Activation Of Inositol-requiring Enzyme Research Articles

Insulin-degrading enzyme inhibition increases the unfolded protein response and favours lipid accumulation in the liver.

Nonalcoholic fatty liver disease refers to liver pathologies, ranging from steatosis to steatohepatitis, with fibrosis ultimately leading to cirrhosis and hepatocellular carcinoma. Although several mechanisms have been suggested, including insulin resistance, oxidative stress, and inflammation, its pathophysiology remains imperfectly understood. Over the last decade, a dysfunctional unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress emerged as one of the multiple driving factors. In parallel, growing evidence suggests that insulin-degrading enzyme (IDE), a highly conserved and ubiquitously expressed metallo-endopeptidase originally discovered for its role in insulin decay, may regulate ER stress and UPR. We investigated, by genetic and pharmacological approaches, in vitro and in vivo, whether IDE modulates ER stress-induced UPR and lipid accumulation in the liver. We found that IDE-deficient mice display higher hepatic triglyceride content along with higher inositol-requiring enzyme 1 (IRE1) pathway activation. Upon induction of ER stress by tunicamycin or palmitate in vitro or in vivo, pharmacological inhibition of IDE, using its inhibitor BDM44768, mainly exacerbated ER stress-induced IRE1 activation and promoted lipid accumulation in hepatocytes, effects that were abolished by the IRE1 inhibitors 4μ8c and KIRA6. Finally, we identified that IDE knockout promotes lipolysis in adipose tissue and increases hepatic CD36 expression, which may contribute to steatosis. These results unravel a novel role for IDE in the regulation of ER stress and development of hepatic steatosis. These findings pave the way to innovative strategies modulating IDE to treat metabolic diseases.

Computer-aided peptide-based drug design for inositol-requiring enzyme 1

Inositol-requiring enzyme 1 (IRE1), an endoplasmic reticulum (ER) transmembrane protein with both kinase and endoribonuclease activities, plays an essential role during ER stress and its subsequent unfolded protein response (UPR). Recent evidence shows IRE1 signaling contributes to tumorigenesis and cancer progression, pointing to the therapeutic importance of this conserved arm of the UPR. Here, we employed different computational tools to design and predict short peptides with the capability of disrupting IRE1 dimerization/oligomerization, as a strategy for inhibiting its Kinase and RNase activities. A mutation-based peptide library was constructed using mCSM-PPI2 and OSPREY 3.0. The molecular interaction analyses between the designed peptides and IRE1 protein were conducted using the HADDOCK 2.2 online server, followed with molecular dynamics analysis by the GROMACS 2020 package. We then selected short peptide candidates that exhibited high affinity and best predicted physicochemical properties in complex with IRE1. Finally, online servers, such as ToxinPred and AllerTop, were used to identify the best peptide candidates that showed no significant allergenic or cytotoxic properties. These rational designed peptides with the capability of binding to IRE1 oligomerization domain can be considered as potential drug candidates for disrupting IRE1 activity in cancer and related diseases, pending for further validation by in silico and experimental studies.

TXNIP Links Anticipatory Unfolded Protein Response to Estrogen Reprogramming Glucose Metabolism in Breast Cancer Cells.

Estrogen and estrogen receptor (ER) play a fundamental role in breast cancer. To support the rapid proliferation of ER+ breast cancer cells, estrogen increases glucose uptake and reprograms glucose metabolism. Meanwhile, estrogen/ER activates the anticipatory unfolded protein response (UPR) preparing cancer cells for the increased protein production required for subsequent cell proliferation. Here, we report that thioredoxin-interacting protein (TXNIP) is an important regulator of glucose metabolism in ER+ breast cancer cells, and estrogen/ER increases glucose uptake and reprograms glucose metabolism via activating anticipatory UPR and subsequently repressing TXNIP expression. In 2 widely used ER+ breast cancer cell lines, MCF7 and T47D, we showed that MCF7 cells express high TXNIP levels and exhibit mitochondrial oxidative phosphorylation (OXPHOS) phenotype, while T47D cells express low TXNIP levels and display aerobic glycolysis (Warburg effect) phenotype. Knockdown of TXNIP promoted glucose uptake and Warburg effect, while forced overexpression of TXNIP inhibited glucose uptake and Warburg effect. We further showed that estrogen represses TXNIP expression and activates UPR sensor inositol-requiring enzyme 1 (IRE1) via ER in the breast cancer cells, and IRE1 activity is required for estrogen suppression of TXNIP expression and estrogen-induced cell proliferation. Our study suggests that TXNIP is involved in estrogen-induced glucose uptake and metabolic reprogramming in ER+ breast cancer cells and links anticipatory UPR to estrogen reprogramming glucose metabolism.

Structural and molecular bases to IRE1 activity modulation.

The Unfolded Protein response is an adaptive pathway triggered upon alteration of endoplasmic reticulum (ER) homeostasis. It is transduced by three major ER stress sensors, among which the Inositol Requiring Enzyme 1 (IRE1) is the most evolutionarily conserved. IRE1 is an ER-resident type I transmembrane protein exhibiting an ER luminal domain that senses the protein folding status and a catalytic kinase and RNase cytosolic domain. In recent years, IRE1 has emerged as a relevant therapeutic target in various diseases including degenerative, inflammatory and metabolic pathologies and cancer. As such several drugs altering IRE1 activity were developed that target either catalytic activity and showed some efficacy in preclinical pathological mouse models. In this review, we describe the different drugs identified to target IRE1 activity as well as their mode of action from a structural perspective, thereby identifying common and different modes of action. Based on this information we discuss on how new IRE1-targeting drugs could be developed that outperform the currently available molecules.

The Sensor IRE1 Couples Stress Detection to Protein Synthesis

Endoplasmic reticulum (ER) stress results in accumulation of misfolded proteins and profoundly regulates cellular protein synthesis. The regulation includes both inhibition of translation of most proteins and upregulation of synthesis of a select group of stress-related polypeptides. This differential regulation has long been attributed to the kinase PERK, but recently the related stress sensor, inositol requiring enzyme 1 (IRE1), was suggested to be linked to translation by virtue of its physical association with both ribosomes1 and the Sec61 translocon2,3. ER stress causes the quiescent IRE1 to dissociate from the inhibitor BiP, self-phosphorylate, oligomerize (an event also called clustering) and activate its RNase activity. Consequently, active IRE1 activates the critical transcription factor XBP1, and also reduces the expression of select other transcripts, to cope with increased ER stress. We previously showed that IRE1 clustering is a response to stress4, but is not related to its RNase activity, raising the question – what is the function of IRE1 clustering? In examining the requirements for IRE clustering we discovered that ongoing protein synthesis and translocation are necessary for both the formation of IRE1 clusters upon ER stress and their maintenance. IRE1 clusters do not develop when cells are treated with either cycloheximide or lactimidomycin, which inhibit nascent chain elongation and ‘freeze’ membrane-bound ribosomes on the ER membrane. If added after clusters were allowed to form under ER stress, both inhibitors caused rapid disappearance of the pre-formed clusters. IRE1 clustering is similarly sensitive to Eeyarestatin I (EEY), an inhibitor of the Sec61 translocon. When translocation is inhibited, IRE1 is dissociated from Sec61 and its interaction with BiP is increased. Concomitantly, more IRE1 is found in high molecular weight species, which perhaps represent ubiquitinylated species. Paradoxically, EEY also induces IRE1 activation (XBP1 splicing). To explain the paradox, we hypothesize the following model: in unstressed conditions, IRE1 binds the translocon and is kept inactive by the interaction with the chaperone BiP. Upon ER stress, different scenarios with distinct IRE1 populations can appear at the same time: 1) IRE1 is released from BiP and clusters on a subset of translocons that are engaged in synthesis of stress-related polypeptides; 2) translocons not engaged in active translocation release IRE1 which is then kept inactive by increased interaction with BiP; 3) some activated IRE1 dimerizes (appearing as non-clustered) and binds the ribosomes that recruit XBP1 mRNA, facilitating its splicing. Acosta-Alvear D et al. eLife 2018 Dec 24;7:e43036. Plumb R et al. eLife 2015 2015 May 20;4:e07426. Sundaram A et al. eLife 2017 May 15;6:e27187. Ricci, D. et al. The FASEB Journal 2019; Sep;33(9):9811-9827.

Diabetic nephropathy in mice is aggravated by the absence of podocyte IRE1 and is correlated with reduced kidney ADH1 expression.

BackgroundInositol-requiring enzyme 1 (IRE1) plays a critical role in attenuating endoplasmic reticulum (ER) stress associated with renal injury which may also be a factor in diabetic nephropathy (DN). Alcohol dehydrogenase type I (ADH1) activity is prominent in the kidney, ADH1 activity is also reported to exert protective effects against ER stress that are not caused by alcohol consumption. However, the role of IRE1 in DN and the correlation between IRE1 and ADH1 activity remain unclear.MethodsIRE1α floxed mice (Ire1f/f) of C57BL/6J background were established and crossbred with Ire1αf/f mice to produce podocyte-specific IRE1α knockout mice. Male db/db mice (C57BLKS/J-leprdb/leprdb mice) were used as a DN model. Male mice were made diabetic by injection of streptozotocin. pLKO.1-based vectors encoding short hairpin RNA (shRNA) specific to the IRE1α gene were transfected into HEK293T cells to knockdown IRE1α in mouse podocytes. ELISA, Masson’s staining, and electron microscopy were performed to analyze the development of DN. The ADH1 expression was assayed by qPCR and western blot.ResultsWe found that IRE activity was increased in the glomeruli of DN mouse models. In contrast, ADH1 expression was decreased in these models and mice with podocyte-specific disruption of IRE1 (PKO mice). PKO mice that were made diabetic using strepto­zotocin exhibited accelerated proteinuria, enhanced glomerular fibrosis, and podocyte cell death. In addition, in cultured podocytes, the knockdown of IRE1 downregulated the ADH1 mRNA expression and induced ER stress, consistent with the result of PKO mice, while its detrimental effects were reversed by ADH1 overexpression.ConclusionsActivation of IRE1 in podocytes serves to limit the progress of DN. The dependence of kidney ADH1 expression on podocyte IRE1 further suggests that ADH1 activity may play an important role downstream of IRE1 in protecting against DN.

Aripiprazole Cytotoxicity Coincides with Activation of the Unfolded Protein Response in Human Hepatic Cells.

Schizophrenia is a mental disease that results in decreased life expectancy and well-being by promoting obesity and sedentary lifestyles. Schizophrenia is treated by antipsychotic drugs. Although the second-generation antipsychotics (SGA), Olanzapine and Aripiprazole, are more effective in treating schizophrenia, they display a higher risk of metabolic side effects, mostly by development of diabetes and insulin resistance, weight gain, and dyslipidemia. Endoplasmic reticulum (ER) stress is induced when ER homeostasis of lipid biosynthesis and protein folding is impaired. This leads to the activation of the unfolded protein response (UPR), a signaling cascade that aims to restore ER homeostasis or initiate cell death. Chronic conditions of ER stress in the liver are associated with diabetes and perturbed lipid metabolism. These metabolic dysfunctions resemble the pharmacological side effects of SGAs. We therefore investigated whether SGAs promote the UPR in human and mouse hepatocytes. We observed full-fledged activation of ER stress by Aripiprazole not by Olanzapine. This occurred at low micromolar concentrations and to variable intensities in different cell types, such as hepatocellular carcinoma, melanoma, and glioblastoma. Mechanistically, Aripiprazole caused depletion of ER calcium, leading to activation of inositol-requiring enzyme 1 (IRE1)and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), two major transducers of the UPR. Cells underwent apoptosis with Aripiprazole treatment, which coincided with UPR induction, and this effect was reduced by adding glutathione without affecting UPR itself. Deletion of IRE1 from HepG2, a human liver cancer cell line, protected cells from Aripiprazole toxicity. Our study reveals for the first time a cytotoxic effect of Aripiprazole that involves the induction of ER stress. SIGNIFICANCE STATEMENT: The antischizophrenic drug Aripiprazole exerts cytotoxic properties at high concentrations. This study shows that this cytotoxicity is associated with the induction of endoplasmic reticulum (ER) stress and IRE1 activation, mechanisms involved in diet-induced obesity. Aripiprazole induced ER stress and calcium mobilization from the ER in human and mouse hepatocytes. Our study highlights a new mechanism of Aripiprazole that is not related to its effect on dopamine signaling.

Peptidomimetic-based identification of FDA-approved compounds inhibiting IRE1 activity.

Inositol-requiring enzyme 1 (IRE1) is a bifunctional serine/threonine kinase and endoribonuclease that is a major mediator of the unfolded protein response (UPR) during endoplasmic reticulum (ER) stress. Tumour cells experience ER stress due to adverse environmental cues such as hypoxia or nutrient shortage and high metabolic/protein-folding demand. To cope with those stresses, cancer cells utilise IRE1 signalling as an adaptive mechanism. Here, we report the discovery of the FDA-approved compounds methotrexate, cefoperazone, folinic acid and fludarabine phosphate as IRE1 inhibitors. These were identified through a structural exploration of the IRE1 kinase domain using IRE1 peptide fragment docking and further optimisation and pharmacophore development. The inhibitors were verified to have an impact on IRE1 activity invitro and were tested for their ability to sensitise human cell models of glioblastoma multiforme (GBM) to chemotherapy. We show that all molecules identified sensitise glioblastoma cells to the standard-of-care chemotherapy temozolomide (TMZ).

Intracellular proliferation of Anaplasma phagocytophilum is promoted via modulation of endoplasmic reticulum stress signaling in host cells.

Anaplasma phagocytophilum, an obligate intracellular bacterium that propagates within host granulocytes, is considered to modify the host intracellular environment for pathogenesis. However, the mechanism(s) underlying such host modifications remain unclear. Here, we aimed to investigate the relation between A. phagocytophilum and endoplasmic reticulum (ER) stress in THP-1 cells. A. phagocytophilum activated the three ER stress sensors: inositol-requiring enzyme-1 (IRE1), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor-6 (ATF6). IRE1 activation occurred immediately after host cell invasion by A. phagocytophilum; however, the activated IRE1-induced splicing of X-box-binding protein 1 was not promoted during A. phagocytophilum infection. This suppression was sustained even after the doxycycline-mediated elimination of intracellular A. phagocytophilum. IRE1 knockdown accelerated A. phagocytophilum-induced apoptosis and decreased intracellular A. phagocytophilum. These data suggest that A. phagocytophilum utilizes IRE1 activation to promote its own intracellular proliferation. Moreover, PERK and ATF6 partially mediated A. phagocytophilum-induced apoptosis by promoting the expression of CCAAT/enhancer-binding protein homologous protein, which induces the transcription of several proapoptotic genes. Thus, A. phagocytophilum possibly manipulates the host ER stress signals to facilitate intracellular proliferation and infection of surrounding cells before/after host cell apoptosis.

Quantitative microscopy reveals dynamics and fate of clustered IRE1α

The endoplasmic reticulum (ER) membrane-resident stress sensor inositol-requiring enzyme 1 (IRE1) governs the most evolutionarily conserved branch of the unfolded protein response. Upon sensing an accumulation of unfolded proteins in the ER lumen, IRE1 activates its cytoplasmic kinase and ribonuclease domains to transduce the signal. IRE1 activity correlates with its assembly into large clusters, yet the biophysical characteristics of IRE1 clusters remain poorly characterized. We combined superresolution microscopy, single-particle tracking, fluorescence recovery, and photoconversion to examine IRE1 clustering quantitatively in living human and mouse cells. Our results revealed that: 1) In contrast to qualitative impressions gleaned from microscopic images, IRE1 clusters comprise only a small fraction (∼5%) of the total IRE1 in the cell; 2) IRE1 clusters have complex topologies that display features of higher-order organization; 3) IRE1 clusters contain a diffusionally constrained core, indicating that they are not phase-separated liquid condensates; 4) IRE1 molecules in clusters remain diffusionally accessible to the free pool of IRE1 molecules in the general ER network; 5) when IRE1 clusters disappear at later time points of ER stress as IRE1 signaling attenuates, their constituent molecules are released back into the ER network and not degraded; 6) IRE1 cluster assembly and disassembly are mechanistically distinct; and 7) IRE1 clusters' mobility is nearly independent of cluster size. Taken together, these insights define the clusters as dynamic assemblies with unique properties. The analysis tools developed for this study will be widely applicable to investigations of clustering behaviors in other signaling proteins.

Gallotannin attenuates 2‑deoxy‑D‑glucose‑induced dedifferentiation and endoplasmic reticulum stress through inhibition of inositol‑requiring enzyme 1 downstream p38 kinase pathway in chondrocytes.

Gallotannin (GT) is a class of polyphenols with antioxidant, anticancer, and antiviral activities. 2‑Deoxy‑D‑glucose (2DG), a glucose‑derived molecule, can inhibit glucose metabolism and induce endoplasmic reticulum (ER) stress. GT in primary‑cultured chondrocytes enhances expression of typeII collagen, an indicator of differentiation, and cyclooxygenase‑2 (COX‑2), which mediates inflammatory reactions. In contrast, 2DG reduces typeII collagen and COX‑2 expression while driving ER‑stress‑induced unglycosylation. In the present study, it was investigated whether GT could attenuate 2DG‑induced dedifferentiation and ER‑stress. Following treatment with GT and 2DG, chondrocytes were assessed using western blotting, RT‑PCR, immunofluorescence, and alcian blue staining. GT restored typeII collagen expression that was reduced by 2DG, inhibited ER‑stress‑induced COX‑2 unglycosylation, and induced COX‑2 expression. The expression of a glucose‑regulated protein, GRP78, which is an indicator of reduced ER‑stress, was decreased. To link the GT signaling pathway with pathways that inhibit 2DG‑induced dedifferentiation and ER‑stress, inhibitors were treated in chondrocytes. The results revealed that, among the different signaling pathways triggered by ER‑stress, the p38 kinase pathway was involved in the inositol‑requiring enzyme 1 (IRE1) downstream signaling pathway. Following inhibition of the IRE1 pathway, typeII collagen expression was increased and COX‑2 expression was decreased. In addition, after examining the splicing of X‑box binding protein 1 (XBP‑1) which is dependent on IRE1 activation induced by ER‑stress, it was revealed that GT inhibited the increase of XBP‑1s after splicing due to 2DG‑induced ER stress. GT in chondrocytes inhibited 2DG‑induced dedifferentiation and ER‑stress‑induced COX‑2 unglycosylation while regulating differentiation and inflammation via the ER‑stress‑induced p38 kinase pathway downstream from the IRE1 pathway.

Endoplasmic Reticulum Stress Signaling Pathways: Activation and Diseases.

Secretory and membrane proteins are folded in the endoplasmic reticulum (ER) prior to their exit. When ER function is disturbed by exogenous and endogenous factors, such as heat shock, ultraviolet radiation, hypoxia, or hypoglycemia, the misfolded proteins may accumulate, promoting ER stress. To rescue this unfavorable situation, the unfolded protein response is activated to reduce misfolded proteins within the ER. Upon ER stress, the ER transmembrane sensor molecules inositol-requiring enzyme 1 (IRE1), RNA-dependent protein kinase (PKR)-like ER kinase (PERK), and activating transcription factor 6, are activated. Here, we discuss the mechanisms of PERK and IRE1 activation and describe two working models for ER stress initiation: the BiP-dependent model and the ligand-driven model. ER stress activation has been linked to multiple diseases, including cancers, Alzheimer's disease, and diabetes. Thus, the regulation of ER stress may provide potential therapeutic targets for these diseases.

Intrinsic Structural Features of the Human IRE1α Transmembrane Domain Sense Membrane Lipid Saturation

Activation of inositol-requiring enzyme (IRE1α) is anindispensable step in remedying the cellular stress associated with lipid perturbation in the endoplasmic reticulum (ER) membrane. IRE1α is a single-spanning ER transmembrane protein possessing both kinase and endonuclease functions, and its activation can be fully achieved through the dimerization and/or oligomerization process. How IRE1α senses membrane lipid saturation remains largely unresolved. Using both computational and experimental tools, we systematically investigated the dimerization process of the transmembrane domain (TMD) of IRE1α and found that, with help of the serine 450 residue, the conserved tryptophan 457 residue buttresses the core dimerization interface of IRE1α-TMD. BiFC (bimolecular fluorescence complementation) experiments revealed that mutation on these residues abolished the saturated fatty acid-induced dimerization in the ER membrane and subsequently inactivated IRE1α activity invivo. Therefore, our results suggest that the structural elements of IRE1α-TMD serve as a key sensor that detects membrane aberrancy.

Carbon monoxide releasing molecule-3 alleviates neuron death after spinal cord injury via inflammasome regulation.

BackgroundGenetic overexpression or pharmacological activation of heme oxygenase (HO) are identified as potential therapeutic target for spinal cord injury (SCI); however, the role of carbon monoxide (CO), which is a major product of haem degenerated by HO, in SCI remains unknown. Applying hemin or chemicals which may regulate HO expression or activity to increase CO production are inadequate to elaborate the direct role of CO. Here, we assessed the effect of CO releasing molecule-3 (CORM-3), the classical donor of CO, in SCI and explained its possible protective mechanism.MethodsRat SCI model was performed with a vascular clip (30 g) compressing at T9 vertebral level for 1 min and CO was delivered immediately after SCI by CORM-3. The neurological deficits and neuron survival were assessed. Inflammasome and inositol-requiring enzyme 1 (IRE1) pathway were measured by western blot and immunofluorescence. For in vitro study, oxygen glucose deprivation (OGD) simulated the SCI-inflammasome change in cultured the primary neurons.FindingsCORM-3 suppressed inflammasome signaling and pyroptosis occurrence, which consequently alleviated neuron death and improved motor functional recovery following SCI. As a pivotal sensor involving in endoplasmic reticulum stress-medicated inflammasome signaling, IRE1 and its downstream X-box binding protein 1 (XBP1) were activated in SCI tissues as well as in OGD neurons; while inhibition of IRE1 by STF-083010 in SCI rats or by si-RNA in OGD neurons suppressed inflammasome signaling and pyroptosis. Interestingly, the SCI/OGD-stimulated IRE1 activation was attenuated by CORM-3 treatment.InterpretationsCO may alleviate neuron death and improve motor functional recovery in SCI through IRE1 regulation, and administration of CO could be a promising therapeutic strategy for SCI.

Arabidopsis GAAP1 and GAAP3 Modulate the Unfolded Protein Response and the Onset of Cell Death in Response to ER Stress.

The function of human Golgi antiapoptotic proteins (GAAPs) resembles that of BAX inhibitor-1, with apoptosis inhibition triggered by intrinsic and extrinsic stimuli. However, little is known about the function of GAAP-related proteins in plants. Here, we studied Arabidopsis GAAP1 and GAAP3 and found that they were localized on the cellular membrane, including the endoplasmic reticulum (ER) membrane. The function of GAAP1/GAAP3 in ER-stress response was tested, and results showed that single or double mutation in GAAP1 and GAAP3 reduced plant survival and enhanced cell death under ER stress. The expression of both genes was induced by various abiotic stress signals. Quantitative real-time polymerase chain reaction analysis showed that GAAP1/GAAP3 level affected the expression pattern of the unfolded-protein response (UPR) signaling pathway genes upon prolonged ER stress. The mutation in both GAAP1 and GAAP3 genes promoted and enhanced UPR signaling when confronted with mild ER stress. Moreover, GAAP1/GAAP3 inhibited cell death caused by ER stress and promoted plant-growth recovery by turning down inositol-requiring enzyme 1 (IRE1) signaling after ER stress had been relieved. Co-immunoprecipitation (Co-Ip) and BiFC assays showed that GAAP1/GAAP3 interacted with IRE1. These data suggested that GAAP1/GAAP3 played dual roles in the negative regulation of IRE1 activity and anti-programmed cell death.

AKT-mTOR signaling modulates the dynamics of IRE1 RNAse activity by regulating ER-mitochondria contacts

Inositol Requiring Enzyme-1 (IRE1) is the most conserved transducer of the Unfolded Protein Response (UPR), a surveillance mechanism that ensures homeostasis of the endoplasmic reticulum (ER) in eukaryotes. IRE1 activation orchestrates adaptive responses, including lipid anabolism, metabolic reprogramming, increases in protein folding competency, and ER expansion/remodeling. However, we still know surprisingly little regarding the principles by which this ER transducer is deactivated upon ER stress clearance. Here we show that Protein Kinase B-mechanistic Target of Rapamycin (PKB/AKT-mTOR) signaling controls the dynamics of IRE1 deactivation by regulating ER-mitochondria physical contacts and the autophosphorylation state of IRE1. AKT-mTOR-mediated attenuation of IRE1 activity is important for ER remodelling dynamics and cell survival in the face of recursive, transient ER stress. Our observations suggest that IRE1 attenuation is an integral component of anabolic programmes regulated by AKT-mTOR. We suggest that AKT-mTOR activity is part of a ‘timing mechanism’ to deactivate IRE1 immediately following engagement of the UPR, in order to limit prolonged IRE1 RNAse activity that could lead to damaging inflammation or apoptosis.

Resveratrol protects against early polymicrobial sepsis-induced acute kidney injury through inhibiting endoplasmic reticulum stress-activated NF-κB pathway.

Resveratrol, a polyphenol compound derived from various edible plants, protects against sepsis-induced acute kidney injury (AKI) via its anti-inflammatory activity, but the underlying mechanisms remain largely unknown. In this study, a rat model of sepsis was established by cecal ligation and puncture (CLP), 30 mg/kg resveratrol was intraperitoneally administrated immediately after the CLP operation. HK-2 cells treated by 1 μg/ml lipopolysaccharide, 0.2 μM tunicamycin, 2.5 mM irestatin 9389 and 20 μM resveratrol were used for in vitro study. The results demonstrated that resveratrol significantly improved the renal function and tubular epithelial cell injury and enhanced the survival rate of CLP-induced rat model of sepsis, which was accompanied by a substantial decrease of the serum content and renal mRNA expressions of TNF-α, IL-1β and IL-6. In addition, resveratrol obviously relieved the endoplasmic reticulum stress, inhibited the phosphorylation of inositol-requiring enzyme 1(IRE1) and nuclear factor-κB (NF-κB) in the kidney. In vitro studies showed that resveratrol enhanced the cell viability, reduced the phosphorylation of NF-κB and production of inflammatory factors in lipopolysaccharide and tunicamycin-induced HK-2 cells through inhibiting IRE1 activation. Taken together, administration of resveratrol as soon as possible after the onset of sepsis could protect against septic AKI mainly through inhibiting IRE1-NF-κB pathway-triggered inflammatory response in the kidney. Resveratrol might be a readily translatable option to improve the prognosis of sepsis.

Divergence and Conservation of the Major UPR Branch IRE1-bZIP Signaling Pathway across Eukaryotes.

The unfolded protein response (UPR) is crucial to life by regulating the cellular response to the stress in the endoplasmic reticulum (ER) imposed by abiotic and biotic cues such as heat shock and viral infection. The inositol requiring enzyme 1 (IRE1) signaling pathway activated by the IRE1-mediated unconventional splicing of HAC1 in yeast, bZIP60 in plants and XBP1 in metazoans, is the most ancient branch of the UPR. In this study, we systematically examined yeast IRE1p-HAC1, plant IRE1A/IRE1B-bZIP60 and human hIRE1-XBP1 pairs. We found that, unlike bZIP60, XBP1 is unable to functionally swap HAC1p in yeast, and that the inter-species heterotypic interactions among HAC1p, bZIP60 and XBP1 are not permitted. These data demonstrate evolutionary divergence of the downstream signaling of IRE1-bZIP. We also discovered that the dual cytosolic domains of plant IRE1s act in vivo in a mechanism consistent with IRE1p and hIRE1, and that plant IRE1B not only interacts with IRE1p but also forms typical IRE1 dynamic foci in yeast. Thus, the upstream components of the IRE1 signaling branch including IRE1 activation and action mechanisms are highly conserved. Taken together these data advance the molecular understanding of evolutionary divergence and conservation of the IRE1 signaling pathway across kingdoms.

Endoplasmic reticulum stress up-regulates Nedd4-2 to induce autophagy.

The accumulation of unfolded proteins within the endoplasmic reticulum (ER) causes ER stress and activation of unfolded protein response (UPR). This response can trigger ER-associated degradation and autophagy, which clear unfolded proteins and restore protein homeostasis. Recently, it has become clear that ubiquitination plays an important role in the regulation of autophagy. In the present study, we investigated how the E3 ubiquitin ligase neural precursor cell-expressed, developmentally down-regulated protein 4-2 (Nedd4-2) interacts with ER stress and autophagy. In mice, we found that an increase in the expression of Nedd4-2, which was concomitant with the activation of the UPR and autophagy, was caused by a prolonged high-fructose and high-fat diet that induces ER stress in the liver. Pharmacologic induction of ER stress also led to an increase in Nedd4-2 expression in cultured cells, which was coincident with UPR and autophagy activation. The inhibition of inositol-requiring enzyme 1 significantly suppressed Nedd4-2 expression. Moreover, increased Nedd4-2 expression in vivo was closely associated with the activation of inositol-requiring enzyme 1 and increased expression of the spliced form of X-box binding protein 1. Furthermore, knockdown of Nedd4-2 in cultured cells suppressed both basal autophagy and ER stress-induced autophagy, whereas overexpression of Nedd4-2-induced autophagy. Taken together, our findings provide evidence that Nedd4-2 is up-regulated in response to ER stress by the spliced form of X-box binding protein 1 and that this is important in the induction of an appropriate autophagic response.-Wang, H. Sun, R.-Q., Camera, D., Zeng, X.-Y., Jo, E., Chan, S. M. H., Herbert, T. P., Molero, J. C., Ye, J.-M. Endoplasmic reticulum stress up-regulates Nedd4-2 to induce autophagy.

A highly sensitive assay of IRE1 activity using the small luciferase NanoLuc: Evaluation of ALS-related genetic and pathological factors

Activation of inositol-requiring enzyme 1 (IRE1) due to abnormal conditions of the endoplasmic reticulum (ER) is responsible for the cleavage of an unspliced form of X-box binding protein 1 (uXBP1), producing its spliced form (sXBP1). To estimate IRE1 activation, several analytical procedures using green fluorescence protein and firefly luciferase have been developed and applied to clarify the roles of IRE1-XBP1 signaling pathways during development and disease progression. In this study, we established a highly sensitive assay of IRE1 activity using a small luciferase, NanoLuc, which has approximately 100-fold higher activity than firefly luciferase. The NanoLuc reporter, which contained a portion of the spliced region of XBP1 upstream of NanoLuc, was highly sensitive and compatible with several types of cell lines. We found that NanoLuc was secreted into the extracellular space independent of the ER-Golgi pathway. The NanoLuc activity of an aliquot of culture medium from the neuroblastoma-spinal neuron hybrid cell line NSC-34 reflected the toxic stimuli-induced elevation of intracellular activity well. Using this technique, we evaluated the effects of several genetic and pathological factors associated with the onset and progression of amyotrophic lateral sclerosis (ALS) on NanoLuc reporter activity. Under our experimental conditions, inhibition of ER-Golgi transport by the overexpression of mutant Sar1 activated luciferase activity, whereas the co-expression of mutant SOD1 or the C-terminal fragment of TDP-43 (TDP-25) did not. The addition of homocysteine elevated the reporter activity; however, we did not observe any synergistic effect due to the overexpression of the mutant genes described above. Taken together, these data show that our analytical procedure is highly sensitive and convenient for screening useful compounds that modulate IRE1-XBP1 signaling pathways as well as for estimating IRE1 activation in several pathophysiological diseases.