The Small Heat Shock Protein IbpA of Escherichia coli Cooperates with IbpB in Stabilization of Thermally Aggregated Proteins in a Disaggregation Competent State
2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês
10.1074/jbc.m412706200
ISSN1083-351X
AutoresMarcelina Matuszewska, Dorota Kuczyńska‐Wiśnik, Ewa Laskowska, Krzysztof Liberek,
Tópico(s)Bee Products Chemical Analysis
ResumoThe small heat shock proteins are ubiquitous stress proteins proposed to increase cellular tolerance to heat shock conditions. We isolated IbpA, the Escherichia coli small heat shock protein, and tested its ability to keep thermally inactivated substrate proteins in a disaggregation competent state. We found that the presence of IbpA alone during substrate thermal inactivation only weakly influences the ability of the bi-chaperone Hsp70-Hsp100 system to disaggregate aggregated substrate. Similar minor effects were observed for IbpB alone, the other E. coli small heat shock protein. However, when both IbpA and IbpB are simultaneously present during substrate inactivation they efficiently stabilize thermally aggregated proteins in a disaggregation competent state. The properties of the aggregated protein substrates are changed in the presence of IbpA and IbpB, resulting in lower hydrophobicity and the ability of aggregates to withstand sizing chromatography conditions. IbpA and IbpB form mixed complexes, and IbpA stimulates association of IbpB with substrate. The small heat shock proteins are ubiquitous stress proteins proposed to increase cellular tolerance to heat shock conditions. We isolated IbpA, the Escherichia coli small heat shock protein, and tested its ability to keep thermally inactivated substrate proteins in a disaggregation competent state. We found that the presence of IbpA alone during substrate thermal inactivation only weakly influences the ability of the bi-chaperone Hsp70-Hsp100 system to disaggregate aggregated substrate. Similar minor effects were observed for IbpB alone, the other E. coli small heat shock protein. However, when both IbpA and IbpB are simultaneously present during substrate inactivation they efficiently stabilize thermally aggregated proteins in a disaggregation competent state. The properties of the aggregated protein substrates are changed in the presence of IbpA and IbpB, resulting in lower hydrophobicity and the ability of aggregates to withstand sizing chromatography conditions. IbpA and IbpB form mixed complexes, and IbpA stimulates association of IbpB with substrate. The proper conformation of proteins is challenged by stress conditions. Exposure to extreme heat shock conditions results in a massive aggregation of proteins inside both prokaryotic and eukaryotic cells (1.Parsell D.A. Kowal A.S. Singer M.A. Lindquist S. Nature. 1994; 373: 475-478Crossref Scopus (751) Google Scholar, 2.Laskowska E. Kuczyńska-Wiśnik D. Skórko-Glonek J. Taylor A. Mol. Microbiol. 1996; 22: 555-571Crossref PubMed Scopus (116) Google Scholar, 3.Mogk A. Tomoyasu T. Goloubinoff P. Rüdiger S. Röder D. Langen H. Bukau B. EMBO J. 1999; 18: 6934-6949Crossref PubMed Scopus (520) Google Scholar). Chaperones from the Hsp100 family, that is ClpB in Escherichia coli and Hsp104 in the yeast Saccharomyces cerevisiae, were implicated in the disaggregation reaction, because aggregated proteins were not eliminated in either clpB or HSP104 deletion strains (1.Parsell D.A. Kowal A.S. Singer M.A. Lindquist S. Nature. 1994; 373: 475-478Crossref Scopus (751) Google Scholar, 2.Laskowska E. Kuczyńska-Wiśnik D. Skórko-Glonek J. Taylor A. Mol. Microbiol. 1996; 22: 555-571Crossref PubMed Scopus (116) Google Scholar, 3.Mogk A. Tomoyasu T. Goloubinoff P. Rüdiger S. Röder D. Langen H. Bukau B. EMBO J. 1999; 18: 6934-6949Crossref PubMed Scopus (520) Google Scholar). Additionally, the clpB and HSP104 gene products were identified as factors conferring thermotolerance in E. coli and S. cerevisiae (4.Squires C.L. Pendersen S. Ross B. Squires C. J. Bacteriol. 1991; 173: 4254-4262Crossref PubMed Google Scholar, 5.Kitagawa M. Wada C. Yoshioka S. Yura T. J. Bacteriol. 1991; 173: 4247-4253Crossref PubMed Google Scholar, 6.Sanchez Y. Lindquist S. Science. 1990; 248: 1112-1115Crossref PubMed Scopus (672) Google Scholar, 7.Sanchez Y. Taulien J. Borkovich K.A. Lindquist S. EMBO J. 1992; 11: 2357-2364Crossref PubMed Scopus (473) Google Scholar). However, in vitro studies on the reactivation of aggregated proteins showed that chaperones from the Hsp100 family alone are not sufficient for disaggregation and refolding. Other chaperone proteins are also involved in this process. E. coli Hsp70 (DnaK) and its cochaperones (DnaJ and GrpE) cooperate with ClpB and form a bi-chaperone system capable of efficient disaggregation of aggregated proteins (3.Mogk A. Tomoyasu T. Goloubinoff P. Rüdiger S. Röder D. Langen H. Bukau B. EMBO J. 1999; 18: 6934-6949Crossref PubMed Scopus (520) Google Scholar, 8.Goloubinoff P. Mogk A. Zvi A.P.B. Tomayasu T. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13732-13737Crossref PubMed Scopus (507) Google Scholar, 9.Zolkiewski M. J. Biol. Chem. 1999; 274: 28083-28086Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Analogous Hsp100-Hsp70 bi-chaperone systems able to disaggregate denatured protein substrates in vitro were established using chaperones from other bacterial species (10.Motohashi K. Watanabe T. Yohda M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7184-7189Crossref PubMed Scopus (225) Google Scholar), as well as from yeast cytosol (11.Glover J.R. Lindquist S. Cell. 1998; 94: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar) and mitochondria (12.Krzewska J. Langer T. Liberek K. FEBS Lett. 2001; 489: 92-96Crossref PubMed Scopus (124) Google Scholar, 13.Germaniuk A. Liberek K. Marszalek J. J. Biol. Chem. 2002; 277: 27801-27808Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). However, the efficiency of refolding reaction catalyzed by these bi-chaperone systems depends strongly on the physical properties of protein aggregates. It was proposed that small heat shock proteins (sHsps) 1The abbreviations used are: sHsp, small heat shock protein(s); IbpA, inclusion body associated protein A; IbpB, inclusion body associated protein B; MDH, malate dehydrogenase; bis-ANS, 1,1′-bi(4-anilino)-naphtalene-5,5′-disulfonic acid. associate with aggregated proteins and change their physical properties in such a way that chaperone-mediated disaggregation and refolding become much more efficient (14.Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (659) Google Scholar, 15.Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (639) Google Scholar, 16.Veinger L. Diamant S. Buchner J. Goloubinoff P. J. Biol. Chem. 1998; 273: 11032-11037Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 17.Lee G.J. Vierling E. Plant Physiol. 2000; 122: 189-198Crossref PubMed Scopus (377) Google Scholar, 18.Mogk A. Deuerling E. Vorderwulbecke S. Vierling E. Bukau B. Mol. Microbiol. 2003; 50: 585-595Crossref PubMed Scopus (304) Google Scholar, 19.Mogk A. Schlieker C. Friedrich K.L. Schönfeld H.-J. Vierling E. Bukau B. J. Biol. Chem. 2003; 278: 31033-31042Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). However, little is known about the molecular mechanism of these processes. Small heat shock proteins are widely distributed both in prokaryotes and eukaryotes. Members of this diverse protein family are characterized by relatively low monomeric molecular masses (15–43 kDa) and a conserved stretch of ∼100 amino acid residues (reviewed in Refs. 20.Haslbeck M. Cell. Mol. Life Sci. 2002; 59: 1649-1657Crossref PubMed Scopus (249) Google Scholar and 21.Narberhaus F. Microbiol. Molecular Biol. Rev. 2002; 66: 64-93Crossref PubMed Scopus (474) Google Scholar). This so-called α-crystallin domain displays sequence similarities to the vertebrate eye lens protein α-crystallin, which prevents protein precipitation and cataract formation in the eye lens. One of the most striking features of sHsps is their organization in large oligomeric structures (reviewed in Refs. 20.Haslbeck M. Cell. Mol. Life Sci. 2002; 59: 1649-1657Crossref PubMed Scopus (249) Google Scholar and 21.Narberhaus F. Microbiol. Molecular Biol. Rev. 2002; 66: 64-93Crossref PubMed Scopus (474) Google Scholar). The interactions between sHsp subunits in these oligomers are highly dynamic, and under physiological conditions, rapid exchange between subunits of these oligomers has been observed (22.Bova M.P. Ding. L. L. Horwitz J. Fung B.K. J. Biol. Chem. 1997; 272: 29511-29517Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 23.Bova M.P. McHaourab H.S. Han Y. Fung B.K. J. Biol. Chem. 2000; 275: 38921-38929Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 24.Friedrich K.L. Giese K.C. Buan N.R. Vierling E. J. Biol. Chem. 2004; 279: 1080-1089Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Moreover, the temperature induces changes in the oligomerization state of sHsps complexes (20.Haslbeck M. Cell. Mol. Life Sci. 2002; 59: 1649-1657Crossref PubMed Scopus (249) Google Scholar, 21.Narberhaus F. Microbiol. Molecular Biol. Rev. 2002; 66: 64-93Crossref PubMed Scopus (474) Google Scholar) essential for their chaperone activity (25.Haslbeck M. Walke S. Stromer T. Ehrnsperger M. White H.E. Chen S. Saibil H. Buchner J. EMBO J. 1999; 18: 6744-6751Crossref PubMed Scopus (386) Google Scholar, 26.Van Montfort R.L.M. Basha E. Friedrich K.L. Slingsby C. Vierling E. Nat. Struct. Biol. 2001; 8: 1025-1030Crossref PubMed Scopus (641) Google Scholar, 27.Giese K.C. Vierling E. J. Biol. Chem. 2002; 277: 46310-46318Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Surprisingly, in many cases deletion of genes encoding sHsps does not result in strong phenotypic effects, making it difficult to clearly assign them a function in the chaperone network. It has been reported, however, that overproduction of sHsps conveys thermotolerance in a number of organisms and cell types, suggesting the involvement of sHsps in the control of protein aggregation and disaggregation processes upon heat shock (28.Landry J. Chretien P. Lambert H. Hickey E. Weber L.A. J. Cell Biol. 1989; 109: 7-15Crossref PubMed Scopus (600) Google Scholar, 29.Van den Ijssel P.R. Overkamp P. Knauf U. Gaestel M. de Jong W.W. FEBS Lett. 1994; 355: 54-56Crossref PubMed Scopus (104) Google Scholar, 30.Nakamoto H. Suzuki N. Roy S.K. FEBS Lett. 2000; 483: 169-174Crossref PubMed Scopus (92) Google Scholar, 31.Kitagawa M. Matsumura Y. Tsuchido T. FEMS Microbiol. Lett. 2000; 184: 165-171Crossref PubMed Google Scholar). Several in vitro studies have reported that sHsps can prevent the aggregation of heat-denatured proteins (32.Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1763) Google Scholar, 33.Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar, 34.Studer S. Narberhaus F. J. Biol. Chem. 2000; 275: 37212-37218Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, in vivo studies (2.Laskowska E. Kuczyńska-Wiśnik D. Skórko-Glonek J. Taylor A. Mol. Microbiol. 1996; 22: 555-571Crossref PubMed Scopus (116) Google Scholar, 35.Allen S.P. Polazzi J.O. Gierse J.K. Easton A.M. J. Bacteriol. 1993; 174: 6938-6947Crossref Google Scholar, 36.Basha E. Lee G.J. Breci L.A. Hausrath A.C. Buan N.R. Giese K.C. Vierling E. J. Biol. Chem. 2004; 279: 7566-7575Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) show that sHsps are rather localized in the insoluble protein fractions of stressed cells, which questions their preventive role during heat stress conditions. Two members of the sHsp family, IbpA and IbpB, are present in E. coli. The IbpA and IbpB proteins are 48% identical at the amino acid sequence level (37.Chuang S.E. Burland V. Plunkett G. Daniels D.L. Blattner F.R. Gene (Amst.). 1993; 134: 1-6Crossref PubMed Scopus (138) Google Scholar), and both were identified as proteins associated with inclusion bodies (35.Allen S.P. Polazzi J.O. Gierse J.K. Easton A.M. J. Bacteriol. 1993; 174: 6938-6947Crossref Google Scholar). IbpA/IbpB were also found in aggregated protein fractions following heat stress (2.Laskowska E. Kuczyńska-Wiśnik D. Skórko-Glonek J. Taylor A. Mol. Microbiol. 1996; 22: 555-571Crossref PubMed Scopus (116) Google Scholar). The deletion of the ibpAB genes results in a weak phenotype manifested only at extreme temperatures (38.Kuczyńska-Wiśnik D. Kêdzierska S. Matuszewska E. Lund P. Taylor A. Lipińska B. Laskowska E. Microbiology. 2002; 148: 1757-1765Crossref PubMed Scopus (87) Google Scholar). Prolonged incubation of such mutant cells at 50 °C results in a decrease of viability, which correlates with an increase in protein aggregation (38.Kuczyńska-Wiśnik D. Kêdzierska S. Matuszewska E. Lund P. Taylor A. Lipińska B. Laskowska E. Microbiology. 2002; 148: 1757-1765Crossref PubMed Scopus (87) Google Scholar). To date, only IbpB has been studied in detail. Purified IbpB forms 2- to 3-MDa oligomers, which, upon exposure to high temperature, dissociate into smaller ∼600-kDa structures (39.Shearstone J.R. Baneyx F. J. Biol. Chem. 1999; 274: 9937-9945Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 40.Kitagawa M. Miyakawa M. Matsumara Y. Tsuchido T. Eur. J. Biochem. 2002; 269: 2907-2917Crossref PubMed Scopus (90) Google Scholar). The importance of these structural changes is not well understood. IbpB protein was shown in vitro to suppress thermal aggregation of model substrate proteins in a concentration-dependent manner (39.Shearstone J.R. Baneyx F. J. Biol. Chem. 1999; 274: 9937-9945Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Moreover, IbpB was also tested for the ability to cooperate with chaperones from the Hsp70 and Hsp100 families in substrate disaggregation and refolding (16.Veinger L. Diamant S. Buchner J. Goloubinoff P. J. Biol. Chem. 1998; 273: 11032-11037Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 18.Mogk A. Deuerling E. Vorderwulbecke S. Vierling E. Bukau B. Mol. Microbiol. 2003; 50: 585-595Crossref PubMed Scopus (304) Google Scholar). The presence of IbpB during the heat inactivation of both malate dehydrogenase (MDH) and lactate dehydrogenase results in more efficient reactivation of these enzymes by the Hsp70 system (DnaK/DnaJ/GrpE) (16.Veinger L. Diamant S. Buchner J. Goloubinoff P. J. Biol. Chem. 1998; 273: 11032-11037Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar) as well as by the bi-chaperone Hsp70-Hsp100 system (ClpB-DnaK/DnaJ/GrpE) (18.Mogk A. Deuerling E. Vorderwulbecke S. Vierling E. Bukau B. Mol. Microbiol. 2003; 50: 585-595Crossref PubMed Scopus (304) Google Scholar, 19.Mogk A. Schlieker C. Friedrich K.L. Schönfeld H.-J. Vierling E. Bukau B. J. Biol. Chem. 2003; 278: 31033-31042Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). However, the details, if any, of cooperation between IbpB and the ClpB-DnaK/DnaJ/GrpE system are unknown. No such detailed studies have been performed for the other E. coli sHsp, IbpA. Only recently, IbpA with a hexahistidine tag at its N terminus was isolated and shown to form multimers (40.Kitagawa M. Miyakawa M. Matsumara Y. Tsuchido T. Eur. J. Biochem. 2002; 269: 2907-2917Crossref PubMed Scopus (90) Google Scholar). Similarly to IbpB, it also has a protective effect on several tested substrate proteins subjected to thermal and oxidative stress (40.Kitagawa M. Miyakawa M. Matsumara Y. Tsuchido T. Eur. J. Biochem. 2002; 269: 2907-2917Crossref PubMed Scopus (90) Google Scholar). However, it was not determined if the presence of IbpA during thermal inactivation of substrate protein results in subsequent efficient disaggregation by the ClpB-DnaK/DnaJ/GrpE chaperones. It is also not known whether IbpA interacts with IbpB and if these small chaperones cooperate in the modification of aggregates during thermal denaturation of substrate proteins. Here, we present results which answer the above questions. We observed that the presence of purified IbpA alone during thermal inactivation very weakly influences the ability of the bi-chaperone system (ClpB-DnaK/DnaJ/GrpE) to disaggregate aggregated substrate. A similar minor effect was observed for purified IbpB alone. However, when both IbpA and IbpB are simultaneously present during substrate inactivation, efficient stabilization of thermally aggregated proteins in a disaggregation competent state was observed, indicating that both sHsps work together as an integrated system. Protein Purification—The IbpA protein was overproduced in the MC4100ΔibpA/B strain transformed with the pUC18-derived pCA plasmid bearing the ibpA gene, under control of the ptac promoter (38.Kuczyńska-Wiśnik D. Kêdzierska S. Matuszewska E. Lund P. Taylor A. Lipińska B. Laskowska E. Microbiology. 2002; 148: 1757-1765Crossref PubMed Scopus (87) Google Scholar). Bacteria overproducing IbpA protein were lysed in a French press (Aminco) in buffer A (50 mm Tris-HCl, pH 7.4, 10% (v/v) glycerol, 1 mm dithiothreitol, 100 mm KCl). The bacterial lysate was clarified for 30 min at 26,000 rpm in a Beckman 30.5 rotor. The supernatant was discarded, and the pellet was resuspended in buffer A containing 2 m urea. After 1-h incubation on ice followed by centrifugation (30 min at 26,000 rpm in a Beckman 30.5 rotor), the pellet was resuspended in buffer A containing 6 m urea. After another 1-h incubation on ice and centrifugation (30 min at 26,000 rpm in a Beckman 30.5 rotor), the protein extract was loaded onto a Q-Sepharose column equilibrated with buffer A containing 6 m urea. The proteins bound to this column were eluted with a KCl gradient (100–300 mm) in buffer A supplemented with 6 m urea. Fractions containing pure IbpA, eluted at the end of the gradient, were pooled together. The urea present in the protein preparation was slowly dialyzed out by sequential dialysis to buffer A containing 4 m, 2 m, 1 m, and no urea. The IbpB protein was overproduced in the MC4100ΔibpA/B strain transformed with the pUC18 derived pCB plasmid bearing the ibpB gene under control of the ptac promoter (38.Kuczyńska-Wiśnik D. Kêdzierska S. Matuszewska E. Lund P. Taylor A. Lipińska B. Laskowska E. Microbiology. 2002; 148: 1757-1765Crossref PubMed Scopus (87) Google Scholar). IbpB was purified using the protocol described previously (39.Shearstone J.R. Baneyx F. J. Biol. Chem. 1999; 274: 9937-9945Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). To obtain the IbpB and IbpA proteins carrying a hexahistidine tag at the N terminus, pETHis-B and pETHis-A plasmids were constructed by cloning the ibpB or ibpA genes into the NdeI and BamHI sites of the pET28b+ vector (Novagen). The His-IbpB and His-IbpA proteins encoded by these plasmids have an additional 20-amino acid residues, which includes the hexahistidine tag, at the N terminus. These fusion proteins were overproduced in the BL21(DE3) ΔibpA/B strain. The purification of His-IbpB and His-IbpA was based on the interaction of the His tag with Ni-NTA-agarose under denaturing conditions. Published protocols were used for the purification of Escherichia coli DnaK, DnaJ, GrpE (41.Zylicz M. Ang D. Liberek K. Georgopoulos C. EMBO J. 1989; 8: 1601-1608Crossref PubMed Scopus (226) Google Scholar), and ClpB (42.Woo K.M. Kim K.L. Goldberg A.L. Ha D.B. Chung C.H. J. Biol. Chem. 1992; 267: 20429-20434Abstract Full Text PDF PubMed Google Scholar). Firefly luciferase (E 1701) was purchased from Promega. Malate dehydrogenase (MDH) was purchased from Sigma (410-13). Protein concentrations were determined with the Bradford (Bio-Rad) assay system, using bovine serum albumin as a standard. Molar concentrations are given assuming a hexameric structure for ClpB and a monomeric structure for the rest of the proteins. Luciferase Inactivation and Refolding Experiments—For inactivation, luciferase (1.5 μm) in buffer B (50 mm Tris-HCl, pH 7.4, 150 mm KCl, 20 mm magnesium acetate, 5 mm dithiothreitol) was incubated at different temperatures for 10 min in the presence of IbpA and IbpB as stated in the figure legends. For disaggregation and refolding, the inactivated luciferase (42 nm) was incubated at 25 °C in buffer B supplemented with ATP (5 mm), ATP regeneration system (10 mm phosphocreatine and 100 μg/ml phosphocreatine kinase), and chaperone proteins (3 μm DnaK, 0.24 μm DnaJ, 0.3 μm GrpE, and 3 μm ClpB) as indicated. Following 1 h of incubation, luciferase activity was determined in a Beckman scintillation counter using the Luciferase Assay System (Promega). In the control experiment it was shown that luciferase activity increases linearly with time during refolding for at least 1 h. MDH Inactivation and Refolding Experiments—Experiments were essentially performed as described previously (16.Veinger L. Diamant S. Buchner J. Goloubinoff P. J. Biol. Chem. 1998; 273: 11032-11037Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). MDH (2 μm) in buffer C (50 mm Tris-HCl, pH 7.4, 150 mm KCl, 2 mm dithiothreitol, 20 mm MgCl2) was inactivated at the indicated temperatures in the presence of IbpA (2 μm) and IbpB (7.8 μm). The disaggregation and refolding reaction was performed with MDH (90 nm) in buffer C supplemented with ATP (5 mm), an ATP regeneration system (10 mm phosphocreatine and 100 μg/ml phosphocreatine kinase) and chaperone proteins (3 μm DnaK, 0.24 μm DnaJ, 0.3 μm GrpE, and 3 μm ClpB) at 25 °C. MDH activity was measured as described before (43.Diamant S. Azem A. Weiss C. Goloubinoff P. J. Biol. Chem. 1995; 270: 28387-28391Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), at 25 °C in 150 mm potassium phosphate buffer, pH 7.5, containing 2 mm dithiothreitol, 0.5 mm oxaloacetate, and 0.28 mm NADH. Sizing Chromatography of Protein Complexes—Luciferase or MDH, following denaturation at different temperatures in the presence of IbpA and IbpB when indicated, was subjected to sizing chromatography on a Sepharose 4BCl column (0.5 × 15 cm) equilibrated in buffer C. Proteins present in fractions (100 μl) following sizing chromatography were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Chromatography resin from the sizing column was removed and discarded after each separation to avoid any contamination of the resin with the aggregated proteins. Isolation of Protein Complexes by Interaction with Ni-NTA-agarose Resin—His-IbpB (6 μm) and IbpA (6 μm), or His-IbpA (6 μm) and IbpB (6 μm), and MDH (8.6 μm), as indicated, were incubated in buffer C lacking dithiothreitol at different temperatures in a 35-μl reaction volume. After 20 min of incubation, Ni-NTA-agarose (20 μl) was added to the reaction mixture, and the mixture was incubated for an additional 10 min. Next, the supernatant was discarded, and the Ni-NTA-agarose was washed several times with buffer containing 50 mm Tris-HCl, pH 8.0, 300 mm NaCl, and 20 mm imidazole. Proteins bound to the resin were eluted with the same buffer containing 300 mm imidazole and subjected to SDS-PAGE followed by staining with Coomassie Brilliant Blue. Purification of IbpA Protein—Two small heat shock proteins, namely IbpA and IbpB, were identified in E. coli. IbpB has been purified and studied previously (39.Shearstone J.R. Baneyx F. J. Biol. Chem. 1999; 274: 9937-9945Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), but, until now, no detailed biochemical studies had been performed on IbpA. IbpA was overproduced in E. coli and purified to homogeneity (Fig. 1). Previous studies showed that IbpA, either induced by heat shock conditions (38.Kuczyńska-Wiśnik D. Kêdzierska S. Matuszewska E. Lund P. Taylor A. Lipińska B. Laskowska E. Microbiology. 2002; 148: 1757-1765Crossref PubMed Scopus (87) Google Scholar) or overproduced (39.Shearstone J.R. Baneyx F. J. Biol. Chem. 1999; 274: 9937-9945Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), was found in the fast sedimenting fraction of cellular extracts containing membranes and aggregated proteins, but not in the soluble fractions. Therefore, we extracted IbpA from these aggregates by dissolving them in 6 m urea. Further steps of the purification were performed under denaturing conditions. At the final step of the purification urea was slowly dialyzed out from the IbpA preparation. Following such a procedure, soluble IbpA protein was obtained. The purified IbpA formed high molecular weight oligomeric structures that deoligomerized into smaller structures following incubation at 48 °C (results not shown). Presence of Both IbpA and IbpB during Thermal Denaturation of Substrate Protein Is Required for Subsequent Reactivation by ClpB-K/J/E System—Previous work (14.Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (659) Google Scholar, 15.Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (639) Google Scholar, 16.Veinger L. Diamant S. Buchner J. Goloubinoff P. J. Biol. Chem. 1998; 273: 11032-11037Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 17.Lee G.J. Vierling E. Plant Physiol. 2000; 122: 189-198Crossref PubMed Scopus (377) Google Scholar, 18.Mogk A. Deuerling E. Vorderwulbecke S. Vierling E. Bukau B. Mol. Microbiol. 2003; 50: 585-595Crossref PubMed Scopus (304) Google Scholar, 19.Mogk A. Schlieker C. Friedrich K.L. Schönfeld H.-J. Vierling E. Bukau B. J. Biol. Chem. 2003; 278: 31033-31042Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) defined the sHsps as factors, which when present during substrate protein denaturation, increase the ability of chaperones from the Hsp100 and Hsp70 families to refold these aggregates into active protein. We used this assay to investigate the chaperone activity of purified IbpA. Firefly luciferase was thermally denatured at 48 °C in the presence or absence of IbpA, followed by the addition of ClpB (Hsp100) and DnaK/DnaJ/GrpE (Hsp70 and cochaperones). Following 1-h reactivation at 25 °C, the luciferase activity was tested. The presence of IbpA during luciferase denaturation did not increase the efficiency of its refolding by the ClpB-K/J/E bi-chaperone system, although a wide range of IbpA concentrations was tested for a possible stabilizing effect (Fig. 2A). Because both ibpA and ibpB are located in one operon in E. coli (37.Chuang S.E. Burland V. Plunkett G. Daniels D.L. Blattner F.R. Gene (Amst.). 1993; 134: 1-6Crossref PubMed Scopus (138) Google Scholar) and it was shown that sHsps from other bacteria form heterooligomers (34.Studer S. Narberhaus F. J. Biol. Chem. 2000; 275: 37212-37218Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), it is plausible that IbpA and IbpB cooperate in substrate stabilization during thermal stress. Therefore, we purified IbpB (Fig. 1) and included it in our in vitro reactivation experiments. The presence of IbpB alone during luciferase denaturation only slightly increased (from 14% to 33%) the efficiency of luciferase refolding by the ClpB-K/J/E chaperone system (Fig. 2B). Moreover, this effect was observed only for high concentrations of IbpB. However, when both IbpB and IbpA were added simultaneously to the reaction, a significant increase in luciferase activity was observed following reactivation by the ClpB-K/J/E chaperones (Fig. 2). To find the optimal concentrations of IbpA and IbpB necessary for this stabilizing effect, titrations of both small chaperones were performed. The most efficient reactivation was observed for ∼2 μm IbpA (Fig. 2A) and ∼7.8 μm IbpB (Fig. 2B). Therefore these concentrations of sHsps were used throughout this report in reactivation experiments. We also tested at which step of the denaturation/reactivation process the simultaneous presence of IbpA and IbpB was required. IbpA/IbpB when present during either the thermal inactivation step or the reactivation step, and in the absence of the ClpB-K/J/E bi-chaperone system, were not able to protect or to recover luciferase activity (Fig. 2C). Moreover, thermal denaturation followed by addition of IbpA/B and bi-chaperone system to the reactivation mixture did not result in an efficiency of reactivation higher than that observed for the bi-chaperone system alone. Only when the IbpA/B proteins were present during the thermal denaturation step, followed by subsequent addition of the bi-chaperone system, was the efficient recovery of luciferase activity observed (Fig. 2C). Thus we concluded that the presence of both IbpA and IbpB during thermal inactivation of luciferase is required for efficient reactivation mediated by the bi-chaperone Hsp70-Hsp100 system. Simultaneous Presence of IbpA and IbpB Results in Stabilization of the Substrate in a Disaggregation Competent State— Because in vivo studies identified the IbpA/B proteins as factors responsible for increasing E. coli thermotolerance (31.Kitagawa M. Matsumura Y. Tsuchido T. FEMS Microbiol. Lett. 2000; 184: 165-171Crossref PubMed Google Scholar), we decided to test the ability of IbpA/B to protect substrate proteins over a wide range of temperatures. First, we tested if IbpA/B are able to maintain the enzymatic activity of luciferase exposed to different temperatures. To this end, luciferase was incubated at the indicated temperatures for 10 min in the presence of IbpA/B, and its activity was assayed directly after incubation. No protective effect of IbpA/B was observed at high temperatures (above 42 °C). However, under mild heat shock conditions (36–40 °C), the minor level of luciferase protection was observed, and the luciferase activity was detected at the level of 20–30% of native control (Fig. 3A). Next, we tested how different temperatures affect the ability of IbpA and IbpB alone or in combination to maintain luciferase in a state competent for bi-chaperone mediated disaggregation and refolding (Fig. 3B). First we incubated luciferase alone at 36–52 °C. Following its thermal inactivation, the Hsp100-Hsp70 bi-chaperone system was added for reactivation. Bi-chaperone-dependent reactivation was not eff
Referência(s)