Yeast Mpd1p reveals the structural diversity of the protein disulfide isomerase family

Oxidoreductases belonging to the protein disulfide isomerase (PDI) family promote proper disulfide bond formation in substrate proteins in the endoplasmic reticulum. In plants and metazoans, new family members continue to be identified and assigned to various functional niches. PDI-like proteins typically contain tandem thioredoxin-fold domains. The limited information available suggested that the relative orientations of these domains may be quite uniform across the family, and structural models based on this assumption are appearing. However, the X-ray crystal structure of the yeast PDI family protein Mpd1p, described here, demonstrates the radically different domain orientations and surface properties achievable with multiple copies of the thioredoxin fold. A comparison of Mpd1p with yeast Pdi1p expands our perspective on the contexts in which redox-active motifs are presented in the PDI family.     

Structural insight into the dimerization of human protein disulfide isomerase

Protein disulfide isomerases (PDIs) are responsible for catalyzing the proper oxidation and isomerization of disulfide bonds of newly synthesized proteins in the endoplasmic reticulum (ER). Here, it is shown that human PDI (PDIA1) dimerizes in vivo and proposed that the dimerization of PDI has physiological relevance by autoregulating its activity. The crystal structure of the dimeric form of noncatalytic bb′ domains of human PDIA1 determined to 2.3 Å resolution revealed that the formation of dimers occludes the substrate binding site and may function as a mechanism to regulate PDI activity in the ER.     

A novel disulphide switch mechanism in Ero1alpha balances ER oxidation in human cells

Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox-driven shutdown mechanism of Ero1alpha, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1alpha in cells arises from the formation of a disulphide bond-identified by mass spectrometry--between the active-site Cys(94) (connected to Cys(99) in the active enzyme) and Cys(131). Competition between substrate thiols and Cys(131) creates a feedback loop where activation of Ero1alpha is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1alpha-Cys131Ala or the isoform Ero1beta, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis.     

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.     

Critical roles of protein disulfide isomerases in balancing proteostasis in the nervous system

Protein disulfide isomerases (PDIs) constitute a family of oxidoreductases promoting redox protein folding and quality control in the endoplasmic reticulum. PDIs catalyze disulfide bond formation, isomerization, and reduction, operating in concert with molecular chaperones to fold secretory cargoes in addition to directing misfolded proteins to be refolded or degraded. Importantly, PDIs are emerging as key components of the proteostasis network, integrating protein folding status with central surveillance mechanisms to balance proteome stability according to cellular needs. Recent advances in the field driven by the generation of new mouse models, human genetic studies, and omics methodologies, in addition to interventions using small molecules and gene therapy, have revealed the significance of PDIs to the physiology of the nervous system. PDIs are also implicated in diverse pathologies, ranging from neurodevelopmental conditions to neurodegenerative diseases and traumatic injuries. Here, we review the principles of redox protein folding in the ER with a focus on current evidence linking genetic mutations and biochemical alterations to PDIs in the etiology of neurological conditions.     

Export of a cysteine-free misfolded secretory protein from the endoplasmic reticulum for degradation requires interaction with protein disulfide isomerase

Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.     

A proposed reaction mechanism for rice NADPH thioredoxin reductase C, an enzyme with protein disulfide reductase activity

NADPH thioredoxin reductase C (NTRC) is an interesting NTR with a thioredoxin (Trx) domain at the C-terminus, able to conjugate both activities for 2-Cys peroxiredoxin (Prx) reduction. NTRC is dimeric in the presence of NADPH and interacted with dimeric 2-Cys Prx through the Trx module by a mixed disulfide between Cys377 of NTRC and Cys61 of the 2-Cys Prx. NTRC variants of both NTR and Trx active sites were inactive, but 1:1 mixtures of both variants allowed partial recovery of activity suggesting inter-subunit transfer of electrons during catalysis. Based on these results we propose a model for the reaction mechanism of NTRC.

Structured summary

MINT-7017333: 2cys Prx (uniprotkb:Q6ER94) and 2cys Prx (uniprotkb:Q6ER94) bind (MI:0407) by molecular sieving (MI:0071)MINT-7017101, MINT-7017183: NTRC (uniprotkb:Q70G58) and 2cys Prx (uniprotkb:Q6ER94) bind (MI:0407) by enzymatic studies (MI:0415)     

Overexpression of thioredoxin reductase 1 inhibits migration of HEK-293 cells

Background information. TrxR (thioredoxin reductase), in addition to protecting against oxidative stress, plays a role in the redox regulation of intracellular signalling pathways controlling, among others, cell proliferation and apoptosis. The aim of the present study was to determine whether TrxR1 is involved in the regulation of cell migration. Results. Stably transfected HEK‐293 (human embryonic kidney) cells which overexpress cytosolic TrxR1 (HEK‐TrxR15 and HEK‐TrxR11 cells) were used in the present study. We found that the stimulation of cell motility induced by PKC (protein kinase C) activators, PMA and DPhT (diphenyltin), was inhibited significantly in the HEK‐TrxR15 and HEK‐TrxR11 cells compared with control cells. The overexpression of TrxR1 also inhibited characteristic morphological changes and reorganization of the F‐actin cytoskeleton induced by PMA and DPhT. In addition, the selective activation of PKCδ by DPhT was inhibited in cells that overexpressed cytosolic TrxR1. Furthermore, rottlerin, a selective inhibitor of PKCδ, and PKCδ siRNA (small interfering RNA), suppressed the morphological changes induced by DPhT in the control cells. Conclusions. The overexpression of TrxR1 inhibits migration of HEK‐293 cells stimulated with PMA and DPhT. Moreover, our observations suggest that this effect is mediated by the inhibition of PKCδ activation.     

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.     

Glutathione reductase and thioredoxin reductase at the crossroad: the structure of Schistosoma mansoni thioredoxin glutathione reductase

Thioredoxin glutathione reductase (TGR) is a key flavoenzyme expressed by schistosomes that bridges two detoxification pathways crucial for the parasite survival in the host's organism. In this article we report the crystal structure (at 2.2 A resolution) of TGR from Schistosoma mansoni (SmTGR), deleted in the last two residues. The structure reveals the peculiar architecture of this chimeric enzyme: the small Glutaredoxin (Grx) domain at the N-terminus is joined to the large thioredoxin reductase (TR) one via an extended complementary surface, involving residues not conserved in the Grx superfamily; the TR domain interacts with an identical partner via its C-terminal domain, forming a dimer with a twisted "W" shape. Although lacking the penultimate Selenocysteine residue (Sec), the enzyme is still able to reduce oxidized glutathione. These data update the interpretation of the interdomain communication in TGR enzymes. The possible function of this enzyme in pathogenic parasites is discussed.     

A fluorescence-based assay for the reductase activity of protein disulfide isomerase

We report on a new spectrofluorimetric assay for the measurement of reductase activity of proteins belonging to the superfamily of thioredoxins such as protein disulfide isomerase (PDI). The assay relies on the preparation of a fluorescence-quenched substrate easily accessible in two steps through functional group transformations of the peptide Gly-Cys-Asp. In the first step fluorescein isothiocyanate is linked to the Gly-NH(2) terminus and in the second step the Cys-SH groups are converted into a disulfide bond. Both intermediate and final substrate have been fully characterized by mass spectrometric and nuclear magnetic resonance measurements. Dimethyl sulfoxide is here reported to be a mild oxidizing agent allowing us to obtain in good overall yield the assay substrate in a single synthetic step. A reliable estimation of PDI reductase activity is obtained via the detection of a strong fluorescence enhancement after enzymatic reduction. Moreover, our assay provides further support for the key role played by thioredoxin reductase in enabling disulfide reductase activity of PDI.     

Synthesis of peptide substrates for mammalian thioredoxin reductase.

Mammalian thioredoxin reductase (TR) catalyzes the reduction of the redox-active disulfide bond of thioredoxin (Trx) and is similar in structure and mechanism to glutathione reductase except for a C-terminal 16-amino acid extension containing a rare vicinal selenylsulfide bond. This vicinal selenylsulfide bond is essentially a substrate for the enzyme's N-terminal redox center. Here we report the synthesis of peptide substrates for the truncated enzyme missing the C-terminal redox center. We developed a procedure for the synthesis of peptides containing cyclic vicinal disulfide/selenylsulfide bonds as well as their corresponding acyclic heterodimers. Vicinal disulfide bonds form eight-membered ring structures and are difficult to synthesize owing to their propensity to dimerize during oxidation. Our procedure makes use of two key improvements for on-resin disulfide bond formation presented previously by Galande and coworkers (Galande AK, Weissleder R, Tung C-H. An effective method of on-resin disulfide bond formation in peptides. J. Comb. Chem. 2005; 7: 174-177). First, the addition of an amine base to the deprotection solution allows the complete removal of the StBu group, allowing it to be replaced with a 5-Npys group. The second enhancement is the direct use of a Cys(Mob) or Sec(Mob) derivative as the nucleophilic partner instead of utilizing a naked sulfhydryl or selenol. These improvements result in the formation of a vicinal disulfide (or selenylsulfide) bond in high purity and yield. A direct comparison with the Galande procedure is presented. We also present a novel strategy for the formation of an acyclic, interchain selenylsulfide-linked peptide (linking H-PTVTGC-OH and H-UG-OH). Cysteine analogs of the cyclic and acyclic peptides were also synthesized. The results show that the ring structure contributes a factor of 52 to the rate, but the presence of selenium in the peptide is more important to catalysis than the presence of the ring.     

Structure of the Catalytic aa Fragment of the Protein Disulfide Isomerase ERp72

Protein disulfide isomerases (PDIs) are responsible for catalyzing the proper oxidation and isomerization of disulfide bonds of newly synthesized proteins in the endoplasmic reticulum (ER). The ER contains many different PDI-like proteins. Some, such as PDI, are general enzymes that directly recognize misfolded proteins while others, such as ERp57 and ERp72, have more specialized roles. Here, we report the high-resolution X-ray crystal structure of the N-terminal portion of ERp72 (also known as CaBP2 or PDI A4), which contains two a⁰a catalytic thioredoxin-like domains. The structure shows that the a⁰ domain contains an additional N-terminal β-strand and a different conformation of the β5-α4 loop relative to other thioredoxin-like domains. The structure of the a domain reveals that a conserved arginine residue inserts into the hydrophobic core and makes a salt bridge with a conserved glutamate residue in the vicinity of the catalytic site. A structural model of full-length ERp72 shows that all three catalytic sites roughly face each other and positions the adjacent hydrophobic patches that are likely involved in protein substrate binding.     

Methods to identify the substrates of thiol‐disulfide oxidoreductases

The formation of a disulfide bond is a critical step in the folding of numerous secretory and membrane proteins and catalyzed in vivo. A variety of mechanisms and protein structures have evolved to catalyze oxidative protein folding. Those enzymes that directly interact with a folding protein to accelerate its oxidative folding are mostly thiol‐disulfide oxidoreductases that belong to the thioredoxin superfamily. The enzymes of this class often use a CXXC active‐site motif embedded in their thioredoxin‐like fold to promote formation, isomerization, and reduction of a disulfide bond in their target proteins. Over the past decade or so, an increasing number of substrates of the thiol‐disulfide oxidoreductases that are present in the ER of mammalian cells have been discovered, revealing that the enzymes play unexpectedly diverse physiological functions. However, functions of some of these enzymes still remain unclear due to the lack of information on their substrates. Here, we review the methods used by researchers to identify the substrates of these enzymes and provide data that show the importance of using trichloroacetic acid in sample preparation for the substrate identification, hoping to aid future studies. We particularly focus on successful studies that have uncovered physiological substrates and functions of the enzymes in the periplasm of Gram‐negative bacteria and the endoplasmic reticulum of mammalian cells. Similar approaches should be applicable to enzymes in other cellular compartments or in other organisms.     

Jasmonic acid‐inducible TSA1 facilitates ER body formation

Members of the Brassicales contain an organelle, the endoplasmic reticulum (ER) body, which is derived from the ER. Recent studies have shed light on the biogenesis of the ER body and its physiological role in plants. However, formation of the ER body and its physiological role are not fully understood. Here, we investigated the physiological role of TSK‐associating protein 1 (TSA1), a close homolog of NAI2 that is involved in ER body formation, and provide evidence that it is involved in ER body biogenesis under wound‐related stress conditions. TSA1 is N‐glycosylated and localizes to the ER body as a luminal protein. TSA1 was highly induced by the plant hormone, methyl jasmonate (MeJA). Ectopic expression of TSA1:GFP induced ER body formation in root tissues of transgenic Arabidopsis thaliana and in leaf tissues of Nicotiana benthamiana. TSA1 and NAI2 formed a heterocomplex and showed an additive effect on ER body formation in N. benthamiana. MeJA treatment induced ER body formation in leaf tissues of nai2 and tsa1 plants, but not nai2/tsa1 double‐mutant plants. However, constitutive ER body formation was altered in young seedlings of nai2 plants but not tsa1 plants. Based on these results, we propose that TSA1 plays a critical role in MeJA‐induced ER body formation in plants.     

Novel Roles of the Non-catalytic Elements of Yeast Protein-disulfide Isomerase in Its Interplay with Endoplasmic Reticulum Oxidoreductin 1

The formation of disulfide bonds in the endoplasmic reticulum (ER) of eukaryotic cells is catalyzed by the sulfhydryl oxidase, ER oxidoreductin 1 (Ero1), and protein-disulfide isomerase (PDI). PDI is oxidized by Ero1 to continuously introduce disulfides into substrates, and feedback regulates Ero1 activity by manipulating the regulatory disulfides of Ero1. In this study we find that yeast Ero1p is enzymatically active even with its regulatory disulfides intact, and further activation of Ero1p by reduction of the regulatory disulfides requires the reduction of non-catalytic Cys⁹⁰-Cys⁹⁷ disulfide in Pdi1p. The principal client-binding site in the Pdi1p b′ domain is necessary not only for the functional Ero1p-Pdi1p disulfide relay but also for the activation of Ero1p. We also demonstrate by complementary activation assays that the regulatory disulfides in Ero1p are much more stable than those in human Ero1α. These new findings on yeast Ero1p-Pdi1p interplay reveal significant differences from our previously identified mode of human Ero1α-PDI interplay and provide insights into the evolution of the eukaryotic oxidative protein folding pathway.     

(Arg)3 within the N-terminal domain of glucosidase I contains ER targeting information but is not required absolutely for ER localization

Glucosidase I is an endoplasmic reticulum (ER) type II membrane enzyme that cleaves the distal alpha1,2-glucose of the asparagine-linked GlcNAc2-Man9-Glc3 precursor. To identify sequence motifs responsible for ER localization, we prepared a protein chimera by transferring the cytosolic and transmembrane domain of glucosidase I to the luminal domain of Golgi-Man9-mannosidase. The GIM9 hybrid was overexpressed in COS 1 cells as an ER-resident protein that displayed alpha1,2-mannosidase activity, excluding the possibility that the glucosidase I-specific domains interfere with folding of the Man9-mannosidase catalytic domain. After substitution of the Args in position 7, 8, or 9 relative to the N-terminus by leucine, the GIM9 mutants were transported to the cell surface indicating that the (Arg)3 sequence functions as an ER-targeting motif. Cell surface expression was also observed after substitution of Arg-7 or Arg-8 but not Arg-9 in GIM9 by either lysine or histidine. Thus the side chain structure, including its positive charge, appears to be essential for signal function. Analysis of the N-linked glycans suggests that the (Arg)3 sequence mediates ER localization through Golgi-to-ER retrograde transport. Glucosidase I remained localized in the ER after truncation or mutation of the N-terminal (Arg)3 signal, in contrast to comparable GIM9 mutants. ER localization was also observed with an M9GI chimera consisting of the cytosolic and transmembrane domain of Man9-mannosidase and the glucosidase I catalytic domain. ER-specific targeting information must therefore be provided by sequence motifs contained within the glucosidase I luminal domain. This structural information appears to direct ER localization by retention rather than by retrieval, as concluded from N-linked Man9-GlcNAc2 being the major glycan released from the wild-type enzyme.     

Over-expression of methionine sulfoxide reductase A in the endoplasmic reticulum increases resistance to oxidative and ER stresses

MsrA and MsrB catalyze the reduction of methionine-S- suifoxide and methionine-R-sulfoxide, respectively, to methionine in different cellular compartments of mammalian cells. One of the three MsrBs, MsrB3, is an endoplasmic reticulum （ER）-type enzyme critical for stress resistance including oxidative and ER stresses. However, there is no evidence for the presence of an ER-type MsrA or the ER local- ization of MsrA. In this work, we developed an ER-targeted recombinant MsrA construct and investigated the potential effects of methionine-S-sulfoxide reduction in the ER on stress resistance. The ER-targeted MsrA construct contained the N-terminal ER-targeting signal peptide of human MsrB3A （MSPRRSLPRPLSLCLSLCLCLCLAAALGSAQ） and the C-terminal ER-retention signal sequence （KAEL）. The over-expression of ER-targeted MsrA significantly increased cellular resistance to H202-induced oxidative stress. The ER-targeted MsrA over-expression also significantly enhanced resistance to dithiothreitol-induced ER stress; however, it had no positive effects on the resistance to ER stresses induced by tunicamycin and thapsigargin. Collectively, our data suggest that methionine-S-sulfoxide reduction in the ER compartment plays a protective role against oxidative and ER stresses.