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Background: The impact of a high-salt (HS) diet on metabolic disturbances in individuals with coronary heart disease remains unclear. The arachidonic acid (AA) metabolic pathway is closely linked to the development of cardiometabolic diseases and atherosclerotic cardiovascular diseases. Furthermore, endoplasmic reticulum stress (ERS) has emerged as a major contributor to cardiometabolic diseases. AA-related inflammation and ERS are hypothesized to play a role in HS diet-induced coronary remodeling., Methods: Rats were subjected to an HS diet for 4 weeks, and the serum concentration of AA was measured via enzyme-linked immunosorbent assay. Immunofluorescence staining and vascular tension measurements were conducted on coronary arteries. In addition, AA-stimulated coronary artery smooth muscle cells (CASMCs) were treated with ERS inhibitors to explore the underlying pathway involved., Results: Increased susceptibility to myocardial infarction in the HS diet-fed rats was accompanied by increased serum AA concentrations and increased expression of the key AA metabolic enzyme cyclooxygenase-2 (COX-2). AA incubation weakened the contraction of denuded coronary arteries, reduced the expression of contraction markers, and increased the fluorescence intensity of synthetic and ERS response markers in coronary arteries. Further investigation of CASMCs revealed that AA-induced phenotypic transformation was mediated via the ERS pathway., Conclusions: ERS and AA were found to be stimulated in CASMCs following an HS diet. AA triggers an ERS response through COX-2 catalysis, and the downstream inositol requiring enzyme 1 - X-box binding protein-1 - osteopontin pathway may contribute to the AA-induced phenotypic transformation of CASMCs, resulting in dysfunctional coronary tension. This study may provide potential therapeutic targets for cardiovascular diseases associated with excessive AA-derived ERS., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Gefitinib, a selective EGFR-tyrosine kinase inhibitor, exhibits potent cytotoxic effects on colorectal cancer cells, though its precise mechanisms are not fully understood. In this study, we demonstrated that gefitinib induces a dose-dependent cytotoxic response in Caco-2 cells, characterized by disrupted microtubule networks, impaired migration, and reduced viability. Gefitinib triggered apoptosis, as indicated by increased levels of cleaved caspase-3, PARP, and elevated late apoptosis rates. Mechanistically, gefitinib-induced endoplasmic reticulum (ER) stress, marked by the upregulation of IRE1α, CHOP, and ATF4. ER stress inhibition by 4-PBA significantly reduced apoptosis and restored mitochondrial membrane potential (MMP). Additionally, gefitinib-induced apoptosis was mediated through the mitochondrial pathway, reflected by the modulation of Bcl-2 family proteins, including the upregulation of Bax and Bim. Inhibition of the IRE1α-mediated JNK/p38 MAPK pathway further mitigated gefitinib-induced apoptosis and restored MMP. These findings highlight the critical role of ER stress and the IRE1α-JNK/p38 MAPK axis in gefitinib-induced mitochondrial apoptosis, offering potential therapeutic targets for colorectal cancer., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Ethical approval: Not applicable. Consent to participate: Not Applicable. Consent for publication: All authors consent to the publication of this., (© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The Cullin-3 E3 ligase adaptor protein SPOP targets proteins for ubiquitination and proteasomal degradation. We previously established the β-cell transcription factor (TF) and human diabetes gene PDX1 as an SPOP substrate, suggesting a functional role for SPOP in the β cell. Here, we generated a β-cell-specific Spop deletion mouse strain ( Spop βKO ) and found that Spop is necessary to prevent aberrant basal insulin secretion and for maintaining glucose-stimulated insulin secretion through impacts on glycolysis and glucose-stimulated calcium flux. Integration of proteomic, TF-regulatory gene network, and biochemical analyses identified XBP1 as a functionally important SPOP substrate in pancreatic β cells. Furthermore, loss of SPOP strengthened the IRE1α-XBP1 axis of unfolded protein response (UPR) signaling. ER stress promoted proteasomal degradation of SPOP, supporting a model whereby SPOP fine-tunes XBP1 activation during the UPR. These results position SPOP as a regulator of β-cell function and proper UPR activation., (© 2025 Oguh et al.; Published by Cold Spring Harbor Laboratory Press.)

Hematopoietic stem cells must mitigate myriad stressors throughout their lifetime to ensure normal blood cell generation. Here, we uncover unfolded protein response stress sensor inositol-requiring enzyme-1α (IRE1α) signaling in hematopoietic stem and progenitor cells (HSPCs) as a safeguard against myeloid leukemogenesis. Activated in part by an NADPH oxidase-2 mechanism, IRE1α-induced X-box binding protein-1 (XBP1) mediated repression of pro-leukemogenic programs exemplified by the Wnt-β-catenin pathway. Transcriptome analysis and genome-wide mapping of XBP1 targets in HSPCs identified an '18-gene signature' of XBP1-repressed β-catenin targets that were highly expressed in acute myeloid leukemia (AML) cases with worse prognosis. Accordingly, IRE1α deficiency cooperated with a myeloproliferative oncogene in HSPCs to cause a lethal AML in mice, while genetic induction of XBP1 suppressed the leukemia stem cell program and activity of patient-derived AML cells. Thus, IRE1α-XBP1 signaling safeguards the integrity of the blood system by restricting pro-leukemogenic programs in HSPCs., Competing Interests: Competing interests: L.H.G. is a former Director of Bristol Myers Squibb and the Waters Corporation and currently serves on the Board of Directors of GlaxoSmithKline Pharmaceuticals and Analog Devices. L.H.G. also serves on the scientific advisory boards of Repare Therapeutics, Abpro Therapeutics and Kaleido Therapeutics. A.M.M. receives research funding from Janssen, Daiichi Sankyo and Sanofi; has consulted for Epizyme, Constellation, BMI and Exo-Therapeutics; and is a scientific advisor to KDAC. A.V. is a current employee of Volastra Therapeutics. All other authors declare no competing interests., (© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

In white adipose tissue, disturbed creatine metabolism through reduced creatine kinase B (CKB) transcription contributes to obesity-related inflammation. However, the mechanisms regulating CKB expression in human white adipocytes remain unclear. By screening conditions perturbed in obesity, we identified endoplasmic reticulum (ER) stress as a key suppressor of CKB transcription across multiple cell types. Through follow-up studies, we found that ER stress through the IRE1-XBP1s pathway, promotes CKB promoter methylation via the methyltransferase DNMT3A. This epigenetic change represses CKB transcription, shifting metabolism towards glycolysis and increasing the production of the pro-inflammatory chemokine CCL2. We validated our findings in vivo, demonstrating that individuals living with obesity show an inverse relationship between CKB expression and promoter methylation in white adipocytes, along with elevated CCL2 secretion. Overall, our study uncovers a regulatory axis where ER stress drives inflammation in obesity by reducing CKB abundance, and consequently altering the bioenergetic state of the cell., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Objectives: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for cirrhosis and hepatocellular carcinoma, for which there is currently no effective treatment. This study aimed to investigate the regulatory mechanism between endoplasmic reticulum stress (ER stress) and pyroptosis in the liver under the context of MASH., Methods and Results: Pyroptosis was examined in both in vivo and in vitro ER stress models. The expression levels of nucleotide-binding oligomerisation domain-like receptor protein 3 (NLRP3), gasdermin D (GSDMD), caspase-1, IL-1β, and IL-18 tended to increase, and "ASC specks" colocalised with the swollen ER in living cells. However, in the pyroptotic model, increased ER stress was not observed. Moreover, the overexpression of inositol-requiring enzyme 1α (IRE-1α), one of the main ER stress sensors, led to increases in the levels of NLRP3 and GSDMD. However, after IRE-1α was blocked by chemical inhibitors or siRNAs, pyroptosis was also abrogated. These data showed that ER stress regulated pyroptosis through IRE-1α. Furthermore, the immunoprecipitation results clearly indicated that GSDMD efficiently bound to IRE-1α when ER stress was stimulated. In the MASH model, IRE-1α was specifically inhibited by pharmacological or genetic methods, which improved the pathology of MASH by alleviating ER stress and pyroptosis. In patients with MASH, both ER stress markers and pyroptosis markers including IRE-1α, glucose-regulated protein 78, GSDMD/GSDMD-N, p20, and NLRP3, are highly expressed in the liver., Conclusions: This study revealed that ER stress may regulate pyroptosis through IRE-1α-GSDMD pathway, which accelerates the progression of MASH. These findings may offer new insights for the treatment of MASH., (© 2025 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Melanocytes protect the body from ultraviolet radiation by synthesizing melanin. Tyrosinase, a key enzyme in melanin production, accumulates in the endoplasmic reticulum (ER) during melanin synthesis, potentially causing ER stress. However, regulating ER function for melanin synthesis has been less studied than controlling Tyrosinase activity., Objective: This study investigates the regulatory mechanisms of melanin production, focusing on ER stress and the ER stress-induced response., Methods: B16 mouse melanoma cells induced to undergo melanogenesis were treated with unfolded protein response (UPR) inhibitors or chemical chaperones, and their effects on melanogenesis were analyzed., Results: During melanogenesis in B16 cells stimulated by alpha-melanocyte-stimulating hormone (α-MSH), ER stress and UPR activation occurred, accompanied by increased Tyrosinase protein. Reducing IRE1 and ATF6 branch activity lowered melanin levels, while chemical chaperone treatment restored melanin production and increased Tyrosinase levels., Conclusion: UPR activation, linked to elevated Tyrosinase levels, influences melanin production during melanogenesis. Modulating UPR can regulate melanin synthesis and provides a potential new approach for treating pigmentation disorders., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: We investigated the mechanism of Dexmedetomidine (Dex) in infant rats with brain injury., Methods: The infant rats underwent brain injury modelling. The motor function, spatial learning and memory abilities in rats, and the hippocampal CA1 region Nissl body level and apoptosis were evaluated by behavioural tests and histological stainings. Levels of the hippocampal CA1 region p-IRE1α, nuclear/cytoplasmic p65, CHOP, Bax and Bcl-2 proteins were determined by Western blot., Results: Propofol anaesthesia caused brain injury in infant rats. Dex increased the hippocampal CA1 region Nissl body level, abated cell apoptosis, reduced p-IRE1α, ATF6, p-PERK/PERK and CHOP levels, decreased the Bax protein level, elevated the Bcl-2 protein level, and alleviated brain injury in infant rats. After ERS induction and the NF-κB pathway inhibition, the hippocampal CA1 region nuclear/cytoplasmic p65 ratio, CHOP level, and apoptosis were reduced in infant rats with brain injury treated with Dex, while the learning and memory abilities of rats were enhanced., Conclusion: Dex reduced the hippocampal CA1 region cell apoptosis and enhanced learning and memory abilities by inhibiting the ERS-mediated IRE1α/NF-κB/CHOP pathway, thereby alleviating brain injury in infant rats.

In recent years, it has been generally accepted that metal-based nanoparticles (NPs) may induce stress in the endoplasmic reticulum (ER), a key organelle where protein folding occurs. We examined ER stress in immortalized human cerebral microvascular cells (hCMEC/D3) after exposure to silver-NPs (Ag-NPs)- and copper oxide-NPs (CuO-NPs) induced toxicity at < 10 nm and < 40 nm or < 50 nm diameters, respectively. In cytotoxicity assessments, cells were exposed to different CuO-NPs (5-400 µg/mL) or Ag-NPs (1-10 µg/mL) concentration ranges for 24 h and 72 h, and tetrazole salt reduction assays (EZ4U) were performed. Also, Ag-NP or CuO-NP effects on cell proliferation, apoptosis (caspase 3/7 assays), and ER stress and cell morphology were evaluated. In ER stress assessments, RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1a), and others stress factor mRNA levels were determined after 24 h treatment using Real-Time PCR. Increased stress sensors (IRE1a, PERK, and ATF6) mRNA levels were observed after exposure to Ag-NPs (< 10 and < 40 nm) or CuO-NPs (< 50 nm). We investigated the expression of tight junction (TJ) proteins (barrier junctions) and showed that both types of NP reduced of OCLN gene expression. Morphological changes were observed after Ag-NP or CuO-NP exposure using holotomographic microscopy. Our data suggest that Ag- and CuO-NPs should undergo future in vitro and in vivo toxicology studies, especially for downstream biomedical application and occupational risk assessments., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Humans are widely exposed to phthalates, a common chemical plasticizer. Previous cohort studies have revealed that maternal exposure to monobutyl phthalate (MBP), a key metabolite of phthalates, is associated with neurodevelopmental defects. However, the molecular mechanism remains unclear. Here, we demonstrate that maternal exposure to MBP enhances neural stem cell (NSC) differentiation into astrocytes with highly expressed C3 and LCN2 in mouse offspring, resulting in increased synapse phagocytosis and cognitive dysfunction. Mechanistically, we find that MBP exposure activates the IRE1α/XBP1s (spliced XBP1) stress response pathway, which regulates key genes involved in astrocyte differentiation (SOX9 and ATF3) and reactivity (C3 and LCN2). Conditional knockout or pharmacological inhibition of IRE1α markedly inhibits NSC differentiation into astrocytes and astrocyte reactivity, attenuates synapse phagocytosis, and improves cognitive function. This phenotype is further recapitulated in a human brain organoid model. Together, these findings unveil the molecular mechanism underlying the neurodevelopmental deficits caused by a widespread environmental pollutant., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The essential homotetrameric endoribonuclease RNase E of Escherichia coli participates in global RNA turnover as well as stable RNA maturation. The protomer's N-terminal half (residues 1-529) bears the catalytic, allosteric, and tetramerization domains, including the active site residues D303 and D346. The C-terminal half (CTH, residues 530-1061) is dispensable for viability. We have previously described a phenomenon of recessive resurrection in RNase E that requires the CTH, wherein the wild-type homotetramer apparently displays nearly identical activity in vivo as a heterotetramer comprising three catalytically dead subunits (with D303A or D346A substitutions) and one wild-type subunit. Here, we show that recessive resurrection is exhibited even in dimeric RNase E with the CTH, and that it is largely dependent on the presence of a membrane-targeting-sequence motif (residues 565-582). A single F575E substitution also impaired recessive resurrection, whereas other CTH motifs (such as those for binding of RNA or of partner proteins) were dispensable. The phenomenon was independent of RNA 5'-monophosphate sensing by the enzyme. We propose that membrane-anchoring of RNase E renders it processive for endoribonucleolytic action, and that recessive resurrection and dominant negativity associated with mutant protomers are mutually exclusive manifestations of, respectively, processive and distributive catalytic mechanisms in a homo-oligomeric enzyme., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA endonucleases are the rate-limiting initiator of decay for many bacterial mRNAs. However, the positions of cleavage and their sequence determinants remain elusive even for the well-studied Bacillus subtilis. Here we present two complementary approaches-transcriptome-wide mapping of endoribonucleolytic activity and deep mutational scanning of RNA cleavage sites-that reveal distinct rules governing the specificity among B. subtilis endoribonucleases. Detection of RNA terminal nucleotides in both 5'- and 3'-exonuclease-deficient cells revealed >103 putative endonucleolytic cleavage sites with single-nucleotide resolution. We found a surprisingly weak consensus for RNase Y targets, a contrastingly strong primary sequence motif for EndoA targets, and long-range intramolecular secondary structures for RNase III targets. Deep mutational analysis of RNase Y cleavage sites showed that the specificity is governed by many disjointed sequence features. Our results highlight the delocalized nature of mRNA stability determinants and provide a strategy for elucidating endoribonuclease specificity in vivo., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The CRISPR-derived endoribonuclease Csy4 is a popular tool for controlling transgene expression in various therapeutically relevant settings, but adverse effects potentially arising from non-specific RNA cleavage remains largely unexplored. Here, we report a split-Csy4 architecture that was carefully optimized for in vivo usage. First, we separated Csy4 into two independent protein moieties whose full catalytic activity can be restored via various constitutive or conditional protein dimerization systems. Next, we show that introduction of split-Csy4 into human cells caused a substantially reduced extent in perturbation of the endogenous transcriptome when directly compared to full-length Csy4. Inspired by these results, we went on to use such split-Csy4 module to engineer inducible CRISPR- and translation-level gene switches regulated by the FDA-approved drug grazoprevir. This work provides valuable resource for Csy4-related biomedical research and discusses important issues for the development of clinically eligible regulation tools., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nonalcoholic fatty liver disease (NAFLD), characterized by hepatic lipid deposition, is one of the most prevalent chronic metabolic disorders globally, and its pharmaceutical treatments are still limited. Excessive lipid accumulation triggers endoplasmic reticulum (ER) stress and autophagy flux dysfunction, which are important mechanisms for NAFLD. Trehalose (Tre), a natural disaccharide, has been identified to reduce hepatic steatosis and glucose intolerance. However, its underlying mechanisms for NAFLD remain unclear. In this study, a high-fat-diet (HFD)-induced mouse NAFLD model and a saturated fatty acid palmitic acid (PA)-stimulated cell model were constructed. The results indicated that Tre supplementation ameliorated hepatocyte lipid deposition in vitro , as well as hepatic steatosis and hyperlipidemia in vivo . Mechanistically, Tre alleviated both autophagy flux dysfunction and endoplasmic reticulum (ER) stress. Under the stimulation of HFD or PA, Tre remarkably increased the expression and nucleic translocation of the lysosomal master protein transcription factor EB (TFEB), while decreasing the accumulation of p62 and also decreasing the ER stress markers (inositol-requiring enzyme 1 (IRE1α), XBP-1, CHOP, and BIP). Similar results were observed in an ER stressor tunicamycin (TM)-induced in vivo and in vitro models. In addition, the transcriptomic analysis of NAFLD patients revealed significant differences in ER stress-related and autophagy-related biomarkers, including TFEB, ATG7, IRE1α, and CHOP. Molecular docking results demonstrated a strong affinity between Tre and both IRE1α and TFEB. Overall, Tre protected hepatocytes from lipotoxicity-related ER stress and autophagy dysfunction, and its regulatory effect on the IRE1α-TFEB signaling pathway may be a critical mechanism. These findings suggest that Tre, as a bioactive substance with significant medicinal potential, holds considerable promise for drug development and clinical application in treating NAFLD.

Ribonucleoprotein (RNP) condensates partition RNA and protein into multiple liquid phases. The multiphasic feature of condensate-enriched components creates experimental challenges for distinguishing membraneless condensate functions from the surrounding dilute phase. We combined fluorescence lifetime imaging microscopy (FLIM) with phasor plot filtering and segmentation to resolve condensates from the dilute phase. Condensate-specific lifetimes were used to track protein-protein interactions by measuring FLIM-Förster resonance energy transfer (FRET). We used condensate FLIM-FRET to evaluate whether mRNA decapping complex subunits can form decapping-competent interactions within P-bodies. Condensate FLIM-FRET revealed the presence of core subunit interactions within P-bodies under basal conditions and the disruption of interactions between the decapping enzyme (Dcp2) and a critical cofactor (Dcp1A) during oxidative stress. Our results show a context-dependent plasticity of the P-body interaction network, which can be rewired within minutes in response to stimuli. Together, our FLIM-based approaches provide investigators with an automated and rigorous method to uncover and track essential protein-protein interaction dynamics within RNP condensates in live cells., (© 2024 Fahim et al.)

Coronaviruses evade detection by the host immune system with the help of the endoribonuclease Nsp15, which regulates levels of viral double stranded RNA by cleaving 3' of uridine (U). While prior structural data shows that to cleave double stranded RNA, Nsp15's target U must be flipped out of the helix, it is not yet understood whether Nsp15 initiates flipping or captures spontaneously flipped bases. We address this gap by designing fluorinated double stranded RNA substrates that allow us to directly relate a U's sequence context to both its tendency to spontaneously flip and its susceptibility to cleavage by Nsp15. Through a combination of nuclease assays, 19 F NMR spectroscopy, mass spectrometry, and single particle cryo-EM, we determine that Nsp15 acts most efficiently on unpaired Us, particularly those that are already flipped. Across sequence contexts, we find Nsp15's cleavage efficiency to be directly related to that U's tendency to spontaneously flip. Overall, our findings unify previous characterizations of Nsp15's cleavage preferences, and suggest that activity of Nsp15 during infection is partially driven by bulged or otherwise relatively accessible Us that appear at strategic positions in the viral RNA., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Citrus grandis fruit is a famous traditional Chinese medicine with various bioactivities, including cardioprotective effects. Polysaccharides are one of the key active ingredients responsible for its cardioprotective effects. This study aimed to investigate the structure and cardioprotective effect of a homogeneous polysaccharide from C. grandis fruit (CGP80-1) and explore its mechanism against myocardial ischemia-reperfusion (MI/R) injury. Structure analysis showed that CGP80-1 (11,917 Da) is an arabinan with compact coil chain conformation, containing →5)-α-L-Araf-(1→, →3,5)-α-L-Araf-(1→, and →2,3,5)-α-L-Araf-(1→ as the backbone, as well as →5)-α-L-Araf-(1→ and t-α-L-Araf as side-chains substituted at the C2 and C3 positions. Pharmacological experiments showed that pre-treatment with CGP80-1 could effectively alleviate MI/R injury by improving endogenous antioxidant enzymes and cardiac enzymes, reducing reactive oxygen species levels, and regulating apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. The protective effects were correlated with the Nrf2/Keap1 and IRE1/GRP78 signaling pathways. Further analysis of structure-activity relationships revealed that the myocardial protection effects of CGP80-1 might be attributed to its appropriate molecular weight, high arabinose content, and unique compact coil chain conformation. Overall, our results provide insight into the chemical structure of CGP80-1 and its mechanism of action, suggesting that CGP80-1 could be a candidate drug for myocardial protection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hypoxic-ischemic encephalopathy (HIE) is a perinatal injury caused by cerebral hypoxia and reduced blood perfusion. Microglia activation-induced neuroinflammatory injury is a leading cause of neuron loss and brain injury. Efficient treatment strategies are still required further investigation. Our study is aimed to investigate the role of IRE1-XBP1 inhibitor 4μ8С in HIE. Rat pups (7 d) were used to establish HIE model using unilateral carotid artery ligation and hypoxia. A series of experiments including Western blot, Morris water maze test, TTC staining, RT-qPCR, TUNEL staining, and immunofluorescence staining were operated to evaluate the role of 4μ8С in HIE. 4μ8С treatment effectively reduced phosphorylated IRElα and XBP1 protein levels. 4μ8С treatment improves cognition and learning abilities of HIE rats. 4μ8С treatment alleviated brain infarction and cell apoptosis in HIE rats. 4μ8С treatment inhibited NLRP3 inflammasome activation-mediated microglia activation and inflammatory response. In conclusion, 4μ8С suppressed microglia and NLRP3 inflammasome activation by inactivating IRE1/XBP1 axis during HIE development, which revealed IRE1α inhibition as a novel mechanism for neuron protection.

This study aimed to investigate the effects of curcumin-carbon dot conjugates (CUR-CD) on periodontitis. Porphyromonas gingivalis lipopolysaccharide (LPS) was used to establish periodontitis mode in vivo and in vitro. Histological analysis was conducted using hematoxylin and eosin (HE) staining. Bone morphogenetic protein 2 (BMP2) and secreted phosphoprotein 1 (OPN) expression was determined using immunohistochemistry. mRNA levels were detected using reverse transcription quantitative real-time PCR (RT-qPCR). Protein expression was determined using Western blot. N6-methyladenosine (m6A) enrichment was determined using methylated RNA immunoprecipitation (MeRIP) assay. Cytokine release was determined using enzyme-linked immunosorbent assay (ELISA) assay. Osteogenic differentiation was detected using alkaline phosphatase (ALP) staining. The results showed that CUR-CD inhibited the inflammatory response, as well as promoted bone healing in vivo and osteogenic differentiation in vitro. Moreover, CUR-CD downregulated methyltransferase 3 (METTL3), which inhibited m6A modification of endoplasmic reticulum to nucleus signaling 1 (IRElα) and downregulated IRElα expression. However, overexpression of IRElα reversed the effects of CUR-CD, stimulating inflammatory response and inhibiting bone healing and osteogenic differentiation. Collectively, CUR-CD inhibits the progression of periodontitis via downregulating IRElα. Therefore, CUR-CD may be an alternative strategy for periodontitis.

The neuroprotective function of ginsenoside Rb1 (GRb1) in cerebral ischemia-reperfusion (I/R) was lately emphasized. However, whether GRb1 plays a regulatory role on endoplasmic reticulum (ER) stress-associated pathway in cerebral I/R damage is still unclear. The aim of this study is to explore the function of GRb1 in cerebral ischemia-induced ER stress and the underlying mechanism related to IRE1/TRAF2/JNK pathway. Longa method, cerebral infarct volume, and HE staining were used to evaluate the efficacy of GRb1 in mice with a mouse model of middle cerebral artery occlusion reperfusion (MCAO/R). We also investigated the effect and mechanism of GRb1 against ischemic stroke using in vitro oxygen-glucose deprivation reperfusion (OGD/R) model. We found that GRb1 could improve neurological scores, infarct volume, and histological injury in ischemic mice. Ischemic attack also activated neuronal apoptosis and ER stress, and this effect was attenuated by GRb1. In addition, GRb1 significantly reduced I/R-induced IRE1-TRAF2 interaction, IRE1, and JNK phosphorylation. The present study also confirmed that GRb1 significantly improved OGD/R-induced PC12 cells injury. GRb1 could decrease ER stress in OGD/R-injured PC12 cells, which was reflected by the decreased expression of GRP78 and CHOP. The ER stress inducer tunicamycin partially prevented the effects of GRb1 on cell viability, ER stress, and apoptosis after OGD/R, whereas the ER stress inhibitor 4-PBA exerted the opposite effect. Moreover, GRb1 markedly decreased IRE1-TRAF2 interaction, IRE1, and JNK phosphorylation in the presence of OGD/R insult. Furthermore, JNK inhibitor SP600125 and IRE1 inhibitor DBSA pretreatment further promoted the inhibition of GRb1 on ER stress induction and cell damage induced by OGD/R. Molecular docking further elucidated that the mechanism by which GRb1 improves cerebral ischemia maybe related to its direct binding to the kinase domain of IRE1, which in turn inhibited the phosphorylation of IRE1. Collectively, these results demonstrated that GRb1 reduced ischemic stroke-induced apoptosis through the ER stress-associated IRE1/TRAF2/JNK pathway and GRb1 has the potential as a protective drug for the treatment of cerebral ischemia., Competing Interests: Declarations. Ethics approval and consent to participate: The present study was approved by The Institutional Animal Care and Use Committee, and the Animal Ethics Committee at Anhui University of Chinese Medicine approved the study (Ethics Certificate Number: 2023047). Consent for publication: All authors have approved for publication. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Non-alcoholic fatty liver disease (NAFLD) is currently the most common type of chronic liver disease. Semaglutide is a glucose-lowering drug administered for the treatment of type 2 diabetes mellitus (T2DM) and is clinically effective in the treatment of NAFLD. X-box binding protein 1 (XBP1) is related to the pathogenesis of both NAFLD and T2DM. The aim of the present study was to demonstrate whether the underlying mechanism of semaglutide treatment for NAFLD is via downregulation of the inositol-requiring transmembrane kinase/endonuclease-1α (IRE1α)-XBP1-CCAAT/enhancer binding protein α (C/EBPα) signaling pathway in macrophages., Methods: In the present study, NAFLD cell modeling was induced by oleic acid (0.4 mm) and palmitic acid (0.2 mm). Hepatocytes (AML12) and macrophages (RAW264.7) were co-cultured in 6-well Transwell plates. Semaglutide (60 or 140 nm) was administrated for 24 h, while pioglitazone (2 μm) and toyocamycin (200 nm) were used as a positive control drug and a XBP1 inhibitor, respectively. Autophagy and apoptosis of AML12 cells were detected by transmission electron microscopy and Western blotting (WB). Hepatocyte steatosis was evaluated by adopting total intracellular triglyceride determination, analysis of the relative expression of proteins and genes associated with lipid metabolism and hepatocyte Oil red O staining. Detection of inflammation factors was conducted by ELISA and WB. To explore the underlying mechanism of NAFLD treatment with semaglutide, the relative expression of related proteins and genes were tested., Results: Our study demonstrated that semaglutide treatment improved autophagy and inhibited apoptosis of hepatocytes, while notably ameliorating steatosis of hepatocytes. In addition, inflammation was attenuated in the NAFLD cell co-culture model after semaglutide administration. Semaglutide also significantly reduced the protein and gene expression levels of the IRE1α-XBP1-C/EBPα signaling pathway in macrophages., Conclusion: Semaglutide partially ameliorated NAFLD by downregulating the IRE1α-XBP1-C/EBPα signaling pathway in macrophages. These findings may provide a potential theoretical basis for semaglutide therapy for NAFLD., (© 2024 S. Karger AG, Basel.)

Inhalation exposure to airborne fine particulate matter (aerodynamic diameter: <2.5 µm, PM 2.5 ) is known to cause metabolic dysfunction-associated steatohepatitis (MASH) and the associated metabolic syndrome. Hepatic lipid accumulation and inflammation are the key characteristics of MASH. However, the mechanism by which PM 2.5 exposure induces lipid accumulation and inflammation in the liver remains to be further elucidated. In this study, we revealed that inhalation exposure to PM 2.5 induces nitrosative stress in mouse livers by suppressing hepatic S -nitrosoglutathione reductase activities, which leads to S -nitrosylation modification of the primary unfolded protein response (UPR) transducer inositol-requiring 1 α (IRE1α), an endoplasmic reticulum-resident protein kinase and endoribonuclease (RNase). S -nitrosylation suppresses the RNase activity of IRE1α and subsequently decreases IRE1α-mediated splicing of the mRNA encoding X-box binding protein 1 (XBP1) and IRE1α-dependent degradation of select microRNAs (miRNAs), including miR-200 family members, miR-34, miR-223, miR-155, and miR-146, in the livers of the mice exposed to PM 2.5 . Elevation of IRE1α-target miRNAs, due to impaired IRE1α RNase activity by PM 2.5 -triggered S -nitrosylation, leads to decreased expression of the major regulators of fatty acid oxidation, lipolysis, and anti-inflammatory response, including XBP1, sirtuin 1, peroxisome proliferator-activated receptor α, and peroxisome proliferator-activated receptor γ, in the liver, which account at least partially for hepatic lipid accumulation and inflammation in mice exposed to airborne PM 2.5 . In summary, our study revealed a novel pathway by which PM 2.5 causes cytotoxicity and promotes MASH-like phenotypes through inducing hepatic nitrosative stress and S -nitrosylation of the primary UPR transducer and subsequent elevation of select miRNAs involved in metabolism and inflammation in the liver. NEW & NOTEWORTHY Exposure to fine airborne particulate matter PM 2.5 causes metabolic dysfunction-associated steatohepatitis characterized by hepatic steatosis, inflammation, and fibrosis. Here, we discovered that inhalation exposure to environmental PM 2.5 induces nitrosative stress in livers by suppressing hepatic S -nitrosoglutathione reductase activities, which leads to S -nitrosylation of the unfolded protein response transducer IRE1α. S -nitrosylation decreases IRE1α-dependent degradation of miRNAs in the livers of mice exposed to PM 2.5 , leading to downregulation of major regulators of energy metabolism and anti-inflammatory response.

Mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors are widely applied to maintain pluripotency, while prolonged MEK inhibition compromises the developmental potential of mouse embryonic stem cells (ESCs). To understand the mechanism of MEK in pluripotency maintenance, we first demonstrated that MEK regulates gene expression at post-transcriptional steps. Consistently, many of the 66 MEK substrates identified by quantitative phosphoproteomics analysis are involved in RNA processing. We further confirmed that MEK1 phosphorylates S563 of DCP1A, an mRNA decapping cofactor and processing body (P body) component. DCP1A, as well as two other P body components, EDC4 and DCP2, are required for the self-renewal and differentiation of ESCs, indicating the role of P bodies in ESCs. Dephosphorylation of DCP1A S563 facilitates both self-renewal and differentiation of ESCs through promoting P body formation and RNA storage. In summary, our study identified 66 MEK substrates supporting the extracellular signal-regulated kinase (ERK)-independent function of MEK and revealed that DCP1A, phosphorylated by MEK, regulates ESC self-renewal and differentiation through modulating P body formation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Inositol-requiring enzyme 1α (IRE1α) is a sensor of endoplasmic reticulum (ER) stress and drives ER stress response pathways. Activated IRE1α exhibits RNase activity and cleaves mRNA encoding X-box binding protein 1, a transcription factor that induces the expression of genes that maintain ER proteostasis for cell survival. Previously, we showed that IRE1α undergoes S -nitrosylation, a post-translational modification induced by nitric oxide (NO), resulting in reduced RNase activity. Therefore, S -nitrosylation of IRE1α compromises the response to ER stress, making cells more vulnerable. We conducted virtual screening and cell-based validation experiments to identify compounds that inhibit the S -nitrosylation of IRE1α by targeting nitrosylated cysteine residues. We ultimately identified a compound (1ACTA) that selectively inhibits the S -nitrosylation of IRE1α and prevents the NO-induced reduction of RNase activity. Furthermore, 1ACTA reduces the rate of NO-induced cell death. Our research identified S -nitrosylation as a novel target for drug development for IRE1α and provides a suitable screening strategy.

Objective: Endoplasmic reticulum (ER) stress plays important roles not only in stress avoidance, but also in cell differentiation and maturation, cell proliferation, and promotion of bone formation. This study aimed to investigate the involvement of ER stress in the onset of pulpitis., Methods: Immunohistochemical analysis was conducted on human teeth extracted for orthodontic reasons. The effects of tunicamycin (TM), an inducer of ER stress, lipopolysaccharide (LPS), and 4μ8c, an inhibitor of inositol-requiring enzyme 1 (IRE1) on cultured human dental pulp cells (hDPCs) were also examined., Results: The expressions of two ER stress markers, X-box binding protein (XBP)-1 and binding immunoglobulin protein (BiP)/78 kDa glucose-regulated protein (GRP78), were found in the human pulp tissues of a decayed tooth that had not developed irreversible acute pulpitis, but not in an impacted tooth without inflammation in pulp tissue. Both TM and LPS increased the mRNA levels of XBP-1, interleukin (IL)-6, and IL-8, whereas TM, but not LPS, enhanced the mRNA expression of BiP/GRP78 in hDPCs. 4μ8c significantly suppressed the increased level of XBP-1 by LPS., Conclusion: This study is the first to demonstrate that XBP-1, in addition to inflammatory cytokines, may participate in the onset of pulpitis through IRE1. These findings provide a more comprehensive understanding of pulpitis pathogenesis through the cooperation of ER stress and inflammatory cytokines.

Conditions of endoplasmic reticulum (ER) stress reduce protein synthesis by provoking translation regulation, governed by the eIF2α kinase PERK. When PERK is inhibited during ER stress, retention of a selective subset of glycoproteins occurs, a phenomenon we termed selective ER retention (sERr). sERr clients are enriched with tyrosine kinase receptors (RTKs), which form large molecular weight disulfide bonded complexes in the ER. The protein disulfide isomerase ERp44 promotes sERr and increases the size of sERr complexes. Here we show that sERr is reversible upon washout. Pulse chase analyses show that upon recovery, only a small fraction of the sERr complexes disintegrates and contributes to the matured proteins, while most are newly synthesized. Sequential inductions of sERr and washouts demonstrate an accelerated recovery that is dependent on the unfolded protein response transducer IRE1. Since IRE1 regulates the expression level PDIA6, we analyzed its contribution to sERr. We found that PDIA6 and ERp44 constitutively interact by disulfides and have opposite effects on resumed recovery of trafficking following removal of sERr conditions. Deletion of ERp44 accelerates, while deletion of PDIA6 slows down recovery with a minimal effect on total protein synthesis. ERp44 is a primary interactor with sERr clients. When missing, PDIA6 partitions more into sERr complexes. Deletion of the tumor suppressor PTEN, which induces RTK signaling, promoted sERr formation kinetics, and accelerated the recovery, suggesting feedback between RTKs signaling and sERr. This study suggests that sERr, should develop physiologically or pathologically, is counteracted by adaptation responses that involve IRE1 and PDIA6., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Neuroinflammation is a key destructive pathophysiological process in early brain injury (EBI) following subarachnoid hemorrhage (SAH). Recent studies have discovered that endoplasmic reticulum stress-related inflammatory pathways include the IRE1α-TRAF2-NF-κB pathway, PERK-eIF2α-NF-κB pathway, and ATF6-AKT -NF-κB pathway leading to neuroinflammatory response. Neuritin is a neurotrophin that is involved in neuronal plasticity and regeneration. Studies have suggested that Neuritin has a vital role in reducing neuroinflammation, and can also decrease the expression of proteins related to endoplasmic reticulum stress following SAH. This suggests that Neuritin could be a potential therapeutic target for SAH and other neurological conditions. However, the regulatory mechanisms of Neuritin in ER stress-related inflammatory pathways after SAH are not yet fully understood. In this work, we discovered that the activation of ER stress-related inflammatory pathways leads to neuroinflammation, which further aggravates neuronal apoptosis after SAH. Our findings indicate that Neuritin overexpression play a neuroprotective role by inhibiting IRE1α-TRAF2-NF-κB pathway, PERK-eIF2α-NF-κB pathway, and ATF6-AKT-NF-κB pathway associated with endoplasmic reticulum stress. These inhibitory effects on neuroinflammation ultimately reduce nerve cell apoptosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Autophagy is a catabolic process involved in different cellular functions. However, the molecular pathways governing its potential roles in different cell types remain poorly understood. We investigated the role of autophagy in the context of proteotoxic stress in two central nervous system cell types: the microglia-like cell line BV2 and the neuronal-like cell line N2a. Proteotoxic stress, induced by proteasome inhibition, produced early apoptosis in BV2 cells, due in part to a predominant activation of the PERK-CHOP pathway. In contrast, N2a cells showcased greater resistance and robust induction of the IRE1α-sXbp1 arm of the UPR. We also demonstrated that proteotoxic stress activated autophagy in both cell lines but with different kinetics and cellular functions. In N2a cells, autophagy restored cellular proteostasis, while in BV2 cells, it participated in regulating phagocytosis. Finally, proteotoxic stress predominantly activated the mTORC2-AKT-FOXO1-β-catenin pathway in BV2 cells, while N2a cells preferentially induced the PDK1-AKT-FOXO3 axis. Collectively, our findings suggest that proteotoxic stress triggers cell-specific responses in microglia and neurons, with different physiological outcomes.

IFN-β is a potent antiviral cytokine and the first member of the type I IFN family of cytokines to be induced during the antiviral response. IFN-β plays an essential protective role in host defense against virus infections, as well as a pathogenic role in numerous autoimmune and autoinflammatory disorders. However, contemporary tools to study the induction, kinetics, and behavior of IFN-β are lacking. In this study, we describe a knockin Ifnb-IRES-TdTomato-Cre reporter mouse to track IFN-β-expressing cells. We demonstrate pathway-specific induction of the TdTomato reporter and show that the linked Cre recombinase enables permanent marking of cells that express IFN-β. We identify a robust MAVS-dependent IFN-β response in lung epithelial cells following Sendai virus infection in vivo. Finally, we find that activation of RNase L in macrophages by RNA ligands of the RIG-I-like receptors prevents protein translation of IFN-β and the TdTomato reporter. Our mouse model provides a powerful tool to study the biology of type I IFN induction and the antiviral response., (Copyright © 2024 by The American Association of Immunologists, Inc.)

During virus infection, the activation of the antiviral endoribonuclease, ribonuclease L (RNase L), by a unique ligand 2'-5'-oilgoadenylate (2-5A) causes the cleavage of single-stranded viral and cellular RNA targets, restricting protein synthesis, activating stress response pathways, and promoting cell death to establish broad antiviral effects. The immunostimulatory dsRNA cleavage products of RNase L activity (RL RNAs) recruit diverse dsRNA sensors to activate signaling pathways to amplify interferon (IFN) production and activate inflammasome, but the sensors that promote cell death are not known. In this study, we found that DEAH-box polypeptide 15 (DHX15) and retinoic acid-inducible gene I (Rig-I) are essential for apoptosis induced by RL RNAs and require mitochondrial antiviral signaling (MAVS), c-Jun amino terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) for caspase-3-mediated intrinsic apoptosis. In RNase L-activated cells, DHX15 interacts with Rig-I and MAVS, and cells lacking MAVS expression were resistant to apoptosis. RL RNAs induced the transcription of genes for IFN and proinflammatory cytokines by interferon regulatory factor 3 (IRF-3) and nuclear factor kB (NF-kB), while cells lacking both DHX15 and Rig-I showed a reduced induction of cytokines. However, apoptotic cell death is independent of both IRF-3 and NF-kB, suggesting that cytokine and cell death induction by RL RNAs are uncoupled. The RNA binding of both DHX15 and Rig-I is required for apoptosis induction, and the expression of both single proteins in cells lacking both DHX15 and Rig-I is insufficient to promote cell death by RL RNAs. Cell death induced by RL RNAs suppressed Coxsackievirus B3 (CVB3) replication, and inhibiting caspase-3 activity or cells lacking IRF-3 showed that the induction of apoptosis directly resulted in the CVB3 antiviral effect, and the effects were independent of the role of IRF-3.

Chemotherapy is often combined with immune checkpoint inhibitor (ICIs) to enhance immunotherapy responses. Despite the approval of chemo-immunotherapy in multiple human cancers, many immunologically cold tumors remain unresponsive. The mechanisms determining the immunogenicity of chemotherapy are elusive. Here, we identify the ER stress sensor IRE1α as a critical checkpoint that restricts the immunostimulatory effects of taxane chemotherapy and prevents the innate immune recognition of immunologically cold triple-negative breast cancer (TNBC). IRE1α RNase silences taxane-induced double-stranded RNA (dsRNA) through regulated IRE1-dependent decay (RIDD) to prevent NLRP3 inflammasome-dependent pyroptosis. Inhibition of IRE1α in Trp53 -/- TNBC allows taxane to induce extensive dsRNAs that are sensed by ZBP1, which in turn activates NLRP3-GSDMD-mediated pyroptosis. Consequently, IRE1α RNase inhibitor plus taxane converts PD-L1-negative, ICI-unresponsive TNBC tumors into PD-L1 high immunogenic tumors that are hyper-sensitive to ICI. We reveal IRE1α as a cancer cell defense mechanism that prevents taxane-induced danger signal accumulation and pyroptotic cell death., Competing Interests: Declaration of interests M.F.R. receives research funding from Pfizer and Genentech and consulting fees from Novartis, Seagen, Macrogenics, and AstraZeneca. M.F.R. is the PI of clinical trial NCT03950570. X.C. reports previous research funding from Fosun Pharma. M.J.E. holds patents and receives income from Veracyte on the PAM50-based products, Prosigna., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Lesions and stable secondary structures in mRNA severely impact the translation efficiency, causing ribosome stalling and collisions. Prokaryotic ribosomal proteins Rps3, Rps4 and Rps5, located in the mRNA entry tunnel, form the mRNA helicase center and unwind stable mRNA secondary structures during translation. However, the mechanism underlying the detection of lesions on translating mRNA is unclear. We used Cryo-EM, biochemical assays, and knockdown experiments to investigate the apurinic/apyrimidinic (AP) endoribonuclease activity of bacterial ribosomes on AP-site containing mRNA. Our biochemical assays show that Rps3, specifically the 130RR131 motif, is important for recognizing and performing the AP-endoribonuclease activity. Furthermore, structural analysis revealed cleaved mRNA product in the 30S ribosome entry tunnel. Additionally, knockdown studies in Mycobacterium tuberculosis confirmed the protective role of Rps3 against oxidative and UV stress. Overall, our results show that prokaryotic Rps3 recognizes and processes AP-sites on mRNA via a novel mechanism that is distinct from eukaryotes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The global health crisis caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) urges the development of new antiviral agents with broad coronavirus coverage. Due to its key role in viral evasion from the host innate immune response, the coronavirus Nsp15 uridine-specific endoribonuclease (EndoU) is of high interest as a drug target. Considering that the isatin scaffold is well-known for its versatile pharmacological properties, we synthesized and evaluated a series of compounds carrying an isatin core. The initial compounds were selected on the basis of in silico predictions. After biochemical assays showed moderate inhibition of SARS-CoV-2 EndoU-mediated RNA cleavage, structural analogues were rationally designed to enhance the interaction with the target. This included the incorporation of a nitrile group since this dipole can improve ADME and facilitate polar interactions with proteins and can operate as hydroxy or carboxy surrogate. A straightforward solvent free and green, microwave-assisted synthetic process was established to achieve the development of the different target compounds. The best compound exhibited inhibitory activity in enzymatic EndoU assays, and reduced the SARS-CoV-2 viral RNA load by almost 68,000-fold in the low micromolar range similarly to the established antiviral agent GS-441524., Competing Interests: Declaration of competing interest All authors have approved the final submission to the journal. The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Masson SAS.)

Human Schlafen 11 (SLFN11) is sensitizing cells to DNA damaging agents by irreversibly blocking stalled replication forks, making it a potential predictive biomarker in chemotherapy. Furthermore, SLFN11 acts as a pattern recognition receptor for single-stranded DNA (ssDNA) and functions as an antiviral restriction factor, targeting translation in a codon-usage-dependent manner through its endoribonuclease activity. However, the regulation of the various SLFN11 functions and enzymatic activities remains enigmatic. Here, we present cryo-electron microscopy (cryo-EM) structures of SLFN11 bound to tRNA-Leu and tRNA-Met that give insights into tRNA binding and cleavage, as well as its regulation by phosphorylation at S219 and T230. SLFN11 phosphomimetic mutant S753D adopts a monomeric conformation, shows ATP binding, but loses its ability to bind ssDNA and shows reduced ribonuclease activity. Thus, the phosphorylation site S753 serves as a conformational switch, regulating SLFN11 dimerization, as well as ATP and ssDNA binding, while S219 and T230 regulate tRNA recognition and nuclease activity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The endoplasmic reticulum (ER) is central to the processing of luminal, transmembrane, and secretory proteins, and maintaining a functional ER is essential for organismal physiology and health. Increased protein-folding load on the ER causes ER stress, which activates quality control mechanisms to restore ER function and protein homeostasis. Beyond protein quality control, mRNA decay pathways have emerged as potent ER fidelity regulators, but their mechanistic roles in ER quality control and their interrelationships remain incompletely understood. Herein, we review ER-associated RNA decay pathways - including regulated inositol-requiring enzyme 1α (IRE1α)-dependent mRNA decay (RIDD), nonsense-mediated mRNA decay (NMD), and Argonaute-dependent RNA silencing - in ER homeostasis, and highlight the intricate coordination of ER-targeted RNA and protein decay mechanisms and their association with antiviral defense., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ovarian cancer, a leading cause of gynecological cancer deaths globally, poses significant treatment challenges. Cisplatin (CDDP) is the first treatment choice for ovarian cancer and it is initially effective. However, 80% of ovarian cancer patients eventually relapse and develop resistance, resulting in chemotherapy failure. Therefore, finding new treatment combinations to overcome ovarian cancer resistance can provide a new tactic to improve the ovarian cancer patients' survival rate. We first identified activation of the Unfolded Protein Response (UPR) in CDDP-resistant ovarian cancer cells, implicating the IRE1α/XBP1 pathway in promoting resistance. Our findings demonstrate that inhibiting IRE1α signaling can re-sensitizes resistant cells to CDDP in vivo and in vitro, suggesting that IRE1α inhibitor used in conjunction with CDDP presumably could merge as a novel therapeutic strategy. Here, our research highlights the critical role of IRE1α signaling in mediating CDDP resistance, and paves the way for improved treatment options through combinatorial therapy., Competing Interests: Declarations. Animal ethical: All animals mentioned in this article were purchased from the Animal Center of East China Normal University. Animal care and maintenance complied with the guidelines of the Animal Investigation Committee of the Institute of Biomedical Sciences, East China Normal University. Competing interests: The authors declare no competing interests., (© 2024. Springer Nature Switzerland AG.)

Porcine epidemic diarrhea virus (PEDV) small envelope protein (E) plays important roles in virus budding, assembly, and release. Our previous study found that PEDV E protein localizes in the endoplasmic reticulum (ER) to trigger the unfolded protein response (UPR). However, how UPR is directly regulated by PEDV E protein remains elusive. Thus, in this study, we investigated the expression of ER chaperone glucose-regulated protein 78 (GRP78) and activations of the three main UPR signaling pathways to elucidate the underlying mechanisms of UPR triggered by PEDV E protein. The results showed that over-expression of PEDV E protein increased expression of GRP78 and induced stronger phosphorylation of both protein kinase RNA-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α), as well as caused the significant degradation of activating transcription factor 6 (ATF6), in both dose- and time-dependent manners. However, PEDV E protein did not induce UPR through the inositol-requiring enzyme 1 (IRE1) signaling pathway, as revealed by the splicing of XBP1 remaining unaffected and unchanged when PEDV E protein was overexpressed. Taken together, these results demonstrate that PEDV E protein induces UPR through activation of both PERK and ATF6 pathways rather than IRE1 signaling. This study not only provides mechanistic details of UPR induced by the PEDV E protein, but also provides insights into these new biologic functions to help us better understand the interactions between PEDV and host cells., Competing Interests: Declarations Competing interest The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Previous studies showed that hyperhomocysteinemia (HHcy) induced endothelial dysfunction by endoplasmic reticulum (ER) stress induction and autophagy stimulation. This study aimed to determine the effect of hydrogen sulfide (H 2 S) in homocysteine (Hcy)-induced endothelial dysfunction and observe the possible mechanism involved. Male Wistar rats (160-180g) were used and randomly divided into four groups: Control group, HHcy group, HHcy+Sodium hydrosulfide (NaHS) group and NaHS group. Rats were fed with 2% high methionine diet for 8 weeks to set up HHcy model. Plasma concentration of Hcy was measured by ELISA. Endothelium-dependent and non-endothelium-dependent vasodilation of rat renal arteries were determined by myograph. The protein expression of cystathionine-γ-lyase (CSE), ER stress- and autophagy-related proteins in renal arteries or human umbilical vein endothelial cells (HUVECs) were analyzed by western blotting. The endothelial function was impaired in HHcy rats and HUVECs. NaHS supplementation could improve the ACh-induced vasodilation, however it was eliminated by ER stress inducer Tunicamycin (TM) or autophagy inducer Rapamycin. Western blotting in renal arteries showed that Glucose-regulated protein 78 (GRP78) and three branches of ER stress (p-IRE1α, p-PERK, ATF6) , p-JNK1+p-JNK2 were downregulated, simultaneously the autophagy marker Beclin1, LC3BII/LC3BI ratio were decreased and p62 was increased with NaHS treatment in HHcy rats. In HUVECs, IRE1α-JNK induced autophagy was involved in HHcy-induced endothelial dysfunction, while NaHS stimulation reversed the protein expression in IRE1α/JNK-autophagy pathway with Hcy incubation. This study might suggest that endothelial dysfunction induced by HHcy might be correlated with IRE1α-JNK-autophagy axis pathway, which was suppressed by exogenous supplementation of H 2 S donor, NaHS., (Copyright © 2024. Published by Elsevier Inc.)

Tauroursodeoxycholic acid (TUDCA) is a naturally occurring hydrophilic bile acid that alleviates endoplasmic reticulum (ER) stress and inhibits apoptosis, thereby protecting cells. Previous studies have shown that enterovirus 71 (EV71) infection modulates ER stress and induces autophagy to assist viral replication. This study observed the effects of TUDCA pretreatment on HeLa and Vero cells infected with EV71, finding that TUDCA inhibits EV71 replication in TUDCA pretreated HeLa and Vero cells in a dose-dependent manner. We found that TUDCA pretreatment inhibited EV71 replication by regulating three branches of UPR, that is up-regulated ATF6, down-regulated both PERK and IRE1. The results also indicated that autophagy which is downstream of UPR, was inhibited either. The results indicate that TUDCA inhibits EV71 replication by regulating UPR sensor proteins and autophagy following ER stress., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

RNA stability, important for eukaryotic gene expression, is thought to depend on deadenylation rates, with shortened poly(A) tails triggering decapping and 5' to 3' degradation. In contrast to this view, recent large-scale studies indicate that the most unstable mRNAs have, on average, long poly(A) tails. To clarify the role of deadenylation in mRNA decay, we first modeled mRNA poly(A) tail kinetics and mRNA stability in yeast. Independent of deadenylation rates, differences in mRNA decapping rates alone were sufficient to explain current large-scale results. To test the hypothesis that deadenylation and decapping are uncoupled, we used rapid depletion of decapping and deadenylation enzymes and measured changes in mRNA levels, poly(A) length and stability, both transcriptome-wide and with individual reporters. These experiments revealed that perturbations in poly(A) tail length did not correlate with variations in mRNA stability. Thus, while deadenylation may be critical for specific regulatory mechanisms, our results suggest that for most yeast mRNAs, it is not critical for mRNA decapping and degradation., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Maturation of human mitochondrial tRNA is essential for cellular energy production, yet the underlying mechanisms remain only partially understood. Here, we present several cryo-EM structures of the mitochondrial RNase Z complex (ELAC2/SDR5C1/TRMT10C) bound to different maturation states of mitochondrial tRNA His , showing the molecular basis for tRNA-substrate selection and catalysis. Our structural insights provide a molecular rationale for the 5'-to-3' tRNA processing order in mitochondria, the 3'-CCA antideterminant effect, and the basis for sequence-independent recognition of mitochondrial tRNA substrates. Furthermore, our study links mutations in ELAC2 to clinically relevant mitochondrial diseases, offering a deeper understanding of the molecular defects contributing to these conditions., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Chlorpyrifos (CPF) is one of the organophosphorus pesticides widely used throughout the world. Epidemiological studies suggested a link between CPF exposure and neurologic disorders, while the molecular mechanisms remain inconclusive. In the present study, we investigated the impacts of chlorpyrifos-oxon (CPO), the major toxic CPF metabolite, on cell apoptosis, and explored possible mechanism associated with endoplasmic reticulum (ER) stress in SH-SY5Y cells. Results showed that CPO exposure induced dose-dependent apoptosis and expression of ER stress-related proteins in SH-SY5Y cells. Pretreatment with 4-PBA (an ER stress inhibitor) effectively inhibited the expression of GRP78, GRP94, p-IRE1α, and XBP1-s, and apoptotic events. Pretreatment with STF-083010 (an IRE1α inhibitor) partially attenuated CPO-induced apoptosis. In addition, CPO exposure significantly evoked the generation of reactive oxygen species (ROS) which could be eliminated by pretreatment of 4-PBA. Of note, buffering the ROS generation with antioxidant NAC had little impact on the expression of p-IRE1α, and only partially attenuated CPO-induced apoptosis. In contrast, co-pretreatment with NAC and STF-083010 effectively inhibited CPO-induced apoptotic events. Collectively, our results indicate that CPO exposure exerts neuronal cytotoxicity via ER stress downstream-regulated IRE1α/XBP1 signaling pathway and ROS generation-triggered apoptosis. These findings highlight the role of ER stress in CPF-induced neurotoxicity, and provide a promising target for the intervention of organophosphate-associated neurodegenerative diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

MHO7 (6-epi-ophiobolin G), a novel component extracted from a mangrove fungus, exhibits significant anticancer effects against breast cancer. However, the precise mechanism underlying the anticancer effects of MHO7 in prostate cancer (PCa) is yet to be fully elucidated. Therefore, this study was undertaken to assess the effect of MHO7 on PCa cells and elucidate its underlying mechanism. A series of in vitro experiments were conducted, including Cell Counting Kit-8, and plate clone formation assays, flow cytometry analysis, electron microscopy, immunofluorescence staining, western blotting, and molecular dynamics simulation. Additionally, in vivo tumor xenograft models were employed. Our findings revealed that MHO7 could induce cellular autophagy at low concentration (2 μM) and apoptosis at relatively high concentration (4 and 8 μM), leading to significant PCa cell growth inhibition. Furthermore, MHO7 triggered endoplasmic reticulum (ER) stress, which subsequently stimulated autophagy and apoptosis via IRE1α/XBP-1s signaling pathway activation. Notably, IRE1α knockdown markedly reduced MHO7-induced autophagy and apoptosis. Moreover, MHO7 targeted the IRE1α protein, thereby enhancing its stability. MHO7 also exhibited substantial anticancer activity in tumor xenograft models. Our study revealed that MHO7 holds considerable potential as an anticancer agent against PCa, attributable to its activation of ER stress-induced autophagy and apoptosis at different concentrations, facilitated by the upregulation of IRE1α expression., (© 2024 John Wiley & Sons Ltd.)

Precisely sensing and guiding cell-state transitions via the conditional genetic activation of appropriate differentiation factors is challenging. Here we show that desired cell-state transitions can be guided via genetically encoded sensors, whereby endogenous cell-state-specific miRNAs regulate the translation of a constitutively transcribed endoribonuclease, which, in turn, controls the translation of a gene of interest. We used this approach to monitor several cell-state transitions, to enrich specific cell types and to automatically guide the multistep differentiation of human induced pluripotent stem cells towards a haematopoietic lineage via endothelial cells as an intermediate state. Such conditional activation of gene expression is durable and resistant to epigenetic silencing and could facilitate the monitoring of cell-state transitions in physiological and pathological conditions and eventually the 'rewiring' of cell-state transitions for applications in organoid-based disease modelling, cellular therapies and regenerative medicine., Competing Interests: Competing interests: The Massachusetts Institute of Technology has filed a patent application on behalf of the inventors (R.W., D. Mishra, E. Pery, W.X. and L.W.) of the endoRNase-based miRNA sensor design described (US provisional application no. 63/164,282). The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

RNA-sensing TLRs are strategically positioned in the endolysosome to detect incoming nonself RNA. RNase T2 plays a critical role in processing long, structured RNA into short oligoribonucleotides that engage TLR7 or TLR8. In addition to its positive regulatory role, RNase T2 also restricts RNA recognition through unknown mechanisms, as patients deficient in RNase T2 suffer from neuroinflammation. Consistent with this, mice lacking RNase T2 exhibit interferon-dependent neuroinflammation, impaired hematopoiesis, and splenomegaly. However, the mechanism by which RNase T2 deficiency unleashes inflammation in vivo remains unknown. Here, we report that the inflammatory phenotype found in Rnaset2-/- mice is completely reversed in the absence of TLR13, suggesting aberrant accumulation of an RNA ligand for this receptor. Interestingly, this TLR13-driven inflammatory phenotype is also fully present in germ-free mice, suggesting a role for RNase T2 in limiting erroneous TLR13 activation by an as yet unidentified endogenous ligand. These results establish TLR13 as a potential self-sensor that is kept in check by RNase T2., (© 2025 Gomez-Diaz et al.)

Coronavirus endoribonuclease Nsp15 contributes to the evasion of host innate immunity by suppressing levels of viral dsRNA. Nsp15 cleaves both ssRNA and dsRNA in vitro with a strong preference for unpaired or bulged U residues, and its activity is stimulated by divalent ions. Here, we systematically quantified effects of RNA sequence and structure context that define its specificity. The results show that sequence preference for U↓A/G, observed previously, contributes only ca. 2-fold to kcat/Km. In contrast, dsRNA structure flanking a bulged U residue increases kcat/Km by an order of magnitude compared to ssRNA while base pairing in dsRNA essentially blocks cleavage. Despite enormous differences in multiple turnover kinetics, the effect of RNA structure on the cleavage step is minimal. Surprisingly, although divalent ion activation of Nsp15 is widely considered to be important for its biological function, the effect on kcat/Km is only ∼2-fold and independent of RNA structure. These results reveal a specificity landscape dominated by RNA structure and provide a quantitative framework for identifying interactions that underlie specificity, determining mechanisms of inhibition and resistance and defining targets important for coronavirus biology., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recent years have led to the identification of a number of enzymes responsible for RNA decapping. This has provided a basis for further research to identify their role, dependency and substrate specificity. However, the multiplicity of these enzymes and the complexity of their functions require advanced tools to study them. Here, we report a high-throughput fluorescence intensity assay based on RNA aptamers designed as substrates for decapping enzymes. Using a library of differently capped RNA probes we generated a decapping susceptibility heat map, which confirms previously reported substrate specificities of seven tested hydrolases and uncovers novel. We have also demonstrated the utility of our assay for evaluating inhibitors of viral decapping enzymes and performed kinetic studies of the decapping process. The assay may accelerate the characterization of new decapping enzymes, enable high-throughput screening of inhibitors and facilitate the development of molecular tools for a better understanding of RNA degradation pathways., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Acute lung injury or acute respiratory distress syndrome (ALI/ARDS) is a devastating clinical syndrome with high incidence and mortality rates. IRE1α-XBP1 pathway is one of the three major signaling axes of endoplasmic reticulum stress that is involved in inflammation, metabolism, and immunity. The role and potential mechanisms of IRE1α-XBP1 axis in ALI/ARDS has not well understood., Methods: The ALI murine model was established by intratracheal administration of lipopolysaccharide (LPS). Hematoxylin and eosin (H&E) staining and analysis of bronchoalveolar lavage fluid (BALF) were used to evaluate degree of lung injury. Inflammatory responses were assessed by ELISA and RT-PCR. Apoptosis was evaluated using TUNEL staining and western blot. Moreover, western blot, immunohistochemistry, and immunofluorescence were applied to test expression of IRE1α, XBP1, NLRP3, TXNIP, IL-1β, ERK1/2 and NF-κB p65., Results: The expression of IRE1α significantly increased after 24 h of LPS treatment. Inhibition of the IRE1α-XBP1 axis with 4µ8C notably improved LPS-induced lung injury and inflammatory infiltration, reduced the levels of IL-6, IL-1β, and TNF-α, and decreased cell apoptosis as well as the activation of the NLRP3 inflammasome. Besides, in LPS-stimulated Beas-2B cells, both 4µ8C and knockdown of XBP1 diminished the mRNA levels of IL-6 and IL-1B, inhibited cell apoptosis and reduced the protein levels of TXNIP, NLRP3 and secreted IL-1β. Mechanically, the phosphorylation and nuclear translocation of ERK1/2 and p65 were significantly suppressed by 4µ8C and XBP1 knockdown., Conclusions: In summary, our findings suggest that IRE1α-XBP1 axis is crucial in the pathogenesis of ALI/ARDS, whose suppression could mitigate the pulmonary inflammatory response and cell apoptosis in ALI through the TXNIP/NLRP3 inflammasome and ERK/p65 signaling pathway. Our study may provide new evidence that IRE1α-XBP1 may be a promising therapeutic target for ALI/ARDS., Competing Interests: Declarations. Ethics approval and consent to participate: This study followed the national guidelines and protocols of the National Institutes of Health and was approved by the Animal Care Committee of Fudan University Zhongshan Hospital (ID:2022-09). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Ribosome biogenesis is a complex process requiring multiple precursor ribosomal RNA (rRNA) cleavage steps. In archaea, the full set of ribonucleases (RNases) involved in rRNA processing remains to be discovered. A previous study suggested that FAU-1, a conserved protein containing an RNase G/E-like protein domain fused to a domain of unknown function (DUF402), acts as an RNase in archaea. However, the molecular basis of this activity remained so far elusive. Here, we report two X-ray crystallographic structures of RNase G/E-like-DUF402 hybrid proteins from Pyrococcus furiosus and Sulfolobus acidocaldarius, at 2.1 and 2.0 Å, respectively. The structures highlight a structural homology with the 5' RNA recognition domain of Escherichia coli RNase E but no homology with other known catalytic nuclease domains. Surprisingly, we demonstrate that the C-terminal domain of this hybrid protein, annotated as a putative diphosphatase domain, harbors the RNase activity. Our functional analysis also supports a model by which the RNase G/E-like domain acts as a regulatory subunit of the RNase activity. Finally, in vivo experiments in Haloferax volcanii suggest that this RNase participates in the maturation of pre-16S rRNA. Together, our study defines a new RNase family, which we termed the RNase W family, as the first archaea-specific contributor to archaeal ribosome biogenesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nucleotide-binding Oligomerization Domain 1 (NOD1) is a cytosolic pattern recognition receptor that senses specific bacterial peptidoglycan moieties, leading to the induction of inflammatory response. Besides, sensing peptidoglycan, NOD1 has been reported to sense metabolic disturbances including the ER stress-induced unfolded protein response (UPR). However, the underpinning crosstalk between the NOD1 activating microbial ligands and the metabolic cues to alter metabolic response is not yet comprehensively defined. Here, we show that underlying ER stress aggravated peptidoglycan-induced NOD1-mediated inflammatory response in hepatoma cells. The HepG2 cells, undergoing ER stress induced by thapsigargin exhibited an amplified inflammatory response induced by peptidoglycan ligand of NOD1 (i.e. iE-DAP). This aggravated inflammatory response disrupted lipid and glucose metabolism, characterized by de novo lipogenic response, and increased gluconeogenesis in HepG2 cells. Further, we characterized that the aggravation of NOD1-induced inflammatory response was dependent on inositol-requiring enzyme 1-α (IRE1-α) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) activation, in conjunction with calcium flux. Altogether, our findings suggest that differential UPR activation makes liver cells more sensitive towards bacterial-derived ligands to pronounce inflammatory response in a NOD1-dependent manner that impairs hepatic nutrient metabolism., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare that are relevant to the content of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)