Both chaperone and isomerase functions of protein disulfide isomerase are essential for acceleration of the oxidative refolding and reactivation of dimeric alkaline protease inhibitor

Oxidative refolding of the dimeric alkaline protease inhibitor (API) from Streptomyces sp. NCIM 5127 has been investigated. We demonstrate here that both isomerase and chaperone functions of the protein folding catalyst, protein disulfide isomerase (PDI), are essential for efficient refolding of denatured-reduced API (dr-API). Although the role of PDI as an isomerase and a chaperone has been reported for a few monomeric proteins, its role as a foldase in refolding of oligomeric proteins has not been demonstrated hitherto. Spontaneous refolding and reactivation of dr-API in redox buffer resulted in 45% to 50% reactivation. At concentrations <0.25 microM, reactivation rates and yields of dr-API are accelerated by catalytic amounts of PDI through its isomerase activity, which promotes disulfide bond formation and rearrangement. dr-API is susceptible to aggregation at concentrations >25 microM, and a large molar excess of PDI is required to enhance reactivation yields. PDI functions as a chaperone by suppressing aggregation and maintains the partially unfolded monomers in a folding-competent state, thereby assisting dimerization. Simultaneously, isomerase function of PDI brings about regeneration of native disulfides. 5-Iodoacetamidofluorescein-labeled PDI devoid of isomerase activity failed to enhance the reactivation of dr-API despite its intact chaperone activity. Our results on the requirement of a stoichiometric excess of PDI and of presence of PDI in redox buffer right from the initiation of refolding corroborate that both the functions of PDI are essential for efficient reassociation, refolding, and reactivation of dr-API.     

Chaperone activity of DsbC.

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 vitro reactivation and suppressing aggregation of denatured D-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 and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides and S-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.     

Catalytic activity and chaperone function of human protein-disulfide isomerase are required for the efficient refolding of proinsulin.

Protein-disulfide isomerase (PDI) catalyzes the formation, rearrangement, and breakage of disulfide bonds and is capable of binding peptides and unfolded proteins in a chaperone-like manner. In this study we examined which of these functions are required to facilitate efficient refolding of denatured and reduced proinsulin. In our model system, PDI and also a PDI mutant having only one active site increased the rate of oxidative folding when present in catalytic amounts. PDI variants that are completely devoid of isomerase activity are not able to accelerate proinsulin folding, but can increase the yield of refolding, indicating that they act as a chaperone. Maximum refolding yields, however, are only achieved with wild-type PDI. Using genistein, an inhibitor for the peptide-binding site, the ability of PDI to prevent aggregation of folding proinsulin was significantly suppressed. The present results suggest that PDI is acting both as an isomerase and as a chaperone during folding and disulfide bond formation of proinsulin.     

Catalysis of creatine kinase refolding by protein disulfide isomerase involves disulfide cross-link and dimer to tetramer switch

Protein disulfide isomerase (PDI) functions as an isomerase to catalyze thiol:disulfide exchange, as a chaperone to assist protein folding, and as a subunit of prolyl-4-hydroxylase and microsomal triglyceride transfer protein. At a lower concentration of 0.2 microm, PDI facilitated the aggregation of unfolded rabbit muscle creatine kinase (CK) and exhibited anti-chaperone activity, which was shown to be mainly due to the hydrophobic interactions between PDI and CK and was independent of the cross-linking of disulfide bonds. At concentrations above 1 microm, PDI acted as a protector against aggregation but an inhibitor of reactivation during CK refolding. The inhibition effect of PDI on CK reactivation was further characterized as due to the formation of PDI-CK complexes through intermolecular disulfide bonds, a process involving Cys-36 and Cys-295 of PDI. Two disulfide-linked complexes containing both PDI and CK were obtained, and the large, soluble aggregates around 400 kDa were composed of 1 molecule of tetrameric PDI and 2 molecules of inactive intermediate dimeric CK, whereas the smaller one, around 200 kDa, was formed by 1 dimeric PDI and 1 dimeric CK. To our knowledge this is the first study revealing that PDI could switch its conformation from dimer to tetramer in its functions as a foldase. According to the observations in this research and our previous study of the folding pathways of CK, a working model was proposed for the molecular mechanism of CK refolding catalyzed by PDI.     

DsbG, a protein disulfide isomerase with chaperone activity

DsbG, a protein disulfide isomerase present in the periplasm of Escherichia coli, is shown to function as a molecular chaperone. Stoichiometric amounts of DsbG are sufficient to prevent the thermal aggregation of two classical chaperone substrate proteins, citrate synthase and luciferase. DsbG was also shown to interact with refolding intermediates of chemically denatured citrate synthase and prevents their aggregation in vitro. Citrate synthase reactivation experiments in the presence of DsbG suggest that DsbG binds with high affinity to early unstructured protein folding intermediates. DsbG is one of the first periplasmic proteins shown to have general chaperone activity. This ability to chaperone protein folding is likely to increase the effectiveness of DsbG as a protein disulfide isomerase.     

Efficient folding of the insect neuropeptide eclosion hormone by protein disulfide isomerase.

Eclosion hormone is an insect neuropeptide that consists of 62 amino acid residues including three disulfide bonds. We have previously reported its hypothetical 3D structure consisting mainly of three alpha-helices. In this paper, we report the effects of chaperone proteins on the refolding of denatured eclosion hormone in a redox buffer containing reduced and oxidized glutathione. Urea-denatured eclosion hormone was spontaneously reactivated within 1 min with a yield of more than 90%, while beta-mercaptoethanol-denatured eclosion hormone was reactivated in a few minutes with a yield of 75%. Under the same experimental conditions, eclosion hormone treated with beta-mercaptoethanol and urea was reactivated slowly with a yield of 47% over a period of 2 h. Protein disulfide isomerase, a eucaryotic chaperone protein, markedly increased the reactivation yield and rate of the totally denatured hormone. GroE oligomers slightly improved the reactivation yield but peptidyl prolyl isomerase had no influence on yield or rate. We propose that the folding pathway of eclosion hormone involves at least two rate-limiting steps, and that protein disulfide isomerase is likely to be involved in the folding in insect neuronal cells.     

The acidic C-terminal domain stabilizes the chaperone function of protein disulfide isomerase

Protein disulfide isomerase (PDI, EC 5.3.4.1) is a chaperone and catalyzes the formation and rearrangement of disulfide bonds in proteins. Domain c-(463-491), containing 18 acidic residues, is an interesting and important C-terminal extension of PDI. In this study, the PDI mutant abb'a', in which domain c is truncated, was used to investigate the relationship between the C-terminal structure and chaperone function. Reactivation and light-scattering experiments show that both wild-type PDI and abb'a' interact with lactate dehydrogenase (LDH, EC 1.1.1.27), which tends to self-aggregate during reactivation. The interaction enhances reactivation of LDH and reduces aggregation. According to these results, it seems as if domain c might be dispensable to the chaperone function of PDI. However, abb'a' is prone to self-aggregation and causes increased aggregation of LDH during thermal denaturation. In contrast, wild-type PDI remains active as a chaperone under these conditions and prevents self-aggregation of LDH. Furthermore, measurements of intrinsic fluorescence and difference absorbance during denaturation show that abb'a' is much more labile to heat or guanidine hydrochloride denaturation than wild-type PDI. This suggests that domain c is required for the stabilization and maintenance of the chaperone function of PDI under extreme conditions.     

Thermodynamics of the folding of D-glyceraldehyde-3-phosphate dehydrogenase assisted by protein disulfide isomerase studied by microcalorimetry.

Thermodynamics of the refolding of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assisted by protein disulfide isomerase (PDI), a molecular chaperone, has been studied by isothermal microcalorimetry at different molar ratios of PDI/GAPDH and temperatures using two thermodynamic models proposed for chaperone-substrate binding and chaperone-assisted substrate folding, respectively. The binding of GAPDH folding intermediates to PDI is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong enthalpy-entropy compensation and weak temperature dependence of Gibbs free energy change. A large negative heat-capacity change of the binding, -156 kJ.mol(-1).K(-1), at all temperatures examined indicates that hydrophobic interaction is a major force for the binding. The binding stoichiometry shows one dimeric GAPDH intermediate per PDI monomer. The refolding of GAPDH assisted by PDI is a largely exothermic reaction at 15.0-25.0 degrees C. With increasing temperature from 15.0 to 37.0 degrees C, the PDI-assisted reactivation yield of denatured GAPDH upon dilution decreases. At 37.0 degrees C, the spontaneous reactivation, PDI-assisted reactivation and intrinsic molar enthalpy change during the PDI-assisted refolding of GAPDH are not detected.     

Peptide disulfide RKCGCFF facilitates oxidative protein refolding by mimicking protein disulfide isomerase

This communication reports a new design of peptide disulfide, RKCGCFF, for facilitating oxidative protein refolding. The new design mimics the properties of protein disulfide isomerase (PDI) by introducing hydrophobic and positively charged patches into the two terminals of disulfide CGC. RKCGCFF was found more effective than the traditional oxidant oxidized glutathione (GSSG) as well as its counterpart, RKCGC, in facilitating the oxidative refolding of lysozyme. More importantly, RKCGCFF could improve lysozyme refolding yield at a high concentration (0.7 mg/mL). The research proved that incorporation of hydrophobic and charged patches into the CGC disulfide made the oxidant more similar to PDI in structure and properties.     

GroEL and protein disulfide isomerase each binds with folding intermediates of D-glyceraldehyde-3-phosphate dehydrogenase released from complexes formed with the other

Simultaneous presence of two chaperones, GroEL and protein disulfide isomerase (PDI), assists the reactivation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in an additive way. Delayed addition of chaperones to the refolding solution after dilution of denatured GAPDH indicates an interaction with intermediates formed mainly in the first 5 min for PDI and formed within a longer time period for GroEL-ATP. The above indicate that the two chaperones interact with different folding intermediates of GAPDH. After delayed addition of one chaperone to the refolding mixture containing the other at 4°C, GroEL binds with all GAPDH intermediates dissociated from PDI, and PDI interacts with the intermediates released from GroEL during the first 10–20 min. It is suggested that the GAPDH folding intermediates released from the chaperone-bound complex are still partially folded so as to be rebound by the other chaperone. The above results clearly support the network model of GroEL and PDI.     

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.     

Protein disulfide isomerase isomerizes non-native disulfide bonds in human proinsulin independent of its peptide-binding activity

Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI-aba'c) where the b' domain was partially deleted, thus abolishing peptide binding but maintaining a PDI-like redox potential. PDI-aba'c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI-aba'c isomerizes non-native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates.     

蛋白质二硫键异构酶相关蛋白A的表达及性质的研究

克隆了Aspergillus niger T21中的蛋白质二硫键异构酶相关蛋白A（PRPA）基因,并将它插入pET23b表达载体。在E. coli中表达时,PRPA占菌体总蛋白的34%。经过超声破细胞、硫酸铵分级沉淀和离子交换层析获得了纯度大于90%的重组蛋白。PRPA有二硫键异构酶活性。在PRPA存在下,变性和还原的溶菌酶复性率和复性速度降低,电泳结果表明溶菌酶聚集增多。荧光结果表明PRPA表面有较多的疏水基团。     

Chaperone-mediated refolding of recombinant prochymosin

It has been verified that prochymosin is characterized by a two-stage refolding: dilution of unfolded protein into pH 11 buffer followed by neutralization at pH 8; the high-pH step is indispensable. Here we demonstrate that one-stage refolding around pH 8 can be achieved when GroE or 10-fold molar excess (rather than catalytic concentration) of protein disulfide isomerase (PDI) over prochymosin is present. The helping effect varies with the oxidation states of prochymosin. GroE and PDI increase the reactivation of the unfolded, partially reduced and the unfolded, oxidized prochymosin from 5% to 40% and from 50% to 100%, respectively. For the unfolded and fully reduced prochymosin, GroE does not have a positive effect, whereas PDI promotes renaturation from 2% to 28%. Based on our previous and present observations, we propose that at pH 8 there may be two kinds of incorrect interactions within and between prochymosin polypeptides leading to unproductive pathways: one prevents disulfide rearrangement, which can be avoided by high pH; the other interferes with acquisition of native conformation, which can be relieved by GroE and PDI.     

Oxidative protein folding in vitro: a study of the cooperation between quiescin-sulfhydryl oxidase and protein disulfide isomerase

The flavin-dependent quiescin-sulfhydryl oxidase (QSOX) inserts disulfide bridges into unfolded reduced proteins with the reduction of molecular oxygen to form hydrogen peroxide. This work investigates how QSOX and protein disulfide isomerase (PDI) cooperate in vitro to generate native pairings in two unfolded reduced proteins: ribonuclease A (RNase, four disulfide bonds and 105 disulfide isomers of the fully oxidized protein) and avian riboflavin binding protein (RfBP, nine disulfide bonds and more than 34 million corresponding disulfide pairings). Experiments combining avian or human QSOX with up to 200 muM avian or human reduced PDI show that the isomerase is not a significant substrate of QSOX. Both reduced RNase and RfBP can be efficiently refolded in an aerobic solution containing micromolar concentrations of reduced PDI and nanomolar levels of QSOX without any added oxidized PDI or glutathione redox buffer. Refolding of RfBP is followed continuously using the complete quenching of the fluorescence of free riboflavin that occurs on binding to apo-RfBP. The rate of refolding is half-maximal at 30 muM reduced PDI when the reduced client protein (1 muM) is used in the presence of 30 nM QSOX. The use of high concentrations of PDI, in considerable excess over the folding protein client, reflects the concentration prevailing in the lumen of the endoplasmic reticulum and allows the redox poise of these in vitro experiments to be set with oxidized and reduced PDI. In the absence of either QSOX or redox buffer, the fastest refolding of RfBP is accomplished with excess reduced PDI and just enough oxidized PDI to generate nine disulfides in the protein client. These in vitro experiments are discussed in terms of current models for oxidative folding in the endoplasmic reticulum.     

Domain a' of Bombyx mori protein disulfide isomerase has chaperone activity

Protein disulfide isomerase (PDI) is an endoplasmic reticulum (ER)-localized multifunctional enzyme that can function as a disulfide oxidase, a reductase, an isomerase, and a chaperone. The domain organization of PDI is abb'xa'c, with two catalytic (CxxC) motifs and a KDEL ER retention motif. The members of the PDI family exhibit differences in tissue distribution, specificity, and intracellular localization. We previously identified and characterized the PDI of Bombyx mori (bPDI) as a thioredoxin-like protein that shares primary sequence homology with other PDIs. Here we compare the reactivation of inactivated rRNase and sRNase by bPDI and three bPDI mutants, and show that bPDI has mammalian PDI-like activity. On its own, the N-terminal a domain does not retain this activity, but the a' domain does. This is the first report of chaperone activity only in the a' domain, but not in the a domain.     

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer

The velocity of the oxidative renaturation of reduced ribonuclease A catalyzed by protein disulfide isomerase (PDI) is strongly dependent on the composition of a glutathione/glutathione disulfide redox buffer. As with the uncatalyzed, glutathione-mediated oxidative folding of ribonuclease, the steady-state velocity of the PDI-catalyzed reaction displays a distinct optimum with respect to both the glutathione (GSH) and glutathione disulfide (GSSG) concentrations. Optimum activity is observed at [GSH] = 1.0 mM and [GSSG] = 0.2 mM. The apparent kcat at saturating RNase concentration is 0.46 +/- 0.05 mumol of RNase renatured min-1 (mumol of PDI)-1 compared to the apparent first-order rate constant for the uncatalyzed reaction of 0.02 +/- 0.01 min-1. Changes in GSH and GSSG concentration have a similar effect on the rate of both the PDI-catalyzed and uncatalyzed reactions except under the more oxidizing conditions employed, where the catalytic effectiveness of PDI is diminished. The ratio of the velocity of the catalyzed reaction to that of the uncatalyzed reaction increases as the quantity [GSH]2/[GSSG] increases and approaches a constant, limiting value at [GSH]2/[GSSG] greater than 1 mM, suggesting that a reduced, dithiol form of PDI is required for optimum activity. As long as the glutathione redox buffer is sufficiently reducing to maintain PDI in an active form [( GSH]2/[GSSG] greater than 1 mM), the rate acceleration provided by PDI is reasonably constant, although the actual rate may vary by more than an order of magnitude. PDI exhibits half of the maximum rate acceleration at a [GSH]2/[GSSG] of 0.06 +/- 0.01 mM.     

Chaperone-like activity of peptidyl-prolyl cis-trans isomerase during creatine kinase refolding

Porcine kidney 18 kD peptidyl-prolyl cis-trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 microM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration-dependent manner. PPIase exhibited chaperone-like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.     

Protein disulfide isomerase-mediated cell-free assembly of recombinant interleukin-12 p40 homodimers.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two subunits, p35 and p40. The disulfide-linked homodimer (p40)2 has been shown to be a potent IL-12 antagonist. In the present study, the p40 subunit was refolded from Escherichia coli inclusion bodies. Formation of (p40)2 was greatly increased in a redox buffer containing reduced and oxidized glutathione, but was not significantly affected by the cosolvents urea, GdnHCl or Chaps. While protein disulfide isomerase (PDI), GroEL/ES or DnaK/J/GrpE suppressed aggregation during refolding of p40, only DnaK/J/GrpE and PDI enhanced p40 dimerization. Oxidative assembly of p40 into (p40)2 by PDI, but not suppression of aggregation, was strongly dependent on inclusion of BSA in the refolding buffer. It is concluded that both chaperone-like and disulfide isomerase effects are essential for correct folding of p40 into dimers.