Protein Kinase RNA-like Endoplasmic Reticulum Research Articles

Intracellular Ca2+ signaling and ORAI calcium release-activated calcium modulator 1 are associated with hepatic lipidosis in dairy cattle.

Fatty liver is a common metabolic disorder afflicting dairy cows during the periparturient period and is closely associated with endoplasmic reticulum (ER) stress. The onset of ER stress in humans and mice alters hepatic lipid metabolism, but it is unknown if such event contributes to fatty liver in dairy cows soon after parturition. ORAI calcium release-activated calcium modulator 1 (ORAI1) is a key component of the store-operated Ca2+ entry mechanism regulating cellular Ca2+ balance. The purpose of this study was to investigate the role of ORAI1 on hepatic lipidosis via ER stress in dairy cows. Liver tissue biopsies were collected from Holstein cows diagnosed as healthy (n = 6) or with hepatic lipidosis (n = 6). Protein and mRNA abundance of ER stress-related targets, lipogenic targets, or the transcription regulator SREBP1 and ORAI1 were greater in cows with lipidosis. In vitro, hepatocytes were isolated from four healthy female calves and used for culture with a 1.2 mM mixture of fatty acids (oleic, linoleic, palmitic, stearic, and palmitoleic acid) for various times (0, 3, 6, 9, or 12 h). As incubation time progressed, increases in concentration of Ca2+ and abundance of protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α), and activating transcription factor-6 (ATF6) protein in response to exogenous fatty acids underscored a mechanistic link among Ca2+, fatty acids, and ER stress. In a subsequent study, hepatocytes were transfected with small interfering RNA (siORAI1) or the ORAI1 inhibitor BTP2 for 48 h or 2 h followed by a challenge with the 1.2 mM mixture of fatty acids for 6 h. Compared with control group, silencing or inhibition of ORAI1 led to decreased abundance of fatty acid synthesis (FASN, SREBP1, and ACACA) and ER stress-related proteins in bovine hepatocytes. Overall, data suggested that NEFA through ORAI1 regulate intracellular Ca2+ signaling, induce ER stress, and lead to lipidosis in isolated hepatocytes.

Endoplasmic reticulum unfolded protein response modulates the adaptation of Trachemys scripta elegans in salinity water

Trachemys scripta elegans, as a freshwater invasive species, can survive and lay eggs in brackish water, which may lead to the expansion of its potential invasion range due to freshwater salinization. Our previous studies have shown that high salinity leads to the accumulation of serum lipid content, which may induce endoplasmic reticulum stress (ERS) in the turtle. To better understand whether ERS is triggered by salinity, and in turn whether the turtles promote the protection mechanism, we exposed the turtles to the freshwater (CK), 5‰ salinity water (S5) and 15‰ salinity water (S15), and sampled at 6 h, 24 h and 30 d. 13 differentially expressed genes (DEGs) related to ERS pathways were found in the comparison of CK vs. S15 by transcriptomics analysis. Then, the mRNA and protein expression of ERS and its related activation pathways were further investigated. ERS marker glucose regulated protein 78 kD (GRP78) increased significantly (p < 0.05) in both the transcript and protein levels after exposure to 15‰ salinity water, which clearly indicated that salinity could induce ERS in T. s. elegans. Meanwhile, the three unfolded protein response (UPR) including transducers protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α) and activating transcription factor-6 (ATF6) were promoted by salinity, suggesting that the turtle might promote physiological process to eliminate damaged cells and cope with unfolded proteins accumulation induced by ERS. Our results provide new insight into the mechanism of salinity adaptation in T. s. elegans and salt-tolerant biological invasion.

Zinc, Selenium and Chromium Supplementation Improves Insulin Resistance by Preventing Hepatic Endoplasmic Reticulum Stress in Diet-Induced Gestational Diabetes Rats

Abstract Objectives Gestational diabetes mellitus (GDM) is one of the most common pregnancy complications and results in adverse outcomes for pregnant women and their offspring. Endoplasmic reticulum (ER) stress is associated with insulin resistance and implicates in the development of GDM. Zinc, selenium and chromium have been shown to maintain glucose homeostasis via multiple mechanisms, but how these trace elements affect the insulin resistance and ER stress in GDM are largely unknown. Methods A GDM rat model was induced by feeding female Sprague-Dawley (SD) rats a high-fat (45%) and sucrose (HFS) diet, while zinc (10 mg/kg.bw), selenium (20 ug/kg.bw), chromium (20 ug/kg.bw) were daily supplemented alone or in combination from 6 weeks before mating to the end of lactation period. Maternal metabolic parameters, hepatic ER stress and insulin signaling were analyzed. Results The results showed that zinc, selenium and chromium supplementation dramatically alleviated HFS-induced glucose intolerance and oxidative stress during entire experiment period. Hepatic ER stress as well as the unfolded protein response (UPR) was activated in GDM rats, characterized by the up-regulation of glucose-regulated protein 78 (GRP78), phosphorylated the protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), and the inositol-requiring enzyme 1α (p-IRE1α). Zinc, selenium and chromium supplementation significantly prevented this activation, by which contributes to the promotion of the phosphorylated protein kinase B (p-AKT) related insulin signaling and maintenance of glucose homeostasis. Conclusions Zinc, selenium and chromium supplementation may be a promising way to prevent the development of GDM by alleviating hepatic ER stress. Funding Sources This work was financially supported by the Angel Nutritech Nutrition Fund (AF2017003) and the National Natural Science Foundation of China (NSFC, 81373006 and 81973043).

PERK/ATF4-Dependent ZFAS1 Upregulation Is Associated with Sorafenib Resistance in Hepatocellular Carcinoma Cells.

Sorafenib, a multi-kinase inhibitor, is the first-line treatment for advanced hepatocellular carcinoma (HCC) patients. However, this drug only provides a short improvement of patients’ overall survival, and drug resistance is commonly developed. Thus, the identification of resistant factor(s) or biomarker(s) is needed to develop more efficient therapeutic strategies. Long, non-coding RNAs (lncRNAs) have recently been viewed as attractive cancer biomarkers and drive many important cancer phenotypes. A lncRNA, ZFAS1 (ZNFX1 antisense RNA 1) has been found to promote HCC metastasis. This study found that sorafenib induced ZFAS1 expression specifically in sorafenib-resistant HCC cells. Although ZFAS1 knockdown did not restore the sensitivity of HCC cells to sorafenib, its expression may act as a resistant biomarker for sorafenib therapy. Bioinformatics analysis predicted that sorafenib tended to induce pathways related to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in sorafenib-resistant HCC cells. In vitro experimental evidence suggested that sorafenib induced protein kinase RNA-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-dependent ZFAS1 expression, and sorafenib resistance could be overcome by PERK/ATF inhibitors. Therefore, PERK/ATF4/ZFAS1 signaling axis might be an attractive therapeutic and prognostic biomarker for sorafenib therapy in HCC.

ER stress and its PERK branch enhance TCR-induced activation in regulatory T cells

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.

Identification of Two Novel Compound Heterozygous EIF2AK3 Mutations Underlying Wolcott-Rallison Syndrome in a Chinese Family.

Objective: Wolcott–Rallison syndrome is a rare autosomal recessive inheritance disorder caused by the defectiveness of eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3), which encodes the PKR-like endoplasmic reticulum kinase (PERK). Defect in EIF2AK3 results in a permanent diabetes in early infancy or newborn period, a tendency to develop skeletal fractures and other associated disorders such as severe liver and renal dysfunction, and central hypothyroidism. Two patients with Wolcott–Rallison syndrome-like manifestations in a Chinese family and family members were genetically analyzed to identify if any variations that occurred in EIF2AK3, which may cause Wolcott–Rallison syndrome.Methods: Whole-exome sequencing (WES) was performed to identify genetic variations, and Sanger sequencing was conducted to verify the identified variations in the family members with Wolcott–Rallison syndrome (WRS) clinical manifestations. Several bioinformatics tools were employed to predict the effect of EIF2AK3 variations on the protein function. The impact on PERK protein was analyzed by sequential analysis and evolution conservation study.Results: Two novel EIF2AK3 heterozygous single base variations (c.2818C>T and c.2980G>C) were detected in the proband. PERK has two functional domains: one is regulatory domain (aa 1–576), and the other is catalytic domain (aa 577–1,115). Both variations are missense mutations and locate in catalytic domain of PERK; c.2818C>T resulted in a residue substitution of proline for serine at amino acid site 940 (p.Pro940Ser), and variation c.2980G>C caused an amino acid change at position 994 from glutamic acid to glutamine (p.Glu994Gln). These novel missense variations may affect the physiological functions of PERK protein.Conclusions: Two novel compound heterozygous EIF2AK3 variations (c.2818C>T, p.Pro940Ser and c.2980G>C, p.Glu994Gln) were found in a Chinese family. The identification of the variations and verification of their pathogenicity extended the variation spectrum of EIF2AK3 variations causing Wolcott–Rallison syndrome and enriched valuable information for precise medical intervention for Wolcott–Rallison syndrome in China.

Endoplasmic reticulum stress promotes epithelial‑mesenchymal transition via the PERK signaling pathway in paraquat‑induced pulmonary fibrosis.

Pulmonary fibrosis is the primary reason for mortality in patients with paraquat (PQ) poisoning. Our previous study demonstrated that epithelial-mesenchymal transition (EMT) had a role in PQ-induced pulmonary fibrosis. However, the role of endoplasmic reticulum (ER) stress in PQ-induced EMT remains clear. The present study aimed to determine the role of ER stress in EMT in PQ-induced pulmonary fibrosis. A549 and RLE-6TN cells were incubated with LY294002 (a PI3K inhibitor) or transfected with protein kinase RNA-like ER kinase (PERK) small interfering RNA (si) for 24 h prior to being exposed to PQ. Next, the expression levels of ER stress-related proteins, PI3K/AKT/GSK-3β signaling pathway-related proteins and EMT-related markers were analyzed by performing western blotting, reverse transcription-quantitative PCR and immunofluorescence assays. The results of the present study revealed that the protein expression levels of PERK, phosphorylated (p)-PERK, p-eukaryotic initiation factor 2 (eIF2)α were significantly upregulated in the PQ group, whereas p-PI3K, p-AKT and p-GSK-3β were significantly upregulated in the sicontrol + PQ group compared with the sicontrol group. In vitro, following transfection with siPERK or treatment with the PI3K inhibitor, the protein expression levels of E-cadherin (an epithelial marker) were upregulated, whereas the protein expression levels of α-SMA (a mesenchymal marker) were downregulated. Immunofluorescence analysis revealed that the levels of E-cadherin were markedly upregulated, whereas the levels of α-SMA were notably downregulated following transfection with siPERK compared with the sicontrol group. The results of wound healing assay demonstrated that cell migration in the siPERK + PQ group was markedly decreased compared with the sicontrol + PQ group. These indicated that PQ-induced EMT was suppressed after silencing PERK. The expression levels of p-GSK-3β, p-AKT and p-PI3K were also markedly downregulated in the siPERK + PQ group compared with the sicontrol + PQ group. In conclusion, the findings of the present study suggested that ER stress may promote EMT through the PERK signaling pathway in PQ-induced pulmonary fibrosis. Thus, ER stress may represent a potential therapeutic target for PQ-induced pulmonary fibrosis.

Salubrinal Exposes Anticancer Properties in Inflammatory Breast Cancer Cells by Manipulating the Endoplasmic Reticulum Stress Pathway.

The endoplasmic reticulum (ER) regulates protein folding, post-translational modifications, lipid synthesis, and calcium signaling to attenuate the accumulation of misfolded proteins causing ER stress and maintains cellular homeostasis. The tumor microenvironment is rich in soluble cytokines, chemokines, growth, and angiogenic factors and can drive the ER’s abnormal functioning in healthy cells. Cancer cells adapt well to the tumor microenvironment induced ER stress. We identified that the inflammatory breast cancer (IBC) cells abundantly express osteoprotegerin (OPG) and their tumor microenvironment is rich in OPG protein. OPG also called osteoclast differentiation factor/osteoclastogenesis inhibitory factor (OCIF) is a soluble decoy receptor for receptor activator of nuclear factor-kappa B ligand (RANKL). Employing mass spectrometry analysis, we identified a set of ER chaperones associated with OPG in IBC cell lysates (SUM149PT, SUM1315MO2) compared to healthy human mammary epithelial cells (HMEC). Proximity ligation assay (PLA) and immunoprecipitation assay validated the interaction between OPG and ER chaperone and master regulator of unfolded protein response (UPR) GRP78/BiP (glucose-regulated protein/Binding immunoglobulin protein). We detected remarkably high gene expression of CCAAT enhancer-binding protein homologous protein (CHOP), inositol-requiring enzyme 1 (IRE1α), protein disulfide-isomerase (PDI), PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4), X-box binding protein 1 (XBP-1) and growth arrest and DNA damage-inducible protein (GADD34) in SUM149PT and SUM190PT cells when compared to HMEC. Similarly, tissue sections of human IBC expressed high levels of ER stress proteins. We evaluated cell death and apoptosis upon Salubrinal and phenylbutyrate treatment in healthy and IBC cells by caspase-3 activity and cleaved poly (ADP-ribose) polymerase (PARP) protein assay. IBC (SUM149PT and SUM190PT) cells were chemosensitive to Salubrinal treatment, possibly via inhibition in OPG secretion, upregulating ATF4, and CHOP, thus ultimately driving caspase-3 mediated IBC cell death. Salubrinal treatment upregulated PDI, which connects ER stress to oxidative stress. We observed increased ROS production and reduced cell proliferation of Salubrinal treated IBC cells. Treatment with antioxidants could rescue IBC cells from ROS and aborted cell proliferation. Our findings implicate that manipulating ER stress with Salubrinal may provide a safer and tailored strategy to target the growth of inflammatory and aggressive forms of breast cancer.

Crizotinib and Doxorubicin Cooperatively Reduces Drug Resistance by Mitigating MDR1 to Increase Hepatocellular Carcinoma Cells Death.

As the sixth most lethal cancers worldwide, hepatocellular carcinoma (HCC) has been treated with doxorubicin (Dox) for decades. However, chemotherapy resistance, especially for Dox is an even more prominent problem due to its high cardiotoxicity. To find a regimen to reduce Dox resistance, and identify the mechanisms behind it, we tried to identify combination of drugs that can overcome drug resistance by screening tyrosine kinase inhibitor(s) with Dox with various HCC cell lines in vitro and in vivo. We report here that combination of Crizo and Dox has a synergistic effect on inducing HCC cell death. Accordingly, Crizo plus Dox increases Dox accumulation in nucleus 3-16 times compared to Dox only; HCC cell death enhanced at least 50% in vitro and tumor weights reduced ranging from 35 to 65%. Combining these two drugs reduces multiple drug resistance 1 (MDR1) protein as a result of activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK), which phosphorylates eIF2α, leading to protein translational repression. Additionally, PERK stimulation activates C-Jun terminal kinase (JNK), resulting in accumulation of unfused autophagosome to enhance autophagic cell death via Poly-ADP-ribosyltransferase (PARP-1) cleavage. When the activity of PERK or JNK is blocked, unfused autophagosome is diminished, cleaved PARP-1 is reduced, and cell death is abated. Therefore, Crizo plus Dox sensitize HCC drug resistance by engaging PERK-p- eIF2α-MDR1, and kill HCC cells by engaging PERK-JNK- autophagic cell death pathways. These newly discovered mechanisms of Crizo plus Dox not only provide a potential treatment for HCC but also point to an approach to overcome MDR1 related drug resistance in other cancers.

Thioredoxin-1 Is a Target to Attenuate Alzheimer-Like Pathology in Diabetic Encephalopathy by Alleviating Endoplasmic Reticulum Stress and Oxidative Stress.

Varying degrees of central nervous system neuropathy induced by diabetes mellitus (DM) contribute to a cognitive disorder known as diabetic encephalopathy (DE), which is also one of the independent risk factors for Alzheimer’s disease (AD). Endoplasmic reticulum stress (ERS) plays a critical role in the occurrence and development of DE and AD. However, its molecular mechanism remains largely unknown. This study aims to investigate whether thioredoxin-1 (Trx-1) could alleviate DE and AD through ERS, oxidative stress (OS) and apoptosis signaling pathways. Mice were randomly divided into a wild-type group (WT-NC), a streptozotocin (STZ)-treated DM group (WT-DM), a Trx-1-TG group (TG-NC) and a Trx-1-TG DM group (TG-DM). Diabetic animals showed an increase in the time spent in the target quadrant and the number of platform crossings as well as AD-like behavior in the water maze experiment. The immunocontent of the AD-related protein Tau and the levels of cell apoptosis, β-amyloid (Aβ) plaque formation and neuronal degeneration in the hippocampus of the diabetic group were increased. Some key factors associated with ERS, such as protein disulfide isomerase (PDI), glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1α (IRE1α), tumor necrosis factor receptor-associated factor 2 (TRAF2), apoptosis signal-regulating kinase-1 (ASK1), c-Jun N-terminal kinase (JNK), protein kinase RNA (PKR)-like ER kinase (PERK), and C/EBP homologous protein (CHOP), were upregulated, and other factors related to anti-oxidant stress, such as nuclear factor erythroid 2-related factor (Nrf2), were downregulated in the DM group. Moreover, DM caused an increase in the immunocontents of caspase-3 and caspase-12. However, these changes were reversed in the Trx-1-tg DM group. Therefore, we conclude that Trx-1 might be a key factor in alleviating DE and AD by regulating ERS and oxidative stress response, thus preventing apoptosis.

Disruption of endoplasmic reticulum homeostasis exacerbates liver injury in clinically ketotic cows

Disruption of endoplasmic reticulum (ER) homeostasis, a condition termed "ER stress," contributes to the development of liver injury in nonruminants. Because liver injury is a prominent pathological feature associated with overproduction of ketone bodies in dairy cows with ketosis, understanding the ER stress state and its functional consequences on liver injury is of particular interest. Here, 30 multiparous cows (within 3 wk postpartum) classified based on blood β-hydroxybutyrate (BHB) as healthy (n = 15, BHB <0.6 mM) or clinically ketotic (n = 15, BHB >3.0 mM) were used. Compared with healthy cows, ketotic cows had greater levels of serum fatty acids and activities of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyl transferase, and glutamate dehydrogenase but lower serum glucose. Furthermore, dairy cows with ketosis had greater protein abundance of ER stress markers in liver tissue, including protein kinase RNA-like ER kinase (PERK), inositol-requiring protein-1α (IRE1α), and cleaved activating transcription factor-6 (ATF6). Cows with ketosis also had higher mRNA levels of hepatic 78-kDa glucose-regulated protein (GRP78) and spliced X-box binding protein 1 (sXBP1). These data confirmed an enhanced ER stress state in clinically ketotic cows. To explore whether enhanced hepatic ER stress was induced by elevated ketone bodies and the possible contribution of ER stress to liver injury, in vitro experiments were then performed using isolated primary calf hepatocytes treated with incremental concentrations of BHB (0, 0.6, 1.2, 3.0, and 4.8 mM) for 12 h with or without overexpression of GRP78 (the master regulator of unfolded protein response). Phosphorylation levels of PERK and IRE1α proteins, level of cleaved ATF6 protein, and mRNA abundance of GRP78 and sXBP1 in hepatocytes increased after treatment with high (3.0 and 4.8 mM) BHB, indicating a mechanistic link between excessive BHB and enhanced hepatic ER stress. Furthermore, treatment with 3.0 and 4.8 mM BHB markedly elevated activities of aspartate aminotransferase and alanine aminotransferase in cell supernatant, indicating exacerbated hepatocyte damage after ER stress was enhanced. Overexpression of GRP78 attenuated both BHB-induced ER stress and the ensuing cellular damage, suggesting that hepatocyte damage caused by excessive BHB can be mediated via enhanced ER stress. Overall, the present study revealed that ER stress may exacerbate liver injury development in clinically ketotic cows, underscoring the biological relevance of this pathway in the context of liver injury.

Berberine Improves Behavioral and Cognitive Deficits in a Mouse Model of Alzheimer's Disease via Regulation of β-Amyloid Production and Endoplasmic Reticulum Stress.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by β-amyloid (Aβ), neurofibrillary tangles, and neuronal cell death. Aggressive Aβ accumulation accelerates senile plaque formation and perturbs endoplasmic reticulum (ER) function. Aβ accumulation-induced changes stimulate the unfolded protein response (UPR), which can trigger neuronal apoptosis. Protein kinase RNA-like endoplasmic reticulum kinase (PERK), whose activation is stress-dependent, increases the phosphorylation of eukaryotic translation initiation factor-2α (eIF2α). eIF2α promotes the synthesis of β-site APP cleavage enzyme 1 (BACE1), which in turn facilitates Aβ generation and subsequent neuronal apoptosis. In this study, we investigated whether berberine could improve cognitive deficits in the triple-transgenic mouse model of Alzheimer's disease (3 × Tg AD) mice. Our results revealed that berberine treatment may inhibit PERK/eIF2α signaling-mediated BACE1 translation, thus reducing Aβ production and resultant neuronal apoptosis. Further, berberine may have neuroprotective effects, via attenuation of ER stress and oxidative stress. In sum, our study demonstrates the therapeutic potential of berberine for treating AD.

Hepatocyte steatosis inhibits hepatitis B virus secretion via induction of endoplasmic reticulum stress.

The effects of hepatocyte steatosis on hepatitis B virus (HBV) DNA replication and HBV-related antigen secretion are incompletely understood. The aims of this study are to explore the effects and mechanism of hepatocyte steatosis on HBV replication and secretion. Stearic acid (SA) and oleic acid (OA) were used to induce HepG2.2.15 cell steatosis in this study. The expressions of glucose-regulated protein 78 (GRP78), phosphorylation of protein kinase R-like endoplasmic reticulum (ER) kinase (p-PERK), and eukaryotic translation initiation factor 2α (p-eIF2α) were detected by Western blotting (WB). HBV DNA, HBsAg, and HBeAg in the supernatant were determined by real-time fluorescent polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay. Intracellular HBV DNA, HBsAg level, and HBV RNA were measured by real-time fluorescent PCR, WB, and real-time quantitative reverse transcriptase-PCR, respectively. The results showed that SA and OA significantly increased intracellular lipid droplets and triglyceride levels. SA and OA significantly induced GRP78, p-PERK, and p-eIF2α expressions from 24 to 72 h. 4-phenylbutyric acid (PBA) alleviated ER stress induced by SA. SA promoted intracellular HBsAg and HBV DNA accumulation; however, it inhibited the transcript of HBV 3.5 kb mRNA and S mRNA. The secretion of HBsAg and HBV DNA inhibited by SA or OA could be partially restored by pretreatment with PBA but not by inhibiting GRP78 expression with siRNA. Hepatocyte steatosis inhibits HBsAg and HBV DNA secretion via induction of ER stress in hepatocytes, but not via induction of GRP78.

PERK Is Critical for Alphavirus Nonstructural Protein Translation

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.

Endoplasmic reticulum stress regulates the intestinal stem cell state through CtBP2

Enforcing differentiation of cancer stem cells is considered as a potential strategy to sensitize colorectal cancer cells to irradiation and chemotherapy. Activation of the unfolded protein response, due to endoplasmic reticulum (ER) stress, causes rapid stem cell differentiation in normal intestinal and colon cancer cells. We previously found that stem cell differentiation was mediated by a Protein kinase R-like ER kinase (PERK) dependent arrest of mRNA translation, resulting in rapid protein depletion of WNT-dependent transcription factor c-MYC. We hypothesize that ER stress dependent stem cell differentiation may rely on the depletion of additional transcriptional regulators with a short protein half-life that are rapidly depleted due to a PERK-dependent translational pause. Using a novel screening method, we identify novel transcription factors that regulate the intestinal stem cell fate upon ER stress. ER stress was induced in LS174T cells with thapsigargin or subtilase cytotoxin (SubAB) and immediate alterations in nuclear transcription factor activity were assessed by the CatTFRE assay in which transcription factors present in nuclear lysate are bound to plasmid DNA, co-extracted and quantified using mass-spectrometry. The role of altered activity of transcription factor CtBP2 was further examined by modification of its expression levels using CAG-rtTA3-CtBP2 overexpression in small intestinal organoids, shCtBP2 knockdown in LS174T cells, and familial adenomatous polyposis patient-derived organoids. CtBP2 overexpression organoids were challenged by ER stress and ionizing irradiation. We identified a unique set of transcription factors with altered activation upon ER stress. Gene ontology analysis showed that transcription factors with diminished binding were involved in cellular differentiation processes. ER stress decreased CtBP2 protein expression in mouse small intestine. ER stress induced loss of CtBP2 expression which was rescued by inhibition of PERK signaling. CtBP2 was overexpressed in mouse and human colorectal adenomas. Inducible CtBP2 overexpression in organoids conferred higher clonogenic potential, resilience to irradiation-induced damage and a partial rescue of ER stress-induced loss of stemness. Using an unbiased proteomics approach, we identified a unique set of transcription factors for which DNA-binding activity is lost directly upon ER stress. We continued investigating the function of co-regulator CtBP2, and show that CtBP2 mediates ER stress-induced loss of stemness which supports the intestinal stem cell state in homeostatic stem cells and colorectal cancer cells.

Thiopurines activate an antiviral unfolded protein response that blocks influenza A virus glycoprotein accumulation.

Influenza A viruses (IAVs) utilize host shutoff mechanisms to limit antiviral gene expression and redirect translation machinery to the synthesis of viral proteins. Previously, we showed that IAV replication is sensitive to protein synthesis inhibitors that block translation initiation and induce formation of cytoplasmic condensates of untranslated messenger ribonucleoprotein complexes called stress granules (SGs). In this study, using an image-based high-content screen, we identified two thiopurines, 6-thioguanine (6-TG) and 6-thioguanosine (6-TGo), that triggered SG formation in IAV-infected cells and blocked IAV replication in a dose-dependent manner without eliciting SG formation in uninfected cells. 6-TG and 6-TGo selectively disrupted the synthesis and maturation of IAV glycoproteins hemagglutinin (HA) and neuraminidase (NA) without affecting the levels of the viral RNAs that encode them. By contrast, these thiopurines had minimal effect on other IAV proteins or the global host protein synthesis. Disruption of IAV glycoprotein accumulation by 6-TG and 6-TGo correlated with activation of unfolded protein response (UPR) sensors activating transcription factor-6 (ATF6), inositol requiring enzyme-1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), leading to downstream UPR gene expression. Treatment of infected cells with the chemical chaperone 4-phenylbutyric acid diminished thiopurine-induced UPR activation and partially restored the processing and accumulation of HA and NA. By contrast, chemical inhibition of the integrated stress response downstream of PERK restored accumulation of NA monomers but did not restore processing of viral glycoproteins. Genetic deletion of PERK enhanced the antiviral effect of 6-TG without causing overt cytotoxicity, suggesting that while UPR activation correlates with diminished viral glycoprotein accumulation, PERK could limit the antiviral effects of drug-induced ER stress. Taken together, these data indicate that 6-TG and 6-TGo are effective host-targeted antivirals that trigger the UPR and selectively disrupt accumulation of viral glycoproteins.IMPORTANCESecreted and transmembrane proteins are synthesized in the endoplasmic reticulum (ER), where they are folded and modified prior to transport. Many viruses rely on the ER for the synthesis and processing of viral glycoproteins that will ultimately be incorporated into viral envelopes. Viral burden on the ER can trigger the unfolded protein response (UPR). Much remains to be learned about how viruses co-opt the UPR to ensure efficient synthesis of viral glycoproteins. Here, we show that two FDA-approved thiopurine drugs, 6-TG and 6-TGo, induce the UPR, which represents a previously unrecognized effect of these drugs on cell physiology. This thiopurine-mediated UPR activation blocks influenza virus replication by impeding viral glycoprotein accumulation. Our findings suggest that 6-TG and 6-TGo may have broad antiviral effect against enveloped viruses that require precise tuning of the UPR to support viral glycoprotein synthesis.

Periplocin and cardiac glycosides suppress the unfolded protein response

The unfolded protein response (UPR) controls protein homeostasis through transcriptional and translational regulation. However, dysregulated UPR signaling has been associated with the pathogenesis of many human diseases. Therefore, the compounds modulating UPR may provide molecular insights for these pathologies in the context of UPR. Here, we screened small-molecule compounds that suppress UPR, using a library of Myanmar wild plant extracts. The screening system to track X-box binding protein 1 (XBP1) splicing activity revealed that the ethanol extract of the Periploca calophylla stem inhibited the inositol-requiring enzyme 1 (IRE1)-XBP1 pathway. We isolated and identified periplocin as a potent inhibitor of the IRE1-XBP1 axis. Periplocin also suppressed other UPR axes, protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Examining the structure–activity relationship of periplocin revealed that cardiac glycosides also inhibited UPR. Moreover, periplocin suppressed the constitutive activation of XBP1 and exerted cytotoxic effects in the human multiple myeloma cell lines, AMO1 and RPMI8226. These results reveal a novel suppressive effect of periplocin or the other cardiac glycosides on UPR regulation, suggesting that these compounds will contribute to our understanding of the pathological or physiological importance of UPR.

Targeting cancer cell kinase PERK incites antitumor immunity by inducing immunogenic cell death and reprograming tumor-linked immunosuppressive myelopoiesis

Abstract The tumor microenvironment incites sustained endoplasmic reticulum (ER) stress in infiltrating T and myeloid cells, which impairs their antitumor potential through intrinsic activation of PKR-like endoplasmic reticulum kinase (PERK). Nonetheless, the immunomodulatory role of PERK activation in the tumor cell compartment remains unknown. Using multiple melanoma models, we established that PERK ablation (PERKKO) in cancer cells culminates in caspase-independent immunogenic cell death (ICD) in response to ER stress. In vivo, PERKKO cells underwent ICD and released immunostimulatory factors including HMGB1, ATP, calreticulin and type I interferons which provoked a dramatic anti-tumor response and induction of the abscopal effect. Immunologically, PERKKO tumor cells undergoing ICD spurred influx of CCR2-dependent monocyte-derived dendritic cells (MoDCs) and tumor infiltration of activated antigen-specific T cells. Moreover, PERK deletion in tumor cells triggered systemic expansion of myeloid precursors and promoted their differentiation into MoDCs via type I interferon signaling. Notably, MoDC-induced T cell responses in PERKKO tumors impaired B16-F10 tumor progression and enabled complete rejection of BrafV600E-SM1 tumors. Substantiating the translational potential of our work, inhibition of PERK in melanoma-bearing hosts resulted in significant anti-tumor effects. Also, TCGA analyses of melanoma patients showed that PERK signaling or repressed ICD marker expression coincided with shorter overall survival. Collectively, these results demonstrate that targeting PERK in tumor cells can offer the dual benefit of inducing ICD and reprogramming myelopoiesis to drive protective antitumor immunity.

ER‐associated Regulation of Astrocyte Mitochondrial Function during METH Exposure and HIV‐1 Infection

Astrocytes are key regulators of central nervous system (CNS) health and neuronal function. Astrocyte mitochondrial dysfunction, such as induced by METH and HIV-1, threatens the provision of essential metabolic and antioxidant support to neurons. Thus, delineating regulatory pathways that can be targeted to prevent aberrant mitochondria homeostasis in astrocytes will be imperative for ensuring neuronal fitness/survival against CNS pathologies. Direct contact sites between the endoplasmic reticulum (ER) and the mitochondria, termed mitochondrial associated membranes (MAMs), are central hubs for regulating several cellular processes required for homeostasis, including mitochondrial metabolic activity. In fact, the transfer of Ca2+ from the ER to mitochondria is essential for mitochondrial bioenergetics. Recent investigations have also identified unique, yet ill-defined contributions of the three unfolded protein response (UPR) arms, beyond their classical ER stress functions, in regulating MAM tethering and/or signaling. Briefly, protein kinase RNA-like endoplasmic reticulum kinase (PERK) has been determined as a key regulator for MAM tethering, inositol-requiring kinase 1 (IRE1α) is implicated in regulating MAM-mediated Ca2+ transfer, and activating transcription factor 6 (ATF6) is suspected to participate in MAM formation as it is known to mediate ER elongation and lipid homeostasis. However, these regulatory mechanisms have not yet been fully elucidated. The current investigation examined changes in astrocyte mitochondrial function, ER stress, Ca2+ signaling, and the regulation of MAMs in response to METH exposure and HIV-1 infection. Then, we explored the role of ER-associated mechanisms in regulating astrocyte mitochondrial function. The effects of METH treatment were evaluated in both acute and chronic paradigms while responses to chronic HIV-1 infection was examined using a pseudotyped HIV-1 virion able to infect astrocytes. Astrocyte metabolic status was determined using Seahorse extracellular flux analyzer for a real-time assessment of cellular metabolism in addition to quantifying the expression profiles of essential metabolites. Changes in the expression of unfolded protein response (UPR) and MAM mediators were determined using RT-PCR and protein expression assays. Finally, pharmacological inhibition of the UPR pathways were used to delineate the ER-associated regulatory mechanisms mediating the changes in mitochondria bioenergetics. Our studies demonstrate increased astrocyte metabolic capacity in response to chronic METH exposure and HIV-1 infection which corresponded to increased expression of UPR/MAM mediators. Moreover, pharmacological inhibition of specifically IRE1α impaired astrocyte mitochondrial activity. These findings illustrate the importance of ER-mitochondria communication in regulating astrocyte mitochondrial function and identifies a possible mechanism to manipulate the metabolic and antioxidant coupling between astrocytes and neurons during METH/HIV-1 pathogenesis.

Pridopidine reduces mutant huntingtin-induced endoplasmic reticulum stress by modulation of the Sigma-1 receptor.

The endoplasmic reticulum (ER)-localized Sigma-1 receptor (S1R) is neuroprotective in models of neurodegenerative diseases, among them Huntington disease (HD). Recent clinical trials in HD patients and preclinical studies in cellular and mouse HD models suggest a therapeutic potential for the high-affinity S1R agonist pridopidine. However, the molecular mechanisms of the cytoprotective effect are unclear. We have previously reported strong induction of ER stress by toxic mutant huntingtin (mHtt) oligomers, which is reduced upon sequestration of these mHtt oligomers into large aggregates. Here, we show that pridopidine significantly ameliorates mHtt-induced ER stress in cellular HD models, starting at low nanomolar concentrations. Pridopidine reduced the levels of markers of the three branches of the unfolded protein response (UPR), showing the strongest effects on the PKR-like endoplasmic reticulum kinase (PERK) branch. The effect is S1R-dependent, as it is abolished in cells expressing mHtt in which the S1R was deleted using CRISPR/Cas9 technology. mHtt increased the level of the detergent-insoluble fraction of S1R, suggesting a compensatory cellular mechanism that responds to increased ER stress. Pridopidine further enhanced the levels of insoluble S1R, suggesting the stabilization of activated S1R oligomers. These S1R oligomeric species appeared in ER-localized patches, and not in the mitochondria-associated membranes nor the ER-derived quality control compartment. The colocalization of S1R with the chaperone BiP was significantly reduced by mHtt, and pridopidine restored this colocalization to normal, unstressed levels. Pridopidine increased toxic oligomeric mHtt recruitment into less toxic large sodium dodecyl sulfate-insoluble aggregates, suggesting that this in turn reduces ER stress and cytotoxicity.