Peptidase Activity of the Escherichia coli Hsp31 Chaperone

Abstract

Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys184, His185, and Asp213 catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His85, His122, and Glu90 metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8–12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDS-polyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase. Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys184, His185, and Asp213 catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His85, His122, and Glu90 metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8–12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDS-polyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase. Every organism responds to a sudden increase in the environmental temperature by the overexpression of a set of highly conserved heat shock proteins (1Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 631-677Crossref PubMed Scopus (4419) Google Scholar, 2Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3121) Google Scholar). Most of these heat shock proteins function either as molecular chaperones, assisting in protein folding and renaturation or as proteases which degrade proteins that are beyond rescue. Hsp31, 1The abbreviations used are: Hsp, heat shock protein; HPLC, high performance liquid chromatography; PMSF, phenylmethylsulfonyl fluoride; amc, 7-amino-4-methylcoumarin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. the hchA gene product (formerly known as YedU), is a heat-inducible homodimeric protein of 31-kDa subunits, which was recently shown to exhibit molecular chaperone activity (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar, 4Sastry M.S. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). It promotes the functional folding of citrate synthase, α-glucosidase, and alcohol dehydrogenase. It also prevents the aggregation at 43 °C of citrate synthase and alcohol dehydrogenase and interacts specifically with unfolded proteins (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar, 4Sastry M.S. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Although Hsp31 does not exhibit any ATPase activity, some of its chaperone activities are partially inhibited by ATP (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar, 4Sastry M.S. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The crystal structure of Hsp31 was solved at 1.6 Å resolution and revealed a system of hydrophobic patches, canyons, and grooves, which may stabilize partially unfolded protein substrates and explain its chaperone activity (5Quigley P.M. Korotkov K. Baneyx F. Hol W.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3137-3142Crossref PubMed Scopus (104) Google Scholar). A putative Cys-His-Asp catalytic triad that is only accessible through a small pocket with a length of 14 Å, and an entrance of about 4 Å was also revealed, suggesting that Hsp31 might present a protease or more probably a peptidase activity (5Quigley P.M. Korotkov K. Baneyx F. Hol W.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3137-3142Crossref PubMed Scopus (104) Google Scholar, 6Zhao Y. Liu D. Kaluarachchi W.D. Bellamy H.D. White M.A. Fox R.O. Protein Sci. 2003; 12: 2303-2311Crossref PubMed Scopus (27) Google Scholar, 7Lee S.J. Kim S.J. Kim I.K. Ko J. Jeong C.S. Kim G.H. Park C. Kang S.O. Suh P.G. Lee H.S. Cha S.S. J. Biol. Chem. 2003; 278: 44552-44559Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 8Quigley P.M. Korotkov K. Baneyx F. Hol W.G. Protein Sci. 2004; 13: 269-277Crossref PubMed Scopus (22) Google Scholar). Although initial attempts to determine whether Hsp31 functions as a protease or peptidase were unsuccessful (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar, 4Sastry M.S. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), it was recently reported that Hsp31 displays very weak protease activity and a peptidase activity against Ala and Gly conjugated to a fluorogenic group, but the physiological relevance of this observation remains unclear (7Lee S.J. Kim S.J. Kim I.K. Ko J. Jeong C.S. Kim G.H. Park C. Kang S.O. Suh P.G. Lee H.S. Cha S.S. J. Biol. Chem. 2003; 278: 44552-44559Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). In the present study, we show that Hsp31 is a broad specificity aminopeptidase with a preference for alanine and basic amino acids and that it is inhibited by cysteine protease inhibitors and by the metal ion complexing agents EDTA and 1,10-phenantroline. Furthermore, we show that an Hsp31-deficient mutant accumulates increased amounts of 8–12-mer peptides, which can be processed in vitro to smaller peptides by purified Hsp31. Finally, we report that Hsp31 specifically interacts with ClpA, GroEL, EF-Tu, and tryptophanase. Our results suggest that, besides its chaperone activity, Hsp31 plays an active role in the downstream processing of peptides generated by the ATP-dependent proteases. Hsp31 Expression and Purification—The Escherichia coli B/r strain AD494 (ara leu7967 lacX74 phoA phoR-malF3 F′[lac+ (lacIq) pro] trxB::kan [DE3] pLysS was purchased from Novagen. It was transformed with plasmid pINTYEDU, which encodes the complete Hsp31/YedU protein to a at its N terminus (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar). were in in the of and by (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar). The Hsp31 protein to the was purified by on a purification with an from as (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar). The Hsp31 protein was as by SDS-polyacrylamide It was at °C in with The C185A mutant was and purified as (7Lee S.J. Kim S.J. Kim I.K. Ko J. Jeong C.S. Kim G.H. Park C. Kang S.O. Suh P.G. Lee H.S. Cha S.S. J. Biol. Chem. 2003; 278: 44552-44559Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). of the Hsp31-deficient of the hcha gene Hsp31 was the system Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The is to the gene in E. coli an plasmid plasmid the Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google the system was The basic is to a with a gene that is generated by by with to the or downstream of the gene and for the of is by in these Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). was used as the for of the hcha mutant of the in the hcha gene is not to have a on the of the downstream of hcha for a putative and for a are in the The of the hcha mutant is of the of E. coli gene Y. Y. K. A. and for and of Hsp31 in was as (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar), and were by and were as in (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar, PubMed Google of Hsp31 were SDS-polyacrylamide either or and proteolytic activity was by from or PubMed Google Scholar). and was also electrophoresis as by C. E. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). of citrate synthase, and was by of the of the protein and of Hsp31 at °C in for the was with and the were to of and were by of product on a reverse in in and with a of and aminopeptidase activities were by the of from the fluorogenic amino or peptides as (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar, D. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). of Hsp31 was at °C for in in which the of product is as a function of with in in a of and at at was of the with activity was by the of or as (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar, Kim Lee Kim PubMed Scopus Google The of was at The of Hsp31 was from its of from and Hsp31-deficient were to and of for to in of The were by with and in for of peptides C. J. Biol. PubMed Scopus Google Scholar). The was for at and the was by of of the peptide was on a with and with the at a of were were by and at the molecular peptides from the were by in and in the or in the of Hsp31, and was a in molecular and were used as molecular of molecular peptides from the were by and in in was by reverse The was in in and with a of in at a of and the were by by at and by or as in C. J. Biol. PubMed Scopus Google Scholar). of Hsp31 by Purification—The Hsp31 protein to a was a and purified as the F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3121) Google Scholar). An E. coli crude extract was this Hsp31 The was with and proteins specifically were with The proteins were by and the that were not by two were by mass spectrometry. of these two was with the and the with the to an E. coli peptidase of in synthase and all were from and were reagent The molecular for was from The fluorogenic peptides were from either or of of reported (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar), we not any proteolytic activity of Hsp31 toward or a similar to that used for the of protease activity PubMed Google Scholar), in which the was in the results were with both SDS-polyacrylamide and not protein as and were not by Hsp31 in a in of or Park Kang J. Biol. PubMed Scopus Google Scholar), of ATP and suggesting that Hsp31 does not function as a broad specificity protease not not any significant activity of Hsp31 in a as Hsp31 in an of a with purified Hsp31, in for into and in the and with not was also with the C185A mutant in the suggesting that it results from a proteolytic activity. of the cleavage specificity of Hsp31 for Hsp31 of the substrates and substrates were with a in that that Hsp31 is a broad specificity aminopeptidase alanine and basic amino revealed that Hsp31 displays for Ala Lys and Arg in that The of Hsp31 are of the of as of as U. J. R. PubMed Scopus Google or as the activity of D. D. 2003; PubMed Scopus Google Scholar, D. A. D. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), but are than the aminopeptidase activity of the aminopeptidase of E. coli D. D. 2003; PubMed Scopus Google Scholar, D. A. D. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). the that small amounts of were for these aminopeptidase we purified Hsp31 on a The alanine and aminopeptidase activities were in the as the Hsp31 protein suggesting that Hsp31 is for these activities results were purified Hsp31 was on a not with was with and of the activity with of the activity with was by Hsp31, suggesting that Hsp31 has activity and were by Hsp31 not A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar). the were also in the of ATP and and similar results not of Hsp31 and of the of the aminopeptidase activity of Hsp31 in the of inhibitors known to cysteine proteases and proteases and and completely the of Hsp31 to degrade Hsp31 is also inhibited by In the protease on Hsp31. the aminopeptidase activity of Hsp31 was completely abolished in a C185A mutant, suggesting that, in with crystallographic is the active of Hsp31. inhibitors EDTA and a of the Hsp31 that the putative His85, His122, and Glu90 metal-binding site of Hsp31 revealed by crystallographic Y. Liu D. Kaluarachchi W.D. Bellamy H.D. White M.A. Fox R.O. Protein Sci. 2003; 12: 2303-2311Crossref PubMed Scopus (27) Google is important for its aminopeptidase activity. Hsp31 inhibitors are from is inhibited by but not by EDTA by iodoacetamide D. D. 2003; PubMed Scopus Google Scholar, D. A. D. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). of and ATP on the Hsp31 Hsp31 is a heat shock protein and displays chaperone activity, we that its aminopeptidase activity might be revealed at high in a of that of the from chaperone to protease of at and C. A. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the Hsp31 aminopeptidase activity on temperature the of °C displays a with a of suggesting that, is not an of Hsp31 by heat shock The aminopeptidase activity of Hsp31 was and was and In crystallographic of Hsp31, a ion was by His85, and with this Hsp31 is completely inhibited by which has a high for Furthermore, in the of the alanine aminopeptidase activity of Hsp31 was inhibited to of the and it be to by to by but not by or at suggesting that is the metal of Hsp31. ATP interacts with Hsp31 and some of its chaperone M.S. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), we the aminopeptidase activities of Hsp31 as in the in the of ATP and and found that ATP was any not of an Hsp31-deficient the that the of Hsp31 to peptide the was The Hsp31-deficient and parental were for at °C in the of The were and and the that was in was C. J. Biol. PubMed Scopus Google Scholar). The from the two were to on a a similar of with a molecular mass of were by and to reverse on a with and with a in shown in the of peptides from the Hsp31-deficient strain is from that of the The Hsp31-deficient strain accumulates a of peptides and and peptides and The of peptides in the Hsp31-deficient strain was by the from the were for terminus and peptide in as in C. J. Biol. PubMed Scopus Google The results of this that the on the is and all the is increased by in that the present in the strain is a of The of and in an of the length of peptides in length from to and results were for peptides of the strain that, as shown in was a of peptides and a of 8–12-mer the of Hsp31 on these peptides, the from the from the Hsp31-deficient strain was to on a column, and with purified Hsp31. shown in the of peptides from the Hsp31-deficient strain as a broad to molecular mass peptides the of these peptides is in with that by for in the of purified Hsp31, is a in the of high molecular peptides and a increase in the of molecular peptides suggesting that Hsp31 a peptidase on these not increase peptide which that the of is in vivo for their complete not can from the results in this that Hsp31 is in the processing of 8–12-mer peptides the of the peptides by ATP-dependent both in vitro and in The of the Hsp31-deficient strain was similar to that of its parental strain, either in or not Furthermore, was increase in the of the mutant from to results are not only peptidase display a or an increased increased results from a in the of proteins to amino acids for the of C. J. Biol. PubMed Scopus Google Scholar). Proteins with used purification and mass to the proteins that specifically interact with Hsp31. A protein of Hsp31 to a was on a as An E. coli crude extract was this Hsp31 in the of and the was with the the proteins were with were by protein were by by mass and identified by Hsp31 specifically proteins which were as the ATPase component ClpA of the ClpAP protease F. D. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), the chaperone F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3121) Google Scholar), elongation factor A. Richarme G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (72) Google Scholar, Y. U. K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), and tryptophanase that to and D. J. J. PubMed Google Scholar). of these proteins was on a or on an is E. coli peptidase in not The Hsp31 and ClpA is in with its function as an aminopeptidase in the downstream processing of peptides by as ClpAP or The Hsp31 and is consistent with the chaperone and peptidase function of Hsp31, the chaperone has also been in F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3121) Google Scholar). The Hsp31 and can be for by the recently chaperone functions of A. Richarme G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (72) Google Scholar, Y. U. K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). a for the of Hsp31 with it is that tryptophanase is in E. coli against and D. J. J. PubMed Google interacting with component of the ClpAP ATP-dependent component of the chaperone elongation protein in a show in this that Hsp31 does not display any significant proteolytic activity but displays an aminopeptidase activity that is against peptide substrates with alanine or basic amino acids at N terminus and is inhibited by and Furthermore, an Hsp31-deficient strain accumulates of 8–12-mer peptides than its parental strain, and Hsp31 can these peptides to smaller peptides, which that it is in the processing of 8–12-mer peptides both in vitro and in The proteolytic activity of Hsp31 is with protein substrates used for protease as or and Furthermore, proteolytic activity be in the of amounts of of the protein by temperature or by of that have the to the activity of proteases. is consistent with which that Hsp31 does not display any proteolytic activity (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar, 4Sastry M.S. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google a very weak proteolytic activity of Hsp31 toward bovine serum albumin has been reported in (7Lee S.J. Kim S.J. Kim I.K. Ko J. Jeong C.S. Kim G.H. Park C. Kang S.O. Suh P.G. Lee H.S. Cha S.S. J. Biol. Chem. 2003; 278: 44552-44559Abstract Full Text Full Text PDF PubMed Scopus (200) Google It is also consistent with the of the Cys-His-Asp catalytic triad of Hsp31, through a small pocket 14 Å with an entrance of about 4 Å in (5Quigley P.M. Korotkov K. Baneyx F. Hol W.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3137-3142Crossref PubMed Scopus (104) Google Scholar, 6Zhao Y. Liu D. Kaluarachchi W.D. Bellamy H.D. White M.A. Fox R.O. Protein Sci. 2003; 12: 2303-2311Crossref PubMed Scopus (27) Google Scholar, 7Lee S.J. Kim S.J. Kim I.K. Ko J. Jeong C.S. Kim G.H. Park C. Kang S.O. Suh P.G. Lee H.S. Cha S.S. J. Biol. Chem. 2003; 278: 44552-44559Abstract Full Text Full Text PDF PubMed Scopus (200) Google that to the of Hsp31 to peptides than protein Hsp31 functions as a broad specificity which alanine and basic amino substrates than The aminopeptidase activity of Hsp31 of which is of the of as of protein U. J. R. PubMed Scopus Google is than that of of the E. coli aminopeptidase D. D. 2003; PubMed Scopus Google Scholar, D. A. D. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). have shown that the aminopeptidase activities to Hsp31 are not by by purification of Hsp31 and by a of Hsp31 and in with Hsp31, is not inhibited by EDTA or iodoacetamide but inhibited by D. D. 2003; PubMed Scopus Google Scholar, D. A. D. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Hsp31 does not display any significant activity, it does not and peptides at their N terminus does it any activity, as reported (3Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar). The of and has been reported by Lee (7Lee S.J. Kim S.J. Kim I.K. Ko J. Jeong C.S. Kim G.H. Park C. Kang S.O. Suh P.G. Lee H.S. Cha S.S. J. Biol. Chem. 2003; 278: 44552-44559Abstract Full Text Full Text PDF PubMed Scopus (200) Google but not that of and The aminopeptidase activity of Hsp31 is inhibited by the cysteine protease iodoacetamide and is completely abolished in the C185A is in with crystallographic results suggesting that is the active of Hsp31 (5Quigley P.M. Korotkov K. Baneyx F. Hol W.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3137-3142Crossref PubMed Scopus (104) Google Scholar). inhibitors and also Hsp31. that the putative His85, His122, and Glu90 metal-binding site revealed by crystallographic is important for Hsp31 activity Y. Liu D. Kaluarachchi W.D. Bellamy H.D. White M.A. Fox R.O. Protein Sci. 2003; 12: 2303-2311Crossref PubMed Scopus (27) Google Scholar). is the only metal ion that is to Hsp31 by suggesting that it is the of Hsp31, as in crystallographic Y. Liu D. Kaluarachchi W.D. Bellamy H.D. White M.A. Fox R.O. Protein Sci. 2003; 12: 2303-2311Crossref PubMed Scopus (27) Google Scholar). Hsp31 a on in with which displays a from chaperone to protease and with an increase in proteolytic activity in that temperature C. A. Full Text Full Text PDF PubMed Scopus Google Scholar). It that Hsp31 plays an important physiological function in peptide an Hsp31-deficient strain accumulates amounts of 8–12-mer peptides than its parental strain, and purified Hsp31 can these peptides to smaller peptides in vitro of mutant C. J. Biol. PubMed Scopus Google Scholar), a mutant, and a mutant J. PubMed Google also small peptides in It is that peptides are by ATP-dependent proteases in the protein and in E. coli S. S. PubMed Scopus Google and the in D. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google and both the processing of 8–12-mer peptides by Hsp31 and its with ClpA the of its in the processing of peptides from The of 8–12-mer peptides in the Hsp31-deficient strain, the of more as that Hsp31 can degrade of peptides that are not by or that it in the of peptides by purification of a crude E. coli extract on an Hsp31 to the of ClpA, GroEL, EF-Tu, and which specifically interact with Hsp31. of these proteins with two either the used for Hsp31 purification or the E. coli peptidase The Hsp31 and the chaperone that Hsp31, which might function as a chaperone M.S. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), unfolded proteins to the chaperone for reported a Hsp31 and the chaperone Baneyx F. 2003; Scopus Google Scholar), and it is that a also with the chaperone The Hsp31 and elongation factor can be in of the recently function of elongation as and proteins A. Richarme G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (72) Google Scholar, Y. U. K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google it has been that of functions as a component of the protein Y. U. K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), and a Hsp31 and be for a The Hsp31 and ClpA the that Hsp31 is in the is of the ATP-dependent proteases a function in protein which peptides of amino acids that are by as and Hsp31 J. PubMed Google Scholar). The that ATP-dependent proteases and a of peptides from proteases to It is that Hsp31 interacts with that tryptophanase in the D. J. J. PubMed Google Scholar). The is in not only but also and cysteine for It which can be to and both of which The Hsp31 chaperone might specifically tryptophanase against these A. for with the