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PubMed1994

Denatured state of ovalbumin in high concentrations of urea as evaluated by disulfide rearrangement analysis.

To investigate the highly denatured state of ovalbumin (molecular mass of 42.7 kDa, four cysteine sulfhydryls and one cystine disulfide) using the disulfide rearrangement approach, we established the peptide-mapping procedure using a cysteine-labeling technique with a fluorescent dye that allows the quantitative analyses for the disulfide-involved half-cystines. Ovalbumin denatured at a low protein concentration in 8-10 M urea, in which the protein showed complete unfolding as evaluated by far-UV CD spectra, was analyzed for the disulfide-involved half-cystines using the peptide-mapping procedure. Data clearly showed that the number of free sulfhydryls and intrachain disulfides were four and one, respectively, but that all six half-cystines are labeled with the dye. These results strongly suggested that 15 disulfide isomers that are theoretically possible for a molecule having one disulfide and four sulfhydryls are all generated during the denaturation. The quantitative data for the ratios of the observed labeling values for the six half-cystines, relative to the overall labeling values, were consistent with the view that the distribution of the 15 possible disulfide isomers at equilibrium depends on the number of amino acid residues separating the two half-cystines to the power of -1.9 to -2.0. Essentially, the same non-gaussian chain nature was also observed with kinetic data for sulfhydryl-disulfide exchanges after denaturation of native ovalbumin.

Amino Acid Sequence↗
PubMed1994

Formation of mitogenically active PDGF-B dimer does not require interchain disulfide bonds.

Platelet-derived growth factor (PDGF), a major mitogen for mesenchymal cells, is a disulfide-bonded dimer of two subunit polypeptides named A and B. All of the three possible dimeric forms, i.e. AA, BB, and AB, exist in nature. The dimeric structure has been presumed to be necessary for biological activity, since reduction of the dimer results in loss of activity and simultaneous conversion to monomeric form as determined by SDS-gel electrophoresis. However, reduction of the native molecule destroys intrachain, as well as interchain, disulfide bonds, and it is possible that the former rather than the latter are critical for proper conformation of the active protein. We show here that PDGF-B polypeptides in which all 8 cysteines or the 2nd, 4th, 5th, and 8th cysteines have been mutated to serines fail to form covalent dimers and possess dramatically less mitogenic activity than native PDGF-BB. Another mutant, PDGF-B(C2,4S), in which just the 2 cysteines involved in interchain disulfides were converted to serine, ran as a monomer on SDS-polyacrylamide gels as expected. Somewhat unexpectedly, however, the mitogenic activity of the PDGF-B(C2,4S) analog was similar to the activity of wild-type PDGF-BB disulfide-bonded dimer under physiological conditions. The activity of the analog was more sensitive to the effect of low pH than was the activity of wild-type PDGF-BB. Molecular weight analysis utilizing light scattering and sedimentation equilibrium demonstrated that the PDGF-B(C2,4S) analog exists as a noncovalent dimer at pH 4-7 but dissociates to a monomer at pH 2.5. Disulfide analysis of the mutant protein demonstrated that the intrachain disulfide bonds are the same as those formed in wild-type PDGF-BB homodimers. We conclude that proper formation of intrachain disulfide bonds is critical to maintaining the correct conformation of PDGF monomers, but that appropriately folded monomers can associate into active noncovalent dimers in the absence of interchain disulfide bonds. Interchain disulfide bonds thus appear to increase the stability of the PDGF dimer rather than being crucial to its existence.

Amino Acid Sequence↗
PubMed1984

Pantethine and cystamine deplete cystine from cystinotic fibroblasts via efflux of cysteamine-cysteine mixed disulfide.

Children suffering from cystinosis, a genetic disease characterized by high levels of lysosomal cystine, are currently being treated with cysteamine to lower the cystine levels in their cells. In fibroblasts from these patients, cysteamine and its disulfide, cystamine, are equally effective in lowering cystine levels. We recently reported that pantethine, a dietary precursor of coenzyme A, depletes cystine from cultured, cystinotic fibroblasts as effectively as cystamine. To determine the mechanism of action of pantethine, and of cystamine, we have compared the fate of [35S]cystine-derived metabolites in the presence and absence of these agents. The results indicate that the ability of pantethine to deplete cystine resides in its being a metabolic precursor of cysteamine. Furthermore, both pantethine and cystamine act by generating the mixed disulfide of cysteamine and cysteine in the lysosomes, which is then rapidly excreted from the cells. The fall in intracellular [35S]cystine caused by these agents was not accompanied by a comparable increase in any intracellular metabolite; rather, it could be accounted for by the appearance of mixed disulfide in the medium. There was no accumulation of mixed disulfide in the cells. Radioactivity in cytoplasmic glutathione was, however, increased by cystamine or pantethine. Thus, cysteamine (formed intracellularly in these experiments) undergoes thiol-disulfide exchange with cystine in the lysosomes, producing cysteamine-cysteine mixed disulfide and free cysteine, which enter the cytoplasm. The free cysteine is available to several pathways, including oxidation to the disulfide or the mixed disulfide, and synthesis of glutathione. The mixed disulfide is excreted from the cell, which ultimately depletes the cell of its excess cystine.

Cells, Cultured↗
PubMed1991

Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme.

Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 265, 7570-7575). Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91. The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding. Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities. Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule. The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond. These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.

Amino Acid Sequence↗
PubMed1981

Inactivation of hydroxymethylglutaryl-CoA reductase from yeast by coenzyme A disulfide.

The time-dependent inactivation of hydroxymethylglutaryl-CoA reductase from yeast by solutions of hydroxymethylglutaryl-CoA and CoASH is due to the rapid inactivation of the enzyme by oxidized CoA (CoA disulfide) present at trace levels in solutions of hydroxymethylglutaryl-CoA and CoASH. Solutions of hydroxymethylglutaryl-CoA or CoASH incubated for 1.5 h with 10 mM dithiothreitol at pH 7.0, 22 degrees C, do not inactivate the enzyme. Inactivation of hydroxymethylglutaryl-CoA reductase is rapid and complete at concentrations of CoA disulfide comparable to those measured in solutions of hydroxymethylglutaryl-CoA and CoASH. Inactivation of the enzyme by CoA disulfide may be reversed by treating the inactive enzyme with 10 mM dithiothreitol at pH 7.0 Both the inactivation of the enzyme by CoA disulfide and reactivation by dithiothreitol are inhibited by hydroxymethylglutaryl-CoA. Other disulfides such as Ellman's reagent and glutathione disulfide also inactivate the enzyme. A thio-disulfide exchange reaction with a sulfhydryl group on the enzyme forming a mixed disulfide or an intramolecular protein disulfide could account for the enzyme inactivation. The normal function of the sulfhydryl group involved in the inactivation of the enzyme is unknown.

Coenzyme A↗
PubMed1995

Efficient catalysis of disulfide formation during protein folding with a single active-site cysteine.

Protein disulfide isomerases (PDIs) catalyze disulfide bond formation during protein folding in vivo and are essential for viability in eukaryotic cells. They share the active-site sequence C-X-X-C that forms a catalytic disulfide. The recent finding that the EUG1 protein, a PDI-related yeast protein, with C-X-X-S sequence at its active sites can complement PDI-deficiency raised the general question of whether disulfide-isomerase activity is essential for cell viability or whether PDI variants with single active-site thiol groups can be catalytically active as disulfide isomerases. We investigated the function of the catalytic cysteine residues in DsbA, a PDI-related protein required for disulfide formation in the periplasmic space of Escherichia coli, by replacing C30 and C33 with alanine. While the mutant C30A and the double mutant CC30/33AA are inactive, C33A catalyzes disulfide-interchange reactions and oxidative renaturation of the reduced, unfolded thrombin inhibitor hirudin with close to wild-type efficiency. Thus, the single active-site thiol group of C30 is sufficient for disulfide-isomerase activity of the DsbA protein.

Base Sequence↗
PubMed1994

Enzymatic catalysis of disulfide formation.

The formation of disulfide bridges in membrane and secretory proteins occurs during the protein folding process in the endoplasmic reticulum of eukaryotic cells and the periplasm of prokaryotes. The formation of disulfide bridges is facilitated by the thiol-disulfide oxidoreductases, protein disulfide isomerase (PDI) in eukaryotes and dsbA in prokaryotes. Structure-function analysis of these soluble proteins demonstrates that their active sites are sequences with similarity to the active site of the redox protein thioredoxin. Although these active sites share homology with thioredoxin, it is evident that other structural determinants change the redox characteristics of these proteins to enable them to facilitate the formation of correct disulfide bridging in the nascent protein. The analysis of structure-function relationships of PDI and dsbA have indicated that these thiol-disulfide oxidoreductases act as protein oxidants to facilitate the formation of disulfides during the folding process. The ability of PDI and dsbA to catalyze the formation and/or isomerization of disulfide bridging establishes their usefulness in facilitating the in vitro and in vivo folding of proteins. Protocols for the purification and assay of PDI activity have been described. Systems for the expression of PDI in Escherichia coli and Spodoptera frugiperda cells have been developed which may prove useful in the expression of recombinant proteins.

Amino Acid Sequence↗
PubMed2003

DsbA and DsbC-catalyzed oxidative folding of proteins with complex disulfide bridge patterns in vitro and in vivo.

Oxidative protein folding in the periplasm of Escherichia coli is catalyzed by the thiol-disulfide oxidoreductases DsbA and DsbC. We investigated the catalytic efficiency of these enzymes during folding of proteins with a very complex disulfide pattern in vivo and in vitro, using the Ragi bifunctional inhibitor (RBI) as model substrate. RBI is a 13.1 kDa protein with five overlapping disulfide bonds. We show that reduced RBI can be refolded quantitatively in glutathione redox buffers in vitro and spontaneously adopts the single correct conformation out of 750 possible species with five disulfide bonds. Under oxidizing redox conditions, however, RBI folding is hampered by accumulation of a large number of intermediates with non-native disulfide bonds, while a surprisingly low number of intermediates accumulates under optimal or reducing redox conditions. DsbC catalyzes folding of RBI under all redox conditions in vitro, but is particularly efficient in rearranging buried, non-native disulfide bonds formed under oxidizing conditions. In contrast, the influence of DsbA on the refolding reaction is essentially restricted to reducing redox conditions where disulfide formation is rate limiting. The effects of DsbA and DsbC on folding of RBI in E.coli are very similar to those observed in vitro. Whereas overexpression of DsbA has no effect on the amount of correctly folded RBI, co-expression of DsbC enhanced the efficiency of RBI folding in the periplasm of E.coli about 14-fold. Addition of reduced glutathione to the growth medium together with DsbC overexpression further increased the folding yield of RBI in vivo to 26-fold. This shows that DsbC is the bacterial enzyme of choice for improving the periplasmic folding yields of proteins with very complex disulfide bond patterns.

Binding Sites↗
PubMed1997

Human IgG is substrate for the thioredoxin system: differential cleavage pattern of interchain disulfide bridges in IgG subclasses.

Thioredoxin, a 12,000 mol. wt protein with two redox-active cysteine residues, together with thioredoxin reductase and NADPH, may reduce protein disulfides and thereby act as a molecular probe of their structure and reactivity. Interchain and intrachain disulfides are structural elements in all immunoglobulins and therefore potential substrates for the reduced thioredoxin, Trx(SH)2. It was investigated whether such disulfides are cleaved in human polyclonal IgG and IgG subclass myeloma proteins by both the human and the Escherichia coli thioredoxin systems. The reactions were monitored spectrophotometrically as oxidation of NADPH at 340 nm, and by following the kinetics of the cleavage patterns with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Human IgG was a substrate for both prokaryotic and eukaryotic Trx(SH)2, which directly reduced IgG disulfides in a time and dose-dependent manner. Stoichiometric analyses indicated near-complete reduction of mainly inter-heavy light chain and inter-heavy chain disulfides, and SDS PAGE corroborated that the buried intrachain disulfides were left intact. The kinetic studies showed that IgG1, IgG3 and IgG4 were readily reduced into heavy and light chains via the formation of half-molecules with slightly slower kinetics for IgG4. In sharp contrast, IgG2 was not cleaved at all, even with increased thioredoxin concentrations or reduction times. A small but significant NADPH consumption by IgG2 myeloma proteins suggested reduction of a labile interchain or surface-exposed mixed disulfide. Consistent results were obtained for several IgG myeloma proteins within each subclass. The structural and functional importance of interchain disulfides in immunoglobulins suggests physiological implications of the thioredoxin system.

Disulfides↗
PubMed1991

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: pre-steady-state kinetics and the utilization of the oxidizing equivalents of the isomerase.

At low concentrations of a glutathione redox buffer, the protein disulfide isomerase (PDI) catalyzed oxidative renaturation of reduced ribonuclease A exhibits a rapid but incomplete activation of ribonuclease, which precedes the steady-state reaction. This behavior can be attributed to a GSSG-dependent partitioning of the substrate, reduced ribonuclease, between two classes of thiol/disulfide redox forms, those that can be converted to active ribonuclease at low concentrations of GSH and those that cannot. With catalytic concentrations of PDI and near stoichiometric concentrations of glutathione disulfide, approximately 4 equiv (2 equiv of ribonuclease disulfide) of GSH are formed very rapidly followed by a slower formation of GSH, which corresponds to an additional 2 disulfide bond equiv. The rapid formation of RNase disulfide bonds and the subsequent rearrangement of incorrect disulfide isomers to active RNase are both catalyzed by PDI. In the absence of GSSG or other oxidants, disulfide bond equivalents of PDI can be used to form disulfide bonds in RNase in a stoichiometric reaction. In the absence of a glutathione redox buffer, the rate of reduced ribonuclease regeneration increases markedly with increasing PDI concentrations below the equivalence point; however, PDI in excess over stoichiometric concentrations inhibits RNase regeneration.

Enzyme Activation↗
PubMed2002

Thiol-disulfide exchange in an immunoglobulin-like fold: structure of the N-terminal domain of DsbD.

Escherichia coli DsbD transports electrons across the plasma membrane, a pathway that leads to the reduction of protein disulfide bonds. Three secreted thioredoxin-like factors, DsbC, DsbE, and DsbG, reduce protein disulfide bonds whereby an active site C-X-X-C motif is oxidized to generate a disulfide bond. DsbD catalyzes the reduction of the disulfide of DsbC, DsbE, and DsbG but not of the thioredoxin-like oxidant DsbA. The reduction of DsbC, DsbE, and DsbG occurs by transport of electrons from cytoplasmic thioredoxin to the C-terminal thioredoxin-like domain of DsbD (DsbD(C)). The N-terminal domain of DsbD, DsbD(N), acts as a versatile adaptor in electron transport and is capable of forming disulfides with oxidized DsbC, DsbE, or DsbG as well as with reduced DsbD(C). Isolated DsbD(N) is functional in electron transport in vitro. Crystallized DsbD(N) assumes an immunoglobulin-like fold that encompasses two active site cysteines, C103 and C109, forming a disulfide bond between beta-strands. The disulfide of DsbD(N) is shielded from the environment and capped by a phenylalanine (F70). A model is discussed whereby the immunoglobulin fold of DsbD(N) may provide for the discriminating interaction with thioredoxin-like factors, thereby triggering movement of the phenylalanine cap followed by disulfide rearrangement.

Amino Acid Sequence↗
PubMed1998

Reactivity of the human thioltransferase (glutaredoxin) C7S, C25S, C78S, C82S mutant and NMR solution structure of its glutathionyl mixed disulfide intermediate reflect catalytic specificity.

Human thioltransferase (TTase) is a 12 kDa thiol-disulfide oxidoreductase that appears to play a critical role in maintaining the redox environment of the cell. TTase acts as a potent and specific reducing agent for protein-S-S-glutathione mixed disulfides (protein-SSG) likely formed during oxidative stress or as redox intermediates in signal transduction pathways. Accordingly, the catalytic cycle of thioltransferase itself involves a covalent glutathionyl enzyme disulfide intermediate (TTase-C22-SSG). To understand the molecular basis of TTase specificity for the glutathione moiety, we engineered a quadruple Cys to Ser mutant of human TTase (C7S, C25S, C78S, and C82S) which retains only the active site cysteine residue (C22), and we solved its high-resolution NMR solution structure in the mixed disulfide intermediate with glutathione (QM-TTase-SSG). This mutant which cannot form a C22-S-S-C25 intramolecular disulfide displays the same catalytic efficiency (Vmax/KM) and specificity for glutathionyl mixed disulfide substrates as wild-type TTase, indicating that the Cys-25-SH moiety is not required for catalysis or glutathionyl specificity. The structure of human thioltransferase is characterized by a thioredoxin-like fold which comprises a four-stranded central beta-sheet flanked on each side by alpha-helices. The disulfide-adducted glutathione in the TTase-SSG complex has an extended conformation and is localized in a cleft near the protein surface encompassing the residues from helices-alpha2,alpha3, the active site loop, and the loop connecting helix-alpha3 and strand-beta3. Numerous van der Waals and electrostatic interactions between the protein and the glutathione moiety are identified as contributing to stabilization of the complex and confering the substrate specificity. Comparison of the human thioltransferase with other thiol-disulfide oxidoreductases reveals structural and functional differences.

Amino Acid Sequence↗
PubMed1986

Thioredoxin-catalyzed refolding of disulfide-containing proteins.

Thioredoxin, a known catalyst for reducing protein disulfides, was shown to catalyze efficiently the refolding of pancreatic RNase either from the reduced, denatured form or from the scrambled form containing oxidized but incorrectly paired disulfides. Thioredoxin was 1000-fold more efficient on a molar basis than the model dithiol, dithiothreitol, in reactivating reduced, denatured RNase, suggesting that thioredoxin acts as an efficient catalyst for disulfide interchange. Starting with reduced, denatured RNase, enzyme activity was recovered quantitatively with a t1/2 of 30 hr with 100 microM thioredoxin compared to only a 10-20% recovery of activity in the control using air oxidation. Oxygen further stimulated the effectiveness of thioredoxin severalfold. Thioredoxin was most effective in reactivating inactive scrambled RNase, which contained mispaired disulfides, showing a t1/2 of 2 hr. Reduced thioredoxin was optimal for catalyzing disulfide interchange in scrambled RNase, whereas oxidized thioredoxin was required for reactivation of the reduced, denatured species. Optimal reactivation of scrambled RNase required a mixture of reduced and oxidized thioredoxin. Addition of reduced thioredoxin after initiating refolding of reduced denatured RNase with oxidized glutathione effected a rapid reactivation of RNase, suggesting a two-step model for protein refolding in which the monothiol catalyzes the rapid initial formation of protein disulfides and thioredoxin catalyzes the second step of disulfide interchange. Arguments are presented suggesting that thioredoxin may serve an in vivo role analogous to the protein disulfide-isomerase (EC 5.3.4.1).

Bacterial Proteins↗
PubMed1999

Functional analysis of paralogous thiol-disulfide oxidoreductases in Bacillus subtilis.

The in vivo formation of disulfide bonds, which is critical for the stability and/or activity of many proteins, is catalyzed by thiol-disulfide oxidoreductases. In the present studies, we show that the Gram-positive eubacterium Bacillus subtilis contains three genes, denoted bdbA, bdbB, and bdbC, for thiol-disulfide oxidoreductases. Escherichia coli alkaline phosphatase, containing two disulfide bonds, was unstable when secreted by B. subtilis cells lacking BdbB or BdbC, and notably, the expression levels of bdbB and bdbC appeared to set a limit for the secretion of active alkaline phosphatase. Cells lacking BdbC also showed decreased stability of cell-associated forms of E. coli TEM-beta-lactamase, containing one disulfide bond. In contrast, BdbA was not required for the stability of alkaline phosphatase or beta-lactamase. Because BdbB and BdbC are typical membrane proteins, our findings suggest that they promote protein folding at the membrane-cell wall interface. Interestingly, pre-beta-lactamase processing to its mature form was stimulated in cells lacking BdbC, suggesting that the unfolded form of this precursor is a preferred substrate for signal peptidase. Surprisingly, cells lacking BdbC did not develop competence for DNA uptake, indicating the involvement of disulfide bond-containing proteins in this process. Unlike E. coli and yeast, none of the thiol-disulfide oxidoreductases of B. subtilis was required for growth in the presence of reducing agents. In conclusion, our observations indicate that BdbB and BdbC have a general role in disulfide bond formation, whereas BdbA may be dedicated to a specific process.

Alkaline Phosphatase↗
PubMed2000

Disulfide bonds are generated by quinone reduction.

The chemistry of disulfide exchange in biological systems is well studied. However, very little information is available concerning the actual origin of disulfide bonds. Here we show that DsbB, a protein required for disulfide bond formation in vivo, uses the oxidizing power of quinones to generate disulfides de novo. This is a novel catalytic activity, which to our knowledge has not yet been described. This catalytic activity is apparently the major source of disulfides in vivo. We developed a new assay to characterize further this previously undescribed enzymatic activity, and we show that quinones get reduced during the course of the reaction. DsbB contains a single high affinity quinone-binding site. We reconstitute oxidative folding in vitro in the presence of the following components that are necessary in vivo: DsbA, DsbB, and quinone. We show that the oxidative refolding of ribonuclease A is catalyzed by this system in a quinone-dependent manner. The disulfide isomerase DsbC is required to regain ribonuclease activity suggesting that the DsbA-DsbB system introduces at least some non-native disulfide bonds. We show that the oxidative and isomerase systems are kinetically isolated in vitro. This helps explain how the cell avoids oxidative inactivation of the disulfide isomerization pathway.

Bacterial Proteins↗
PubMed2005

Catalysis of thiol/disulfide exchange. Glutaredoxin 1 and protein-disulfide isomerase use different mechanisms to enhance oxidase and reductase activities.

Glutaredoxin (Grx) and protein-disulfide isomerase (PDI) are members of the thioredoxin superfamily of thiol/disulfide exchange catalysts. Thermodynamically, rat PDI is a 600-fold better oxidizing agent than Grx1 from Escherichia coli. Despite that, Grx1 is a surprisingly good protein oxidase. It catalyzes protein disulfide formation in a redox buffer with an initial velocity that is 30-fold faster than PDI. Catalysis of protein and peptide oxidation by the individual catalytic domains of PDI and by a Grx1-PDI chimera show that differences in active site chemistry are fundamental to their oxidase activity. Mutations in the active site cysteines reveal that Grx1 needs only one cysteine to catalyze rapid substrate oxidation, whereas PDI requires both cysteines. Grx1 is a good oxidase because of the high reactivity of a Grx1-glutathione mixed disulfide, and PDI is a good oxidase because of the high reactivity of the disulfide between the two active site cysteines. As a protein disulfide reductase, Grx1 is also superior to PDI. It catalyzes the reduction of nonnative disulfides in scrambled ribonuclease and protein-glutathione mixed disulfides 30-180 times faster than PDI. A multidomain structure is necessary for PDI to catalyze effective protein reduction; however, placing Grx1 into the PDI multidomain structure does not enhance its already high reductase activity. Grx1 and PDI have both found mechanisms to enhance active site reactivity toward proteins, particularly in the kinetically difficult direction: Grx1 by providing a reactive glutathione mixed disulfide to supplement its oxidase activity and PDI by utilizing its multidomain structure to supplement its reductase activity.

Animals↗
PubMed1998

Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins.

Cytoplasmic proteins do not generally contain structural disulfide bonds, although certain cytoplasmic enzymes form such bonds as part of their catalytic cycles. The disulfide bonds in these latter enzymes are reduced in Escherichia coli by two systems; the thioredoxin pathway and the glutathione/glutaredoxin pathway. However, structural disulfide bonds can form in proteins in the cytoplasm when the gene (trxB) for the enzyme thioredoxin reductase is inactivated by mutation. This disulfide bond formation can be detected by assessing the state of the normally periplasmic enzyme alkaline phosphatase (AP) when it is localized to the cytoplasm. Here we show that the formation of disulfide bonds in cytoplasmic AP in the trxB mutant is dependent on the presence of two thioredoxins in the cell, thioredoxins 1 and 2, the products of the genes trxA and trxC, respectively. Our evidence supports a model in which the oxidized forms of these thioredoxins directly catalyze disulfide bond formation in cytoplasmic AP, a reversal of their normal role. In addition, we show that the recently discovered thioredoxin 2 can perform many of the roles of thioredoxin 1 in vivo, and thus is able to reduce certain essential cytoplasmic enzymes. Our results suggest that the three most effective cytoplasmic disulfide-reducing proteins are thioredoxin 1, thioredoxin 2 and glutaredoxin 1; expression of any one of these is sufficient to support aerobic growth. Our results help to explain how the reducing environment in the cytoplasm is maintained so that disulfide bonds do not normally occur.

Alkaline Phosphatase↗
PubMed2001

Interchain disulfide bonds promote protein cross-linking during protein folding.

We provide evidence that in vitro protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. Oxidative refolding and thermal unfolding of ribonuclease A, lysozyme, and protein disulfide isomerase led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis. Chemical modification of free amino groups in these proteins or unfolding at pH < 7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links; no dimers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol. Similarly, the Cys326Ser point mutation in protein disulfide isomerase abrogated its ability to cross-link into homodimers. Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-links and interchain isopeptide cross-links. No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of thermal denaturation temperature.

Animals↗