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• W2012000393 abstract "Several Escherichia coli proteins participate in protein disulfide bond formation. Among them, DsbA is the primary factor that oxidizes target cysteines. Biochemical evidence indicates that DsbC has disulfide isomerization activity. To study intracellular functions of DsbA and DsbC, we used an alkaline phosphatase mutant, PhoA[SCCC], with the most amino-terminal cysteine replaced by serine. It was found that the remaining 3 cysteines in PhoA[SCCC] form a disulfide bond of incorrect as well as correct combinations. An aberrant disulfide bond was preferentially formed in wild-type cells, which was converted slowly to the normal disulfide bond. This conversion did not occur in the dsbC-disrupted cells. Overproduction of DsbC stimulated the formation of the correct disulfide bond. In contrast, the inefficiently formed disulfide bonds in the dsbA-disrupted cells, and the more efficiently formed disulfide bonds in the same strain in the presence of oxidized glutathione were mostly in the correct form. These results suggest that the DsbA-catalyzed reaction can be too rapid for some proteins. DsbA may simply oxidize available pairs of cysteines, which happen to be in an incorrect combination in the case of PhoA[SCCC]. In contrast, DsbC stimulates the formation of correct disulfide bonds and corrects previously introduced aberrant ones. Thus, DsbC acts to isomerize disulfide bonds in vivo. Several Escherichia coli proteins participate in protein disulfide bond formation. Among them, DsbA is the primary factor that oxidizes target cysteines. Biochemical evidence indicates that DsbC has disulfide isomerization activity. To study intracellular functions of DsbA and DsbC, we used an alkaline phosphatase mutant, PhoA[SCCC], with the most amino-terminal cysteine replaced by serine. It was found that the remaining 3 cysteines in PhoA[SCCC] form a disulfide bond of incorrect as well as correct combinations. An aberrant disulfide bond was preferentially formed in wild-type cells, which was converted slowly to the normal disulfide bond. This conversion did not occur in the dsbC-disrupted cells. Overproduction of DsbC stimulated the formation of the correct disulfide bond. In contrast, the inefficiently formed disulfide bonds in the dsbA-disrupted cells, and the more efficiently formed disulfide bonds in the same strain in the presence of oxidized glutathione were mostly in the correct form. These results suggest that the DsbA-catalyzed reaction can be too rapid for some proteins. DsbA may simply oxidize available pairs of cysteines, which happen to be in an incorrect combination in the case of PhoA[SCCC]. In contrast, DsbC stimulates the formation of correct disulfide bonds and corrects previously introduced aberrant ones. Thus, DsbC acts to isomerize disulfide bonds in vivo. Disulfide bonds are found in many extracytosolic proteins in all organisms and contribute to folding and stability of these proteins. While disulfide bond formation is a simple reaction of oxidation of cysteine residues, and it can be reproduced in vitro under appropriate conditions (1Anfinsen C.B. Science. 1973; 181: 223-230Crossref PubMed Scopus (5079) Google Scholar), recent studies established that it does not occur effectively in vivo without the aid of other proteins (2Bardwell J.C.A. Mol. Microbiol. 1994; 14: 199-205Crossref PubMed Scopus (197) Google Scholar). In Escherichia coli, a periplasmic protein, DsbA, is required for disulfide bond formation in vivo (3Bardwell J.C.A. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (223) Google Scholar). It directly oxidizes cysteines on the target proteins in vitro(5Zapun A. Creighton T.E. Biochemistry. 1994; 33: 5202-5211Crossref PubMed Scopus (88) Google Scholar, 6Akiyama Y. Kamitani S. Kusukawa N. Ito K. J. Biol. Chem. 1992; 267: 22440-22445Abstract Full Text PDF PubMed Google Scholar). It has a thioredoxin-like Cys30-X-X-Cys motif characteristically found in disulfide oxidoreductases (7Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar).DsbB, an integral membrane protein, is also required for the processes (8Bardwell J.C.A. Lee J.-O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1038-1042Crossref PubMed Scopus (357) Google Scholar). The role of DsbB is to reoxidize DsbA to enable its catalytic turnover (8Bardwell J.C.A. Lee J.-O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1038-1042Crossref PubMed Scopus (357) Google Scholar, 9Kishigami S. Kanaya E. Kikuchi M. Ito K. J. Biol. Chem. 1995; 270: 17072-17074Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 10Guilhot C. Jander G. Martin L.N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9895-9899Crossref PubMed Scopus (128) Google Scholar, 11Kishigami S. Ito K. Genes Cells. 1996; 1: 201-208Crossref PubMed Scopus (47) Google Scholar). Genes dsbC and dsbD(dipZ) also encodes factors involved in disulfide bond metabolism (12Missiakas D. Georgopoulos C. Raina S. EMBO J. 1994; 13: 2013-2020Crossref PubMed Scopus (190) Google Scholar, 13Missiakas D. Schwager F. Raina S. EMBO J. 1995; 14: 3415-3424Crossref PubMed Scopus (169) Google Scholar, 14Shevchik V.E. Condemine G. Robert-Baudouy J. EMBO J. 1994; 13: 2007-2012Crossref PubMed Scopus (130) Google Scholar, 15Crooke H. Cole J. Mol. Microbiol. 1995; 15: 1139-1150Crossref PubMed Scopus (114) Google Scholar). DsbD is a membrane protein with a thioredoxin-like motif in the periplasmic domain, and it may have a regulatory role of conferring reducing power to the periplasm. DsbC is a periplasmic protein with 4 cysteines among which Cys98 and Cys101 forms a thioredoxin-like motif. Creighton and his colleagues (16Zapun A. Missiakas D. Raina S. Creighton T.E. Biochemistry. 1995; 34: 5075-5089Crossref PubMed Scopus (220) Google Scholar) characterized the redox activity of DsbC using a model substrate. They showed that while DsbA merely oxidized cysteines on the substrate, DsbC efficiently isomerized preformed disulfide bonds.Bacterial alkaline phosphatase, a periplasmic protein, is a dimer of the phoA gene product (PhoA) with two intramolecular disulfide bonds (Cys168-Cys178 and Cys286-Cys336) (17Bradshaw R.A. Cancedda F. Ericsson L.H. Neumann P.E. Piccoli S.P. Schlesinger M.J. Shriefer K. Walsh K.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3473-3477Crossref PubMed Scopus (143) Google Scholar). Disulfide bond formation is essential for the correct folding of this enzyme (3Bardwell J.C.A. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (223) Google Scholar, 18Derman A.I. Beckwith J. J. Bacteriol. 1991; 173: 7719-7722Crossref PubMed Scopus (160) Google Scholar, 19Akiyama Y. Ito K. J. Biol. Chem. 1993; 286: 8146-8150Google Scholar). We found that, of the two disulfide bonds in PhoA, the carboxyl-terminal one (Cys286-Cys336) is required and sufficient for the active conformation of this enzyme (20Sone M. Kishigami S. Yoshihisa T. Ito K. J. Biol. Chem. 1997; 272: 6174-6178Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Thus, a mutant form of PhoA, termed PhoA[SSCC], with the two NH2-terminally located cysteines replaced by serine is as active as the wild-type enzyme, although it is no longer resistant to a protease. Interestingly, the presence of an additional cysteine at residue 178 lowered the enzymatic activity significantly (20Sone M. Kishigami S. Yoshihisa T. Ito K. J. Biol. Chem. 1997; 272: 6174-6178Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). We show here that this mutant PhoA, termed PhoA[SCCC], forms an aberrant disulfide bond among Cys178, Cys286, and Cys336.Using this unique experimental system, we investigated into thein vivo roles played by DsbA and DsbC. It was found that DsbA principally introduced an aberrant disulfide bond into PhoA[SCCC], whereas DsbC stimulated the eventual formation of the correct disulfide bond in vivo. Thus, DsbC functions, in concert with DsbA, as a disulfide isomerase in vivo.DISCUSSIONArtifacts in the determination of in vivo redox states of proteins can be minimized by examining them after acid denaturation (23Pollitt S. Zalkin H. J. Bacteriol. 1983; 153: 27-32Crossref PubMed Google Scholar, 27Kishigami S. Akiyama Y. Ito K. FEBS Lett. 1995; 364: 55-58Crossref PubMed Scopus (68) Google Scholar), the method employed in this study. Of the two disulfide bonds of PhoA, the amino-terminal disulfide (Cys168-Cys178) constrains a loop of only 9 amino acids, while the carboxyl-terminally located disulfide (Cys286-Cys336) constrains a loop of 49 amino acids. The fast migration of the oxidized PhoA in SDS-PAGE can essentially be ascribed to the Cys286-Cys336disulfide bond (Fig. 1). Since any incorrect disulfide bonds that can be formed in PhoA should connect cysteines that flank at least 107 amino acids (in the case of Cys178-Cys286), they are expected to confer even more increased mobility in gel electrophoresis. The ox2 species we observed in PhoA[SCCC] meets this expectation, although we have not determined whether it contains Cys178-Cys286 or Cys178-Cys336 disulfide bond.Why does PhoA[SCCC] form an aberrant disulfide bond in the presence of DsbA? Disulfide bond formation is essential for the folding of alkaline phosphatase in vivo (3Bardwell J.C.A. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (223) Google Scholar) and in vitro(19Akiyama Y. Ito K. J. Biol. Chem. 1993; 286: 8146-8150Google Scholar), triggering the subsequent folding and dimerization reactions (19Akiyama Y. Ito K. J. Biol. Chem. 1993; 286: 8146-8150Google Scholar). The fact that PhoA[SSCC] has almost 100% enzymatic activity (20Sone M. Kishigami S. Yoshihisa T. Ito K. J. Biol. Chem. 1997; 272: 6174-6178Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) indicates that the PhoA molecule without the NH2-terminal disulfide bond retains the ability to position the active site residues in proper geometry, but this event is incomplete until the carboxyl-terminal disulfide bond has been formed. In PhoA[SCCC], kinetic competition may occur between different combinations of the 3 cysteines for disulfide bond formation, and it should be modulated by ongoing folding reactions (28Frech C. Wunderlich M. Glockshuber R. Schmid F.X. Biochemistry. 1996; 35: 11386-11395Crossref PubMed Scopus (15) Google Scholar). Thus, extremely rapid disulfide bond formation may be more random than slower disulfide bond formation. Furthermore, DsbA may be so potent and efficient that it introduces or initiates to introduce a disulfide while a substrate polypeptide chain is still in the process of membrane translocation; the first translocating cysteine, Cys178, will be committed for disulfide bond formation when it reaches the periplasm and forms a transient disulfide with the reactive Cys30 residue (29Nelson J.W. Creighton T.E. Biochemistry. 1994; 33: 5974-5983Crossref PubMed Scopus (223) Google Scholar, 30Zapun A. Cooper L. Creighton T.E. Biochemistry. 1994; 33: 1907-1914Crossref PubMed Scopus (55) Google Scholar, 31Grauschopf U. Winther J.R. Korber P. Zander T. Dallinger P. Bardwell J.C.A. Cell. 1995; 83: 947-955Abstract Full Text PDF PubMed Scopus (276) Google Scholar) of DsbA. In contrast, “spontaneous” or glutathione-driven disulfide bond formation in the absence of DsbA will be less efficient and slower such that the correct combination is preferred due to the local folding properties of the polypeptide chain. DsbA-dependent formation of aberrant disulfide bonds was implicated previously (but not demonstrated) in a system where a foreign protein was expressed in E. coli (32Alksne L.E. Keenney D. Rasmussen B.A. J. Bacteriol. 1995; 177: 462-464Crossref PubMed Google Scholar, 33Alksne L.E. Rasmussen B.A. J. Bacteriol. 1996; 178: 4306-4309Crossref PubMed Google Scholar).We obtained two kinds of results that suggest that DsbC functions to stimulate the formation of the correct disulfide bond. First, slow conversion occurs from the aberrant to the correct disulfide bonds of pulse-labeled PhoA[SCCC] in wild-type cells, but not in thedsbC deletion strain. This observation clearly indicates that DsbC-dependent isomerization of disulfide bond occurs in the cell. Second, overproduction of DsbC in thedsbA + cells enhanced the rapid formation of the correct disulfide bond. This observation could be interpreted in terms of either very rapid isomerization in the presence of excess DsbC orde novo introduction of the correct disulfide bond in the presence of excess DsbC. In view of the biochemical demonstration of isomerization activity of DsbC (16Zapun A. Missiakas D. Raina S. Creighton T.E. Biochemistry. 1995; 34: 5075-5089Crossref PubMed Scopus (220) Google Scholar), we think it reasonable to assume the rapid isomerization. This is consistent with the observation (Fig.3, D and H) that DsbA is required for DsbC to exhibit the correct disulfide-introducing function.Recently, Rietsch et al. (34Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) reported in vivoresults that a dsbC disruption had no effect on OmpA (normally with a single disulfide bond), that it resulted in accumulation of a small amount of reduced PhoA 4Although these authors suggest that it was an aberrant form, we believe that it must have been the reduced form because of the discussion already given about the electrophoretic mobilities of PhoA. That it disappeared when the culture received dithiothreitol before pulse labeling (34Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) could be explained. For instance, if dithiothreitol can access the periplasm freely but iodoacetamide is somehow restricted, the low concentration of dithiothreitol might antagonize the action of iodoacetamide added after sampling, leaving free cysteines unmodified, which will then be oxidized after cell pelleting and solubilization in SDS. , and that it resulted in greatly diminished production of active urokinase (normally with 12 disulfide bonds). Although these results are consistent with their interpretation that DsbC has a disulfide isomerizing role in vivo, the evidence for the aberrant disulfide bond formation in the absence of DsbC was inconclusive.Our results provide some additional information about the DsbC functions in vivo. Excess DsbC was inhibitory against the background disulfide bond formation that occurs inefficiently in the absence of DsbA. Probably, the reducing character of DsbC might dominate the air oxidation. It was also found, however, that excess DsbC can exhibit DsbA-like function when GSSG was supplemented to the medium. This is consistent with another finding of Rietsch et al. (34Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) that the suppression of dsbA null mutation by loss-of-function dsbD (dipZ) mutations requires the functional DsbC. They proposed that DsbD (DipZ) together with thioredoxin normally keeps DsbC in the partially reduced state, and the loss of reducing factor leads to the production of oxidized DsbC, which in turn substitutes for DsbA. This model is consistent with our results; excess DsbC is oxidized by GSSG and then it substitutes for DsbA.We have now demonstrated that the principal role of DsbC is to facilitate disulfide bonds that are formed between correct pairs of cysteines. This demonstration was made possible by using a unique model protein, PhoA[SCCC]. The results of Rietsch et al. (34Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) that the formation of active urokinase, a natural (but still foreign toE. coli) protein with multiple disulfide bonds, depends absolutely on DsbC also supports this notion. These in vivoresults establish that the cellular role of DsbC lies in the isomerization of disulfide bonds until the protein has been folded into a stable conformation. Disulfide bonds are found in many extracytosolic proteins in all organisms and contribute to folding and stability of these proteins. While disulfide bond formation is a simple reaction of oxidation of cysteine residues, and it can be reproduced in vitro under appropriate conditions (1Anfinsen C.B. Science. 1973; 181: 223-230Crossref PubMed Scopus (5079) Google Scholar), recent studies established that it does not occur effectively in vivo without the aid of other proteins (2Bardwell J.C.A. Mol. Microbiol. 1994; 14: 199-205Crossref PubMed Scopus (197) Google Scholar). In Escherichia coli, a periplasmic protein, DsbA, is required for disulfide bond formation in vivo (3Bardwell J.C.A. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (223) Google Scholar). It directly oxidizes cysteines on the target proteins in vitro(5Zapun A. Creighton T.E. Biochemistry. 1994; 33: 5202-5211Crossref PubMed Scopus (88) Google Scholar, 6Akiyama Y. Kamitani S. Kusukawa N. Ito K. J. Biol. Chem. 1992; 267: 22440-22445Abstract Full Text PDF PubMed Google Scholar). It has a thioredoxin-like Cys30-X-X-Cys motif characteristically found in disulfide oxidoreductases (7Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). DsbB, an integral membrane protein, is also required for the processes (8Bardwell J.C.A. Lee J.-O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1038-1042Crossref PubMed Scopus (357) Google Scholar). The role of DsbB is to reoxidize DsbA to enable its catalytic turnover (8Bardwell J.C.A. Lee J.-O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1038-1042Crossref PubMed Scopus (357) Google Scholar, 9Kishigami S. Kanaya E. Kikuchi M. Ito K. J. Biol. Chem. 1995; 270: 17072-17074Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 10Guilhot C. Jander G. Martin L.N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9895-9899Crossref PubMed Scopus (128) Google Scholar, 11Kishigami S. Ito K. Genes Cells. 1996; 1: 201-208Crossref PubMed Scopus (47) Google Scholar). Genes dsbC and dsbD(dipZ) also encodes factors involved in disulfide bond metabolism (12Missiakas D. Georgopoulos C. Raina S. EMBO J. 1994; 13: 2013-2020Crossref PubMed Scopus (190) Google Scholar, 13Missiakas D. Schwager F. Raina S. EMBO J. 1995; 14: 3415-3424Crossref PubMed Scopus (169) Google Scholar, 14Shevchik V.E. Condemine G. Robert-Baudouy J. EMBO J. 1994; 13: 2007-2012Crossref PubMed Scopus (130) Google Scholar, 15Crooke H. Cole J. Mol. Microbiol. 1995; 15: 1139-1150Crossref PubMed Scopus (114) Google Scholar). DsbD is a membrane protein with a thioredoxin-like motif in the periplasmic domain, and it may have a regulatory role of conferring reducing power to the periplasm. DsbC is a periplasmic protein with 4 cysteines among which Cys98 and Cys101 forms a thioredoxin-like motif. Creighton and his colleagues (16Zapun A. Missiakas D. Raina S. Creighton T.E. Biochemistry. 1995; 34: 5075-5089Crossref PubMed Scopus (220) Google Scholar) characterized the redox activity of DsbC using a model substrate. They showed that while DsbA merely oxidized cysteines on the substrate, DsbC efficiently isomerized preformed disulfide bonds. Bacterial alkaline phosphatase, a periplasmic protein, is a dimer of the phoA gene product (PhoA) with two intramolecular disulfide bonds (Cys168-Cys178 and Cys286-Cys336) (17Bradshaw R.A. Cancedda F. Ericsson L.H. Neumann P.E. Piccoli S.P. Schlesinger M.J. Shriefer K. Walsh K.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3473-3477Crossref PubMed Scopus (143) Google Scholar). Disulfide bond formation is essential for the correct folding of this enzyme (3Bardwell J.C.A. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (223) Google Scholar, 18Derman A.I. Beckwith J. J. Bacteriol. 1991; 173: 7719-7722Crossref PubMed Scopus (160) Google Scholar, 19Akiyama Y. Ito K. J. Biol. Chem. 1993; 286: 8146-8150Google Scholar). We found that, of the two disulfide bonds in PhoA, the carboxyl-terminal one (Cys286-Cys336) is required and sufficient for the active conformation of this enzyme (20Sone M. Kishigami S. Yoshihisa T. Ito K. J. Biol. Chem. 1997; 272: 6174-6178Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Thus, a mutant form of PhoA, termed PhoA[SSCC], with the two NH2-terminally located cysteines replaced by serine is as active as the wild-type enzyme, although it is no longer resistant to a protease. Interestingly, the presence of an additional cysteine at residue 178 lowered the enzymatic activity significantly (20Sone M. Kishigami S. Yoshihisa T. Ito K. J. Biol. Chem. 1997; 272: 6174-6178Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). We show here that this mutant PhoA, termed PhoA[SCCC], forms an aberrant disulfide bond among Cys178, Cys286, and Cys336. Using this unique experimental system, we investigated into thein vivo roles played by DsbA and DsbC. It was found that DsbA principally introduced an aberrant disulfide bond into PhoA[SCCC], whereas DsbC stimulated the eventual formation of the correct disulfide bond in vivo. Thus, DsbC functions, in concert with DsbA, as a disulfide isomerase in vivo. DISCUSSIONArtifacts in the determination of in vivo redox states of proteins can be minimized by examining them after acid denaturation (23Pollitt S. Zalkin H. J. Bacteriol. 1983; 153: 27-32Crossref PubMed Google Scholar, 27Kishigami S. Akiyama Y. Ito K. FEBS Lett. 1995; 364: 55-58Crossref PubMed Scopus (68) Google Scholar), the method employed in this study. Of the two disulfide bonds of PhoA, the amino-terminal disulfide (Cys168-Cys178) constrains a loop of only 9 amino acids, while the carboxyl-terminally located disulfide (Cys286-Cys336) constrains a loop of 49 amino acids. The fast migration of the oxidized PhoA in SDS-PAGE can essentially be ascribed to the Cys286-Cys336disulfide bond (Fig. 1). Since any incorrect disulfide bonds that can be formed in PhoA should connect cysteines that flank at least 107 amino acids (in the case of Cys178-Cys286), they are expected to confer even more increased mobility in gel electrophoresis. The ox2 species we observed in PhoA[SCCC] meets this expectation, although we have not determined whether it contains Cys178-Cys286 or Cys178-Cys336 disulfide bond.Why does PhoA[SCCC] form an aberrant disulfide bond in the presence of DsbA? Disulfide bond formation is essential for the folding of alkaline phosphatase in vivo (3Bardwell J.C.A. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (223) Google Scholar) and in vitro(19Akiyama Y. Ito K. J. Biol. Chem. 1993; 286: 8146-8150Google Scholar), triggering the subsequent folding and dimerization reactions (19Akiyama Y. Ito K. J. Biol. Chem. 1993; 286: 8146-8150Google Scholar). The fact that PhoA[SSCC] has almost 100% enzymatic activity (20Sone M. Kishigami S. Yoshihisa T. Ito K. J. Biol. Chem. 1997; 272: 6174-6178Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) indicates that the PhoA molecule without the NH2-terminal disulfide bond retains the ability to position the active site residues in proper geometry, but this event is incomplete until the carboxyl-terminal disulfide bond has been formed. In PhoA[SCCC], kinetic competition may occur between different combinations of the 3 cysteines for disulfide bond formation, and it should be modulated by ongoing folding reactions (28Frech C. Wunderlich M. Glockshuber R. Schmid F.X. Biochemistry. 1996; 35: 11386-11395Crossref PubMed Scopus (15) Google Scholar). Thus, extremely rapid disulfide bond formation may be more random than slower disulfide bond formation. Furthermore, DsbA may be so potent and efficient that it introduces or initiates to introduce a disulfide while a substrate polypeptide chain is still in the process of membrane translocation; the first translocating cysteine, Cys178, will be committed for disulfide bond formation when it reaches the periplasm and forms a transient disulfide with the reactive Cys30 residue (29Nelson J.W. Creighton T.E. Biochemistry. 1994; 33: 5974-5983Crossref PubMed Scopus (223) Google Scholar, 30Zapun A. Cooper L. Creighton T.E. Biochemistry. 1994; 33: 1907-1914Crossref PubMed Scopus (55) Google Scholar, 31Grauschopf U. Winther J.R. Korber P. Zander T. Dallinger P. Bardwell J.C.A. Cell. 1995; 83: 947-955Abstract Full Text PDF PubMed Scopus (276) Google Scholar) of DsbA. In contrast, “spontaneous” or glutathione-driven disulfide bond formation in the absence of DsbA will be less efficient and slower such that the correct combination is preferred due to the local folding properties of the polypeptide chain. DsbA-dependent formation of aberrant disulfide bonds was implicated previously (but not demonstrated) in a system where a foreign protein was expressed in E. coli (32Alksne L.E. Keenney D. Rasmussen B.A. J. Bacteriol. 1995; 177: 462-464Crossref PubMed Google Scholar, 33Alksne L.E. Rasmussen B.A. J. Bacteriol. 1996; 178: 4306-4309Crossref PubMed Google Scholar).We obtained two kinds of results that suggest that DsbC functions to stimulate the formation of the correct disulfide bond. First, slow conversion occurs from the aberrant to the correct disulfide bonds of pulse-labeled PhoA[SCCC] in wild-type cells, but not in thedsbC deletion strain. This observation clearly indicates that DsbC-dependent isomerization of disulfide bond occurs in the cell. Second, overproduction of DsbC in thedsbA + cells enhanced the rapid formation of the correct disulfide bond. This observation could be interpreted in terms of either very rapid isomerization in the presence of excess DsbC orde novo introduction of the correct disulfide bond in the presence of excess DsbC. In view of the biochemical demonstration of isomerization activity of DsbC (16Zapun A. Missiakas D. Raina S. Creighton T.E. Biochemistry. 1995; 34: 5075-5089Crossref PubMed Scopus (220) Google Scholar), we think it reasonable to assume the rapid isomerization. This is consistent with the observation (Fig.3, D and H) that DsbA is required for DsbC to exhibit the correct disulfide-introducing function.Recently, Rietsch et al. (34Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) reported in vivoresults that a dsbC disruption had no effect on OmpA (normally with a single disulfide bond), that it resulted in accumulation of a small amount of reduced PhoA 4Although these authors suggest that it was an aberrant form, we believe that it must have been the reduced form because of the discussion already given about the electrophoretic mobilities of PhoA. That it disappeared when the culture received dithiothreitol before pulse labeling (34Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) could be explained. For instance, if dithiothreitol can access the periplasm freely but iodoacetamide is somehow restricted, the low concentration of dithiothreitol might antagonize the action of iodoacetamide added after sampling, leaving free cysteines unmodified, which will then be oxidized after cell pelleting and solubilization in SDS. , and that it resulted in greatly diminished production of active urokinase (normally with 12 disulfide bonds). Although these results are consistent with their interpretation that DsbC has a disulfide isomerizing role in vivo, the evidence for the aberrant disulfide bond formation in the absence of DsbC was inconclusive.Our results provide some additional information about the DsbC functions in vivo. Excess DsbC was inhibitory against the background disulfide bond formation that occurs inefficiently in the absence of DsbA. Probably, the reducing character of DsbC might dominate the air oxidation. It was also found, however, that excess DsbC can exhibit DsbA-like function when GSSG was supplemented to the medium. This is consistent with another finding of Rietsch et al. (34Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) that the suppression of dsbA null mutation by loss-of-function dsbD (dipZ) mutations requires the functional DsbC. They proposed that DsbD (DipZ) together with thioredoxin normally keeps DsbC in the partially reduced state, and the loss of reducing factor leads to the production of oxidized DsbC, which in turn substitutes for DsbA. This model is consistent with our results; excess DsbC is oxidized by GSSG and then it substitutes for DsbA.We have now demonstrated that the principal role of DsbC is to facilitate disulfide bonds that are formed between correct pairs of cysteines. This demonstration was made possible by using a unique model protein, PhoA[SCCC]. The results of Rietsch et al. (34Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) that the formation of active urokinase, a natural (but still foreign toE. coli) protein with multiple disulfide bonds, depends absolutely on DsbC also supports this notion. These in vivoresults establish that the cellular role of DsbC lies in the isomerization of disulfide bonds until the protein has been folded into a stable conformation. Artifacts in the determination of in vivo redox states of proteins can be minimized by examining them after acid denaturation (23Pollitt S. Zalkin H. J. Bacteriol. 1983; 153: 27-32Crossref PubMed Google Scholar, 27Kishigami S. Akiyama Y. Ito K. FEBS Lett. 1995; 364: 55-58Crossref PubMed Scopus (68) Google Scholar), the method employed in this study. Of the two disulfide bonds of PhoA, the amino-terminal disulfide (Cys168-Cys178) constrains a loop of only 9 amino acids, while the carboxyl-terminally located disulfide (Cys286-Cys336) constrains a loop of 49 amino acids. The fast migration of the oxidized PhoA in SDS-PAGE can essentially be ascribed to the Cys286-Cys336disulfide bond (Fig. 1). Since any incorrect disulfide bonds that can be formed in PhoA should connect cysteines that flank at least 107 amino acids (in the case of Cys178-Cys286), they are expected to confer even more increased mobility in gel electrophoresis. The ox2 species we observed in PhoA[SCCC] meets this expectation, although we have not determined whether it contains Cys178-Cys286 or Cys178-Cys336 disulfide bond. Why does PhoA[SCCC] form an aberrant disulfide bond in the presence of DsbA? Disulfide bond formation is essential for the folding of alkaline phosphatase in vivo (3Bardwell J.C.A. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (223) Google Scholar) and in vitro(19Akiyama Y. Ito K. J. Biol. Chem. 1993; 286: 8146-8150Google Scholar), triggering the subsequent folding and dimerization reactions (19Akiyama Y. Ito K. J. Biol. Chem. 1993; 286: 8146-8150Google Scholar). The fact that PhoA[SSCC] has almost 100% enzymatic activity (20Sone M. Kishigami S. Yoshihisa T. Ito K. J. Biol. Chem. 1997; 272: 6174-6178Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) indicates that the PhoA molecule without the NH2-terminal disulfide bond retains the ability to position the active site residues in proper geometry, but this event is incomplete until the carboxyl-terminal disulfide bond has been formed. In PhoA[SCCC], kinetic competition may occur between different combinations of the 3 cysteines for disulfide bond formation, and it should be modulated by ongoing folding reactions (28Frech C. Wunderlich M. Glockshuber R. Schmid F.X. Biochemistry. 1996; 35: 11386-11395Crossref PubMed Scopus (15) Google Scholar). Thus, extremely rapid disulfide bond formation may be more random than slower disulfide bond formation. Furthermore, DsbA may be so potent and efficient that it introduces or initiates to introduce a disulfide while a substrate polypeptide chain is still in the process of membrane translocation; the first translocating cysteine, Cys178, will be committed for disulfide bond formation when it reaches the periplasm and forms a transient disulfide with the reactive Cys30 residue (29Nelson J.W. Creighton T.E. Biochemistry. 1994; 33: 5974-5983Crossref PubMed Scopus (223) Google Scholar, 30Zapun A. Cooper L. Creighton T.E. Biochemistry. 1994; 33: 1907-1914Crossref PubMed Scopus (55) Google Scholar, 31Grauschopf U. Winther J.R. Korber P. Zander T. Dallinger P. Bardwell J.C.A. Cell. 1995; 83: 947-955Abstract Full Text PDF PubMed Scopus (276) Google Scholar) of DsbA. In contrast, “spontaneous” or glutathione-driven disulfide bond formation in the absence of DsbA will be less efficient and slower such that the correct combination is preferred due to the local folding properties of the polypeptide chain. DsbA-dependent formation of aberrant disulfide bonds was implicated previously (but not demonstrated) in a system where a foreign protein was expressed in E. coli (32Alksne L.E. Keenney D. Rasmussen B.A. J. Bacteriol. 1995; 177: 462-464Crossref PubMed Google Scholar, 33Alksne L.E. Rasmussen B.A. J. Bacteriol. 1996; 178: 4306-4309Crossref PubMed Google Scholar). We obtained two kinds of results that suggest that DsbC functions to stimulate the formation of the correct disulfide bond. First, slow conversion occurs from the aberrant to the correct disulfide bonds of pulse-labeled PhoA[SCCC] in wild-type cells, but not in thedsbC deletion strain. This observation clearly indicates that DsbC-dependent isomerization of disulfide bond occurs in the cell. Second, overproduction of DsbC in thedsbA + cells enhanced the rapid formation of the correct disulfide bond. This observation could be interpreted in terms of either very rapid isomerization in the presence of excess DsbC orde novo introduction of the correct disulfide bond in the presence of excess DsbC. In view of the biochemical demonstration of isomerization activity of DsbC (16Zapun A. Missiakas D. Raina S. Creighton T.E. Biochemistry. 1995; 34: 5075-5089Crossref PubMed Scopus (220) Google Scholar), we think it reasonable to assume the rapid isomerization. This is consistent with the observation (Fig.3, D and H) that DsbA is required for DsbC to exhibit the correct disulfide-introducing function. Recently, Rietsch et al. (34Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) reported in vivoresults that a dsbC disruption had no effect on OmpA (normally with a single disulfide bond), that it resulted in accumulation of a small amount of reduced PhoA 4Although these authors suggest that it was an aberrant form, we believe that it must have been the reduced form because of the discussion already given about the electrophoretic mobilities of PhoA. That it disappeared when the culture received dithiothreitol before pulse labeling (34Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) could be explained. For instance, if dithiothreitol can access the periplasm freely but iodoacetamide is somehow restricted, the low concentration of dithiothreitol might antagonize the action of iodoacetamide added after sampling, leaving free cysteines unmodified, which will then be oxidized after cell pelleting and solubilization in SDS. , and that it resulted in greatly diminished production of active urokinase (normally with 12 disulfide bonds). Although these results are consistent with their interpretation that DsbC has a disulfide isomerizing role in vivo, the evidence for the aberrant disulfide bond formation in the absence of DsbC was inconclusive. Our results provide some additional information about the DsbC functions in vivo. Excess DsbC was inhibitory against the background disulfide bond formation that occurs inefficiently in the absence of DsbA. Probably, the reducing character of DsbC might dominate the air oxidation. It was also found, however, that excess DsbC can exhibit DsbA-like function when GSSG was supplemented to the medium. This is consistent with another finding of Rietsch et al. (34Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) that the suppression of dsbA null mutation by loss-of-function dsbD (dipZ) mutations requires the functional DsbC. They proposed that DsbD (DipZ) together with thioredoxin normally keeps DsbC in the partially reduced state, and the loss of reducing factor leads to the production of oxidized DsbC, which in turn substitutes for DsbA. This model is consistent with our results; excess DsbC is oxidized by GSSG and then it substitutes for DsbA. We have now demonstrated that the principal role of DsbC is to facilitate disulfide bonds that are formed between correct pairs of cysteines. This demonstration was made possible by using a unique model protein, PhoA[SCCC]. The results of Rietsch et al. (34Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) that the formation of active urokinase, a natural (but still foreign toE. coli) protein with multiple disulfide bonds, depends absolutely on DsbC also supports this notion. These in vivoresults establish that the cellular role of DsbC lies in the isomerization of disulfide bonds until the protein has been folded into a stable conformation. ACKNOWLEDGEMENTS We thank Dr. John Joly for generously providing the dsbC deletion strain, Dr. Guy Condemine for pDS30, and Kiyoko Mochizuki for laboratory supplies. Differential in vivo roles played by DsbA and DsbC in the formation of protein disulfide bonds.Journal of Biological ChemistryVol. 273Issue 42PreviewPlasmid pMS002 and its derivatives used in the above two publications proved to contain an additional mutation for a Ser-401 → Cys substitution within PhoA. We traced this mutation back to the phoA plasmid (provided by others) that was used to substitute the amplified segment, as described in the first publication (page 6174, “Experimental Procedures”). Given this fact, we eliminated this unwanted mutation from most of the plasmid constructions and repeated key experiments presented in both publications. Full-Text PDF Open Access" @default.
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• W2012000393 title "Differential in Vivo Roles Played by DsbA and DsbC in the Formation of Protein Disulfide Bonds" @default.
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