Bacitracin inhibits the reductive activity of protein disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain

The peptide antibiotic bacitracin is widely used as an inhibitor of protein disulfide isomerase (PDI) to demonstrate the role of the protein-folding catalyst in a variety of molecular pathways. Commercial bacitracin is a mixture of at least 22 structurally related peptides. The inhibitory activity of individual bacitracin analogs on PDI is unknown. For the present study, we purified the major bacitracin analogs, A, B, H, and F, and tested their ability to inhibit the reductive activity of PDI by use of an insulin aggregation assay. All analogs inhibited PDI, but the activity (IC(50) ) ranged from 20 μm for bacitracin F to 1050 μm for bacitracin B. The mechanism of PDI inhibition by bacitracin is unknown. Here, we show, by MALDI-TOF/TOF MS, a direct interaction of bacitracin with PDI, involving disulfide bond formation between an open thiol form of the bacitracin thiazoline ring and cysteines in the substrate-binding domain of PDI.     

Active Site Mutations in Yeast Protein Disulfide Isomerase Cause Dithiothreitol Sensitivity and a Reduced Rate of Protein Folding in the Endoplasmic Reticulum

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a′, each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a′ domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth.     

Juglone prevents human platelet aggregation through inhibiting Akt and protein disulfide isomerase

Background/PurposeJuglone, a natural compound widely found in Juglandaceae plants, has been suggested as a potential drug candidate for treating cancer, inflammation, and diabetic vascular complications. In the present study, the antiplatelet effect and underlying mechanisms of juglone were investigated for the first time.Study design/methodsHuman platelet aggregation and activation were measured by turbidimetric aggregometry, flow cytometry, and Western blotting. In vitro antithrombotic activity of juglone was assessed using collagen-coated flow chambers under whole-blood flow conditions. The effect of juglone on protein disulfide isomerase (PDI) activity was determined by the dieosin glutathione disulfide assay.ResultsJuglone (1 – 5 μM) inhibited platelet aggregation and glycoprotein (GP) IIb/IIIa activation caused by various agonists. In a whole blood flow chamber system, juglone reduced thrombus formation on collagen-coated surfaces under arterial shear rates. Juglone abolished intracellular Ca²⁺ elevation and protein kinase C activation caused by collagen, but had no significant effect on that induced by G protein-coupled receptor agonists. In contrast, Akt activation caused by various agonists were inhibited in juglone-treated platelets. Additionally, juglone showed inhibitory effects on both recombinant human PDI and platelet surface PDI at concentrations similar to those needed to prevent platelet aggregation.ConclusionJuglone exhibits potent in vitro antiplatelet and antithrombotic effects that are associated with inhibition of Akt activation and platelet surface PDI activity.     

Protein disulfide isomerase homolog TrPDI2 contributing to cellobiohydrolase production in Trichoderma reesei

The majority of the cysteine residues in the secreted proteins form disulfide bonds via protein disulfide isomerase (PDI)-mediated catalysis, stabilizing the enzyme activity. The role of PDI in cellulase production is speculative, as well as the possibility of PDI as a target for improving enzyme production efficiency of Trichoderma reesei, a widely used producer of enzyme for the production of lignocellulose-based biofuels and biochemicals. Here, we report that a PDI homolog, TrPDI2 in T. reesei exhibited a 36.94% and an 11.81% similarity to Aspergillus niger TIGA and T. reesei PDI1, respectively. The capability of TrPDI2 to recover the activity of reduced and denatured RNase by promoting refolding verified its protein disulfide isomerase activity. The overexpression of Trpdi2 increased the secretion and the activity of CBH1 at the early stage of cellulase induction. In addition, both the expression level and redox state of TrPDI2 responded to cellulase induction in T. reesei, providing sustainable oxidative power to ensure cellobiohydrolase maturation and production. The results suggest that TrPDI2 may contribute to cellobiohydrolase secretion by enhancing the capability of disulfide bond formation, which is essential for protein folding and maturation.     

Synthesis and biological evaluation of anti-cancer agents that selectively inhibit Her2 over-expressed breast cancer cell growth via down-regulation of Her2 protein

Compound JCC76 selectively inhibited the proliferation of human epidermal growth factor 2 (Her2) over-expressed breast cancer cells. In the current study, a ligand based structural optimization was performed to generate new analogs, and we identified derivatives 16 and 17 that showed improved activity and selectivity against Her2 positive breast cancer cells. A structure activity relationship (SAR) was summarized. Compounds 16 and 17 were also examined by western blot assay to check their effect on Her2 protein. The results reveal that the compounds could decrease the Her2 protein, which explains their selectivity to Her2 over-expressed breast cancer cells. Furthermore, the compounds inhibited the chaperone activity of small chaperone protein that could stabilize Her2 protein.     

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.     

Gaussia princeps luciferase: a bioluminescent substrate for oxidative protein folding

Gaussia princeps luciferase (GLuc) generates an intense burst of blue light when exposed to coelenterazine in the absence of ATP. Here we show that this 5‐disulfide containing enzyme can be used as a facile and convenient substrate for studies of oxidative protein folding. Reduced GLuc (rGLuc), with 10 free cysteine residues, is completely inactive as a luciferase but >60% bioluminescence activity, compared to controls, can be recovered using a range of oxidizing regimens in the absence of the exogenous shuffling activity of protein disulfide isomerase (PDI). The sulfhydryl oxidase QSOX1 can be assayed using rGLuc in a simple bioluminescence plate reader format. Similarly, low concentrations of rGLuc can be oxidized by millimolar levels of dehydroascorbate, hydrogen peroxide or much lower concentrations of sodium tetrathionate. The oxidative refolding of rGLuc in the presence of a range of glutathione redox buffers is only marginally accelerated by micromolar levels of PDI. This modest rate enhancement probably results from a relatively simple disulfide connectivity in native GLuc; reflecting two homologous domains each carrying two disulfide bonds with a single interdomain disulfide. When GLuc is reoxidized under denaturing conditions the resulting scrambled protein (sGLuc) can be used in a sensitive bioluminescence assay for reduced PDI in the absence of added exogenous thiols. Finally, the general facility by which rGLuc can recover bioluminescent activity in vitro provides a sensitive method for the assessment of inhibitors of oxidative protein folding.     

Characterization of the S-denitrosation activity of protein disulfide isomerase

S-nitrosoglutathione (GSNO) denitrosation activity of recombinant human protein disulfide isomerase (PDI) has been kinetically characterized by monitoring the loss of the S-NO absorbance, using a NO electrode, and with the aid of the fluorogenic NOx probe 2,3-diaminonaphthalene. The initial rates of denitrosation as a function of [GSNO] displayed hyperbolic behavior irrespective of the method used to monitor denitrosation. The Km values estimated for GSNO were 65 +/- 5 microm and 40 +/- 10 microm for the loss in the S-NO bond and NO production (NO electrode or 2,3-diaminonaphthalene), respectively. Hemoglobin assay provided additional evidence that the final product of PDI-dependent GSNO denitrosation was NO*. A catalytic mechanism, involving a nitroxyl disulfide intermediate stabilized by imidazole (His160 a-domain or His589 a'-domain), which after undergoing a one-electron oxidation decomposes to yield NO plus dithiyl radical, has been proposed. Evidence for the formation of thiyl/dithiyl radicals during PDI-catalyzed denitrosation was obtained with 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl. Evidence has also been obtained showing that in a NO- and O2-rich environment, PDI can form N2O3 in its hydrophobic domains. This "NO-charged PDI" can perform intra- and intermolecular S-nitrosation reactions similar to that proposed for serum albumin. Interestingly, reduced PDI was able to denitrosate S-nitrosated PDI (PDI-SNO) resulting in the release of NO. PDI-SNO, once formed, is stable at room temperature in the absence of reducing agent over the period of 2 h. It has been established that PDI is continuously secreted from cells that are net producers of NO-like endothelial cells. The present demonstration that PDI can be S-nitrosated and that PDI-SNO can be denitrosated by PDI suggests that this enzyme could be intimately involved in the transport of intracellular NO equivalents to the cell surface as well as the previous demonstration of PDI in the transfer of S-nitrosothiol-bound NO to the cytosol.     

Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction

Soluble guanylyl cyclase (sGC) is a heterodimeric nitric oxide (NO) receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI) interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT) and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed.     

A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.     

Disulfide Bond Formation in the Mammalian Endoplasmic Reticulum

The formation of disulfide bonds between cysteine residues occurs during the folding of many proteins that enter the secretory pathway. As the polypeptide chain collapses, cysteines brought into proximity can form covalent linkages during a process catalyzed by members of the protein disulfide isomerase family. There are multiple pathways in mammalian cells to ensure disulfides are introduced into proteins. Common requirements for this process include a disulfide exchange protein and a protein oxidase capable of forming disulfides de novo. In addition, any incorrect disulfides formed during the normal folding pathway are removed in a process involving disulfide exchange. The pathway for the reduction of disulfides remains poorly characterized. This work will cover the current knowledge in the field and discuss areas for future investigation.One of the characteristics of proteins that enter the secretory pathway is that they frequently contain covalent linkages called disulfide bonds within and between constituent polypeptide chains. The presence of these linkages is thought to confer stability when secreted proteins are exposed to the extracellular milieu or when membrane proteins are recycled through acidic endocytic compartments. In addition to structural disulfides it is now clear that a number of proteins use the formation and breaking of disulfides as a mechanism for regulation of activity (Schwertassek et al. 2007). Hence, it is important that we have a clear understanding of how correct disulfides are formed within proteins both during the protein folding process and to regulate protein function. The focus of this article will be on how correct disulfides are introduced into proteins within the secretory pathway, specifically within the endoplasmic reticulum (ER) during folding and assembly.The formation of disulfides within polypeptides begins as the protein is being cotranslationally translocated into the ER (Chen et al. 1995). The initial collapse of the polypeptide and formation of secondary structure brings cysteine residues into close enough proximity for them to form disulfides. Correct disulfide formation requires enzymes to both introduce disulfides between proximal cysteines and to reduce disulfides that form during folding but that are not present in the final native structure (Jansens et al. 2002). In addition, proteins that do not fold correctly are targeted for degradation and may require their disulfides to be broken before dislocation across the ER membrane into the cytosol (Ushioda et al. 2008). Hence, there must be a reduction and oxidation pathway present in the ER to ensure that native disulfides form and nonnative disulfides are broken during protein folding.Central to both reduction and oxidation pathways is the protein disulfide isomerase (PDI) family of enzymes (Ellgaard and Ruddock 2005) that are capable of exchanging disulfides with their substrate proteins (Fig. 1). Whether disulfide exchange results in the formation or breaking of a disulfide depends on the relative stability of the disulfides in the enzyme and substrate. To drive the formation of disulfides, the PDI family member must itself be oxidized. It is now clear that there are a number of ways for the disulfide exchange proteins to be oxidized by specific oxidases. Importantly, these oxidases do not introduce disulfides into nascent polypeptide chains; rather, they specifically oxidize members of the PDI family. The exception to this rule is the enzyme quiescin sulfydryl oxidase (QSOX; see below). The pathway for disulfide reduction is not as well characterized. It is known that the PDI family members can be reduced by the low molecular mass thiol glutathione (GSH) (Chakravarthi and Bulleid 2004; Jessop and Bulleid 2004; Molteni et al. 2004) but no enzymatic process for reduction has been identified. The glutathione redox balance within the ER is significantly more oxidized than in the cytosol (Hwang et al. 1992; Dixon et al. 2008), indicating that GSH is actively oxidized to glutathione disulfide either during the reduction of PDI family members or by reducing disulfides in nascent polypeptides directly. However, there is currently no clear indication as to how glutathione disulfide is itself reduced.Open in a separate windowFigure 1.PDI family of enzymes catalyzes disulfide exchange reactions in the endoplasmic reticulum. Nascent polypeptide chains are cotranslationally translocated across the ER membrane whereupon cysteines in close proximity can form disulfides. The reaction is catalyzed by members of the PDI family (depicted as PDI) by a disulfide exchange reaction resulting in the reduction of the PDI active site. If nonnative disulfides are formed these can be reduced by the reverse disulfide exchange reaction, resulting in the oxidation of the PDI active site.Both the formation and breaking of disulfides can be thought of as electron transport pathways that require suitable electron acceptors or donors to drive the flow of electrons. For the purposes of this article the two pathways will be discussed separately, but it should be appreciated that each pathway occurs within the same organelle so the possibility of crossover between them is real. Whether futile redox reactions occur between the pathways is unclear but any kinetic segregation of the pathways will be highlighted where it is known to occur.     

Entamoeba histolytica: biochemical characterization of a protein disulfide isomerase

Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. Here, we report the biochemical characterization of a PDI enzyme from the protozoan parasite Entamoeba histolytica (EhPDI). Our results show that EhPDI behaves mainly as an oxidase/isomerase and can be inhibited by bacitracin, a known PDI inhibitor; moreover, it exhibits chaperone-like activity. Albeit its physiological role in the life style of the parasite (including virulence and survival) remains to be studied, EhPDI could represent a potential drug target for anti-amebic therapy.     

A second protein disulfide isomerase plays a protective role against nitrosative and nutritional stresses in Schizosaccharomyces pombe

In the present work, a second gene encoding protein disulfide isomerase (PDI2) was cloned and characterized from Schizosaccharomyces pombe, and its regulation was studied. The structural gene encoding PDI2 was amplified from the genomic DNA using PCR, and ligated into the E. coli-yeast shuttle vector pRS316 to generate the recombinant plasmid pYPDI2. The determined DNA sequence carries 2,578 bp and is able to encode a protein of 726 amino acid sequence with CGAC at the putative active site. The fission yeast cells harboring pYPDI2 contained 1.62- and 2.73-fold higher PDI activity than the control yeast cells in exponential and stationary phases, respectively, indicating that the cloned gene is in vivo functioning. The PDI2 mRNA levels in both vector control and pYPDI2-containing yeast cells were found to be significantly higher in the stationary phase than in the exponential phase, suggesting that expression of the PDI2 gene is under stationary control. The yeast cells harboring pYPDI2 showed enhanced survival on minimal media plates containing nitric oxide (NO)-generating sodium nitroprusside (SNP) and no nitrogen. The synthesis of β-galactosidase from the PDI2-lacZ fusion gene was markedly enhanced in the Pap1-positive KP1 cells by SNP and nitrogen starvation. However, the enhancement in the synthesis of β-galactosidase from the PDI2-lacZ fusion gene by SNP and nitrogen starvation appeared to be relatively reduced in the Pap1-negative TP108-3C cells than in the Pap1-positive KP1 cells. The PDI2 mRNA level was elevated by SNP and nitrogen starvation in the Pap1-positive cells but not in the Pap1-negative cells. In brief, the S. pombe PDI2 plays a protective role against nitrosative and nutritional stresses, and is positively regulated by NO and nitrogen starvation in a Pap1-dependent manner.     

Protein disulfide isomerase is cleaved by caspase-3 and -7 during apoptosis

Apoptotic signals are typically accompanied by activation of aspartate-specific cysteine proteases called caspases, and caspase-3 and -7 play crucial roles in the execution of apoptosis. Previously, using the proteomic approach, protein disulfide isomerase (PDI) was found to be a candidate substrate of caspase-7. This abundant 55 kDa protein introduces disulfide bonds into proteins (via its oxidase activity) and catalyzes the rearrangement of incorrect disulfide bonds (via its isomerase activity). PDI is abundant in the ER but is also found in non-ER locations. In this study we demonstrated that PDI is cleaved by caspase-3 and -7 in vitro. In addition, in vivo experiment showed that it is cleaved during etoposide-induced apoptosis in HL-60 cells. Subcellular fractionation showed that PDI was also present in the cytosol. Furthermore, only cytosolic PDI was clearly digested by caspase-3 and -7. It was also confirmed by confocal image analysis that PDI and caspase-7 partially co-localize in both resting and apoptotic MCF-7 cells. Overexpression of cytosolic PDI (ER retention sequence deleted) inhibited cell death after an apoptotic stimulus. These data indicate that cytosolic PDI is a substrate of caspase-3 and -7, and that it has an anti-apoptotic action.     

Reduction-reoxidation cycles contribute to catalysis of disulfide isomerization by protein-disulfide isomerase

Protein-disulfide isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding. This process can be error-prone in its early stages, and any incorrect disulfides that form must be rearranged to their native configuration. When the second cysteine (CGHC) in the PDI active site is mutated to Ser, the isomerase activity drops by 7-8-fold, and a covalent intermediate with the substrate accumulates. This led to the proposal that the second active site cysteine provides an escape mechanism, preventing PDI from becoming trapped with substrates that isomerize slowly (Walker, K. W., and Gilbert, H. F. (1997) J. Biol. Chem. 272, 8845-8848). Escape also reduces the substrate, and if it is invoked frequently, disulfide isomerization will involve cycles of reduction and reoxidation in preference to intramolecular isomerization of the PDI-bound substrate. Using a gel-shift assay that adds a polyethylene glycol-conjugated maleimide of 5 kDa for each sulfhydryl group, we find that PDI reduction and oxidation are kinetically competent and essential for isomerization. Oxidants inhibit isomerization and oxidize PDI when a redox buffer is not present to maintain the PDI redox state. Reductants also inhibit isomerization as they deplete oxidized PDI. These rapid cycles of PDI oxidation and reduction suggest that PDI catalyzes isomerization by trial and error, reducing disulfides and oxidizing them in a different configuration. Disulfide reduction-reoxidation may set up critical folding intermediates for intramolecular isomerization, or it may serve as the only isomerization mechanism. In the absence of a redox buffer, these steady-state reduction-oxidation cycles can balance the redox state of PDI and support effective catalysis of disulfide isomerization.     

Molecular Characterization and Expression Profiling of the Protein Disulfide Isomerase Gene Family in Brachypodium distachyon L

Protein disulfide isomerases (PDI) are involved in catalyzing protein disulfide bonding and isomerization in the endoplasmic reticulum and functions as a chaperone to inhibit the aggregation of misfolded proteins. Brachypodium distachyon is a widely used model plant for temperate grass species such as wheat and barley. In this work, we report the first molecular characterization, phylogenies, and expression profiles of PDI and PDI-like (PDIL) genes in B. distachyon in different tissues under various abiotic stresses. Eleven PDI and PDIL genes in the B. distachyon genome by in silico identification were evenly distributed across all five chromosomes. The plant PDI family has three conserved motifs that are involved in catalyzing protein disulfide bonding and isomerization, but a different exon/intron structural organization showed a high degree of structural differentiation. Two pairs of genes (BdPDIL4-1 and BdPDIL4-2; BdPDIL7-1 and BdPDIL7-2) contained segmental duplications, indicating each pair originated from one progenitor. Promoter analysis showed that Brachypodium PDI family members contained important cis-acting regulatory elements involved in seed storage protein synthesis and diverse stress response. All Brachypodium PDI genes investigated were ubiquitously expressed in different organs, but differentiation in expression levels among different genes and organs was clear. BdPDIL1-1 and BdPDIL5-1 were expressed abundantly in developing grains, suggesting that they have important roles in synthesis and accumulation of seed storage proteins. Diverse treatments (drought, salt, ABA, and H₂O₂) induced up- and down-regulated expression of Brachypodium PDI genes in seedling leaves. Interestingly, BdPDIL1-1 displayed significantly up-regulated expression following all abiotic stress treatments, indicating that it could be involved in multiple stress responses. Our results provide new insights into the structural and functional characteristics of the plant PDI gene family.     

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.     

Functional in vitro analysis of the ERO1 protein and protein-disulfide isomerase pathway

Oxidative protein folding in the endoplasmic reticulum is supported by efficient electron relays driven by enzymatic reactions centering on the ERO1-protein-disulfide isomerase (PDI) pathway. A controlled in vitro oxygen consumption assay was carried out to analyze the ERO1-PDI reaction. The results showed the pH-dependent oxidation of PDI by ERO1α. Among several possible disulfide bonds regulating ERO1α activity, Cys(94)-Cys(131) and Cys(99)-Cys(104) disulfide bonds are dominant regulators by excluding the involvement of the Cys(85)-Cys(391) disulfide in the regulation. The fine-tuned species specificity of the ERO1-PDI pathway was demonstrated by functional in vitro complementation assays using yeast and mammalian oxidoreductases. Finally, the results provide experimental evidence for the intramolecular electron transfer from the a domain to the a' domain within PDI during its oxidation by ERO1α.     

Tissue Distribution of ERp61 and Association of Its Increased Expression with IgG Production in Hybridoma Cells

A protein of molecular weight 60 kDa was purified from the culture medium of a murine colon carcinoma cell line, colon26, and its partial amino-acid sequence determined. Extremely high homology was found with the deduced sequence from cDNA of rat ERp61, earlier found to be an endoplasmic reticulum (ER)-resident protein with redox activity and a similar structure to protein disulfide isomerase (PDI). Western blotting analysis showed that colon26 cells secrete a significant amount of ERp61 into culture medium, although most remains intracellular. The thiol:protein disulfide oxidoreductase activity of the purified mouse ERp61 was demonstrated by insulin-reduction assay. The ER location of the protein in fibroblasts was immunocytochemically confirmed by double staining for ERp61 and another ER-resident protein, PDI or Hsp47. Immunohistochemical studies of murine tissues showed a ubiquitous distribution of ERp61 in a wide variety of cell types. However, it was particularly abundant in plasma cells, mucus-secreting cells in various tissues, neuroendocrine cells including neurons, and follicular epithelia of thyroid gland that actively synthesize and secrete proteins containing cysteine residues. Furthermore, a high correlation was observed between intracellular amounts of ERp61 and immunoglobulin production by hybridoma cells. These results indicate that ERp61 may be involved in disulfide bond formation for such proteins.