Eukaryotic Translation Initiation Factor 2alpha Research Articles

Abstract P2-16-05: Targeting EZH2 to overcome chemoresistance in Triple Negative Breast Cancers Employing Combinatorial Approach

Abstract Objective: Triple Negative Breast Cancers (TNBC) are highly invasive and 46% of patients develop distant metastases, leading to higher mortality. Chemotherapy remains the most efficacious option, however, there is still a largely unmet need to identify novel therapeutic targets in TNBC to increase treatment options and improve patient outcomes, survival. Enhancer of zeste homologue 2 (EZH2), a member of the catalytic subunit of the polycomb repressive complex 2, is a histone methyltransferase and its canonical function is to methylate lysine 27 of histone 3. EZH2 is a potential driver of TNBC metastasis, and its high expression strongly associates with the TNBC phenotype as compared with other molecular subtypes of breast cancer. Despite the advancement in the discovery of inhibitors for EZH2 that attenuate its catalytic activity. Currently, several EZH2 inhibitors are under development and undergoing clinical trials and these compounds have been proved to be effective in the treatment of hematological malignancies, sarcomas and malignant rhabdoid tumors. However these inhibitors do not affect the intrinsic protein stability of EZH2, but typically competes with the cofactor S-adenosylmethionine (SAM) and binds to the SET domain of EZH2. Hence, the EZH2 inhibitors are only effective for some malignant blood tumors, and have poor efficacy for solid tumors, such as TNBC. Several studies have shown the involvement of neurotransmitter dopamine in proliferation, apoptosis, tumor angiogenesis, and drug resistance among different cancers, including the breast1. Dopamine D1 receptor activation in TNBC cell line induces apoptosis, autophagy and phosphorylation of eukaryotic translation initiation factor 2-alpha (eIF2a)2. Also, D1R agonists inhibits the invasion of breast cancer cell lines MDA-MB-231 and BT-20 and regress mammary tumors3. The oncogenic nature of EZH2 in driving aggressiveness in TNBC cells led us to simultaneously target dopamine D1 receptor and EZH2 to completely ablate tumor growth and metastasis. Methods: Schrodinger protein modeling software was employed for docking studies. TNBC cells MDA-MB-231 cells were treated with the EZH2 inhibitor GSK126 and/or D1R agonists A77636 and SKF38393 for assessing cell viability, migration. Immuno precipitation to investigate if the combination could disrupt the PRC2 complex in TNBC cells. To model tumor burden and the effects of combination therapy on tumor growth in vitro, we tested the combinatory effect of GSK126 and D1R agonists in a 3D culture system of MDA-MB-231 cells encapsulated in calcium-alginate microgels seeded from a microfluidic droplet generator. Cell death was determined by Yo-pro-Propidium Iodide staining. Result: In silico analysis confirmed that D1R agonists exhibited strong stabilization of protein structures upon binding to the EZH2 catalytic active site, albeit with slightly weaker affinity than GSK126. Combination treatment with GSK126 and dopamine agonists A77636 or SKF 38393 led to significant and synergistic inhibition of cell viability (p< 0.01), migration, invasion, and EZH2 activity (p< 0.01) in TNBC cells. In addition, MDA-MB-231 cells formed tumor spheroids that were subjected to the EZH2 inhibitor alongside dopamine agonists, indicating tumor reduction relative to single agent treatment in 3D culture (p < 0.0001). The combination also led to increased necrotic cell death (p < 0.05). Immunoprecipitation of EZH2 in the presence of GSK126 and SKF38393 and A77636 dissociated the physical interaction between EZH2, EED, and SUZ12 in MDA-MB-231 cells. Conclusion: Our data suggest that the combination of GSK126 and Dopamine D1 agonists synergistically inhibits TNBC proliferation by disrupting EZH2 functions leading to necrotic cell death. (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). 1) PMID: 21531818 2) PMID: 29773888 3) PMID: 26477316 Citation Format: Eswar Shankar, Sridhar SNC, Xilal Y. Rima, Prem Kushwaha, Shiv Verma, Gautam K. Sarathy, Anagh kulkarni, Dharmaraja Allimuthu, Eduardo Reátegui, Sanjay Gupta, Bhuvaneswari Ramaswamy. Targeting EZH2 to overcome chemoresistance in Triple Negative Breast Cancers Employing Combinatorial Approach. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-05.

A CASE REPORT ON WOLCOTT RALLISON SYNDROME

A Case Report of Wolcott–Rallison syndrome, it is a rare, autosomal recessive disorder with infancy-onset diabetes mellitus, multiple epiphyseal dysplasia, osteopenia, mental retardation or developmental delay, and hepatic and renal dysfunction as main clinical findings. Patients have most common history of consanguineous marriage. Patients with WRS have mutations in the EIF2AK3 gene, which encodes the eukaryotic translation initiation factor 2-alpha kinase 3. Most common cause of death is fulminant hepatitis in early childhood.The EIKF2AK3 gene codes for PERK (pancreatic endoplasmic reticulum kinase),an explanation for the spectrum of symptoms.PERK is associated with the activity of beta cells in the pancreas.A broad range of bodily systems is affected, including pancreas, kidney, liver,bone, and nervous system, because of deficient stress response to improperly folded proteins inside the endoplasmic reticulum. A two months old male child born out of non-consanguineous marriage, Hindu by religion, presented with complain of fever since 4 days, respiratory distress since 12 hours. Patient was admitted and on regular blood sugar screening on arrival was high. Repeat random blood sugar was also elevated.Arterial blood gas was done which was suggested metabolic acidosis.Due to persistent high blood sugar,insulin drip was started and HbA1c was done which was 11 gm% and other investigations done. Diagnosis of neonatal diabetes mellitus was kept and managed accordingly. Studies for other endocrinopathies and autoimmune disease done which was negative. Child was having normal growth and development according to age. Genetic studies for neonatal diabetes was planned and EIKF2AK3 gene mutation was found homozygous. Both the parents were heterozygous for the mutation. So diagnosis was kept and regular follow up was advised.Key-words:Infant,Neonatal Diabetes,Endocrinopathy

BRCA1 mediates protein homeostasis through the ubiquitination of PERK and IRE1.

Tumors with BRCA1 mutations have poor prognoses due to genomic instability. Yet this genomic instability has risks and BRCA1-deficient (def) cancer cells must develop pathways to mitigate these risks. One such risk is the accumulation of unfolded proteins in BRCA1-def cancers from increased mutations due to their loss of genomic integrity. Little is known about how BRCA1-def cancers survive their genomic instability. Here we show that BRCA1 is an E3 ligase in the endoplasmic reticulum (ER) that targets the unfolded protein response (UPR) stress sensors, Eukaryotic Translation Initiation Factor 2-alpha Kinase 3 (PERK) and Serine/Threonine-Protein Kinase/Endoribonuclease Inositol-Requiring Enzyme 1 (IRE1) for ubiquitination and subsequent proteasome-mediated degradation. When BRCA1 is mutated or depleted, both PERK and IRE1 protein levels are increased, resulting in a constitutively activated UPR. Furthermore, the inhibition of protein folding or UPR signaling markedly decreases the overall survival of BRCA1-def cancer cells. Our findings define a mechanism used by the BRCA1-def cancer cells to survive their increased unfolded protein burden which can be used to develop new therapeutic strategies to treat these cancers.

Bioinformatics analysis combined with experiments to verify potential autophagy genes in wound healing.

BackgroundThe skin is the most exposed tissue and has multiple functions. Wound healing is a major medical problem due to trauma and pathophysiological alterations suffered by patients. The aim of the present study was to search for potential autophagy genes associated with wound healing.MethodsThe GSE168760 dataset was obtained from the Gene Expression Omnibus (GEO) database, and sequencing results were obtained for 14 patient traumas at different time periods. Differentially expressed gene (DEG) analysis, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed. Immune cell and correlation analysis were performed for autophagy genes and DEGs. Peripheral blood was collected from patients at different time periods and Western blot (WB) assay was performed to verify autophagy genes.ResultsA total of 226 DEGs were screened on days 0, 7, and 14, of which 162 genes were upregulated and 64 genes were downregulated. Of these, eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2) and retinoblastoma 1 (RB1) were autophagy-associated genes. The DEGs were mainly involved in response to virus, cellular response to type I interferon Epstein-Barr virus infection, human papillomavirus infection, ribosome, hepatitis B and RIG-I-like. EIF2AK2 and RB1 showed positive correlation with some of the immune cells, and WB showed that EIF2AK2 and RB1 proteins were significantly increased with wound healing.ConclusionsThe comprehensive analysis of GEO data in the present study provides a new theoretical basis for the molecular pathogenesis of trauma healing and potential autophagy-related therapeutic targets.

Stress Granules and Acute Ischemic Stroke: Beyond mRNA Translation.

Ischemic stroke is a leading cause of death and disability worldwide. Following an ischemic insult, cells undergo endoplasmic reticulum (ER) stress, which increases the ER’s protein-folding and degradative capacities and blocks the global synthesis of proteins by phosphorylating the eukaryotic translation initiation factor 2-alpha (eIF2α). Phosphorylation of eIF2α is directly related to the dynamics of stress granules (SGs), which are membraneless organelles composed of RNA-binding proteins and mRNA. SGs play a critical role in mRNA metabolism and translational control. Other translation factors are also linked to cellular pathways, including SG dynamics following a stroke. Because the formation of SGs is closely connected to mRNA translation, it is interesting to study the relationship between SG dynamics and cellular outcome in cases of ischemic damage. Therefore, in this review, we focus on the role of SG dynamics during cerebral ischemia.

Wolcott Rallison Syndrome: Beyond Neonatal Diabetes

Background: Wolcott–Rallison Syndrome (WRS) is a rare autosomal recessive disorder characterized by permanent neonatal diabetes mellitus, skeletal dysplasia, hepatic dysfunction, and other systemic associations. Clinical Description: A 3-month-old infant with a history of fever and poor oral intake presented with severe dehydration, acidosis and 3+ urine ketones and was diagnosed to have sepsis and diabetic ketoacidosis (DKA). He also developed acute kidney injury (AKI) with blood urea 118 mg/dL and serum creatinine 1.5 mg/dL. Management: The child was ventilated, stabilized, and managed for DKA with fluids and insulin as per guidelines. AKI was managed with peritoneal dialysis. Genetic analysis revealed homozygous mutation in eukaryotic translation initiation factor 2-alpha kinase 3 gene consistent with the diagnosis of WRS. A close follow-up was kept with regular screening for other associated manifestations. Central hypothyroidism was detected first followed by skeletal dysplasia and chronic kidney disease. Growth retardation and developmental delay are also present. Conclusion: Neonatal diabetes cases need an early genetic work up and watchful follow-up for the manifestation of other possible associated features.

Microcystin-LR induces ferroptosis in intestine of common carp (Cyprinus carpio)

Previous studies provide comprehensive evidence of the environmental hazards and intestinal toxicity of microcystin-LR (MC-LR) exposure. However, little is known about the mechanisms underlying the injury of intestine exposed to MC-LR. Juvenile common carp (Cyprinus carpio) were exposed to MC-LR (0 and 10 μg/L) for 15 days. The results suggest that organic anion-transporting polypeptides 3a1, 4a1, 2b1, and 1d1 mediate MC-LR entry into intestinal tissues. Lesion morphological features (vacuolization, deformation and dilation of the endoplasmic reticulum [ER], absence of mitochondrial cristae in mid-intestine), up-regulated mRNA expressions of ER stress (eukaryotic translation initiation factor 2-alpha kinase 3, endoplasmic reticulum to nucleus signaling 1, activating transcription factor [ATF] 6, ATF4, DNA damage-inducible transcript 3), iron accumulation, and down-regulated activity of glutathione peroxidase (GPx) and glutathione (GSH) content were all typical characteristics of ferroptosis in intestinal tissue following MC-LR exposure. GSH levels in intestinal tissue corroborated as the most influential GSH/GPx 4- related metabolic pathway in response to MC-LR exposure. Verrucomicrobiota, Planctomycetes, Bdellovibrionota, Firmicutes and Cyanobacteria were correlated with the ferroptosis-related GSH following MC-LR exposure. These findings provide new perspectives of the ferroptosis mechanism of MC-LR-induced intestinal injury in the common carp.

Pulmonary veno-occlusive disease in childhood-a rare disease not to be missed.

Pulmonary veno-occlusive disease (PVOD) is a rare disease leading to pulmonary hypertension and potentially death related to right heart failure and/or respiratory insufficiency. Clinical symptoms are heterogenous and nonspecific: fatigue, decreased exercise tolerance, shortness of breath on exertion, cough, dizziness, chest pain with exercise, palpitations, syncope, as well as nonspecific symptoms such as headache, poor appetite, pallor or perioral cyanosis. Mutations in the EIF2AK4 (eukaryotic translation initiation factor 2-alpha kinase 4) have been recently described, other risk factors include exposure to organic solvent and trichloroethylene, tobacco exposure and chemotherapy. Echocardiography helps to estimate right ventricular systemic pressure, but further diagnostic workup includes cardiac catheterization to confirm pulmonary hypertension and increased pulmonary vascular resistance. High-resolution computed tomography reveals typical findings: centrilobular ground-glass nodules or opacities, septal lines, thickened interlobular septa, mosaic perfusion, and lymphadenopathy. Histology remains the gold standard, but carries risks for the patient. Proper workup is essential in order to avoid incorrect diagnosis. Pulmonary hypertension targeted treatment has been used in patients with PVOD, however, experience is limited, vasodilatory effects on pulmonary vasculature may lead to deterioration of the patients and should be used with great caution. Lung transplantation is currently the only valid treatment option for patients with PVOD. With prolonged waiting time and progression of the disease mechanical support could be considered.

Modulation of Salubrinal-Mediated Endoplasmic Reticulum Stress in an Experimental Subarachnoid Hemorrhage Model

Perfusion abnormalities due to vasospasm remain a major cause of morbidity and mortality in subarachnoid hemorrhage (SAH). Despite a large number of clinical trials, therapeutic options with strong evidence for prevention and treatment of cerebral vasospasm are rare. In this study, we aimed to evaluate the neuroprotective effect of salubrinal (SLB) in endoplasmic reticulum stress-induced apoptosis, a catastrophic consequence of vasospasm. Thirty-two Wistar albino rats were divided into 4 groups of 8 rats each: control group, SAH, SAH+SLB, and SAH+nimodipine (NMN). In the SAH+SLB group, intraperitoneal SLB (1 mg/kg dose) administered 30 minutes after establishment of SAH, and in the SAH+NMN group, intraperitoneal NMN (0.1 mg/kg dose) was also administered 30 minutes after SAH. Higher total antioxidant status level, lower oxidative stress index, and significantly higher vascular endothelial growth factor-A (VEGF-A) level were detected in the SAH+SLB and SAH+NMN groups compared with the SAH group. There was a significant increase in eukaryotic translation initiation factor-2 alpha (elF2α) level in the SAH+SLB group compared with the SAH group. Histopathological evaluation revealed decrease in the subarachnoid hemorrhagic area, as well as in cortical edema and apoptotic bodies in the SAH+SLB and SAH+NMN groups. There was a significant decrease in caspase-3 staining in the SAH+SLB group, and the levels were significantly less in the SAH+NMN group than the SAH and SAH+SLB groups. SLB, selective inhibitor of eIF2α dephosphorylation, and NMN, a calcium channel blocker, can ameliorate SAH-induced damage. Inhibition of eIF2α dephosphorylation and enhanced VEGF-A production with SLB may protect brain tissue from apoptosis.

The PKR/P38/RIPK1 Signaling Pathway as a Therapeutic Target in Alzheimer's Disease.

Neuropathological lesions in Alzheimer’s disease (AD) include amyloid plaques formed by the accumulation of amyloid peptides, neurofibrillary tangles made of hyperphosphorylated tau protein, synaptic and neuronal degenerations, and neuroinflammation. The cause of AD is unknown, but according to the amyloid hypothesis, amyloid oligomers could lead to the activation of kinases such as eukaryotic translation initiation factor 2-alpha kinase 2 (PKR), p38, and receptor-interacting serine/threonine-protein kinase 1 (RIPK1), which all belong to the same stress-activated pathway. Many toxic kinase activations have been described in AD patients and in experimental models. A p38 mitogen-activated protein kinase inhibitor was recently tested in clinical trials but with unsuccessful results. The complex PKR/P38/RIPK1 (PKR/dual specificity mitogen-activated protein kinase kinase 6 (MKK6)/P38/MAP kinase-activated protein kinase 2 (MK2)/RIPK1) is highly activated in AD brains and in the brains of AD transgenic animals. To delineate the implication of this pathway in AD, we carried out a search on PubMed including PKR/MKK6/p38/MK2/RIPK1, Alzheimer, and therapeutics. The involvement of this signaling pathway in the genesis of AD lesions, including Aβ accumulations and tau phosphorylation as well as cognitive decline, is demonstrated by the reports described in this review. A future combination strategy with kinase inhibitors should be envisaged to modulate the consequences for neurons and other brain cells linked to the abnormal activation of this pathway.

The Role of Oxymatrine in Amelioration of Acute Lung Injury Subjected to Myocardial I/R by Inhibiting Endoplasmic Reticulum Stress in Diabetic Rats.

Background Oxymatrine (OMT) is the primary pharmacological component of Sophora flavescens Aiton., which has been shown to possess potent antifibrotic, antioxidant, and anti-inflammatory activities. The aim of the present study was to clarify the protective mechanism of OMT on acute lung injury (ALI) subjected to myocardial ischemia/reperfusion (I/R). Methods A myocardial I/R-induced ALI model was achieved in diabetic rats by occluding the left anterior descending coronary artery for 1 h, followed by reperfusion for 1 h. The levels of inflammatory factors (tumor necrosis factor-α, interleukin- (IL-) 6, and IL-17) in bronchoalveolar lavage fluid were assessed using commercially available kits. The index of myocardial injury, including the detection of cardiac troponin I (cTnI), cardiac troponin T (cTnT), lactate dehydrogenase (LDH), and creatine kinase-MB (CK-MB), was also determined using commercially available kits. Hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were used to identify histological changes. The expression levels of endoplasmic reticulum chaperone BiP (GRP78), DNA damage-inducible transcript 3 protein (CHOP), eukaryotic translation initiation factor 2-alpha kinase 3 (PERK), inositol dependent enzyme 1α (IRE1α), ATF6, caspase-3, -9, and-12, Bcl-2, and Bax were determined by Western blotting. The mRNA expression levels of GRP78 and CHOP were detected by reverse transcription-quantitative PCR. Results Myocardial I/R increased the levels of cTnI, cTnT, LDH, and CK-MB in diabetic rats. Damaged and irregularly arranged myocardial cells were also observed, as well as more serious ALI with higher lung injury scores and WET/DRY ratios and lower PaO2. Moreover, the expression of key proteins of endoplasmic reticulum stress (ERS) was increased by I/R injury, including phosphorylated- (p-) PERK, p-IRE1ɑ, and ATF6, as well as decreased levels of apoptosis. These effects were all significantly reversed by OMT treatment. Conclusions OMT protects against ALI subjected to myocardial I/R by inhibiting ERS in diabetic rats.

Molecular mechanisms underlying the effects of the small molecule AMC-04 on apoptosis: Roles of the activating transcription factor 4-C/EBP homologous protein-death receptor 5 pathway

The unfolded protein response (UPR) is an emerging target pathway for cancer treatment owing to its ability to induce cell death. In our previous analysis of UPR-modulating small molecules, we had reported that piperazine oxalate derivative compounds (AMC-01–04) are able to promote increased phosphorylation of eukaryotic translation initiation factor-2 alpha (eIF2α). In this study, we found that AMC-04 induces apoptotic cell death via the activation of UPR in human breast and liver cancer cells. AMC-04 upregulated the expression of activating transcription factor-4 (ATF4)-C/EBP homologous protein (CHOP) and death receptor 5 (DR5) in cancer cells, as revealed by microarray analysis, small-interference RNA assay, and western blotting. From a mechanistic perspective, cytotoxic UPR pathway activation by AMC-04 is mediated by reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (p38 MAPK) signaling. A chemical informatics approach predicted that AMC-04 modulates histone methyltransferase activity. Based on biochemical analysis, the activity of histone methyltransferases, including SUV39H1, SUV39H2, SETDB1, and EHMT1, was inhibited by AMC-04. Furthermore, chemical inhibition of the identified target proteins induced UPR activation and apoptotic cell death, suggesting that inhibition of histone methyltransferases is a promising strategy for cancer therapy. Taken together, we showed that the small molecule AMC-04 modulates epigenetic enzyme activity and mediates the link between cytotoxic UPR and histone modifications.

A novel splice site indel alteration in the EIF2AK3 gene is responsible for the first cases of Wolcott-Rallison syndrome in Hungary

BackgroundWolcott-Rallison Syndrome (WRS) is a rare autosomal recessive disease that is the most common cause of neonatal diabetes in consanguineous families. WRS is caused by various genetic alterations of the Eukaryotic Translation Initiation Factor 2-Alpha Kinase 3 (EIF2AK3) gene.MethodsGenetic analysis of a consanguineous family where two children were diagnosed with WRS was performed by Sanger sequencing. The altered protein was investigated by in vitro cloning, expression and immunohistochemistry.ResultsThe first cases in Hungary, − two patients in one family, where the parents were fourth-degree cousins - showed the typical clinical features of WRS: early onset diabetes mellitus with hyperglycemia, growth retardation, infection-induced multiple organ failure. The genetic background of the disease was a novel alteration in the EIF2AK3 gene involving the splice site of exon 11– intron 11–12 boundary: g.53051_53062delinsTG. According to cDNA sequencing this created a new splice site and resulted in a frameshift and the development of an early termination codon at amino acid position 633 (p.Pro627AspfsTer7). Based on in vitro cloning and expression studies, the truncated protein was functionally inactive. Immunohistochemistry revealed that the intact protein was absent in the islets of pancreas, furthermore insulin expressing cells were also dramatically diminished. Elevated GRP78 and reduced CHOP protein expression were observed in the liver.ConclusionsThe novel genetic alteration causing the absence of the EIF2AK3 protein resulted in insufficient handling of severe endoplasmic reticulum stress, leading to liver failure and demise of the patients.

HDAC6, A Novel Cargo for Autophagic Clearance of Stress Granules, Mediates the Repression of the Type I Interferon Response During Coxsackievirus A16 Infection.

Autophagic cargoes ensure selective autophagy for the recognition and removal of various cytosolic aggregated proteins, damaged organelles, or pathogens. Stress granules (SGs), as antiviral immune complexes, serve a positive role in the type I interferon (IFN) response and can be targeted by autophagy (termed granulophagy). However, the cargo of granulophagy remains elusive, and it is still unknown whether granulophagy plays a role in viral infection. Here, we found that histone deacetylase 6 (HDAC6), a component of viral RNA-induced SGs, is a novel granulophagic cargo that is recognized by p62/Sequestosome 1 (SQSTM1) and mediates the degradation of SGs in coxsackievirus A16 (CA16)-infected cells. CA16 viral RNA activated the protein kinase RNA-activated (PKR)/eukaryotic translation initiation factor 2-alpha (eIF2α) pathway to promote SG assembly. The SGs were degraded by CA16-triggered autophagy via the interaction between the ubiquitin-associated (UBA) domain of p62 and the ubiquitin-binding domain (UBD) of HDAC6, which was bridged by a poly-ubiquitin chain. We also found that granulophagy repressed the type I interferon response and facilitated viral replication. These results suggest that HDAC6 might be the first identified granulophagic cargo and granulophagy could be a strategy that viruses apply to repress the antiviral immune response.

Cardioprotective effect of Rosa canina L. methanolic extract on heat shock induced cardiomyocyte injury: An experimental study.

Introduction: Overexposure to heat conditions can affect the functioning of the cardiovascular system and may promote cardiovascular disorders. Heat shock induced myocardial injury via increasing endoplasmic reticulum response-mediated apoptosis. This study investigated the impact of pretreatment with Rosa canina (RC), a natural antioxidant, on myocardial damage induced by heat stress exposure and underlying mechanisms in cardiomyocytes in rats. Methods: Sixty adult male Wistar rats were allocated into five groups, including Control: received normal saline (NS), Heat Stress (HS), and HS+RC groups. Animals in the HS groups were subjected to heat stress (43 ºC) for 15 minutes once a day for two weeks. Animals in the HS+RC groups received three doses of RC (250, 500, and 1000 mg/mL) one hour before being subjected to heat shock. The endoplasmic reticulum (ER) transmembrane kinases, including PKR-like endoplasmic reticulum kinase (PERK), immunoreactivity of CCAAT/enhancer-binding protein homologous protein (CHOP), and eukaryotic translation initiation factor 2-alpha (eIF2α) as well as caspase 8 were detected by Western blot. The levels of reactive oxygen species (ROS) were assessed. Moreover, histopathological changes and apoptosis were also assayed in the heart tissue by using histopathological and TUNEL assays. Results: Heat exposure increased the level of ROS and induced oxidative damage in the heart tissue. The results demonstrated that RC administration decreased the overproduction of ROS induced by heat stress in cardiomyocytes. Moreover, heat stress up regulated the expression of p-PERK, p-eIF2α,and CHOP protein while pretreatment with RC decreased expression of ER stress-related markers in cardiomyocytes. Besides, RC diminished heat stress-induced cellular damage and apoptosis associated with inhibition of caspase 8 activation, a pro-apoptotic protein in cardiomyocytes. Conclusion: These findings indicate that RC exerts a protective effect on heart tissue, at least in part,through inactivation of PERK/eIF2α/CHOP pathway or inhibition of ER stress and oxidative stress triggeredapoptosis in cardiomyocytes induced by heat stress.

Induction of the unfolded protein response (UPR) suppresses porcine reproductive and respiratory syndrome virus (PRRSV) replication

Replication of most RNA viruses is closely associated with the endoplasmic reticulum (ER) within permissive cells. As such, viruses often induce tremendous amounts of stress on cells during viral replication. To cope with the stress, cells initiate the unfolded protein response (UPR) to promote cellular survival. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically important swine pathogen, was previously shown to induce cellular stress at late stages post-infection resulting in the formation of stress granules (SGs). Here in this study, we demonstrate that PRRSV also induces additional cellular response pathways, including the UPR. Confocal microscopy analysis demonstrated significant morphological changes in the ER of PRRSV-infected cells, indicative of pronounced ER stress. Further investigation revealed induction of all three branches of the UPR, including eukaryotic translation initiation factor 2-alpha kinase 3 (PERK), serine-threonine protein kinase/endoribonuclease (IRE1), and cyclic AMP-dependent transcription factor (ATF6). Activation of these sensors resulted in significant transcriptional upregulation of downstream UPR effectors. Additionally, UPR activation was shown to be detrimental to PRRSV replication, as treatment of cells with chemical ER stress inducers potently suppressed viral replication and RNA synthesis. Further investigation into the molecular mechanisms of UPR suppression of PRRSV replication revealed that PERK exacerbates the PRRSV-induced cytokine response. Collectively, these results demonstrate that PRRSV infection induces UPR activation through all three branches, and that UPR signaling may play a role in PRRSV pathogenesis. The results of this study further our understanding of the underlying molecular mechanisms of PRRSV replication and host-pathogen interactions.

The PKR-Like Endoplasmic Reticulum Kinase Promotes the Dissemination of Myc-Induced Leukemic Cells.

Hyperactive oncogenic Myc stimulates protein synthesis that induces the unfolded protein response, which requires the function of the eukaryotic translation initiation factor 2-alpha kinase 3, also known as protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). Activated PERK acts to limit mRNA translation, enable proper protein folding, and restore the homeostasis in the endoplasmic reticulum. Given that Myc activation contributes to many types of lymphoid and myeloid human leukemias, we used a mouse model to examine the importance of PERK in development and progression of Myc-induced leukemias. We found that genetic ablation of Perk does not suppress the generation of the leukemic cells in the bone marrow. However, the cell-autonomous Perk deficiency restricts the dissemination of leukemic cells into peripheral blood, lymph nodes, and vital peripheral organs. Whereas the loss of the IFNAR1 chain of type I IFN receptor stimulated leukemia, Perk ablation did not stabilize IFNAR1, suggesting that PERK stimulates the leukemic cells' dissemination in an IFNAR1-independent manner. We discuss the rationale for using PERK inhibitors against Myc-driven leukemias. IMPLICATIONS: The role of PERK in dissemination of Myc-induced leukemic cells demonstrated in this study argues for the use of PERK inhibitors against leukemia progression.

A Novel SNP in EIF2AK4 Gene Is Associated with Thermal Tolerance Traits in Chinese Cattle.

Simple SummaryChina harbors two lineages of cattle (Bos taurus and Bos indicus) that display pronounced geographical distribution differences. Northern Chinese cattle predominantly belong to B. taurus and southern Chinese cattle belong to B. indicus. Both B. taurus and B. indicus contribute to the admixture of cattle in central China. Thermal stress induces oxidative stress and DNA damage in mammals. In general, B. indicus are more resistant to thermal stress than B. taurus. Eukaryotic translation initiation factor 2-alpha kinase 4 (EIF2AK4), which pertains to the family of serine–threonine kinase, is a candidate gene for thermal stress. However, the effects of the bovine EIF2AK4 gene on the thermal tolerance traits of Chinese cattle breeds remain unknown. Our results suggest that a variant of the EIF2AK4 gene is associated with thermal tolerance traits in Chinese cattle.Eukaryotic translation initiation factor 2-alpha kinase 4 (EIF2AK4, also known as GCN2), which pertains to the family of serine–threonine kinase, is involved in oxidative stress and DNA damage repair. A missense single-nucleotide polymorphism (SNP) (NC_037337.1 g.35615224 T > G) in exon 6 of the EIF2AK4 gene which encodes a p.Ile205Ser substitution was observed in the Bovine Genome Variation Database and Selective Signatures (BGVD). The purpose of the current study is to determine the allelic frequency distribution of the locus and analyze its association with thermal tolerance in Chinese indigenous cattle. In our study, the allelic frequency distribution of the missense mutation (NC_037337.1 g.35615224 T > G) in Chinese cattle was analyzed by sequencing 1105 individuals of 37 breeds including 35 Chinese indigenous cattle breeds and two exotic breeds. In particular, association analysis was carried out between the genotypes and three environmental parameters including annual mean temperature (T), relative humidity (RH), and temperature–humidity index (THI). The frequency of the mutant allele G (NC_037337.1 g.35615224 T > G) gradually decreased from the southern cattle groups to the northern cattle groups, whereas the frequency of the wild-type allele T showed an opposite pattern, consistent with the distribution of indicine and taurine cattle in China. In accordance with the association analysis, genotypes were significantly associated with T (P < 0.01), RH (P < 0.01), and THI (P < 0.01), suggesting that the cattle with genotype GG were found in regions with higher T, RH, and THI. Thus, our results suggest that the mutation (NC_037337.1 g.35615224 T > G) of the EIF2AK4 gene is associated with thermal tolerance traits in Chinese cattle.

Induction of endoplasmic reticulum stress might be responsible for defective autophagy in cadmium-induced prostate carcinogenesis

Earlier, we reported that chronic cadmium (Cd)-exposure to prostate epithelial (RWPE-1) cells causes defective autophagy, which leads to the transformation of a malignant phenotype in both in vitro and in vivo models. However, the upstream events responsible for defective autophagy are yet to be delineated. The present study suggests that chronic Cd exposure induces endoplasmic reticulum (ER) stress that triggers the phosphorylation of stress transducers [protein kinase R-like ER Kinase- (PERK), eukaryotic translation initiation factor 2-alpha- (eIF2-α) and Activating Transcription Factor 4 –(ATF-4)], resulting in defective autophagy that protects Cd-exposed RWPE-1 cells. On the other hand, inhibition of the ATF4 stress inducer by siRNA blocked the Cd-induced defective autophagy in transforming cells. While dissecting the upstream activators of ER stress, we found that increased expression of reactive oxygen species (ROS) is responsible for ER stress in Cd-exposed RWPE-1 cells. Overexpression of antioxidants (SOD1/SOD2) mitigates Cd-induced ROS that results in inhibition of ER stress and autophagy in prostate epithelial cells. These results suggest that the induction of ROS and subsequent ER stress are responsible for defective autophagy in Cd-induced transformation in prostate epithelial cells.

EIF2AK3 novel mutation in a child with early-onset diabetes mellitus, a case report

BackgroundWolcott-Rallison syndrome (WRS) is caused by a biallelic mutation in the gene encoding eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3) on chromosome 2p11.2. This condition is characterized by permanent early-onset diabetes mellitus, epiphyseal dysplasia, and hepatic dysfunction. We report a patient with WRS born to a consanguineous marriage due to a novel biallelic frameshift mutation in the EIF2AK3 gene.Case presentationOur patient was a 2-year-and-6-month-old Yemeni girl born to consanguineous parents who was diagnosed with neonatal diabetes at 20 days of age. She presented with chronic diarrhea and liver dysfunction. The child was normocephalic and exhibited failure to thrive and hepatomegaly with no skeletal deformities. Further investigations revealed microcytic anemia, liver impairment and primary hypothyroidism. Genetic testing confirmed the diagnosis of WRS via identification of a novel biallelic frameshift mutation in the EIF2AK3 gene. During her hospital stay, she went into septic shock and developed multi-organ failure, including fulminant hepatic failure. She unfortunately died within 2 weeks of her hospital stay.ConclusionsWolcott-Rallison syndrome is recognized as the most common cause of early-onset diabetes in infants born to consanguineous marriages. Screening for genetic mutations in EIF2AK3 is recommended for establishing early diagnosis, providing genetic counselling, and predicting the development of additional clinical features, most importantly hepatic failure. Hence, this screening is important for guiding optimal management and improving patient outcome.