bims-unfpre Biomed News
on Unfolded protein response
Issue of 2021‒06‒06
eleven papers selected by
Susan Logue
University of Manitoba

  1. Mucosal Immunol. 2021 Jun 01.
      Barrier epithelial cells lining the mucosal surfaces of the gastrointestinal and respiratory tracts interface directly with the environment. As such, these tissues are continuously challenged to maintain a healthy equilibrium between immunity and tolerance against environmental toxins, food components, and microbes. An extracellular mucus barrier, produced and secreted by the underlying epithelium plays a central role in this host defense response. Several dedicated molecules with a unique tissue-specific expression in mucosal epithelia govern mucosal homeostasis. Here, we review the biology of Inositol-requiring enzyme 1β (IRE1β), an ER-resident endonuclease and paralogue of the most evolutionarily conserved ER stress sensor IRE1α. IRE1β arose through gene duplication in early vertebrates and adopted functions unique from IRE1α which appear to underlie the basic development and physiology of mucosal tissues.
  2. Biochem Biophys Res Commun. 2021 May 28. pii: S0006-291X(21)00845-7. [Epub ahead of print]563 8-14
      Although accumulating evidence indicates participation of endoplasmic reticulum (ER) stress pathway or the unfolded protein response (UPR) in immunity, there still exists little information linking ER stress to regulatory T cells (Tregs). To evaluate the potential contribution of the UPR, we tested the effects of thapsigargin (TG), an ER stress inducer, on the function of Tregs. Here we reported that TG stimulation induced the up-regulation of the endoplasmic reticulum (ER)-stress chaperon Glucose-Regulated Protein 78 (GRP78), which is a master regulator of the UPR, the phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) and the induction of activating transcription factor 4 (ATF4), which are both protein kinase R (PKR)-like ER kinase (PERK) downstream targets in Tregs. Simultaneously, we demonstrated that, under ER stress conditions, Tregs presented enhanced functional activity upon TCR stimulation, as illustrated with forkhead box transcription factor (Foxp3) expression, interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) production and suppressive functional analysis. Notably, pretreatment with GSK2656157, a potent and selective PERK inhibitor, markedly diminished the TG-induced hyperresponsiveness of Tregs upon T cell antigen receptor (TCR) stimulation. Therefore, our findings illustrated the inter-connection and coordination of the evolutionarily conserved ER stress response and TCR signaling in Tregs and uncover a critical new role of the PERK branch of UPR in the regulation of Tregs function.
    Keywords:  ER stress; GRP78; PERK; Tregs; UPR
  3. Cancers (Basel). 2021 May 28. pii: 2649. [Epub ahead of print]13(11):
      Multiple myeloma is a genetically complex hematologic neoplasia in which malignant plasma cells constantly operate at the maximum limit of their unfolded protein response (UPR) due to a high secretory burden of immunoglobulins and cytokines. The endoplasmic reticulum (ER) resident protein disulfide isomerase, PDIA1 is indispensable for maintaining structural integrity of cysteine-rich antibodies and cytokines that require accurate intramolecular disulfide bond arrangement. PDIA1 expression analysis from RNA-seq of multiple myeloma patients demonstrated an inverse relationship with survival in relapsed or refractory disease, supporting its critical role in myeloma persistence. Using a structure-guided medicinal chemistry approach, we developed a potent, orally bioavailable small molecule PDIA1 inhibitor CCF642-34. The inhibition of PDIA1 overwhelms the UPR in myeloma cells, resulting in their apoptotic cell death at doses that do not affect the normal CD34+ hematopoietic stem and progenitor cells. Bortezomib resistance leads to increased PDIA1 expression and thus CCF642-34 sensitivity, suggesting that proteasome inhibitor resistance leads to PDIA1 dependence for proteostasis and survival. CCF642-34 induces acute unresolvable UPR in myeloma cells, and oral treatment increased survival of mice in the syngeneic 5TGM1 model of myeloma. Results support development of CCF642-34 to selectively target the plasma cell program and overcome the treatment-refractory state in myeloma.
    Keywords:  ER stress; ERMM; IRMM; UPR; protein disulfide isomerase PDIA1
  4. Cell Death Discov. 2021 Jun 03. 7(1): 131
      Altered protein homeostasis is associated with neurodegenerative diseases and acute brain injury induced under energy depletion conditions such as ischemia. The accumulation of damaged or unfolded proteins triggers the unfolded protein response (UPR), which can act as a homeostatic response or lead to cell death. However, the factors involved in turning and adaptive response into a cell death mechanism are still not well understood. Several mechanisms leading to brain injury induced by severe hypoglycemia have been described but the contribution of the UPR has been poorly studied. Cell responses triggered during both the hypoglycemia and the glucose reinfusion periods can contribute to neuronal death. Therefore, we have investigated the activation dynamics of the PERK and the IRE1α branches of the UPR and their contribution to neuronal death in a model of glucose deprivation (GD) and glucose reintroduction (GR) in cortical neurons. Results show a rapid activation of the PERK/p-eIF2α/ATF4 pathway leading to protein synthesis inhibition during GD, which contributes to neuronal adaptation, however, sustained blockade of protein synthesis during GR promotes neuronal death. On the other hand, IRE1α activation occurs early during GD due to its interaction with BAK/BAX, while ASK1 is recruited to IRE1α activation complex during GR promoting the nuclear translocation of JNK and the upregulation of Chop. Most importantly, results show that IRE1α RNase activity towards its splicing target Xbp1 mRNA occurs late after GR, precluding a homeostatic role. Instead, IRE1α activity during GR drives neuronal death by positively regulating ASK1/JNK activity through the degradation of 14-3-3 θ mRNA, a negative regulator of ASK and an adaptor protein highly expressed in brain, implicated in neuroprotection. Collectively, results describe a novel regulatory mechanism of cell death in neurons, triggered by the downregulation of 14-3-3 θ mRNA induced by the IRE1α branch of the UPR.
  5. Biochim Biophys Acta Proteins Proteom. 2021 May 27. pii: S1570-9639(21)00086-8. [Epub ahead of print]1869(9): 140680
      Beta-cell death and dysfunction are involved in the development of type 1 and 2 diabetes. ER-stress impairs beta-cells function resulting in pro-apoptotic stimuli that promote cell death. Hence, the identification of protective mechanisms in response to ER-stress could lead to novel therapeutic targets and insight in the pathology of these diseases. Here, we report the identification of proteins involved in dysregulated pathways upon thapsigargin treatment of MIN6 cells. Utilizing quantitative proteomics we identified upregulation of proteins involved in protein folding, unfolded protein response, redox homeostasis, proteasome processes associated with endoplasmic reticulum and downregulation of TCA cycle, cellular respiration, lipid metabolism and ribosome assembly processes associated to mitochondria and eukaryotic initiation translation factor components. Subsequently, pro-inflammatory cytokine treatment was performed to mimic pathological changes observed in beta-cells during diabetes. Cytokines induced ER stress and impaired mitochondrial function in beta-cells corroborating the results obtained with the proteomic approach. HSPB1 levels are increased by prolactin on pancreatic beta-cells and this protein is a key factor for cytoprotection although its role has not been fully elucidated. Here we show that while up-regulation of HSPB1 was able to restore the mitochondrial dysfunction induced by beta-cells' exposure to inflammatory cytokines, silencing of this chaperone abrogated the beneficial effects promoted by PRL. Taken together, our results outline the importance of HSPB1 to mitigate beta-cell dysfunction. Further studies are needed to elucidate its role in diabetes.
    Keywords:  Diabetes; ER stress; HSPB1; Mass spectrometry; Mitochondrial bioenergetics; Pancreatic beta cells; Proteomics; UPR
  6. Cells. 2021 May 12. pii: 1178. [Epub ahead of print]10(5):
      Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide with limited treatment options. Biomarker-based active phenolic flavonoids isolated from medicinal plants might shed some light on potential therapeutics for treating HCC. 3,3'-diindolylmethane (DIM) is a unique biologically active dimer of indole-3-carbinol (I3C), a phytochemical compound derived from Brassica species of cruciferous vegetables-such as broccoli, kale, cabbage, and cauliflower. It has anti-cancer effects on various cancers such as breast cancer, prostate cancer, endometrial cancer, and colon cancer. However, the molecular mechanism of DIM involved in reducing cancer risk and/or enhancing therapy remains unknown. The aim of the present study was to evaluate anti-cancer and therapeutic effects of DIM in human hepatoma cell lines Hep3B and HuhCell proliferation was measured with MTT and trypan blue colony formation assays. Migration, invasion, and apoptosis were measured with Transwell assays and flow cytometry analyses. Reactive oxygen species (ROS) intensity and the loss in mitochondrial membrane potential of Hep3B and Huh7 cells were determined using dihydroethidium (DHE) staining and tetramethylrhodamine ethyl ester dye. Results showed that DIM significantly suppressed HCC cell growth, proliferation, migration, and invasion in a concentration-dependent manner. Furthermore, DIM treatment activated caspase-dependent apoptotic pathway and suppressed epithelial-mesenchymal transition (EMT) via ER stress and unfolded protein response (UPR). Taken together, our results suggest that DIM is a potential anticancer drug for HCC therapy by targeting ER-stress/UPR.
    Keywords:  DIM; EMT; ER stress; apoptosis; hepatocellular carcinoma; unfolded protein response
  7. Cancers (Basel). 2021 May 12. pii: 2341. [Epub ahead of print]13(10):
      The proteasome is an appealing target for anticancer therapy and the proteasome inhibitor bortezomib has been approved for the treatment of several types of malignancies. However, the molecular mechanisms underlying cancer cell resistance to bortezomib remain poorly understood. In the current article, we investigate how modulation of the eIF2α-ATF4 stress pathway affects hepatoma cell response to bortezomib. Transcriptome profiling revealed that many ATF4 transcriptional target genes are among the most upregulated genes in bortezomib-treated HepG2 human hepatoma cells. While pharmacological enhancement of the eIF2α-ATF4 pathway activity results in the elevation of the activities of all branches of the unfolded protein response (UPR) and sensitizes cells to bortezomib toxicity, the suppression of ATF4 induction delays bortezomib-induced cell death. The pseudokinase TRIB3, an inhibitor of ATF4, is expressed at a high basal level in hepatoma cells and is strongly upregulated in response to bortezomib. To map genome-wide chromatin binding loci of TRIB3 protein, we fused a Flag tag to endogenous TRIB3 in HepG2 cells and performed ChIP-Seq. The results demonstrate that TRIB3 predominantly colocalizes with ATF4 on chromatin and binds to genomic regions containing the C/EBP-ATF motif. Bortezomib treatment leads to a robust enrichment of TRIB3 binding near genes induced by bortezomib and involved in the ER stress response and cell death. Disruption of TRIB3 increases C/EBP-ATF-driven transcription, augments ER stress and cell death upon exposure to bortezomib, while TRIB3 overexpression enhances cell survival. Thus, TRIB3, colocalizing with ATF4 and limiting its transcriptional activity, functions as a factor increasing resistance to bortezomib, while pharmacological over-activation of eIF2α-ATF4 can overcome the endogenous restraint mechanisms and sensitize cells to bortezomib.
    Keywords:  Tribbles; cell death; chemotherapy resistance; integrated stress response; proteasome inhibitor
  8. Diabetologia. 2021 May 29.
      AIMS/HYPOTHESIS: Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred to as mitochondrial dynamics, to adapt to nutritional demands. Evidence suggests that impaired mitochondrial dynamics leads to metabolic abnormalities such as non-alcoholic steatohepatitis (NASH) phenotypes. However, how mitochondrial dynamics are involved in the development of NASH is poorly understood. This study aimed to elucidate the role of mitochondrial fission factor (MFF) in the development of NASH.METHODS: We created mice with hepatocyte-specific deletion of MFF (MffLiKO). MffLiKO mice fed normal chow diet (NCD) or high-fat diet (HFD) were evaluated for metabolic variables and their livers were examined by histological analysis. To elucidate the mechanism of development of NASH, we examined the expression of genes related to endoplasmic reticulum (ER) stress and lipid metabolism, and the secretion of triacylglycerol (TG) using the liver and primary hepatocytes isolated from MffLiKO and control mice.
    RESULTS: MffLiKO mice showed aberrant mitochondrial morphologies with no obvious NASH phenotypes during NCD, while they developed full-blown NASH phenotypes in response to HFD. Expression of genes related to ER stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic TG secretion was downregulated, with reduced hepatic TG secretion in MffLiKO mice in vivo and in primary cultures of MFF-deficient hepatocytes in vitro. Furthermore, thapsigargin-induced ER stress suppressed TG secretion in primary hepatocytes isolated from control mice.
    CONCLUSIONS/INTERPRETATION: We demonstrated that ablation of MFF in liver provoked ER stress and reduced hepatic TG secretion in vivo and in vitro. Moreover, MffLiKO mice were more susceptible to HFD-induced NASH phenotype than control mice, partly because of ER stress-induced apoptosis of hepatocytes and suppression of TG secretion from hepatocytes. This study provides evidence for the role of mitochondrial fission in the development of NASH.
    Keywords:  ER stress; Lipid metabolism; MFF; Mitochondrial dynamics; NASH
  9. Biochim Biophys Acta Mol Cell Res. 2021 Jun 01. pii: S0167-4889(21)00128-2. [Epub ahead of print] 119074
      Endoplasmic reticulum (ER) stress can initiate autophagy via unfolded protein response (UPR). As a key downstream gene of UPR, DDIT3/CHOP is expressed in chondrocytes. However, the regulation mechanism of DDIT3/CHOP on autophagy in chondrocytes remains unclear. In this study, the expression levels of autophagic markers Beclin1 and LC3B were found to decrease while p62 increase in the tibial growth plate and costal primary chondrocytes from DDIT3/CHOP cKO mice. In vitro, overexpressing DDIT3/CHOP induced autophagy in ATDC5 chondrocytes, displaying an elevated immunofluorescence signal of LC3B and elevated numbers of autophagosome and autolysosomes. Analysis of the gain- and loss-of-function indicated that the protein level of Beclin1 and the ratio of LC3BII/I increased in DDIT3/CHOP overexpression cells, whereas decreased in DDIT3/CHOP knockdown cells. The decreased level of p62 and additional accumulation of LC3BII caused by chloroquine (CQ) further indicated that DDIT3/CHOP enhanced autophagic flux. Mechanistically, we found that DDIT3/CHOP binds directly to the promoter of SIRT1 to promote its expression by CHIP, qRT-PCR, and Western blot analysis. In addition, SIRT1 enhanced autophagic activity in ATDC5 cells, and inhibition or activation of SIRT1 partially reversed the effect of overexpressing or downregulating DDIT3/CHOP on autophagy. Furthermore, AKT signaling was found to be responsible for DDIT3/CHOP-regulated autophagy in ATDC5 cells. SIRT1 knockdown reversed the effect of DDIT3/CHOP overexpression on AKT signaling. In conclusion, our data clarifies that DDIT3/CHOP promotes autophagy in ATDC5 chondrocytes through the SIRT1-AKT pathway. These results were also confirmed in the primary chondrocytes.
    Keywords:  AKT signaling; DDIT3/CHOP; SIRT1; autophagy; chondrocytes
  10. Cells Dev. 2021 May 28. pii: S2667-2901(21)00034-6. [Epub ahead of print] 203687
      Bone marrow mesenchymal stem cells (BMSCs) have strong proliferative ability and multi-directional differentiation potential. Osteoarthritis is a degenerative joint disease that is closely related to the loss of osteogenic differentiation function of BMSCs. Autophagy, plays a crucial role in the maintenance of cellular functions, but its regulatory mechanism during the osteogenic differentiation of BMSCs remains unclear. In this study, we analyzed the differential gene networks and pathways during BMSC osteogenesis using bioinformatics, and further validated the regulatory roles of autophagy during the osteogenic differentiation of BMSCs in inflammatory condition in vitro. We found that Tumor necrosis factor alpha (TNF-α) treatment led to actin cytoskeleton rearrangements and inhibited osteogenic differentiation in BMSCs. In addition, TNF-α enhanced Rho-associated protein kinase 1 (ROCK1) expression and decreased autophagy activation. ROCK1 knockdown reduced Endoplasmic Reticulum stress (ER stress) and promoted autophagy, resulting reversion of osteogenic differentiation in BMSCs under inflammatory condition. Rapamycin reversed the TNF-α-induced decrease in osteogenesis of BMSCs, assessed by alkaline phosphatase (ALP) activity and Alizarin staining. Autophagy treated with inhibitor 3-Methyladenine (3-MA) further increased TNF-α-induced osteogenesis inhibition of BMSCs. Collectively, these results indicate that ER stress and dysfunction of autophagy promote inflammation-induced bone loss through the activation of ROCK1 signaling in BMSCs.
    Keywords:  BMSCs; ER stress/autophagy; Osteogenic differentiation; ROCK 1; TNF-α
  11. Viruses. 2021 May 12. pii: 892. [Epub ahead of print]13(5):
      Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis. Previous work indicated that VEEV infection induced early growth response 1 (EGR1) expression, leading to cell death via the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) arm of the unfolded protein response (UPR) pathway. Loss of PERK prevented EGR1 induction and decreased VEEV-induced death. The results presented within show that loss of PERK in human primary astrocytes dramatically reduced VEEV and eastern equine encephalitis virus (EEEV) infectious titers by 4-5 log10. Loss of PERK also suppressed VEEV replication in primary human pericytes and human umbilical vein endothelial cells, but it had no impact on VEEV replication in transformed U87MG and 293T cells. A significant reduction in VEEV RNA levels was observed as early as 3 h post-infection, but viral entry assays indicated that the loss of PERK minimally impacted VEEV entry. In contrast, the loss of PERK resulted in a dramatic reduction in viral nonstructural protein translation and negative-strand viral RNA production. The loss of PERK also reduced the production of Rift Valley fever virus and Zika virus infectious titers. These data indicate that PERK is an essential factor for the translation of alphavirus nonstructural proteins and impacts multiple RNA viruses, making it an exciting target for antiviral development.
    Keywords:  PERK; Venezuelan equine encephalitis virus (VEEV); alphavirus; eastern equine encephalitis virus (EEEV); translation