Periplasmic protein thiol:disulfide oxidoreductases of Escherichia coli

Disulfide bond formation is part of the folding pathway for many periplasmic and outer membrane proteins that contain structural disulfide bonds. In Escherichia coli, a broad variety of periplasmic protein thiol:disulfide oxidoreductases have been identified in recent years, which substantially contribute to this pathway. Like the well-known cytoplasmic thioredoxins and glutaredoxins, these periplasmic protein thiol:disulfide oxidoreductases contain the conserved C-X-X-C motif in their active site. Most of them have a domain that displays the thioredoxin-like fold. In contrast to the cytoplasmic system, which consists exclusively of reducing proteins, the periplasmic oxidoreductases have either an oxidising, a reducing or an isomerisation activity. Apart from understanding their physiological role, it is of interest to learn how these proteins interact with their target molecules and how they are recycled as electron donors or acceptors. This review reflects the recently made efforts to elucidate the sources of oxidising and reducing power in the periplasm as well as the different properties of certain periplasmic protein thiol:disulfide oxidoreductases of E. coli.     

The human PDI family: versatility packed into a single fold

The enzymes of the protein disulfide isomerase (PDI) family are thiol-disulfide oxidoreductases of the endoplasmic reticulum (ER). They contain a CXXC active-site sequence where the two cysteines catalyze the exchange of a disulfide bond with or within substrates. The primary function of the PDIs in promoting oxidative protein folding in the ER has been extended in recent years to include roles in other processes such as ER-associated degradation (ERAD), trafficking, calcium homeostasis, antigen presentation and virus entry. Some of these functions are performed by non-catalytic members of the family that lack the active-site cysteines. Regardless of their function, all human PDIs contain at least one domain of approximately 100 amino acid residues with structural homology to thioredoxin. As we learn more about the individual proteins of the family, a complex picture is emerging that emphasizes as much their differences as their similarities, and underlines the versatility of the thioredoxin fold. Here, we primarily explore the diversity of cellular functions described for the human PDIs.     

Thioredoxins and glutaredoxins as facilitators of protein folding

Thiol-disulfide oxidoreductase systems of bacterial cytoplasm and eukaryotic cytosol favor reducing conditions and protein thiol groups, while bacterial periplasm and eukaryotic endoplasmatic reticulum provide oxidizing conditions and a machinery for disulfide bond formation in the secretory pathway. Oxidoreductases of the thioredoxin fold superfamily catalyze steps in oxidative protein folding via protein-protein interactions and covalent catalysis to act as chaperones and isomerases of disulfides to generate a native fold. The active site dithiol/disulfide of thioredoxin fold proteins is CXXC where variations of the residues inside the disulfide ring are known to increase the redox potential like in protein disulfide isomerases. In the catalytic mechanism thioredoxin fold proteins bind to target proteins through conserved backbone-backbone hydrogen bonds and induce conformational changes of the target disulfide followed by nucleophilic attack by the N-terminally located low pK(a) Cys residue. This generates a mixed disulfide covalent bond which subsequently is resolved by attack from the C-terminally located Cys residue. This review will focus on two members of the thioredoxin superfamily of proteins known to be crucial for maintaining a reduced intracellular redox state, thioredoxin and glutaredoxin, and their potential functions as facilitators and regulators of protein folding and chaperone activity.     

Oxidative folding and reductive activities of EhPDI, a protein disulfide isomerase from Entamoeba histolytica

PDI enzymes are oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. We have previously identified an E. histolytica PDI enzyme (EhPDI) that exhibits oxidase activity in vivo. However, little is known about the specific role of its redox-related structural features on the enzymatic activity. Here, we have studied the in vivo oxidative folding of EhPDI by mutagenic analysis and functional complementation assays as well as the in vitro oxidative folding and reductive activities by comparative kinetics using functional homologues in standard assays. We have found that the active-site cysteine residues of the functional domains (Trx-domains) are essential for catalysis of disulfide bond formation in polypeptides and proteins, such as the bacterial alkaline phosphatase. Furthermore, we have shown that the recombinant EhPDI enzyme has some typical properties of PDI enzymes: oxidase and reductase activities. These activities were comparable to those observed for other functional equivalents, such as bovine PDI or bacterial thioredoxin, under the same experimental conditions. These findings will be helpful for further studies intended to understand the physiological role of EhPDI.     

A thiol‐disulfide oxidoreductase of the Gram‐positive pathogen Corynebacterium diphtheriae is essential for viability,pilus assembly,toxin production and virulence

The Gram‐positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane‐bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein‐folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol‐disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin‐like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature‐sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.     

Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway.     

Oxidative protein folding in the plant endoplasmic reticulum

For most of the proteins synthesized in the endoplasmic reticulum (ER), disulfide bond formation accompanies protein folding in a process called oxidative folding. Oxidative folding is catalyzed by a number of enzymes, including the family of protein disulfide isomerases (PDIs), as well as other proteins that supply oxidizing equivalents to PDI family proteins, like ER oxidoreductin 1 (Ero1). Oxidative protein folding in the ER is a basic vital function, and understanding its molecular mechanism is critical for the application of plants as protein production tools. Here, I review the recent research and progress related to the enzymes involved in oxidative folding in the plant ER. Firstly, nine groups of plant PDI family proteins are introduced. Next, the enzymatic properties of plant Ero1 are described. Finally, the cooperative folding by multiple PDI family proteins and Ero1 is described.     

Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm.

The thiol/disulfide oxidoreductase DsbA is the strongest oxidant of the thioredoxin superfamily and is required for efficient disulfide bond formation in the periplasm of Escherichia coli. To determine the importance of the redox potential of the final oxidant in periplasmic protein folding, we have investigated the ability of the most reducing thiol/disulfide oxidoreductase, E.coli thioredoxin, of complementing DsbA deficiency when secreted to the periplasm. In addition, we secreted thioredoxin variants with increased redox potentials as well as the catalytic a-domain of human protein disulfide isomerase (PDI) to the periplasm. While secreted wild-type thioredoxin and the most reducing thioredoxin variant could not replace DsbA, all more oxidizing thioredoxin variants as well as the PDI a-domain could complement DsbA deficiency in a DsbB-dependent manner. There is an excellent agreement between the activity of the secreted thioredoxin variants in vivo and their ability to oxidize polypeptides fast and quantitatively in vitro. We conclude that the redox potential of the direct oxidant of folding proteins and in particular its reactivity towards reduced polypeptides are crucial for efficient oxidative protein folding in the bacterial periplasm.     

Cell-free synthesis of proteins that require disulfide bonds using glucose as an energy source

The primary objective of this work was to create a cell-free protein synthesis extract that produces proteins requiring disulfide bonds while using glucose as an energy source. We attempted to avoid using iodoacetamide (IAM) to stabilize the required oxidizing thiol redox potential, since previous IAM pretreatments prevented glucose utilization apparently by inactivating glyceraldehyde 3-phosphate dehydrogenase (G-3PDH). Instead, the glutathione reductase (Gor)-mediated disulfide reductase system was disabled by deleting the gor gene from the KC6 cell-extract source strain. The thioredoxin reductase (TrxB)-mediated system was disabled by first adding a purification tag to the trxB gene in the chromosome to create strain KGK10 and then by affinity removal of the tagged TrxB. This was expected to result in a cell extract devoid of all disulfide reductase activity, but this was not the case. Although the concentration of IAM required to stabilize oxidized glutathione in the KGK10 extract could be reduced 20-fold, IAM pretreatment was still required to avoid disulfide reduction. Nonetheless, active urokinase and murine granulocyte macrophage-colony stimulating factor (mGM-CSF) were produced in reactions with KGK10 extract either with affinity removal of TrxB or with 50 microM IAM pretreatment. With the less intensive IAM pretreatment, glucose could be used as an energy source in a production system that promotes oxidative protein folding. This new protocol offers an economically feasible cell-free system for the production of secreted mammalian proteins as human therapeutics or vaccines.     

On the role of the cis-proline residue in the active site of DsbA

In addition to the Cys-Xaa-Xaa-Cys motif at position 30-33, DsbA, the essential catalyst for disulfide bond formation in the bacterial periplasm shares with other oxidoreductases of the thioredoxin family a cis-proline in proximity of the active site residues. In the variant DsbA(P151A), this residue has been changed to an alanine, an almost isosteric residue which is not disposed to adopt the cis conformation. The substitution strongly destabilized the structure of DsbA, as determined by the decrease in the free energy of folding. The pKa of the thiol of Cys30 was only marginally decreased. Although in vivo the variant appeared to be correctly oxidized, it exhibited an activity less than half that of the wild-type enzyme with respect to the folding of alkaline phosphatase, used as a reporter of the disulfide bond formation in the periplasm. DsbA(P151A) crystallized in a different crystal form from the wild-type protein, in space group P2(1) with six molecules in the asymmetric unit. Its X-ray structure was determined to 2.8 A resolution. The most significant conformational changes occurred at the active site. The loop 149-152 adopted a new backbone conformation with Ala151 in a trans conformation. This rearrangement resulted in the loss of van der Waals interactions between this loop and the disulfide bond. His32 from the Cys-Xaa-Xaa-Cys sequence presented in four out of six molecules in the asymmetric unit a gauche conformation not observed in the wild-type protein. The X-ray structure and folding studies on DsbA(P151A) were consistent with the cis-proline playing a major role in the stabilization of the protein. A role for the positioning of the substrate is discussed. These important properties for the enzyme function might explain the conservation of this residue in DsbA and related proteins possessing the thioredoxin fold.     

Protein thiol modifications visualized in vivo

Thiol-disulfide interconversions play a crucial role in the chemistry of biological systems. They participate in the major systems that control the cellular redox potential and prevent oxidative damage. In addition, thiol-disulfide exchange reactions serve as molecular switches in a growing number of redox-regulated proteins. We developed a differential thiol-trapping technique combined with two-dimensional gel analysis, which in combination with genetic studies, allowed us to obtain a snapshot of the in vivo thiol status of cellular proteins. We determined the redox potential of protein thiols in vivo, identified and dissected the in vivo substrate proteins of the major cellular thiol-disulfide oxidoreductases, and discovered proteins that undergo thiol modifications during oxidative stress. Under normal growth conditions most cytosolic proteins had reduced cysteines, confirming existing dogmas. Among the few partly oxidized cytosolic proteins that we detected were proteins that are known to form disulfide bond intermediates transiently during their catalytic cycle (e.g., dihydrolipoyl transacetylase and lipoamide dehydrogenase). Most proteins with highly oxidized thiols were periplasmic proteins and were found to be in vivo substrates of the disulfide-bond-forming protein DsbA. We discovered a substantial number of redox-sensitive cytoplasmic proteins, whose thiol groups were significantly oxidized in strains lacking thioredoxin A. These included detoxifying enzymes as well as many metabolic enzymes with active-site cysteines that were not known to be substrates for thioredoxin. H₂O₂-induced oxidative stress resulted in the specific oxidation of thiols of proteins involved in detoxification of H₂O₂ and of enzymes of cofactor and amino acid biosynthesis pathways such as thiolperoxidase, GTP-cyclohydrolase I, and the cobalamin-independent methionine synthase MetE. Remarkably, a number of these proteins were previously or are now shown to be redox regulated.     

Glutathione directly reduces an oxidoreductase in the endoplasmic reticulum of mammalian cells

The formation of disulfide bonds is an essential step in the folding of many glycoproteins and secretory proteins. Non-native disulfide bonds are often formed between incorrect cysteine residues, and thus the cell has dedicated a family of oxidoreductases that are thought to isomerize non-native bonds. For an oxidoreductase to be capable of performing isomerization or reduction reactions, it must be maintained in a reduced state. Here we show that most of the oxidoreductases are predominantly reduced in vivo. Following oxidative stress the oxidoreductases are quickly reduced, demonstrating that a robust reductive pathway is in place in mammalian cells. Using ERp57 as a model we show that the reductive pathway is cytosol-dependent and that the component responsible for the reduction of the oxidoreductases is the low molecular mass thiol glutathione. In addition, ERp57 is not reduced following oxidative stress when inhibitors of glutathione synthesis or glutathione reduction are added to cells. Glutathione directly reduces ERp57 at physiological concentrations in vitro, and biotinylated glutathione forms a mixed disulfide with ERp57 in microsomes. Our results demonstrate that glutathione plays a direct role in the isomerization of disulfide bonds by maintaining the mammalian oxidoreductases in a reduced state.     

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding‐induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond‐containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild‐type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding‐induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide‐stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra‐cytosolic compartment, like the periplasm.     

Characterization of Escherichia coli thioredoxin variants mimicking the active-sites of other thiol/disulfide oxidoreductases.

Thiol/disulfide oxidoreductases like thioredoxin, glutaredoxin, DsbA, or protein disulfide isomerase (PDI) share the thioredoxin fold and a catalytic disulfide bond with the sequence Cys-Xaa-Xaa-Cys (Xaa corresponds to any amino acid). Despite their structural similarities, the enzymes have very different redox properties, which is reflected by a 100,000-fold difference in the equilibrium constant (K(eq)) with glutathione between the most oxidizing member, DsbA, and the most reducing member, thioredoxin. Here we present a systematic study on a series of variants of thioredoxin from Escherichia coli, in which the Xaa-Xaa dipeptide was exchanged by that of glutaredoxin, PDI, and DsbA. Like the corresponding natural enzymes, all thioredoxin variants proved to be stronger oxidants than the wild-type, with the order wild-type < PDI-type < DsbA-type < glutaredoxin-type. The most oxidizing, glutaredoxin-like variant has a 420-fold decreased value of K(eq), corresponding to an increase in redox potential by 75 mV. While oxidized wild-type thioredoxin is more stable than the reduced form (delta deltaG(ox/red) = 16.9 kJ/mol), both redox forms have almost the same stability in the variants. The pH-dependence of the reactivity with the alkylating agent iodoacetamide proved to be the best method to determine the pKa value of thioredoxin's nucleophilic active-site thiol (Cys32). A pKa of 7.1 was measured for Cys32 in the reduced wild-type. All variants showed a lowered pKa of Cys32, with the lowest value of 5.9 for the glutaredoxin-like variant. A correlation of redox potential and the Cys32 pKa value could be established on a quantitative level. However, the predicted correlation between the measured delta deltaG(ox/red) values and Cys32 pKa values was only qualitative.     

蛋白质二硫键异构酶家族的结构与功能

蛋白质二硫键异构酶（protein disulfide isomerase,PDI）家族是一类在内质网中起作用的巯基-二硫键氧化还原酶.它们通常含有CXXC（Cys-Xaa-Xaa-Cys,CXXC）活性位点,活性位点的两个半胱氨酸残基可催化底物二硫键的形成、异构及还原.所有PDI家族成员包含至少一个约100个氨基酸残基的硫氧还蛋白同源结构域.PDI家族的主要职能是催化内质网中新生肽链的氧化折叠,另外在内质网相关的蛋白质降解途径（ERAD）、蛋白质转运、钙稳态、抗原提呈及病毒入侵等方面也起重要作用.     

叶绿体硫氧还蛋白系统的调节机制

硫氧还蛋白(Trx)属于巯基-二硫键氧化还原酶家族, 通过作用于底物蛋白侧链2个半胱氨酸残基之间的二硫键(还原、异构和转移)来调控胞内蛋白的结构和功能。叶绿体Trx系统包括Trx及Trx类似蛋白、铁氧还蛋白(Fd)依赖的硫氧还蛋白还原酶(FTR)和还原型烟酰腺嘌呤二核苷磷酸(NADPH)依赖的硫氧还蛋白还原酶C (NTRC)。除了基质蛋白酶类活性变化及叶绿体蛋白的转运受Trx系统调控之外, 在叶绿体中还存在1条跨类囊体膜的还原势传递途径, 把基质Trx的还原势经跨膜转运蛋白介导, 最终传递给类囊体腔蛋白。FTR和NTRC共同作用维持叶绿体的氧化还原平衡。该文对叶绿体硫氧还蛋白系统的调节机制进行了综述, 同时讨论了叶绿体硫氧还蛋白系统对维持植物光合效率的重要意义。     

Oxidative protein folding: From thiol–disulfide exchange reactions to the redox poise of the endoplasmic reticulum

This review examines oxidative protein folding within the mammalian endoplasmic reticulum (ER) from an enzymological perspective. In protein disulfide isomerase-first (PDI-first) pathways of oxidative protein folding, PDI is the immediate oxidant of reduced client proteins and then addresses disulfide mispairings in a second isomerization phase. In PDI-second pathways the initial oxidation is PDI-independent. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Strategies and challenges are discussed for determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDI_red:PDI_ox. The preponderance of evidence suggests that the mammalian ER is more reducing than first envisaged. The average redox state of major PDI-family members is largely to almost totally reduced. These observations are consistent with model studies showing that oxidative protein folding proceeds most efficiently at a reducing redox poise consistent with a stoichiometric insertion of disulfides into client proteins. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, this review concludes with an elaboration of a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC–MS–MS methods. Consortia of oxidoreductases that are in redox equilibrium can then be identified and compared to the glutathione redox poise of the ER to gain a more detailed understanding of the factors that influence oxidative protein folding within the secretory compartment.     

蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。