Turning a disulfide isomerase into an oxidase: DsbC mutants that imitate DsbA

There are two distinct pathways for disulfide formation in prokaryotes. The DsbA-DsbB pathway introduces disulfide bonds de novo, while the DsbC-DsbD pathway functions to isomerize disulfides. One of the key questions in disulfide biology is how the isomerase pathway is kept separate from the oxidase pathway in vivo. Cross-talk between these two systems would be mutually destructive. To force communication between these two systems we have selected dsbC mutants that complement a dsbA null mutation. In these mutants, DsbC is present as a monomer as compared with dimeric wild-type DsbC. Based on these findings we rationally designed DsbC mutants in the dimerization domain. All of these mutants are able to rescue the dsbA null phenotype. Rescue depends on the presence of DsbB, the native re-oxidant of DsbA, both in vivo and in vitro. Our results suggest that dimerization acts to protect DsbC's active sites from DsbB-mediated oxidation. These results explain how oxidative and reductive pathways can co-exist in the periplasm of Escherichia coli.     

The oxidase DsbA folds a protein with a nonconsecutive disulfide

One of the last unsolved problems of molecular biology is how the sequential amino acid information leads to a functional protein. Correct disulfide formation within a protein is hereby essential. We present periplasmic ribonuclease I (RNase I) from Escherichia coli as a new endogenous substrate for the study of oxidative protein folding. One of its four disulfides is between nonconsecutive cysteines. In general view, the folding of proteins with nonconsecutive disulfides requires the protein disulfide isomerase DsbC. In contrast, our study with RNase I shows that DsbA is a sufficient catalyst for correct disulfide formation in vivo and in vitro. DsbA is therefore more specific than generally assumed. Further, we show that the redox potential of the periplasm depends on the presence of glutathione and the Dsb proteins to maintain it at-165 mV. We determined the influence of this redox potential on the folding of RNase I. Under the more oxidizing conditions of dsb(-) strains, DsbC becomes necessary to correct non-native disulfides, but it cannot substitute for DsbA. Altogether, DsbA folds a protein with a nonconsecutive disulfide as long as no incorrect disulfides are formed.     

DsbA and DsbC affect extracellular enzyme formation in Pseudomonas aeruginosa

DsbA and DsbC proteins involved in the periplasmic formation of disulfide bonds in Pseudomonas aeruginosa were identified and shown to play an important role for the formation of extracellular enzymes. Mutants deficient in either dsbA or dsbC or both genes were constructed, and extracellular elastase, alkaline phosphatase, and lipase activities were determined. The dsbA mutant no longer produced these enzymes, whereas the lipase activity was doubled in the dsbC mutant. Also, extracellar lipase production was severely reduced in a P. aeruginosa dsbA mutant in which an inactive DsbA variant carrying the mutation C34S was expressed. Even when the lipase gene lipA was constitutively expressed in trans in a lipA dsbA double mutant, lipase activity in cell extracts and culture supernatants was still reduced to about 25%. Interestingly, the presence of dithiothreitol in the growth medium completely inhibited the formation of extracellular lipase whereas the addition of dithiothreitol to a cell-free culture supernatant did not affect lipase activity. We conclude that the correct formation of the disulfide bond catalyzed in vivo by DsbA is necessary to stabilize periplasmic lipase. Such a stabilization is the prerequisite for efficient secretion using the type II pathway.     

A new Escherichia coli gene, dsbG, encodes a periplasmic protein involved in disulphide bond formation, required for recycling DsbA/DsbB and DsbC redox proteins

We have identified and functionally characterized a new Escherichia coli gene, dsbG , whose product is involved in disulphide bond formation in the periplasm. The dsbG gene was cloned from a multicopy plasmid library lacking the dsbB redox protein-encoding gene. Multicopy dsbG -carrying clones were selected, since they allowed E. coli to grow at lethal concentrations of dithiothreitol. In a complementary genetic approach, point mutations were independently obtained and mapped to the dsbG gene. Such mutations led simultaneously to a dithiothreitol-sensitive phenotype and an increased σ^E-dependent heat shock response, which reflects the presence of misfolded proteins in the extracytoplasm. In agreement with these observations, dsbG mutants were shown to accumulate reduced forms of a variety of disulphide bond-containing proteins in the periplasm. This DsbG defect could be rescued by addition to the growth medium of either oxidized dithiothreitol or cystine, or by overexpression of the dsbA or dsbB genes. DsbG is synthesized as a precursor form of 27.5 kDa and processed to a 25.7 kDa mature species located in the periplasm. DsbG was overproduced, purified to homogeneity and shown to have redox properties of thiol–disulphide oxidoreductases in vitro . Replacement of the first Cys residue of the predicted active site, Phe–(Xaa)₄–Cys–Pro–Tyr–Cys by Ala, completely inactivated DsbG protein function. Taken together, all our results demonstrate that DsbG acts in vivo as an efficient thiol–disulphide oxidase. In addition, dsbG is the first member of the dsb family for which null mutations are conditionally lethal and can be propagated only if supplemented with oxidants in the growth medium. We propose that the main role of DsbG is to maintain the proper redox balance between the DsbA/DsbB and DsbC systems.     

The requirement for DsbA in pullulanase secretion is independent of disulphide bond formation in the enzyme

Results from previous studies have suggested that an intramolecular disulphide bond in the exoprotein pullulanase is needed for its recognition and transport across the outer membrane. This interpretation of the data is shown here to be incorrect: pullulanase devoid of all potential disulphide bonds is secreted with apparently the same efficiency as the wild-type protein. Furthermore, the periplasmic disulphide bond, oxidoreductase DsbA, previously shown to catalyse the formation of a disulphide bond in pullulanase and to decrease its transit time in the periplasm, is shown here to be required for the rapid secretion of pullulanase devoid of disulphide bonds. Several possible explanations for the role of DsbA in pullulanase secretion are discussed.     

The binding site of bisphenol A to protein disulphide isomerase

Protein disulphide isomerase (PDI) has been isolated as a binding protein of bisphenol A (BPA) in the rat brain. In this study, we determined binding sites of BPA to PDI and characterized the binding site. First, we identified the BPA-binding domain with ab, b'a'c, a, b, b' and a'c fragment peptides of PDI by surface plasmon resonance spectroscopy. BPA interacted with ab, b'a 'c, a and b', suggesting that a and b' domains are important in their interaction. Second, ab, b'a'c, a,b,b',a', abb'a', abb', b'a', Δb' and a'c fragment peptides were used for their isomerase activity with RNase as a substrate. BPA could inhibit the activity of peptide fragments including b', suggesting that b' domain contributes to inhibition of catalytic activity of PDI by BPA. Next, we investigated the BPA-binding capacity of PDI by amino acid substitution. PDI lost the BPA-binding activity by the mutation of H258 and mutation of Q245 and N300 also decreased its activity. Furthermore, acidic condition increased the BPA-binding activity of PDI. These results suggest that the charge of these amino acid especially, H258, is important for the BPA to bind to PDI.     

Oxidative refolding of lysozyme assisted by DsbA, DsbC and the GroEL apical domain immobilized in cellulose

Expression of recombinant proteins in Escherichia coli often leads to formation of inclusion bodies (IB). If a recombinant protein contains one or more disulfide bonds, protein refolding and thiol oxidation reactions are required to recover its biological activity. Previous studies have demonstrated that molecular chaperones and foldases assist with the in vitro protein refolding. However, their use has been limited by the stoichiometric amount required for the refolding reaction. In search of alternatives to facilitate the use of these folding biocatalysts in this study, DsbA, DsbC, and the apical domain of GroEL (AD) were fused to the carbohydrate-binding module CBD_Cex of Cellulomonas fimi. The recombinant proteins were purified and immobilized in cellulose and used to assist the oxidative refolding of denatured and reduced lysozyme. The assisted refolding yields obtained with immobilized folding biocatalysts were at least twice of those obtained in the spontaneous refolding, suggesting that the AD, DsbA, and DsbC immobilized in cellulose might be useful for the oxidative refolding of recombinant proteins that are expressed as inclusion bodies. In addition, the spontaneous or assisted refolding kinetics data fitted well (r² > 0.9) to a previously reported lysozyme refolding model. The estimated refolding (k _N) and aggregation (k _A) constants were consistent with the hypothesis that foldases assisted the oxidative refolding of lysozyme by decreasing protein aggregation rather than increasing the refolding rate.     

Copper stress causes an in vivo requirement for the Escherichia coli disulfide isomerase DsbC

In Escherichia coli, the periplasmic disulfide oxidoreductase DsbA is thought to be a powerful but nonspecific oxidant, joining cysteines together the moment they enter the periplasm. DsbC, the primary disulfide isomerase, likely resolves incorrect disulfides. Given the reliance of protein function on correct disulfide bonds, it is surprising that no phenotype has been established for null mutations in dsbC. Here we demonstrate that mutations in the entire DsbC disulfide isomerization pathway cause an increased sensitivity to the redox-active metal copper. We find that copper catalyzes periplasmic disulfide bond formation under aerobic conditions and that copper catalyzes the formation of disulfide-bonded oligomers in vitro, which DsbC can resolve. Our data suggest that the copper sensitivity of dsbC- strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfides in the absence of functional DsbC. Absence of functional DsbA augments the deleterious effects of copper on a dsbC- strain, even though the dsbA- single mutant is unaffected by copper. This may indicate that DsbA successfully competes with copper and forms disulfide bonds more accurately than copper does. These findings lead us to a model in which DsbA may be significantly more accurate in disulfide oxidation than previously thought, and in which the primary role of DsbC may be to rearrange incorrect disulfide bonds that are formed during certain oxidative stresses.     

Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli

DsbC is one of five Escherichia coli proteins required for disulfide bond formation and is thought to function as a disulfide bond isomerase during oxidative protein folding in the periplasm. DsbC is a 2 x 23 kDa homodimer and has both protein disulfide isomerase and chaperone activity. We report the 1.9 A resolution crystal structure of oxidized DsbC where both Cys-X-X-Cys active sites form disulfide bonds. The molecule consists of separate thioredoxin-like domains joined via hinged linker helices to an N-terminal dimerization domain. The hinges allow relative movement of the active sites, and a broad uncharged cleft between them may be involved in peptide binding and DsbC foldase activities.     

Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemi and Escherichia coli with disulfide isomerase activity.

We identified and characterized an Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation that prevents disulfide bond formation in periplasmic proteins. This gene, dsbC, codes for a 24 kDa periplasmic protein that contains a characteristic active site sequence of disulfide isomerases, Phe-X-X-X-X-Cys-X-X-Cys. Besides the active site, DsbC has no homology with DsbA, thioredoxin or eukaryotic protein disulfide isomerase and it could define a new subfamily of disulfide isomerases. Purified DsbC protein is able to catalyse insulin oxidation in a dithiothreitol dependent manner. The E.coli gene xprA codes for a protein functionally equivalent to DsbC. The in vivo function of DsbC seems to be the formation of disulfide bonds in proteins. The presence of XprA could explain the residual disulfide isomerase activity existing in dsbA mutants. Re-oxidation of XprA does not seem to occur through DsbB, the protein that probably re-oxidizes DsbA.     

The human protein disulphide isomerase family: substrate interactions and functional properties

The process of disulphide bond formation in the endoplasmic reticulum of eukaryotic cells was one of the first mechanisms of catalysed protein folding to be discovered. Protein disulphide isomerase (PDI) is now known to catalyse all of the reactions that are involved in native disulphide bond formation, but despite more than 40 years of study, its mechanism of action is still not fully understood. This review discusses recent advances in our understanding of the human PDI family of enzymes and focuses on their functional properties, substrate interactions and some recently identified family members.     

Crystal structure of the outer membrane protein RcsF, a new substrate for the periplasmic protein-disulfide isomerase DsbC

The bacterial Rcs phosphorelay is a stress-induced defense mechanism that controls the expression of numerous genes, including those for capsular polysaccharides, motility, and virulence factors. It is a complex multicomponent system that includes the histidine kinase (RcsC) and the response regulator (RcsB) and also auxiliary proteins such as RcsF. RcsF is an outer membrane lipoprotein that transmits signals from the cell surface to RcsC. The physiological signals that activate RcsF and how RcsF interacts with RcsC remain unknown. Here, we report the three-dimensional structure of RcsF. The fold of the protein is characterized by the presence of a central 4-stranded β sheet, which is conserved in several other proteins, including the copper-binding domain of the amyloid precursor protein. RcsF, which contains four conserved cysteine residues, presents two nonconsecutive disulfides between Cys(74) and Cys(118) and between Cys(109) and Cys(124), respectively. These two disulfides are not functionally equivalent; the Cys(109)-Cys(124) disulfide is particularly important for the assembly of an active RcsF. Moreover, we show that formation of the nonconsecutive disulfides of RcsF depends on the periplasmic disulfide isomerase DsbC. We trapped RcsF in a mixed disulfide complex with DsbC, and we show that deletion of dsbC prevents the activation of the Rcs phosphorelay by signals that function through RcsF. The three-dimensional structure of RcsF provides the structural basis to understand how this protein triggers the Rcs signaling cascade.     

Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli

We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.     

The disulfide bond isomerase DsbC is activated by an immunoglobulin-fold thiol oxidoreductase: crystal structure of the DsbC-DsbDalpha complex

The Escherichia coli disulfide bond isomerase DsbC rearranges incorrect disulfide bonds during oxidative protein folding. It is specifically activated by the periplasmic N-terminal domain (DsbDalpha) of the transmembrane electron transporter DsbD. An intermediate of the electron transport reaction was trapped, yielding a covalent DsbC-DsbDalpha complex. The 2.3 A crystal structure of the complex shows for the first time the specific interactions between two thiol oxidoreductases. DsbDalpha is a novel thiol oxidoreductase with the active site cysteines embedded in an immunoglobulin fold. It binds into the central cleft of the V-shaped DsbC dimer, which assumes a closed conformation on complex formation. Comparison of the complex with oxidized DsbDalpha reveals major conformational changes in a cap structure that regulates the accessibility of the DsbDalpha active site. Our results explain how DsbC is selectively activated by DsbD using electrons derived from the cytoplasm.     

Loss of PML cooperates with mutant p53 to drive more aggressive cancers in a gender-dependent manner

p53 mutations and downregulation of promyelocytic leukemia (PML) are common genetic alterations in human cancers. In healthy cells these two key tumor suppressors exist in a positive regulatory loop, promoting cell death and cellular senescence. However, the influence of their interplay on tumorigenesis has not been explored directly in vivo. The contribution of PML to mutant p53 driven cancer was evaluated in a mouse model harboring a p53 mutation (p53^{wild-type/R172H}) that recapitulates a frequent p53 mutation (p53^R175H) in human sporadic and Li-Fraumeni cancers. These mice with PML displayed perturbation of the hematopoietic compartment, manifested either as lymphoma or extramedullary hematopoiesis (EMH). EMH was associated with peripheral blood leucocytosis and macrocytic anemia, suggestive of myeloproliferative- myelodysplastic overlap. In contrast, a complete loss of PML from these mice resulted in a marked alteration in tumor profile. While the incidence of lymphomas was unaltered, EMH was not detected and the majority of mice succumbed to sarcomas. Further, males lacking PML exhibited a high incidence of soft tissue sarcomas and reduced survival, while females largely developed osteosarcomas, without impact on survival. Together, these findings demonstrate that PML is an important tumor suppressor dictating disease development in a pertinent mouse model of human cancer.

Key Points: (1) A mutant p53 allele disrupts hematopoiesis in mice, by promoting lymphomas and myeloproliferative / myelodysplastic overlap. (2) Coincidental p53 allele mutation and PML loss shifts the tumor profile toward sarcoma formation, which is paralleled in human leiomyosarcomas (indicated by immunohistochemistry; IHC).     

De novo design and evolution of artificial disulfide isomerase enzymes analogous to the bacterial DsbC

The Escherichia coli disulfide isomerase, DsbC is a V-shaped homodimer with each monomer comprising a dimerization region that forms part of a putative peptide-binding pocket and a thioredoxin catalytic domain. Disulfide isomerases from prokaryotes and eukaryotes exhibit little sequence homology but display very similar structural organization with two thioredoxin domains facing each other on top of the dimerization/peptide-binding region. To aid the understanding of the mechanistic significance of thioredoxin domain dimerization and of the peptide-binding cleft of DsbC, we constructed a series of protein chimeras comprising unrelated protein dimerization domains fused to thioredoxin superfamily enzymes. Chimeras consisting of the dimerization domain and the alpha-helical linker of the bacterial proline cis/trans isomerase FkpA and the periplasmic oxidase DsbA gave rise to enzymes that catalyzed the folding of multidisulfide substrate proteins in vivo with comparable efficiency to E. coli DsbC. In addition, expression of FkpA-DsbAs conferred modest resistance to CuCl2, a phenotype that depends on disulfide bond isomerization. Selection for resistance to elevated CuCl2 concentrations led to the isolation of FkpA-DsbA mutants containing a single amino acid substitution that changed the active site of the DsbA domain from CPHC into CPYC, increasing the similarity to the DsbC active site (CGYC). Unlike DsbC, which is resistant to oxidation by DsbB-DsbA and does not normally catalyze disulfide bond formation under physiological conditions, the FkpA-DsbA chimeras functioned both as oxidases and isomerases. The engineering of these efficient artificial isomerases delineates the key features of catalysis of disulfide bond isomerization and enhances our understanding of its evolution.     

Crystallization and initial crystallographic analysis of the disulfide bond isomerase DsbC in complex with the alpha domain of the electron transporter DsbD

The protein disulfide bond isomerase DsbC catalyzes the rearrangement of incorrect disulfide bonds during oxidative protein folding in the periplasm of Escherichia coli. The active site cysteines of DsbC are maintained in the active reduced form by the transmembrane electron transporter DsbD. DsbD obtains electrons from the cytoplasm, transports them across the inner membrane, and passes them onto periplasmic substrates, such as DsbC. The electron transport process involves several thiol disulfide exchange reactions between different classes of thiol oxidoreductase. We were able to trap the final electron transport reaction using active site mutants yielding a stable DsbC-DsbDalpha complex. This disulfide cross-linked complex was purified to homogeneity and crystallized. Dehydration of the tetragonal crystals changed the unit cell dimensions from a approximately b = 73 A, c = 267.5 A to a = b = 68.9 A, c = 230.3 A, reducing the cell volume by 23% and the solvent content from 55 to 41%. Crystal dehydration and cryo-cooling improved the diffraction quality of the crystals from 7 to 2.3 A resolution.     

Cotranslational glycosylation of proteins in systems depleted of protein disulphide isomerase.

The role of protein disulphide isomerase (PDI) and other resident proteins of the endoplasmic reticulum (ER) lumen in co- and post-translational modification of secretory proteins has been studied in experiments on translation in vitro. We have devised procedures for extracting the lumenal content proteins of dog pancreas microsomal vesicles by alkaline buffer, or detergent washing, and for reconstitution of the depleted membrane fraction. When microsomal membranes are depleted of content by washing at pH 9.1, they are able to co-translationally glycosylate human interferon-gamma (IFN-gamma) and yeast pro-alpha-factor and the products appear to be identical to those produced by control microsomes. However, when microsomal membranes are depleted of content by washing with saponin they are still able to co-translationally translocate and glycosylate human IFN-gamma, but the products were of higher apparent Mr than those generated by control microsomes. When saponin-washed microsomal membranes were reconstituted with homogeneous protein disulphide isomerase (PDI), the generated vesicles gave the same pattern of co-translationally glycosylated IFN-gamma as saponin-washed microsomal membranes lacking PDI. These results are discussed in relation to the roles of resident ER proteins in co-translational modification; they suggest that PDI is not an essential component of the machinery of co-translational N-glycosylation, but that detergent washing may inactivate or remove some ER glycosidases.