Protein Kinase R (PKR) as a Novel dsRNA Sensor in Antiviral Innate Immunity.

Protein kinase R (PKR), a key double-stranded RNA (dsRNA)-activated sensor, is pivotal for cellular responses to diverse stimuli. This protocol delineates a comprehensive methodological framework employing single luciferase assays, yeast assays, immunoblot assays, and quantitative PCR (qPCR) to discern and validate PKR activities and their downstream impacts on NF-κB-activating signaling pathways. These methodologies furnish a systematic approach to unraveling the role of PKR as a dsRNA sensor and effector in antiviral innate immunity, enabling in-depth analyses of dsRNA sensor activities.

Small GTP-binding protein GDP dissociation stimulator influences cisplatin-induced acute kidney injury via PERK-dependent ER stress.

Cisplatin is a common anticancer drug, but its frequent nephrotoxicity limits its clinical use. Small GTP-binding protein GDP dissociation stimulator (smgGDS), a small GTPase chaperone protein, was considerably downregulated during cisplatin-induced acute kidney injury (CDDP-AKI), especially in renal tubular epithelial cells. SmgGDS-knockdown mice was established and found that smgGDS knockdown promoted CDDP-AKI, as demonstrated by an increase in serum creatine, blood urea nitrogen levels and the appearance of tubular patterns. RNA sequencing suggested that protein kinase RNA-like ER kinase (PERK), which bridges mitochondria-associated ER membranes, was involved in smgGDS knockdown following CDDP-AKI, and then identified that smgGDS knockdown increased phosphorylated-PERK in vivo and in vitro. Furthermore, we confirmed that smgGDS deficiency aggravated apoptosis and ER stress in vivo and in vitro. And the ER stress inhibitor 4-Phenylbutyric acid and the inhibition of PERK phosphorylation mitigated smgGDS deficiency-induced ER stress related apoptosis following cisplatin treatment, while the eIF2α phosphorylation inhibitor could not reverse the smgGDS deficiency accelerated cell death. Furthermore, the over-expression of smgGDS could reverse the ER stress and apoptosis caused by CDDP. Overall, smgGDS regulated PERK-dependent ER stress and apoptosis, thereby influencing renal damage. This study identified a target for diagnosing and treating cisplatin-induced acute kidney injury.

Targeting NLRP3 inhibits AML progression by inducing PERK/eIF2-mediated apoptosis.

BACKGROUND: Acute myeloid leukemia (AML) is characterized by the abnormal proliferation of myeloid precursor cells and presents significant challenges in treatment due to its heterogeneity. Recently, the NLRP3 inflammasome has emerged as a potential contributor to AML pathogenesis, although its precise mechanisms remain poorly understood. METHODS: Public genome datasets were utilized to evaluate the expression of NLRP3 inflammasome-related genes (IL-1ß, IL-18, ASC, and NLRP3) in AML patients compared to healthy individuals. CRISPR/Cas9 technology was employed to generate NLRP3-deficient MOLM-13 AML cells, followed by comprehensive characterization using real-time PCR, western blotting, FACS analysis, and transmission electron and immunofluorescence microscopy. Proteomic analyses were conducted to identify NLRP3-dependent alterations in protein levels, with a focus on the eIF2 kinase PERK-mediated signaling pathways. Additionally, in vivo studies were performed using a leukemic mouse model to elucidate the pathogenic role of NLRP3 in AML. RESULTS: Elevated expression of NLRP3 was significantly associated with diminished overall survival in AML patients. Genetic deletion, pharmacological inhibition and silencing by RNA interference of NLRP3 led to decreased AML cell survival through the induction of apoptosis. Proteomic analyses uncovered NLRP3-dependent alterations in protein translation, characterized by enhanced eIF2α phosphorylation in NLRP3-deficient AML cells. Moreover, inhibition of PERK-mediated eIF2α phosphorylation reduced apoptosis by downregulating pro-apoptotic Bcl-2 family members. In vivo studies demonstrated reduced leukemic burden in mice engrafted with NLRP3 knockout AML cells, as evidenced by alleviated leukemic symptoms. CONCLUSION: Our findings elucidate the involvement of the NLRP3/PERK/eIF2 axis as a novel driver of AML cell survival. Targeting NLRP3-induced signaling pathways, particularly through the PERK/eIF2 axis, presents a promising therapeutic strategy for AML intervention. These insights into the role of the NLRP3 inflammasome offer potential avenues for improving the prognosis and treatment outcomes of AML patients.

Green tea polyphenols alleviate TBBPA-induced gastric inflammation and apoptosis by modulating the ROS-PERK/IRE-1/ATF6 pathway in mouse models.

Green tea polyphenols (GTP), an important phytochemical in the daily human diet, bind to various cellular receptors and exert anti-inflammatory and antioxidant benefits. The environmental contaminant tetrabromobisphenol A (TBBPA) enters the digestive system through multiple pathways, resulting in oxidative stress (OS), gastroenteritis, and mucosal injury. The aim of this study was to explore the molecular mechanisms of TBBPA-induced gastritis in mice treated with GTP in vivo and in an in vitro model. The results showed that exposure to TBBPA increased reactive oxygen species (ROS) levels, activated oxidative stress (OS) induced endoplasmic reticulum stress (ERS), and the expression of endoplasmic reticulum stress-related factors (e.g., GRP78, PERK, IRE-1, ATF-6, etc.) increased. The inflammatory pathway NF-κB was activated, and the pro-inflammatory factors TNF-α, IL-1ß, and IL-6 increased, while triggering a cascade reaction mediated by caspase-3. However, the addition of GTP could inhibit OS, restore the balance of endoplasmic reticulum homeostasis, and improve the inflammatory infiltration and apoptosis of gastric mucosal epithelial cells. Therefore, GTP alleviated ERS, reduced inflammation and apoptosis, and restored the gastric mucosal barrier by alleviating TBBPA-induced OS in mouse gastric tissues and GES-1 cells. This provides basic information for exploring the antioxidant mechanism of GTP and further investigating the toxic effects of TBBPA on mouse gastric mucosa.

Time-Dependent Potentiation of the PERK Branch of UPR by GPR68 Offers Protection in Brain Ischemia.

BACKGROUND: In ischemia, acidosis occurs in/around injured tissue and parallels disease progression. Therefore, targeting an acid-sensitive receptor offers unique advantages in achieving the spatial and temporal specificity required for therapeutic interventions. We previously demonstrated that increased expression of GPR68 (G protein-coupled receptor 68), a proton-sensitive G protein-coupled receptor, mitigates ischemic brain injury. Here, we investigated the mechanism underlying GPR68-dependent protection. METHODS: We performed biochemical and molecular analyses to examine poststroke signaling. We used in vitro brain slice cultures and in vivo mouse transient middle cerebral artery occlusion (tMCAO) models to investigate ischemia-induced injuries. RESULTS: GPR68 deletion reduced PERK (protein kinase R-like ER kinase) expression in mouse brain. Compared with the wild-type mice, the GPR68-/- (knockout) mice exhibited a faster decline in eIF2α (eukaryotic initiation factor-2α) phosphorylation after tMCAO. Ogerin, a positive modulator of GPR68, stimulated eIF2α phosphorylation at 3 to 6 hours after tMCAO, primarily in the ipsilateral brain tissue. Consistent with the changes in eIF2α phosphorylation, Ogerin enhanced tMCAO-induced reduction in protein synthesis in ipsilateral brain tissue. In organotypic cortical slices, Ogerin reduced pH 6 and oxygen-glucose deprivation-induced neurotoxicity. Following tMCAO, intravenous delivery of Ogerin reduced brain infarction in wild-type but not knockout mice. Coapplication of a PERK inhibitor abolished Ogerin-induced protection. Delayed Ogerin delivery at 5 hours after tMCAO remained protective, and Ogerin has a similar protective effect in females. Correlated with these findings, tMCAO induced GPR68 expression at 6 hours, and Ogerin alters post-tMCAO proinflammatory/anti-inflammatory cytokine/chemokine expression profile. CONCLUSIONS: These data demonstrate that GPR68 potentiation leads to neuroprotection, at least in part, through enhancing PERK-eIF2α activation in ischemic tissue but has little impact on healthy tissue.

Modulating the unfolded protein response with ISRIB mitigates cisplatin ototoxicity.

Cisplatin is a commonly used chemotherapy agent with a nearly universal side effect of sensorineural hearing loss. The cellular mechanisms underlying cisplatin ototoxicity are poorly understood. Efforts in drug development to prevent or reverse cisplatin ototoxicity have largely focused on pathways of oxidative stress and apoptosis. An effective treatment for cisplatin ototoxicity, sodium thiosulfate (STS), while beneficial when used in standard risk hepatoblastoma, is associated with reduced survival in disseminated pediatric malignancy, highlighting the need for more specific drugs without potential tumor protective effects. The unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways have been shown to be involved in the pathogenesis of noise-induced hearing loss and cochlear synaptopathy in vivo, and these pathways have been implicated broadly in cisplatin cytotoxicity. This study sought to determine whether the UPR can be targeted to prevent cisplatin ototoxicity. Neonatal cochlear cultures and HEK cells were exposed to cisplatin, and UPR marker gene expression and cell death measured. Treatment with ISRIB (Integrated Stress Response InhIBitor), a drug that activates eif2B and downregulates the pro-apoptotic PERK/CHOP pathway of the UPR, was tested for its ability to reduce apoptosis in HEK cells, hair-cell death in cochlear cultures, and hearing loss using an in vivo mouse model of cisplatin ototoxicity. Finally, to evaluate whether ISRIB might interfere with cisplatin chemoeffectiveness, we tested it in head and neck squamous cell carcinoma (HNSCC) cell-based assays of cisplatin cytotoxicity. Cisplatin exhibited a biphasic, non-linear dose-response of cell death and apoptosis that correlated with different patterns of UPR marker gene expression in HEK cells and cochlear cultures. ISRIB treatment protected against cisplatin-induced hearing loss and hair-cell death, but did not impact cisplatin's cytotoxic effects on HNSCC cell viability, unlike STS. These findings demonstrate that targeting the pro-apoptotic PERK/CHOP pathway with ISRIB can mitigate cisplatin ototoxicity without reducing anti-cancer cell effects, suggesting that this may be a viable strategy for drug development.

EIF2AK2 protein targeted activation of AIM2-mediated PANoptosis promotes sepsis-induced acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) frequently occurs as a complication of sepsis. PANoptosis refers to a type of inflammatory programmed cell death that exhibits key characteristics of apoptosis, necroptosis, and pyroptosis. Here, we evaluated the role of absent in melanoma 2 (AIM2) and eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2) in septic AKI. METHODS: A septic AKI model was created through cecal ligation and puncture (CLP), while an in vitro model was developed using lipopolysaccharide (LPS)-stimulated HK2 cells. Hematoxylin and eosin (HE), Periodic acid-Schiff (PAS), and TUNEL staining were conducted to assess kidney injury in mice. Levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were detected by kits. Gene expression was detected utilizing RT-qPCR, and Western blot was used to test protein levels. Immunofluorescence was employed to measure EIF2AK2 and AIM2 expression in mouse kidney tissue. Lactate dehydrogenase (LDH) activity assay was conducted to evaluate cytotoxicity. Co-immunoprecipitation (Co-IP) was performed to verify the binding relationship between EIF2AK2 and AIM2. RESULTS: AIM2 expression was increased in the renal tissue of mice subjected to CLP. Activation of the inflammasome and PANoptosis were observed in the renal tissue of CLP mice. AIM2 depletion attenuated PANoptosis in LPS-treated HK-2 cells. Additionally, EIF2AK2 could directly target AIM2, leading to a positive regulation of AIM2 expression. Notably, EIF2AK2 induced PANoptosis through upregulating AIM2 in HK-2 cells stimulated by LPS. CONCLUSIONS: Our results revealed the important role of EIF2AK2-induced AIM2 upregulation in the activation of PANoptosis during septic AKI.

Renal tissue from CLP mice exhibited an increase in AIM2 expression.Renal tissue from CLP mice demonstrated inflammasome activation and PANoptosis.AIM2 silencing reduced PANoptosis in LPS-treated HK-2 cells.EIF2AK2 directly targeted AIM2 and positively regulated its expression.EIF2AK2 promoted PANoptosis via AIM2 in LPS-triggered HK-2 cells.

Rotenone-induced cell apoptosis via endoplasmic reticulum stress and PERK-eIF2α-CHOP signalling pathways in TM3 cells.

Rotenone (ROT), a widely used natural pesticide, has an uncertain effect on reproductive toxicity. In this study, we used 20 mice distributed randomly into four groups, with each group receiving ROT doses of 0, 2, 4, and 8 mg/kg/day for 28 days. The results demonstrated that ROT induced significant testicular damage, including impaired spermatogenesis, inhibition of testosterone synthesis, and apoptosis of Leydig cells. Additionally, ROT disrupted the normal ultrastructure of the endoplasmic reticulum (ER) in testicular tissue, leading to ER stress in Leydig cells. To further explore whether ROT-induced apoptosis in Leydig cells is related to ER stress, the mouse Leydig cell line (TM3 cells) was treated with ROT at 0, 250, 500, and 1000 nM. ROT inhibited TM3 cell viability, induced cytotoxicity, and reduced testosterone content in the culture supernatants. Furthermore, ROT treatment triggered apoptosis in TM3 cells by activating ER stress and the PERK-eIF2α-CHOP signalling pathway. Pre-treatment of TM3 cells exposed to ROT with the ER stress inhibitor 4-phenylbutyric acid (4-PBA) alleviated these effects, decreasing apoptosis and preserving testosterone levels. Further intervention with the PERK inhibitor GSK2606414 reduced ROT-induced apoptosis and testosterone reduction by inhibiting PERK activity. In summary, ROT-induced male reproductive toxicity is specifically driven by apoptosis, with the PERK-eIF2α-CHOP signalling pathway activated by ER stress playing a crucial role in the apoptosis of Leydig cells triggered by ROT.

Interaction Between the PERK/ATF4 Branch of the Endoplasmic Reticulum Stress and Mitochondrial One-Carbon Metabolism Regulates Neuronal Survival After Intracerebral Hemorrhage.

Recent investigations have revealed that oxidative stress can lead to neuronal damage and disrupt mitochondrial and endoplasmic reticulum functions after intracerebral hemorrhage (ICH). However, there is limited evidence elucidating their role in maintaining neuronal homeostasis. Metabolomics analysis, RNA sequencing, and CUT&Tag-seq were performed to investigate the mechanism underlying the interaction between the PERK/ATF4 branch of the endoplasmic reticulum stress (ERS) and mitochondrial one-carbon (1C) metabolism during neuronal resistance to oxidative stress. The association between mitochondrial 1C metabolism and the PERK/ATF4 branch of the ERS after ICH was investigated using transcription factor motif analysis and co-immunoprecipitation. The findings revealed interactions between the GRP78/PERK/ATF4 and mitochondrial 1C metabolism, which are important in preserving neuronal homeostasis after ICH. ATF4 is an upstream transcription factor that directly regulates the expression of 1C metabolism genes. Additionally, the GRP78/PERK/ATF4 forms a negative regulatory loop with MTHFD2 because of the interaction between GRP78 and MTHFD2. This study presents evidence of disrupted 1C metabolism and the occurrence of ERS in neurons post-ICH. Supplementing exogenous NADPH or interfering with the PERK/ATF4 could reduce symptoms related to neuronal injuries, suggesting new therapeutic prospects for ICH.

17-AAG Induces Endoplasmic Reticulum Stress-mediated Apoptosis in Breast Cancer Cells, Possibly Through PERK/eIF2α Up-regulation.

BACKGROUND/AIM: Breast cancer is the most predominant type of cancer affecting women worldwide and the current therapeutic treatment for breast cancer patients is not adequately effective. This study aimed to investigate the mechanism of 17-AAG, a heat shock protein (HSP90) inhibitor, as a treatment for inducing breast cancer cell apoptosis. MATERIALS AND METHODS: The pharmacology network was employed to examine the correlation of 17-AAG with the gene expression profiles of breast cancer, obtained by Gene Expression Profiling Interactive Analysis (GEPIA). MTT and flow cytometry were utilized to investigate cell proliferation and cell apoptosis, respectively. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay and western blot analysis were employed to examine the correlation between cellular oxidant levels and protein expression. Immunofluorescence staining was utilized to confirm the protein localization and assess DNA damage. RESULTS: The pharmacological network analysis revealed that HSP90 serves as the common target connecting 17-AAG and breast cancer genes. Treatment with 17-AAG significantly increased cell apoptosis. Moreover, the treatment resulted in up-regulation of cellular oxidant levels and PERK/eIF2α expression. In line with these, protein localization after treatment revealed an increase in DNA damage, correlating with higher ER stress levels. Furthermore, GEPIA demonstrated that PERK and eIF2α expression were significantly higher in breast invasive carcinoma compared to other tumor types. CONCLUSION: HSP90 emerges as a potential target for inducing apoptosis in breast cancer cells by disrupting protein homeostasis in the endoplasmic reticulum, possibly through PERK/eIF2α up-regulation. 17-AAG, an HSP90 inhibitor, may therefore potentially hold an alternative therapeutic strategy for breast cancer treatment.

An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

Adar null mutant mouse embryos die with aberrant double-stranded RNA (dsRNA)-driven interferon induction, and Adar Mavs double mutants, in which interferon induction is prevented, die soon after birth. Protein kinase R (Pkr) is aberrantly activated in Adar Mavs mouse pup intestines before death, intestinal crypt cells die, and intestinal villi are lost. Adar Mavs Eifak2 (Pkr) triple mutant mice rescue all defects and have long-term survival. Adenosine deaminase acting on RNA 1 (ADAR1) and PKR co-immunoprecipitate from cells, suggesting PKR inhibition by direct interaction. AlphaFold studies on an inhibitory PKR dsRNA binding domain (dsRBD)-kinase domain interaction before dsRNA binding and on an inhibitory ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA provide a testable model of the inhibition. Wild-type or editing-inactive human ADAR1 expressed in A549 cells inhibits activation of endogenous PKR. ADAR1 dsRNA binding is required for, but is not sufficient for, PKR inhibition. Mutating the ADAR1 dsRBD3-PKR contact prevents co-immunoprecipitation, ADAR1 inhibition of PKR activity, and co-localization of ADAR1 and PKR in cells.

[Research progress on the molecular mechanism of endoplasmic reticulum stress-mediated macrophage polarization].

The endoplasmic reticulum (ER) is an essential organelle that maintains intracellular Ca2+ homeostasis, folds newly synthesized secreted and membrane proteins, and facilitates post-translational protein modifications. Misfolding and aggregation of unfolded proteins within the ER lumen can activate endoplasmic reticulum stress (ERS), which in turn activates three different downstream signaling pathways: protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α(IRE-1α), and activating transcription factor 6 (ATF6). These pathways affect cell survival, differentiation, and phenotype transition. Recent studies have shown a close interaction between the downstream signaling cascade of ERS and the signaling pathways that induce macrophage polarization towards pro-inflammatory M1 type (IFN-γ and LPS) and anti-inflammatory M2 type (IL-4 and IL-10). However, the specific molecular mechanisms underlying these interactions are complex and intriate. The article summarizes the primary mechanisms by which ERS mediates macrophage polarization, focusing on discussing the molecular mechanisms by which three different downstream signals of ERS affect macrophage polarization.

[Myxovirus resistance protein A (MxA) induces the expression of interferon-stimulated genes in HepG2 cells by enhancing the activity of the interferon-stimulated response element (ISRE)].

Objective To explore the effects of Myxovirus resistance protein A (MxA) on the Janus kinase/Signal transducer and activator of transcription (JAK/STAT) pathway in HepG2 cells. Methods HepG2 cells were transfected with the pcDNA3.1-Flag-MxA construct, and subsequent localization and expression of the MxA protein were detected through immunofluorescence cytochemistry. The presence of MxA protein was further confirmed by using Western blot analysis. Following transfection with MxA small interfering RNA (si-MxA) and subsequent treatment with alpha interferon (IFN-α), real-time fluorescent quantitative PCR was employed to measure the mRNA levels of myxovirus resistance protein A (MxA), protein kinase R (PKR), and oligoadenylate synthase (OAS). Western blot analysis was used to detect the protein expression of MxA, PKR, OAS, signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (pSTAT1), STAT2, phosphorylated STAT2 (p-STAT2) and interferon regulatory factor 9 (IRF9). Additionally, pcDNA3.1-Flag-MxA and pISRE-TA-luc were co-transfected into HepG2 and HepG2.2.15 cells, respectively, to assess the activity of the interferon-stimulated response element (ISRE) by using a luciferase activity assay. Results MxA protein was expressed in both the cytoplasm and nucleus of HepG2 cells, with higher expression levels in the cytoplasm than in the nucleus. Knocking down MxA expression in HepG2 cells did not affect the expression of STAT1, p-STAT1, STAT2, p-STAT2, and IRF9 proteins induced by IFN-α, but significantly reduced the expression of antiviral proteins PKR and OAS. Overexpression of MxA in HepG2 cells enhanced ISRE activity and increased the expression of PKR and OAS proteins, but this effect was inhibited in HepG2.2.15 cells. Conclusion MxA induces the expression of antiviral proteins by enhancing the activity of the JAK/STAT signaling pathway ISRE.

PERK-ATAD3A interaction provides a subcellular safe haven for protein synthesis during ER stress.

Endoplasmic reticulum (ER) stress induces the repression of protein synthesis throughout the cell. Attempts to understand how localized stress leads to widespread repression have been limited by difficulties in resolving translation rates at the subcellular level. Here, using live-cell imaging of reporter mRNA translation, we unexpectedly found that during ER stress, active translation at mitochondria was significantly protected. The mitochondrial protein ATPase family AAA domain-containing protein 3A (ATAD3A) interacted with protein kinase RNA-like endoplasmic reticulum kinase (PERK) and mediated this effect on localized translation by competing for binding with PERK's target, eukaryotic initiation factor 2 (eIF2). PERK-ATAD3A interactions increased during ER stress, forming mitochondria-ER contact sites. Furthermore, ATAD3A binding attenuated local PERK signaling and rescued the expression of some mitochondrial proteins. Thus, PERK-ATAD3A interactions can control translational repression at a subcellular level, mitigating the impact of ER stress on the cell.

The making of a nucleic acid sensor at the dawn of jawed vertebrate evolution.

Self and nonself discrimination is fundamental to immunity. However, it remains largely enigmatic how the mechanisms of distinguishing nonself from self originated. As an intracellular nucleic acid sensor, protein kinase R (PKR) recognizes double-stranded RNA (dsRNA) and represents a crucial component of antiviral innate immunity. Here, we combine phylogenomic and functional analyses to show that PKR proteins probably originated from a preexisting kinase protein through acquiring dsRNA binding domains at least before the last common ancestor of jawed vertebrates during or before the Silurian period. The function of PKR appears to be conserved across jawed vertebrates. Moreover, we repurpose a protein closely related to PKR proteins into a putative dsRNA sensor, recapturing the making of PKR. Our study illustrates how a nucleic acid sensor might have originated via molecular tinkering with preexisting proteins and provides insights into the origins of innate immunity.

Integrated stress response activator halofuginone protects mice from diabetes-like phenotypes.

The integrated stress response (ISR) is a vital signaling pathway initiated by four kinases, PERK, GCN2, HRI and PKR, that ensure cellular resilience and protect cells from challenges. Here, we investigated whether increasing ISR signaling could rescue diabetes-like phenotypes in a mouse model of diet-induced obesity (DIO). We show that the orally available and clinically approved GCN2 activator halofuginone (HF) can activate the ISR in mouse tissues. We found that daily oral administration of HF increases glucose tolerance whilst reducing weight gain, insulin resistance, and serum insulin in DIO mice. Conversely, the ISR inhibitor GSK2656157, used at low doses to optimize its selectivity, aggravates glucose intolerance in DIO mice. Whilst loss of function mutations in mice and humans have revealed that PERK is the essential ISR kinase that protects from diabetes, our work demonstrates the therapeutic value of increasing ISR signaling by activating the related kinase GCN2 to reduce diabetes phenotypes in a DIO mouse model.

PKR activation-induced mitochondrial dysfunction in HIV-transgenic mice with nephropathy.

HIV disease remains prevalent in the USA and chronic kidney disease remains a major cause of morbidity in HIV-1-positive patients. Host double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a sensor for viral dsRNA, including HIV-1. We show that PKR inhibition by compound C16 ameliorates the HIV-associated nephropathy (HIVAN) kidney phenotype in the Tg26 transgenic mouse model, with reversal of mitochondrial dysfunction. Combined analysis of single-nucleus RNA-seq and bulk RNA-seq data revealed that oxidative phosphorylation was one of the most downregulated pathways and identified signal transducer and activator of transcription (STAT3) as a potential mediating factor. We identified in Tg26 mice a novel proximal tubular cell cluster enriched in mitochondrial transcripts. Podocytes showed high levels of HIV-1 gene expression and dysregulation of cytoskeleton-related genes, and these cells dedifferentiated. In injured proximal tubules, cell-cell interaction analysis indicated activation of the pro-fibrogenic PKR-STAT3-platelet-derived growth factor (PDGF)-D pathway. These findings suggest that PKR inhibition and mitochondrial rescue are potential novel therapeutic approaches for HIVAN.

Unfolded proteins in the mitochondria activate HRI and inhibit mitochondrial protein translation.

The mitochondrial unfolded protein response (UPRmt) is triggered through eIF2α phosphorylation in mammals. However, the mechanisms of UPRmt activation and the influence of eIF2α phosphorylation on mitochondrial protein translation remain unclear. In this study, we confirmed that the UPRmt is a rapid and specific stress response that occurs through pharmacological induction of eIF2α phosphorylation, along with the phosphorylation of eIF2α, ATF4, and CHOP. Moreover, with the upregulation of the expression of some chaperones, cytochrome P450 enzymes, and DDIT4, as determined by RNA-Seq and ribosome profiling, eIF2α phosphorylation was found to be essential for the expression of ATF4 and CHOP, after which ATF4 trafficked into the nucleus and initiated CHOP expression. In addition, the generation of ROS and mitochondrial morphology were not affected by the GTPP-induced UPRmt. Furthermore, we investigated the mechanism by which HRI kinase-mediated UPRmt is induced by mitochondrial unfolded proteins via CRISPR-Cas9 technology, mitochondrial recruitment of HRI and interaction with other proteins. Moreover, we confirmed that mitochondrial protein translation and mitochondrial protein import were inhibited through eIF2α phosphorylation with the accumulation of unfolded mitochondrial proteins. These findings reveal the molecular mechanism of the UPRmt and its impact on cellular protein translation, which will offer novel insights into the functions of the UPRmt, including its implications for human disease and pathobiology.

Targeting PERK and GRP78 in colorectal cancer: Genetic insights and novel therapeutic approaches.

Colorectal cancer (CRC) ranks among the leading causes of cancer-related deaths worldwide. Enhancing CRC diagnosis and prognosis requires the development of improved biomarkers and therapeutic targets. Emerging evidence suggests that the unfolded protein response (UPR) plays a pivotal role in CRC progression, presenting new opportunities for diagnosis, treatment, and prevention. This study hypothesizes that genetic variants in endoplasmic reticulum (ER) stress response genes influence CRC susceptibility. We examined the frequencies of SNPs in PERK (rs13045) and GRP78/BiP (rs430397) within a South Iranian cohort. We mapped the cellular and molecular features of PERK and GRP78 genes in colorectal cancer, observing their differential expressions in tumor and metastatic tissues. We constructed co-expression and protein-protein interaction networks and performed gene set enrichment analysis, highlighting autophagy as a significant pathway through KEGG. Furthermore, the study included 64 CRC patients and 60 control subjects. DNA extraction and genotyping were conducted using high-resolution melting (HRM) analysis. Significant differences in PERK and GRP78 expressions were observed between CRC tissues and controls. Variations in PERK and GRP78 genotypes were significantly correlated with CRC risk. Utilizing a Multi-Target Directed Ligands approach, a dual PERK/GRP78 inhibitor was designed and subjected to molecular modeling studies. Docking experiments indicated high-affinity binding between the proposed inhibitor and both genes, PERK and GRP78, suggesting a novel therapy for CRC. These findings highlight the importance of understanding genetic backgrounds in different populations to assess CRC risk. Polymorphisms in UPR signaling pathway elements may serve as potential markers for predicting CRC susceptibility, paving the way for personalized therapeutic strategies.

Trimethylamine N-oxide induces non-alcoholic fatty liver disease by activating the PERK.

Nonalcoholic fatty liver disease (NAFLD) is a liver disease causing different progressive pathological changes. Trimethylamine N-oxide (TMAO), a product of gut microbiota metabolism, is a specific agonist of the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway, one of the endoplasmic reticulum stress (ERS) pathways. TMAO has been associated with the occurrence and development of NAFLD based on the results of previous studies, but whether the simple consumption of TMAO can directly induce NAFLD and its underlying mechanism remain unclear. To investigate this question, we constructed an animal model in which adult male zebrafish were fed a controlled diet containing 1 % or 3 % TMAO for 20 weeks. Eventually, we observed that TMAO caused lipid accumulation, inflammatory infiltration, liver injury and liver fibrosis in zebrafish livers; meanwhile, the PERK signaling pathway was activated in the zebrafish livers. This finding was further confirmed in HepG2 cells and hepatic stellate cells models. In conclusion, this study found that TMAO directly induced different pathological states of NAFLD in zebrafish liver, and the activation of PERK pathway is an important mechanism, which may provide crucial strategies for the diagnosis and treatment of NAFLD.