Unfolded protein response in chronic obstructive pulmonary disease: smoking, aging and disease: a SAD trifecta

Cigarette smoke (CS) is a risk factor for the development of chronic obstructive pulmonary disease (COPD). Oxidative stress is an immediate result of CS exposure and has the ability to modify cellular proteins. The endoplasmic reticulum (ER) is a compartment where early steps of synthesis and folding of membrane and secretory proteins takes place. Oxidative stress has been shown to interfere with protein folding in the ER and elicits the unfolded protein response (UPR). The UPR is a massive endoplasmic reticulum to the nucleus and the cellular kinase cascades signaling pathway. The UPR triggers a series of intracellular events that aim to help cells overcome the consequences of the stress or eliminate rogue cells by altering expression of genes involved in anti-oxidant defense, cell cycle progression, inflammation, and apoptosis. Recent data demonstrate that CS induces the UPR in vitro and in vivo. The timing of UPR induction in smokers and the mechanism of CS-induced UPR are areas of active investigation. The role of UPR in the protection of smoker's lungs from CS-induced oxidative stress, and its contribution to CS-induced apoptosis and inflammation, is beginning to emerge. This review discusses recent data about UPR in COPD and summarizes findings on UPR that have potential relevance to COPD.     

Oxidative protein folding in vitro: a study of the cooperation between quiescin-sulfhydryl oxidase and protein disulfide isomerase

The flavin-dependent quiescin-sulfhydryl oxidase (QSOX) inserts disulfide bridges into unfolded reduced proteins with the reduction of molecular oxygen to form hydrogen peroxide. This work investigates how QSOX and protein disulfide isomerase (PDI) cooperate in vitro to generate native pairings in two unfolded reduced proteins: ribonuclease A (RNase, four disulfide bonds and 105 disulfide isomers of the fully oxidized protein) and avian riboflavin binding protein (RfBP, nine disulfide bonds and more than 34 million corresponding disulfide pairings). Experiments combining avian or human QSOX with up to 200 muM avian or human reduced PDI show that the isomerase is not a significant substrate of QSOX. Both reduced RNase and RfBP can be efficiently refolded in an aerobic solution containing micromolar concentrations of reduced PDI and nanomolar levels of QSOX without any added oxidized PDI or glutathione redox buffer. Refolding of RfBP is followed continuously using the complete quenching of the fluorescence of free riboflavin that occurs on binding to apo-RfBP. The rate of refolding is half-maximal at 30 muM reduced PDI when the reduced client protein (1 muM) is used in the presence of 30 nM QSOX. The use of high concentrations of PDI, in considerable excess over the folding protein client, reflects the concentration prevailing in the lumen of the endoplasmic reticulum and allows the redox poise of these in vitro experiments to be set with oxidized and reduced PDI. In the absence of either QSOX or redox buffer, the fastest refolding of RfBP is accomplished with excess reduced PDI and just enough oxidized PDI to generate nine disulfides in the protein client. These in vitro experiments are discussed in terms of current models for oxidative folding in the endoplasmic reticulum.     

The Reduction Potential of the Active Site Disulfides of Human Protein Disulfide Isomerase Limits Oxidation of the Enzyme by Ero1��

Disulfide formation in newly synthesized proteins entering the mammalian endoplasmic reticulum is catalyzed by protein disulfide isomerase (PDI), which is itself thought to be directly oxidized by Ero1α. The activity of Ero1α is tightly regulated by the formation of noncatalytic disulfides, which need to be broken to activate the enzyme. Here, we have developed a novel PDI oxidation assay, which is able to simultaneously determine the redox status of the individual active sites of PDI. We have used this assay to confirm that when PDI is incubated with Ero1α, only one of the active sites of PDI becomes directly oxidized with a slow turnover rate. In contrast, a deregulated mutant of Ero1α was able to oxidize both PDI active sites at an equivalent rate to the wild type enzyme. When the active sites of PDI were mutated to decrease their reduction potential, both were now oxidized by wild type Ero1α with a 12-fold increase in activity. These results demonstrate that the specificity of Ero1α toward the active sites of PDI requires the presence of the regulatory disulfides. In addition, the rate of PDI oxidation is limited by the reduction potential of the PDI active site disulfide. The inability of Ero1α to oxidize PDI efficiently likely reflects the requirement for PDI to act as both an oxidase and an isomerase during the formation of native disulfides in proteins entering the secretory pathway.     

Oxidative protein folding and unfolded protein response elicit differing redox regulation in endoplasmic reticulum and cytosol of yeast

Oxidative protein folding can exceed the cellular secretion machinery, inducing the unfolded protein response (UPR). Sustained endoplasmic reticulum (ER) stress leads to cell stress and disease, as described for Alzheimer, Parkinson, and diabetes mellitus, among others. It is currently assumed that the redox state of the ER is optimally balanced for formation of disulfide bonds using glutathione as the main redox buffer and that UPR causes a reduction of this organelle. The direct effect of oxidative protein folding in the ER, however, has not yet been dissected from UPR regulation. To measure in vivo redox conditions in the ER and cytosol of the yeast model organism Pichia pastoris we targeted redox-sensitive roGFP variants to the respective organelles. Thereby, we clearly demonstrate that induction of the UPR causes reduction of the cytosol in addition to ER reduction. Similarly, a more reduced redox state of the cytosol, but not of the ER, is observed during oxidative protein folding in the ER without UPR induction, as demonstrated by overexpressing genes of disulfide bond-rich secretory proteins such as porcine trypsinogen or protein disulfide isomerase (PDI1) and ER oxidase (ERO1). Cytosolic reduction seems not to be caused by the action of glutathione reductase (GLR1) and could not be compensated for by overexpression of cytosolic glutathione peroxidase (GPX1). Overexpression of GPX1 and PDI1 oxidizes the ER and increases the secretion of correctly folded proteins, demonstrating that oxidative protein folding per se is enhanced by a more oxidized ER and is counterbalanced by a more reduced cytosol. As the total glutathione concentration of these strains does not change significantly, but the ratio of GSH to GSSG is altered, either transport or redox signaling between the glutathione pools of ER and cytosol is assumed. These data clearly demonstrate that protein folding and ER stress have a severe impact on the cytosolic redox balance, which may be a major factor during development of folding-related diseases.     

Depletion of Cyclophilins B and C Leads to Dysregulation of Endoplasmic Reticulum Redox Homeostasis

Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This “hyperoxidation” phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins.     

Oxidative protein folding in eukaryotes: mechanisms and consequences

The endoplasmic reticulum (ER) provides an environment that is highly optimized for oxidative protein folding. Rather than relying on small molecule oxidants like glutathione, it is now clear that disulfide formation is driven by a protein relay involving Ero1, a novel conserved FAD-dependent enzyme, and protein disulfide isomerase (PDI); Ero1 is oxidized by molecular oxygen and in turn acts as a specific oxidant of PDI, which then directly oxidizes disulfide bonds in folding proteins. While providing a robust driving force for disulfide formation, the use of molecular oxygen as the terminal electron acceptor can lead to oxidative stress through the production of reactive oxygen species and oxidized glutathione. How Ero1p distinguishes between the many different PDI-related proteins and how the cell minimizes the effects of oxidative damage from Ero1 remain important open questions.     

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.     

Ero1-α and PDIs constitute a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases

Ero1-α and endoplasmic reticulum (ER) oxidoreductases of the protein disulfide isomerase (PDI) family promote the efficient introduction of disulfide bonds into nascent polypeptides in the ER. However, the hierarchy of electron transfer among these oxidoreductases is poorly understood. In this paper, Ero1-α–associated oxidoreductases were identified by proteomic analysis and further confirmed by surface plasmon resonance. Ero1-α and PDI were found to constitute a regulatory hub, whereby PDI induced conformational flexibility in an Ero1-α shuttle cysteine (Cys99) facilitated intramolecular electron transfer to the active site. In isolation, Ero1-α also oxidized ERp46, ERp57, and P5; however, kinetic measurements and redox equilibrium analysis revealed that PDI preferentially oxidized other oxidoreductases. PDI accepted electrons from the other oxidoreductases via its a′ domain, bypassing the a domain, which serves as the electron acceptor from reduced glutathione. These observations provide an integrated picture of the hierarchy of cooperative redox interactions among ER oxidoreductases in mammalian cells.     

An Additional Function of the Rough Endoplasmic Reticulum Protein Complex Prolyl 3-Hydroxylase 1·Cartilage-associated Protein·Cyclophilin B: THE CXXXC MOTIF REVEALS DISULFIDE ISOMERASE ACTIVITY IN VITRO*

Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.     

Ero1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum.

Native protein disulfide bond formation in the endoplasmic reticulum (ER) requires protein disulfide isomerase (PDI) and Ero1p. Here we show that oxidizing equivalents flow from Ero1p to substrate proteins via PDI. PDI is predominantly oxidized in wild-type cells but is reduced in an ero1-1 mutant. Direct dithiol-disulfide exchange between PDI and Ero1p is indicated by the capture of PDI-Ero1p mixed disulfides. Mixed disulfides can also be detected between PDI and the ER precursor of carboxypeptidase Y (CPY). Further, PDI1 is required for the net formation of disulfide bonds in newly synthesized CPY, indicating that PDI functions as an oxidase in vivo. Together, these results define a pathway for protein disulfide bond formation in the ER. The PDI homolog Mpd2p is also oxidized by Ero1p.     

The yeast EUG1 gene encodes an endoplasmic reticulum protein that is functionally related to protein disulfide isomerase.

The product of the EUG1 gene of Saccharomyces cerevisiae is a soluble endoplasmic reticulum protein with homology to both the mammalian protein disulfide isomerase (PDI) and the yeast PDI homolog encoded by the essential PDI1 gene. Deletion or overexpression of EUG1 causes no growth defects under a variety of conditions. EUG1 mRNA and protein levels are dramatically increased in response to the accumulation of native or unglycosylated proteins in the endoplasmic reticulum. Overexpression of the EUG1 gene allows yeast cells to grow in the absence of the PDI1 gene product. Depletion of the PDI1 protein in Saccharomyces cerevisiae causes a soluble vacuolar glycoprotein to accumulate in its endoplasmic reticulum form, and this phenotype is only partially relieved by the overexpression of EUG1. Taken together, our results indicate that PDI1 and EUG1 encode functionally related proteins that are likely to be involved in interacting with nascent polypeptides in the yeast endoplasmic reticulum.     

Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin

Cholera toxin is assembled from two subunits in the periplasm of Vibrio cholerae and disassembled in the analogous compartment of target cells, the lumen of the endoplasmic reticulum (ER), before a fragment of it, the A1 chain, is transported into the cytosol. We show that protein disulfide isomerase (PDI) in the ER lumen functions to disassemble and unfold the toxin once its A chain has been cleaved. PDI acts as a redox-driven chaperone; in the reduced state, it binds to the A chain and in the oxidized state it releases it. Our results explain the pathway of cholera toxin, suggest a role for PDI in retrograde protein transport into the cytosol, and indicate that PDI can act as a novel type of chaperone, whose binding and release of substrates is regulated by a redox, rather than an ATPase, cycle.     

The expression of protein disulfide isomerase from Litopenaeus vannamei hemocytes is regulated by bacterial inoculation

A partial clone encoding a member of the protein disulfide isomerase (PDI) was isolated from a Litopenaeus vannamei hemocyte cDNA library. The 5′-end sequence was obtained by RACE. The complete sequence encodes for a 502-residues protein that contains two thioredoxin domains and the typical endoplasmic reticulum retention KDEL motif. Shrimp PDI is highly similar to the homologue protein described in both vertebrates and invertebrates. Changes in the shrimp PDI mRNA expression were observed after injection of Vibrio alginolyticus, suggesting that PDI is implicated in the immune defense system. This is the first report of a PDI in crustaceans.     

Glutathione limits Ero1-dependent oxidation in the endoplasmic reticulum

Many proteins of the secretory pathway contain disulfide bonds that are essential for structure and function. In the endoplasmic reticulum (ER), Ero1 alpha and Ero1 beta oxidize protein disulfide isomerase (PDI), which in turn transfers oxidative equivalents to newly synthesized cargo proteins. However, oxidation must be limited, as some reduced PDI is necessary for disulfide isomerization and ER-associated degradation. Here we show that in semipermeable cells, PDI is more oxidized, disulfide bonds are formed faster, and high molecular mass covalent protein aggregates accumulate in the absence of cytosol. Addition of reduced glutathione (GSH) reduces PDI and restores normal disulfide formation rates. A higher GSH concentration is needed to balance oxidative folding in semipermeable cells overexpressing Ero1 alpha, indicating that cytosolic GSH and lumenal Ero1 alpha play antagonistic roles in controlling the ER redox. Moreover, the overexpression of Ero1 alpha significantly increases the GSH content in HeLa cells. Our data demonstrate tight connections between ER and cytosol to guarantee redox exchange across compartments: a reducing cytosol is important to ensure disulfide isomerization in secretory proteins.     

Molecular bases of cyclic and specific disulfide interchange between human ERO1alpha protein and protein-disulfide isomerase (PDI)

In the endoplasmic reticulum (ER) of human cells, ERO1α and protein-disulfide isomerase (PDI) constitute one of the major electron flow pathways that catalyze oxidative folding of secretory proteins. Specific and limited PDI oxidation by ERO1α is essential to avoid ER hyperoxidation. To investigate how ERO1α oxidizes PDI selectively among more than 20 ER-resident PDI family member proteins, we performed docking simulations and systematic biochemical analyses. Our findings reveal that a protruding β-hairpin of ERO1α specifically interacts with the hydrophobic pocket present in the redox-inactive PDI b'-domain through the stacks between their aromatic residues, leading to preferred oxidation of the C-terminal PDI a'-domain. ERO1α associated preferentially with reduced PDI, explaining the stepwise disulfide shuttle mechanism, first from ERO1α to PDI and then from oxidized PDI to an unfolded polypeptide bound to its hydrophobic pocket. The interaction of ERO1α with ERp44, another PDI family member protein, was also analyzed. Notably, ERO1α-dependent PDI oxidation was inhibited by a hyperactive ERp44 mutant that lacks the C-terminal tail concealing the substrate-binding hydrophobic regions. The potential ability of ERp44 to inhibit ERO1α activity may suggest its physiological role in ER redox and protein homeostasis.     

Pathways for protein disulphide bond formation

The folding of many secretory proteins depends upon the formation of disulphide bonds. Recent advances in genetics and cell biology have outlined a core pathway for disulphide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. In this pathway, oxidizing equivalents flow from the recently identified ER membrane protein Ero1p to secretory proteins via protein disulphide isomerase (PDI). Contrary to prior expectations, oxidation of glutathione in the ER competes with oxidation of protein thiols. Contributions of PDI homologues to the catalysis of oxidative folding will be discussed, as will similarities between eukaryotic and prokaryotic disulphide-bond-forming systems.     

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.     

Is protein disulfide isomerase a redox-dependent molecular chaperone?

Protein disulfide isomerase (PDI) is a multifunctional protein catalysing the formation of disulfide bonds, acting as a molecular chaperone and being a component of the enzymes prolyl 4-hydroxylase (P4H) and microsomal triglyceride transfer protein. The role of PDI as a molecular chaperone or polypeptide-binding protein is mediated primarily through an interaction of substrates with its b' domain. It has been suggested that this binding is regulated by the redox state of PDI, with association requiring the presence of glutathione, and dissociation the presence of glutathione disulfide. To determine whether this is the case, we investigated the ability of PDI to bind to a folding polypeptide chain within a functionally intact endoplasmic reticulum and to be dissociated from the alpha-subunit of P4H in vitro in the presence of reducing or oxidizing agents. Our results clearly demonstrate that binding of PDI to these polypeptides is not regulated by its redox state. We also demonstrate that the dissociation of PDI from substrates observed in the presence of glutathione disulfide can be explained by competition for the peptide-binding site on PDI.     

Protein disulfide isomerase inhibits endoplasmic reticulum stress response and apoptosis via its oxidoreductase activity in colorectal cancer

Protein disulfide isomerase (PDI), a principal endoplasmic reticulum resident oxidoreductase chaperone, is known to play a role in malignancies. This study aims to explore the molecular mechanism by which PDI regulates endoplasmic reticulum stress and the apoptosis signaling pathway in colorectal cancer (CRC). We determined the expression of PDI in CRC tissues and adjacent normal tissues. Gain- and loss- of function assays were conducted to evaluate the effects of PDI on oxidative stress, endoplasmic reticulum stress, and apoptosis in CRC cells, as reflected by hydrogen peroxide (H₂O₂) level and the expression of related proteins. PDI protein expression was upregulated in CRC tissues. Small molecule inhibitor of PDI or PDI knockdown reduced CRC cell viability and induced apoptosis. Overexpression of wild-type PDI augmented the viability of CRC cells and inhibited endoplasmic reticulum stress response and apoptosis. Small molecule inhibitor of PDI or PDI knockdown increased intracellular H₂O₂ level and activated apoptosis signaling pathway, which could be reversed by wild-type PDI restoration. Moreover, the catalytic active site of C-terminal of PDI was found to be indispensable for the regulatory effects of PDI on H₂O₂ levels, apoptosis and cell viability in CRC cells. Collectively, PDI inhibits endoplasmic reticulum stress and apoptosis of CRC cells through its oxidoreductase activity, thereby promoting the malignancy of CRC.