bims-unfpre Biomed News
on Unfolded protein response
Issue of 2022‒05‒15
nineteen papers selected by
Susan Logue
University of Manitoba
  1. The Hsp90 Chaperone Regulates p38 Activation During ER Stress.
  2. IL-10 Attenuates ER-Stress Induced Apoptosis in Doxorubicin Treated Cardiomyocytes.
  3. TLR4 Promotes ER stress and Unfolded Protein Response in Hypertension-Induced Kidney Injury.
  4. Cyclosporine A but not tacrolimus induces pro-apoptotic endoplasmic reticulum stress in renal tubular cells.
  5. ROS and Endoplasmic Reticulum Stress in Pulmonary Disease.
  6. The Unfolded Protein Response Regulates ER-phagy in Fibrogenic Hepatic Stellate Cells.
  7. Common signaling principles and interconnectivity in the ISR-UPR networks.
  8. Mitochondrial ROS in Slc4a11 KO Corneal Endothelial Cells Lead to ER Stress.
  9. Differential Behavior of 4E-BP1 and 4E-BP2 Under ER Stress.
  10. Endoplasmic Reticulum (ER) Stress and Its Role in Pancreatic β-Cell Dysfunction and Senescence in Type 2 Diabetes.
  11. The Molecular Chaperone, GRP170, Protects Against Acute Kidney Injury and ER Stress in Mice.
  12. Rhomboid pseudoproteases: An Achilles heel's for BCL-2/IP3R-dependent resistance to ER stress-induced cell death.
  13. Activation of Sigma-1 Receptor Alleviates ER-Associated Cell Death and Microglia Activation in Traumatically Injured Mice.
  14. Disruption of Proteostasis Causes IRE1 Mediated Reprogramming of Alveolar Epithelial Cells in Lung Fibrosis.
  15. The Acyltransferase Gpc1 is Both a Target and an Effector of the Unfolded Protein Response (UPR).
  16. New insights into IRE1α activation and function in anti-tumor immunity.
  17. A novel and important role of UFM1-binding protein 1 (UFBP1) in the regulation of ER and cardiac homeostasis.
  18. The Epithelial-Specific ER Stress Sensor IRE1β Enables Host-Microbiota Crosstalk to Affect Colon Goblet Cell Development.
  19. Conformational Dynamics Linking Kinase and Endonuclease Domains Explain the Divergent Function of IRE1 Paralogues.
  1. FASEB J. 2022 May;36 Suppl 1
    The Hsp90 Chaperone Regulates p38 Activation During ER Stress.
    Madeleine R Enos, Jay L Brewster.
      Diseases such as diabetes, Alzheimer's disease, and Parkinson's disease can be characterized by the presence of endoplasmic reticulum stress. The endoplasmic reticulum is an organelle that plays a major role in protein synthesis, but an accumulation of misfolded proteins within the ER lumen causes the organelle to release a stress signal through the unfolded protein response (UPR). Despite being present in many disorders, there are alternative signaling routes within the UPR branches that are not fully understood. Perk dimerization initiates one of three main signaling cascades in the UPR by phosphorylating eIF2α and causing a cell-wide pause in mRNA translation; however, a study has shown that outside of the established pathway, activation of Perk results in p38 activation, which induces apoptosis during prolonged ER stress. The mechanism responsible for p38 activation is still unknown. One route for elucidating the regulation of p38 activation during ER stress is the Hsp90 chaperone. The Hsp90 chaperone complex sequesters p38 in the cytoplasm and prevents its auto-activation under normal conditions, so we hypothesized that Perk activation may mediate post-translational modification of the Hsp90 complex in a manner that would release p38, allowing it to activate. In this study, Tunicamycin-induced ER stress and Hsp90 inhibition were applied to BHK21 fibroblasts, a cell line in which ER stress has been well characterized, and dissociation of the p38-Hsp90 complex was measured through immunoblot and co-immunoprecipitation analysis. Initial immunoblot results from 50nM and 200nM of tunicamycin reconfirm that moderate doses of tunicamycin induced p38 activation in a dose dependent manner. Co-immunoprecipitation of Hsp90 reconfirmed that tunicamycin-induced ER stress causes p38 to dissociate from Hsp90. Previous experiments with co-immunoprecipitation of p38 by Jingyi Chen revealed that treatment with 50nM of tunicamycin resulted in 0.3 decrease in fold induction after 15 minutes. Unexpectedly, we found that inhibition of Hsp90 causes it to exhibit increased binding of p38 with an peak average fold increase of 7 after treatment with 5uM of Hsp90-I (17-AAG) for 30 minutes, which suggests Hsp90 as a potential regulator for p38. Because p38 plays an important role in the induction of many intracellular signaling pathways, learning ways to regulate its actions within the context of ER stress may be beneficial in mediating symptoms of disease.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R6362
  2. FASEB J. 2022 May;36 Suppl 1
    IL-10 Attenuates ER-Stress Induced Apoptosis in Doxorubicin Treated Cardiomyocytes.
    Akshi Malik, Ashim K Bagchi, Davinder S Jassal, Pawan K Singal.
      Despite its antitumor efficacy, use of Doxorubicin (Dox) is limited by its cardiotoxic side effects. In Dox-induced cardiomyopathy (DIC), a causal role of oxidative stress (OS) has gained a lot of traction. Among other subcellular changes, Dox causes vacuolization of the cytoplasm and dilation of endoplasmic reticulum (ER). Dox also causes an excessive production of reactive oxygen species as well as imbalance in redox state both of which can affect ER homeostasis and protein folding. An accumulation of unfolded/misfolded proteins triggers the unfolded protein response (UPR) in the ER, which activates transcriptional and translation pathways involving: a) Protein-kinase like endoplasmic reticulum kinase (PERK); b) activating transcription factor 6α (ATF6α); and c) inositol requiring kinase1α (IRE1α) (Fig.1). Thus leading to the activation of ER chaperons, protein foldases and induce the expression of various genes, including X-box binding protein 1 (XBP1). UPR plays an important role in the attenuation of protein translation, upregulation of chaperones and antioxidant expression, and thus maintains ER homeostasis. However, if UPR fails to restore ER homeostasis, ER stress-initiated apoptotic signaling pathways are activated via CCAAT/enhancer homologous protein (CHOP), caspase-12 activation, phosphorylation of c-JUN NH2-terminal kinase (JNK) and pro-apoptotic gene expression. Understanding the role of these pathways of the ER under Dox treatment will highlight potential targets for new interventions as an adjunct therapy to restore ER functioning. In this study on isolated rat cardiomyocytes, we analyzed ER-stress markers, spliced XBP1 mRNA, and ER-apoptotic proteins. We also assessed protective effects of Interleukin-10 (IL-10) against Dox initiated ER-induced apoptosis and overall ER-stress. Dox significantly decreased the viability of cardiomyocytes and upregulated ER stress markers (GRP78, GRP94, and PDI). Interestingly, the exogenous administration of IL-10, one hour prior to the exposure of cardiomyocytes to Dox for 24hrs, was found to be effective in suppressing the expressions of ER-stress markers as well as ER-related apoptotic proteins and preserved cell viability. Additionally, significant changes in downstream targets of IRE1α, XBP1 in IL-10 treatment were recorded. IL-10 significantly increased the expression of unspliced XBP1 (U), an inhibitor of spliced XBP1(S) particularly in Dox-treated cells. This was followed by a reduction in the UPR mediated cell death in cardiomyocytes confirmed by a decrease in expression of GRP78, GRP94, and spliced XBP1 (S). IRE1 α arm of UPR initiates phosphorylation of JNK, which was reduced by IL-10 in Dox treated cells. Dox also initiated activation of procaspase-12, which is known to activate caspase-3 and apoptosis. IL-10 significantly reduced the activation of procaspase-12 and cell death. These data suggest that IL-10 provides protection against Dox-induced cardiotoxicity by restoring ER homeostasis.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2735
  3. FASEB J. 2022 May;36 Suppl 1
    TLR4 Promotes ER stress and Unfolded Protein Response in Hypertension-Induced Kidney Injury.
    Sathnur Pushpakumar, Subir Kumar Juin, Jaden Whitehead, Utpal Sen.
      Hypertension is the second leading cause of chronic kidney injury in the world. Endoplasmic reticulum (ER) is an important cell organelle that is involved in the synthesis, folding, and modification of various proteins to maintain homeostasis. ER stress is reported in hypertension-induced damage of kidney, heart and brain. Further, TLR4 activation and signaling is linked to the development and progression of hypertension induced damage in various organs; however, the interplay between TLR4 and ER stress in renal pathology remains unknown. In the present study, we investigated whether TLR4 activation contributes to renal ER stress and inflammation in Angiotensin-II (Ang-II) induced hypertension and whether TLR4 deficiency protects the kidney by suppressing ER stress. C3H/HeouJ (Normal TLR4, TLR4N) and C3H/HeJ (Dysfunctional TLR4, TLR4M) aged 12-14 weeks were treated with Ang-II (1000 ng/Kg/min. x 28 days) using osmotic pumps. We found renal function was impaired in TLR4N mice compared to TLR4M. The expression of ER stress markers (ATF6, p-IRE1α and p-PERK), renal inflammation (TNFα, IL-1β, CCL2) and transcription factor p-NFkB was upregulated in TLR4N kidneys compared to TLR4M. Further, ER stress in TLR4N mice was associated with mRNA changes in GRP78, sXBP1, ATF4, CHOP genes that are involved in unfolded protein response. In TLR4N mice, renal inflammation was predominant in the tubular area and ER stress was observed in both glomerular and tubular area. TLR4M mice showed reduced ER stress and inflammation in the kidney. Taken together, our results suggest significant interaction between TLR4 and ER stress that may act as an obligatory step in mediating renal inflammation and damage, and TLR4 deficiency attenuates injury by regulating ER stress and unfolded protein response in Ang-II-induced hypertension.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3963
  4. FASEB J. 2022 May;36 Suppl 1
    Cyclosporine A but not tacrolimus induces pro-apoptotic endoplasmic reticulum stress in renal tubular cells.
    Duygu E Yilmaz, Karin Kirschner, Hasan Demirci, Nina Himmerkus, Sebastian Bachmann, Kerim Mutig.
      Calcineurin inhibitors (CNI), cyclosporine A (CsA) or tacrolimus (Tac), belong to the first-line immunosuppressive strategies in organ-transplanted patients. Both CNI may exert renal side effects, which are typically stronger in patients receiving CsA. Continued clinical demand for CsA requires improved understanding of its nephrotoxicity. Calcineurin inhibition by CsA occurs via complexes with cyclophilins, whereas Tac recruits FKBP12 instead. We hypothesized that impaired chaperone function of cyclophilins may aggravate CsA cytotoxicity due to induction of endoplasmic reticulum (ER) stress and pro-apoptotic unfolded protein response (UPR). To address this hypothesis, effects of CsA vs. Tac (10 µM for 6 h) on the UPR signaling were evaluated in cultured human embryonic kidney cells (HEK293), human renal proximal tubular (PT) epithelial cells (HRPTEpC), and isolated rat PTs using quantitative PCR, immunoblotting, and immunofluorescence staining. An established ER stress inducer, thapsigargin (Tg) served as a positive control. CsA and Tg, but not Tac, induced ER stress and pro-apoptotic UPR in HEK293 cells, which was reflected by increased levels of key UPR products (BiP, CHOP, spliced XBP1, and phosphorylated IRE1a) and cleaved caspase 3 (cCas3). Similar effects were observed in HRPTEpC cells and isolated rat PTs. Knockdown of cyclophilin A or B isoforms in HEK293 cells using siRNA augmented CHOP and cCas3 to an extent comparable with CsA treatment suggesting relevance of cyclophilin activity for intact proteostasis. Application of chemical chaperones, TUDCA or 4-PBA, alleviated the CsA-induced UPR suggesting that boosting the protein folding may have protective effects against CsA cytotoxicity. Along the same line, genetic suppression of UPR in HEK293 cells by CRISPR/Cas9-mediated deletion of the key ER stress sensors, PERK or ATF6, blunted the pro-apoptotic UPR in response to CsA. In summary, these results suggest that nephrotoxic effects of CsA are partially mediated by suppression of cyclophilins, ER stress, and pro-apoptotic UPR. Pharmacological modulation of UPR bears the potential to alleviate CsA nephrotoxicity.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3571
  5. Front Pharmacol. 2022 ;13 879204
    ROS and Endoplasmic Reticulum Stress in Pulmonary Disease.
    Xiangning Cui, Yang Zhang, Yingdong Lu, Mi Xiang.
      Pulmonary diseases are main causes of morbidity and mortality worldwide. Current studies show that though specific pulmonary diseases and correlative lung-metabolic deviance own unique pathophysiology and clinical manifestations, they always tend to exhibit common characteristics including reactive oxygen species (ROS) signaling and disruptions of proteostasis bringing about accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER). ER is generated by the unfolded protein response. When the adaptive unfolded protein response (UPR) fails to preserve ER homeostasis, a maladaptive or terminal UPR is engaged, leading to the disruption of ER integrity and to apoptosis, which is called ER stress. The ER stress mainly includes the accumulation of misfolded and unfolded proteins in lumen and the disorder of Ca2+ balance. ROS mediates several critical aspects of the ER stress response. We summarize the latest advances in of the UPR and ER stress in the pathogenesis of pulmonary disease and discuss potential therapeutic strategies aimed at restoring ER proteostasis in pulmonary disease.
    Keywords:  er stress; oxidative stress; pulmonary disease; reactive oxygen spieces; unfolded protein response
    DOI:  https://doi.org/10.3389/fphar.2022.879204
  6. FASEB J. 2022 May;36 Suppl 1
    The Unfolded Protein Response Regulates ER-phagy in Fibrogenic Hepatic Stellate Cells.
    Zachary Hanquier, Yvonne Thomason, Jessica L Maiers.
      INTRODUCTION: Liver injury activates Hepatic Stellate Cells (HSCs) which secrete fibrogenic proteins such as collagen I to promote scarring. Increased translation of the collagen I precursor procollagen I by HSCs causes ER stress due to 30% of nascent procollagen I failing to fold correctly, placing a burden on the ER; however, it is unclear how HSCs adapt to this stress. ER stress activates the Unfolded Protein Response (UPR) which signals through pathways mediated by Activating Transcription Factor 6α (ATF6α), Inositol Requiring Enzyme 1 (IRE1α), or Protein Kinase R-like ER kinase (PERK). ATF6α or IRE1α inhibition limits HSC activation and promotes apoptosis in vitro, while deletion of ATF6α or IRE1α limits fibrogenesis and reduces HSC number in vivo. While it is clear that the UPR plays a crucial role in HSC activation and survival, the mechanisms that facilitate this role are unknown. Recent work shows that misfolded procollagen I can undergo ER-phagy, where receptors on the ER recruit autophagic membranes to engulf portions of the ER containing misfolded proteins and target them for degradation. ER-phagy can be activated by ER stress, but the fibrogenic role and regulation of ER-phagy in HSCs is unknown. We hypothesized that UPR induction of ER-phagy targets misfolded procollagen I for degradation, thus promoting HSC survival and fibrogenesis.METHODS: Expression of ER-phagy receptors (Cell-cycle progression gene 1 (CCPG1), Family with sequence similarity 134B (FAM134B), and Atlastin 3 (ATL3)) was assessed in 1) livers from patients with advanced fibrosis or controls (GSE25097), 2) murine livers harvested from age- and sex-matched mice following bile-duct ligation (3 weeks) or sham controls; and 3) primary hHSCs or mHSCs, or immortalized hHSCs (LX-2) following TGFβ treatment (2ng/mL, 24h). ER-phagic flux was measured in LX-2 cells expressing a fluorescent ER-phagy reporter (RAMP4-GFP-mCherry), with RAMP4 as a known ER-phagic cargo. UPR signaling was disrupted using inhibitors targeting ATF6α (6µM Ceapin-A7) or IRE1α (0.5µM 4µ8C), or RNAi targeting PERK. CCPG1 or FAM134B were knocked out from LX-2 cells using CRISPR-Cas9. HSC activation and UPR signaling were measured by qPCR and Western blot.
    RESULTS: Expression of CCPG1 and ATL3 increased in fibrotic human livers compared to controls, while CCPG1 mRNA, and FAM134B protein and mRNA levels increased in fibrotic mouse livers compared to controls. In vitro activation of primary hHSCs or mHSCs also increased CCPG1, FAM134B, and ATL3 protein and mRNA levels, as well as increased ER-phagic flux in LX-2 cells. Regulation of ER-phagic flux and expression of ER-phagy receptors was UPR-dependent, with inhibition of ATF6α or IRE1α blocking TGFβ-induced ER-phagic flux, while PERK knockdown increased ER-phagic flux. Interestingly, CCPG1 or FAM134B loss did not impact HSC activation, or induce the pro-apoptotic UPR.
    CONCLUSIONS: ER-phagy receptors increased in fibrotic human and murine livers, and TGFβ upregulated ER-phagy receptors and increased ER-phagic flux in HSCs through UPR-dependent mechanisms. Deletion of CCPG1 or FAM134B did not impact HSC activation, suggesting redundancy between ER-phagic receptors. Future studies will focus on understanding the fibrogenic role of ER-phagy in HSCs.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4963
  7. FASEB J. 2022 May;36 Suppl 1
    Common signaling principles and interconnectivity in the ISR-UPR networks.
    Diego Acosta-Alvear.
      The integrated stress response (ISR) and the unfolded protein response (UPR) are conserved signaling networks governed by stress sensor kinases. Four ISR sensor kinases, GCN2, HRI, PERK, and PKR, phosphorylate eIF2α in response to multiple stresses, leading to a global protein synthesis shutdown coupled to the translation of select mRNAs, which results in an adaptive remodeling of the proteome. Besides PERK, which the ISR and the UPR share, the UPR relies on two additional ER stress sensors, the kinase/RNase IRE1 and the membrane-tethered transcription factor ATF6, to induce adaptive signaling programs that reinstate ER homeostasis. However, if the stress is unrelenting, both the ISR and the UPR can switch to drive cell death. The mechanistic commonalities and crosstalk between the ISR and UPR remain an active area of investigation. In this talk, I will share new results that support common signaling principles of the ISR and UPR sensor kinases and reveal a new layer of communication between the ISR and the UPR. Specifically, we found that dynamic clustering is a prominent feature of PKR activation reminiscent of the high-order assemblies of IRE1 and PERK observed during ER stress. Surprisingly, PKR clusters excluded eIF2α, and mutations in PKR that disrupt cluster assembly enhanced eIF2α phosphorylation, suggesting that PKR clusters act as enzyme sinks that control enzyme-substrate interactions by limiting PKR-eIF2α encounters. Moreover, stress-free activation of PKR induced a master cell death program dependent on ISR-driven expression of DR5 (death receptor 5), as occurs during unmitigable ER stress. Remarkably, stress-free activation of the ISR selectively activated IRE1 independent of sensing unfolded proteins in the ER lumen, and treatment with the small-molecule ISR inhibitor ISRIB reversed it. Our data provide new mechanistic insights into two fundamental aspects of stress sensor kinase signaling: (1) dynamic clustering of stress sensors may provide the means to fine-tune stress responses, and (2) the ISR selectively activates IRE1, thus coupling the ISR and the UPR outside their common node PERK.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0I138
  8. Front Cell Dev Biol. 2022 ;10 878395
    Mitochondrial ROS in Slc4a11 KO Corneal Endothelial Cells Lead to ER Stress.
    Rajalekshmy Shyam, Diego G Ogando, Joseph A Bonanno.
      Recent studies from Slc4a11 -/- mice have identified glutamine-induced mitochondrial dysfunction as a significant contributor toward oxidative stress, impaired lysosomal function, aberrant autophagy, and cell death in this Congenital Hereditary Endothelial Dystrophy (CHED) model. Because lysosomes are derived from endoplasmic reticulum (ER)-Golgi, we asked whether ER function is affected by mitochondrial ROS in Slc4a11 KO corneal endothelial cells. In mouse Slc4a11 -/- corneal endothelial tissue, we observed the presence of dilated ER and elevated expression of ER stress markers BIP and CHOP. Slc4a11 KO mouse corneal endothelial cells incubated with glutamine showed increased aggresome formation, BIP and GADD153, as well as reduced ER Ca2+ release as compared to WT. Induction of mitoROS by ETC inhibition also led to ER stress in WT cells. Treatment with the mitochondrial ROS quencher MitoQ, restored ER Ca2+ release and relieved ER stress markers in Slc4a11 KO cells in vitro. Systemic MitoQ also reduced BIP expression in Slc4a11 KO endothelium. We conclude that mitochondrial ROS can induce ER stress in corneal endothelial cells.
    Keywords:  ERAD (ER associated protein degradation); MitoQ; ROS—reactive oxygen species; SLC4A11 ammonia transporter; corneal endothelial cells; er stress
    DOI:  https://doi.org/10.3389/fcell.2022.878395
  9. FASEB J. 2022 May;36 Suppl 1
    Differential Behavior of 4E-BP1 and 4E-BP2 Under ER Stress.
    Shruthi Gobbooru, Woon-Kai Low.
      eIF4E-binding proteins (4E-BPs) are a family of translational repressors, when unphosphorylated or hypo-phosphorylated, can bind to eIF4E and inhibit eukaryotic cap-dependent translation initiation. The 3 4E-BPs (4E-BP1, 4E-BP2 and 4E-BP3) are not isoforms but are often regarded alike in terms of inhibiting translation initiation. They are not clearly differentiated in terms of function. Not only do they originate from different genes on different chromosomes, but they also have distinct mRNA structure. We hypothesized a potential for their differential regulation under cell stress. During cell stress, 4E-BPs need to be upregulated to stop protein synthesis and conserve cellular energy. 4E-BP1 promoter has CARE (C/Ebp-ATF Response Element) in its promoter region, that helps it respond to ER stress in an ATF4-dependent manner. 4E-BP2 on the other hand, does not have any CARE elements in its promoter, hence ideally should remain unchanged under ER stress. Gene expression was observed through qPCR and protein levels were studied using western blotting. We observed a nearly 4-fold increase in 4E-BP1 mRNA levels under ER stress in sync with ATF4 mRNA levels, whereas 4E-BP2 mRNA levels didn't change significantly. 4E-BP1 protein levels increased under ER stress whereas 4E-BP2 protein levels decreased under ER stress. We observed selective proteasomal degradation of 4E-BP2 under ER stress, which recovered when treated with proteasome inhibitor MG132. The reason behind this selective degradation needs to be further elucidated. These findings shed a new light on the behavior of 4E-BP2 under cell stress which has been, until now, treated synonymous to 4E-BP1 in the cellular landscape.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2603
  10. Int J Mol Sci. 2022 Apr 27. pii: 4843. [Epub ahead of print]23(9):
    Endoplasmic Reticulum (ER) Stress and Its Role in Pancreatic β-Cell Dysfunction and Senescence in Type 2 Diabetes.
    Ji-Hye Lee, Jaemin Lee.
      An increased life span and accompanying nutritional affluency have led to a rapid increase in diseases associated with aging, such as obesity and type 2 diabetes, imposing a tremendous economic and health burden on society. Pancreatic β-cells are crucial for controlling glucose homeostasis by properly producing and secreting the glucose-lowering hormone insulin, and the dysfunction of β-cells determines the outcomes for both type 1 and type 2 diabetes. As the native structure of insulin is formed within the endoplasmic reticulum (ER), ER homeostasis should be appropriately maintained to allow for the proper metabolic homeostasis and functioning of β-cells. Recent studies have found that cellular senescence is critically linked with cellular stresses, including ER stress, oxidative stress, and mitochondrial stress. These studies implied that β-cell senescence is caused by ER stress and other cellular stresses and contributes to β-cells' dysfunction and the impairment of glucose homeostasis. This review documents and discusses the current understanding of cellular senescence, β-cell function, ER stress, its associated signaling mechanism (unfolded protein response), and the effect of ER stress on β-cell senescence and dysfunction.
    Keywords:  ER stress; cellular senescence; endoplasmic reticulum; insulin; islet amyloid polypeptide; pancreatic beta cell; type 2 diabetes
    DOI:  https://doi.org/10.3390/ijms23094843
  11. FASEB J. 2022 May;36 Suppl 1
    The Molecular Chaperone, GRP170, Protects Against Acute Kidney Injury and ER Stress in Mice.
    Teresa M Buck, Aidan Porter, Wily G Ruiz, Dennis R Clayton, Stephanie M Mutchler, Evan C Ray, Allison L Marciszyn, Lubika J NKashama, Arohan R Subramanya, Sebastien Gingras, Thomas Kleyman, Gerard Apodaca, Linda M Hendershot, Jeffrey L Brodsky.
      GRP170 is an Hsp70-like, molecular chaperone localized to the endoplasmic reticulum (ER). Two separate functions have been described for GRP170. First, GRP170 acts as a nucleotide exchange factor (co-chaperone) for the ER lumenal, Hsp70, BiP. Second, GRP170 possess "holdase" activity, and independently binds to aggregation prone regions of proteins to maintain solubility. We previously demonstrated that GRP170 regulates the quality control of the epithelial sodium channel, ENaC. ENaC is responsible for sodium reabsorption in the distal nephron and regulates salt/water homeostasis, and therefore, blood pressure. To better understand how GRP170 impacts kidney function we generated an inducible, nephron specific, GRP170 KO mouse. Loss of GRP170 results in rapid weight/volume loss, electrolyte imbalance and significantly elevated aldosterone levels. The GRP170 KO animals also demonstrate many of the hallmarks of acute kidney injury (AKI) including elevated plasma BUN and creatinine levels. Loss of GRP170 also results in induction of the unfolded protein response (UPR) as shown by upregulation of UPR targets by qPCR (sXbp1, BiP, CHOP and ATF4) and western blotting. We hypothesize that sustained induction of the UPR leads to kidney injury associated with our model, alternatively, misregulation of ion channel trafficking and volume loss may contribute to the pathogenic phenotype. To begin to understand how loss of an ER localized chaperone results in profound kidney injury we treated our GRP170 KO mice with the UPR inhibitor, TUDCA, or a high-salt diet. Preliminary data suggest both UPR induction and electrolyte imbalance may contribute to kidney injury associated with loss of GRP170.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4334
  12. Cell Calcium. 2022 Apr 30. pii: S0143-4160(22)00067-7. [Epub ahead of print]104 102593
    Rhomboid pseudoproteases: An Achilles heel's for BCL-2/IP3R-dependent resistance to ER stress-induced cell death.
    Fernanda O Lemos, Ian de Ridder, Geert Bultynck, Jan B Parys.
      Anti-apoptotic BCL-2 targets and inhibits IP3 receptors (IP3Rs), thereby preventing apoptosis by limiting ER-mitochondrial Ca2+ flux. Recently, Dulloo et al. (2022), Nat. Comm. 13:1257[6] revealed a novel role for rhomboid pseudoproteases in ER stress-induced apoptosis by stripping BCL-2 from IP3Rs, thereby enabling pro-apoptotic Ca2+ signaling.
    Keywords:  Apoptosis; BCL-2; ER stress; IP(3)R; Rhomboid pseudoproteases
    DOI:  https://doi.org/10.1016/j.ceca.2022.102593
  13. J Clin Med. 2022 Apr 22. pii: 2348. [Epub ahead of print]11(9):
    Activation of Sigma-1 Receptor Alleviates ER-Associated Cell Death and Microglia Activation in Traumatically Injured Mice.
    Mingming Shi, Liang Liu, Xiaobin Min, Liang Mi, Yan Chai, Fanglian Chen, Jianhao Wang, Shuyuan Yue, Jianning Zhang, Quanjun Deng, Xin Chen.
      BACKGROUND: Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) is associated with neuroinflammation and subsequent cell death following traumatic brain injury (TBI). The sigma-1 receptor (Sig-1R) acts as a dynamic pluripotent modulator of fundamental cellular processes at the mitochondria-associated membranes (MAMs). The activation of Sig-1R is neuroprotective in a variety of central nervous system diseases, but its impact on ER stress induced by traumatic brain injury is not known. This study investigated the role of Sig-1R in regulating the ER stress-mediated microglial activation and programmed cell death (apoptosis and pyroptosis) induced by TBI.METHODS: Ten human brain tissues were obtained from The Tianjin Medical University General Hospital. Four normal brain tissues were obtained from patients who underwent surgery for cerebral vascular malformation, through which peripheral brain tissues were isolated. Six severe TBI tissues were from patients with brain injury caused by accidents. None of the patients had any other known neurological disorders. Mice with Sig-1R deletion using CRISPR technology were subjected to controlled cortical impact-induced injury. In parallel, wild type C57BL/6J mice were analyzed for outcomes after they were exposed to TBI and received the Sig-1R agonist PRE-084 (10 mg/kg daily for three days) either alone or in combination with the Sig-1R antagonist BD-1047 (10 mg/kg).
    RESULTS: The expression of Sig-1R and the 78 kDa glucose-regulated protein, a known UPR marker, were significantly elevated in the injured cerebral tissues from TBI patients and mice subjected to TBI. PRE-084 improved neurological function, restored the cerebral cortical perfusion, and ameliorated and brain edema in C57BL/6J mice subjected to TBI by reducing endoplasmic reticulum stress-mediated apoptosis, pyroptosis, and microglia activation. The effect of PRE-084 was abolished in mice receiving Sig-1R antagonist BD-1047.
    CONCLUSIONS: ER stress and UPR were upregulated in TBI patients and mice subjected to TBI. Sig-1R activation by the exogenous activator PRE-084 attenuated microglial cells activation, reduced ER stress-associated programmed cell death, and restored cerebrovascular and neurological function in TBI mice.
    Keywords:  BD-1047; PRE-084; apoptosis; cerebrovascular function; endoplasmic reticulum stress; microglia activation; pyroptosis; sigma-1 receptor; traumatic brain injury
    DOI:  https://doi.org/10.3390/jcm11092348
  14. FASEB J. 2022 May;36 Suppl 1
    Disruption of Proteostasis Causes IRE1 Mediated Reprogramming of Alveolar Epithelial Cells in Lung Fibrosis.
    Jeremy Katzen, Luis Rodriguez, Yaniv Tomer, Apoorva Babu, Ming Zhao, Aditi Murthy, Michael Morley, Surafel Mulugeta, Timothy Pelura, Glenn Rosen, Michaell F Beers.
      Pulmonary Fibrosis (PF), a devastating lung disease with rising prevalence, is defined by a physiologic defect in gas exchange owing to two cardinal pathological features: intrusion of myofibroblasts into the distal lung parenchyma and an alteration of normal alveolar epithelial composition most clearly defined by a loss of the squamous alveolar epithelial type 1 cell (AEC1) and the presence of a recently identified aberrant alveolar epithelial "transitional state" enriched in profibrotic mediators. While this transitional cell state has been identified transiently in the epithelium of murine lung injury models, the mechanism by which it arises in human fibrotic lung disease is unknown. We previously reported the in vivo modeling of PF utilizing inducible, knock-in expression of a clinical Surfactant Protein C (SP-C) mutation in alveolar type 2 cells (AEC2). Expression of the SP-C mutation in the adult mouse AEC2s led to activation of Unfolded Protein Response (UPR) signaling pathways, endoplasmic reticulum (ER) stress and spontaneous fibrosis providing proof of concept for disruption to proteostasis as a proximal driver of PF. We hypothesized that disruption of AEC2 protein quality control and specifically UPR signaling causes cell autonomous AEC2 reprogramming to the transitional state in the absence of an exogenous lung injury. Using two clinical SP-C mutation models we discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop the recently identified transitional cell state. Using single cell RNA sequencing of the epithelium in our murine models we identify that UPR activated AEC2s develop the transitional cell phenotype in the absence of an exogenous lung injury. Through organoid based modeling we validated that this state arises de novo from intrinsic AEC2 dysfunction. The cell autonomous AEC2 reprogramming is mediated through IRE1 signaling as use of a novel IRE1 inhibitor attenuated the development of the transitional cell state and diminished AEC2 driven recruitment of granulocytes, alveolitis, and lung injury arising from the loss of proteostasis. We further show that this transitional state persists into the fibrotic phase of our murine models and is enriched in pro-fibrotic mediators. These findings identify AEC2 proteostasis, and specifically IRE1 signaling, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3918
  15. FASEB J. 2022 May;36 Suppl 1
    The Acyltransferase Gpc1 is Both a Target and an Effector of the Unfolded Protein Response (UPR).
    Victoria L Hrach, Laura Nelson, Will King, Shane Conklin, Jana Patton-Vogt.
      The unfolded protein response (UPR) is sensitive to both proteotoxic stress and membrane bilayer stress. These stresses are sensed by the ER transmembrane protein Ire1. When activated, Ire1 uses its endonuclease activity to splice HAC1mRNA, producing a mature transcription factor that binds to UPR elements (UPREs) in the promoters of target genes. Hac1 targets include not only genes involved in protein folding, secretion, and degradation, but also a subset of lipid metabolic genes. One aspect of lipid metabolism is the deacylation of phosphatidylcholine (PC) by phospholipases to produce glycerophosphocholine (GPC). In Saccharomyces cerevisiae, GPC can be reacylated in a novel two-step process catalyzed first by GPC acyltransferase Gpc1, followed by acylation of the lyso-PC molecule by Ale1. This metabolic cycle has been termed the PC deacylation/reacylation pathway (PC-DRP). In prior studies, loss of Gpc1 was shown to result in an increase in di-unsaturated PC species at the expense of mono-unsaturated PC species, indicating a role for PC-DRP in PC acyl chain remodeling. Here, we probe the role of Gpc1 as both a target and an effector of the UPR. Exposure to the UPR-inducing compounds tunicamycin, DTT, and canavanine results in an increase in GPC1 message that is dependent upon the UPR transcriptional activator Hac1. The importance of this increased expression to cellular function is illustrated by the finding that cells lacking Gpc1 exhibit increased sensitivity to those compounds. In a converse set of experiments, we show that that loss of GPC1 results in upregulation of the UPR as measured by expression of the ER chaperone KAR2. Consistent with these findings, we show that Gpc1 primarily co-localizes with the endoplasmic reticulum.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R5072
  16. J Cell Biol. 2022 Jun 06. pii: e202205019. [Epub ahead of print]221(6):
    New insights into IRE1α activation and function in anti-tumor immunity.
    Susan E Logue, Adrienne M Gorman, Afshin Samali.
      Logue, Gorman, and Samali highlight a study by Guttman and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202111068) that shows exogenous antigen peptides imported into the ER can activate the ER stress sensor IRE1α, attenuating cross-presentation by dendritic cells.
    DOI:  https://doi.org/10.1083/jcb.202205019
  17. FASEB J. 2022 May;36 Suppl 1
    A novel and important role of UFM1-binding protein 1 (UFBP1) in the regulation of ER and cardiac homeostasis.
    Varsha Tandra, Wenxia Ma, Aizhen Zhang, Juan Ayala, Honglin Li, Huabo Su, Jie Li.
      Protein modifications by ubiquitin and ubiquitin-like proteins are emerging as an important mechanism regulating heart function. UFM1 (ubiquitin-fold modifier 1) is a novel ubiquitin-like protein that modifies protein targets via UFM1-specific conjugation enzymes. Dysregulation of UFM1 modification, termed ufmylation, has been linked to multiple human diseases but its importance in the heart remains unclear. We have previously reported that ufmylation is upregulated in compensated, hypertrophic mouse hearts but decreased in failing human hearts. Inhibition of ufmylation by targeted ablation of the E3 UFM1 ligase 1 (UFL1) in mouse hearts resulted in dilated cardiomyopathy during ageing and increased the propensity to heart failure in response to hemodynamic stress. However, how ufmylation exerts its cardioprotective effect remains unclear. Here, we report that UFBP1 (UFM1 binding protein 1), an ER-resident ufmylation target, is an important downstream effector of ufmylation in the heart. While deletion of UFBP1 in mouse hearts has no impact on ufmylation, the knockout (KO) mice developed dilated cardiomyopathy at resting condition, as indicated by left ventricular wall thinning, chamber dilatation and significantly decreased cardiac contractility at 6 months of age. Moreover, loss of UFBP1 exacerbated pressure overload-induced cardiac remodeling and dysfunction, recapitulating multiple aspects of the phenotypes of UFL1-deficient hearts. Mechanistically, UFL1 controls the expression of UFBP1. Depletion of both UFL1 and UFBP1 in cultured cardiomyocytes aggravated ER stress-induced cell injury. Furthermore, excess ER stress is implicated in UFBP1KO hearts and is aggravated with the progression of cardiomyopathy. Interestingly, ER stress inducers upregulated the expression of ufmylation pathway components at both transcript and protein levels in cultured cardiomyocytes and mouse hearts, indicating an intimate cross-talk between ufmylation and ER stress. Collectively, our data identify a novel UFM1-UFL1-UFBP1 axis in constraining pathological cardiac remodeling and maintaining ER homeostasis.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R5358
  18. FASEB J. 2022 May;36 Suppl 1
    The Epithelial-Specific ER Stress Sensor IRE1β Enables Host-Microbiota Crosstalk to Affect Colon Goblet Cell Development.
    Michael J Grey, Heidi De Luca, Beth A McCormick, Jerrold R Turner, Wayne I Lencer.
      Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express IRE1β (Ern2), a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor IRE1α. How IRE1β functions at the host-environment interface and why a second IRE1 paralogue evolved remain incompletely understood. Using conventionally raised and germ-free Ern2-/- mice, we found that IRE1β was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced IRE1β expression. IRE1β acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although IRE1α can also splice Xbp1 mRNA, it did not act redundantly to IRE1β in this context. By regulating assembly of the colon mucus layer, IRE1β further shaped the composition of the gut microbiota. Mice lacking IRE1β had a dysbiotic microbial community that failed to induce goblet cell development when transferred into germ-free wild type mice. These results show that IRE1β evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R1937
  19. FASEB J. 2022 May;36 Suppl 1
    Conformational Dynamics Linking Kinase and Endonuclease Domains Explain the Divergent Function of IRE1 Paralogues.
    Michael J Grey, Dylan Jainchill, Mariska S Simpson, Wayne I Lencer.
      The ER stress sensors IRE1α and IRE1β have dual kinase and endonuclease activities that mediate cellular proteostasis by splicing Xbp1 mRNA and degrading other ER-targeted transcripts. Although the two paralogues share a high degree of sequence homology, the epithelial cell-specific paralogue IRE1β, which is expressed at mucosal surfaces, has impaired kinase activity and phosphorylation that restrict stress-induced activation of the endonuclease domain. To uncover structural features that explain these differences, we used normal mode analysis to build dynamic correlation networks describing active and inactive conformations of IRE1 kinase-endonuclease domains. For human IRE1α, we identified a fingerprint of intra- and interdomain dynamic couplings that distinguish between the two conformations. Mutations that disrupted IRE1α endonuclease activity perturbed the dynamic networks of the active conformation and enhanced features of an inactive conformation, thus validating our approach. Strikingly, the dynamic networks of human IRE1β when modeled in an active conformation resembled the inactive IRE1α network. A set of non-conserved amino acids were found to distinguish between the dynamic networks linking kinase-endonuclease domains for the two IRE1 paralogues. In most cases, however, these divergent network-determining amino acids were found only in IRE1β sequences from mammals. In lower vertebrates, the key amino acids and the dynamic couplings of IRE1α active state were conserved in IRE1β. Thus, IRE1β evolved distinct conformational dynamics and enzymatic activity for a role in mucus barrier function that is unique to higher vertebrates.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R1925
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