Protein Kinase RNA-like Endoplasmic Reticulum Research Articles

Protein overexpression by adeno-associated virus-based gene therapy products in cardiomyocytes induces endoplasmic reticulum stress and myocardial degeneration in mice.

Gene therapy (GT) products created using adeno-associated virus (AAV) vectors tend to exhibit toxicity via immune reactions, but other mechanisms of toxicity remain incompletely understood. We examined the cardiotoxicity of an overexpressed transgenic protein. Male C57BL/6J mice were treated with a single intravenous dose of product X, an AAV-based GT product, at 2.6 × 1013 vg/kg. Necropsies were performed at 24 h, 7 days, and 14 days after dosing. Pathological examination and gene expression analysis were performed on the heart. Histopathologically, hypertrophy and vacuolar degeneration of cardiomyocytes and fibrosis were observed 14 days after dosing. Immunohistochemistry for endoplasmic reticulum (ER) stress-related proteins revealed increased positive reactions for glucose-regulated protein 78 and C/EBPR homologous protein in cardiomyocytes 7 days after dosing, without histopathological abnormalities. Fourteen days after dosing, some cardiomyocytes showed positivity for PKR-like endoplasmic reticulum kinase and activating transcription factor 4 expression. Ultrastructurally, increases in the ER and cytosol were observed in cardiomyocytes 7 days after dosing, along with an increase in the number of Golgi apparatus compartments 14 days after dosing. The tissue concentration of the transgene product protein increased 7 days after dosing. Gene expression analysis showed upregulation of ER stress-related genes 7 days after dosing, suggesting activation of the PKR-like ER kinase pathway of the unfolded protein reaction (UPR). Thus, the cardiotoxicity induced by product X was considered to involve cell damage caused by the overexpression of the product protein accompanied by UPR. Marked UPR activation may also cause toxicity of AAV-based GT products.

Protein Kinases in Obesity, and the Kinase-Targeted Therapy.

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified as dual-specificity kinases and dual-specificity phosphatases. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases and play an important role in obesity. Impairment of insulin signaling in obesity is largely mediated by the activation of the inhibitor of kappa B-kinase beta and the c-Jun N-terminal kinase (JNK). Oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular levels. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. To alleviate lipotoxicity and insulin resistance, promising targets are pharmacologically inhibited. Nifedipine, calcium channel blocker, stimulates lipogenesis and adipogenesis by downregulating AMPK and upregulating mTOR, which thereby enhances lipid storage. Contrary to the nifedipine, metformin activates AMPK, increases fatty acid oxidation, suppresses fatty acid synthesis and deposition, and thus alleviates lipotoxicity. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2 alpha kinase (PERK), and activating transcription factor-6. The transcriptional regulation of adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. An increase in intracellular oxidative stress can promote PKC-β activation. Activated PKC-β induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhance triglyceride accumulation and lipotoxicity. Liraglutide attenuates mitochondrial dysfunction and reactive oxygen species generation. Co-treatment of antiobesity and antidiabetic herbal compound, berberine with antipsychotic drug olanzapine decreases the accumulation of triglyceride. While low-dose rapamycin, metformin, amlexanox, thiazolidinediones, and saroglitazar protect against insulin resistance, glucagon-like peptide-1 analog liraglutide inhibits palmitate-induced inflammation by suppressing mTOR complex 1 (mTORC1) activity and protects against lipotoxicity.

Experimental periodontitis induced hypoadiponectinemia by IRE1α-mediated endoplasmic reticulum stress in adipocytes

BackgroudHypoadiponectinemia is the important cause of insulin resistance. Recent studies have shown that periodontitis is associated with hypoadiponectinemia. The purpose of this study was to investigate the effect of periodontitis-induced endoplasmic reticulum stress (ERS) in visceral adipocytes on hypoadiponectinemia.MethodsRat periodontitis models were established by local ligation with silk around the bilateral maxillary second molars. Porphyromonas gingivalis-lipopolysaccharid (P.g-LPS) was also used to stimulate the visceral adipocytes in vitro. The protein expression levels of glucose regulated protein 78 (GRP78), inositol-requiring protein 1α (IRE1α), protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6) and adiponectin were detected. IRE1α lentiviruses were transfected into visceral adipocytes in vitro, and an IRE1α inhibitor (KIRA6) was injected in epididymal adipose tissue of rats to detect and verify the effect of ERS on adiponectin expression in visceral adipocytes in vivo.ResultsHypoadiponectinemia was observed in periodontitis rat, and the expression levels of ERS key proteins GRP78 and the phosphorylation levels of IRE1α (p-IRE1α)/IRE1α in visceral adipocytes were increased, while the expression levels of adiponectin protein were decreased. After KIRA6 injection into epididymal adipose tissue of rats with periodontitis, adiponectin levels in visceral adipocytes increased, and serum adiponectin levels recovered to a certain extent. The protein expression levels of GRP78 and p-IRE1α/IRE1α were increased and adiponectin protein expression was decreased in P.g-LPS-induced visceral adipocytes. Overexpression of IRE1α further inhibited adiponectin expression in P.g-LPS-stimulated visceral adipocytes, and conversely, IRE1α inhibition restored adiponectin expression.ConclusionsOur findings suggest that periodontitis induces ERS in visceral adipocytes leading to hypoadiponectinemia. IRE1α is a key protein regulating adiponectin expression in visceral adipocytes.

Atypical cell death and insufficient matrix organization in long-bone growth plates from Tric-b-knockout mice

TRIC-A and TRIC-B proteins form homotrimeric cation-permeable channels in the endoplasmic reticulum (ER) and nuclear membranes and are thought to contribute to counterionic flux coupled with store Ca2+ release in various cell types. Serious mutations in the TRIC-B (also referred to as TMEM38B) locus cause autosomal recessive osteogenesis imperfecta (OI), which is characterized by insufficient bone mineralization. We have reported that Tric-b-knockout mice can be used as an OI model; Tric-b deficiency deranges ER Ca2+ handling and thus reduces extracellular matrix (ECM) synthesis in osteoblasts, leading to poor mineralization. Here we report irregular cell death and insufficient ECM in long-bone growth plates from Tric-b-knockout embryos. In the knockout growth plate chondrocytes, excess pro-collagen fibers were occasionally accumulated in severely dilated ER elements. Of the major ER stress pathways, activated PERK/eIF2α (PKR-like ER kinase/ eukaryotic initiation factor 2α) signaling seemed to inordinately alter gene expression to induce apoptosis-related proteins including CHOP (CCAAT/enhancer binding protein homologous protein) and caspase 12 in the knockout chondrocytes. Ca2+ imaging detected aberrant Ca2+ handling in the knockout chondrocytes; ER Ca2+ release was impaired, while cytoplasmic Ca2+ level was elevated. Our observations suggest that Tric-b deficiency directs growth plate chondrocytes to pro-apoptotic states by compromising cellular Ca2+-handling and exacerbating ER stress response, leading to impaired ECM synthesis and accidental cell death.

Involvement of spinal NADPH oxidase 4 and endoplasmic reticulum stress in morphine-tolerant rats.

Morphine tolerance (MT) is currently a challenging issue related to intractable pain treatment. Studies have shown that reactive oxygen species (ROSs) derived from NADPH oxidase (NOX) and produced in response to endoplasmic reticulum (ER) stress participate in MT development. However, which NOX subtype initiates ER stress during MT development is unclear. NOX4 is mainly expressed on intracellular membranes, such as the ER and mitochondrial membranes, and its sole function is to produce ROS. Whether NOX4 is activated during MT development and the mechanisms underlying the association between NOX4 and ER stress during this process still need to be confirmed. In our study, we used the classic morphine-tolerant rat model and evaluated the analgesic effect of intrathecally injected morphine through a hot water tail-flick assay. Our research demonstrated for the first time that chronic morphine administration upregulates NOX4 expression in the spinal cord by activating three ER stress sensors, protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6), subsequently leading to the activation of microtubule-associated protein 1 light chain 3 b (LC3B) and P62 (a well-known autophagy marker) in GABAergic neurons. Our results may suggest that regulating NOX4 and the key mechanism underlying ER stress or autophagy may be a promising strategy to treat and prevent MT development.

Dibromoacetonitrile induced autophagy by mediating the PERK signalling pathway and ROS interaction in HT22 cell

Dibromoacetonitrile (DBAN) is a high-risk haloacetonitrile (HAN) generated as a byproduct of chloramine disinfection in drinking water. DBAN-induced neurotoxicity in mouse hippocampal neuronal cells (HT22) and mammals was observed to be related to reactive oxygen species (ROS). ROS, endoplasmic reticulum stress (ERS) and autophagy play crucial roles in regulating a variety of cellular processes. However, whether ERS and autophagy are associated with HAN-responsive apoptosis remains unclear. This study indicated that DBAN (10 μM, 24 h) activated the ERS protein kinase like endoplasmic reticulum kinase (PERK) signaling pathway. The ERS inhibitor 4-phenylbutyric acid (4-PBA) reversed DBAN-inhibited cell viability and alleviated DBAN-induced apoptosis in HT22 cell, indicating that activation of the ERS PERK pathway mediates DBAN induced cytotoxicity. Moreover, DBAN activated autophagy. The autophagy inhibitor 3-methyladenine(3-MA) reversed DBAN-inhibited cell viability and alleviated DBAN-induced apoptosis in HT22 cell, suggesting that autophagy activation mediates DBAN-induced cell toxicity. Notably, the results showed that 4-PBA inhibited DBAN-activated autophagy, demonstrating that ERS–PERK promotes DBAN-induced cellular autophagy. Pretreatment with antioxidant N-acetylcysteine (NAC) inhibited the increase in ROS production and the activation of ERS, and protected cells from toxicity. Furthermore, 4-PBA pretreatment reduced the increase in ROS production, indicating that the ROS and PERK promote each other and form a positive feedback loop. ROS also promoted DBAN-induced autophagy. In summary, our findings indicate that DBAN induced autophagy by mediating the PERK signalling pathway and ROS interaction, leading to HT22 cell damage. Accordingly, targeting these pathogenic mechanisms may provide a potential target and theoretical basis for preventing and improving HAN-induced neurotoxicity.

Ascomylactam C Induces an Immunogenic Cell Death Signature via Mitochondria-Associated ER Stress in Lung Cancer and Melanoma.

Ascomylactam C (AsC) is a new 13-membered-ring macrocyclic alkaloid, which was first isolated and identified in 2019 from the secondary metabolites of the mangrove endophytic fungus Didymella sp. CYSK-4 in the South China Sea. AsC has been found to have a broad-spectrum cytotoxic activity. However, the antitumor effects in vivo and mechanisms of AsC remain unclear. The aim of this study was to describe the effects of AsC on lung cancer and melanoma cells and to explore the antitumor molecular mechanism of AsC. In vitro, we used plate colony formation experiments and demonstrated the ability of AsC to inhibit low-density tumor growth. An Annexin V/PI cell apoptosis detection experiment revealed that AsC induced tumor cell apoptosis. In vivo, AsC suppressed the tumor growth of LLC and B16F10 allograft significantly in mice, and promoted the infiltration of CD4+ T and CD8+ T cells in tumor tissues. Mechanistically, by analyses of Western blotting, immunofluorescence and ELISA analysis, we found that AsC increased ROS formation, induced endoplasmic reticulum (ER) stress, activated the protein kinase RNA-like ER kinase (PERK)/eukaryotic translation initiation factor (eIF2α)/activating transcription factor 4 (ATF4)/C/EBP homologous protein (CHOP) signaling pathway, and induced immunogenic cell death (ICD) of tumor cells. Our results suggest that AsC may be a potentially promising antitumor drug candidate.

Formylpeptide receptor 1 contributes to epidermal barrier dysfunction-induced skin inflammation through NOD-like receptor C4-dependent keratinocyte activation.

Skin barrier dysfunction may both initiate and aggravate skin inflammation. However, the mechanisms involved in the inflammation process remain largely unknown. We sought to determine how skin barrier dysfunction enhances skin inflammation and molecular mechanisms. Skin barrier defect mice were established by tape stripping or topical use of acetone on wildtype mice, or filaggrin deficiency. RNA-Seq was employed to analyse the differentially expressed genes in mice with skin barrier defects. Primary human keratinocytes were transfected with formylpeptide receptor (FPR)1 or protein kinase R-like endoplasmic reticulum (ER) kinase (PERK) small interfering RNA to examine the effects of these gene targets. The expressions of inflammasome NOD-like receptor (NLR)C4, epidermal barrier genes and inflammatory mediators were evaluated. Mechanical (tape stripping), chemical (acetone) or genetic (filaggrin deficiency) barrier disruption in mice amplified the expression of proinflammatory genes, with transcriptomic profiling revealing overexpression of formylpeptide receptor (Fpr1) in the epidermis. Treatment with the FPR1 agonist fMLP in keratinocytes upregulated the expression of the NLRC4 inflammasome and increased interleukin-1β secretion through modulation of ER stress via the PERK-eIF2α-C/EBP homologous protein pathway. The activation of the FPR1-NLRC4 axis was also observed in skin specimens from old healthy individuals with skin barrier defect or elderly mice. Conversely, topical administration with a FPR1 antagonist, or Nlrc4 silencing, led to the normalization of barrier dysfunction and alleviation of inflammatory skin responses in vivo. In summary, our findings show that the FPR1-NLRC4 inflammasome axis is activated upon skin barrier disruption and may explain exaggerated inflammatory responses that are observed in disease states characterized by epidermal dysfunction. Pharmacological inhibition of FPR1 or NLRC4 represents a potential therapeutic target.

A review on the mechanisms underlying the antitumor effects of natural products by targeting the endoplasmic reticulum stress apoptosis pathway.

Cancer poses a substantial risk to human life and wellbeing as a result of its elevated incidence and fatality rates. Endoplasmic reticulum stress (ERS) is an important pathway that regulates cellular homeostasis. When ERS is under- or overexpressed, it activates the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-, inositol-requiring enzyme 1 (IRE1)- and activating transcription Factor 6 (ATF6)-related apoptotic pathways to induce apoptosis. Tumor cells and microenvironment are susceptible to ERS, making the modulation of ERS a potential therapeutic approach for treating tumors. The use of natural products to treat tumors has substantially progressed, with various extracts demonstrating antitumor effects. Nevertheless, there are few reports on the effectiveness of natural products in inducing apoptosis by specifically targeting and regulating the ERS pathway. Further investigation and elaboration of its mechanism of action are still needed. This paper examines the antitumor mechanism of action by which natural products exert antitumor effects from the perspective of ERS regulation to provide a theoretical basis and new research directions for tumor therapy.

Pan-Inhibition of Protein Disulfide Isomerase Caused Cell Death through Disrupting Cellular Proteostasis in Pancreatic Ductal Adenocarcinoma Cells.

The protein disulfide isomerase (PDI) family is a group of thioredoxin endoplasmic reticulum (ER)-resident enzymes and molecular chaperones that play crucial roles in the correct folding of proteins. PDIs are upregulated in multiple cancer types and are considered a novel target for cancer therapy. In this study, we found that a potent pan-PDI inhibitor, E64FC26, significantly decreased the proliferation of pancreatic ductal adenocarcinoma (PDAC) cells. As expected, E64FC26 treatment increased ER stress and the unfolded protein response (UPR), as evidenced by upregulation of glucose-regulated protein, 78-kDa (GRP78), phosphorylated (p)-PKR-like ER kinase (PERK), and p-eukaryotic initiation factor 2α (eIF2α). Persistent ER stress was found to lead to apoptosis, ferroptosis, and autophagy, all of which are dependent on lysosomal functions. First, there was little cleaved caspase-3 in E64FC26-treated cells according to Western blotting, but a higher dose of E64FC26 was needed to induce caspase activity. Then, E64FC26-induced cell death could be reversed by adding the iron chelator, deferoxamine, and the reactive oxygen species scavengers, ferrostatin-1 and N-acetylcysteine. Furthermore, the autophagosome-specific marker, light chain 3B (LC3B)-II, increased, but the autolysosome marker, sequestosome 1 (SQSTM1)/p62, was not degraded in E64FC26-treated cells. Using the FUW mCherry-LC3 plasmid and acridine orange staining, we also discovered a lower number of acidic vesicles, such as autolysosomes and mature lysosomes, in E64FC26-treated cells. Finally, E64FC26 treatment increased the cathepsin L precursor (pre-CTSL) but decreased mature CTSL expression according to Western blotting, indicating a defective lysosome. These results suggested that the PDI inhibitor, E64FC26, might initially impede proper folding of proteins, and then induce ER stress and disrupt proteostasis, subsequently leading to lysosomal defects. Due to defective lysosomes, the extents of apoptosis and ferroptosis were limited, and fusion with autophagosomes was blocked in E64FC26-treated cells. Blockade of autolysosomal formation further led to the autophagic cell death of PDAC cells.

The Unfolded Protein Response in a Murine Model of Alzheimer's Disease: Looking for Predictors.

Alzheimer's disease (AD) represents the most frequent type of dementia worldwide, and aging is the most important risk factor for the sporadic form of the pathology. The endoplasmic reticulum (ER), the main cellular actor involved in proteostasis, appears significantly compromised in AD due to the accumulation of the β-amyloid (Aβ) protein and the phosphorylated Tau protein. Increasing protein misfolding activates a specific cellular response known as Unfolded Protein Response (UPR), which orchestrates the recovery of ER function. The aim of the present study was to investigate the role of UPR in a murine model of AD induced by intracerebroventricular (i.c.v.) injection of Aβ1-42 oligomers at 3 or 18 months. The oligomer injection in aged animals induced memory impairment, oxidative stress, and the depletion of glutathione reserve. Furthermore, the RNA sequencing and the bioinformatic analysis performed showed the enrichment of several pathways involved in neurodegeneration and protein regulations. The analysis highlighted the significant dysregulation of the protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α) and activating transcription factor 6 (ATF-6). In turn, ER stress affected the PI3K/Akt/Gsk3β and MAPK/ERK pathways, highlighting Mapkapk5 as a potential marker, whose regulation could lead to the definition of new pharmacological and neuroprotective strategies to counteract AD.

A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease.

JOURNAL/nrgr/04.03/01300535-202408000-00037/figure1/v/2023-12-16T180322Z/r/image-tiff Endoplasmic reticulum stress and mitochondrial dysfunction play important roles in Parkinson's disease, but the regulatory mechanism remains elusive. Prohibitin-2 (PHB2) is a newly discovered autophagy receptor in the mitochondrial inner membrane, and its role in Parkinson's disease remains unclear. Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is a factor that regulates cell fate during endoplasmic reticulum stress. Parkin is regulated by PERK and is a target of the unfolded protein response. It is unclear whether PERK regulates PHB2-mediated mitophagy through Parkin. In this study, we established a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease. We used adeno-associated virus to knockdown PHB2 expression. Our results showed that loss of dopaminergic neurons and motor deficits were aggravated in the MPTP-induced mouse model of Parkinson's disease. Overexpression of PHB2 inhibited these abnormalities. We also established a 1-methyl-4-phenylpyridine (MPP+)-induced SH-SY5Y cell model of Parkinson's disease. We found that overexpression of Parkin increased co-localization of PHB2 and microtubule-associated protein 1 light chain 3, and promoted mitophagy. In addition, MPP+ regulated Parkin involvement in PHB2-mediated mitophagy through phosphorylation of PERK. These findings suggest that PHB2 participates in the development of Parkinson's disease by interacting with endoplasmic reticulum stress and Parkin.

Thromboxane A2/thromboxane A2 receptor axis facilitates hepatic insulin resistance and steatosis through endoplasmic reticulum stress in non-alcoholic fatty liver disease.

Defective insulin signalling and dysfunction of the endoplasmic reticulum (ER), driven by excessive lipid accumulation in the liver, is a characteristic feature in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Thromboxane A2 (TXA2 ), an arachidonic acid metabolite, is significantly elevated in obesity and plays a crucial role in hepatic gluconeogenesis and adipose tissue macrophage polarization. However, the role of liver TXA2 /TP receptors in insulin resistance and lipid metabolism is largely unknown. TP receptor knockout (TP-/- ) mice were generated and fed a high-fat diet for 16 weeks. Insulin sensitivity, ER stress responses and hepatic lipid accumulation were assessed. Furthermore, we used primary hepatocytes to dissect the mechanisms by which the TXA2 /TP receptor axis regulates insulin signalling and hepatocyte lipogenesis. TXA2 was increased in diet-induced obese mice, and depletion of TP receptors in adult mice improved systemic insulin resistance and hepatic steatosis. Mechanistically, we found that the TXA2 /TP receptor axis disrupts insulin signalling by activating the Ca2+ /calcium calmodulin-dependent kinase II γ (CaMKIIγ)-protein kinase RNA-like endoplasmic reticulum kinase (PERK)-C/EBP homologous protein (Chop)-tribbles-like protein 3 (TRB3) axis in hepatocytes. In addition, our results revealed that the TXA2 /TP receptor axis directly promoted lipogenesis in primary hepatocytes and contributed to Kupffer cell inflammation. The TXA2 /TP receptor axis facilitates insulin resistance through Ca2+ /CaMKIIγ to activate PERK-Chop-TRB3 signalling. Inhibition of hepatocyte TP receptors improved hepatic steatosis and inflammation. The TP receptor is a new therapeutic target for NAFLD and metabolic syndrome.

Abstract 13301: TRIM29 Causes Pathogenesis of Viral Myocarditis by Inducing PERK-Mediated Endoplasmic Reticulum Stress and Reactive Oxygen Species Responses

Introduction: Viral myocarditis is an inflammatory disease of the myocardium which contributes to a significant fraction of sudden death in children and young adults. The ongoing coronavirus disease 19 pandemic emphasizes the need for understanding the pathogenesis mechanisms and potential treatment strategies for viral myocarditis. Methods: To understand the role of the novel E3 ligase TRIM29 in controlling viral myocarditis, wild-type and TRIM29 knockout mice were infected with coxsackievirus B3 (CVB3) for inducing viral myocarditis. Mice survival and heart function were monitored by transthoracic echocardiography, and hearts were harvested for histology and immunohistochemistry analysis. Real-time PCR, western blotting, co-immunoprecipitation, enzyme-linked immunoassay, flow cytometry, over-expression and knockdown techniques were used to understand the pathogenic mechanisms of TRIM29 in regulating endoplasmic reticulum stress response after virus infection in this study. Results: We found that TRIM29 was highly induced by cardiotropic viruses, including CVB3 and encephalomyocarditis (EMCV), and promoted protein kinase RNA-like endoplasmic reticulum kinase (PERK) mediated endoplasmic reticulum (ER) stress, apoptosis, and reactive oxygen species (ROS) responses that restrict viral replication in cardiomyocytes in vitro . TRIM29 deficiency protected mice from viral myocarditis by promoting cardiac antiviral functions and reducing PERK-mediated inflammation and immunosuppressive cells in vivo . Mechanistically, TRIM29 interacted with PERK to catalyze SUMOylation of PERK for maintaining its stability, thereby promoting PERK-mediated signaling pathways. Conclusions: Our data provide the first evidence that TRIM29-mediated PERK signaling pathways cause pathogenesis of viral myocarditis and identify the TRIM29-PERK axis as a potential therapeutic target for treating viral induced cardiovascular diseases.

Evaluation of the dietary L-valine on fish growth and intestinal health after infection with Aeromonas veronii

Endoplasmic reticulum stress and subsequent apoptosis play a pivotal role in bacterial infection in intestine. The objective of this study was to assess the influence of dietary L-valine (Val) on growth, antioxidant capacity, and endoplasmic reticulum stress-mediated apoptosis in the intestine of juvenile largemouth bass Micropterus punctulatus following an Aeromonas veronii infection. A total of 720 fish were randomly assigned to six groups and provided with varying levels of Val for 77 days. Subsequently, they were challenged with A. veronii. Our results revealed that dietary Val: (1) increased percent weight gain (PWG), specific growth rate (SGR), feed intake (FI), feed efficiency (FE), protein efficiency ratio (PER), protein production value (PPV), lipid production value (LPV), ash production value (APV) and survival rate (SR). Additionally, it alleviated A. veronii-induced intestinal injury; (2) improved intestinal antioxidant capacity by enhancing the mRNA levels of superoxide dismutase 1 (SOD1), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and glutamate-cysteine ligase catalytic subunit (GCLC) via the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway; (3) reduced intestinal DNA fragments and down-regulated glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), protein kinase-like ER kinase (PERK), eukaryotic Initiation Factor 2 (eIF2α), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), caspase 9, and caspase 3 mRNA levels through modulating the PERK/eIF2α signaling pathway. These findings suggest that optimal dietary Val enhances the growth performance, antioxidant capacity, and mitigates A. veronii-induced intestinal injury. Finally, the mitigates optimal Val requirement of largemouth bass (37.99–162.92 g) is 21.50 g/kg diet, corresponding to 46.64 g/kg protein.

Acute Endoplasmic Reticulum Stress Suppresses Hepatic Gluconeogenesis by Stimulating MAPK Phosphatase 3 Degradation.

Drug-induced liver injury (DILI) is a widespread and harmful disease, and is closely linked to acute endoplasmic reticulum (ER) stress. Previous reports have shown that acute ER stress can suppress hepatic gluconeogenesis and even leads to hypoglycemia. However, the mechanism is still unclear. MAPK phosphatase 3 (MKP-3) is a positive regulator for gluconeogenesis. Thus, this study was conducted to investigate the role of MKP-3 in the suppression of gluconeogenesis by acute ER stress, as well as the regulatory role of acute ER stress on the expression of MKP-3. Results showed that acute ER stress induced by tunicamycin significantly suppressed gluconeogenesis in both hepatocytes and mouse liver, reduced glucose production level in hepatocytes, and decreased fasting blood glucose level in mice. Additionally, the protein level of MKP-3 was reduced by acute ER stress in both hepatocytes and mouse liver. Mkp-3 deficiency eliminated the inhibitory effect of acute ER stress on gluconeogenesis in hepatocytes. Moreover, the reduction effect of acute ER stress on blood glucose level and hepatic glucose 6-phosphatase (G6pc) expression was not observed in the liver-specific Mkp-3 knockout mice. Furthermore, activation of protein kinase R-like ER kinase (PERK) decreased the MKP-3 protein level, while inactivation of PERK abolished the reduction effect of acute ER stress on the MKP-3 protein level in hepatocytes. Taken together, our study suggested that acute ER stress could suppress hepatic gluconeogenesis by stimulating MKP-3 degradation via PERK, at least partially. Thus, MKP-3 might be a therapeutic target for DILI-related hypoglycemia.

Omega-3 Fatty Acids Inhibit Endoplasmic Reticulum (ER) Stress in Human Coronary Artery Endothelial Cells

The biological effects of fatty acids differ by their structure. Saturated fatty acids and trans-fatty acids are recognized as promoters of coronary artery disease (CAD), while monounsaturated and omega-3 fatty acids may have salutary effects. Since cellular stress is recognized as a fundamental driver of CAD, the effect of these fatty acids on endoplasmic reticulum (ER) stress in human coronary artery endothelial cells (HCAECs) was measured using the ER stress-responsive alkaline phosphatase (ES-TRAP) assay. Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and eicosapentaenoic acid ethyl ester (EPA-EE) suppressed ER stress induced with pharmacologic (tunicamycin) and physiologic (high-dextrose concentration) ER stress inducers. In tunicamycin-treated cells, DHA reduced the expression of unfolded protein response (UPR) markers such as phosphorylation of inositol requiring enzyme 1α (IRE1α) and protein kinase R-like endoplasmic reticulum kinase (PERK) and increased activating transcription factor 6 (ATF6) and glucose regulated protein 78 (GRP78) expression. Similarly, treatment with both oleic acid and arachidonic acid, but not elaidic acid (a trans-fatty acid), suppressed both tunicamycin and high-dextrose-induced ER stress while treatment with saturated fatty acids (C14 : 0, C16 : 0, and C18 : 0) enhanced both tunicamycin and high-dextrose-induced ER stress. The latter fatty acids at higher concentrations caused cytotoxicity. These results indicate that omega-3 fatty acids as well as select unsaturated fatty acids and arachidonic acid suppress ER stress in HCAEC.

Duck Tembusu virus NS3 protein induces apoptosis by activating the PERK/PKR pathway and mitochondrial pathway.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that replicates well in mosquito, bird, and mammalian cells. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in the serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and poses a threat to mammalian health. Thus, understanding the pathogenic mechanism of DTMUV is crucial for identifying potential antiviral targets. In this study, we discovered that NS3 can induce the mitochondria-mediated apoptotic pathway through the PERK/PKR pathway; it can also interact with voltage-dependent anion channel 2 to induce apoptosis. Our findings provide a theoretical basis for understanding the pathogenic mechanism of DTMUV infection and identifying potential antiviral targets and may also serve as a reference for exploring the pathogenesis of other flaviviruses.

ATF5-regulated Mitochondrial Unfolded Protein Response Attenuates Neuronal Damage in Epileptic Rat by Reducing Endoplasmic Reticulum Stress Through Mitochondrial ROS.

Endoplasmic reticulum (ER) dysfunction caused by excessive ER stress is a crucial mechanism underlying seizures-induced neuronal injury. Studies have shown that mitochondrial reactive oxygen species (ROS) are closely related to ER stress, and our previous study showed that activating transcription factor 5 (ATF5)-regulated mitochondrial unfolded protein response (mtUPR) modulated mitochondrial ROS generation in a hippocampal neuronal culture model of seizures. However, the effects of ATF5-regulated mtUPR on ER stress and the underlying mechanisms remain uncertain in epilepsy. In this study, ATF5 upregulation by lentivirus infection attenuated seizures-induced neuronal damage and apoptosis in a rat model of pilocarpine-induced epilepsy, whereas ATF5 downregulation by lentivirus infection had the opposite effects. ATF5 upregulation potentiated mtUPR by increasing the expression of mitochondrial chaperone heat shock protein 60 (HSP60) and caseinolytic protease proteolytic subunit (ClpP) and reducing mitochondrial ROS generation in pilocarpine-induced seizures in rats. Additionally, upregulation of ATF5 reduced the expression of glucose-regulated protein 78 (GRP78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), suggesting suppression of ER stress; Moreover, ATF5 upregulation attenuated apoptosis-related proteins such as B-cell lymphoma-2 (BCL2) downregulation, BCL2-associated X (BAX) and cleaved-caspase-3 upregulation. However, ATF5 downregulation exerted the opposite effects. Furthermore, pretreatment with the mitochondria-targeted antioxidant mito-TEMPO attenuated the harmful effects of ATF5 downregulation on ER stress and neuronal apoptosis by reducing mitochondrial ROS generation. Overall, our study suggested that ATF5-regulated mtUPR exerted neuroprotective effects against pilocarpine-induced seizures in rats and the underlying mechanisms might involve mitochondrial ROS-mediated ER stress.

Knockdown of NUPR1 inhibits angiogenesis in lung cancer through IRE1/XBP1 and PERK/eIF2α/ATF4 signaling pathways.

The stress response molecule nuclear protein‑1 (NUPR1) is essential for the growth of multiple types of human malignant tumor cells. However, the significance of NUPR1 in lung cancer is still not entirely elucidated. Therefore, this study is aimed to explore the function and underlying mechanisms of NUPR1 in lung cancer. NUPR1 mRNA and protein levels in lung cancer cell lines (A549 or H1299 cells) were silenced through siRNA transfection and western blot observed its successful infection efficiency. Then, using tube formation and wound healing experiments, the effects of si-NUPR1 on angiogenesis and migration of human umbilical vein endothelial cells (HUVEC) were examined, respectively, which indicated inhibitory effects on the angiogenesis and migration of HUVEC. Vascular endothelial growth factor A (VEGFA), a vital molecule in angiogenesis, was detected by PCR and western blot assays, manifesting NUPR1 knockdown represses VEGFA expression. Furthermore, the knockdown of NUPR1 may reduce angiogenesis by lowering VEGFA expression through inositol-requiring enzyme 1 (IRE1)/X box binding protein 1 (XBP1) and protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2 A (eIF2α)/recombinant activating transcription factor 4 (ATF4) signaling pathways in A549 or H1299 cells. In conclusion, these findings demonstrated that NUPR1 knockdown inhibits angiogenesis in A549 and H1299 cells through IRE1/XBP1 and PERK/eIF2α/ATF4 signaling pathways, indicating that NUPR1 could represent a novel lung cancer therapeutic target.