Induction Of Endoplasmic Reticulum Stress Research Articles

Zelenirstat Inhibits N-Myristoyltransferases to Disrupt Src Family Kinase Signalling and Oxidative Phosphorylation Killing Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a hematological malignancy with limited treatment options and a high likelihood of recurrence after chemotherapy. We studied N-myristoylation, the myristate modification of proteins linked to survival signaling and metabolism, as a potential therapeutic target for AML. N-myristoylation is catalyzed by two N-myristoyltransferases (NMTs), NMT1 and NMT2, with varying expressions in AML cell lines and patient samples. We identified NMT2 expression as a marker for AML patient survival, and low NMT2 expression was associated with poor outcomes. We used the first-in-class pan-NMT inhibitor, zelenirstat, to investigate the role of N-myristoylation in AML. Zelenirstat effectively inhibits myristoylation in AML cell lines and patient samples, leading to degradation of Src family kinases (SFKs), induction of endoplasmic reticulum (ER) stress, apoptosis, and cell death. Zelenirstat was well tolerated in vivo and reduced the leukemic burden in an ectopic AML cell line and in multiple orthotopic AML patient-derived xenograft models. The leukemia stem cell (LSC)-enriched fractions of the hierarchical OCI-AML22 model were highly sensitive to myristoylation inhibition. Zelenirstat also impairs mitochondrial complex I and oxidative phosphorylation, which are critical for LSC survival. These findings suggest that targeting N-myristoylation with zelenirstat represents a novel therapeutic approach for AML, with promise in patients with currently poor outcomes.

Unstressing the Reticulum: Nutritional Strategies for Modulating Endoplasmic Reticulum Stress in Obesity.

The progression of obesity involves several molecular mechanisms that are closely associated with the pathophysiological response of the disease. Endoplasmic reticulum (ER) stress is one such factor. Lipotoxicity disrupts endoplasmic reticulum homeostasis in the context of obesity. Furthermore, it induces ER stress by activating several signaling pathways via inflammatory responses and oxidative stress. ER performs crucial functions in protein synthesis and lipid metabolism; thus, triggers such as lipotoxicity can promote the accumulation of misfolded proteins in the organelle. The accumulation of these proteins can lead to metabolic disorders and chronic inflammation, resulting in cell death. Thus, alternatives, such as flavonoids, amino acids, and polyphenols that are associated with antioxidant and anti-inflammatory responses have been proposed to attenuate this response by modulating ER stress via the administration of nutrients and bioactive compounds. Decreasing inflammation and oxidative stress can reduce the expression of several ER stress markers and improve clinical outcomes through the management of obesity, including the control of body weight, visceral fat, and lipid accumulation. This review explores the metabolic changes resulting from ER stress and discusses the role of nutritional interventions in modulating the ER stress pathway in obesity.

Insulin-degrading enzyme inhibition increases the unfolded protein response and favours lipid accumulation in the liver.

Nonalcoholic fatty liver disease refers to liver pathologies, ranging from steatosis to steatohepatitis, with fibrosis ultimately leading to cirrhosis and hepatocellular carcinoma. Although several mechanisms have been suggested, including insulin resistance, oxidative stress, and inflammation, its pathophysiology remains imperfectly understood. Over the last decade, a dysfunctional unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress emerged as one of the multiple driving factors. In parallel, growing evidence suggests that insulin-degrading enzyme (IDE), a highly conserved and ubiquitously expressed metallo-endopeptidase originally discovered for its role in insulin decay, may regulate ER stress and UPR. We investigated, by genetic and pharmacological approaches, in vitro and in vivo, whether IDE modulates ER stress-induced UPR and lipid accumulation in the liver. We found that IDE-deficient mice display higher hepatic triglyceride content along with higher inositol-requiring enzyme 1 (IRE1) pathway activation. Upon induction of ER stress by tunicamycin or palmitate in vitro or in vivo, pharmacological inhibition of IDE, using its inhibitor BDM44768, mainly exacerbated ER stress-induced IRE1 activation and promoted lipid accumulation in hepatocytes, effects that were abolished by the IRE1 inhibitors 4μ8c and KIRA6. Finally, we identified that IDE knockout promotes lipolysis in adipose tissue and increases hepatic CD36 expression, which may contribute to steatosis. These results unravel a novel role for IDE in the regulation of ER stress and development of hepatic steatosis. These findings pave the way to innovative strategies modulating IDE to treat metabolic diseases.

Advances in the Effects of Heat Stress on Ovarian Granulosa Cells: Unveiling Novel Ferroptosis Pathways

Heat stress has been one of the key research areas for researchers due to the wide-ranging effects and complex mechanisms of action of its stress product reactive oxygen species (ROS). The aim of this paper is to comprehensively review and summarize the effects of heat stress on ovarian granulosa cells and their mechanism of action. We systematically reviewed the effects of heat stress on ovarian granulosa cells, including intracellular steroid hormone changes, oxidative stress, apoptosis, and mitochondrial function. Meanwhile, this paper discusses in detail several major mechanisms by which heat stress induces apoptosis in ovarian granulosa cells, such as through the activation of apoptosis-related genes, induction of endoplasmic reticulum stress, and the mitochondrial pathway. In addition, we analyzed the mechanism of ferroptosis in ovarian granulosa cells under heat stress conditions, summarized the potential association between heat stress and ferroptosis in light of the existing literature, and explored the key factors in the mechanism of action of heat stress, such as the signaling pathways of Nrf2/Keap1, HSPs, and JNK, and analyzed their possible roles in the process of ferroptosis. Finally, this paper provides an outlook on the future research direction, describing the possible interaction between heat stress and ferroptosis, with a view to providing a theoretical basis for further understanding and revealing the complex mechanism of ferroptosis occurrence in ovarian granulosa cells under heat stress.

Endoplasmic reticulum stress alters myelin associated protein expression and extracellular vesicle composition in human oligodendrocytes

Myelination of the central nervous system is mediated by specialized glial cells called oligodendrocytes (OLs). Multiple sclerosis (MS) is characterized by loss of myelination and subsequent clinical symptoms that can severely impact the quality of life and mobility of those affected by the disease. The major protein components of myelin sheaths are synthesized in the endoplasmic reticulum (ER), and ER stress has been observed in patients with MS. Extracellular vesicles (EVs) have been shown to carry bioactive cargo and have the potential to be utilized as noninvasive biomarkers for various diseases. In the current study, we sought to determine how ER stress in OLs affected the production of key myelination proteins and EV release and composition. To achieve this, tunicamycin was used to induce ER stress in a human oligodendroglioma cell line and changes in myelination protein expression and markers of autophagy were assessed. EVs were also separated from the conditioned cell culture media through size exclusion chromatography and characterized. Significant reductions in the expression of myelination proteins and alterations to autophagosome formation were observed in cells undergoing ER stress. EVs released from these cells were slightly smaller relative to controls, and had strong expression of LC3B. We also observed significant upregulation of miR-29a-3p in ER stress EVs when compared to controls. Taken together, these data suggest that ER stress negatively impacts production of key myelination proteins and induces cells to release EVs that may function to preemptively activate autophagic pathways in neighboring cells.

The cytoplasmic-nuclear transport of DDX3X promotes immune-mediated liver injury in mice regulated by endoplasmic reticulum stress

Immune-mediated liver injury is a common characteristic of various liver diseases, including autoimmune and viral hepatitis. Here, we investigated the role of DEAD-box helicase 3, X-linked (DDX3X) in immune-mediated liver injury. Liver injury was induced in C57BL/6J mice via concanavalin A (Con A). DDX3X hepatocyte-specific knockout (DDX3XΔHep) mice and control (DDX3Xfl/fl) mice were utilized to investigate the role of DDX3X in liver injury. Primary hepatocytes were treated with tunicamycin (TM) to induce ER stress in vitro. The expression of DDX3X in patients with various liver diseases was evaluated. Hepatic DDX3X expression increased, and DDX3X translocated from the cytoplasm to the nucleus during Con A-induced liver injury. DDX3X deficiency ameliorated mouse liver injury and reduced ER stress in liver tissue. The inhibition of ER stress with 4-PBA significantly attenuated liver injury while decreasing DDX3X levels in liver tissue. However, the upregulation of hepatic DDX3X expression reversed Con A-induced liver injury and negated the protective effect of 4-PBA. Mechanistically, the nuclear translocation of DDX3X promoted ER stress-induced apoptosis through the transcriptional induction of CHOP. Moreover, DDX3X was elevated and translocated into the nucleus in patients with HBV-LF and AIH. Additionally, serum DDX3X levels markedly increased in patients with HBV-LF, and a consistent decrease in DDX3X was associated with a good prognosis. The cytoplasmic-to-nuclear translocation of DDX3X promotes ER stress-induced apoptosis, which is an obligatory step that drives hepatic necrosis and tissue damage. Notably, DDX3X is a potential therapeutic target for immune-mediated liver injury.

Isovalerylspiramycin I suppresses small cell lung cancer proliferation via ATR/CHK1 mediated DNA damage response and PERK/eIF2α/ATF4/CHOP mediated ER stress

Small cell lung cancer (SCLC) urgently needs new therapeutic approaches. We found that the antibiotic-derived compound Isovalerylspiramycin I (ISP-I) has potent anti-tumor activity against SCLC cell lines H1048 and DMS53 both in vitro and in vivo. ISP-I induced apoptosis, G2/M phase cell cycle arrest, and mitochondrial respiratory chain dysfunction in both cell lines. Comprehensive RNA sequencing revealed that the anti-SCLC effects of ISP-I were primarily attributed to ATR/CHK1-mediated DNA damage response and PERK/eIF2α/ATF4/CHOP-mediated ER stress. Importantly, the induction of DNA damage, ER stress, and apoptosis by ISP-I was mitigated by the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC), underscoring the critical role of ROS in the anti-SCLC mechanism of ISP-I. Moreover, ISP-I treatment induced immunogenic cell death (ICD) in SCLC cells, as evidenced by increased adenosine triphosphate (ATP) secretion, elevated release of high-mobility group box 1 (HMGB1), and enhanced exposure of calreticulin (CRT) on the cell surface. Additionally, network pharmacology analysis, combined with cellular thermal shift assay (CETSA) and cycloheximide (CHX) chase experiments, demonstrated that ISP-I acted as a ligand for apurinic/apyrimidinic endonuclease 1 (APEX1) and promoted its degradation, leading to the accumulation of ROS. In conclusion, our findings elucidate the multifaceted mechanisms underlying the anti-cancer effects of ISP-I, highlighting its potential as a promising therapeutic candidate for SCLC treatment.

Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development

Disruption of endoplasmic reticulum (ER) homeostasis, i.e., ER stress, is intrinsically linked with lipid metabolism disorders in dairy cows. Caveolin 1 (CAV1) is a ubiquitously-expressed membrane-associated scaffolding protein involved in regulating the secretory pathway within the ER. Whether inhibiting the activity of CAV1 affects the ER and its potential role in hepatic lipid deposition in dairy cows is unknown. Biopsies of liver tissue from Holstein cows (days in milk: median = 13 d, range = 5 to 21) diagnosed as healthy (n = 6, hepatic TAG levels < 1%, milk production: median = 38.9 kg/day, Interquartile range = 38.0 and 40.8) or suffering from fatty liver (n = 6, hepatic TAG levels > 5%, milk production: median = 36.6 kg/day, Interquartile range = 35.7 and 38.1) revealed that fatty liver was associated with lower abundance of CAV1 gene and protein, higher phosphorylation (p) levels of PERK and IRE1α, and increased abundance of ATF6, GRP78, CHOP protein, and several unfolded protein response (UPR) genes (ATF4, sXBP1, and GRP78). Proteins related to de novo fatty acid synthesis, including ACC1, SREBP-1c, PPARγ, and downstream targets genes of SREBP1 (ACACA and FASN) also had greater abundance. This in vivo analysis highlighted a mechanistic link between CAV1 protein abundance, ER stress, and lipid metabolism during fatty liver. A mechanistic study was then performed in vitro with primary hepatocytes isolated from 5 healthy calves (weight, 40-45 kg; 1 d old). Initially, hepatocytes were treated with FFA (1.2 mM) for 1, 3, 6, or 12 h. FFA treatment reduced CAV1 protein abundance linearly while it reduced abundance of ER stress-related proteins, p-IRE1α, p-PERK, GRP78, ATF6, and CHOP. Proteins related to de novo fatty acid synthesis (ACC1, SREBP-1c, PPARγ) also increased linearly, and lipid droplets accumulated progressively over time following FFA treatment. Subsequently, to assess the role of CAV1 in FFA-induced ER stress and de novo fatty acid synthesis, hepatocytes were transfected with pCMV-CAV1 (cattle)-3 × FLAG-Neo (pc-CAV1) plasmid to overexpress CAV1 or with siRNA to silence CAV1 (siCAV1) transcription. Overexpression of CAV1 alleviated ER stress by reducing levels of p-PERK and p-IRE1α, as well as the protein abundance of ATF6, GRP78, CHOP, and several UPR genes (GRP78, ATF4, and sXBP1). Similarly, CAV1 overexpression decreased protein abundance of ACC1, SREBP-1c, PPARγ, and downstream targets genes of SREBP1 (ACACA and FASN). Conversely, silencing CAV1 exacerbated FFA-induced ER stress and de novo fatty acid synthesis. Considering the negative role of FFA-induced ER stress on lipid accumulation in hepatocytes, a second in vitro experiment involved hepatocytes treated with 0.5 μg/mL tunicamycin (TM, a typical ER stress inducer) for 24 h with or without overexpressing CAV1 (pc-CAV1). Overexpressing caveolin 1 reversed TM-induced increases in mRNA and protein associated with ER stress and de novo fatty acid synthesis. Furthermore, use of hepatocytes transfected with pc-CAV1 for 48 h and subjected to co-immunoprecipitation revealed that CAV1 interacts with IRE1α and ATF6. Overall, the data suggest that CAV1 may help reduce hepatic ER stress and mitigate fatty acid synthesis by binding to and inhibiting IRE1α and ATF6 signaling.

Selective killing of castration-resistant prostate cancer cells by formycin A via the ATF4-CHOP axis.

Prostate cancer is initially androgen-dependent but often relapses to an androgen-independent state called castration-resistant prostate cancer (CRPC). Currently approved therapies have limited efficacy against CRPC, highlighting the need for novel therapeutic strategies. To address this need, we conducted a drug screen in our previously established aggressive CRPC cell model. We found that formycin A induced cell death in CRPC model cells but not in parental prostate cancer cells. In addition, formycin A upregulated death receptor 5 through the induction of endoplasmic reticulum stress, activating the "extrinsic" apoptosis pathway in CRPC model cells. Moreover, formycin A showed invivo antitumor efficacy against CRPC xenografts in castrated nude mice. Thus, our findings highlight the potential of formycin A as a CRPC therapeutic.

The Role of Protein Disulfide Isomerase Inhibitors in Cancer Therapy.

Protein disulfide isomerase (PDI) is a member of the mercaptan isomerase family, primarily located in the endoplasmic reticulum (ER). At least 21 PDI family members have been identified. PDI plays a key role in protein folding, correcting misfolded proteins, and catalyzing disulfide bond formation, rearrangement, and breaking. It also acts as a molecular chaperone. Dysregulation of PDI activity is thus linked to diseases such as cancer, infections, immune disorders, thrombosis, neurodegenerative diseases, and metabolic disorders. In particular, elevated intracellular PDI levels can enhance cancer cell proliferation, metastasis, and invasion, making it a potential cancer marker. Cancer cells require extensive protein synthesis, with disulfide bond formation by PDI being a critical producer. Thus, cancer cells have higher PDI levels than normal cells. Targeting PDI can induce ER stress and activate the Unfolded Protein Response (UPR) pathway, leading to cancer cell apoptosis. This review discusses the structure and function of PDI, PDI inhibitors in cancer therapy, and the limitations of current inhibitors, proposing especially future directions for developing new PDI inhibitors.

PCSK1N as a tumor size marker and an ER stress response protein in corticotroph pituitary adenomas.

Silent corticotroph adenoma (SCA) exhibits more tumor aggressiveness features than functioning adenomas (FCA). We aimed to investigate PCSK1N expression in CA and examine if ER stress-induced responses affect cell survival in a corticotroph tumor cell model. Clinical and imaging characteristics were recorded in 33 patients with FCA (20 women, 11 macroadenomas) and 18 SCA (8 women, all macroadenomas). Gene expression of proopiomelanocortin (POMC), T-box transcription factor 19(TBX19)/TPIT, proprotein convertase subtilisin/kexin type 1(PCSK1)/PC1/3, and its inhibitor PCSK1N, was measured by RT-qPCR in adenoma tissue.Mouse pituitary corticotroph tumor (AtT-20) cells were treated with tanespimycin (17-AAG), a HSP90 chaperone inhibitor, to induce ER stress, followed by gene and protein analyses. POMC, TPIT, and PCSK1 expression were higher, whereas PCSK1N was lower in FCA compared to SCA. PCSK1N correlated with POMC (rs= -0.514, p <0.001), TPIT (rs= -0.386, p = 0.005), PCSK1 (rs= -0.3691, p = 0.008), and tumor largest diameter (rs= 0.645, p <0.001), in all CA. Induction of ER stress by 17-AAG in AtT-20 cells led to a decrease of POMC and an increase of PCSK1N gene expression at 24h. Moreover, a downregulation of cell cycle, apoptosis, and senescence pathways, and alterations in cell adhesion and cytoskeleton were observed at the protein level. PCSK1N is higher in SCA compared with FCA, and associated with corticotroph cell markers and tumor size. PCSK1N is likely to be part of the adaptive response to ER stress, potentially conferring a survival advantage to the corticotroph tumor cell in conjunction with other proteins.

SIK2 Controls the Homeostatic Character of the POMC Secretome Acutely in Response to Pharmacological ER Stress Induction.

The neuronal etiology of obesity is centered around a diet-induced inflammatory state in the arcuate nucleus of the hypothalamus, which impairs the functionality of pro-opiomelanocortine neurons (POMCs) responsible for whole-body energy homeostasis and feeding behavior. Intriguingly, systemic salt inducible kinase 2 (SIK2) knockout mice demonstrated reduced food intake and energy expenditure along with modestly dysregulated metabolic parameters, suggesting a causal link between the absence of SIK2 activity in POMCs and the observed phenotype. To test this hypothesis, we conducted a comparative secretomics study from POMC neurons following pharmacologically induced endoplasmic reticulum (ER) stress induction, a hallmark of metabolic inflammation and POMC dysregulation in diet-induced obese (DIO) mice. Our data provide significant in vitro evidence for the POMC-specific SIK2 activity in controlling energy metabolism and feeding in DIO mice by regulating the nature of the related POMC secretome. Our data also suggest that under physiological stress conditions, SIK2 may act as a gatekeeper for the secreted inflammatory factors and signaling molecules critical for cellular survival and energy homeostasis. On the other hand, in the absence of SIK2, the gate opens, leading to a surge of inflammatory cytokines and apoptotic cues concomitant with the dysregulation of POMC neurons.

Metabolism of primary high-grade serous ovarian carcinoma (HGSOC) cells under limited glutamine or glucose availability

BackgroundHigh-grade serous ovarian carcinoma (HGSOC) is the most common and aggressive subtype of epithelial ovarian carcinoma. It is primarily diagnosed at stage III or IV when the 5-year survival rate ranges between 20% and 40%. Here, we aimed to validate the hypothesis, based on HGSOC cell lines, that proposed the existence of two distinct groups of HGSOC cells with high and low oxidative phosphorylation (OXPHOS) metabolism, respectively, which are associated with their responses to glucose and glutamine withdrawal.MethodsWe isolated and cultivated primary cancer cell cultures from HGSOC and nontransformed ovarian fibroblasts from the surrounding ovarium of 45 HGSOC patients. We tested the metabolic flexibility of the primary cells, particularly in response to glucose and glutamine depletion, analyzed and modulated endoplasmic reticulum stress, and searched for indices of the existence of previously reported groups of HGSOC cells with high and low OXPHOS metabolism.ResultsThe primary HGSOC cells did not form two groups with high and low OXPHOS that responded differently to glucose and glutamine availabilities in the cell culture medium. Instead, they exhibited a continuum of OXPHOS phenotypes. In most tumor cell isolates, the responses to glucose or glutamine withdrawal were mild and surprisingly correlated with those of nontransformed ovarian fibroblasts from the same patients. The growth of tumor-derived cells in the absence of glucose was positively correlated with the lipid trafficking regulator FABP4 and was negatively correlated with the expression levels of HK2 and HK1. The correlations between the expression of electron transport chain (ETC) proteins and the oxygen consumption rates or extracellular acidification rates were weak. ER stress markers were strongly expressed in all the analyzed tumors. ER stress was further potentiated by tunicamycin but not by the recently proposed ER stress inducers based on copper(II)-phenanthroline complexes. ER stress modulation increased autophagy in tumor cell isolates but not in nontransformed ovarian fibroblasts.ConclusionsAnalysis of the metabolism of primary HGSOC cells rejects the previously proposed hypothesis that there are distinct groups of HGSOC cells with high and low OXPHOS metabolism that respond differently to glutamine or glucose withdrawal and are characterized by ETC protein levels.

FicD sensitizes cellular response to glucose fluctuations in mouse embryonic fibroblasts

During homeostasis, the endoplasmic reticulum (ER) maintains productive transmembrane and secretory protein folding that is vital for proper cellular function. The ER-resident HSP70 chaperone, binding immunoglobulin protein (BiP), plays a pivotal role in sensing ER stress to activate the unfolded protein response (UPR). BiP function is regulated by the bifunctional enzyme filamentation induced by cyclic-AMP domain protein (FicD) that mediates AMPylation and deAMPylation of BiP in response to changes in ER stress. AMPylated BiP acts as a molecular rheostat to regulate UPR signaling, yet little is known about the molecular consequences of FicD loss. In this study, we investigate the role of FicD in mouse embryonic fibroblast (MEF) response to pharmacologically and metabolically induced ER stress. We find differential BiP AMPylation signatures when comparing robust chemical ER stress inducers to physiological glucose starvation stress and recovery. Wildtype MEFs respond to pharmacological ER stress by down-regulating BiP AMPylation. Conversely, BiP AMPylation in wildtype MEFs increases upon metabolic stress induced by glucose starvation. Deletion of FicD results in widespread gene expression changes under baseline growth conditions. In addition, FicD null MEFs exhibit dampened UPR signaling, altered cell stress recovery response, and unconstrained protein secretion. Taken together, our findings indicate that FicD is important for tampering UPR signaling, stress recovery, and the maintenance of secretory protein homeostasis.

An Essential Role for Calnexin in ER-Phagy and the Unfolded Protein Response.

ER-phagy is a specialized form of autophagy, defined by the lysosomal degradation of ER subdomains. ER-phagy has been implicated in relieving the ER from misfolded proteins during ER stress upon activation of the unfolded protein response (UPR). Here, we identified an essential role for the ER chaperone calnexin in regulating ER-phagy and the UPR in neurons. We showed that chemical induction of ER stress triggers ER-phagy in the somata and axons of primary cultured motoneurons. Under basal conditions, the depletion of calnexin leads to an enhanced ER-phagy in axons. However, upon ER stress induction, ER-phagy did not further increase in calnexin-deficient motoneurons. In addition to increased ER-phagy under basal conditions, we also detected an elevated proteasomal turnover of insoluble proteins, suggesting enhanced protein degradation by default. Surprisingly, we detected a diminished UPR in calnexin-deficient early cortical neurons under ER stress conditions. In summary, our data suggest a central role for calnexin in orchestrating both ER-phagy and the UPR to maintain protein homeostasis within the ER.

African swine fever virus modulates the endoplasmic reticulum stress-ATF6-calcium axis to facilitate viral replication

ABSTRACT African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boar, which threatens the global pig industry. Endoplasmic reticulum (ER) is a multifunctional signaling organelle in eukaryotic cells that is involved in protein synthesis, processing, posttranslational modification and quality control. As intracellular parasitic organisms, viruses have evolved several strategies to modulate ER functions to favor their life cycles. We have previously demonstrated that the differentially expressed genes associated with unfolded protein response (UPR), which represents a response to ER stress, are significantly enriched upon ASFV infection. However, the correlation between the ER stress or UPR and ASFV replication has not been illuminated yet. Here, we demonstrated that ASFV infection induces ER stress both in target cells and in vivo, and subsequently activates the activating transcription factor 6 (ATF6) branch of the UPR to facilitate viral replication. Mechanistically, ASFV infection disrupts intracellular calcium (Ca2+) homeostasis, while the ATF6 pathway facilitates ASFV replication by increasing the cytoplasmic Ca2+ level. More specifically, we demonstrated that ASFV infection triggers ER-dependent Ca2+ release via the inositol triphosphate receptor (IP3R) channel. Notably, we showed that the ASFV B117L protein plays crucial roles in ER stress and the downstream activation of the ATF6 branch, as well as the disruption of Ca2+ homeostasis. Taken together, our findings reveal for the first time that ASFV modulates the ER stress-ATF6-Ca2+ axis to facilitate viral replication, which provides novel insights into the development of antiviral strategies for ASFV.

Targeting stress induction of GRP78 by cardiac glycoside oleandrin dually suppresses cancer and COVID-19

BackgroundDespite recent therapeutic advances, combating cancer resistance remains a formidable challenge. The 78-kilodalton glucose-regulated protein (GRP78), a key stress-inducible endoplasmic reticulum (ER) chaperone, plays a crucial role in both cancer cell survival and stress adaptation. GRP78 is also upregulated during SARS-CoV-2 infection and acts as a critical host factor. Recently, we discovered cardiac glycosides (CGs) as novel suppressors of GRP78 stress induction through a high-throughput screen of clinically relevant compound libraries. This study aims to test the possibility that agents capable of blocking stress induction of GRP78 could dually suppress cancer and COVID-19.ResultsHere we report that oleandrin (OLN), is the most potent among the CGs in inhibiting acute stress induction of total GRP78, which also results in reduced cell surface and nuclear forms of GRP78 in stressed cells. The inhibition of stress induction of GRP78 is at the post-transcriptional level, independent of protein degradation and autophagy and may involve translational control as OLN blocks stress-induced loading of ribosomes onto GRP78 mRNAs. Moreover, the human Na+/K+-ATPase α3 isoform is critical for OLN suppression of GRP78 stress induction. OLN, in nanomolar range, enhances apoptosis, sensitizes colorectal cancer cells to chemotherapeutic agents, and reduces the viability of patient-derived colon cancer organoids. Likewise, OLN, suppresses GRP78 expression and impedes tumor growth in an orthotopic breast cancer xenograft model. Furthermore, OLN blocks infection by SARS-CoV-2 and its variants and enhances existing anti-viral therapies. Notably, GRP78 overexpression mitigates OLN-mediated cancer cell apoptotic onset and suppression of virus release.ConclusionOur findings validate GRP78 as a target of OLN anti-cancer and anti-viral activities. These proof-of-principle studies support further investigation of OLN as a readily accessible compound to dually combat cancer and COVID-19.

ER-associated degradation ligase HRD1 links ER stress to DNA damage repair by modulating the activity of DNA-PKcs

Proteostasis and genomic integrity are respectively regulated by the endoplasmic reticulum-associated protein degradation (ERAD) and DNA damage repair signaling pathways, with both pathways essential for carcinogenesis and drug resistance. How these signaling pathways coordinate with each other remains unexplored. We found that ER stress specifically induces the DNA-PKcs-regulated nonhomologous end joining (NHEJ) pathway to amend DNA damage and impede cell death. Intriguingly, sustained ER stress rapidly decreased the activity of DNA-PKcs and DNA damage accumulated, facilitating a switch from adaptation to cell death. This DNA-PKcs inactivation was caused by increased KU70/KU80 protein degradation. Unexpectedly, the ERAD ligase HRD1 was found to efficiently destabilize the classic nuclear protein HDAC1 in the cytoplasm, by catalyzing HDAC1's polyubiquitination at lysine 74, at a late stage of ER stress. By abolishing HDAC1-mediated KU70/KU80 deacetylation, HRD1 transmits ER signals to the nucleus. The resulting enhanced KU70/KU80 acetylation provides binding sites for the nuclear E3 ligase TRIM25, resulting in the promotion of polyubiquitination and the degradation of KU70/KU80 proteins. Both in vitro and in vivo cancer models showed that genetic or pharmacological inhibition of HADC1 or DNA-PKcs sensitizes colon cancer cells to ER stress inducers, including the Food and Drug Administration-approved drug celecoxib. The antitumor effects of the combined approach were also observed in patient-derived xenograft models. These findings identify a mechanistic link between ER stress (ERAD) in the cytoplasm and DNA damage (NHEJ) pathways in the nucleus, indicating that combined anticancer strategies may be developed that induce severe ER stress while simultaneously inhibiting KU70/KU80/DNA-PKcs-mediated NHEJ signaling.

Endoplasmic reticulum-targeting fluorescence turn-on probe for nitric oxide detection in living cells and serum samples

Nitric oxide (NO) is an important gas signaling molecule, and endoplasmic reticulum (ER) stress induced by NO may be related to the pathogenesis of many diseases. Therefore, the development of ER-targeted fluorescent probes for NO is of great significance to investigate the relationship between ER stress and NO concentration changes in related diseases. Herein, an ER-targeted fluorescent probe (ER-Np) for sensing NO was constructed. ER-Np was served as an excellent tool for detection NO with high selectivity, sensitivity and ER-targetable ability. Moreover, fluorescence imaging experiments indicated that ER-Np is capable of imaging NO in living cells. Impressively, visualization of endogenous NO production during dithiothreitol (DTT)-induced ER stress in living cells was successfully observed. In addition, we found that serum NO levels were upregulated in epilepsy children, which opens up a new avenue for further understanding the relationship between the diagnostic of epilepsy.

ER stress modulates Kv1.5 channels via PERK branch in HL-1 atrial myocytes: Relevance to atrial arrhythmogenesis and the effect of tetramethylpyrazine

Endoplasmic reticulum (ER) stress is implicated in cardiac arrhythmia whereas the associated mechanisms remain inadequately understood. Kv1.5 channels are essential for atrial repolarization. Whether ER stress affects Kv1.5 channels is unknown. This study aimed to elucidate the response of Kv1.5 channels to ER stress by clarifying the unfolded protein response (UPR) branch responsible for the channel modulation. In addition, the effect of tetramethylpyrazine (TMP) on Kv1.5 channels was studied. Patch clamp and western-blot results revealed that exposure of HL-1 atrial myocytes to ER stress inducer tunicamycin upregulates Kv1.5 expression, increases Kv1.5 channel current (IKur) (14.91 ± 1.11 vs. 6.11 ± 1.31 pA/pF, P < 0.001), and shortened action potential duration (APD) (APD90: 82.79 ± 5.25 vs.121.11 ± 6.72 ms, P < 0.01), which could be reverted by ER stress inhibitors. Blockade of the PERK branch while not IRE1 and ATF6 branches of UPR downregulated Kv1.5 expression, accompanied by a decreased IKur (9.03 ± 0.99 pA/pF) and a prolonged APD90 (113.69 ± 4.41 ms) (P < 0.01). PERK-mediated increases of Kv1.5 expression and IKur were also observed in HL-1 cells incubated with thapsigargin. TMP suppressed the enhancement of IKur (10.52 ± 0.97 vs. 17.52 ± 2.25 pA/pF, P < 0.05), prevented the shortening of APD (APD90: 110.16 ± 5.36 vs. 84.84 ± 4.58 ms, P < 0.05), and inhibited the upregulation of Kv1.5 triggered by ER stress. Our study suggests that ER stress induces upregulation and activation of Kv1.5 channels in atrial myocytes through the PERK branch of UPR. TMP prevents Kv1.5 upregulation/activation and the resultant APD shortening by inhibiting ER stress. These results may shed light on the mechanisms of atrial arrhythmogenesis and the antiarrhythmic effect of the traditional Chinese herb TMP.