Protein disulfide isomerases exploit synergy between catalytic and specific binding domains

Protein disulfide isomerases (PDIs) catalyse the formation of native disulfide bonds in protein folding pathways. The key steps involve disulfide formation and isomerization in compact folding intermediates. The high-resolution structures of the a and b domains of PDI are now known, and the overall domain architecture of PDI and its homologues can be inferred. The isolated a and a′ domains of PDI are good catalysts of simple thiol–disulfide interchange reactions but require additional domains to be effective as catalysts of the rate-limiting disulfide isomerizations in protein folding pathways. The b′ domain of PDI has a specific binding site for peptides and its binding properties differ in specificity between members of the PDI family. A model of PDI function can be deduced in which the domains function synergically: the b′ domain binds unstructured regions of polypeptide, while the a and a′ domains catalyse the chemical isomerization steps.     

Different Interaction Modes for Protein-disulfide Isomerase (PDI) as an Efficient Regulator and a Specific Substrate of Endoplasmic Reticulum Oxidoreductin-1α (Ero1α)

Protein-disulfide isomerase (PDI) and sulfhydryl oxidase endoplasmic reticulum oxidoreductin-1α (Ero1α) constitute the pivotal pathway for oxidative protein folding in the mammalian endoplasmic reticulum (ER). Ero1α oxidizes PDI to introduce disulfides into substrates, and PDI can feedback-regulate Ero1α activity. Here, we show the regulatory disulfide of Ero1α responds to the redox fluctuation in ER very sensitively, relying on the availability of redox active PDI. The regulation of Ero1α is rapidly facilitated by either a or a′ catalytic domain of PDI, independent of the substrate binding domain. On the other hand, activated Ero1α specifically binds to PDI via hydrophobic interactions and preferentially catalyzes the oxidation of domain a′. This asymmetry ensures PDI to function simultaneously as an oxidoreductase and an isomerase. In addition, several PDI family members are also characterized to be potent regulators of Ero1α. The novel modes for PDI as a competent regulator and a specific substrate of Ero1α govern efficient and faithful oxidative protein folding and maintain the ER redox homeostasis.     

Generating an Unfoldase from Thioredoxin-like Domains

Protein-disulfide isomerase (PDI), an endoplasmic reticulum (ER)-resident protein, is primarily known as a catalyst of oxidative protein folding but also has a protein unfolding activity. We showed previously that PDI unfolds the cholera toxin A1 (CTA1) polypeptide to facilitate the ER-to-cytosol retrotranslocation of the toxin during intoxication. We now provide insight into the mechanism of this unfoldase activity. PDI includes two redox-active (a and a′) and two redox-inactive (b and b′) thioredoxin-like domains, a linker (x), and a C-terminal domain (c) arranged as abb′xa′c. Using recombinant PDI fragments, we show that binding of CTA1 by the continuous PDIbb′xa′ fragment is necessary and sufficient to trigger unfolding. The specific linear arrangement of bb′xa′ and the type a domain (a′ versus a) C-terminal to bb′x are additional determinants of activity. These data suggest a general mechanism for the unfoldase activity of PDI: the concurrent and specific binding of bb′xa′ to particular regions along the CTA1 molecule triggers its unfolding. Furthermore, we show the bb′ domains of PDI are indispensable to the unfolding reaction, whereas the function of its a′ domain can be substituted partially by the a′ domain from ERp57 (abb′xa′c) or ERp72 (ca°abb′xa′), PDI-like proteins that do not unfold CTA1 normally. However, the bb′ domains of PDI were insufficient to convert full-length ERp57 into an unfoldase because the a domain of ERp57 inhibited toxin binding. Thus, we propose that generating an unfoldase from thioredoxin-like domains requires the bb′(x) domains of PDI followed by an a′ domain but not preceded by an inhibitory a domain.Protein-disulfide isomerase (PDI)² is a multifunctional protein that resides in the endoplasmic reticulum (ER) lumen of all eukaryotic cells (reviewed in Ref. 1). Mammalian PDI was first identified as a catalyst of oxidative protein folding (2), but it is now also known to mediate viral infection (3, 4), antigen processing (5), collagen assembly (6), and ER-associated degradation (7–9). To participate in this variety of cellular processes, PDI performs multiple activities. For example, during oxidative protein folding, PDI catalyzes the oxidation and isomerization of disulfide bonds and induces conformational changes in non-native polypeptides (10). Independently of redox chemistry, PDI is a molecular chaperone, binding polypeptides to prevent their aggregation (11–13). PDI also acts as a structural subunit of the prolyl 4-hydroxylase (P4H) and microsomal triglyceride transfer protein complexes; however, this function is similar to its chaperone activity (14–19). In contrast to its protein folding activities, PDI unfolds the catalytic A1 polypeptide of cholera toxin (CTA1) in preparation for the retrotranslocation of the toxin from the ER lumen into the cytosol (8, 20).Cholera toxin (CT) is a pathogenic factor that causes secretory diarrhea in animals (reviewed in Ref. 21). The holotoxin includes a single catalytic A subunit (CTA) and a homopentameric B subunit (CTB) joined noncovalently (22). Upon secretion from the bacterium Vibrio cholerae, CTA is cleaved into the A1 and A2 polypeptides, which are joined by a disulfide bond and noncovalent interactions (22, 23). To intoxicate a cell, CTB binds the ganglioside GM1 on the surface of the cell, and the holotoxin is transported in a retrograde manner to the ER lumen (24). In the ER, CTA is reduced to generate CTA1, and PDI unfolds and dissociates CTA1 from the holotoxin (20). The unfolded toxin is subsequently transported across the ER membrane (25, 26). Upon reaching the cytosol, CTA1 refolds and induces toxicity (27, 28).We showed previously that PDI acts as a redox-dependent chaperone to unfold CTA1 (20). In the reduced state of PDI, it binds and unfolds the toxin. Subsequent oxidation of PDI by ER oxidase 1 causes PDI to release unfolded CTA1 (25). Aside from this information, nothing is known about the mechanism of the unfolding activity of PDI.PDI is a modular protein comprising two a-type thioredoxin-like domains (a and a′), two b-type thioredoxin-like domains (b and b′), a flexible linker (x), and an extended C-terminal domain (c) arranged as abb′xa′c (29–31). The a-type domains are characterized by the presence of the catalytic sequence CXXC and are therefore redox-active, whereas the b-type domains lack this sequence and are redox-inactive (32). The thioredoxin-like domains of PDI differ from each other in primary structure despite having a common fold. The crystal structure of yeast PDI shows the bb′ domains form a rigid base from which the a-type domains extend like flexible arms (33, 34). This base is thought to be the core of a substrate-binding groove formed by all four thioredoxin-like domains (30, 33, 35).To understand the mechanism of the unfoldase activity of PDI, we analyzed the contribution of each domain to the ability of PDI to bind and unfold CTA1 using recombinant PDI fragments. Unfolded CTA1 was detected by an established in vitro trypsin sensitivity assay that relies on tryptic cleavage sites hidden in the folded toxin to be exposed in the unfolded toxin (20). Because CTA1 likely mimics a misfolded host cell protein for its recognition and unfolding by PDI (22, 36, 37), this study has implications for how PDI unfolds endogenous misfolded proteins in preparation for their retrotranslocation and subsequent ER-associated degradation.There are nearly 20 mammalian PDI-like proteins, characterized by the presence of one or more thioredoxin-like domains and ER localization (reviewed in Refs. 38, 39). We previously demonstrated that two PDI-like proteins, ERp72 and ERp57, do not facilitate CTA1 retrotranslocation (8). In contrast to PDI, ERp72 retains CTA1 in the ER and either stabilizes its native conformation or renders it more compact (8). To understand how these structurally homologous proteins are functionally unique, we tested whether the various thioredoxin-like domains of ERp57 and ERp72 could functionally replace the corresponding PDI domains to unfold CTA1. Thus, in addition to suggesting a general mechanism for the unfoldase activity of PDI, our data indicate functional similarities and differences among thioredoxin-like domains of PDI family proteins.     

Redox-Dependent Domain Rearrangement of Protein Disulfide Isomerase Coupled with Exposure of Its Substrate-Binding Hydrophobic Surface

Protein disulfide isomerase (PDI) is a major protein in the endoplasmic reticulum, operating as an essential folding catalyst and molecular chaperone for disulfide-containing proteins by catalyzing the formation, rearrangement, and breakage of their disulfide bridges. This enzyme has a modular structure with four thioredoxin-like domains, a, b, b′, and a′, along with a C-terminal extension. The homologous a and a′ domains contain one cysteine pair in their active site directly involved in thiol-disulfide exchange reactions, while the b′ domain putatively provides a primary binding site for unstructured regions of the substrate polypeptides. Here, we report a redox-dependent intramolecular rearrangement of the b′ and a′ domains of PDI from Humicola insolens, a thermophilic fungus, elucidated by combined use of nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS) methods. Our NMR data showed that the substrates bound to a hydrophobic surface spanning these two domains, which became more exposed to the solvent upon oxidation of the active site of the a′ domain. The hydrogen-deuterium exchange and relaxation data indicated that the redox state of the a′ domain influences the dynamic properties of the b′ domain. Moreover, the SAXS profiles revealed that oxidation of the a′ active site causes segregation of the two domains. On the basis of these data, we propose a mechanistic model of PDI action; the a′ domain transfers its own disulfide bond into the unfolded protein accommodated on the hydrophobic surface of the substrate-binding region, which consequently changes into a “closed” form releasing the oxidized substrate.     

Functional properties of the two redox-active sites in yeast protein disulphide isomerase in vitro and in vivo

Protein folding catalysed by protein disulphide isomerase (PDI) has been studied both in vivo and in vitro using different assays. PDI contains a CGHC active site in each of its two catalytic domains (a and a'). The relative importance of each active site in PDI from Saccharomyces cerevisiae (yPDI) has been analysed by exchanging the active-site cysteine residues for serine residues. The activity of the mutant forms of yPDI was determined quantitatively by following the refolding of bovine pancreatic trypsin inhibitor in vitro. In this assay the activity of the wild-type yPDI is quite similar to that of human PDI, both in rearrangement and oxidation reactions. However, while the a domain active site of the human enzyme is more active than the a'-site, the reverse is the case for yPDI. This prompted us to set up an assay to investigate whether the situation would be different with a native yeast substrate, procarboxypeptidase Y. In this assay, however, the a' domain active site also appeared to be much more potent than the a-site. These results were unexpected, not only because of the difference with human PDI, but also because analysis of folding of procarboxypeptidase Y in vivo had shown the a-site to be most important. We furthermore show that the apparent difference between in vivo and in vitro activities is not due to catalytic contributions from the other PDI homologues found in yeast.     

BiP/Kar2p serves as a molecular chaperone during carboxypeptidase Y folding in yeast

Although transiently associated with numerous newly synthesized proteins, BiP has not been shown to be an essential component directly linked to the folding and oligomerization of newly synthesized proteins in the endoplasmic reticulum. To determine whether it is needed as a molecular chaperone, we analyzed the maturation of an endogenous yeast glycoprotein, carboxypeptidase Y (CPY) in several yeast strains with temperature-sensitive mutations in BiP. These kar2 mutant strains have previously been found to be defective in translocation at the nonpermissive temperature (Vogel, J. P., L. M. Misra, and M. D. Rose, 1990. J. Cell Biol, 110:1885-1895). To circumvent the translocation block, we used DTT at permissive temperature to delay folding and intracellular transport. We then followed the maturation of the ER- retained CPY after shifting to the nonpermissive temperature and dilution of the DTT. Without the functional chaperone, CPY aggregated, failed to be oxidized, and remained in the ER. In contrast to wild-type cells, in which BiP binding was transient with no more than 10-15% of labeled CPY associated at any time, 30-100% of the CPY remained associated with BiP in the mutant strains. In a heterozygous diploid strain, CPY matured and exited the ER normally. Taken together, the results provide clear evidence that BiP plays a critical role as a molecular chaperone in CPY folding.     

Modulation of an Active-Site Cysteine pKa Allows PDI to Act as a Catalyst of both Disulfide Bond Formation and Isomerization

Protein disulfide isomerase (PDI) plays a central role in disulfide bond formation in the endoplasmic reticulum. It is implicated both in disulfide bond formation and in disulfide bond reduction and isomerization. To be an efficient catalyst of all three reactions requires complex mechanisms. These include mechanisms to modulate the pK_a values of the active-site cysteines of PDI. Here, we examined the role of arginine 120 in modulating the pK_a values of these cysteines. We find that arginine 120 plays a significant role in modulating the pK_a of the C-terminal active-site cysteine in the a domain of PDI and plays a role in determining the reactivity of the N-terminal active-site cysteine but not via direct modulation of its pK_a. Mutation of arginine 120 and the corresponding residue, arginine 461, in the a′ domain severely reduces the ability of PDI to catalyze disulfide bond formation and reduction but enhances the ability to catalyze disulfide bond isomerization due to the formation of more stable PDI-substrate mixed disulfides. These results suggest that the modulation of pK_a of the C-terminal active cysteine by the movement of the side chain of these arginine residues into the active-site locales has evolved to allow PDI to efficiently catalyze both oxidation and isomerization reactions.     

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.     

Effects of signal sequences and folding accessory proteins on extracellular expression of carboxypeptidase Y in recombinant Saccharomyces cerevisiae

Carboxypeptidase Y (CPY) is a yeast vacuolar protease with useful applications including C-terminal sequencing of peptides and terminal modification of target proteins. To overexpress CPY with the pro-sequence (proCPY) encoded by the Saccharomyces cerevisiae PRC1 gene in recombinant S. cerevisiae, the proCPY gene was combined with the gene coding for a signal sequence of S. cerevisiae mating factor α (MFα), invertase (SUC2), or Kluyveromyces marxianus inulinase (INU1). Among the three constructs, the MFα signal sequence gave the best specific activity of extracellular CPY. To enhance the CPY expression level, folding accessory proteins of Kar2p, Pdi1p and Ero1p located in the S. cerevisiae endoplasmic reticulum were expressed individually and combinatorially. A single expression of Kar2p led to a 28 % enhancement in extracellular CPY activity, relative to the control strain of S. cerevisiae CEN.PK2-1D/p426Gal1-MFαCPY. Coexpression of Kar2p, Pdi1p and Ero1p gave a synergistic effect on CPY expression, of which activity was 1.7 times higher than that of the control strain. This work showed that engineering of signal sequences and protein-folding proteins would be helpful to overexpress yeast proteins of interest.     

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.     

Discrimination between native and non-native disulfides by protein-disulfide isomerase

The folding assistant and chaperone protein-disulfide isomerase (PDI) catalyzes disulfide formation, reduction, and isomerization of misfolded proteins. PDI substrates are not restricted to misfolded proteins; PDI catalyzes the dithiothreitol (DTT)-dependent reduction of native ribonuclease A, microbial ribonuclease, and pancreatic trypsin inhibitor, suggesting that an ongoing surveillance by PDI can test even native disulfides for their ability to rearrange. The mechanism of reduction is consistent with an equilibrium unfolding of the substrate, attack by the nucleophilic cysteine of PDI followed by direct attack of DTT on a covalent intermediate between PDI and the substrate. For native proteins, the rate constants for PDI-catalyzed reduction correlate very well with the rate constants for uncatalyzed reduction by DTT. However, the rate is weakly correlated with disulfide stability, surface exposure, or local disorder in the crystal. Compared with native proteins, scrambled ribonuclease is a much better substrate for PDI than predicted from its reactivity with DTT; however, partially reduced bovine pancreatic trypsin inhibitor (des(14-38)) is not. An extensively unfolded polypeptide may be required by PDI to distinguish native from non-native disulfides.     

Tobacco Mesophyll Protoplasts Synthesize 1,3-beta-Glucanase, Chitinases, and "Osmotins" during in Vitro Culture

Tobacco (Nicotiana tabacum) mesophyll protoplasts synthesize six basic proteins (a, a′, a₁, b, b′, and c) which are undetectable in the leaf and whose synthesis is reduced by auxin (Y Meyer, L Aspart, Y Chartier [1984] Plant Physiol 75: 1027-1033). Polypeptides a, a′, and a₁ were shown to have similar mobilities on two-dimensional electrophoresis as one 1,3-β-glucanase and two chitinases from tobacco mosaic virus-infected leaves. In immunoblotting experiments, polypeptide a was recognized by specific antibodies raised against the 1,3-β-glucanase and a′ and a₁ reacted with anti-chitinase antibodies. Similarly, b and b′ comigrated with osmotin and its neutral counterpart, two proteins characteristic of salt-adapted tobacco cells, and reacted with anti-osmotin antibodies. In addition it has been shown that 1,3-β-glucanase and chitinase activities increased at the same time as a, a′, and a₁ accumulated in cultivated protoplasts. Finally, polypeptide c was also detected in tobacco mosaic virus-infected leaves but could not be identified as any of the pathogenesis-related proteins characterized so far in tobacco. Thus, cultivated tobacco protoplasts synthesize and accumulate typical stress proteins.     

Phylogenetic Conservation and Homology Modeling Help Reveal a Novel Domain within the Budding Yeast Heterochromatin Protein Sir1

The yeast Sir1 protein's ability to bind and silence the cryptic mating-type locus HMRa requires a protein-protein interaction between Sir1 and the origin recognition complex (ORC). A domain within the C-terminal half of Sir1, the Sir1 ORC interaction region (Sir1OIR), and the conserved bromo-adjacent homology (BAH) domain within Orc1, the largest subunit of ORC, mediate this interaction. The structure of the Sir1OIR-Orc1BAH complex is known. Sir1OIR and Orc1BAH interacted with a high affinity in vitro, but the Sir1OIR did not inhibit Sir1-dependent silencing when overproduced in vivo, suggesting that other regions of Sir1 helped it bind HMRa. Comparisons of diverged Sir1 proteins revealed two highly conserved regions, N1 and N2, within Sir1's poorly characterized N-terminal half. An N-terminal portion of Sir1 (residues 27 to 149 [Sir1^27-149]) is similar in sequence to the Sir1OIR; homology modeling predicted a structure for Sir1^27-149 in which N1 formed a submodule similar to the known Orc1BAH-interacting surface on Sir1. Consistent with these findings, two-hybrid assays indicated that the Sir1 N terminus could interact with BAH domains. Amino acid substitutions within or near N1 or N2 reduced full-length Sir1's ability to bind and silence HMRa and to interact with Orc1BAH in a two-hybrid assay. Purified recombinant Sir1 formed a large protease-resistant structure within which the Sir1OIR domain was protected, and Orc1BAH bound Sir1OIR more efficiently than full-length Sir1 in vitro. Thus, the Sir1 N terminus exhibited both positive and negative roles in the formation of a Sir1-ORC silencing complex. This functional duality might contribute to Sir1's selectivity for silencer-bound ORCs in vivo.     

Oxidative Activity of Yeast Ero1p on Protein Disulfide Isomerase and Related Oxidoreductases of the Endoplasmic Reticulum

The sulfhydryl oxidase Ero1 oxidizes protein disulfide isomerase (PDI), which in turn catalyzes disulfide formation in proteins folding in the endoplasmic reticulum (ER). The extent to which other members of the PDI family are oxidized by Ero1 and thus contribute to net disulfide formation in the ER has been an open question. The yeast ER contains four PDI family proteins with at least one potential redox-active cysteine pair. We monitored the direct oxidation of each redox-active site in these proteins by yeast Ero1p in vitro. In this study, we found that the Pdi1p amino-terminal domain was oxidized most rapidly compared with the other oxidoreductase active sites tested, including the Pdi1p carboxyl-terminal domain. This observation is consistent with experiments conducted in yeast cells. In particular, the amino-terminal domain of Pdi1p preferentially formed mixed disulfides with Ero1p in vivo, and we observed synthetic lethality between a temperature-sensitive Ero1p variant and mutant Pdi1p lacking the amino-terminal active-site disulfide. Thus, the amino-terminal domain of yeast Pdi1p is on a preferred pathway for oxidizing the ER thiol pool. Overall, our results provide a rank order for the tendency of yeast ER oxidoreductases to acquire disulfides from Ero1p.     

Sulfhydryl oxidation, not disulfide isomerization, is the principal function of protein disulfide isomerase in yeast Saccharomyces cerevisiae

Protein disulfide isomerase (PDI) is an essential protein folding assistant of the eukaryotic endoplasmic reticulum that catalyzes both the formation of disulfides during protein folding (oxidase activity) and the isomerization of disulfides that may form incorrectly (isomerase activity). Catalysis of thiol-disulfide exchange by PDI is required for cell viability in Saccharomyces cerevisiae, but there has been some uncertainty as to whether the essential role of PDI in the cell is oxidase or isomerase. We have studied the ability of PDI constructs with high oxidase activity and very low isomerase activity to complement the chromosomal deletion of PDI1 in S. cerevisiae. A single catalytic domain of yeast PDI (PDIa') has 50% of the oxidase activity but only 5% of the isomerase activity of wild-type PDI in vitro. Titrating the expression of PDI using the inducible/repressible GAL1-10 promoter shows that the amount of wild-type PDI protein needed to sustain a normal growth rate is 60% or more of the amount normally expressed from the PDI1 chromosomal location. A single catalytic domain (PDIa') is needed in molar amounts that are approximately twice as high as those required for wild-type PDI, which contains two catalytic domains. This comparison suggests that high (>60%) PDI oxidase activity is critical to yeast growth and viability, whereas less than 6% of its isomerase activity is needed.     

Cooperative Protein Folding by Two Protein Thiol Disulfide Oxidoreductases and ERO1 in Soybean

Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (Ero1s) supply oxidizing equivalent to the active centers of PDI. In this study, we expressed recombinant soybean Ero1 (GmERO1a) and found that GmERO1a oxidized multiple soybean ER oxidoreductases, in contrast to mammalian Ero1s having a high specificity for PDI. One of these ER oxidoreductases, GmPDIM, associated in vivo and in vitro with GmPDIL-2, was unable to be oxidized by GmERO1a. We therefore pursued the possible cooperative oxidative folding by GmPDIM, GmERO1a, and GmPDIL-2 in vitro and found that GmPDIL-2 synergistically accelerated oxidative refolding. In this process, GmERO1a preferentially oxidized the active center in the a′ domain among the a, a′, and b domains of GmPDIM. A disulfide bond introduced into the active center of the a′ domain of GmPDIM was shown to be transferred to the active center of the a domain of GmPDIM and the a domain of GmPDIM directly oxidized the active centers of both the a or a′ domain of GmPDIL-2. Therefore, we propose that the relay of an oxidizing equivalent from one ER oxidoreductase to another may play an essential role in cooperative oxidative folding by multiple ER oxidoreductases in plants.In eukaryotes, many secretory and membrane proteins fold via disulfide bond formation in the endoplasmic reticulum (ER). Seed storage proteins of major crops, such as wheat, corn, rice, and beans, which are important protein sources for humans and domestic animals, are synthesized in the ER of the endosperm or cotyledon. A number of seed storage proteins fold by the formation of intramolecular disulfide bonds (oxidative folding) and are transported to and accumulate in protein bodies (Kermode and Bewley, 1999; Jolliffe et al., 2005). In contrast to normally folded proteins, misfolded and unfolded proteins are retained in the ER and degraded by an ER-associated degradation or vacuolar system (Smith et al., 2011; Pu and Bassham, 2013). Therefore, quick and efficient oxidative folding of nascent seed storage proteins is needed for their accumulation in protein bodies.During this process, protein disulfide isomerase (PDI; EC 5.3.4.1) and other ER protein thiol disulfide oxidoreductases (ER oxidoreductases) are thought to catalyze the formation and isomerization of disulfide bonds in nascent proteins (Hatahet and Ruddock, 2009; Feige and Hendershot, 2011; Lu and Holmgren, 2014). After phylogenetic analysis of the Arabidopsis genome, 10 classes of ER oxidoreductases (classes I–X) were identified (Houston et al., 2005). Among them, class I ER oxidoreductase, a plant PDI ortholog, has been studied in a wide variety of plants. Class I ER oxidoreductases have two catalytically active domains a and a′, containing active centers composed of Cys-Gly-His-Cys and two catalytically inactive domains b and b′. An Arabidopsis ortholog of class I ER oxidoreductases is required for proper seed development and regulates the timing of programmed cell death by chaperoning and inhibiting Cys proteases (Andème Ondzighi et al., 2008). OaPDI, a PDI from Oldenlandia affinis, a coffee family (Rubiaceae) plant, is involved in the folding of knotted circular proteins (Gruber et al., 2007). The rice ortholog (PDIL1-1) was suggested to be involved in the maturation of the major seed storage protein glutelin (Takemoto et al., 2002). Furthermore, rice PDIL1-1 plays a role in regulatory activities for various proteins that are essential for the synthesis of grain components as determined by analysis of a T-DNA insertion mutant (Satoh-Cruz et al., 2010).The oxidative refolding ability of class I ER oxidoreductases was confirmed in recombinant soybean (GmPDIL-1) and wheat proteins produced by an Escherichia coli expression system established from cDNAs (Kamauchi et al., 2008; Kimura et al., 2015).Class II and III ER oxidoreductases have an a–b–b′–a′ domain structure. Class II ER oxidoreductases have an acidic amino acid-rich sequence in the N-terminal region ahead of the a domain. Recombinant soybean (GmPDIL-2) and wheat class II ER oxidoreductases have oxidative refolding activities similar to that of class I (Kamauchi et al., 2008; Kimura et al., 2015). Class III ER oxidoreductases contain the nonclassical redox-center Cys-X-X-Ser/Cys motifs, as opposed to the more traditional CGHC sequence, in the a and a′ domains. Recombinant soybean (GmPDIL-3) and wheat proteins lack oxidative refolding activity in vitro (Iwasaki et al., 2009; Kimura et al., 2015). Class IV ER oxidoreductases are unique to plants and have an a–a′–ERp29 domain structure, which is homologous to the C-terminal domain of mammalian ERp29 (Demmer et al., 1997).Recombinant soybean class IV ER oxidoreductases (GmPDIS-1 and GmPDIS-2) and wheat class IV ER oxidoreductase possess an oxidative refolding activity that is weaker than that of classes I and II (Wadahama et al., 2007; Kimura et al., 2015). Class V ER oxidoreductases are plant orthologs of mammalian P5 and have an a–a′–b domain structure. A rice class V ER oxidoreductase, consisting of PDIL2 and PDIL3, plays an important role in the accumulation of the seed storage protein Cys-rich 10-kD prolamin (crP10; Onda et al., 2011). Recombinant soybean class V ER oxidoreductase, GmPDIM and wheat class V ER oxidoreductase possess an oxidative refolding activity similar to that of class IV (Wadahama et al., 2008; Kimura et al., 2015). In the soybean, GmPDIL-1, GmPDIL-2, GmPDIM, GmPDIS-1, and GmPDIS-2 were found to associate transiently with a seed storage precursor protein, proglycinin, in the ER of the cotyledon by coimmunoprecipitation experiments, suggesting that multiple ER oxidoreductases are involved in the folding of the nascent proglycinin.The disulfide bond in the active center of ER oxidoreductases is reduced as a result of catalyzing disulfide bond formation in an unfolded protein. The reduced active center of PDI was discovered to be oxidized again by ER oxidoreductin-1 (Ero1p) in yeast (Frand and Kaiser, 1998; Pollard et al., 1998). Ero1p orthologs are present universally in eukaryotes. Yeast and flies have a single copy of the ERO1 gene, which is essential for survival (Frand and Kaiser, 1998; Pollard et al., 1998; Tien et al., 2008). Mammals have two genes encoding Ero1-α (Cabibbo et al., 2000) and Ero1-β (Pagani et al., 2000) that function as major disulfide donors to nascent proteins in the ER, but are not critical for survival (Zito et al., 2010). Domain a of yeast PDI is the most favored substrate of yeast Ero1p (Vitu et al., 2010), whereas a′ of human PDI is specifically oxidized by human Ero1-α (Chambers et al., 2010) and Ero1-β (Wang et al., 2011). Electrons from Cys residues of the active centers of PDI are transferred to oxygen by Ero1 (Tu and Weissman, 2004; Sevier and Kaiser, 2008). The reaction mechanisms of yeast Ero1p and human Ero1s have been intensively investigated; their regulation by PDI has been extensively studied as well (Tavender and Bulleid, 2010; Araki and Inaba, 2012; Benham et al., 2013; Ramming et al., 2015). Only rice Ero1 (OsERO1) has been identified as a plant ortholog of Ero1p (Onda et al., 2009). OsERO1 is necessary for disulfide bond formation in rice endosperm. The formation of native disulfide bonds in the major seed storage protein proglutelin was demonstrated to depend upon OsERO1 by RNAi knockdown experiments. However, no plant protein thiol disulfide oxidoreductases that are oxidized by a plant Ero1 ortholog have been identified to date.In this study, we show that multiple soybean ER oxidoreductases can be activated by a soybean Ero1 ortholog (GmERO1a). In addition, we propose a synergistic mechanism by which GmPDIM and GmPDIL-2 cooperatively fold unfolded proteins using oxidizing equivalents provided by GmERO1 in vitro.     

The contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum

In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 +/- 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization.     

pH-Dependent mismatch discrimination of oligonucleotide duplexes containing 2'-deoxytubercidin and 2- or 7-substituted derivatives: protonated base pairs formed between 7-deazapurines and cytosine

Oligonucleotides incorporating 2′-deoxytubercidin (1a), its 2-amino derivative 2a and related 2-, or 7-substituted analogs (1d, 2b–d, 3 and 4) are synthesized. For this purpose, a series of novel phosphoramidites are prepared and employed in solid-phase synthesis. Hybridization experiments performed with 12mer duplexes indicate that 7-halogenated nucleosides enhance the duplex stability both in antiparallel and parallel DNA, whereas 2-fluorinated 7-deaza-2′-deoxyadenosine residues destabilize the duplex structure. The 7-deazaadenine nucleosides 1a, 1d and their 2-amino derivatives 2a–d form stable base pairs with dT but also with dC and dG. The mispairing with dC is pH-dependent. Ambiguous base pairing is observed at pH 7 or under acid conditions, whereas base discrimination occurs in alkaline medium (pH 8.0). This results from protonated base pairs formed between 1a or 2a and dC under neutral or acid condition, which are destroyed in alkaline medium. It is underlined by the increased basicity of the pyrrolo[2,3-d]pyrimidine nucleosides over that of the parent purine compounds (pK_a values: 1a = 5.30; 2a = 5.71; dA = 3.50).