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Unfolded Protein Response (unfolded + protein_response)
Selected AbstractsThe unfolded protein response is required to maintain the integrity of the endoplasmic reticulum, prevent oxidative stress and preserve differentiation in , -cellsDIABETES OBESITY & METABOLISM, Issue 2010R. J. Kaufman Diabetes is an epidemic of worldwide proportions caused by , -cell failure. Nutrient fluctuations and insulin resistance drive , -cells to synthesize insulin beyond their capacity for protein folding and secretion and thereby activate the unfolded protein response (UPR), an adaptive signalling pathway to promote cell survival upon accumulation of unfolded protein in the endoplasmic reticulum (ER). Protein kinase-like endoplasmic reticulum kinase (PERK) signals one component of the UPR through phosphorylation of eukaryotic initiation factor 2 on the , -subunit (eIF2,) to attenuate protein synthesis, thereby reducing the biosynthetic burden. , -Cells uniquely require PERK-mediated phosphorylation of eIF2, to preserve cell function. Unabated protein synthesis in , -cells is sufficient to initiate a cascade of events, including oxidative stress, that are characteristic of , -cell failure observed in type 2 diabetes. In contrast to acute adaptive UPR activation, chronic activation increases expression of the proapoptotic transcription factor CAAT/enhancer-binding protein homologous protein (CHOP). Chop deletion in insulin-resistant mice profoundly increases , -cell mass and prevents , -cell failure to forestall the progression of diabetes. The findings suggest an unprecedented link by which protein synthesis and/or misfolding in the ER causes oxidative stress and should encourage the development of novel strategies to treat diabetes. [source] Inositol-requiring 1/X-box-binding protein 1 is a regulatory hub that links endoplasmic reticulum homeostasis with innate immunity and metabolismEMBO MOLECULAR MEDICINE, Issue 6 2010Randal J. Kaufman Abstract Inositol-requiring 1 (IRE1)/X-box-binding protein 1 (XBP1)-mediated signalling represents the most conserved branch of the unfolded protein response. A series of recent studies reveal novel and potentially ancient roles for this pathway in the coordination of metabolic and immune responses. [source] Endoplasmic reticulum stress and the unfolded protein response are linked to synergistic IFN-, induction via X-box binding protein 1EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2008Judith A. Smith Dr. Abstract Type,I IFN are strongly induced upon engagement of certain pattern recognition receptors by microbial products, and play key roles in regulating innate and adaptive immunity. It has become apparent that the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), in addition to restoring ER homeostasis, also influences the expression of certain inflammatory cytokines. However, the extent to which UPR signaling regulates type,I IFN remains unclear. Here we show that cells undergoing a UPR respond to TLR4 and TLR3 ligands, and intracellular dsRNA, with log-fold greater IFN-, induction. This synergy is not dependent on autocrine type,I IFN signaling, but unexpectedly requires the UPR transcription factor X-box binding protein,1 (XBP-1). Synergistic IFN-, induction also occurs in HLA-B27/human ,2m-transgenic rat macrophages exhibiting a UPR as a consequence of HLA-B27 up-regulation, where it correlates with activation of XBP-1 splicing. Together these findings indicate that the cellular response to endogenous ,danger' that disrupts ER homeostasis is coupled to IFN-, induction by XBP-1, which has implications for the immune response and the pathogenesis of diseases involving the UPR. [source] Protein folding and disulfide bond formation in the eukaryotic cellFEBS JOURNAL, Issue 23 2009Denmark), Disulfide Bond Formation 2009 (Elsinore, Meeting report based on the presentations at the European Network Meeting on Protein Folding The endoplasmic reticulum (ER) plays a critical role as a compartment for protein folding in eukaryotic cells. Defects in protein folding contribute to a growing list of diseases, and advances in our understanding of the molecular details of protein folding are helping to provide more efficient ways of producing recombinant proteins for industrial and medicinal use. Moreover, research performed in recent years has shown the importance of the ER as a signalling compartment that contributes to overall cellular homeostasis. Hamlet's castle provided a stunning backdrop for the latest European network meeting to discuss this subject matter in Elsinore, Denmark, from 3 to 5 June 2009. Organized by researchers at the Department of Biology, University of Copenhagen, the meeting featured 20 talks by both established names and younger scientists, focusing on topics such as oxidative protein folding and maturation (in particular in the ER, but also in other compartments), cellular redox regulation, ER-associated degradation, and the unfolded protein response. Exciting new advances were presented, and the intimate setting with about 50 participants provided an excellent opportunity to discuss current key questions in the field. [source] Autoregulation of the HAC1 gene is required for sustained activation of the yeast unfolded protein responseGENES TO CELLS, Issue 2 2004Naoki Ogawa Eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by activating a transcriptional induction program termed the unfolded protein response (UPR). The transcription factor Hac1p responsible for the UPR in Saccharomyces cerevisiae is tightly regulated by a post-transcriptional mechanism. HAC1 mRNA must be spliced in response to ER stress to produce Hac1p, which then activates transcription via direct binding to the cis -acting UPR element (UPRE) present in the promoter regions of its target genes. Here, we show that the HAC1 promoter itself responds to ER stress to induce transcription of its downstream gene, similarly to the KAR2 promoter; the KAR2 gene represents a major target of the UPR. Consistent with this observation, the HAC1 promoter contains an UPRE-like sequence, which is necessary and sufficient for the induction and to which Hac1p binds directly. Cells expressing the HAC1 gene from a mutant HAC1 promoter lacking the HAC1 UPRE could not maintain high levels of either unspliced or spliced HAC1 mRNA and became sensitive to ER stress when insulted for hours. Based on these results, we concluded that autoregulation of the HAC1 genes is required for sustained activation of the UPR and sustained resistance to ER stress. [source] From HLA-B27 to spondyloarthritis: a journey through the ERIMMUNOLOGICAL REVIEWS, Issue 1 2010Robert A. Colbert Summary:, Almost four decades of research into the role of human leukocyte antigen-B27 (HLA-B27) in susceptibility to spondyloarthritis has yet to yield a convincing answer. New results from an HLA-B27 transgenic rat model now demonstrate quite convincingly that CD8+ T cells are not required for the inflammatory phenotype. Discoveries that the HLA-B27 heavy chain has a tendency to misfold during the assembly of class I complexes in the endoplasmic reticulum (ER) and to form aberrant disulfide-linked dimers after transport to the cell surface have forced the generation of new ideas about its role in disease pathogenesis. In transgenic rats, HLA-B27 misfolding generates ER stress and leads to activation of the unfolded protein response, which dramatically enhances the production of interleukin-23 (IL-23) in response to pattern recognition receptor agonists. These findings have led to the discovery of striking T-helper 17 cell activation and expansion in this animal model, consistent with results emerging from humans with spondyloarthritis and the discovery of IL23R as an additional susceptibility gene for ankylosing spondylitis. Together, these results suggest a novel link between HLA-B27 and the T-helper 17 axis through the consequences of protein misfolding and open new avenues of investigation as well as identifying new targets for therapeutic intervention in this group of diseases. [source] The X-box binding protein-1 transcription factor is required for plasma cell differentiation and the unfolded protein responseIMMUNOLOGICAL REVIEWS, Issue 1 2003Neal N. Iwakoshi Summary:, X-box binding protein-1 (XBP-1) is a transcription factor essential for plasma cell differentiation. XBP-1 transcripts are found at high levels in plasma cells from rheumatoid synovium and myeloma cell lines. Lymphoid chimeras deficient in XBP-1 have a profound defect in plasma cell differentiation, with few plasma cells in their periphery and severely reduced serum immunoglobulin levels. When introduced into B-lineage cells, XBP-1 initiates plasma cell differentiation. XBP-1 is also the mammalian homologue of the yeast transcription factor Hac1p, an important component of the unfolded protein response (UPR). The UPR allows cells to tolerate conditions of endoplasmic reticulum (ER) stress caused by misfolded proteins. Studies examining the relationship between plasma cell differentiation, XBP-1, and the UPR demonstrate that this novel signaling system is vital for plasma cell differentiation. Signals that induce plasma cell differentiation and the UPR cooperate via XBP-1 to induce terminal B-cell differentiation. Additionally, XBP-1 plays an important role in the regulation of interleukin-6 production, a cytokine essential for plasma cell survival. [source] Nerve growth factor blocks thapsigargin-induced apoptosis at the level of the mitochondrion viaregulation of BimJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6a 2008E. Szegezdi Abstract This study examined how the neurotrophin, nerve growth factor (NGF), protects PC12 cells against endoplasmic reticulum (ER) stress-induced apoptosis. ER stress was induced using thapsigargin (TG) that inhibits the sarcoplasmic/ER Ca2+ -ATPase pump (SERCA) and depletes ER Ca2+ stores. NGF pre-treatment inhibited translocation of Bax to the mitochondria, loss of mitochondrial transmembrane potential, cytochrome c release, activation of caspases (,3, ,7 and ,9) and apoptosis induction by TG. Notably, TG also caused a marked induction of Bimel mRNA and protein, and knockdown of Bim with siRNA protected cells against TG-induced apoptosis. NGF delayed the induction and increased the phosphorylation of Bimel. NGF-mediated protection was dependent on phosphatidylinositol-3 kinase (PI3K) signalling since all above apoptotic events, including expression and phosphorylation status of Bimel protein, could be reverted by the PI3K inhibitor LY294002. In contrast, NGF had no effect on the TG-mediated induction of the unfolded protein response (increased expression of Grp78, GADD34, splicing of XBP1 mRNA) or ER stress-associated pro-apoptotic responses (induction of C/EBP homologous protein [CHOP], induction and processing of caspase-12). These data indicate that NGF-mediated protection against ER stress-induced apoptosis occurs at the level of the mitochondria by regulating induction and activation of Bim and mitochondrial translocation of Bax. [source] Dysfunction of the unfolded protein response increases neurodegeneration in aged rat hippocampus following proteasome inhibitionAGING CELL, Issue 6 2009María Paz Gavilán Summary Dysfunctions of the ubiquitin proteasome system (UPS) have been proposed to be involved in the aetiology and/or progression of several age-related neurodegenerative disorders. However, the mechanisms linking proteasome dysfunction to cell degeneration are poorly understood. We examined in young and aged rat hippocampus the activation of the unfolded protein response (UPR) under cellular stress induced by proteasome inhibition. Lactacystin injection blocked proteasome activity in young and aged animals in a similar extent and increased the amount of ubiquitinated proteins. Young animals activated the three UPR arms, IRE1,, ATF6, and PERK, whereas aged rats failed to induce the IRE1, and ATF6, pathways. In consequence, aged animals did not induce the expression of pro-survival factors (chaperones, Bcl-XL and Bcl-2), displayed a more sustained expression of pro-apoptotic markers (CHOP, Bax, Bak and JKN), an increased caspase-3 processing. At the cellular level, proteasome inhibition induced neuronal damage in young and aged animals as assayed using Fluorojade-B staining. However, degenerating neurons were evident as soon as 24 h postinjection in aged rats, but it was delayed up to 3 days in young animals. Our findings show evidence supporting age-related dysfunctions in the UPR activation as a potential mechanism linking protein accumulation to cell degeneration. An imbalance between pro-survival and pro-apoptotic proteins, because of noncanonical activation of the UPR in aged rats, would increase the susceptibility to cell degeneration. These findings add a new molecular vision that might be relevant in the aetiology of several age-related neurodegenerative disorders. [source] Changes in endoplasmic reticulum stress proteins and aldolase A in cells exposed to dopamineJOURNAL OF NEUROCHEMISTRY, Issue 1 2008April A. Dukes Abstract In Parkinson's disease, oxidative stress is implicated in protein misfolding and aggregation, which may activate the unfolded protein response by the endoplasmic reticulum (ER). Dopamine (DA) can initiate oxidative stress via H2O2 formation by DA metabolism and by oxidation into DA quinone. We have previously shown that DA quinone induces oxidative protein modification, mitochondrial dysfunction in vitro, and dopaminergic cell toxicity in vivo and in vitro. In this study, we used cysteine- and lysine-reactive fluorescent dyes with 2D difference in-gel electrophoresis, mass spectrometry, and peptide mass fingerprint analysis to identify proteins in PC12 cell mitochondrial-enriched fractions that were altered in abundance following DA exposure (150 ,M, 16 h). Quantitative changes in proteins labeled with fluorescent dyes indicated increases in a subset of proteins after DA exposure: calreticulin, ERp29, ERp99, Grp58, Grp78, Grp94 and Orp150 (149,260%), and decreased levels of aldolase A (39,42%). Changes in levels of several proteins detected by 2D difference in-gel electrophoresis were confirmed by western blot. Using this unbiased proteomics approach, our findings demonstrated that in PC12 cells, DA exposure leads to a cellular response indicative of ER stress prior to the onset of cell death, providing a potential link between DA and the unfolded protein response in the pathogenesis of Parkinson's disease. [source] Endoplasmic reticulum dysfunction , a common denominator for cell injury in acute and degenerative diseases of the brain?JOURNAL OF NEUROCHEMISTRY, Issue 4 2001Wulf Paschen Various physiological, biochemical and molecular biological disturbances have been put forward as mediators of neuronal cell injury in acute and chronic pathological states of the brain such as ischemia, epileptic seizures and Alzheimer's or Parkinson's disease. These include over-activation of glutamate receptors, a rise in cytoplasmic calcium activity and mitochondrial dysfunction. The possible involvement of the endoplasmic reticulum (ER) dysfunction in this process has been largely neglected until recently, although the ER plays a central role in important cell functions. Not only is the ER involved in the control of cellular calcium homeostasis, it is also the subcellular compartment in which the folding and processing of membrane and secretory proteins takes place. The fact that blocking of these processes is sufficient to cause cell damage indicates that they are crucial for normal cell functioning. This review presents evidence that ER function is disturbed in many acute and chronic diseases of the brain. The complex processes taken place in this subcellular compartment are however, affected in different ways in various disorders; whereas the ER-associated degradation of misfolded proteins is affected in Parkinson's disease, it is the unfolded protein response which is down-regulated in Alzheimer's disease and the ER calcium homeostasis that is disturbed in ischemia. Studying the consequences of the observed deteriorations of ER function and identifying the mechanisms causing ER dysfunction in these pathological states of the brain will help to elucidate whether neurodegeneration is indeed caused by these disturbances, and will help to fascilitate the search for drugs capable of blocking the pathological process directly at an early stage. [source] Transgenic mouse and cell culture models demonstrate a lack of mechanistic connection between endoplasmic reticulum stress and tau dysfunctionJOURNAL OF NEUROSCIENCE RESEARCH, Issue 9 2010M.L. Spatara Abstract In vivo aggregation of tau protein is a hallmark of many neurodegenerative disorders, including Alzheimer's disease (AD). Recent evidence has also demonstrated activation of the unfolded protein response (UPR), a cellular response to endoplasmic reticulum (ER) stress, in AD, although the role of the UPR in disease pathogenesis is not known. Here, three model systems were used to determine whether a direct mechanistic link could be demonstrated between tau aggregation and the UPR. The first model system used was SH-SY5Y cells, a neuronal cultured cell line that endogenously expresses tau. In this system, the UPR was activated using chemical stressors, tunicamycin and thapsigargin, but no changes in tau expression levels, solubility, or phosphorylation were observed. In the second model system, wild-type 4R tau and P301L tau, a variant with increased aggregation propensity, were heterologously overexpressed in HEK 293 cells. This overexpression did not activate the UPR. The last model system examined here was the PS19 transgenic mouse model. Although PS19 mice, which express the P301S variant of tau, display severe neurodegeneration and formation of tau aggregates, brain tissue samples did not show any activation of the UPR. Taken together, the results from these three model systems suggest that a direct mechanistic link does not exist between tau aggregation and the UPR. © 2010 Wiley-Liss, Inc. [source] TorsinA in PC12 cells: Localization in the endoplasmic reticulum and response to stressJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2003Jeffrey Hewett Abstract Most cases of early-onset torsion dystonia are caused by deletion of GAG in the coding region of the DYT1 gene encoding torsinA. This autosomal dominant neurologic disorder is characterized by abnormal movements, believed to originate from neuronal dysfunction in the basal ganglia of the human brain. The torsins (torsinA and torsinB) are members of the "ATPases associated with a variety of cellular activities" (AAA+) superfamily of proteins that mediate chaperone and other functions involved in conformational modeling of proteins, protection from stress, and targeting of proteins to cellular organelles. In this study, the intracellular localization and levels of endogenous torsin were evaluated in rat pheochromocytoma PC12 cells following differentiation and stress. TorsinA, apparent MW 37 kDa, cofractionates with markers for the microsomal/endoplasmic reticulum (ER) compartment and appears to reside primarily within the ER lumen based on protease resistance. TorsinA immunoreactivity colocalizes with the lumenal ER protein protein disulfide isomerase (PDI) and extends throughout neurites. Levels of torsinA did not increase notably in response to nerve growth factor-induced differentiation. None of the stress conditions tested, including heat shock and the unfolded protein response, affected torsinA, except for oxidative stress, which resulted in an increase in the apparent MW of torsinA and redistribution to protrusions from the cell surface. These findings are consistent with a relatively rapid covalent modification of torsinA in response to oxidative stress causing a change in state. Mutant torsinA may interfere with and/or compromise ER functions, especially in dopaminergic neurons, which have high levels of torsinA and are intrinsically vulnerable to oxidative stress. © 2003 Wiley-Liss, Inc. [source] The flavonoid tangeretin activates the unfolded protein response and synergizes with imatinib in the erythroleukemia cell line K562MOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 6 2010Sofie Lust Abstract We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region-abelson murine leukemia (Bcr-Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G2/M phase and stimulated an accumulation of the cells in the sub-G0 phase. TAN-induced cell death was evidenced by poly(ADP)-ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl-1 and Bcl-xL. Pretreatment with the pancaspase inhibitor Z-VAD-FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)-associated cell death pathways could be involved. We demonstrated that TAN-induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR-like ER kinase. This was accompanied by enhanced levels of glucose-regulated protein of 78,kDa and of spliced X-box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr-Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib. [source] Chemical stress induces the unfolded protein response in olfactory sensory neuronsTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 10 2010Neeraja Sammeta Abstract More than any other neuron, olfactory sensory neurons are exposed to environmental insults. Surprisingly, their only documented response to damaging stress is apoptosis and subsequent replacement by new neurons. However, they expressed unfolded protein response genes, a transcriptionally regulated defense mechanism activated by many types of insults. The unfolded protein response transcripts Xbp1, spliced Xbp1, Chop (Ddit3), and BiP (Hspa5) were decreased when external access of stressors was reduced by blocking a nostril (naris occlusion). These transcripts and Nrf2 (Nfe2l2) were increased by systemic application of tunicamycin or the selective olfactotoxic chemical methimazole. Methimazole's effects overcame naris occlusion, and the unfolded protein response was independent of odor-evoked neuronal activity. Chemical stress is therefore a major and chronic activator of the unfolded protein response in olfactory sensory neurons. Stress-dependent repression of the antiapoptotic gene Bcl2 was absent, however, suggesting a mechanism for disconnecting the UPR from apoptosis and tolerating a chronic unfolded protein response. Environmental stressors also affect both the sustentacular cells that support the neurons and the respiratory epithelia, because naris occlusion decreased expression of the xenobiotic chemical transformation enzyme Cyp2a5 in sustentacular cells, and both naris occlusion and methimazole altered the abundance of the antibacterial lectin Reg3g in respiratory epithelia. J. Comp. Neurol. 518:1825,1836, 2010. © 2009 Wiley-Liss, Inc. [source] Plant endoplasmin supports the protein secretory pathway and has a role in proliferating tissuesTHE PLANT JOURNAL, Issue 5 2006Eva M. Klein Summary Endoplasmin is a molecular chaperone of the heat-shock protein 90 class located in the endoplasmic reticulum and its activity is poorly characterized in plants. We assessed the ability of endoplasmin to alleviate stress via its transient overexpression in tobacco protoplasts treated with tunicamycin, an inhibitor of glycosylation and inducer of the unfolded protein response (UPR). Endoplasmin supported the secretion of a model secretory protein but was less effective than BiP, the endoplasmic reticulum member of the heat-shock protein 70 family. Consistently, immunoprecipitation experiments with in vivo radioactively labelled proteins using an antiserum prepared against Arabidopsis endoplasmin showed that a much smaller number of newly synthesized polypeptides associated with endoplasmin than with BiP. Synthesis of endoplasmin was enhanced by UPR inducers in tobacco seedlings but not protoplasts. As BiP synthesis was induced in both systems, we conclude that the UPR acts differently, at least in part, on the expression of the two chaperones. Endoplasmin was not detectable in extracts of leaves and stems of the Arabidopsis endoplasmin T-DNA insertion mutant shepherd. However, the chaperone is present, albeit at low levels, in shepherd mutant callus, mature roots and tunicamycin-treated seedlings, demonstrating that the mutation is leaky. Reduced endoplasmin in the shepherd mutant has no effect on BiP protein levels in callus or mature roots, leaves and stems, but is compensated by increased BiP in seedlings. This increase occurs in proliferating rather than expanding leaf cells, indicating an important role for endoplasmin in proliferating plant tissues. [source] HLA,B27 misfolding and the unfolded protein response augment interleukin-23 production and are associated with Th17 activation in transgenic ratsARTHRITIS & RHEUMATISM, Issue 9 2009Monica L. DeLay Objective To determine whether HLA,B27 misfolding and the unfolded protein response (UPR) result in cytokine dysregulation and whether this is associated with Th1 and/or Th17 activation in HLA,B27/human ,2 -microglobulin (Hu,2m),transgenic rats, an animal model of spondylarthritis. Methods Cytokine expression in lipopolysaccharide (LPS),stimulated macrophages was analyzed in the presence and absence of a UPR induced by chemical agents or by HLA,B27 up-regulation. Cytokine expression in colon tissue and in cells purified from the lamina propria was determined by real-time reverse transcription,polymerase chain reaction analysis, and differences in Th1 and Th17 CD4+ T cell populations were quantified after intracellular cytokine staining. Results Interleukin-23 (IL-23) was found to be synergistically up-regulated by LPS in macrophages undergoing a UPR induced by pharmacologic agents or by HLA,B27 misfolding. IL-23 was also increased in the colon tissue from B27/Hu,2m-transgenic rats concurrently with the development of intestinal inflammation, and IL-17, a downstream target of IL-23, exhibited robust up-regulation in a similar temporal pattern. IL-23 and IL-17 transcripts were localized to CD11+ antigen-presenting cells and CD4+ T cells, respectively, from the colonic lamina propria. Colitis was associated with a 6-fold expansion of CD4+ IL-17,expressing T cells. Conclusion The IL-23/IL-17 axis is strongly activated in the colon of B27/Hu,2m-transgenic rats with spondylarthritis-like disease. HLA,B27 misfolding and UPR activation in macrophages can result in enhanced induction of the pro-Th17 cytokine IL-23. These results suggest a possible link between HLA,B27 misfolding and immune dysregulation in this animal model, with implications for human disease. [source] HLA,B27 up-regulation causes accumulation of misfolded heavy chains and correlates with the magnitude of the unfolded protein response in transgenic rats: Implications for the pathogenesis of spondylarthritis-like diseaseARTHRITIS & RHEUMATISM, Issue 1 2007Matthew J. Turner Objective HLA,B27 is implicated in the pathogenesis of spondylarthritis (SpA), yet the molecular mechanisms are incompletely defined. HLA,B27 misfolding has been associated with endoplasmic reticulum stress and activation of the unfolded protein response (UPR) in macrophages from HLA,B27/human ,2 -microglobulin,transgenic (B27-transgenic) rats. This study was performed to assess the mechanisms that drive activation of the HLA,B27,induced UPR and to determine whether splenocytes respond in a similar manner. Methods Splenocytes were isolated and bone marrow macrophages were derived from B27-transgenic and wild-type rats. Cells were treated for up to 24 hours with cytokines that induce class I major histocompatibility complex expression. HLA,B27 expression and misfolding were assessed by real-time reverse transcription,polymerase chain reaction, flow cytometry, and immunoblotting. Activation of the UPR was measured by quantifying UPR target gene expression and X-box binding protein 1 messenger RNA (mRNA) splicing. Results HLA,B27 mRNA up-regulation was accompanied by a dramatic increase in the accumulation of misfolded heavy chains and preceded robust activation of the UPR in macrophages. When macrophages were treated with various cytokines, the magnitude of the UPR correlated strongly with the degree of HLA,B27 up-regulation. In contrast, B27-transgenic splenocytes exhibited only low-level differences in the expression of UPR target genes after exposure to interferon-, or concanavalin A, which resulted in minimal HLA,B27 up-regulation. Conclusion These results suggest that HLA,B27,associated activation of the UPR in macrophages is attributable to the accumulation of misfolded heavy chains, and that certain cell types may be more susceptible to the effects of HLA,B27 misfolding. Strategies that eliminate HLA,B27 up-regulation and/or the accumulation of misfolded heavy chains may be useful in evaluating the role of these events in the pathogenesis of SpA. [source] Engineering of Pichia pastoris for improved production of antibody fragmentsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2006Brigitte Gasser Abstract The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins, including antibody fragments. However, limitations became obvious especially when secreting heterodimeric Fab fragments. Up-to-date, antibody fragments have only been expressed under control of the strong inducible alcohol oxidase 1 (AOX1) promoter, which may stress the cells by excessive transcription. Here, we examined the secretion characteristics of single chain and Fab fragments of two different monoclonal anti-HIV1 antibodies (2F5 and 2G12) with both the AOX1 and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Also, the influences of different secretion leaders and strains were evaluated. Interestingly, secretion was only achieved when using the GAP promoter and the Saccharomyces cerevisiae mating factor , (MF, leader), whereas there was no difference between the two P. pastoris strains. During fed batch fermentation of a 2F5 Fab expressing strain, intracellular retention of Fab heavy chains was observed, while both intact Fab and single light chain molecules were only detected in the supernatants. This led to the conclusion that protein folding and heterodimer assembly in the ER are rate limiting steps in Fab secretion. To alleviate this limitation, S. cerevisiae protein disulfide isomerase (PDI) and the unfolded protein response (UPR) transcription factor HAC1 were constitutively overexpressed in P. pastoris. While the overexpression of HAC1 led to a moderate increase of Fab secretion of 1.3-fold, PDI enabled an increase of the Fab level by 1.9-fold. Hence, the formation of interchain disulfide bonds can be seen as a major rate limiting factor to Fab assembly and subsequent secretion. © 2006 Wiley Periodicals, Inc. [source] Preliminary X-ray crystallographic studies of mouse UPR responsive protein P58(IPK) TPR fragmentACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2008Jiahui Tao Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), which can promote protein folding and misfolded protein degradation and attenuate protein translation and protein translocation into the ER. P58(IPK) has been proposed to function as a molecular chaperone to maintain protein-folding homeostasis in the ER under normal and stressed conditions. P58(IPK) contains nine TPR motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the ER, a P58(IPK) TPR fragment without the C-terminal J-domain was crystallized. The crystals diffract to 2.5,Å resolution using a synchrotron X-ray source. The crystals belong to space group P21, with unit-cell parameters a = 83.53, b = 92.75, c = 84.32,Å, , = 90.00, , = 119.36, , = 90.00°. There are two P58(IPK) molecules in the asymmetric unit, which corresponds to a solvent content of approximately 60%. Structure determination by MAD methods is under way. [source] Regulation of XBP-1 signaling during transient and stable recombinant protein production in CHO cellsBIOTECHNOLOGY PROGRESS, Issue 2 2010Sebastian C. Y. Ku Abstract X-box binding protein 1 (XBP-1) is a key regulator of cellular unfolded protein response (UPR). The spliced isoform of XBP-1, XBP-1S, is a transcription activator, which is expressed only when UPR is induced. However, the impact of recombinant protein production on the regulation of XBP-1 signaling in CHO cells is not well understood. In this report, we cloned the Chinese hamster XBP-1 homolog to aid the investigation of the interplay between protein productivity, culture conditions, and endogenous XBP-1 signaling in CHO cells. Interestingly, expression of XBP-1S is detected in the non-producing and unstressed CHO-K1 cells. Transient expression of recombinant erythropoietin reveals a positive correlation between XBP-1 mRNA abundance and protein production level. However, such a correlation is not observed in batch cultivation of stable producing cell lines. The increased XBP-1 splicing is detected in late-phase cultures, suggesting that induction of XBP-1S may be a result of nutrient limitations or other environmental stresses rather than that of increased intracellular accumulation of recombinant proteins. Our data suggest that XBP-1 is a key determinant for the secretory capacity of CHO cells. Understanding its dynamic regulation hence provides a rational basis for cellular engineering strategies to improve recombinant protein secretion. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Proteomic analysis to characterize differential mouse strain sensitivity to cadmium-induced forelimb teratogenesis,BIRTH DEFECTS RESEARCH, Issue 4 2008Haiyan Chen Abstract BACKGROUND: Cadmium ion (Cd2+) is a ubiquitous environmental contaminant, and it is a potent teratogen in mice. An intraperitoneal dose of 4 mg/kg of CdCl2 at gestational day 9 causes forelimb ectrodactyly in the C57BL/6N mouse strain, but the SWV/Fnn strain is resistant. The objective of this study was to identify differentially displayed proteins in two target tissues for cadmium teratogenesis, and to derive hypotheses regarding the mechanisms involved in the murine strain difference in Cd-induced forelimb ectrodactyly. METHODS: The global proteomics strategy used two-dimensional polyacrylamide gel electrophoresis for protein separation, and MALDI-TOF-MS and LC-MS/MS for protein identification, to compare and identify proteins in forelimb buds and yolk sacs from the two mouse strains following Cd administration. RESULTS: More than 1,000 protein spots were detected by two-dimensional polyacrylamide gel electrophoresis in day 10.0 mouse forelimb buds and yolk sacs. Thirty-eight proteins had identifiable differences in abundance levels in Cd-treated forelimb buds between the two strains. Of those 38 proteins, 14 could be associated with the unfolded protein response process and seven are associated with actin polymerization. The proteins that were found to be differentially abundant between the strains in yolk sacs that were exposed to CdCl2 were predominantly different than the proteins detected differentially in the limb buds of the two strains with an overlap of approximately 20%. CONCLUSIONS: These patterns of differentially displayed proteins rationalize a hypothesis that the differential murine strain response to cadmium-induced forelimb ectrodactyly is due to differences in their pathways for the unfolded protein response and/or actin polymerization. Birth Defects Research (Part A), 2008. © 2008 Wiley-Liss, Inc. [source] 2233: Endoplasmic reticulum stress and inflammation signaling in RPE cellsACTA OPHTHALMOLOGICA, Issue 2010A SALMINEN Pathogenesis of AMD is linked to augmentation of cellular stress, e.g. oxidative and proteotoxic stress, hypoxia and inflammation. All these conditions trigger stress in endoplasmic reticulum (ER) and in that way can disturb the protein quality control in retinal pigment epithelial (RPE) cells. ER stress stimulates the unfolded protein response (UPR) via the activation of IRE1, PERK and ATF6 transducers. The UPR signaling can restore cellular homeostasis but chronic and overwhelming stress can induce inflammatory response via different UPR signaling pathways and lead to apoptotic cell death. Moreover, ER stress is a well-known inducer of vascular endothelial growth factor (VEGF) expression and in AMD, ER stress could provoke neovascularization and the conversion of dry form to wet counterpart. ER stress has a fundamental role in the pathogenesis of several diseases, e.g. in diabetes and neurodegenerative diseases. This lecture will review the recent advance in understanding the inducers of ER stress, present in RPE cells during AMD, and the possible role of ER stress in evoking inflammation and neovascularization during the pathogenesis of AMD. [source] | ||||||||||||||||||||||||||||