A cytosolic surveillance mechanism activates the mitochondrial UPR.

The mitochondrial unfolded protein response (UPRmt) is essential to safeguard mitochondria from proteotoxic damage by activating a dedicated transcriptional response in the nucleus to restore proteostasis1,2. Yet, it remains unclear how the information on mitochondria misfolding stress (MMS) is signalled to the nucleus as part of the human UPRmt (refs. 3,4). Here, we show that UPRmt signalling is driven by the release of two individual signals in the cytosol-mitochondrial reactive oxygen species (mtROS) and accumulation of mitochondrial protein precursors in the cytosol (c-mtProt). Combining proteomics and genetic approaches, we identified that MMS causes the release of mtROS into the cytosol. In parallel, MMS leads to mitochondrial protein import defects causing c-mtProt accumulation. Both signals integrate to activate the UPRmt; released mtROS oxidize the cytosolic HSP40 protein DNAJA1, which leads to enhanced recruitment of cytosolic HSP70 to c-mtProt. Consequently, HSP70 releases HSF1, which translocates to the nucleus and activates transcription of UPRmt genes. Together, we identify a highly controlled cytosolic surveillance mechanism that integrates independent mitochondrial stress signals to initiate the UPRmt. These observations reveal a link between mitochondrial and cytosolic proteostasis and provide molecular insight into UPRmt signalling in human cells.

Noncanonical Form of ERAD Regulates Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy increases demands on protein folding, which causes an accumulation of misfolded proteins in the endoplasmic reticulum (ER). These misfolded proteins can be removed by the adaptive retrotranslocation, polyubiquitylation, and a proteasome-mediated degradation process, ER-associated degradation (ERAD), which, as a biological process and rate, has not been studied in vivo. To investigate a role for ERAD in a pathophysiological model, we examined the function of the functional initiator of ERAD, valosin-containing protein-interacting membrane protein (VIMP), positing that VIMP would be adaptive in pathological cardiac hypertrophy in mice. METHODS: We developed a new method involving cardiac myocyte-specific adeno-associated virus serovar 9-mediated expression of the canonical ERAD substrate, TCRα, to measure the rate of ERAD, ie, ERAD flux, in the heart in vivo. Adeno-associated virus serovar 9 was also used to either knock down or overexpress VIMP in the heart. Then mice were subjected to transverse aortic constriction to induce pressure overload-induced cardiac hypertrophy. RESULTS: ERAD flux was slowed in both human heart failure and mice after transverse aortic constriction. Surprisingly, although VIMP adaptively contributes to ERAD in model cell lines, in the heart, VIMP knockdown increased ERAD and ameliorated transverse aortic constriction-induced cardiac hypertrophy. Coordinately, VIMP overexpression exacerbated cardiac hypertrophy, which was dependent on VIMP engaging in ERAD. Mechanistically, we found that the cytosolic protein kinase SGK1 (serum/glucocorticoid regulated kinase 1) is a major driver of pathological cardiac hypertrophy in mice subjected to transverse aortic constriction, and that VIMP knockdown decreased the levels of SGK1, which subsequently decreased cardiac pathology. We went on to show that although it is not an ER protein, and resides outside of the ER, SGK1 is degraded by ERAD in a noncanonical process we call ERAD-Out. Despite never having been in the ER, SGK1 is recognized as an ERAD substrate by the ERAD component DERLIN1, and uniquely in cardiac myocytes, VIMP displaces DERLIN1 from initiating ERAD, which decreased SGK1 degradation and promoted cardiac hypertrophy. CONCLUSIONS: ERAD-Out is a new preferentially favored noncanonical form of ERAD that mediates the degradation of SGK1 in cardiac myocytes, and in so doing is therefore an important determinant of how the heart responds to pathological stimuli, such as pressure overload.

C9orf72 regulates the unfolded protein response and stress granule formation by interacting with eIF2α.

Rationale: A C9orf72 hexanucleotide repeat expansion (GGGGCC) is the most common genetic origin of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Haploinsufficiency of C9orf72 has been proposed as a possible disease mechanism (loss-of-function mechanism). Additionally, the aberrantly activated unfolded protein response (UPR) and stress granule (SG) formation are associated with the etiopathology of both ALS and FTD. However, the molecular determinants in this pathogenesis are not well characterized. Methods: We performed an immunoprecipitation-mass spectrometry (IP-MS) assay to identify potential proteins interacting with the human C9orf72 protein. We used C9orf72 knockout cell and rat models to determine the roles of C9orf72 in translation initiation and the stress response. Results: Here, we show that C9orf72, which is genetically and pathologically related to ALS and FTD, interacts with eukaryotic initiation factor 2 subunit alpha (eIF2α) and regulates its function in translation initiation. C9orf72 knockout weakens the interaction between eIF2α and eIF2B5, leading to global translation inhibition. Moreover, the loss of C9orf72 results in primary ER stress with activated UPR in rat spleens, which is one of the causes of splenomegaly with inflammation in C9orf72 -/- rats. Finally, C9orf72 delays SG formation by interacting with eIF2α in stressed cells. Conclusions: In summary, these data reveal that C9orf72 modulates translation initiation, the UPR and SG formation, which have implications for understanding ALS/FTD pathogenesis.

PRP-1, a toll-like receptor ligand, upregulates the unfolded protein response in human chondrosarcoma cells.

BACKGROUND: Previous studies showed that proline-rich polypeptide (PRP-1) is a ligand for innate immunity toll-like receptors (TLR), and an inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) which induces the death of chondrosarcoma cancer stem cells (CSC). The aim of this study was to investigate the effect of PRP-1 on the regulation of unfolded protein response (UPR) in human chondrosarcoma cells. MATERIALS AND METHODS: Lysates were prepared from a monolayer (bulk or ALDHhigh population), or spheroids chondrosarcoma cell cultures and treated with PRP-1 or control, followed by protein levels quantification by western blotting and mRNA expression by RT-qPCR of protein-RNA-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), CCAAT-enhancer-binding protein homologous protein (CHOP), activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1α), and X-box binding protein (XBP1). RESULTS: The PRP-1 has been shown to increase the expression of PERK, eIF2α, ATF4, CHOP, ATF6, IRE1α, and XBP1, on both protein and mRNA levels. CONCLUSION: PRP-1 activated UPR branches in monolayer, spheroid, and stem cell populations of human chondrosarcoma.

Inhibiting the IRE1α Axis of the Unfolded Protein Response Enhances the Antitumor Effect of AZD1775 in TP53 Mutant Ovarian Cancer.

Targeting the G2/M checkpoint mediator WEE1 has been explored as a novel treatment strategy in ovarian cancer, but mechanisms underlying its efficacy and resistance remains to be understood. Here, it is demonstrated that the WEE1 inhibitor AZD1775 induces endoplasmic reticulum stress and activates the protein kinase RNA-like ER kinase (PERK) and inositol-required enzyme 1α (IRE1α) branches of the unfolded protein response (UPR) in TP53 mutant (mtTP53) ovarian cancer models. This is facilitated through NF-κB mediated senescence-associated secretory phenotype. Upon AZD1775 treatment, activated PERK promotes apoptotic signaling via C/EBP-homologous protein (CHOP), while IRE1α-induced splicing of XBP1 (XBP1s) maintains cell survival by repressing apoptosis. This leads to an encouraging synergistic antitumor effect of combining AZD1775 and an IRE1α inhibitor MKC8866 in multiple cell lines and preclinical models of ovarian cancers. Taken together, the data reveal an important dual role of the UPR signaling network in mtTP53 ovarian cancer models in response to AZD1775 and suggest that inhibition of the IRE1α-XBP1s pathway may enhance the efficacy of AZD1775 in the clinics.

Arabidopsis TBP-ASSOCIATED FACTOR 12 ortholog NOBIRO6 controls root elongation with unfolded protein response cofactor activity.

Plant root growth is indeterminate but continuously responds to environmental changes. We previously reported on the severe root growth defect of a double mutant in bZIP17 and bZIP28 (bz1728) modulating the unfolded protein response (UPR). To elucidate the mechanism by which bz1728 seedlings develop a short root, we obtained a series of bz1728 suppressor mutants, called nobiro, for rescued root growth. We focused here on nobiro6, which is defective in the general transcription factor component TBP-ASSOCIATED FACTOR 12b (TAF12b). The expression of hundreds of genes, including the bZIP60-UPR regulon, was induced in the bz1728 mutant, but these inductions were markedly attenuated in the bz1728nobiro6 mutant. In view of this, we assigned transcriptional cofactor activity via physical interaction with bZIP60 to NOBIRO6/TAF12b. The single nobiro6/taf12b mutant also showed an altered sensitivity to endoplasmic reticulum stress for both UPR and root growth responses, demonstrating that NOBIRO6/TAF12b contributes to environment-responsive root growth control through UPR.

Sec62 promotes gastric cancer metastasis through mediating UPR-induced autophagy activation.

BACKGROUND AND AIMS: Sec62 is a membrane protein of the endoplasmic reticulum that facilitates protein transport. Its role in cancer is increasingly recognised, but remains largely unknown. We investigated the functional role of Sec62 in gastric cancer (GC) and its underlying mechanism. METHODS: Bioinformatics, tissue microarray, immunohistochemistry (IHC), western blotting (WB), quantitative polymerase chain reaction (qPCR), and immunofluorescence were used to examine the expression of target genes. Transwell, scratch healing assays, and xenograft models were used to evaluate cell migration and invasion. Transmission electron microscopy and mRFP-GFP-LC3 double-labeled adenoviruses were used to monitor autophagy. Co-immunoprecipitation (CO-IP) was performed to evaluate the binding activity between the proteins. RESULTS: Sec62 expression was upregulated in GC, and Sec62 upregulation was an independent predictor of poor prognosis. Sec62 overexpression promoted GC cell migration and invasion both in vitro and in vivo. Sec62 promoted migration and invasion by affecting TIMP-1 and MMP2/9 balance. Moreover, Sec62 could activate autophagy by upregulating PERK/ATF4 expression and binding to LC3II with concomitant FIP200/Beclin-1/Atg5 activation. Furthermore, autophagy blockage impaired the promotive effects of Sec62 on GC cell migration and invasion, whereas autophagy activation rescued the inhibitory effect of Sec62 knockdown on GC metastasis. Notably, Sec62 inhibition combined with autophagy blockage exerted a synergetic anti-metastatic effect in vitro and in vivo. CONCLUSION: Sec62 promotes GC metastasis by activating autophagy and subsequently regulating TIMP-1 and MMP2/9 balance. The activation of autophagy by Sec62 may involve the unfolded protein response (UPR)-related PERK/ATF4 pathway and binding of LC3II during UPR recovery involving FIP200/Beclin-1/Atg5 upregulation. Specifically, the dual inhibition of Sec62 and autophagy may provide a promising therapeutic strategy for GC metastasis.

Targeting the Unfolded Protein Response as a Disease-Modifying Pathway in Dementia.

Dementia is a global medical and societal challenge; it has devastating personal, social and economic costs, which will increase rapidly as the world's population ages. Despite this, there are no disease-modifying treatments for dementia; current therapy modestly improves symptoms but does not change the outcome. Therefore, new treatments are urgently needed-particularly any that can slow down the disease's progression. Many of the neurodegenerative diseases that lead to dementia are characterised by common pathological responses to abnormal protein production and misfolding in brain cells, raising the possibility of the broad application of therapeutics that target these common processes. The unfolded protein response (UPR) is one such mechanism. The UPR is a highly conserved cellular stress response to abnormal protein folding and is widely dysregulated in neurodegenerative diseases. In this review, we describe the basic machinery of the UPR, as well as the evidence for its overactivation and pathogenicity in dementia, and for the marked neuroprotective effects of its therapeutic manipulation in murine models of these disorders. We discuss drugs identified as potential UPR-modifying therapeutic agents-in particular the licensed antidepressant trazodone-and we review epidemiological and trial data from their use in human populations. Finally, we explore future directions for investigating the potential benefit of using trazodone or similar UPR-modulating compounds for disease modification in patients with dementia.

Regulation of the unfolded protein response transducer IRE1α by SERPINH1 aggravates periodontitis with diabetes mellitus via prolonged ER stress.

The hyperglycemic microenvironment induced by diabetes mellitus aggravates the inflammatory response, in which the IRE1α signal transduction pathway of the unfolded protein response (UPR) participates. However, the mechanism by which hyperglycemia regulates the IRE1α signaling pathway and affects endoplasmic reticulum (ER) homeostasis in human gingival epithelium in periodontitis with diabetes mellitus remains unknown. Our current data provide evidence that diabetes mellitus causes a hyperinflammatory response in the gingival epithelium, which accelerates periodontal inflammation. Next, we assessed UPR-IRE1α signaling in periodontitis with diabetes mellitus by examining human clinical gingival epithelium samples from healthy subjects, subjects with periodontitis and subjects with periodontitis with diabetes mellitus and by in vitro challenge of human epithelial cells with a hyperglycemic microenvironment. The results showed that a hyperglycemic microenvironment inhibited the IRE1α/XBP1 axis, decreased the expression of a UPR target gene (GRP78), and ultimately impaired the UPR, causing ER stress to be prolonged or more severe in human gingival epithelium. Subsequently, RNA sequencing (RNA-seq) data was analyzed to investigate the expression of ER-related genes in human gingival epithelium. Experiments verified that the mechanism by which periodontitis is aggravated in individuals with diabetes mellitus may involve decreased SERPINH1 expression. Furthermore, experiments in SERPINH1-knockdown and SERPINH1-overexpression models established in vitro indicated that SERPINH1 might act as an activator of IRE1α, maintaining human gingival epithelium homeostasis and reducing proinflammatory cytokine expression by preventing prolonged ER stress induced by high-glucose conditions. In conclusion, regulation of the UPR transducer IRE1α by SERPINH1 alleviates periodontitis with diabetes mellitus by mitigating prolonged ER stress. This finding provides evidence for the further study of periodontitis with diabetes mellitus.

NHR-80 senses the mitochondrial UPR to rewire citrate metabolism for lipid accumulation in Caenorhabditis elegans.

Mitochondria are known as the powerhouse of the cell. Dysfunction of mitochondria homeostasis induces the mitochondrial unfolded protein response (UPRmt), altering cellular metabolism. How cells sense the UPRmt to rewire metabolism is largely unknown. Here, we show that inactivation of either the citric/tricarboxylic acid (TCA) cycle enzymes aco-2 or idha-1, which encode aconitase and isocitrate dehydrogenase respectively, leads to citrate accumulation. In Caenorhabditis elegans, both in vitro and in vivo, citrate accumulation consequently triggers the UPRmt and also promotes lipid accumulation. The transcription factor DVE-1 binds to the promoter of the nuclear hormone receptor nhr-80 to transactivate its expression. NHR-80 then upregulates lipogenesis and lipid accumulation, shifting excess citrate for use in lipogenesis and for storage as triacylglycerol in lipid droplets. Inactivation of DVE-1 or NHR-80 fully abolishes the citrate-induced lipid accumulation. Therefore, our work uncovers a DVE-1-NHR-80-lipogenesis axis linking the transmission of the mitochondrial stress signal to lipid metabolism.

AMPK-PERK axis represses oxidative metabolism and enhances apoptotic priming of mitochondria in acute myeloid leukemia.

AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.

GrpEL1 regulates mitochondrial unfolded protein response after experimental subarachnoid hemorrhage in vivo and in vitro.

Subarachnoid hemorrhage (SAH) is a hemorrhagic stroke disease with high mortality and disability rates. Neurological recovery in early brain injury (EBI) after SAH is a crucial stage to reduce complications and improve the prognosis of patients. The mitochondrial unfolded protein response (UPRmt) is an essential mitochondrial damage repair process, that degrades aggresomes formed by misfolded proteins. UPRmt is a response to cellular stress and enhances mitochondrial homeostasis. GrpEL1 is a nucleotide exchange factor that assists mtHSP70 in nonnative folding proteins in mitochondria. However, the role of UPRmt and GrpEL1 after SAH is unclear. Western blot, Immunofluorescence, Aggresome staining, JC-1 staining were conducted to detect UPRmt after SAH in vivo and in vitro. The results showed that the UPRmt-related proteins HSP60 and mtHSP70 did not change in the EBI after SAH in vivo and in vitro but increased in the isolated mitochondria. In vitro primary neurons treated with oxyhemoglobin (OxyHb) achieved the same result as MG132 induction, increasing neuron protein aggresomes. The expression of GRPEL1 was unchanged in total protein and mitochondrial protein by Western blot. Co-immunoprecipitation (Co-IP) experiments showed that the GRPEL1-mtHSP70 complex decreased after OxyHb treatment. After GRPEL1 overexpression, the GRPEL1-mtHSP70 complex increased, while aggresome in neurons decreased. JC-1 showed an increased mitochondrial membrane potential, ATP content increased, and Western blot analysis revealed decreased cleaved-Caspase 9, suggesting improved mitochondrial function. In conclusion, the reduced GrpEL1-mtHSP70 complex is an essential factor affecting UPRmt in EBI after SAH. Increasing GrpEL1 promotes GrpEL1 and mtHSP70 binding, promoting the neuronal mitochondrial homeostasis, and might be an essential clinical intervention target for EBI after SAH.

Genetic modulators associated with regulatory surveillance of mitochondrial quality control, play a key role in regulating stress pathways and longevity in C. elegans.

The multi-factorial Parkinson's disease (PD) is known to be associated with mitochondrial dysfunction, endoplasmic reticulum stress, alpha synuclein aggregation and dopaminergic neuronal death, with oxidative stress being a common denominator to these underlying processes. The perception of mitochondria being 'just ATP producing compartments' have been counterpoised as studies, particularly related to PD, have underlined their strong role in cause and progression of the disease. During PD pathogenesis, neurons encounter chronic stress conditions mainly due to failure of Mitochondrial Quality Control (MQC) machinery. To dissect the regulatory understanding of mitochondrial dysfunction during neurological disease progression, we endeavored to identify key regulatory endpoints that control multiple facets of MQC machinery. Our studies, employing transgenic C. elegans strain expressing human α-synuclein, led us to identification of mitochondrial genes nuo-5 (involved in oxidative phosphorylation), F25B4.7 (exhibits ATP transmembrane transporter activity) and C05D11.9 (having ribonuclease activity), which form predicted downstream targets of most elevated and down-regulated mi-RNA molecules. RNAi mediated silencing, gene ontology and functional genomics analysis studies demonstrated their role in modulating major MQC pathways. The attenuated MQC pathways mainly affected clearance of misfolded and aggregated proteins, redox homeostasis and longevity with compromised dopaminergic functions. Overexpression of the mitochondrial genes by 3 beta-hydroxyl steroid, Tomatidine, was found to curtail the redox imbalance thus leading to amelioration of effects associated with PD and an increase in the lifespan of treated nematodes. Therefore, this study unveils the regulatory role of mitochondrial genes as critical modulators of stress control involved in effects associated with PD pathogenesis.

Pharmacological inhibition of translocon is sufficient to alleviate endoplasmic reticulum stress and improve Ca2+ handling and contractile recovery of stunned myocardium.

INTRODUCTION: The function of endoplasmic reticulum (ER), a Ca2+ storage compartment and site of protein folding, is altered by disruption of intracellular homeostasis. Misfolded proteins accumulated in the ER lead to ER stress (ERS), unfolded protein response (UPR) activation and ER Ca2+ loss. Myocardial stunning is a temporary contractile dysfunction, which occurs after brief ischemic periods with minimal or no cell death, being oxidative stress and Ca2+ overload potential underlying mechanisms. Myocardial stunning induces ERS response with negatively impact on the post-ischemic mechanical performance through an unknown mechanism. AIMS: In this study, we explored whether ER Ca2+ efflux through the translocon, a major Ca2+ leak channel, contributes to Ca2+ mishandling and the consequent contractile abnormalities of the stunned myocardium. METHODS: Mechanical performance, cytosolic Ca2+, UPR markers and oxidative state were evaluated in perfused rat/mouse hearts subjected to a brief ischemia followed by reperfusion (I/R) in absence or presence of the translocon inhibitor, emetine (1 µM), comparing its effects with those of the chaperones TUDCA (30 µM) and 4-PBA (3 mM). RESULTS: Emetine treatment precluded the I/R-induced increase in UPR signaling markers and improved the contractile recovery together with a remarkable attenuation in myocardial stiffness when compared to I/R hearts with no drug. This alleviation of I/R-induced mechanical abnormalities was more effective than that obtained with the chemical chaperones, TUDCA and 4-PBA. Moreover, emetine treatment produced a striking improvement in diastolic Ca2+ handling with a partial recovery of the I/R-induced oxidative stress. CONCLUSION: Blocking ER Ca2+ store depletion via translocon suppressed ER stress and improved mechanical performance and diastolic Ca2+ handling of stunned myocardium. Modulation of translocon permeability emerges as a therapeutic approach to face dysfunctional consequences of the I/R injury.

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.

An update on the unfolded protein response in brain ischemia: Experimental evidence and therapeutic opportunities.

After ischemic stroke or cardiac arrest, brain ischemia occurs. Currently, no pharmacologic intervention that targets cellular processes has proven effective in improving neurologic outcome in patients after brain ischemia. Recent experimental research has identified the crucial role of proteostasis in survival and recovery of cells after ischemia. In particular, the unfolded protein response (UPR), a key signaling pathway that safeguards cellular proteostasis, is emerging as a promising therapeutic target for brain ischemia. For some time, the UPR has been known to play a critical role in the pathophysiology of brain ischemia; however, only in the recent years has the field grown substantially, largely due to the extensive use of UPR-specific mouse genetic models and the rapidly expanding availability of pharmacologic tools that target the UPR. In this review, we provide a timely update on the progress in our understanding of the UPR in experimental brain ischemia, and discuss the therapeutic implications of targeting the UPR in ischemic stroke and cardiac arrest.

Ubiquitination of ATF6 by disease-associated RNF186 promotes the innate receptor-induced unfolded protein response.

Properly balancing microbial responses by the innate immune system through pattern recognition receptors (PRRs) is critical for intestinal immune homeostasis. Ring finger protein 186 (RNF186) genetic variants are associated with inflammatory bowel disease (IBD). However, functions for the E3 ubiquitin ligase RNF186 are incompletely defined. We found that upon stimulation of the PRR nucleotide-binding oligomerization domain containing 2 (NOD2) in human macrophages, RNF186 localized to the ER, formed a complex with ER stress sensors, ubiquitinated the ER stress sensor activating transcription factor 6 (ATF6), and promoted the unfolded protein response (UPR). These events, in turn, led to downstream signaling, cytokine secretion, and antimicrobial pathway induction. Importantly, RNF186-mediated ubiquitination of K152 on ATF6 was required for these outcomes, highlighting a key role for ATF6 ubiquitination in PRR-initiated functions. Human macrophages transfected with the rare RNF186-A64T IBD risk variant and macrophages from common rs6426833 RNF186 IBD risk carriers demonstrated reduced NOD2-induced outcomes, which were restored by rescuing UPR signaling. Mice deficient in RNF186 or ATF6 demonstrated a reduced UPR in colonic tissues, increased weight loss, and less effective clearance of bacteria with dextran sodium sulfate-induced injury and upon oral challenge with Salmonella Typhimurium. Therefore, we identified that RNF186 was required for PRR-induced, UPR-associated signaling leading to key macrophage functions; defined that RNF186-mediated ubiquitination of ATF6 was essential for these functions; and elucidated how RNF186 IBD risk variants modulated these outcomes.

Living Dangerously: Protective and Harmful ER Stress Responses in Pancreatic ß-Cells.

Type 2 diabetes (T2D) is a growing cause of poor health, psychosocial burden, and economic costs worldwide. The pancreatic ß-cell is a cornerstone of metabolic physiology. Insulin deficiency leads to hyperglycemia, which was fatal before the availability of therapeutic insulins; even partial deficiency of insulin leads to diabetes in the context of insulin resistance. Comprising only an estimated 1 g or <1/500th of a percent of the human body mass, pancreatic ß-cells of the islets of Langerhans are a vulnerable link in metabolism. Proinsulin production constitutes a major load on ß-cell endoplasmic reticulum (ER), and decompensated ER stress is a cause of ß-cell failure and loss in both type 1 diabetes (T1D) and T2D. The unfolded protein response (UPR), the principal ER stress response system, is critical for maintenance of ß-cell health. Successful UPR guides expansion of ER protein folding capacity and increased ß-cell number through survival pathways and cell replication. However, in some cases the ER stress response can cause collateral ß-cell damage and may even contribute to diabetes pathogenesis. Here we review the known beneficial and harmful effects of UPR pathways in pancreatic ß-cells. Improved understanding of this stress response tipping point may lead to approaches to maintain ß-cell health and function.

Influenza A viruses balance ER stress with host protein synthesis shutoff.

Excessive production of viral glycoproteins during infections poses a tremendous stress potential on the endoplasmic reticulum (ER) protein folding machinery of the host cell. The host cell balances this by providing more ER resident chaperones and reducing translation. For viruses, this unfolded protein response (UPR) offers the potential to fold more glycoproteins. We postulated that viruses could have developed means to limit the inevitable ER stress to a beneficial level for viral replication. Using a relevant human pathogen, influenza A virus (IAV), we first established the determinant for ER stress and UPR induction during infection. In contrast to a panel of previous reports, we identified neuraminidase to be the determinant for ER stress induction, and not hemagglutinin. IAV relieves ER stress by expression of its nonstructural protein 1 (NS1). NS1 interferes with the host messenger RNA processing factor CPSF30 and suppresses ER stress response factors, such as XBP1. In vivo viral replication is increased when NS1 antagonizes ER stress induction. Our results reveal how IAV optimizes glycoprotein expression by balancing folding capacity.