Both the isomerase and chaperone activities of protein disulfide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2.

The spontaneous reactivation yield of acidic phospholipase A2 (APLA2), a protein containing seven disulfide bonds, after reduction and denaturation in guanidine hydrochloride is very low. Protein disulfide isomerase (PDI) markedly increases the reactivation yield and prevents the aggregation of APLA2 during refolding in a redox buffer containing GSH and GSSG. S-methylated PDI (mPDI), with no isomerase but as nearly full chaperone activity as native PDI, has no effect on either the reactivation or aggregation of APLA2. However, the simultaneous presence of PDI and mPDI in molar ratios to APLA2 of 0.1 and 0.9 respectively fully reactivates the denatured enzyme, as does PDI alone at a ratio of 1. At ratios of 0.1 and 0.15 respectively, they completely suppress APLA2 aggregation, as does PDI alone at a ratio of 0.25. Moreover, delayed addition of PDI to the refolding buffer greatly diminished the reactivation yield of APLA2, but this deteriorating effect can be alleviated markedly by the presence of mPDI in the refolding buffer. Without GSSG, mPDI prevents the aggregation of APLA2 during refolding. It is proposed that the in vitro action of PDI as a foldase consists of both isomerase and chaperone activities, and the latter activity can be fully replaced by mPDI.     

Chaperone-Like Manner of Human Neuronal Tau Towards Lactate Dehydrogenase

In our experiments, inactivation of lactate dehydrogenase (LDH, EC1.1.1.27) in the presence of human microtubule-associated tau is observably suppressed during thermal and guanidine hydrochloride (GdnHCl) denaturation. Kinetic studies show tau can prevent LDH from self-aggregation monitored by light scattering during thermal denaturation. On the other hand, neuronal tau promotes reactivation of LDH and suppresses self-aggregation of non-native LDH when GdnHCl solution is diluted. Furthermore, the reactivation yield of LDH decreases significantly with delayed addition of tau. All experiments were completed in the reducing buffer with 1 mM DTT to avoid between tau and LDH forming the covalent bonds during unfolding and refolding. Thus, Tau prevents proteins from misfolding and aggregating into insoluble, nonfunctional inclusions and assists them to refold to reach the stable native state by binding to the exposed hydrophobic patches on proteins instead of by forming or breaking covalent bonds. Additionally, tau remarkably enhances reactivation of GDH (glutamic dehydrogenase, EC 1.4.1.3), another carbohydrate metabolic enzyme, also showing a chaperone-like manner. It suggests that neuronal tau non-specifically functions a chaperone-like protein towards the enzymes of carbohydrate metabolism.     

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.     

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.     

The acidic C-terminal domain of protein disulfide isomerase is not critical for the enzyme subunit function or for the chaperone or disulfide isomerase activities of the polypeptide.

Protein disulfide isomerase (PDI) is a multifunctional polypeptide that acts as a subunit in the animal prolyl 4-hydroxylases and the microsomal triglyceride transfer protein, and as a chaperone that binds various peptides and assists their folding. We report here that deletion of PDI sequences corresponding to the entire C-terminal domain c, previously thought to be critical for chaperone activity, had no inhibitory effect on the assembly of recombinant prolyl 4-hydroxylase in insect cells or on the in vitro chaperone activity or disulfide isomerase activity of purified PDI. However, partially overlapping critical regions for all these functions were identified at the C-terminal end of the preceding thioredoxin-like domain a'. Point mutations introduced into this region identified several residues as critical for prolyl 4-hydroxylase assembly. Circular dichroism spectra of three mutants suggested that two of these mutations may have caused only local alterations, whereas one of them may have led to more extensive structural changes. The critical region identified here corresponds to the C-terminal alpha helix of domain a', but this is not the only critical region for any of these functions.     

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.     

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.     

The excised heat-shock domain of alphaB crystallin is a folded, proteolytically susceptible trimer with significant surface hydrophobicity and a tendency to self-aggregate upon heating

The lens protein, alpha-crystallin, is a molecular chaperone that prevents the thermal aggregation of other proteins. The C-terminal domain of this protein (homologous to domains present in small heat-shock proteins) is implicated in chaperone function, although the domain itself has been reported to show no chaperone activity. Here, we show that the domain can be excised out of the intact alphaB polypeptide and recovered directly in pure form through the transfer of CNBr digests of whole lens homogenates into urea-containing buffer, followed by dialysis-based refolding of digests under acidic conditions and a single gel-filtration purification step. The folded (beta sheet) domain thus obtained is found to be (a) predominantly trimeric, and to display (b) significant surface hydrophobicity, (c) a marked tendency to undergo degradation, and (d) a tendency to aggregate upon heating, and on exposure to UV light. Thus, the twin 'chaperone' features of multimericity and surface hydrophobicity are clearly seen to be insufficient for this domain to function as a chaperone. Since alpha-crystallin interacts with its substrates through hydrophobic interactions, the hydrophobicity of the excised domain indicates that separation of domains may regulate function; at the same time, the fact is also highlighted that surface hydrophobicity is a liability in a chaperone since heating strengthens hydrophobic interactions and can potentially promote self-aggregation. Thus, it would appear that the role of the N-terminal domain in alpha-crystallin is to facilitate the creation of a porous, hollow structural framework of >/=24 subunits in which solubility is effected through increase in the ratio of exposed surface area to buried volume. Trimers of interacting C-terminal domains anchored to this superstructure, and positioned within its interior, might allow hydrophobic surfaces to remain accessible to substrates without compromising solubility.     

The activation mechanism of Hsp26 does not require dissociation of the oligomer

Small heat shock proteins (sHsps) are molecular chaperones that specifically bind non-native proteins and prevent them from irreversible aggregation. A key trait of sHsps is their existence as dynamic oligomers. Hsp26 from Saccharomyces cerevisiae assembles into a 24mer, which becomes activated under heat shock conditions and forms large, stable substrate complexes. This activation coincides with the destabilization of the oligomer and the appearance of dimers. This and results from other groups led to the generally accepted notion that dissociation might be a requirement for the chaperone mechanism of sHsps. To understand the chaperone mechanism of sHsps it is crucial to analyze the relationship between chaperone activity and stability of the oligomer. We generated an Hsp26 variant, in which a serine residue of the N-terminal domain was replaced by cysteine. This allowed us to covalently crosslink neighboring subunits by disulfide bonds. We show that under reducing conditions the structure and function of this variant are indistinguishable from that of the wild-type protein. However, when the cysteine residues are oxidized, the dissociation into dimers at higher temperatures is no longer observed, yet the chaperone activity remains unaffected. Furthermore, we show that the exchange of subunits between Hsp26 oligomers is significantly slower than substrate aggregation and even inhibited in the presence of disulfide bonds. This demonstrates that the rearrangements necessary for shifting Hsp26 from a low to a high affinity state for binding non-native proteins occur without dissolving the oligomer.     

Mechanism of the antichaperone activity of protein disulfide isomerase: facilitated assembly of large, insoluble aggregates of denatured lysozyme and PDI

Protein disulfide isomerase (PDI), a folding catalyst and chaperone can, under certain conditions, facilitate the misfolding and aggregation of its substrates. This behavior, termed antichaperone activity [Puig, A., and Gilbert, H. F., (1994) J. Biol. Chem. 269, 25889] may provide a common mechanism for aggregate formation in the cell, both as a normal consequence of cell function or as a consequence of disease. When diluted from the denaturant, reduced, denatured lysozyme (10-50 microM) remains soluble, although it does aggregate to form an ensemble of species with an average sedimentation coefficient of 23 +/- 5 S (approximately 600 +/- 100 kDa). When low concentrations of PDI (1-5 microM) are present, the majority (80 +/- 8%) of lysozyme molecules precipitate in large, insoluble aggregates, together with 87 +/- 12% of the PDI. PDI-facilitated aggregation occurs even when disulfide formation is precluded by the presence of dithiothreitol (10 mM). Maximal lysozyme-PDI precipitation occurs at a constant lysozyme/PDI ratio of 10:1 over a range of lysozyme concentrations (10-50 microM). Concomitant resolubilization of PDI and lysozyme from these aggregates by increasing concentrations of urea suggests that PDI is an integral component of the mixed aggregate. PDI induces lysozyme aggregation by noncovalently cross-linking 23 S lysozyme species to form aggregates that become so large (approximately 38,000 S) that they are cleared from the analytical ultracentrifuge even at low speed (1500 rpm). The rate of insoluble aggregate formation increases with increasing PDI concentration (although a threshold PDI concentration is observed). However, increasing lysozyme concentration slows the rate of aggregation, presumably by depleting PDI from solution. A simple mechanism is proposed that accounts for these unusual aggregation kinetics as well as the switch between antichaperone and chaperone behavior observed at higher concentrations of PDI.     

Mutations in domain a' of protein disulfide isomerase affect the folding pathway of bovine pancreatic ribonuclease A

Protein disulfide isomerase (PDI, EC 5.3.4.1), an enzyme and chaperone, catalyses disulfide bond formation and rearrangements in protein folding. It is also a subunit in two proteins, the enzyme collagen prolyl 4-hydroxylase and the microsomal triglyceride transfer protein. It consists of two catalytically active domains, a and a', and two inactive ones, b and b', all four domains having the thioredoxin fold. Domain b' contains the primary peptide binding site, but a' is also critical for several of the major PDI functions. Mass spectrometry was used here to follow the folding pathway of bovine pancreatic ribonuclease A (RNase A) in the presence of three PDI mutants, F449R, Delta455-457, and abb', and the individual domains a and a'. The first two mutants contained alterations in the last alpha helix of domain a', while the third lacked the entire domain a'. All mutants produced genuine, correctly folded RNase A, but the appearance rate of 50% of the product, as compared to wild-type PDI, was reduced 2.5-fold in the case of PDI Delta455-457, 7.5-fold to eightfold in the cases of PDI F449R and PDI abb', and over 15-fold in the cases of the individual domains a and a'. In addition, PDI F449R and PDI abb' affected the distribution of folding intermediates. Domains a and a' catalyzed the early steps in the folding but no disulfide rearrangements, and therefore the rate observed in the presence of these individual domains was similar to that of the spontaneous process.     

Substrate-Induced Unfolding of Protein Disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from Its Holotoxin

To generate a cytopathic effect, the catalytic A1 subunit of cholera toxin (CT) must be separated from the rest of the toxin. Protein disulfide isomerase (PDI) is thought to mediate CT disassembly by acting as a redox-driven chaperone that actively unfolds the CTA1 subunit. Here, we show that PDI itself unfolds upon contact with CTA1. The substrate-induced unfolding of PDI provides a novel molecular mechanism for holotoxin disassembly: we postulate the expanded hydrodynamic radius of unfolded PDI acts as a wedge to dislodge reduced CTA1 from its holotoxin. The oxidoreductase activity of PDI was not required for CT disassembly, but CTA1 displacement did not occur when PDI was locked in a folded conformation or when its substrate-induced unfolding was blocked due to the loss of chaperone function. Two other oxidoreductases (ERp57 and ERp72) did not unfold in the presence of CTA1 and did not displace reduced CTA1 from its holotoxin. Our data establish a new functional property of PDI that may be linked to its role as a chaperone that prevents protein aggregation.     

Thioredoxin fold as homodimerization module in the putative chaperone ERp29: NMR structures of the domains and experimental model of the 51 kDa dimer.

BACKGROUND: ERp29 is a ubiquitously expressed rat endoplasmic reticulum (ER) protein conserved in mammalian species. Fold predictions suggest the presence of a thioredoxin-like domain homologous to the a domain of human protein disulfide isomerase (PDI) and a helical domain similar to the C-terminal domain of P5-like PDIs. As ERp29 lacks the double-cysteine motif essential for PDI redox activity, it is suggested to play a role in protein maturation and/or secretion related to the chaperone function of PDI. ERp29 self-associates into 51 kDa dimers and also higher oligomers. RESULTS: 3D structures of the N- and C-terminal domains determined by NMR spectroscopy confirmed the thioredoxin fold for the N-terminal domain and yielded a novel all-helical fold for the C-terminal domain. Studies of the full-length protein revealed a short, flexible linker between the two domains, homodimerization by the N-terminal domain, and the presence of interaction sites for the formation of higher molecular weight oligomers. A gadolinium-based relaxation agent is shown to present a sensitive tool for the identification of macromolecular interfaces by NMR. CONCLUSIONS: ERp29 is the first eukaryotic PDI-related protein for which the structures of all domains have been determined. Furthermore, an experimental model of the full-length protein and its association states was established. It is the first example of a protein where the thioredoxin fold was found to act as a specific homodimerization module, without covalent linkages or supporting interactions by further domains. A homodimerization module similar as in ERp29 may also be present in homodimeric human PDI.     

Conserved residues flanking the thiol/disulfide centers of protein disulfide isomerase are not essential for catalysis of thiol/disulfide exchange.

Protein disulfide isomerase (PDI) catalyzes the oxidative folding of proteins containing disulfide bonds by increasing the rate of disulfide bond rearrangements which normally occur during the folding process. The amino acid sequences of the N- and C-terminal redox active sites (PWCGHCK) in PDI are completely conserved from yeast to man and display considerable identity with the redox-active center of thioredoxin (EWCGPCK). Available data indicate that the two thiol/disulfide centers of PDI can function independently in the isomerase reaction and that the cysteine residues in each active site are essential for catalysis. To evaluate the role of residues flanking the active-site cysteines of PDI in function, a variety of mutations were introduced into the N-terminal active site of PDI within the context of both a functional C-terminal active site and an inactive C-terminal active site in which serine residues replaced C379 and C382. Replacement of non-cysteine residues (W34 to Ser, G36 to Ala, and K39 to Arg) resulted in only a modest reduction in catalytic activity in both the oxidative refolding of RNase A and the reduction of insulin (10-27%), independent of the status of the C-terminal active site. A somewhat larger effect was observed with the H37P mutation where approximately 80% of the activity attributable to the N-terminal domain (approximately 40%) was lost. However, the H37P mutant N-terminal site expressed within the context of an inactive C-terminal domain exhibits 30% activity, approximately 70% of the activity of the N-terminal site alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasticity of Human Protein Disulfide Isomerase: EVIDENCE FOR MOBILITY AROUND THE X-LINKER REGION AND ITS FUNCTIONAL SIGNIFICANCE*

Protein disulfide isomerase (PDI), which consists of multiple domains arranged as abb′xa′c, is a key enzyme responsible for oxidative folding in the endoplasmic reticulum. In this work we focus on the conformational plasticity of this enzyme. Proteolysis of native human PDI (hPDI) by several proteases consistently targets sites in the C-terminal half of the molecule (x-linker and a′ domain) leaving large fragments in which the N terminus is intact. Fluorescence studies on the W111F/W390F mutant of full-length PDI show that its fluorescence is dominated by Trp-347 in the x-linker which acts as an intrinsic reporter and indicates that this linker can move between “capped” and “uncapped” conformations in which it either occupies or exposes the major ligand binding site on the b′ domain of hPDI. Studies with a range of constructs and mutants using intrinsic fluorescence, collision quenching, and extrinsic probe fluorescence (1-anilino-8-naphthalene sulfonate) show that the presence of the a′ domain in full-length hPDI moderates the ability of the x-linker to generate the capped conformation (compared with shorter fragments) but does not abolish it. Hence, unlike yeast PDI, the major conformational plasticity of full-length hPDI concerns the mobility of the a′ domain “arm” relative to the bb′ “trunk” mediated by the x-linker. The chaperone and enzymatic activities of these constructs and mutants are consistent with the interpretation that the reversible interaction of the x-linker with the ligand binding site mediates access of protein substrates to this site.     

The effect of calnexin deletion on the expression level of PDI in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under heat stress conditions

We cultured calnexin-disrupted and wild-type Saccharomyces cerevisiae strains under conditions of heat stress. The growth rate of the calnexin-disrupted yeast was almost the same as that of the wild-type yeast under those conditions. However, the induced mRNA level of the molecular chaperone PDI in the ER was clearly higher in calnexin-disrupted S. cerevisiae relative to the wild type at 37°C, despite being almost the same in the two strains under normal conditions. The western blotting analysis for PDI protein expression in the ER yielded results that show a parallel in their mRNA levels in the two strains. We suggest that PDI may interact with calnexin under heat stress conditions, and that the induction of PDI in the ER can recover part of the function of calnexin in calnexin-disrupted yeast, and result in the same growth rate as in wild-type yeast.     

Human protein-disulfide isomerase is a redox-regulated chaperone activated by oxidation of domain a'

Protein-disulfide isomerase (PDI), with domains arranged as abb'xa'c, is a key enzyme and chaperone localized in the endoplasmic reticulum (ER) catalyzing oxidative folding and preventing misfolding/aggregation of proteins. It has been controversial whether the chaperone activity of PDI is redox-regulated, and the molecular basis is unclear. Here, we show that both the chaperone activity and the overall conformation of human PDI are redox-regulated. We further demonstrate that the conformational changes are triggered by the active site of domain a', and the minimum redox-regulated cassette is located in b'xa'. The structure of the reduced bb'xa' reveals for the first time that domain a' packs tightly with both domain b' and linker x to form one compact structural module. Oxidation of domain a' releases the compact conformation and exposes the shielded hydrophobic areas to facilitate its high chaperone activity. Thus, the study unequivocally provides mechanistic insights into the redox-regulated chaperone activity of human 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.     

ATP‐independent molecular chaperone activity generated under reducing conditions

Molecular chaperones are essential to maintain proteostasis. While the functions of intracellular molecular chaperones that oversee protein synthesis, folding and aggregation, are established, those specialized to work in the extracellular environment are less understood. Extracellular proteins reside in a considerably more oxidizing milieu than cytoplasmic proteins and are stabilized by abundant disulfide bonds. Hence, extracellular proteins are potentially destabilized and sensitive to aggregation under reducing conditions. We combine biochemical and mass spectrometry experiments and elucidate that the molecular chaperone functions of the extracellular protein domain Bri2 BRICHOS only appear under reducing conditions, through the assembly of monomers into large polydisperse oligomers by an intra‐ to intermolecular disulfide bond relay mechanism. Chaperone‐active assemblies of the Bri2 BRICHOS domain are efficiently generated by physiological thiol‐containing compounds and proteins, and appear in parallel with reduction‐induced aggregation of extracellular proteins. Our results give insights into how potent chaperone activity can be generated from inactive precursors under conditions that are destabilizing to most extracellular proteins and thereby support protein stability/folding in the extracellular space.SignificanceChaperones are essential to cells as they counteract toxic consequences of protein misfolding particularly under stress conditions. Our work describes a novel activation mechanism of an extracellular molecular chaperone domain, called Bri2 BRICHOS. This mechanism is based on reducing conditions that initiate small subunits to assemble into large oligomers via a disulfide relay mechanism. Activated Bri2 BRICHOS inhibits reduction‐induced aggregation of extracellular proteins and could be a means to boost proteostasis in the extracellular environment upon reductive stress.