SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2021024369> ?p ?o ?g. }

Showing items 1 to 100 of ±109 with 100 items per page.

W2021024369 endingPage "19605" @default.
W2021024369 startingPage "19601" @default.
W2021024369 abstract "DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitroreactivation and suppressing aggregation of denaturedd-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled andS-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides andS-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI. DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitroreactivation and suppressing aggregation of denaturedd-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled andS-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides andS-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI. protein disulfide isomerase d-glyceraldehyde-3-phosphate dehydrogenase isopropyl-β-d-thiogalactopyranoside scrambled RNase S-carboxymethylated PDI S-carboxymethylated DsbC dithiothreitol 5,5′-dithiobis(2-nitrobenzoic acid) bovine serum albumin guanidine hydrochloride 1-anilino-8-naphthalenesulfonic acid thiol-protein oxidoreductase protein disulfide oxidoreductase glutathione disulfide Disulfide bonds are important for conformational stability and biological activity of proteins and intimately involved in protein folding. The formation of disulfide bonds is now known to be catalyzed by protein disulfide isomerase (PDI)1 in endoplasmic reticulum for eukaryotes (1Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (655) Google Scholar) and by proteins of the Dsb family in periplasm for Gram-negative bacteria (2Missiakas D. Raina S. J. Bacteriol. 1997; 179: 2465-2471Crossref PubMed Scopus (214) Google Scholar). DsbA, a monomeric soluble member of the Dsb family is the primary factor that catalyzes the oxidative formation of disulfide bonds (3Sone M. Akiyama Y. Ito K. J. Biol. Chem. 1997; 272: 10349-10352Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). DsbC is a homodimer with four thiol groups in each 23-kDa subunit, two in the active site -Cys98-Gly-Tyr-Cys101- and the other two at positions Cys141 and Cys163 (4Shevchik V.E. Condemine G. Robert-Baudouy J. EMBO J. 1994; 13: 2007-2012Crossref PubMed Scopus (130) Google Scholar, 5Missiakas D. Georgopoulos C. Raina S. EMBO J. 1994; 13: 2013-2020Crossref PubMed Scopus (190) Google Scholar). DsbC catalyzes the rearrangement of disulfide bonds in concert with DsbA for the formation of native disulfides and has been recognized as a counterpart of eukaryotic PDI (3Sone M. Akiyama Y. Ito K. J. Biol. Chem. 1997; 272: 10349-10352Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 6Zapun A. Missiakas D. Raina S. Creighton T.E. Biochemistry. 1995; 34: 5075-5089Crossref PubMed Scopus (220) Google Scholar, 7Joly J. Swartz J. Biochemistry. 1997; 36: 10067-10072Crossref PubMed Scopus (118) Google Scholar). Secondary structure prediction of DsbC suggests that the molecule appears to consist of two domains (8Frishman D. Biochem. Biophys. Res. Commun. 1996; 219: 686-689Crossref PubMed Scopus (12) Google Scholar). Preliminary x-ray diffraction data of native and selenomethionine DsbC have been reported (9Rybin V. Zapun A. Torronen A. Raina S. Misssiakas D. Creighton T.E. Metcalf P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 1219-1221Crossref PubMed Scopus (6) Google Scholar).Many in vitro and in vivo data have supported that as a foldase, PDI displays both isomerase and chaperone activities (10Wang C.C. Tsou C.L. FASEB J. 1993; 7: 1515-1517Crossref PubMed Scopus (141) Google Scholar, 11Cai H. Wang C.C. Tsou C.L. J. Biol. Chem. 1994; 269: 24550-24552Abstract Full Text PDF PubMed Google Scholar, 12Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 7764-7771Abstract Full Text PDF PubMed Google Scholar, 13Song J.L. Wang C.C. Eur. J. Biochem. 1995; 231: 312-316Crossref PubMed Scopus (136) Google Scholar, 14Hayano T. Hirose M. Kikuchi M. FEBS Lett. 1995; 377: 505-511Crossref PubMed Scopus (84) Google Scholar, 15Lamberg A. Jauhiainen M. Metso J. Ehnholm C. Shoulders C. Scott J. Pihlajaniemi T. Kivirikko K.I. Biochem. J. 1996; 315: 533-536Crossref PubMed Scopus (55) Google Scholar, 16Wang C.C. Guzman N.A. Prolyl Hydroxylase, Protein Disulfide Isomerase, and Other Structurally Related Proteins. Marcel Dekker Inc., New York1997: 315-339Google Scholar, 17Yao Y. Zhou Y.C. Wang C.C. EMBO J. 1997; 16: 651-658Crossref PubMed Scopus (112) Google Scholar, 18Wang C.C. Biochemistry (Moscow). 1998; 63: 407-412PubMed Google Scholar). The chaperone activity of PDI greatly increases its efficiency as a foldase in promoting protein folding and in catalyzing the formation of native disulfide bonds (19Wang C.C. Tsou C.L. FEBS Lett. 1998; 425: 382-384Crossref PubMed Scopus (41) Google Scholar).Most disulfide-bonded proteins in bacteria are located in the periplasm and the outer-membrane or are secreted to extracellular medium through the periplasm as their disulfide bonds can be formed readily in the oxidative periplasm but not in the reductive cytoplasm (2Missiakas D. Raina S. J. Bacteriol. 1997; 179: 2465-2471Crossref PubMed Scopus (214) Google Scholar). The bacterial periplasm is separated from the environment by only the permeable outer membrane and is therefore liable to be affected by environmental fluctuation. However, no classical molecular chaperone has been identified in the periplasm so far (2Missiakas D. Raina S. J. Bacteriol. 1997; 179: 2465-2471Crossref PubMed Scopus (214) Google Scholar). Hence, we are interested in examining whether DsbC, like its eukaryotic counterpart PDI, has chaperone activity to promote the formation of correct disulfide bonds during protein folding. In fact, DsbA has been suggested to function as a chaperone in the maturation of secreted virulence factors of Vibrio cholerae (20Peek J.A. Taylor R.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6210-6214Crossref PubMed Scopus (184) Google Scholar) and in pilus biogenesis of Escherichia coli. (21Jacob-Dubuisson F. Pinkner J. Xu Z. Striker R. Padmanhaban A. Hultgren S.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11552-11556Crossref PubMed Scopus (127) Google Scholar). The high-resolution crystal structure analysis of DsbA (22Guddat L.W. Bardwell J.C.A. Zander T. Martin J.L. Protein Sci. 1997; 6: 1148-1156Crossref PubMed Scopus (77) Google Scholar) has shown that an extensive uncharged surface surrounding the active-site disulfide is responsible for peptide binding. We have also presented evidence for the chaperone activity of DsbA in assisting the in vitro refolding ofd-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (23Zheng W.D. Quan H. Song J.L. Yang S.L. Wang C.C. Arch. Biochem. Biophys. 1997; 337: 326-331Crossref PubMed Scopus (38) Google Scholar). Recently, Darby et al. (24Darby N.J. Raina S. Creighton T.E. Biochemstry. 1998; 37: 783-791Crossref PubMed Scopus (40) Google Scholar) reported more substantial noncovalent interactions between peptides with DsbC than with DsbA.In this report, we propose to show an even stronger chaperone activity of DsbC than that of PDI for protein folding in vitro with possibly different mechanisms for target protein binding.DISCUSSIONIn bacteria periplasm, DsbA and DsbC are the two soluble members of Dsb family directly involved in the formation of disulfide bonds. However, compared with eukaryotic PDI, the in vitroisomerase activity is about 30% for DsbC and only 5% for DsbA (23Zheng W.D. Quan H. Song J.L. Yang S.L. Wang C.C. Arch. Biochem. Biophys. 1997; 337: 326-331Crossref PubMed Scopus (38) Google Scholar).It has been suggested that, in evolution, some enzymes acquired substrate binding ability in a chaperone-like mode to improve their catalytic efficiency, such as PDI, the ATP-dependent proteases, or trigger factors (19Wang C.C. Tsou C.L. FEBS Lett. 1998; 425: 382-384Crossref PubMed Scopus (41) Google Scholar). DsbA has been found to have chaperone activity in vitro (23Zheng W.D. Quan H. Song J.L. Yang S.L. Wang C.C. Arch. Biochem. Biophys. 1997; 337: 326-331Crossref PubMed Scopus (38) Google Scholar) and suggested to function as a chaperone in vivo (20Peek J.A. Taylor R.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6210-6214Crossref PubMed Scopus (184) Google Scholar, 21Jacob-Dubuisson F. Pinkner J. Xu Z. Striker R. Padmanhaban A. Hultgren S.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11552-11556Crossref PubMed Scopus (127) Google Scholar). Recently, the requirement for DsbA in pullulanase secretion has been reported to be unrelated to its ability to catalyze the formation of disulfide bond, but possibly because of its chaperone-like effect on folding of the enzyme and presentation of the secretion signal (33Sauvonnet N. Pugsley A.P. Mol. Microbiol. 1998; 27: 661-667Crossref PubMed Scopus (27) Google Scholar). The peptide binding site of DsbA has been identified by high resolution crystallography (22Guddat L.W. Bardwell J.C.A. Zander T. Martin J.L. Protein Sci. 1997; 6: 1148-1156Crossref PubMed Scopus (77) Google Scholar), and secondary structure prediction of DsbC has suggested a similar structure to that of DsbA in that they both have an extra domain in addition to the thioredoxin-like domain (8Frishman D. Biochem. Biophys. Res. Commun. 1996; 219: 686-689Crossref PubMed Scopus (12) Google Scholar). Kinetic studies on DsbA- and DsbC-catalyzed reactions in simple unfolded peptides have revealed that the interaction between DsbC and its substrate appears to be more substantial than that between DsbA and the same peptide (24Darby N.J. Raina S. Creighton T.E. Biochemstry. 1998; 37: 783-791Crossref PubMed Scopus (40) Google Scholar). The noncovalent peptide binding ability is a prerequisite for a protein to be a chaperone. The isomerase activity of DsbC is low, but as very few periplasmic and outer membrane proteins have more than two disulfides per subunit, hence high isomerase activity may not be needed (7Joly J. Swartz J. Biochemistry. 1997; 36: 10067-10072Crossref PubMed Scopus (118) Google Scholar). The fact, that bacteria possess a large Dsb family composed of at least six members in periplasm (2Missiakas D. Raina S. J. Bacteriol. 1997; 179: 2465-2471Crossref PubMed Scopus (214) Google Scholar, 34Andersen C.L. Matthey-Dupraz A. Missiakas D. Raina S. Mol. Microbiol. 1997; 26: 121-132Crossref PubMed Scopus (91) Google Scholar) makes it possible for some Dsb proteins to function as chaperones and in this way to make up for the deficiency of molecular chaperones in this compartment.In this report, we presented evidence that DsbC indeed shows chaperone activity independent from its isomerase activity more pronounced than PDI in promoting the reactivation of denatured GAPDH and concurrently decreasing aggregation during its refolding. DsbC assists the reactivation of denatured and reduced lysozyme to a higher extent than PDI does even though it has only 30% of the disulfide isomerase activity of PDI, providing further support for the high chaperone activity of DsbC.It is likely that during refolding of denatured GAPDH, the denatured enzyme passes through several intermediates before either spontaneous reactivation or misfolding to form aggregates. The additive effect of DsbC and PDI on GAPDH reactivation suggests that DsbC and PDI may recognize different GAPDH folding intermediates, and interaction with each intermediate contributes to the overall reactivation of GAPDH. The effect of delayed addition of DsbC on GAPDH reactivation suggests that compared with PDI, DsbC may recognize earlier folding intermediates. The fact that DsbC, unlike PDI (12Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 7764-7771Abstract Full Text PDF PubMed Google Scholar, 32Song J.L. Quan H. Wang C.C. Biochem. J. 1997; 328: 841-846Crossref PubMed Scopus (28) Google Scholar), does not display anti-chaperone activity during lysozyme reactivation in HEPES buffer also suggests that DsbC and PDI bind with different intermediates, and this may contribute to the higher efficiency of DsbC in assisting the folding of some proteins as compared with PDI.It is surprising that relatively short peptides, including peptides with 10, 14, and 21 residues, inhibit neither the reductase activity of DsbC nor the assisted reactivation of GAPDH. It appears that the interactions of these peptides with DsbC are not strong enough to compete with the binding of the GAPDH folding intermediates. The fact, that sRNase does inhibit both the assisted reactivation of GAPDH and the PDOR activity of DsbC offers a further hint that DsbC has a large surface for peptide binding. It seems that DsbC has a somehow extended surface for peptide binding so that only a relatively large unfolded peptide is able to compete with the target folding intermediate for binding to DsbC. It was suggested by Darby et al. (24Darby N.J. Raina S. Creighton T.E. Biochemstry. 1998; 37: 783-791Crossref PubMed Scopus (40) Google Scholar) that DsbA binds an extended peptide chain as its substrate binding area is a long cleft and appears to be in a stereochemically restricted form, whereas the binding site of DsbC may favor the binding of a compact form of the peptide molecule. The peptide binding surface of PDI might be constructed by combining sites from several domains to form a large pocket (35Darby N.J. Penka E. Vincentelli R. J. Mol. Biol. 1998; 276: 239-247Crossref PubMed Scopus (152) Google Scholar), and this could also be the case for DsbC. By kinetics comparison, Joly and Swartz (7Joly J. Swartz J. Biochemistry. 1997; 36: 10067-10072Crossref PubMed Scopus (118) Google Scholar) indicated that DsbC has a higher affinity for the misfolded insulin-like growth factor-1 substrate than PDI and DsbA. However, like the effect of native staphylococcal nuclease on the PDI-assisted GAPDH reactivation (29Quan H. Fan G.B. Wang C.C. J. Biol. Chem. 1995; 270: 17078-17080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), native RNase shows little effect on the DsbC-assisted GAPDH reactivation, indicating that DsbC and PDI do not interact with native structures. The details of the topological arrangement of the four domains in the dimeric DsbC and eight domains in dimeric PDI molecules, the peptide binding surface and its relation to the active sites, and the functional implications of such a modular structure can only be clarified by crystal structure analysis.Secondary structure prediction suggested DsbC to be composed of two domains, the C-terminal thioredoxin-like domain and the N-terminal mixed α/β domain of unknown structure (8Frishman D. Biochem. Biophys. Res. Commun. 1996; 219: 686-689Crossref PubMed Scopus (12) Google Scholar). Recent NMR determination of the PDI b domain shows that it also contains the thioredoxin motif, and PDI has thus been considered to consist of active and inactive thioredoxin modules (36Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). In this respect, it is tempting to assume that, in homology to PDI, dimeric DsbC is composed of two active-site containing domains and two domains without the active site sequence. The multiple-domain structure of PDI has been demonstrated to be essential for its high catalytic efficiency and chaperone activity by increasing substrate binding interactions (35Darby N.J. Penka E. Vincentelli R. J. Mol. Biol. 1998; 276: 239-247Crossref PubMed Scopus (152) Google Scholar). The PDI-like modular structure of DsbC might be the structural basis for its PDI-like dual activities. Disulfide bonds are important for conformational stability and biological activity of proteins and intimately involved in protein folding. The formation of disulfide bonds is now known to be catalyzed by protein disulfide isomerase (PDI)1 in endoplasmic reticulum for eukaryotes (1Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (655) Google Scholar) and by proteins of the Dsb family in periplasm for Gram-negative bacteria (2Missiakas D. Raina S. J. Bacteriol. 1997; 179: 2465-2471Crossref PubMed Scopus (214) Google Scholar). DsbA, a monomeric soluble member of the Dsb family is the primary factor that catalyzes the oxidative formation of disulfide bonds (3Sone M. Akiyama Y. Ito K. J. Biol. Chem. 1997; 272: 10349-10352Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). DsbC is a homodimer with four thiol groups in each 23-kDa subunit, two in the active site -Cys98-Gly-Tyr-Cys101- and the other two at positions Cys141 and Cys163 (4Shevchik V.E. Condemine G. Robert-Baudouy J. EMBO J. 1994; 13: 2007-2012Crossref PubMed Scopus (130) Google Scholar, 5Missiakas D. Georgopoulos C. Raina S. EMBO J. 1994; 13: 2013-2020Crossref PubMed Scopus (190) Google Scholar). DsbC catalyzes the rearrangement of disulfide bonds in concert with DsbA for the formation of native disulfides and has been recognized as a counterpart of eukaryotic PDI (3Sone M. Akiyama Y. Ito K. J. Biol. Chem. 1997; 272: 10349-10352Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 6Zapun A. Missiakas D. Raina S. Creighton T.E. Biochemistry. 1995; 34: 5075-5089Crossref PubMed Scopus (220) Google Scholar, 7Joly J. Swartz J. Biochemistry. 1997; 36: 10067-10072Crossref PubMed Scopus (118) Google Scholar). Secondary structure prediction of DsbC suggests that the molecule appears to consist of two domains (8Frishman D. Biochem. Biophys. Res. Commun. 1996; 219: 686-689Crossref PubMed Scopus (12) Google Scholar). Preliminary x-ray diffraction data of native and selenomethionine DsbC have been reported (9Rybin V. Zapun A. Torronen A. Raina S. Misssiakas D. Creighton T.E. Metcalf P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 1219-1221Crossref PubMed Scopus (6) Google Scholar). Many in vitro and in vivo data have supported that as a foldase, PDI displays both isomerase and chaperone activities (10Wang C.C. Tsou C.L. FASEB J. 1993; 7: 1515-1517Crossref PubMed Scopus (141) Google Scholar, 11Cai H. Wang C.C. Tsou C.L. J. Biol. Chem. 1994; 269: 24550-24552Abstract Full Text PDF PubMed Google Scholar, 12Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 7764-7771Abstract Full Text PDF PubMed Google Scholar, 13Song J.L. Wang C.C. Eur. J. Biochem. 1995; 231: 312-316Crossref PubMed Scopus (136) Google Scholar, 14Hayano T. Hirose M. Kikuchi M. FEBS Lett. 1995; 377: 505-511Crossref PubMed Scopus (84) Google Scholar, 15Lamberg A. Jauhiainen M. Metso J. Ehnholm C. Shoulders C. Scott J. Pihlajaniemi T. Kivirikko K.I. Biochem. J. 1996; 315: 533-536Crossref PubMed Scopus (55) Google Scholar, 16Wang C.C. Guzman N.A. Prolyl Hydroxylase, Protein Disulfide Isomerase, and Other Structurally Related Proteins. Marcel Dekker Inc., New York1997: 315-339Google Scholar, 17Yao Y. Zhou Y.C. Wang C.C. EMBO J. 1997; 16: 651-658Crossref PubMed Scopus (112) Google Scholar, 18Wang C.C. Biochemistry (Moscow). 1998; 63: 407-412PubMed Google Scholar). The chaperone activity of PDI greatly increases its efficiency as a foldase in promoting protein folding and in catalyzing the formation of native disulfide bonds (19Wang C.C. Tsou C.L. FEBS Lett. 1998; 425: 382-384Crossref PubMed Scopus (41) Google Scholar). Most disulfide-bonded proteins in bacteria are located in the periplasm and the outer-membrane or are secreted to extracellular medium through the periplasm as their disulfide bonds can be formed readily in the oxidative periplasm but not in the reductive cytoplasm (2Missiakas D. Raina S. J. Bacteriol. 1997; 179: 2465-2471Crossref PubMed Scopus (214) Google Scholar). The bacterial periplasm is separated from the environment by only the permeable outer membrane and is therefore liable to be affected by environmental fluctuation. However, no classical molecular chaperone has been identified in the periplasm so far (2Missiakas D. Raina S. J. Bacteriol. 1997; 179: 2465-2471Crossref PubMed Scopus (214) Google Scholar). Hence, we are interested in examining whether DsbC, like its eukaryotic counterpart PDI, has chaperone activity to promote the formation of correct disulfide bonds during protein folding. In fact, DsbA has been suggested to function as a chaperone in the maturation of secreted virulence factors of Vibrio cholerae (20Peek J.A. Taylor R.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6210-6214Crossref PubMed Scopus (184) Google Scholar) and in pilus biogenesis of Escherichia coli. (21Jacob-Dubuisson F. Pinkner J. Xu Z. Striker R. Padmanhaban A. Hultgren S.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11552-11556Crossref PubMed Scopus (127) Google Scholar). The high-resolution crystal structure analysis of DsbA (22Guddat L.W. Bardwell J.C.A. Zander T. Martin J.L. Protein Sci. 1997; 6: 1148-1156Crossref PubMed Scopus (77) Google Scholar) has shown that an extensive uncharged surface surrounding the active-site disulfide is responsible for peptide binding. We have also presented evidence for the chaperone activity of DsbA in assisting the in vitro refolding ofd-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (23Zheng W.D. Quan H. Song J.L. Yang S.L. Wang C.C. Arch. Biochem. Biophys. 1997; 337: 326-331Crossref PubMed Scopus (38) Google Scholar). Recently, Darby et al. (24Darby N.J. Raina S. Creighton T.E. Biochemstry. 1998; 37: 783-791Crossref PubMed Scopus (40) Google Scholar) reported more substantial noncovalent interactions between peptides with DsbC than with DsbA. In this report, we propose to show an even stronger chaperone activity of DsbC than that of PDI for protein folding in vitro with possibly different mechanisms for target protein binding. DISCUSSIONIn bacteria periplasm, DsbA and DsbC are the two soluble members of Dsb family directly involved in the formation of disulfide bonds. However, compared with eukaryotic PDI, the in vitroisomerase activity is about 30% for DsbC and only 5% for DsbA (23Zheng W.D. Quan H. Song J.L. Yang S.L. Wang C.C. Arch. Biochem. Biophys. 1997; 337: 326-331Crossref PubMed Scopus (38) Google Scholar).It has been suggested that, in evolution, some enzymes acquired substrate binding ability in a chaperone-like mode to improve their catalytic efficiency, such as PDI, the ATP-dependent proteases, or trigger factors (19Wang C.C. Tsou C.L. FEBS Lett. 1998; 425: 382-384Crossref PubMed Scopus (41) Google Scholar). DsbA has been found to have chaperone activity in vitro (23Zheng W.D. Quan H. Song J.L. Yang S.L. Wang C.C. Arch. Biochem. Biophys. 1997; 337: 326-331Crossref PubMed Scopus (38) Google Scholar) and suggested to function as a chaperone in vivo (20Peek J.A. Taylor R.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6210-6214Crossref PubMed Scopus (184) Google Scholar, 21Jacob-Dubuisson F. Pinkner J. Xu Z. Striker R. Padmanhaban A. Hultgren S.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11552-11556Crossref PubMed Scopus (127) Google Scholar). Recently, the requirement for DsbA in pullulanase secretion has been reported to be unrelated to its ability to catalyze the formation of disulfide bond, but possibly because of its chaperone-like effect on folding of the enzyme and presentation of the secretion signal (33Sauvonnet N. Pugsley A.P. Mol. Microbiol. 1998; 27: 661-667Crossref PubMed Scopus (27) Google Scholar). The peptide binding site of DsbA has been identified by high resolution crystallography (22Guddat L.W. Bardwell J.C.A. Zander T. Martin J.L. Protein Sci. 1997; 6: 1148-1156Crossref PubMed Scopus (77) Google Scholar), and secondary structure prediction of DsbC has suggested a similar structure to that of DsbA in that they both have an extra domain in addition to the thioredoxin-like domain (8Frishman D. Biochem. Biophys. Res. Commun. 1996; 219: 686-689Crossref PubMed Scopus (12) Google Scholar). Kinetic studies on DsbA- and DsbC-catalyzed reactions in simple unfolded peptides have revealed that the interaction between DsbC and its substrate appears to be more substantial than that between DsbA and the same peptide (24Darby N.J. Raina S. Creighton T.E. Biochemstry. 1998; 37: 783-791Crossref PubMed Scopus (40) Google Scholar). The noncovalent peptide binding ability is a prerequisite for a protein to be a chaperone. The isomerase activity of DsbC is low, but as very few periplasmic and outer membrane proteins have more than two disulfides per subunit, hence high isomerase activity may not be needed (7Joly J. Swartz J. Biochemistry. 1997; 36: 10067-10072Crossref PubMed Scopus (118) Google Scholar). The fact, that bacteria possess a large Dsb family composed of at least six members in periplasm (2Missiakas D. Raina S. J. Bacteriol. 1997; 179: 2465-2471Crossref PubMed Scopus (214) Google Scholar, 34Andersen C.L. Matthey-Dupraz A. Missiakas D. Raina S. Mol. Microbiol. 1997; 26: 121-132Crossref PubMed Scopus (91) Google Scholar) makes it possible for some Dsb proteins to function as chaperones and in this way to make up for the deficiency of molecular chaperones in this compartment.In this report, we presented evidence that DsbC indeed shows chaperone activity independent from its isomerase activity more pronounced than PDI in promoting the reactivation of denatured GAPDH and concurrently decreasing aggregation during its refolding. DsbC assists the reactivation of denatured and reduced lysozyme to a higher extent than PDI does even though it has only 30% of the disulfide isomerase activity of PDI, providing further support for the high chaperone activity of DsbC.It is likely that during refolding of denatured GAPDH, the denatured enzyme passes through several intermediates before either spontaneous reactivation or misfolding to form aggregates. The additive effect of DsbC and PDI on GAPDH reactivation suggests that DsbC and PDI may recognize different GAPDH folding intermediates, and interaction with each intermediate contributes to the overall reactivation of GAPDH. The effect of delayed addition of DsbC on GAPDH reactivation suggests that compared with PDI, DsbC may recognize earlier folding intermediates. The fact that DsbC, unlike PDI (12Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 7764-7771Abstract Full Text PDF PubMed Google Scholar, 32Song J.L. Quan H. Wang C.C. Biochem. J. 1997; 328: 841-846Crossref PubMed Scopus (28) Google Scholar), does not display anti-chaperone activity during lysozyme reactivation in HEPES buffer also suggests that DsbC and PDI bind with different intermediates, and this may contribute to the higher efficiency of DsbC in assisting the folding of some proteins as compared with PDI.It is surprising that relatively short peptides, including peptides with 10, 14, and 21 residues, inhibit neither the reductase activity of DsbC nor the assisted reactivation of GAPDH. It appears that the interactions of these peptides with DsbC are not strong enough to compete with the binding of the GAPDH folding intermediates. The fact, that sRNase does inhibit both the assisted reactivation of GAPDH and the PDOR activity of DsbC offers a further hint that DsbC has a large surface for peptide binding. It seems that DsbC has a somehow extended surface for peptide binding so that only a relatively large unfolded peptide is able to compete with the target folding intermediate for binding to DsbC. It was suggested by Darby et al. (24Darby N.J. Raina S. Creighton T.E. Biochemstry. 1998; 37: 783-791Crossref PubMed Scopus (40) Google Scholar) that DsbA binds an extended peptide chain as its substrate binding area is a long cleft and appears to be in a stereochemically restricted form, whereas the binding site of DsbC may favor the binding of a compact form of the peptide molecule. The peptide binding surface of PDI might be constructed by combining sites from several domains to form a large pocket (35Darby N.J. Penka E. Vincentelli R. J. Mol. Biol. 1998; 276: 239-247Crossref PubMed Scopus (152) Google Scholar), and this could also be the case for DsbC. By kinetics comparison, Joly and Swartz (7Joly J. Swartz J. Biochemistry. 1997; 36: 10067-10072Crossref PubMed Scopus (118) Google Scholar) indicated that DsbC has a higher affinity for the misfolded insulin-like growth factor-1 substrate than PDI and DsbA. However, like the effect of native staphylococcal nuclease on the PDI-assisted GAPDH reactivation (29Quan H. Fan G.B. Wang C.C. J. Biol. Chem. 1995; 270: 17078-17080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), native RNase shows little effect on the DsbC-assisted GAPDH reactivation, indicating that DsbC and PDI do not interact with native structures. The details of the topological arrangement of the four domains in the dimeric DsbC and eight domains in dimeric PDI molecules, the peptide binding surface and its relation to the active sites, and the functional implications of such a modular structure can only be clarified by crystal structure analysis.Secondary structure prediction suggested DsbC to be composed of two domains, the C-terminal thioredoxin-like domain and the N-terminal mixed α/β domain of unknown structure (8Frishman D. Biochem. Biophys. Res. Commun. 1996; 219: 686-689Crossref PubMed Scopus (12) Google Scholar). Recent NMR determination of the PDI b domain shows that it also contains the thioredoxin motif, and PDI has thus been considered to consist of active and inactive thioredoxin modules (36Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). In this respect, it is tempting to assume that, in homology to PDI, dimeric DsbC is composed of two active-site containing domains and two domains without the active site sequence. The multiple-domain structure of PDI has been demonstrated to be essential for its high catalytic efficiency and chaperone activity by increasing substrate binding interactions (35Darby N.J. Penka E. Vincentelli R. J. Mol. Biol. 1998; 276: 239-247Crossref PubMed Scopus (152) Google Scholar). The PDI-like modular structure of DsbC might be the structural basis for its PDI-like dual activities. In bacteria periplasm, DsbA and DsbC are the two soluble members of Dsb family directly involved in the formation of disulfide bonds. However, compared with eukaryotic PDI, the in vitroisomerase activity is about 30% for DsbC and only 5% for DsbA (23Zheng W.D. Quan H. Song J.L. Yang S.L. Wang C.C. Arch. Biochem. Biophys. 1997; 337: 326-331Crossref PubMed Scopus (38) Google Scholar). It has been suggested that, in evolution, some enzymes acquired substrate binding ability in a chaperone-like mode to improve their catalytic efficiency, such as PDI, the ATP-dependent proteases, or trigger factors (19Wang C.C. Tsou C.L. FEBS Lett. 1998; 425: 382-384Crossref PubMed Scopus (41) Google Scholar). DsbA has been found to have chaperone activity in vitro (23Zheng W.D. Quan H. Song J.L. Yang S.L. Wang C.C. Arch. Biochem. Biophys. 1997; 337: 326-331Crossref PubMed Scopus (38) Google Scholar) and suggested to function as a chaperone in vivo (20Peek J.A. Taylor R.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6210-6214Crossref PubMed Scopus (184) Google Scholar, 21Jacob-Dubuisson F. Pinkner J. Xu Z. Striker R. Padmanhaban A. Hultgren S.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11552-11556Crossref PubMed Scopus (127) Google Scholar). Recently, the requirement for DsbA in pullulanase secretion has been reported to be unrelated to its ability to catalyze the formation of disulfide bond, but possibly because of its chaperone-like effect on folding of the enzyme and presentation of the secretion signal (33Sauvonnet N. Pugsley A.P. Mol. Microbiol. 1998; 27: 661-667Crossref PubMed Scopus (27) Google Scholar). The peptide binding site of DsbA has been identified by high resolution crystallography (22Guddat L.W. Bardwell J.C.A. Zander T. Martin J.L. Protein Sci. 1997; 6: 1148-1156Crossref PubMed Scopus (77) Google Scholar), and secondary structure prediction of DsbC has suggested a similar structure to that of DsbA in that they both have an extra domain in addition to the thioredoxin-like domain (8Frishman D. Biochem. Biophys. Res. Commun. 1996; 219: 686-689Crossref PubMed Scopus (12) Google Scholar). Kinetic studies on DsbA- and DsbC-catalyzed reactions in simple unfolded peptides have revealed that the interaction between DsbC and its substrate appears to be more substantial than that between DsbA and the same peptide (24Darby N.J. Raina S. Creighton T.E. Biochemstry. 1998; 37: 783-791Crossref PubMed Scopus (40) Google Scholar). The noncovalent peptide binding ability is a prerequisite for a protein to be a chaperone. The isomerase activity of DsbC is low, but as very few periplasmic and outer membrane proteins have more than two disulfides per subunit, hence high isomerase activity may not be needed (7Joly J. Swartz J. Biochemistry. 1997; 36: 10067-10072Crossref PubMed Scopus (118) Google Scholar). The fact, that bacteria possess a large Dsb family composed of at least six members in periplasm (2Missiakas D. Raina S. J. Bacteriol. 1997; 179: 2465-2471Crossref PubMed Scopus (214) Google Scholar, 34Andersen C.L. Matthey-Dupraz A. Missiakas D. Raina S. Mol. Microbiol. 1997; 26: 121-132Crossref PubMed Scopus (91) Google Scholar) makes it possible for some Dsb proteins to function as chaperones and in this way to make up for the deficiency of molecular chaperones in this compartment. In this report, we presented evidence that DsbC indeed shows chaperone activity independent from its isomerase activity more pronounced than PDI in promoting the reactivation of denatured GAPDH and concurrently decreasing aggregation during its refolding. DsbC assists the reactivation of denatured and reduced lysozyme to a higher extent than PDI does even though it has only 30% of the disulfide isomerase activity of PDI, providing further support for the high chaperone activity of DsbC. It is likely that during refolding of denatured GAPDH, the denatured enzyme passes through several intermediates before either spontaneous reactivation or misfolding to form aggregates. The additive effect of DsbC and PDI on GAPDH reactivation suggests that DsbC and PDI may recognize different GAPDH folding intermediates, and interaction with each intermediate contributes to the overall reactivation of GAPDH. The effect of delayed addition of DsbC on GAPDH reactivation suggests that compared with PDI, DsbC may recognize earlier folding intermediates. The fact that DsbC, unlike PDI (12Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 7764-7771Abstract Full Text PDF PubMed Google Scholar, 32Song J.L. Quan H. Wang C.C. Biochem. J. 1997; 328: 841-846Crossref PubMed Scopus (28) Google Scholar), does not display anti-chaperone activity during lysozyme reactivation in HEPES buffer also suggests that DsbC and PDI bind with different intermediates, and this may contribute to the higher efficiency of DsbC in assisting the folding of some proteins as compared with PDI. It is surprising that relatively short peptides, including peptides with 10, 14, and 21 residues, inhibit neither the reductase activity of DsbC nor the assisted reactivation of GAPDH. It appears that the interactions of these peptides with DsbC are not strong enough to compete with the binding of the GAPDH folding intermediates. The fact, that sRNase does inhibit both the assisted reactivation of GAPDH and the PDOR activity of DsbC offers a further hint that DsbC has a large surface for peptide binding. It seems that DsbC has a somehow extended surface for peptide binding so that only a relatively large unfolded peptide is able to compete with the target folding intermediate for binding to DsbC. It was suggested by Darby et al. (24Darby N.J. Raina S. Creighton T.E. Biochemstry. 1998; 37: 783-791Crossref PubMed Scopus (40) Google Scholar) that DsbA binds an extended peptide chain as its substrate binding area is a long cleft and appears to be in a stereochemically restricted form, whereas the binding site of DsbC may favor the binding of a compact form of the peptide molecule. The peptide binding surface of PDI might be constructed by combining sites from several domains to form a large pocket (35Darby N.J. Penka E. Vincentelli R. J. Mol. Biol. 1998; 276: 239-247Crossref PubMed Scopus (152) Google Scholar), and this could also be the case for DsbC. By kinetics comparison, Joly and Swartz (7Joly J. Swartz J. Biochemistry. 1997; 36: 10067-10072Crossref PubMed Scopus (118) Google Scholar) indicated that DsbC has a higher affinity for the misfolded insulin-like growth factor-1 substrate than PDI and DsbA. However, like the effect of native staphylococcal nuclease on the PDI-assisted GAPDH reactivation (29Quan H. Fan G.B. Wang C.C. J. Biol. Chem. 1995; 270: 17078-17080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), native RNase shows little effect on the DsbC-assisted GAPDH reactivation, indicating that DsbC and PDI do not interact with native structures. The details of the topological arrangement of the four domains in the dimeric DsbC and eight domains in dimeric PDI molecules, the peptide binding surface and its relation to the active sites, and the functional implications of such a modular structure can only be clarified by crystal structure analysis. Secondary structure prediction suggested DsbC to be composed of two domains, the C-terminal thioredoxin-like domain and the N-terminal mixed α/β domain of unknown structure (8Frishman D. Biochem. Biophys. Res. Commun. 1996; 219: 686-689Crossref PubMed Scopus (12) Google Scholar). Recent NMR determination of the PDI b domain shows that it also contains the thioredoxin motif, and PDI has thus been considered to consist of active and inactive thioredoxin modules (36Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). In this respect, it is tempting to assume that, in homology to PDI, dimeric DsbC is composed of two active-site containing domains and two domains without the active site sequence. The multiple-domain structure of PDI has been demonstrated to be essential for its high catalytic efficiency and chaperone activity by increasing substrate binding interactions (35Darby N.J. Penka E. Vincentelli R. J. Mol. Biol. 1998; 276: 239-247Crossref PubMed Scopus (152) Google Scholar). The PDI-like modular structure of DsbC might be the structural basis for its PDI-like dual activities. We are sincerely grateful to Dr. Rudi Glockshuber, ETH, Switzerland for the generous gift of the cloned DsbC precursor gene. We also thank X. L. Li and X. X. Sun in this laboratory for kindly providing rabbit muscle GAPDH and di-fluoresceinthiocarbamyl-insulin, respectively. We sincerely thank Prof. C. L. Tsou for continuous encouragement, helpful advice, and critical reading of the manuscript." @default.
W2021024369 created "2016-06-24" @default.
W2021024369 creator A5003642180 @default.
W2021024369 creator A5037774305 @default.
W2021024369 creator A5037990173 @default.
W2021024369 creator A5052737424 @default.
W2021024369 creator A5082857859 @default.
W2021024369 creator A5084163607 @default.
W2021024369 date "1999-07-01" @default.
W2021024369 modified "2023-10-17" @default.
W2021024369 title "Chaperone Activity of DsbC" @default.
W2021024369 cites W1508560627 @default.
W2021024369 cites W1543665493 @default.
W2021024369 cites W1547929361 @default.
W2021024369 cites W1559988816 @default.
W2021024369 cites W1584511565 @default.
W2021024369 cites W1830036395 @default.
W2021024369 cites W184948381 @default.
W2021024369 cites W1937700077 @default.
W2021024369 cites W1964623405 @default.
W2021024369 cites W1968295893 @default.
W2021024369 cites W1971068216 @default.
W2021024369 cites W1974436393 @default.
W2021024369 cites W1977294234 @default.
W2021024369 cites W1977885347 @default.
W2021024369 cites W1988219519 @default.
W2021024369 cites W2003231315 @default.
W2021024369 cites W2007245883 @default.
W2021024369 cites W2012000393 @default.
W2021024369 cites W2032599079 @default.
W2021024369 cites W2034811695 @default.
W2021024369 cites W2036503551 @default.
W2021024369 cites W2041250116 @default.
W2021024369 cites W2053951541 @default.
W2021024369 cites W2059604113 @default.
W2021024369 cites W2061563016 @default.
W2021024369 cites W2066765533 @default.
W2021024369 cites W2072597086 @default.
W2021024369 cites W2079220537 @default.
W2021024369 cites W2089484980 @default.
W2021024369 cites W2096086782 @default.
W2021024369 cites W2150149751 @default.
W2021024369 cites W2222366583 @default.
W2021024369 cites W2280036301 @default.
W2021024369 cites W4244425588 @default.
W2021024369 doi "https://doi.org/10.1074/jbc.274.28.19601" @default.
W2021024369 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10391895" @default.
W2021024369 hasPublicationYear "1999" @default.
W2021024369 type Work @default.
W2021024369 sameAs 2021024369 @default.
W2021024369 citedByCount "156" @default.
W2021024369 countsByYear W20210243692012 @default.
W2021024369 countsByYear W20210243692013 @default.
W2021024369 countsByYear W20210243692014 @default.
W2021024369 countsByYear W20210243692015 @default.
W2021024369 countsByYear W20210243692016 @default.
W2021024369 countsByYear W20210243692017 @default.
W2021024369 countsByYear W20210243692018 @default.
W2021024369 countsByYear W20210243692019 @default.
W2021024369 countsByYear W20210243692020 @default.
W2021024369 countsByYear W20210243692021 @default.
W2021024369 countsByYear W20210243692022 @default.
W2021024369 crossrefType "journal-article" @default.
W2021024369 hasAuthorship W2021024369A5003642180 @default.
W2021024369 hasAuthorship W2021024369A5037774305 @default.
W2021024369 hasAuthorship W2021024369A5037990173 @default.
W2021024369 hasAuthorship W2021024369A5052737424 @default.
W2021024369 hasAuthorship W2021024369A5082857859 @default.
W2021024369 hasAuthorship W2021024369A5084163607 @default.
W2021024369 hasBestOaLocation W20210243691 @default.
W2021024369 hasConcept C12554922 @default.
W2021024369 hasConcept C142724271 @default.
W2021024369 hasConcept C185592680 @default.
W2021024369 hasConcept C2775962898 @default.
W2021024369 hasConcept C55493867 @default.
W2021024369 hasConcept C70721500 @default.
W2021024369 hasConcept C71924100 @default.
W2021024369 hasConcept C86803240 @default.
W2021024369 hasConcept C95444343 @default.
W2021024369 hasConceptScore W2021024369C12554922 @default.
W2021024369 hasConceptScore W2021024369C142724271 @default.
W2021024369 hasConceptScore W2021024369C185592680 @default.
W2021024369 hasConceptScore W2021024369C2775962898 @default.
W2021024369 hasConceptScore W2021024369C55493867 @default.
W2021024369 hasConceptScore W2021024369C70721500 @default.
W2021024369 hasConceptScore W2021024369C71924100 @default.
W2021024369 hasConceptScore W2021024369C86803240 @default.
W2021024369 hasConceptScore W2021024369C95444343 @default.
W2021024369 hasIssue "28" @default.
W2021024369 hasLocation W20210243691 @default.
W2021024369 hasOpenAccess W2021024369 @default.
W2021024369 hasPrimaryLocation W20210243691 @default.
W2021024369 hasRelatedWork W1561855879 @default.
W2021024369 hasRelatedWork W2089772414 @default.
W2021024369 hasRelatedWork W2107126967 @default.
W2021024369 hasRelatedWork W2133411779 @default.
W2021024369 hasRelatedWork W2140924459 @default.
W2021024369 hasRelatedWork W2166642778 @default.