4-Phenylbutyric Acid Reduces Endoplasmic Reticulum Stress in Chondrocytes That Is Caused by Loss of the Protein Disulfide Isomerase ERp57.

OBJECTIVE: The integrity of cartilage depends on the correct synthesis of extracellular matrix (ECM) components. In case of insufficient folding of proteins in the endoplasmic reticulum (ER) of chondrocytes, ECM proteins aggregate, ER stress evolves, and the unfolded protein response (UPR) is initiated. By this mechanism, chondrocytes relieve the stress condition or initiate cell death by apoptosis. Especially persistent ER stress has emerged as a pathogenic mechanism in cartilage diseases, such as chondrodysplasias and osteoarthritis. As pharmacological intervention is not available yet, it is of great interest to understand cartilage ER stress in detail and to develop therapeutics to intervene. METHODS: ERp57-deficient chondrocytes were generated by CRISPR/Cas9-induced KO. ER stress and autophagy were studied on mRNA and protein level as well as by transmission electron microscopy (TEM) in chondrocyte micromass or cartilage explant cultures of ERp57 KO mice. Thapsigargin (Tg), an inhibitor of the ER-residing Ca2+-ATPase, and 4-Phenylbutyric acid (4-PBA), a small molecular chemical chaperone, were applied to induce or inhibit ER stress. RESULTS: Our data reveal that the loss of the protein disulfide isomerase ERp57 is sufficient to induce ER stress in chondrocytes. 4-PBA efficiently diffuses into cartilage explant cultures and diminishes excessive ER stress in chondrocytes dose dependently, no matter if it is induced by ERp57 KO or stimulation with Tg. CONCLUSION: ER-stress-related diseases have different sources; therefore, various targets for therapeutic treatment exist. In the future, 4-PBA may be used alone or in combination with other drugs for the treatment of ER-stress-related skeletal disorders in patients.

Platelet Protein Disulfide Isomerase Promotes Glycoprotein Ibα-Mediated Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions.

BACKGROUND: Platelet-neutrophil interactions contribute to vascular occlusion and tissue damage in thromboinflammatory disease. Platelet glycoprotein Ibα (GPIbα), a key receptor for the cell-cell interaction, is believed to be constitutively active for ligand binding. Here, we established the role of platelet-derived protein disulfide isomerase (PDI) in reducing the allosteric disulfide bonds in GPIbα and enhancing the ligand-binding activity under thromboinflammatory conditions. METHODS: Bioinformatic analysis identified 2 potential allosteric disulfide bonds in GPIbα. Agglutination assays, flow cytometry, surface plasmon resonance analysis, a protein-protein docking model, proximity ligation assays, and mass spectrometry were used to demonstrate a direct interaction between PDI and GPIbα and to determine a role for PDI in regulating GPIbα function and platelet-neutrophil interactions. Also, real-time microscopy and animal disease models were used to study the pathophysiological role of PDI-GPIbα signaling under thromboinflammatory conditions. RESULTS: Deletion or inhibition of platelet PDI significantly reduced GPIbα-mediated platelet agglutination. Studies using PDI-null platelets and recombinant PDI or Anfibatide, a clinical-stage GPIbα inhibitor, revealed that the oxidoreductase activity of platelet surface-bound PDI was required for the ligand-binding function of GPIbα. PDI directly bound to the extracellular domain of GPIbα on the platelet surface and reduced the Cys4-Cys17 and Cys209-Cys248 disulfide bonds. Real-time microscopy with platelet-specific PDI conditional knockout and sickle cell disease mice demonstrated that PDI-regulated GPIbα function was essential for platelet-neutrophil interactions and vascular occlusion under thromboinflammatory conditions. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that PDI-GPIbα signaling played a crucial role in tissue damage. CONCLUSIONS: Our results demonstrate that PDI-facilitated cleavage of the allosteric disulfide bonds tightly regulates GPIbα function, promoting platelet-neutrophil interactions, vascular occlusion, and tissue damage under thromboinflammatory conditions.

Protein disulfide isomerase enhances tissue factor-dependent thrombin generation.

Protein disulfide isomerase (PDI) plays an important role in fibrin generation in vivo, but the underlying mechanism remains largely unknown. In this study, using thrombin generation assay (TGA), we investigated whether PDI contributes to tissue factor (TF)-mediated thrombin generation. Human peripheral blood mononuclear cells (PBMCs) were treated with 100 ng/ml lipopolysaccharide (LPS), the expression of TF on cell surface was analyzed by flow cytometry. After incubation with an inhibitory anti-TF antibody, recombinant PDI protein or a PDI inhibitor PACMA31, LPS-stimulated human PBMCs were incubated with human plasma, and thrombin generation was assessed by Ceveron Alpha TGA and a fluorescent thrombin substrate. Bone marrow mononuclear cells isolated from PDI-knockout and wild-type mice were stimulated by LPS, followed by measurement of thrombin generation. LPS stimulation increased expression of TF on PBMCs, and thrombin generation. Inhibitory anti-TF antibody almost completely suppressed thrombin generation of LPS-stimulated PBMCs, suggesting that thrombin generation was TF-dependent. Recombinant PDI protein increased thrombin generation, while PACMA31 attenuated thrombin generation. Compared with control cells, PDI-deficient marrow mononuclear cells had less capacity in thrombin generation. Taken together, these data suggest that PDI enhances TF-dependent thrombin generation.

The Multiple Localized Glyceraldehyde-3-Phosphate Dehydrogenase Contributes to the Attenuation of the Francisella tularensis dsbA Deletion Mutant.

The DsbA homolog of Francisella tularensis was previously demonstrated to be required for intracellular replication and animal death. Disruption of the dsbA gene leads to a pleiotropic phenotype that could indirectly affect a number of different cellular pathways. To reveal the broad effects of DsbA, we compared fractions enriched in membrane proteins of the wild-type FSC200 strain with the dsbA deletion strain using a SILAC-based quantitative proteomic analysis. This analysis enabled identification of 63 proteins with significantly altered amounts in the dsbA mutant strain compared to the wild-type strain. These proteins comprise a quite heterogeneous group including hypothetical proteins, proteins associated with membrane structures, and potential secreted proteins. Many of them are known to be associated with F. tularensis virulence. Several proteins were selected for further studies focused on their potential role in tularemia's pathogenesis. Of them, only the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme of glycolytic pathway, was found to be important for full virulence manifestations both in vivo and in vitro. We next created a viable mutant strain with deleted gapA gene and analyzed its phenotype. The gapA mutant is characterized by reduced virulence in mice, defective replication inside macrophages, and its ability to induce a protective immune response against systemic challenge with parental wild-type strain. We also demonstrate the multiple localization sites of this protein: In addition to within the cytosol, it was found on the cell surface, outside the cells, and in the culture medium. Recombinant GapA was successfully obtained, and it was shown that it binds host extracellular serum proteins like plasminogen, fibrinogen, and fibronectin.

A DsbA-Deficient Periplasm Enables Functional Display of a Protein with Redox-Sensitive Folding on M13 Phage.

The requirements for target protein folding in M13 phage display are largely underappreciated. Here we chose Fbs1, a carbohydrate binding protein, as a model to address this issue. Importantly, folding of Fbs1 is impaired in an oxidative environment. Fbs1 can be displayed on M13 phage using the SRP or Sec pathway. However, the displayed Fbs1 protein is properly folded only when Fbs1 is translocated via the SRP pathway and displayed using Escherichia coli cells with a DsbA-negative periplasm. This study indicates M13 phage display may be improved using a system specifically designed according to the folding requirements of each target protein.

Fibrillin-1 mgΔ(lpn) Marfan syndrome mutation associates with preserved proteostasis and bypass of a protein disulfide isomerase-dependent quality checkpoint.

Fibrillin-1 mutations promote Marfan syndrome (MFS) via complex yet unclear pathways. The roles of endoplasmic reticulum (ER) and the major ER redox chaperone protein disulfide isomerase-A1 in the processing of normal and mutated fibrillin-1 and ensuing protein secretion and/or intracellular retention are unclear. Our results in mouse embryonic fibroblasts bearing the exon-skipping mgΔ(lox-P-neo) (mgΔ(lpn)) mutation, which associates in vivo with MFS and in vitro with disrupted microfibrils, indicate a preserved ER-dependent proteostasis or redox homeostasis. Rather, mutated fibrillin-1 is secreted normally through Golgi-dependent pathways and is not intracellularly retained. Similar results occurred for the C1039G point mutation. In parallel, we provide evidence that PDIA1 physically interacts with fibrillin-1 in the ER. Moreover, siRNA against PDIA1 augmented fibrillin-1 secretion rates in wild-type cells. However, fibrillin-1 with the mgΔ(lpn) mutation bypassed PDI checkpoint delay, while the C1039G mutation did not. This heretofore undisclosed PDIA1-mediated mechanism may be important to control the extracellular availability of function-competent fibrillin-1, an important determinant of disease phenotype. Moreover, our results may reveal a novel, holdase-like, PDI function associated with ER protein quality control.

Depletion of the thiol oxidoreductase ERp57 in tumor cells inhibits proliferation and increases sensitivity to ionizing radiation and chemotherapeutics.

Rapidly growing tumor cells must synthesize proteins at a high rate and therefore depend on an efficient folding and quality control system for nascent secretory proteins in the endoplasmic reticulum (ER). The ER resident thiol oxidoreductase ERp57 plays an important role in disulfide bond formation. Lentiviral, doxycycline-inducible ERp57 knockdown was combined with irradiation and treatment with chemotherapeutic agents. The knockdown of ERp57 significantly enhanced the apoptotic response to anticancer treatment in HCT116 colon cancer cells via a p53-dependent mechanism. Instead of a direct interaction with p53, depletion of ERp57 induced cell death via a selective activation of the PERK branch of the Unfolded Protein Response (UPR). In contrast, apoptosis was reduced in MDA-MB-231 breast cancer cells harboring mutant p53. Nevertheless, we observed a strong reduction of proliferation in response to ERp57 knockdown in both cell lines regardless of the p53 status. Depletion of ERp57 reduced the phosphorylation activity of the mTOR-complex1 (mTORC1) as demonstrated by reduction of p70S6K phosphorylation. Our data demonstrate that ERp57 is a promising target for anticancer therapy due to synergistic p53-dependent induction of apoptosis and p53-independent inhibition of proliferation.

Francisella tularensis type B ΔdsbA mutant protects against type A strain and induces strong inflammatory cytokine and Th1-like antibody response in vivo.

Francisella tularensis subspecies tularensis is a highly virulent intracellular bacterial pathogen, causing the disease tularemia. However, a safe and effective vaccine for routine application against F. tularensis has not yet been developed. We have recently constructed the deletion mutants for the DsbA homolog protein (ΔdsbA/FSC200) and a hypothetical protein IglH (ΔiglH/FSC200) in the type B F. tularensis subsp. holarctica FSC200 strain, which exerted different protection capacity against parental virulent strain. In this study, we further investigated the immunological correlates for these different levels of protection provided by ΔdsbA/FSC200 and ΔiglH/FSC200 mutants. Our results show that ΔdsbA/FSC200 mutant, but not ΔiglH/FSC200 mutant, induces an early innate inflammatory response leading to strong Th1-like antibody response. Furthermore, vaccination with ΔdsbA/FSC200 mutant, but not with ΔiglH/FSC200, elicited protection against the subsequent challenge with type A SCHU S4 strain in mice. An immunoproteomic approach was used to map a spectrum of antigens targeted by Th1-like specific antibodies, and more than 80 bacterial antigens, including novel ones, were identified. Comparison of tularemic antigens recognized by the ΔdsbA/FSC200 post-vaccination and the SCHU S4 post-challenge sera then revealed the existence of 22 novel SCHU S4 specific antibody clones.

Increased expression of ERp57/GRP58 is protective against pancreatic beta cell death caused by autophagic failure.

Autophagy is a tightly regulated self-digestion system. As in other cell types, autophagy plays an essential role in the homeostasis of pancreatic beta cells. However, the mechanisms involved in the deterioration of beta cell function caused by autophagic failure have not yet been fully elucidated. To gain insight into its mechanisms, we compared the protein expression of islets from beta cell-specific Atg7-deficient mice (Atg7(Δß-cell) mice) and their controls (Atg7(f/f) mice). Liquid chromatography/mass spectrometry after 1-dimensional electrophoresis identified the increased expression of ERp57/GRP58 in islets isolated from Atg7(Δß-cell) mice compared with those from Atg7(f/f) mice. The expression level of ERp57 was also elevated in rat insulinoma INS-1 cells by inducible knock-down of the atg7-gene. In Atg7 knock-down INS-1 cells, the suppression of ERp57 expression by siRNA resulted in an increase in the level of cleaved Caspase-3 protein and a decrease in the number of live cells. Furthermore, cell cycle analyses demonstrated that the suppressed expression of ERp57 increased the sub-G1 population. These data reveal that increased expression of ERp57 may contribute to the protection from beta cell death caused by autophagic failure.

The disulfide isomerase ERp57 is required for fibrin deposition in vivo.

BACKGROUND: ERp57 is required for platelet function; however, whether ERp57 contributes to fibrin generation is unknown. METHODS AND RESULTS: Using an inhibitory anti-ERp57 antibody (mAb1), Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice, and mutants of ERp57, we analyzed the function of ERp57 in laser-induced thrombosis. Fibrin deposition was decreased in Pf4-Cre/ERp57(fl/fl) mice, consistent with a role for platelet ERp57 in fibrin generation. Fibrin deposition was further decreased with infusion of mAb1 and in Tie2-Cre/ERp57(fl/fl) mice, consistent with endothelial cells also contributing to fibrin deposition. Infusion of eptibifatide inhibited platelet and fibrin deposition, confirming a role for platelets in fibrin deposition. Infusion of recombinant ERp57 corrected the defect in fibrin deposition but not platelet accumulation, suggesting a direct effect of ERp57 on coagulation. mAb1 inhibited thrombin generation in vitro, consistent with a requirement for ERp57 in coagulation. Platelet accumulation was decreased to similar extents in Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice and normal mice infused with mAb1. Infusion of completely inactivated ERp57 or ERp57 with a non-functional second active site inhibited fibrin deposition and platelet accumulation, indicating that the isomerase activity of the second active site is required for these processes. CONCLUSION: ERp57 regulates thrombosis via multiple targets.

ERp57 modulates mitochondrial calcium uptake through the MCU.

ERp57 participates in the regulation of calcium homeostasis. Although ERp57 modulates calcium flux across the plasma membrane and the endoplasmic reticulum membrane, its functions on mitochondria are largely unknown. Here, we found that ERp57 can regulate the expression of the mitochondrial calcium uniporter (MCU) and modulate mitochondrial calcium uptake. In ERp57-silenced HeLa cells, MCU was downregulated, and the mitochondrial calcium uptake was inhibited, consistent with the effect of MCU knockdown. When MCU was re-expressed in the ERp57 knockdown cells, mitochondrial calcium uptake was restored. Thus, ERp57 is a potent regulator of mitochondrial calcium homeostasis.

Endoplasmic reticulum stress mediates house dust mite-induced airway epithelial apoptosis and fibrosis.

BACKGROUND: The endoplasmic reticulum (ER) stress response participates in many chronic inflammatory and autoimmune diseases. In the current study, we sought to examine the contribution of ER stress transducers in the pathogenesis of three principal facets of allergic asthma: inflammation, airway fibrosis, and airways hyperresponsiveness. METHODS: House Dust Mite (HDM) was used as an allergen for in vitro and in vivo challenge of primary human and murine airway epithelial cells. ER stress transducers were modulated using specific small interfering RNAs (siRNAs) in vivo. Inflammation, airway remodeling, and hyperresponsiveness were measured by total bronchoalveolar lavage (BAL) cell counts, determination of collagen, and methacholine responsiveness in mice, respectively. RESULTS: Challenge of human bronchiolar and nasal epithelial cells with HDM extract induced the ER stress transducer, activating transcription factor 6 α (ATF6α) as well as protein disulfide isomerase, ERp57, in association with activation of caspase-3. SiRNA-mediated knockdown of ATF6α and ERp57 during HDM administration in mice resulted in a decrease in components of HDM-induced ER stress, disulfide mediated oligomerization of Bak, and activation of caspase-3. Furthermore, siRNA-mediated knockdown of ATF6α and ERp57 led to decreased inflammation, airway hyperresponsiveness and airway fibrosis. CONCLUSION: Collectively, our work indicates that HDM induces ER stress in airway epithelial cells and that ATF6α and ERp57 play a significant role in the development of cardinal features of allergic airways disease. Inhibition of ER stress responses may provide a potential therapeutic avenue in chronic asthma and sub-epithelial fibrosis associated with loss of lung function.

Platelet-derived ERp57 mediates platelet incorporation into a growing thrombus by regulation of the αIIbß3 integrin.

The platelet protein disulfide isomerase called ERp57 mediates platelet aggregation, but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis, we generated a megakaryocyte/platelet-specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times with FeCl3-induced carotid artery injury. Using a mesenteric artery thrombosis model, we found decreased incorporation of ERp57-deficient platelets into a growing thrombus. Platelets lacking ERp57 have defective activation of the αIIbß3 integrin and platelet aggregation. The defect in aggregation was corrected by the addition of exogenous ERp57, implicating surface ERp57 in platelet aggregation. Using mutants of ERp57, we demonstrate the second active site targets a platelet surface substrate to potentiate platelet aggregation. Binding of Alexa 488-labeled ERp57 to thrombin-activated and Mn(2+)-treated platelets lacking ß3 was decreased substantially, suggesting a direct interaction of ERp57 with αIIbß3. Surface expression of ERp57 protein and activity in human platelets increased with platelet activation, with protein expression occurring in a physiologically relevant time frame. In conclusion, platelet-derived ERp57 directly interacts with αIIbß3 during activation of this receptor and is required for incorporation of platelets into a growing thrombus.

Effects of Leishmania major clones showing different levels of virulence on infectivity, differentiation and maturation of human dendritic cells.

Leishmania parasites and dendritic cell interactions (DCs) play an essential role in initiating and directing T cell responses and influence disease evolution. These interactions may vary depending on Leishmania species and strains. To evaluate the correlation between Leishmania major (Lm) virulence and in-vitro human DC response, we compared the ability of high (HV) and low virulent (LV) Lm clones to invade, modulate cytokine production and interfere with differentiation of DCs. Clones derived from HV and LV (HVΔlmpdi and LVΔlmpdi), and deleted for the gene coding for a Lm protein disulphide isomerase (LmPDI), probably involved in parasite natural pathogenicity, were also used. Unlike LV, which fails to invade DCs in half the donors, HV promastigotes were associated with a significant increase of the infected cells percentage and parasite burden. A significant decrease of both parameters was observed in HVΔlmpdi-infected DCs, compared to wild-type cells. Whatever Lm virulence, DC differentiation was accompanied by a significant decrease in CD1a expression. Lm clones decreased interleukin (IL)-12p70 production similarly during lipopolysaccharide (LPS)-induced maturation of DCs. LPS stimulation was associated with a weak increase in tumour necrosis factor (TNF)-α and IL-10 productions in HV-, HVΔlmpdi- and LVΔlmpdi-infected DCs. These results indicate that there is a significant variability in the capacity of Lm clones to infect human DCs which depends upon their virulence, probably involving LmPDI protein. However, independently of their virulence, Lm clones were able to down-regulate CD1a expression during DC differentiation and IL-12p70 production during DC maturation, which may favour their survival.

The estrogen-responsive Agr2 gene regulates mammary epithelial proliferation and facilitates lobuloalveolar development.

Agr2 is a putative protein disulfide isomerase (PDI) initially identified as an estrogen-responsive gene in breast cancer cell lines. While Agr2 expression in breast cancer is positively correlated with estrogen receptor (ER) expression, it is upregulated in both hormone dependent and independent carcinomas. Several in vitro and xenograft studies have implicated Agr2 in different oncogenic features of breast cancer; however, the physiological role of Agr2 in normal mammary gland development remains to be defined. Agr2 expression is developmentally regulated in the mammary gland, with maximum expression during late pregnancy and lactation. Using a mammary gland specific knockout mouse model, we show that Agr2 facilitates normal lobuloalveolar development by regulating mammary epithelial cell proliferation; we found no effects on apoptosis in Agr2(-/-) mammary epithelial cells. Consequently, mammary glands of Agr2(-/-) females exhibit reduced expression of milk proteins, and by two weeks post-partum their pups are smaller in size. Utilizing a conditional mouse model, we show that Agr2 constitutive expression drives precocious lobuloalveolar development and increased milk protein expression in the virgin mammary gland. In vitro studies using knock down and overexpression strategies in estrogen receptor positive and negative mammary epithelial cell lines demonstrate a role for Agr2 in estradiol-induced cell proliferation. In conclusion, the estrogen-responsive Agr2, a candidate breast cancer oncogene, regulates epithelial cell proliferation and lobuloalveolar development in the mammary gland. The pro-proliferative effects of Agr2 may explain its actions in early tumorigenesis.

Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulphide bond formation to an Escherichia coli strain lacking DsbA.

Escherichia coli uses the DsbA/DsbB system for introducing disulphide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulphide bond formation but does not entirely eliminate it. Whether such background disulphide bond forming activity is enzyme-catalysed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulphide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulphide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulphide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulphenic acid form and reacts with substrate proteins to form mixed disulphide adducts. DsbC either prevents the formation of these mixed disulphides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyse disulphide bond formation. Although this PspE/DsbC system is not responsible for the background disulphide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system.

Intestinal cell phosphate uptake and the targeted knockout of the 1,25D3-MARRS receptor/PDIA3/ERp57.

We have crossed ERp57(flx/flx) mice with commercially available mice expressing villin-driven cre-recombinase. Enterocytes isolated from 3- to 4-wk-old littermate (LM) male mice responded to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] with enhanced phosphate uptake relative to corresponding controls within 1 min of addition, whereas in cells from targeted knockout (KO) mice, the response was severely blunted. Unlike chick enterocytes, mouse enterocytes did not respond to phorbol ester with enhanced phosphate uptake. However, forskolin, which does not stimulate phosphate uptake in chick intestinal cells, did so in enterocytes isolated from either young male LM or KO mice. Intestinal cells isolated from young female LM mice also responded to 1,25(OH)2D3 with enhanced phosphate uptake within 5 min of hormone addition, whereas cells from KO mice did not. Forskolin also stimulated phosphate uptake in enterocytes from young female KO or LM mice. As with intestinal cells from adult male chickens or rats, cells from adult (8 wk) male LM mice lost the ability to respond to 1,25(OH)2D3) with enhanced phosphate uptake. In contrast, intestinal cells from adult female LM mice did respond with enhanced phosphate uptake within 1 min of steroid hormone addition relative to corresponding controls, and the magnitude of the effect was greater than that observed in enterocytes of young females. Cells isolated from young or adult male or female LM mice failed to respond to 1,25(OH)2D3 with enhanced protein kinase C activity. Finally, we have previously reported that mouse enterocytes have cell surface vitamin D receptor; however preincubation of such cells with anti-vitamin D receptor antibodies demonstrated that the classical receptor is not involved in the rapid 1,25(OH)2D3-stimulated uptake of phosphate.

Action of protein disulfide isomerase on proinsulin exit from endoplasmic reticulum of pancreatic ß-cells.

For insulin synthesis, the proinsulin precursor is translated at the endoplasmic reticulum (ER), folds to include its three native disulfide bonds, and is exported to secretory granules for processing and secretion. Protein disulfide isomerase (PDI) has long been assumed to assist proinsulin in this process. Herein we have examined the effect of PDI knockdown (PDI-KD) in ß-cells. The data establish that upon PDI-KD, oxidation of proinsulin to form native disulfide bonds is unimpaired and in fact enhanced. This is accompanied by improved proinsulin exit from the ER and increased total insulin secretion, with no evidence of ER stress. We provide evidence for direct physical interaction between PDI and proinsulin in the ER of pancreatic ß-cells, in a manner requiring the catalytic activity of PDI. In ß-cells after PDI-KD, enhanced export is selective for proinsulin over other secretory proteins, but the same effect is observed for recombinant proinsulin trafficking upon PDI-KD in heterologous cells. We hypothesize that PDI exhibits unfoldase activity for proinsulin, increasing retention of proinsulin within the ER of pancreatic ß-cells.

Modulation of STIM1 and capacitative Ca2+ entry by the endoplasmic reticulum luminal oxidoreductase ERp57.

STIM1 is an endoplasmic reticulum (ER) membrane Ca(2+) sensor responsible for activation of store-operated Ca(2+) influx. We discovered that STIM1 oligomerization and store-operated Ca(2+) entry (SOC) are modulated by the ER oxidoreductase ERp57. ERp57 interacts with the ER luminal domain of STIM1, with this interaction involving two conserved cysteine residues, C(49) and C(56). SOC is accelerated in the absence of ERp57 and inhibited in C(49) and C(56) mutants of STIM1. We show that ERp57, by ER luminal interaction with STIM1, has a modulatory role in capacitative Ca(2+) entry. This is the first demonstration of a protein involved in ER intraluminal regulation of STIM1.

Specialization of endoplasmic reticulum chaperones for the folding and function of myelin glycoproteins P0 and PMP22.

Peripheral myelin protein 22 (PMP22) and protein 0 (P0) are major peripheral myelin glycoproteins, and mutations in these two proteins are associated with hereditary demyelinating peripheral neuropathies. Calnexin, calreticulin, and ERp57 are critical components of protein quality control responsible for proper folding of newly synthesized glycoproteins. Here, using confocal microscopy, we show that cell surface targeting of P0 and PMP22 is not affected in the absence of the endoplasmic reticulum chaperones. However, the folding and function (adhesiveness) of PMP22 and P0, measured using the adhesion assay, are affected significantly in the absence of calnexin but not in the absence of calreticulin. Deficiency in oxidoreductase ERp57 results in impaired folding and function of P0, a disulfide bond-containing protein, but does not have any effect on folding or function of PMP22 (a protein that does not contain a disulfide bond). We concluded that calnexin and ERp57, but not calreticulin, play an important role in the biology of peripheral myelin proteins PMP22 and P0, and, consequently, these chaperones may contribute to the pathogenesis of peripheral neuropathies and the diversity of these neurological disorders.