Endoplasmic Reticulum Chaperone BiP Research Articles

Endoplasmic reticulum stress response and transcriptional reprogramming.

Endoplasmic reticulum stress response and transcriptional reprogramming.

Sil1, a nucleotide exchange factor for BiP, is not required for antibody assembly or secretion.

Sil1 is a nucleotide exchange factor for the endoplasmic reticulum chaperone BiP, and mutations in this gene lead to Marinesco-Sjögren syndrome (MSS), a debilitating autosomal recessive disease characterized by multisystem defects. A mouse model for MSS was previously produced by disrupting Sil1 using gene-trap methodology. The resulting Sil1Gt mouse phenocopies several pathologies associated with MSS, although its ability to assemble and secrete antibodies, the best-characterized substrate of BiP, has not been investigated. In vivo antigen-specific immunizations and ex vivo LPS stimulation of splenic B cells revealed that the Sil1Gt mouse was indistinguishable from wild-type age-matched controls in terms of both the kinetics and magnitude of antigen-specific antibody responses. There was no significant accumulation of BiP-associated Ig assembly intermediates or evidence that another molecular chaperone system was used for antibody production in the LPS-stimulated splenic B cells from Sil1Gt mice. ER chaperones were expressed at the same level in Sil1WT and Sil1Gt mice, indicating that there was no evident compensation for the disruption of Sil1. Finally, these results were confirmed and extended in three human EBV-transformed lymphoblastoid cell lines from individuals with MSS, leading us to conclude that the BiP cofactor Sil1 is dispensable for antibody production.

Unfolded Protein Response-regulated Drosophila Fic (dFic) Protein Reversibly AMPylates BiP Chaperone during Endoplasmic Reticulum Homeostasis

Drosophila Fic (dFic) mediates AMPylation, a covalent attachment of adenosine monophosphate (AMP) from ATP to hydroxyl side chains of protein substrates. Here, we identified the endoplasmic reticulum (ER) chaperone BiP as a substrate for dFic and mapped the modification site to Thr-366 within the ATPase domain. The level of AMPylated BiP in Drosophila S2 cells is high during homeostasis, whereas the level of AMPylated BiP decreases upon the accumulation of misfolded proteins in the ER. Both dFic and BiP are transcriptionally activated upon ER stress, supporting the role of dFic in the unfolded protein response pathway. The inactive conformation of BiP is the preferred substrate for dFic, thus endorsing a model whereby AMPylation regulates the function of BiP as a chaperone, allowing acute activation of BiP by deAMPylation during an ER stress response. These findings not only present the first substrate of eukaryotic AMPylator but also provide a target for regulating the unfolded protein response, an emerging avenue for cancer therapy.

Preconditioning with Endoplasmic Reticulum Stress Ameliorates Endothelial Cell Inflammation

Endoplasmic Reticulum (ER) stress, caused by disturbance in ER homeostasis, has been implicated in several pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. Our present study aims at understanding the role of ER stress in endothelial cell (EC) inflammation, a critical event in the pathogenesis of acute lung injury (ALI). We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation. The two approaches adopted to abrogate BiP function induced ATF4 protein expression and the phosphorylation of eIF2α, both markers of ER stress, which in turn resulted in blunting the activation of NF-κB, and restoring endothelial barrier integrity. Pretreatment of HPAEC with BiP siRNA inhibited thrombin-induced IκBα degradation and its resulting downstream signaling pathway involving NF-κB nuclear translocation, DNA binding, phosphorylation at serine536, transcriptional activation and subsequent expression of adhesion molecules. However, TNFα-mediated NF-κB signaling was unaffected upon BiP knockdown. In an alternative approach, SubAB-mediated inactivation of NF-κB was independent of IκBα degradation. Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines. In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability. Together our studies indicate that BiP plays a central role in EC inflammation and injury via its action on NF-κB activation and regulation of vascular permeability.

Abstract 3985: Differential activation of unfolded protein response in metastatic vs. non-metastatic osteosarcoma cells following hypoxia and chemotherapeutic stress

Abstract Osteosarcoma (OS) is the most common primary tumor of bone and the second highest cause of cancer-related death in the pediatric age group. Despite significant advances in diagnosis and treatment for this cancer, the overall survival has remained static over the past 20 years. This is due to the fact that little is known about the basic science mechanisms of the pathology of human osteosarcoma as well as the mechanisms of inherent drug resistance of OS. In particular lesser still is known regarding the role of tumor microenvironment that govern growth and progression of osteosarcoma in vivo. Recent clinical reports have demonstrated a negative correlation between tumor hypoxia and overall survival in osteosarcoma patients. In addition to the upregulation of HIFs, it has been shown that hypoxia can trigger an adaptive response such as the unfolded protein response (UPR) that allows tumor cells to avoid therapy-induced death. Using in vitro experimental models of both the non-metastatic (SAOS-2) and metastatic (143-b) osteosarcoma cell lines, we now show that hypoxic signaling in these cells differentially activates a pro-survival mechanism via the up-regulation of the endoplasmic reticulum (ER) chaperone BiP and increased activation of the ER stress-activated eukaryotic translation initiator factor 2alpha kinase RNA-dependent protein kinase-like ER kinase (PERK) and ATF-6, markers of UPR allowing the cells to resist drug toxicity. Furthermore RNA interference and dominant-negative expression studies revealed that both ATF-6 and PERK signaling promote survival and drug resistance of OS cells, Our findings shed light on the unknown mechanisms underlying chemotherapeutic drug resistance in osteosarcoma patients. We propose that hypoxic induction of UPR protects OS cells from stress insults, such as chemotherapy. Our research may lead to novel therapies that seek out and destroy the chemoresistant OS cells within the hypoxic core of tumors, thereby preventing survival and metastasis, and ultimately improving the chances of survival amongst OS patients. Citation Format: Janine Faraj, Pooja Hingorani, Aparna R. Sertil. Differential activation of unfolded protein response in metastatic vs. non-metastatic osteosarcoma cells following hypoxia and chemotherapeutic stress. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3985. doi:10.1158/1538-7445.AM2014-3985

Fenugreek seed extract and its phytocompounds- trigonelline and diosgenin arbitrate their hepatoprotective effects through attenuation of endoplasmic reticulum stress and oxidative stress in type 2 diabetic rats

Fenugreek seed and some of its phytoconstituents are known to possess hypoglycemic and hypolipidemic properties but their mode of action in insulin target tissues remains to be elucidated. As hepatic insulin resistance contributes to the development of type 2 diabetes mellitus (T2DM), the protective effects of fenugreek seed extract (FSE) and two of its phytoconstituents-trigonelline and diosgenin were evaluated in T2DM rats with special focus on changes in endoplasmic reticulum (ER) stress and oxidative stress markers in liver. Trigonelline and diosgenin content in FSE were quantified by HPTLC. T2DM was induced in male Sprague–Dawley rats by feeding high-fat diet and administration of low-dose streptozotocin. T2DM rats exhibited hyperglycemia and significantly reduced serum C-peptide levels. Liver damage in T2DM rats was marked by significantly elevated levels of liver marker enzymes in serum, liver triglycerides (TGs), and reduced liver glycogen and was further confirmed by histological analysis. T2DM rats also displayed twofold–threefold increase in the levels of ER chaperones Bip, protein disulfide isomerase (PDI) as well as ER stress associated proapoptotic markers CHOP, Caspase12 and Caspase3 in liver along with elevated lipid peroxidation (LPO) and reduced antioxidant levels. Promisingly, FSE, trigonelline, and diosgenin were found to exhibit protective effects individually and brought about significant reduction in serum enzymes, liver TGs, LPO, expression of liver ER stress marker proteins, and significant increase in liver glycogen content and antioxidants. Histological analysis also supported their protective effects suggesting that FSE and its phytoconstituents alleviate T2DM associated liver damage by normalizing ER stress and oxidative stress.

Differential Effects of Escherichia coli Subtilase Cytotoxin and Shiga Toxin 2 on Chemokine and Proinflammatory Cytokine Expression in Human Macrophage, Colonic Epithelial, and Brain Microvascular Endothelial Cell Lines

Subtilase cytotoxin (SubAB) is the prototype of a recently emerged family of AB5 cytotoxins produced by Shiga-toxigenic Escherichia coli (STEC). Its mechanism of action involves highly specific A-subunit-mediated proteolytic cleavage of the essential endoplasmic reticulum (ER) chaperone BiP. Our previous in vivo studies showed that intraperitoneal injection of purified SubAB causes a major redistribution of leukocytes and elevated leukocyte apoptosis in mice, as well as profound splenic atrophy. In the current study, we investigated selected chemokine and proinflammatory cytokine responses to treatment with SubAB, a nontoxic derivative (SubAA272B), or Shiga toxin 2 (Stx2) in human macrophage (U937), brain microvascular endothelial (HBMEC), and colonic epithelial (HCT-8) cell lines, at the levels of secreted protein, cell-associated protein, and gene expression. Stx2 treatment upregulated expression of chemokines and cytokines at both the protein and mRNA levels. In contrast, SubAB induced significant decreases in secreted interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in all three tested cell lines and a significant decrease in secreted IL-6 in HBMECs. The downregulation of secreted chemokines or cytokines was not observed in SubAA272B-treated cells, indicating a requirement for BiP cleavage. The downregulation of secreted chemokines and cytokines by SubAB was not reflected at the mRNA and cell-associated protein levels, suggesting a SubAB-induced export defect.

Structural Basis of Subtilase Cytotoxin SubAB Assembly

Pathogenic strains of Escherichia coli produce a number of toxins that belong to the AB5 toxin family, which comprise a catalytic A-subunit that induces cellular dysfunction and a B-pentamer that recognizes host glycans. Although the molecular actions of many of the individual subunits of AB5 toxins are well understood, how they self-associate and the effect of this association on cytotoxicity are poorly understood. Here we have solved the structure of the holo-SubAB toxin that, in contrast to other AB5 toxins whose molecular targets are located in the cytosol, cleaves the endoplasmic reticulum chaperone BiP. SubA interacts with SubB in a similar manner to other AB5 toxins via the A2 helix and a conserved disulfide bond that joins the A1 domain with the A2 helix. The structure revealed that the active site of SubA is not occluded by the B-pentamer, and the B-pentamer does not enhance or inhibit the activity of SubA. Structure-based sequence comparisons with other AB5 toxin family members, combined with extensive mutagenesis studies on SubB, show how the hydrophobic patch on top of the B-pentamer plays a dominant role in binding the A-subunit. The structure of SubAB and the accompanying functional characterization of various mutants of SubAB provide a framework for understanding the important role of the B-pentamer in the assembly and the intracellular trafficking of this AB5 toxin.

HLA–B27 Alters the Response to Tumor Necrosis Factor α and Promotes Osteoclastogenesis in Bone Marrow Monocytes From HLA–B27–Transgenic Rats

To determine whether HLA-B27 expression alters the response of bone marrow monocytes from HLA-B27/human β2 -microglobulin-transgenic (B27-Tg) rats to tumor necrosis factor α (TNFα) and, if so, whether this affects the cells involved in bone homeostasis. Bone marrow monocytes were treated with RANKL or with TNFα to promote osteoclast formation. Osteoclasts were quantified by counting. Gene expression was measured using quantitative polymerase chain reaction analysis, and protein was detected by enzyme-linked immunosorbent assay, immunoblotting, or immunofluorescence. Effects of endogenously produced cytokines on osteoclast formation were determined with neutralizing antibodies. TNFα treatment enhanced osteoclast formation 2.5-fold in HLA-B27-expressing cells as compared to wild-type or to HLA-B7/human β2 -microglobulin-expressing monocytes. TNFα induced ∼4-fold up-regulation of HLA-B27, which was associated with the accumulation of misfolded heavy chains, binding of the endoplasmic reticulum (ER) chaperone BiP, and activation of an ER stress response, which was not seen with HLA-B7. No differences were seen with RANKL-induced osteoclastogenesis. Enhanced interleukin-1α (IL-1α) production from ER-stressed bone marrow monocytes from B27-Tg rats was found to be necessary and sufficient for enhanced osteoclast formation. However, bone marrow monocytes from B27-Tg rats also produced more interferon-β (IFNβ), which attenuated the effect of IL-1α on osteoclast formation. HLA-B27-induced ER stress alters the response of bone marrow monocytes from B27-Tg rats to TNFα, which is associated with enhanced production of IL-1α and IFNβ, cytokines that exhibit opposing effects on osteoclast formation. The altered response of cells expressing HLA-B27 to proinflammatory cytokines suggests that this class I major histocompatibility complex allele may contribute to the pathogenesis of spondyloarthritis and its unique phenotype through downstream effects involving alterations in bone homeostasis.

BiP Negatively Affects Ricin Transport

The AB plant toxin ricin binds both glycoproteins and glycolipids at the cell surface via its B subunit. After binding, ricin is endocytosed and then transported retrogradely through the Golgi to the endoplasmic reticulum (ER). In the ER, the A subunit is retrotranslocated to the cytosol in a chaperone-dependent process, which is not fully explored. Recently two separate siRNA screens have demonstrated that ER chaperones have implications for ricin toxicity. ER associated degradation (ERAD) involves translocation of misfolded proteins from ER to cytosol and it is conceivable that protein toxins exploit this pathway. The ER chaperone BiP is an important ER regulator and has been implicated in toxicity mediated by cholera and Shiga toxin. In this study, we have investigated the role of BiP in ricin translocation to the cytosol. We first show that overexpression of BiP inhibited ricin translocation and protected cells against the toxin. Furthermore, shRNA-mediated depletion of BiP enhanced toxin translocation resulting in increased cytotoxicity. BiP-dependent inhibition of ricin toxicity was independent of ER stress. Our findings suggest that in contrast to what was shown with the Shiga toxin, the presence of BiP does not facilitate, but rather inhibits the entry of ricin into the cytosol.

ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish

ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra.

Sensing of Bacterial Type IV Secretion via the Unfolded Protein Response

ABSTRACTHost cytokine responses to Brucella abortus infection are elicited predominantly by the deployment of a type IV secretion system (T4SS). However, the mechanism by which the T4SS elicits inflammation remains unknown. Here we show that translocation of the T4SS substrate VceC into host cells induces proinflammatory responses. Ectopically expressed VceC interacted with the endoplasmic reticulum (ER) chaperone BiP/Grp78 and localized to the ER of HeLa cells. ER localization of VceC required a transmembrane domain in its N terminus. Notably, the expression of VceC resulted in reorganization of ER structures. In macrophages, VceC was required for B. abortus-induced inflammation by induction of the unfolded protein response by a process requiring inositol-requiring transmembrane kinase/endonuclease 1. Altogether, these findings suggest that translocation of the T4SS effector VceC induces ER stress, which results in the induction of proinflammatory host cell responses during B. abortus infection.

Unfolded Protein Responses With or Without Unfolded Proteins?

The endoplasmic reticulum (ER) is the site of secretory protein biogenesis. The ER quality control (QC) machinery, including chaperones, ensures the correct folding of secretory proteins. Mutant proteins and environmental stresses can overwhelm the available QC machinery. To prevent and resolve accumulation of misfolded secretory proteins in the ER, cells have evolved integral membrane sensors that orchestrate the Unfolded Protein Response (UPR). The sensors, Ire1p in yeast and IRE1, ATF6, and PERK in metazoans, bind the luminal ER chaperone BiP during homeostasis. As unfolded secretory proteins accumulate in the ER lumen, BiP releases, and the sensors activate. The mechanisms of activation and attenuation of the UPR sensors have exhibited unexpected complexity. A growing body of data supports a model in which Ire1p, and potentially IRE1, directly bind unfolded proteins as part of the activation process. However, evidence for an unfolded protein-independent mechanism has recently emerged, suggesting that UPR can be activated by multiple modes. Importantly, dysregulation of the UPR has been linked to human diseases including Type II diabetes, heart disease, and cancer. The existence of alternative regulatory pathways for UPR sensors raises the exciting possibility for the development of new classes of therapeutics for these medically important proteins.

Dietary curcumin counteracts extracellular transthyretin deposition: Insights on the mechanism of amyloid inhibition

The transthyretin amyloidoses (ATTR) are devastating diseases characterized by progressive neuropathy and/or cardiomyopathy for which novel therapeutic strategies are needed. We have recently shown that curcumin (diferuloylmethane), the major bioactive polyphenol of turmeric, strongly suppresses TTR fibril formation in vitro, either by stabilization of TTR tetramer or by generating nonfibrillar small intermediates that are innocuous to cultured neuronal cells.In the present study, we aim to assess the effect of curcumin on TTR amyloidogenesis in vivo, using a well characterized mouse model for familial amyloidotic polyneuropathy (FAP). Mice were given 2% (w/w) dietary curcumin or control diet for a six week period. Curcumin supplementation resulted in micromolar steady-state levels in plasma as determined by LC/MS/MS. We show that curcumin binds selectively to the TTR thyroxine-binding sites of the tetramer over all the other plasma proteins.The effect on plasma TTR stability was determined by isoelectric focusing (IEF) and curcumin was found to significantly increase TTR tetramer resistance to dissociation. Most importantly, immunohistochemistry (IHC) analysis of mice tissues demonstrated that curcumin reduced TTR load in as much as 70% and lowered cytotoxicity associated with TTR aggregation by decreasing activation of death receptor Fas/CD95, endoplasmic reticulum (ER) chaperone BiP and 3-nitrotyrosine in tissues. Taken together, our results highlight the potential use of curcumin as a lead molecule for the prevention and treatment of TTR amyloidosis.

The ADP ribosylation switch in BiP

The endoplasmic reticulum (ER) chaperone BiP binds unfolded proteins to prevent their aggregation and is upregulated following activation of the ER unfolded protein response. BiP ADP ribosylation has been suggested to contribute to the fast adaptation of the ER to the changing levels of unfolded proteins. Consistent with this, Chambers et al. report that BiP ADP ribosylation that is observed in the pancreas of fasted mice (low protein synthesis) is reversed following feeding (high protein synthesis). Moreover, they identify the conserved Arg470 and Arg492 residues as sites for ADP ribosylation in BiP. Interestingly, this modification alters the electrostatic properties of BiP, and mutations that mimic the charge changes induced by ADP ribosylation destabilize BiP–substrate binding. Finally, kinetic studies suggested that BiP ADP ribosylation, which correlates with low levels of newly synthesized proteins, allows protein folding, whereas BiP deribosylation during increased protein synthesis prevents aggregation of unfolded proteins.

Evidence of Endoplasmic Reticulum Stress Mediating Cell Death in Transplanted Human Islets

A key limitation to the success of islet transplantation is islet cell exhaustion and cell death during islet isolation and following transplantation. Endoplasmic reticulum (ER) stress has been identified as an important mechanism in the development of β-cell dysfunction, cell death, and diabetes. This study investigated the role of ER stress in islet loss during human islet isolation and posttransplantation in a diabetic athymic mouse model. Islets were isolated from human organ donor pancreata using intraductal enzymatic dissociation and continuous density gradient purification. ER stress mediators were assessed by Western blot and by RT-PCR. Caspase-3 activity was quantified by a bioluminescent peptide cleavage assay. Normal and streptozotocin-treated diabetic nude mice were transplanted with 2,000 IEQ of human islets under the kidney capsule and the grafts were harvested 3 or 28 days after transplantation. The grafts were analyzed for the presence for ER stress signals by immunohistochemistry. Isolated islets demonstrated higher levels of ER chaperone Bip, ER stress mediators eIF2α, ATF, spliced XBP-1, and CHOP, and also ER stress-associated apoptotic signals like JNK, caspase-3/7, and cleaved PARP. Donor pancreatic tissue did not show expression of any of these ER stress mediators. After transplantation, low expression of only protective ER stress mediators was evident in the grafts from the normal recipients. In contrast, both protective and apoptotic ER stress mediators were highly expressed in the grafts of hyperglycemic mice. ER stress mediators were induced during islet isolation and may contribute to islet apoptosis and cell death. Islet isolation activates ER stress and apoptotic pathways in isolated islets. Hyperglycemia may prolong this ER stress signal in engrafted islets, converting the protective aspects of the ER stress response to a proapoptotic response and thus contribute to deterioration of β-cell function and survival.

Murine Cytomegalovirus Targets Transcription Factor ATF4 To Exploit the Unfolded-Protein Response

The unfolded-protein response (UPR), activated by sensor molecules PERK, ATF6, and IRE1 to resolve endoplasmic reticulum (ER) stress, has emerged as a key target for host cells and viruses to control the infection outcomes. The UPR regulates ER protein folding, controls cell fate upon ER stress, and plays an important role in innate immunity. We and others have shown that human cytomegalovirus (HCMV) modulates the UPR. We show here that murine CMV (MCMV), the widely used CMV model for small animal infection, regulated the UPR in a manner similar to that of HCMV. This modulatory ability was triggered by virion entry and enhanced by viral immediate-early and early gene expression. Thus, while vulnerable at early times, MCMV became resistant to exogenous ER stress at late times of infection. MCMV activated the PERK-ATF4 pathway but only induced a subset of representative ATF4 targets at levels somewhat lower than those by the ER stress inducer tunicamycin. Moreover, MCMV induced ER chaperone Bip but actively blocked IRE1-mediated Xbp1(s) protein accumulation. ATF4 depletion severely attenuated viral growth at a low multiplicity of infection by modestly reducing viral DNA synthesis and more pronouncedly inhibiting late gene transcription. Collectively, we show that the UPR is a conserved target of CMVs and identify ATF4, a key UPR component, as a factor critical for MCMV infection. This work sets the stage for using the MCMV model to explore the role of this stress response in CMV biology, particularly during infection of the host, which is difficult to study in HCMV.

The ER chaperones BiP and Jem1/Scj1 are genetic modifiers of TorsinA, the AAA+ ATPase associated with the neurological disease primary torsion dystonia

Dystonia is a disease characterized by involuntary and sustained muscle contractions that lead to paralysis and abnormal postures. The most severe type of dystonia, early‐onset Torsion dystonia, is associated with a deletion of a glutamate (ΔE) near the C‐terminus in TorsinA. TorsinA is an endoplasmic reticulum (ER) AAA+ ATPase of ill‐defined function, and the reasons that the ΔE variant is associated with the disease are unclear. Further, the penetrance of the disease is highly variable, suggesting that genetic modifiers affect disease severity. In order to identify modifiers of TorsinA stability and folding, we developed a TorsinA expression system in Saccharomyces cerevisiae. Similar to TorsinA in mammalian cells, TorsinA and TorsinAΔE in yeast associate with the luminal side of the ER membrane and are glycosylated. We found that the stability and glycosylation of TorsinA and TorsinAΔE depend on the yeast ER Hsp70 chaperone BiP and Hsp40 co‐chaperones Jem1 and Scj1. Mutations in TorsinA that prevent glycosylation, ATP binding, or ATP hydrolysis, or that eliminate the hydrophobic domain led to BiP‐dependent degradation, and to differences in protease sensitivity and membrane association. Therefore, we have identified conserved ER chaperones that are critical for TorsinA conformation and stability. We are testing these findings in mammalian systems. Supported by the Dystonia Medical Research Foundation.

The sigma-1 receptor protects against cellular oxidative stress and activates antioxidant response elements

Sigma-1 receptors are associated with Alzheimer's disease, major depressive disorders, and schizophrenia. These receptors show progrowth/antiapoptotic properties via their chaperoning functions to counteract ER (endoplasmic reticulum) stress, to block neurodegeneration, and to regulate neuritogenesis. The sigma-1 receptor knock out mouse offered an opportunity to assess possible mechanisms by which the sigma-1 receptor modulates cellular oxidative stress. Nuclear magnetic resonance (NMR) metabolomic screening of the WT (wild type) and sigma-1 KO (knockout) livers was performed to investigate major changes in metabolites that are linked to oxidative stress. Significant changes in protein levels were also identified by two-dimensional (2D) gel electrophoresis and mass spectrometry. Increased levels of the antioxidant protein peroxiredoxin 6 (Prdx6), and the ER chaperone BiP (GRP78) compared to WT littermates were detected. Oxidative stress was measured in WT and sigma-1 KO mouse liver homogenates, in primary hepatocytes and in lung homogenates. Furthermore, sigma-1 receptor mediated activation of the antioxidant response element (ARE) to upregulate NAD(P)H quinone oxidoreductase 1 (NQO1) and superoxide dismutase 1 (SOD1) mRNA expression in COS cells was shown by RT PCR. These novel functions of the sigma-1 receptor were sensitive to well-known sigma ligands via their antagonist/agonist properties.

Attenuation of endoplasmic reticulum stress using the chemical chaperone 4-phenylbutyric acid prevents cardiac fibrosis induced by isoproterenol

Increasing evidence indicates that endoplasmic reticulum (ER) stress is involved in various diseases. In the human heart, ischemia/reperfusion has been correlated to ER stress, and several markers of the unfolded protein response (UPR) participate during cardiac remodeling and fibrosis. Here, we used isoproterenol (ISO) injection as a model for in vivo cardiac fibrosis. ISO induced significant cardiomyocyte loss and collagen deposition in the damaged areas of the endocardium. These responses were accompanied by an increase in the protein levels of the luminal ER chaperones BIP and PDI, as well as an increase in the UPR effector CHOP. The use of the chemical chaperone 4-phenylbutyric acid (4-PBA) prevented the activation of the UPR, the increase in luminal chaperones and also, leads to decreased collagen deposition, cardiomyocyte loss into the damaged zones. Our results suggest that cardiac damage and fibrosis induced in vivo by the beta-adrenergic agonist ISO are tightly related to ER stress signaling pathways, and that increasing the ER luminal folding capacity with exogenously administrated 4-PBA is a powerful strategy for preventing the development of cardiac fibrosis. Additionally, 4-PBA might prevent the loss of cardiomyocytes. Our data suggests that the attenuation of ER stress pathways with pharmacological compounds such as the chemical chaperone 4-PBA can prevent the development of cardiac fibrosis and adverse remodeling.