Fluorescent lifetime imaging of an ER-tuned redox-responsive probe revealed an unanticipated stability of ER thiol redox to fluctuations in unfolded protein load, in contrast with sensitivity to lumenal calcium.
Interfering with disulfide bond formation impedes protein folding and promotes endoplasmic reticulum (ER) stress. Due to limitations in measurement techniques, the relationships of altered thiol redox and ER stress have been difficult to assess. We report that fluorescent lifetime measurements circumvented the crippling dimness of an ER-tuned fluorescent redox-responsive probe (roGFPiE), faithfully tracking the activity of the major ER-localized protein disulfide isomerase, PDI. In vivo lifetime imaging by time-correlated single-photon counting (TCSPC) recorded subtle changes in ER redox poise induced by exposure of mammalian cells to a reducing environment but revealed an unanticipated stability of redox to fluctuations in unfolded protein load. By contrast, TCSPC of roGFPiE uncovered a hitherto unsuspected reductive shift in the mammalian ER upon loss of luminal calcium, whether induced by pharmacological inhibition of calcium reuptake into the ER or by physiological activation of release channels. These findings recommend fluorescent lifetime imaging as a sensitive method to track ER redox homeostasis in mammalian cells.
Reduction of the CHOP target Gadd34 restores motor function in P0S63del mice with demyelinating neuropathy.
P0 glycoprotein is an abundant product of terminal differentiation in myelinating Schwann cells. The mutant P0S63del causes Charcot-Marie-Tooth 1B neuropathy in humans, and a very similar demyelinating neuropathy in transgenic mice. P0S63del is retained in the endoplasmic reticulum of Schwann cells, where it promotes unfolded protein stress and elicits an unfolded protein response (UPR) associated with translational attenuation. Ablation of Chop, a UPR mediator, from S63del mice completely rescues their motor deficit and reduces active demyelination by half. Here, we show that Gadd34 is a detrimental effector of CHOP that reactivates translation too aggressively in myelinating Schwann cells. Genetic or pharmacological limitation of Gadd34 function moderates translational reactivation, improves myelination in S63del nerves, and reduces accumulation of P0S63del in the ER. Resetting translational homeostasis may provide a therapeutic strategy in tissues impaired by misfolded proteins that are synthesized during terminal differentiation.
There is compelling evidence that oligodendrocyte apoptosis, in response to CNS inflammation, contributes significantly to the development of the demyelinating disorder multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Therefore, approaches designed to protect oligodendrocytes would likely have therapeutic value. Activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum (ER) stress increases cell survival under various cytotoxic conditions. Moreover, there is evidence that PERK signaling is activated in oligodendrocytes within demyelinating lesions in MS and EAE. Our previous study demonstrated that CNS delivery of the inflammatory cytokine interferon-γ before EAE onset protected mice against EAE, and this protection was dependent on PERK signaling. In our current study, we sought to elucidate the role of PERK signaling in oligodendrocytes during EAE. We generated transgenic mice that allow for temporally-controlled activation of PERK signaling, in the absence of ER stress, specifically in oligodendrocytes. We demonstrated that persistent activation of PERK signaling was not deleterious to oligodendrocyte viability or the myelin of adult animals. Importantly, we found that enhanced activation of PERK signaling specifically in oligodendrocytes significantly attenuated EAE disease severity, which was associated with reduced oligodendrocyte apoptosis, demyelination, and axonal degeneration. This effect was not the result of an altered degree of the inflammatory response in EAE mice. Our results provide direct evidence that activation of PERK signaling in oligodendrocytes is cytoprotective, protecting mice against EAE.
High fasting serum lipid levels are significant risk factors for atherosclerosis. However, the contributions of postprandial excursions in serum lipoproteins to atherogenesis are less well characterized.
This study aims to delineate whether changes in intestinal lipid absorption associated with loss of inositol requiring enzyme 1β (Ire1β) would affect the development of hyperlipidemia and atherosclerosis in Apoe−/− mice.
Methods and Results
We used Ire1β deficient mice to assess the contribution of intestinal lipid absorption to atherosclerosis. Here we show that Ire1b−/−/Apoe−/− mice contain higher levels of intestinal microsomal triglyceride transfer protein, absorb more lipids, develop hyperlipidemia, and have higher levels of atherosclerotic plaques compared to Apoe−/− mice when fed chow and western diets.
These studies indicate that Ire1β regulates intestinal lipid absorption and that increased intestinal lipoprotein production contributes to atherosclerosis.
Lipid absorption; intestine; atherosclerosis; cholesterol; Ire1β; MTP; ApoB
Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the ‘integrated stress response’ (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders.
The synthesis of proteins is an essential step in many biological processes, including memory, and drugs that inhibit protein synthesis are known to impair memory in rodents. It is thought that the brain needs these proteins to convert short-term memories into long-term memories through a process known as consolidation.
A protein called EIF2α has a key role in the regulation of protein synthesis, and has also been implicated in memory. EIF2α can be activated as a result of being phosphorylated by any of four protein kinases: these are in turn activated by processes that subject cells to stress, such as viral infection, UV light or—in the case of a kinase known as PERK—the accumulation of unfolded proteins in a cellular organelle called the endoplasmic reticulum. Activation of EIF2α downregulates most protein synthesis inside the cell, but upregulates the production of a small number of key regulatory molecules: these changes help cells to cope with whatever stressful event they have just experienced.
To obtain further insight into the cellular stress response, Sidrauski et al. screened a large library of compounds in search of one that inhibits PERK. They identified a molecule—known as ISRIB—which acts downstream of all four protein kinases by reversing the effects of EIF2α phosphorylation. ISRIB is the first molecule shown to have this effect, and thus represents an important tool for investigating the stress response inside cells.
When Sidrauski et al. injected ISRIB into mice, the animals showed improved memory: for example, they learnt to locate a hidden platform in a water maze more rapidly than controls. This suggests that ISRIB could be used to explore the mechanisms that underlie memory consolidation, and possibly even as a memory enhancer. Moreover, given that many tumor cells exploit the cellular stress response to aid their own growth, ISRIB may have potential as a novel chemotherapeutic agent.
eIF2; eIF2B; ATF4; integrated stress response; unfolded protein response; memory consolidation; Human; Mouse; Rat
Inactivating ADP ribosylation of the ER chaperone BiP is a rapidly reversible mechanism for buffering acute changes in unfolded protein load.
Gene expression programs that regulate the abundance of the chaperone BiP adapt the endoplasmic reticulum (ER) to unfolded protein load. However, such programs are slow compared with physiological fluctuations in secreted protein synthesis. While searching for mechanisms that fill this temporal gap in coping with ER stress, we found elevated levels of adenosine diphosphate (ADP)–ribosylated BiP in the inactive pancreas of fasted mice and a rapid decline in this modification in the active fed state. ADP ribosylation mapped to Arg470 and Arg492 in the substrate-binding domain of hamster BiP. Mutations that mimic the negative charge of ADP-ribose destabilized substrate binding and interfered with interdomain allosteric coupling, marking ADP ribosylation as a rapid posttranslational mechanism for reversible inactivation of BiP. A kinetic model showed that buffering fluctuations in unfolded protein load with a recruitable pool of inactive chaperone is an efficient strategy to minimize both aggregation and costly degradation of unfolded proteins.
Background: PERK controls unfolded protein load in the ER and promotes a latent gene expression program whose relative contributions to cell physiology are incompletely understood.
Results: Acute PERK inhibition deregulates protein synthesis and promotes accumulation of misfolded pro-insulin.
Conclusion: PERK contributes to proteostasis acutely.
Significance: The proteostatic activity of PERK can be uncoupled from its latent role in gene expression.
Loss-of-function mutations in EIF2AK3, encoding the pancreatic endoplasmic reticulum (ER) kinase, PERK, are associated with dysfunction of the endocrine pancreas and diabetes. However, to date it has not been possible to uncouple the long term developmental effects of PERK deficiency from sensitization to physiological levels of ER unfolded protein stress upon interruption of PERK modulation of protein synthesis rates. Here, we report that a selective PERK inhibitor acutely deregulates protein synthesis in freshly isolated islets of Langerhans, across a range of glucose concentrations. Acute loss of the PERK-mediated strand of the unfolded protein response leads to rapid accumulation of misfolded pro-insulin in cultured beta cells and is associated with a kinetic defect in pro-insulin processing. These in vitro observations uncouple the latent role of PERK in beta cell development from the regulation of unfolded protein flux through the ER and attest to the importance of the latter in beta cell proteostasis.
Endoplasmic Reticulum Stress; Insulin Synthesis; Pancreatic Islets; Protein Misfolding; Protein Synthesis; Unfolded Protein Response; Insulin Metabolism; Translation Initiation
The response of S. pombe, also known as fission yeast, to misfolded proteins involves mechanisms that have not been observed in other species.
Unfolded Protein Response; Ire1; selective mRNA decay; Bip1 mRNA stabilization; ER homeostasis
Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1α, ERO1β, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H2O2-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.
► Abnormal extracellular matrix in mice compromised in their ER thiol oxidases ► Abnormal procollagen maturation responsive to ascorbate repletion ► Tissue ascorbate depletion and noncanonical scurvy in the mutant mice
Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than two decades indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, we hypothesized that high throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small molecule activators of the apoptotic arm of the UPR to control or kill OSCC. We have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR sub-pathways. A ~66K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of pre-fractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80μM. A series of citrinin derivatives were isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds we examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, we found that patulin at 2.5 – 10μM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34 and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.
unfolded protein response; endoplasmic reticulum stress; cell-based assay; luciferase reporter; natural products
The endoplasmic reticulum-localized transmembrane kinase PERK is one of three major ER stress transducers. The crystal structure of PERK’s kinase domain has been determined to 2.8 Å resolution.
The endoplasmic reticulum (ER) unfolded protein response (UPR) is comprised of several intracellular signaling pathways that alleviate ER stress. The ER-localized transmembrane kinase PERK is one of three major ER stress transducers. Oligomerization of PERK’s N-terminal ER luminal domain by ER stress promotes PERK trans-autophosphorylation of the C-terminal cytoplasmic kinase domain at multiple residues including Thr980 on the kinase activation loop. Activated PERK phosphorylates Ser51 of the α-subunit of translation initiation factor 2 (eIF2α), which inhibits initiation of protein synthesis and reduces the load of unfolded proteins entering the ER. The crystal structure of PERK’s kinase domain has been determined to 2.8 Å resolution. The structure resembles the back-to-back dimer observed in the related eIF2α kinase PKR. Phosphorylation of Thr980 stabilizes both the activation loop and helix αG in the C-terminal lobe, preparing the latter for eIF2α binding. The structure suggests conservation in the mode of activation of eIF2α kinases and is consistent with a ‘line-up’ model for PERK activation triggered by oligomerization of its luminal domain.
UPR; PERK; kinase domains; endoplasmic reticulum; eIF2α kinase; protein translation
While nonsense-mediated RNA decay (NMD) is an established mechanism to rapidly degrade select transcripts, the physiological regulation and biological significance of NMD are not well characterized. We previously demonstrated that NMD is inhibited in hypoxic cells. Here we show that the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) translation initiation factor by a variety of cellular stresses leads to the inhibition of NMD and that eIF2α phosphorylation and NMD inhibition occur in tumors. To explore the significance of this NMD regulation, we used an unbiased approach to identify approximately 750 NMD-targeted mRNAs and found that these mRNAs are overrepresented in stress response and tumor-promoting pathways. Consistent with these findings, the inhibition of NMD promotes cellular resistance to endoplasmic reticulum stress and encourages tumor formation. The transcriptional and translational regulations of gene expression by the microenvironment are established mechanisms by which tumor cells adapt to stress. These data indicate that NMD inhibition by the tumor microenvironment is also an important mechanism to dynamically regulate genes critical for the response to cellular stress and tumorigenesis.
The ability to respond to perturbations in endoplasmic reticulum (ER) function is a fundamentally important property of all cells, but ER stress can also lead to apoptosis. In settings of chronic ER stress, the associated apoptosis may contribute to pathophysiological processes involved in a number of prevalent diseases, including neurodegenerative diseases, diabetes, atherosclerosis and renal disease. The molecular mechanisms linking ER stress to apoptosis are the topic of this review, with emphases on relevance to pathophysiology and integration and complementation among the various apoptotic pathways induced by ER stress.
Endoplasmic reticulum (ER) oxidation 1 (ERO1) transfers disulfides to protein disulfide isomerase (PDI) and is essential for oxidative protein folding in simple eukaryotes such as yeast and worms. Surprisingly, ERO1-deficient mammalian cells exhibit only a modest delay in disulfide bond formation. To identify ERO1-independent pathways to disulfide bond formation, we purified PDI oxidants with a trapping mutant of PDI. PRDX4 stood out in this list, as the related cytosolic peroxiredoxins are known to form disulfides in the presence of hydroperoxides. Mouse embryo fibroblasts lacking ERO1 were intolerant of PRDX4 knockdown. Introduction of wildtype mammalian PRDX4 into the ER rescued the temperature-sensitive phenotype of an ero1 yeast mutation. In the presence of an H2O2 generating system, purified PRDX4 oxidized PDI and reconstituted oxidative folding of RNase A. These observations implicate ER localized PRDX4 in a previously unanticipated, parallel, ERO1-independent pathway that couples hydroperoxide production to oxidative protein folding in mammalian cells.
protein folding; endoplasmic reticulum; hydrogen peroxide; enzyme analysis – in vitro
Mannose-6-phosphate is a common metabolite in glycoconjugate synthesis. In this study, it is revealed to do double duty as a second messenger-like signaling molecule, in a novel pathway that responds to HSV-1 stress to destroy host lipid–linked oligosaccharides otherwise used by the virus to generate glycoprotein components of its envelope.
Mannose-6-phosphate (M6P) is an essential precursor for mannosyl glycoconjugates, including lipid-linked oligosaccharides (LLO; glucose3mannose9GlcNAc2-P-P-dolichol) used for protein N-glycosylation. In permeabilized mammalian cells, M6P also causes specific LLO cleavage. However, the context and purpose of this paradoxical reaction are unknown. In this study, we used intact mouse embryonic fibroblasts to show that endoplasmic reticulum (ER) stress elevates M6P concentrations, leading to cleavage of the LLO pyrophosphate linkage with recovery of its lipid and lumenal glycan components. We demonstrate that this M6P originates from glycogen, with glycogenolysis activated by the kinase domain of the stress sensor IRE1-α. The apparent futility of M6P causing destruction of its LLO product was resolved by experiments with another stress sensor, PKR-like ER kinase (PERK), which attenuates translation. PERK's reduction of N-glycoprotein synthesis (which consumes LLOs) stabilized steady-state LLO levels despite continuous LLO destruction. However, infection with herpes simplex virus 1, an N-glycoprotein-bearing pathogen that impairs PERK signaling, not only caused LLO destruction but depleted LLO levels as well. In conclusion, the common metabolite M6P is also part of a novel mammalian stress-signaling pathway, responding to viral stress by depleting host LLOs required for N-glycosylation of virus-associated polypeptides. Apparently conserved throughout evolution, LLO destruction may be a response to a variety of environmental stresses.
Recent studies have implicated endoplasmic reticulum (ER) stress in insulin resistance associated with caloric excess. In mice placed on a 3-day high fat diet, we find augmented eIF2α signaling, together with hepatic lipid accumulation and insulin resistance. To clarify the role of the liver ER stress-dependent phospho-eIF2α (eIF2α-P) pathway in response to acute caloric excess on liver and muscle glucose and lipid metabolism, we studied transgenic mice in which the hepatic ER stress-dependent eIF2α-P pathway was inhibited by overexpressing a constitutively active C-terminal fragment of GADD34/PPP1R15a, a regulatory subunit of phosphatase that terminates ER stress signaling by phospho-eIF2α. Inhibition of the eIF2α-P signaling in liver led to a decrease in hepatic glucose production in the basal and clamped state, which could be attributed to reduced gluconeogenic gene expression, resulting in reduced basal plasma glucose concentrations. Surprisingly, hepatic eIF2α inhibition also impaired insulin-stimulated muscle and adipose tissue insulin sensitivity. This latter effect could be attributed at least in part by an increase in circulating IGFBP-3 levels in the transgenic animals. In addition, infusion of insulin during a hyperinsulinemic-euglycemic clamp induced conspicuous ER stress in the 3-day high fat diet-fed mice, which was aggravated through continuous dephosphorylation of eIF2α. Together, these data imply that the hepatic ER stress eIF2α signaling pathway affects hepatic glucose production without altering hepatic insulin sensitivity. Moreover, hepatic ER stress-dependent eIF2α-P signaling is implicated in an unanticipated cross-talk between the liver and peripheral organs to influence insulin sensitivity, probably via IGFBP-3. Finally, eIF2α is crucial for proper resolution of insulin-induced ER stress.
Endoplasmic Reticulum (ER); Insulin Resistance; Insulin-like Growth Factor (IGF); Liver Metabolism; Triacylglycerol
The early steps in the biogenesis of secreted and membrane proteins occur in the lumen of the endoplasmic reticulum (ER), where resident proteins that make up the ER machinery assist in their folding, maturation, and complex assembly. Variation in the load of ER client proteins and in the function of the organelle's aforementioned machinery for coping with that load can lead to an imbalance between the two that is referred to as ER stress. This triggers a cellular response, mediated by highly conserved signaling pathways that collectively restore equilibrium to the protein-folding environment in the organelle by increasing the expression of genes that enhance nearly all aspects of ER function, and by transiently repressing the biosynthesis of new client proteins. Evidence accrued over the past 10 years suggests that ER stress and response to it influence the fate of mutant proteins that fold inefficiently, impact on the functionality of cells and tissues that cope with unusual loads of ER client proteins, and intersect with signaling pathways that influence inflammation and cancer biology. Here, we review some of the basic workings of unfolded protein response and relate them to processes that are of potential relevance to pulmonary disease.
chaperones; protein folding; secretion; signal transduction; pulmonary disease
There is increasing evidence that neuron death in neurode-generative diseases, such as Parkinson's disease, is due to the activation of programmed cell death. However, the upstream mediators of cell death remain largely unknown. One approach to the identification of upstream mediators is to perform gene expression analysis in disease models. Such analyses, performed in tissue culture models induced by neurotoxins, have identified up-regulation of CHOP/GADD153, a transcription factor implicated in apoptosis due to endoplasmic reticulum stress or oxidative injury. To evaluate the disease-related significance of these findings, we have examined the expression of CHOP/GADD153 in neurotoxin models of parkinsonism in living animals. Nuclear expression of CHOP protein is observed in developmental and adult models of dopamine neuron death induced by intrastriatal injection of 6-hydroxydopamine (6OHDA) and in models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). CHOP is a mediator of neuron death in the adult 60HDA model because a null mutation results in a reduction in apoptosis. In the chronic MPTP model, however, while CHOP is robustly expressed, the null mutation does not protect from the loss of neurons. We conclude that the role of CHOP depends on the nature of the toxic stimulus. For 6OHDA, an oxidative metabolite of dopamine, it is a mediator of apoptotic death.
apoptosis; endoplasmic reticulum stress; oxidative stress; Parkinson's disease; programmed cell death; substantia nigra
Signaling in the most conserved branch of the endoplasmic reticulum (ER) unfolded protein response (UPR) is initiated by sequence-specific cleavage of the HAC1/XBP1 mRNA by the ER stress-induced kinase-endonuclease IRE1. We have discovered that the flavonol quercetin activates yeast IRE1’s RNase and potentiates activation by ADP, a natural activating ligand that engages the IRE1 nucleotide binding cleft. Enzyme kinetics and the structure of a co-crystal of IRE1 complexed with ADP and quercetin reveal engagement by quercetin of an unanticipated ligand binding pocket at the dimer interface of IRE1’s Kinase Extension Nuclease (KEN) domain. Analytical ultracentrifugation and crosslinking studies support the preeminence of enhanced dimer formation in quercetin’s mechanism of action. These findings hint at the existence of endogenous cytoplasmic ligands that may function alongside stress signals from the ER lumen to modulate IRE1 activity and at the potential for the development of drugs that modify UPR signaling from this unanticipated site.
P58(IPK) may function as an ER molecular chaperone to maintain protein folding homeostasis during Unfolded Protein Responses (UPR). P58(IPK) contains nine TPR motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism how P58(IPK) functions to promote protein folding within ER, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5Å resolution by SAD method. The crystal structure of P58(IPK) revealed three domains (I, II and III) with similar folds and each domain contains three TPR motifs. An In vitro ELISA assay indicates that P58(IPK) acts as a molecular chaperone by interacting with the misfolded proteins such as Luciferase and Rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that may be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK).
Upon antigen stimulation, B lymphocytes differentiate into antibody secreting cells (ASC), most of which undergo apoptosis after a few days of intense Ig production. Differentiation entails expansion of the endoplasmic reticulum (ER) and requires XBP1 but not other elements of the unfolded protein response, like Perk. Moreover, normal and malignant ASC are exquisitely sensitive to proteasome inhibitors, but the underlying mechanisms are poorly understood. Here we analyze the role of CHOP, a transcription factor mediating apoptosis in many cell types that experience high levels of ER stress. CHOP is transiently induced early upon B cell stimulation: covalent IgM aggregates form more readily and IgM secretion is slower in chop-/- cells. Despite these subtle changes, ASC differentiation and lifespan are normal in chop-/- mice. Unlike fibroblasts and other cell types, chop-/- ASC are equally or slightly more sensitive to proteasome inhibitors and ER stressors, implying tissue-specific roles for CHOP in differentiation and stress.
UPR; ER stress; plasma cell differentiation; IgM secretion; proteasome inhibitors
Genetic analyses previously implicated the matrix-localized protease ClpP in signaling the stress of protein misfolding in the mitochondrial matrix to activate nuclear encoded mitochondrial chaperone genes in C. elegans (UPRmt). Here we report that haf-1, a gene encoding a mitochondria-localized ATP-binding cassette protein, is required for signaling within the UPRmt and for coping with misfolded protein stress. Peptide efflux from isolated mitochondria was ATP-dependent and required HAF-1 and the protease ClpP. Defective UPRmt signaling in the haf-1 deleted worms was associated with failure of the bZIP protein, ZC376.7, to localize to nuclei in worms with perturbed mitochondrial protein folding, whereas zc376.7(RNAi) strongly inhibited the UPRmt. These observations suggest a simple model whereby perturbation of the protein-folding environment in the mitochondrial matrix promotes ClpP-mediated generation of peptides whose haf-1-dependent export from the matrix contributes to UPRmt signaling across the mitochondrial inner membrane.
chaperones; unfolded proteins; heat shock; inter-organellar signaling; peptide transport
ERO1-β has an unexpected and selective function for oxidative protein folding in insulin-producing cells.
Mammals have two genes encoding homologues of the endoplasmic reticulum (ER) disulfide oxidase ERO1 (ER oxidoreductin 1). ERO1-β is greatly enriched in the endocrine pancreas. We report in this study that homozygosity for a disrupting allele of Ero1lb selectively compromises oxidative folding of proinsulin and promotes glucose intolerance in mutant mice. Surprisingly, concomitant disruption of Ero1l, encoding the other ERO1 isoform, ERO1-α, does not exacerbate the ERO1-β deficiency phenotype. Although immunoglobulin-producing cells normally express both isoforms of ERO1, disulfide bond formation and immunoglobulin secretion proceed at nearly normal pace in the double mutant. Moreover, although the more reducing environment of their ER protects cultured ERO1-β knockdown Min6 cells from the toxicity of a misfolding-prone mutant Ins2Akita, the diabetic phenotype and islet destruction promoted by Ins2Akita are enhanced in ERO1-β compound mutant mice. These findings point to an unexpectedly selective function for ERO1-β in oxidative protein folding in insulin-producing cells that is required for glucose homeostasis in vivo.
The endoplasmic reticulum (ER) Unfolded Protein Response (UPR) restores equilibrium to the ER, but prolonged expression of the UPR effector CHOP (GADD153) is cytotoxic. We found that ER stress-induced CHOP expression was suppressed by prior engagement of toll-like receptor (TLR) 3 or 4 through a TRIF-dependent pathway. TLR engagement did not suppress phosphorylation of PERK or eIF-2α, which are upstream of CHOP, but phospho-eIF-2α failed to promote translation of the CHOP activator ATF4. In mice subjected to systemic ER stress, pre-treatment with low-dose lipopolysaccharide (LPS), a TLR4 ligand, suppressed CHOP expression and apoptosis in splenic macrophages, renal tubule cells, and hepatocytes, and prevented renal dysfunction and hepatosteatosis. This protective effect of LPS did not occur in Trif−/− mice nor in wild-type mice in which CHOP expression was genetically restored. Thus, TRIF-mediated signals from TLRs selectively attenuate translational activation of ATF4 and its downstream target gene CHOP. We speculate that this mechanism evolved to promote survival of TLR-expressing cells that experience prolonged levels of physiologic ER stress in the course of the host response to invading pathogens.