Browsing by Subject "Unfolded protein response"
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Item A Benzenesulfonamide-based Mitochondrial Uncoupler Induces Endoplasmic Reticulum Stress and Immunogenic Cell Death in Epithelial Ovarian Cancer(American Association for Cancer Research, 2021) Bi, Fangfang; Jiang, Ziyan; Park, Wonmin; Hartwich, Tobias M. P.; Ge, Zhiping; Chong, Kay Y.; Yang, Kevin; Morrison, Madeline J.; Kim, Dongin; Kim, Jaeyeon; Zhang, Wen; Kril, Liliia M.; Watt, David S.; Liu, Chunming; Yang-Hartwich, Yang; Biochemistry and Molecular Biology, School of MedicineEpithelial ovarian cancer (EOC) is a leading cause of death from gynecologic malignancies and requires new therapeutic strategies to improve clinical outcomes. EOCs metastasize in the abdominal cavity through dissemination in the peritoneal fluid and ascites, efficiently adapt to the nutrient-deprived microenvironment, and resist current chemotherapeutic agents. Accumulating evidence suggests that mitochondrial oxidative phosphorylation is critical for the adaptation of EOC cells to this otherwise hostile microenvironment. Although chemical mitochondrial uncouplers can impair mitochondrial functions and thereby target multiple, essential pathways for cancer cell proliferation, traditional mitochondria uncouplers often cause toxicity that precludes their clinical application. In this study, we demonstrated that a mitochondrial uncoupler, specifically 2,5-dichloro-N-(4-nitronaphthalen-1-yl)benzenesulfonamide, hereinafter named Y3, was an antineoplastic agent in ovarian cancer models. Y3 treatment activated AMP-activated protein kinase and resulted in the activation of endoplasmic reticulum stress sensors as well as growth inhibition and apoptosis in ovarian cancer cells in vitro. Y3 was well tolerated in vivo and effectively suppressed tumor progression in three mouse models of EOC, and Y3 also induced immunogenic cell death of cancer cells that involved the release of damage-associated molecular patterns and the activation of antitumor adaptive immune responses. These findings suggest that mitochondrial uncouplers hold promise in developing new anticancer therapies that delay tumor progression and protect ovarian cancer patients against relapse.Item Autophagy participates in the unfolded protein response in Toxoplasma gondii(Oxford University Press, 2017-08-15) Nguyen, Hoa Mai; Berry, Laurence; Sullivan, William J., Jr.; Besteiro, Sébastien; Pharmacology and Toxicology, School of MedicineEnvironmental and genetic perturbations of endoplasmic reticulum (ER) function can lead to the accumulation of unfolded proteins. In these conditions, eukaryotic cells can activate a complex signaling network called the unfolded protein response (UPR) to reduce ER stress and restore cellular homeostasis. Autophagy, a degradation and recycling process, is part of this response. The parasitic protist Toxoplasma gondii is known to be able to activate the UPR upon ER stress, and we now show that this pathway leads to autophagy activation, supporting the idea of a regulated function for canonical autophagy as part of an integrated stress response in the parasites.Item Coordinated signaling of activating transcription factor 6α and inositol-requiring enzyme 1α regulates hepatic stellate cell-mediated fibrogenesis in mice(American Physiological Society, 2021) Xue, Fei; Lu, Jianwen; Buchl, Samuel C.; Sun, Liankang; Shah, Vijay H.; Malhi, Harmeet; Maiers, Jessica L.; Medicine, School of MedicineLiver injury and the unfolded protein response (UPR) are tightly linked, but their relationship differs with cell type and injurious stimuli. UPR initiation promotes hepatic stellate cell (HSC) activation and fibrogenesis, but the underlying mechanisms are unclear. Despite the complexity and overlap downstream of UPR transducers inositol-requiring protein 1α (IRE1α), activating transcription factor 6α (ATF6α), and protein kinase RNA-like ER kinase (PERK), previous research in HSCs primarily focused on IRE1α. Here, we investigated the fibrogenic role of ATF6α or PERK in vitro and HSC-specific UPR signaling in vivo. Overexpression of ATF6α, but not the PERK effector activating transcription factor 4 (ATF4), promoted HSC activation and fibrogenic gene transcription in immortalized HSCs. Furthermore, ATF6α inhibition through Ceapin-A7, or Atf6a deletion, disrupted transforming growth factor β (TGFβ)-mediated activation of primary human hepatic stellate cells (hHSCs) or murine hepatic stellate cells (mHSCs), respectively. We investigated the fibrogenic role of ATF6α in vivo through conditional HSC-specific Atf6a deletion. Atf6aHSCΔ/Δ mice displayed reduced fibrosis and HSC activation following bile duct ligation (BDL) or carbon tetrachloride (CCl4)-induced injury. The Atf6aHSCΔ/Δ phenotype differed from HSC-specific Ire1a deletion, as Ire1aHSCΔ/Δ mice showed reduced fibrogenic gene transcription but no changes in fibrosis compared with Ire1afl/fl mice following BDL. Interestingly, ATF6α signaling increased in Ire1aΔ/Δ HSCs, whereas IRE1α signaling was upregulated in Atf6aΔ/Δ HSCs. Finally, we asked whether co-deletion of Atf6a and Ire1a additively limits fibrosis. Unexpectedly, fibrosis worsened in Atf6aHSCΔ/ΔIre1aHSCΔ/Δ mice following BDL, and Atf6aΔ/ΔIre1aΔ/Δ mHSCs showed increased fibrogenic gene transcription. ATF6α and IRE1α individually promote fibrogenic transcription in HSCs, and ATF6α drives fibrogenesis in vivo. Unexpectedly, disruption of both pathways sensitizes the liver to fibrogenesis, suggesting that fine-tuned UPR signaling is critical for regulating HSC activation and fibrogenesis. NEW & NOTEWORTHY: ATF6α is a critical driver of hepatic stellate cell (HSC) activation in vitro. HSC-specific deletion of Atf6a limits fibrogenesis in vivo despite increased IRE1α signaling. Conditional deletion of Ire1α from HSCs limits fibrogenic gene transcription without impacting overall fibrosis. This could be due in part to observed upregulation of the ATF6α pathway. Dual loss of Atf6a and Ire1a from HSCs worsens fibrosis in vivo through enhanced HSC activation.Item Endoplasmic reticulum stress alters ryanodine receptor function in the murine pancreatic β cell(American Society for Biochemistry and Molecular Biology, 2018-11-12) Yamamoto, Wataru R.; Bone, Robert N.; Sohn, Paul; Syed, Farooq; Reissaus, Christopher A.; Mosley, Amber L.; Wijeratne, Aruna B.; True, Jason D.; Tong, Xin; Kono, Kono; Evans-Molina, Carmella; Biochemistry and Molecular Biology, School of MedicineAlterations in endoplasmic reticulum (ER) calcium (Ca2+) levels diminish insulin secretion and reduce β-cell survival in both major forms of diabetes. The mechanisms responsible for ER Ca2+ loss in β cells remain incompletely understood. Moreover, a specific role for either ryanodine receptor (RyR) or inositol 1,4,5-triphosphate receptor (IP3R) dysfunction in the pathophysiology of diabetes remains largely untested. To this end, here we applied intracellular and ER Ca2+ imaging techniques in INS-1 β cells and isolated islets to determine whether diabetogenic stressors alter RyR or IP3R function. Our results revealed that the RyR is sensitive mainly to ER stress–induced dysfunction, whereas cytokine stress specifically alters IP3R activity. Consistent with this observation, pharmacological inhibition of the RyR with ryanodine and inhibition of the IP3R with xestospongin C prevented ER Ca2+ loss under ER and cytokine stress conditions, respectively. However, RyR blockade distinctly prevented β-cell death, propagation of the unfolded protein response (UPR), and dysfunctional glucose-induced Ca2+ oscillations in tunicamycin-treated INS-1 β cells and mouse islets and Akita islets. Monitoring at the single-cell level revealed that ER stress acutely increases the frequency of intracellular Ca2+ transients that depend on both ER Ca2+ leakage from the RyR and plasma membrane depolarization. Collectively, these findings indicate that RyR dysfunction shapes ER Ca2+ dynamics in β cells and regulates both UPR activation and cell death, suggesting that RyR-mediated loss of ER Ca2+ may be an early pathogenic event in diabetes.Item Evolution of insulin at the edge of foldability and its medical implications(National Academy of Sciences, 2020-11-24) Rege, Nischay K.; Liu, Ming; Yang, Yanwu; Dhayalan, Balamurugan; Wickramasinghe, Nalinda P.; Chen, Yen-Shan; Rahimi, Leili; Guo, Huan; Haataja, Leena; Sun, Jinhong; Ismail-Beigi, Faramarz; Phillips, Nelson B.; Arvan, Peter; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineProteins have evolved to be foldable, and yet determinants of foldability may be inapparent once the native state is reached. Insight has emerged from studies of diseases of protein misfolding, exemplified by monogenic diabetes mellitus due to mutations in proinsulin leading to endoplasmic reticulum stress and β-cell death. Cellular foldability of human proinsulin requires an invariant Phe within a conserved crevice at the receptor-binding surface (position B24). Any substitution, even related aromatic residue TyrB24, impairs insulin biosynthesis and secretion. As a seeming paradox, a monomeric TyrB24 insulin analog exhibits a native-like structure in solution with only a modest decrement in stability. Packing of TyrB24 is similar to that of PheB24, adjoining core cystine B19-A20 to seal the core; the analog also exhibits native self-assembly. Although affinity for the insulin receptor is decreased ∼20-fold, biological activities in cells and rats were within the range of natural variation. Together, our findings suggest that the invariance of PheB24 among vertebrate insulins and insulin-like growth factors reflects an essential role in enabling efficient protein folding, trafficking, and secretion, a function that is inapparent in native structures. In particular, we envision that the para-hydroxyl group of TyrB24 hinders pairing of cystine B19-A20 in an obligatory on-pathway folding intermediate. The absence of genetic variation at B24 and other conserved sites near this disulfide bridge-excluded due to β-cell dysfunction-suggests that insulin has evolved to the edge of foldability. Nonrobustness of a protein's fitness landscape underlies both a rare monogenic syndrome and "diabesity" as a pandemic disease of civilization.Item General control nonderepressible 2 deletion predisposes to asparaginase-associated pancreatitis in mice(American Physiological Society, 2016-06-01) Phillipson-Weiner, Lindsey; Mirek, Emily T.; Wang, Yongping; McAuliffe, W. Geoffrey; Wek, Ronald C.; Anthony, Tracy G.; Biochemistry and Molecular Biology, School of MedicineTreatment with the antileukemic agent asparaginase can induce acute pancreatitis, but the pathophysiology remains obscure. In the liver of mice, eukaryotic initiation factor 2 (eIF2) kinase general control nonderepressible 2 (GCN2) is essential for mitigating metabolic stress caused by asparaginase. We determined the consequences of asparaginase treatment on the pancreata of wild-type (WT, GCN2-intact) and GCN2-deleted (ΔGcn2) mice. Mean pancreas weights in ΔGcn2 mice treated with asparaginase for 8 days were increased (P < 0.05) above all other groups. Histological examination revealed acinar cell swelling and altered staining of zymogen granules in ΔGcn2, but not WT, mice. Oil Red O staining and measurement of pancreas triglycerides excluded lipid accumulation as a contributor to acini appearance. Instead, transmission electron microscopy revealed dilatation of the endoplasmic reticulum (ER) and accumulation of autophagic vacuoles in the pancreas of ΔGcn2 mice treated with asparaginase. Consistent with the idea that loss of GCN2 in a pancreas exposed to asparaginase induced ER stress, phosphorylation of protein kinase R-like ER kinase (PERK) and its substrate eIF2 was increased in the pancreas of asparaginase-treated ΔGcn2 mice. In addition, mRNA expression of PERK target genes, activating transcription factors 4, 3, and 6 (Atf4, Atf3, and Atf6), fibroblast growth factor 21 (Fgf21), heat shock 70-kDa protein 5 (Hspa5), and spliced Xbp1 (sXbp1), as well as pancreas mass, was elevated in the pancreas of asparaginase-treated ΔGcn2 mice. Furthermore, genetic markers of oxidative stress [sirtuin (Sirt1)], inflammation [tumor necrosis factor-α (Tnfα)], and pancreatic injury [pancreatitis-associated protein (Pap)] were elevated in asparaginase-treated ΔGcn2, but not WT, mice. These data indicate that loss of GCN2 predisposes the exocrine pancreas to a maladaptive ER stress response and autophagy during asparaginase treatment and represent a genetic basis for development of asparaginase-associated pancreatitis.Item Inhibition of the Eukaryotic Initiation Factor-2-α Kinase PERK Decreases Risk of Autoimmune Diabetes in Mice(bioRxiv, 2024-06-03) Muralidharan, Charanya; Huang, Fei; Enriquez, Jacob R.; Wang, Jiayi E.; Nelson, Jennifer B.; Nargis, Titli; May, Sarah C.; Chakraborty, Advaita; Figatner, Kayla T.; Navitskaya, Svetlana; Anderson, Cara M.; Calvo, Veronica; Surguladze, David; Mulvihill, Mark J.; Yi, Xiaoyan; Sarkar, Soumyadeep; Oakes, Scott A.; Webb-Robertson, Bobbie-Jo M.; Sims, Emily K.; Staschke, Kirk A.; Eizirik, Decio L.; Nakayasu, Ernesto S.; Stokes, Michael E.; Tersey, Sarah A.; Mirmira, Raghavendra G.; Pediatrics, School of MedicinePreventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves β cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in β cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in β cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-βH1 human β cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.Item Obtaining Functional Proteomics Insights From Thermal Proteome Profiling Through Optimized Melt Shift Calculation and Statistical Analysis With InflectSSP(American Society for Biochemistry and Molecular Biology, 2023) McCracken, Neil A.; Liu, Hao; Runnebohm, Avery M.; Wijeratne, H. R. Sagara; Wijeratne, Aruna B.; Staschke, Kirk A.; Mosley, Amber L.; Biochemistry and Molecular Biology, School of MedicineThermal proteome profiling (TPP) is an invaluable tool for functional proteomics studies that has been shown to discover changes associated with protein–ligand, protein–protein, and protein–RNA interaction dynamics along with changes in protein stability resulting from cellular signaling. The increasing number of reports employing this assay has not been met concomitantly with new approaches leading to advancements in the quality and sensitivity of the corresponding data analysis. The gap between data acquisition and data analysis tools is important to fill as TPP findings have reported subtle melt shift changes related to signaling events such as protein posttranslational modifications. In this study, we have improved the Inflect data analysis pipeline (now referred to as InflectSSP, available at https://CRAN.R-project.org/package=InflectSSP) to increase the sensitivity of detection for both large and subtle changes in the proteome as measured by TPP. Specifically, InflectSSP now has integrated statistical and bioinformatic functions to improve objective functional proteomics findings from the quantitative results obtained from TPP studies through increasing both the sensitivity and specificity of the data analysis pipeline. InflectSSP incorporates calculation of a “melt coefficient” into the pipeline with production of average melt curves for biological replicate studies to aid in identification of proteins with significant melts. To benchmark InflectSSP, we have reanalyzed two previously reported datasets to demonstrate the performance of our publicly available R-based program for TPP data analysis. We report new findings following temporal treatment of human cells with the small molecule thapsigargin that induces the unfolded protein response as a consequence of inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase 2A. InflectSSP analysis of our unfolded protein response study revealed highly reproducible and statistically significant target engagement over a time course of treatment while simultaneously providing new insights into the possible mechanisms of action of the small molecule thapsigargin.Item The Pancreatic ß-cell Response to Secretory Demands and Adaption to Stress(Endocrine Society, 2021-11) Kalwat, Michael A.; Scheuner, Donalyn; Rodrigues-dos-Santos, Karina; Eizirik, Decio L.; Cobb, Melanie H.; Medicine, School of MedicinePancreatic β cells dedicate much of their protein translation capacity to producing insulin to maintain glucose homeostasis. In response to increased secretory demand, β cells can compensate by increasing insulin production capability even in the face of protracted peripheral insulin resistance. The ability to amplify insulin secretion in response to hyperglycemia is a critical facet of β-cell function, and the exact mechanisms by which this occurs have been studied for decades. To adapt to the constant and fast-changing demands for insulin production, β cells use the unfolded protein response of the endoplasmic reticulum. Failure of these compensatory mechanisms contributes to both type 1 and 2 diabetes. Additionally, studies in which β cells are "rested" by reducing endogenous insulin demand have shown promise as a therapeutic strategy that could be applied more broadly. Here, we review recent findings in β cells pertaining to the metabolic amplifying pathway, the unfolded protein response, and potential advances in therapeutics based on β-cell rest.Item Stress and Liver Fibrogenesis: Understanding the Role and Regulation of Stress Response Pathways in Hepatic Stellate Cells(Elsevier, 2023) Hanquier, Zachary; Misra, Jagannath; Baxter, Reese; Maiers, Jessica L.; Medical and Molecular Genetics, School of MedicineStress response pathways are crucial for cells to adapt to physiological and pathologic conditions. Increased transcription and translation in response to stimuli place a strain on the cell, necessitating increased amino acid supply, protein production and folding, and disposal of misfolded proteins. Stress response pathways, such as the unfolded protein response (UPR) and the integrated stress response (ISR), allow cells to adapt to stress and restore homeostasis; however, their role and regulation in pathologic conditions, such as hepatic fibrogenesis, are unclear. Liver injury promotes fibrogenesis through activation of hepatic stellate cells (HSCs), which produce and secrete fibrogenic proteins to promote tissue repair. This process is exacerbated in chronic liver disease, leading to fibrosis and, if unchecked, cirrhosis. Fibrogenic HSCs exhibit activation of both the UPR and ISR, due in part to increased transcriptional and translational demands, and these stress responses play important roles in fibrogenesis. Targeting these pathways to limit fibrogenesis or promote HSC apoptosis is a potential antifibrotic strategy, but it is limited by our lack of mechanistic understanding of how the UPR and ISR regulate HSC activation and fibrogenesis. This article explores the role of the UPR and ISR in the progression of fibrogenesis, and highlights areas that require further investigation to better understand how the UPR and ISR can be targeted to limit hepatic fibrosis progression.