsin, substrate ranged from 0 to 0.1 mm, in the absence or presence of 4 nm SANT2. The catalytic constant, kcat, was determined for 0.2–5.0 nm chymotrypsin in the presence of 0.1 mm Succ-AAPF-pNA. To determine the residual activity of chymotrypsin in the presence of the preformed ACT-SANT2 complex, human plasma ACT (4.0 nm) was preincubated for 20 min at 25 °C in the presence of 0–640 nm His6-HTJ1493–554, His6-HTJ1493–554:W497A, or His6-HTJ1493–554:W497A, W520A and 0.1 mm Succ-AAPF-pNA prior to the addition of chymotrypsin (4.0 nm final concentration). The residual chymotrypsin activity was measured for 5–10 min at 15-s intervals. The percentage of inhibition was normalized to the activity of chymotrypsin alone (100%). Kinetic analysis of ACT inhibition of chymotrypsin in the presence or absence of SANT2 was performed as described (43Hwang S.R. Steineckert B. Toneff T. Bundey R. Logvinova A.V. Goldsmith P. Hook V.Y. Biochemistry. 2002; 41: 10397-10405Crossref PubMed Scopus (30) Google Scholar) with the following modifications. Equimolar concentrations of chymotrypsin and ACT (4 nm) were incubated 0–10 min and the residual chymotrypsin activity was measured at timed intervals after additio
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