Condensation of RNase L promotes its rapid activation in response to viral infection in mammalian cells.

Oligoadenylate synthetase 3 (OAS3) and ribonuclease L (RNase L) are components of a pathway that combats viral infection in mammals. Upon detection of viral double-stranded RNA (dsRNA), OAS3 synthesizes 2'-5'-oligo(A), which activates the RNase domain of RNase L by promoting the homodimerization and oligomerization of RNase L monomers. Activated RNase L rapidly degrades all cellular mRNAs, shutting off several cellular processes. We sought to understand the molecular mechanisms underlying the rapid activation of RNase L in response to viral infection. Through superresolution microscopy and live-cell imaging, we showed that OAS3 and RNase L concentrated into higher-order cytoplasmic complexes known as dsRNA-induced foci (dRIF) in response to dsRNA or infection with dengue virus, Zika virus, or West Nile virus. The concentration of OAS3 and RNase L at dRIF corresponded with the activation of RNase L-mediated RNA decay. We showed that dimerized/oligomerized RNase L concentrated in a liquid-like shell surrounding a core OAS3-dRIF structure and dynamically exchanged with the cytosol. These data establish that the condensation of dsRNA, OAS3, and RNase L into dRIF is a molecular switch that promotes the rapid activation of RNase L upon detection of dsRNA in mammalian cells.

Non-canonical IKKs side with N4BP1 against the family.

The ubiquitin-binding endoribonuclease N4BP1 is a critical immunosuppressor, but the mechanism by which it acts to constrain TLR-induced inflammatory cytokine production has remained unclear. In this issue of Immunity, Gitlin et al. find that N4BP1 works in concert with the non-canonical IκB kinase (IKK) to limit activity of the IKK complex.

N4BP1 coordinates ubiquitin-dependent crosstalk within the IκB kinase family to limit Toll-like receptor signaling and inflammation.

The ubiquitin-binding endoribonuclease N4BP1 potently suppresses cytokine production by Toll-like receptors (TLRs) that signal through the adaptor MyD88 but is inactivated via caspase-8-mediated cleavage downstream of death receptors, TLR3, or TLR4. Here, we examined the mechanism whereby N4BP1 limits inflammatory responses. In macrophages, deletion of N4BP1 prolonged activation of inflammatory gene transcription at late time points after TRIF-independent TLR activation. Optimal suppression of inflammatory cytokines by N4BP1 depended on its ability to bind polyubiquitin chains, as macrophages and mice-bearing inactivating mutations in a ubiquitin-binding motif in N4BP1 displayed increased TLR-induced cytokine production. Deletion of the noncanonical IκB kinases (ncIKKs), Tbk1 and Ikke, or their adaptor Tank phenocopied N4bp1 deficiency and enhanced macrophage responses to TLR1/2, TLR7, or TLR9 stimulation. Mechanistically, N4BP1 acted in concert with the ncIKKs to limit the duration of canonical IκB kinase (IKKα/ß) signaling. Thus, N4BP1 and the ncIKKs serve as an important checkpoint against over-exuberant innate immune responses.

TurboID-Based IRE1 Interactome Reveals Participants of the Endoplasmic Reticulum-Associated Protein Degradation Machinery in the Human Mast Cell Leukemia Cell Line HMC-1.2.

The unfolded protein response is an intricate system of sensor proteins in the endoplasmic reticulum (ER) that recognizes misfolded proteins and transmits information via transcription factors to either regain proteostasis or, depending on the severity, to induce apoptosis. The main transmembrane sensor is IRE1α, which contains cytoplasmic kinase and RNase domains relevant for its activation and the mRNA splicing of the transcription factor XBP1. Mast cell leukemia (MCL) is a severe form of systemic mastocytosis. The inhibition of IRE1α in the MCL cell line HMC-1.2 has anti-proliferative and pro-apoptotic effects, motivating us to elucidate the IRE1α interactors/regulators in HMC-1.2 cells. Therefore, the TurboID proximity labeling technique combined with MS analysis was applied. Gene Ontology and pathway enrichment analyses revealed that the majority of the enriched proteins are involved in vesicle-mediated transport, protein stabilization, and ubiquitin-dependent ER-associated protein degradation pathways. In particular, the AAA ATPase VCP and the oncoprotein MTDH as IRE1α-interacting proteins caught our interest for further analyses. The pharmacological inhibition of VCP activity resulted in the increased stability of IRE1α and MTDH as well as the activation of IRE1α. The interaction of VCP with both IRE1α and MTDH was dependent on ubiquitination. Moreover, MTDH stability was reduced in IRE1α-knockout cells. Hence, pharmacological manipulation of IRE1α-MTDH-VCP complex(es) might enable the treatment of MCL.

RNA degradation in human mitochondria: the journey is not finished.

Mitochondria are vital organelles present in almost all eukaryotic cells. Although most of the mitochondrial proteins are nuclear-encoded, mitochondria contain their own genome, whose proper expression is necessary for mitochondrial function. Transcription of the human mitochondrial genome results in the synthesis of long polycistronic transcripts that are subsequently processed by endonucleases to release individual RNA molecules, including precursors of sense protein-encoding mRNA (mt-mRNA) and a vast amount of antisense noncoding RNAs. Because of mitochondrial DNA (mtDNA) organization, the regulation of individual gene expression at the transcriptional level is limited. Although transcription of most protein-coding mitochondrial genes occurs with the same frequency, steady-state levels of mature transcripts are different. Therefore, post-transcriptional processes are important for regulating mt-mRNA levels. The mitochondrial degradosome is a complex composed of the RNA helicase SUV3 (also known as SUPV3L1) and polynucleotide phosphorylase (PNPase, PNPT1). It is the best-characterized RNA-degrading machinery in human mitochondria, which is primarily responsible for the decay of mitochondrial antisense RNA. The mechanism of mitochondrial sense RNA decay is less understood. This review aims to provide a general picture of mitochondrial genome expression, with a particular focus on mitochondrial RNA (mtRNA) degradation.

Deletion of IRE1α in podocytes exacerbates diabetic nephropathy in mice.

Protein misfolding in the endoplasmic reticulum (ER) of podocytes contributes to the pathogenesis of glomerular diseases. Protein misfolding activates the unfolded protein response (UPR), a compensatory signaling network. We address the role of the UPR and the UPR transducer, inositol-requiring enzyme 1α (IRE1α), in streptozotocin-induced diabetic nephropathy in mice. Diabetes caused progressive albuminuria in control mice that was exacerbated in podocyte-specific IRE1α knockout (KO) mice. Compared to diabetic controls, diabetic IRE1α KO mice showed reductions in podocyte number and synaptopodin. Glomerular ultrastructure was altered only in diabetic IRE1α KO mice; the major changes included widening of podocyte foot processes and glomerular basement membrane. Activation of the UPR and autophagy was evident in diabetic control, but not diabetic IRE1α KO mice. Analysis of human glomerular gene expression in the JuCKD-Glom database demonstrated induction of genes associated with the ER, UPR and autophagy in diabetic nephropathy. Thus, mice with podocyte-specific deletion of IRE1α demonstrate more severe diabetic nephropathy and attenuation of the glomerular UPR and autophagy, implying a protective effect of IRE1α. These results are consistent with data in human diabetic nephropathy and highlight the potential for therapeutically targeting these pathways.

Specific Knockout of Notch-1 in Macrophages Modulate the Progression of Hepatic Insulin Resistance in HFD Fed Mice via Regulating IRE1α-XBP1 Signals.

OBJECTIVE: To develop an intervention based on Notch-1 signalling pathway blockade by investigating the potential application of the neurogenic locus notch homologue protein 1(Notch-1) signalling pathway as a key regulator of chronic inflammation and adipogenesis in the treatment of hepatic insulin resistance (HIR). STUDY DESIGN: Experimental study. Place and Duration of the Study: Animal Laboratory of the Fourth Hospital of Hebei Medical University, Shijiazhuang, China, from April 2021 to June 2022. METHODOLOGY: HIR models were established in Notch-1WT and Notch-1MAC-KO mice by high fat diet (HFD) for 16 weeks. Haematoxylin and eosin (HE) staining and oil red O (ORO) staining were used to detect inflammatory infiltration and lipid accumulation in each group. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of TNF-α and IL-6. Free fatty acid (FFA) and total cholesterol (TC) were measured with relevant kits. Moreover, real-time quantitative polymerase chain reaction (PCR) was performed to detect the relative expressions of F4/80, Mcp1, and CD11b in hepatic tissues. Mass spectrometry was used to analyse the levels of triglyceride (TG), diacylglycerol (DAG) and conformite europeenne (CE) in liver tissue. Western blotting was used to detect the expression of related proteins. RESULTS: Specific knockdown of Notch-1 in macrophages decreases the relative fluorescence intensity of CD68 and attenuates inflammatory infiltration and lipid degeneration. There was no difference in plasma levels of FFA and TG. Specific knockdown of Notch-1 in macrophages decreases the expression of F4/80, Mcp1, and CD11b, as well as the levels of TG, DAG, CE, IL-6, and TNF-α. CONCLUSION: Specific knockout of Notch-1 in macrophages may reduce HIR by inhibiting the IRE1α-XBP1 signalling pathway. KEY WORDS: Hepatic insulin resistance, Macrophages, Notch-1, IRE1α, XBP1.

Bax Inhibitor-1 preserves pancreatic ß-cell proteostasis by limiting proinsulin misfolding and programmed cell death.

The prevalence of diabetes steadily increases worldwide mirroring the prevalence of obesity. Endoplasmic reticulum (ER) stress is activated in diabetes and contributes to ß-cell dysfunction and apoptosis through the activation of a terminal unfolded protein response (UPR). Our results uncover a new role for Bax Inhibitor-One (BI-1), a negative regulator of inositol-requiring enzyme 1 (IRE1α) in preserving ß-cell health against terminal UPR-induced apoptosis and pyroptosis in the context of supraphysiological loads of insulin production. BI-1-deficient mice experience a decline in endocrine pancreatic function in physiological and pathophysiological conditions, namely obesity induced by high-fat diet (HFD). We observed early-onset diabetes characterized by hyperglycemia, reduced serum insulin levels, ß-cell loss, increased pancreatic lipases and pro-inflammatory cytokines, and the progression of metabolic dysfunction. Pancreatic section analysis revealed that BI-1 deletion overburdens unfolded proinsulin in the ER of ß-cells, confirmed by ultrastructural signs of ER stress with overwhelmed IRE1α endoribonuclease (RNase) activity in freshly isolated islets. ER stress led to ß-cell dysfunction and islet loss, due to an increase in immature proinsulin granules and defects in insulin crystallization with the presence of Rod-like granules. These results correlated with the induction of autophagy, ER phagy, and crinophagy quality control mechanisms, likely to alleviate the atypical accumulation of misfolded proinsulin in the ER. In fine, BI-1 in ß-cells limited IRE1α RNase activity from triggering programmed ß-cell death through apoptosis and pyroptosis (caspase-1, IL-1ß) via NLRP3 inflammasome activation and metabolic dysfunction. Pharmaceutical IRE1α inhibition with STF-083010 reversed ß-cell failure and normalized the metabolic phenotype. These results uncover a new protective role for BI-1 in pancreatic ß-cell physiology as a stress integrator to modulate the UPR triggered by accumulating unfolded proinsulin in the ER, as well as autophagy and programmed cell death, with consequences on ß-cell function and insulin secretion. In pancreatic ß-cells, BI-1-/- deficiency perturbs proteostasis with proinsulin misfolding, ER stress, terminal UPR with overwhelmed IRE1α/XBP1s/CHOP activation, inflammation, ß-cell programmed cell death, and diabetes.

IRE1α determines ferroptosis sensitivity through regulation of glutathione synthesis.

Cellular sensitivity to ferroptosis is primarily regulated by mechanisms mediating lipid hydroperoxide detoxification. We show that inositol-requiring enzyme 1 (IRE1α), an endoplasmic reticulum (ER) resident protein critical for the unfolded protein response (UPR), also determines cellular sensitivity to ferroptosis. Cancer and normal cells depleted of IRE1α gain resistance to ferroptosis, while enhanced IRE1α expression promotes sensitivity to ferroptosis. Mechanistically, IRE1α's endoribonuclease activity cleaves and down-regulates the mRNA of key glutathione biosynthesis regulators glutamate-cysteine ligase catalytic subunit (GCLC) and solute carrier family 7 member 11 (SLC7A11). This activity of IRE1α is independent of its role in regulating the UPR and is evolutionarily conserved. Genetic deficiency and pharmacological inhibition of IRE1α have similar effects in inhibiting ferroptosis and reducing renal ischemia-reperfusion injury in mice. Our findings reveal a previously unidentified role of IRE1α to regulate ferroptosis and suggests inhibition of IRE1α as a promising therapeutic strategy to mitigate ferroptosis-associated pathological conditions.

Hepatic IRE1α-XBP1 signaling promotes GDF15-mediated anorexia and body weight loss in chemotherapy.

Platinum-based chemotherapy drugs can lead to the development of anorexia, a detrimental effect on the overall health of cancer patients. However, managing chemotherapy-induced anorexia and subsequent weight loss remains challenging due to limited effective therapeutic strategies. Growth differentiation factor 15 (GDF15) has recently gained significant attention in the context of chemotherapy-induced anorexia. Here, we report that hepatic GDF15 plays a crucial role in regulating body weight in response to chemo drugs cisplatin and doxorubicin. Cisplatin and doxorubicin treatments induce hepatic Gdf15 expression and elevate circulating GDF15 levels, leading to hunger suppression and subsequent weight loss. Mechanistically, selective activation by chemotherapy of hepatic IRE1α-XBP1 pathway of the unfolded protein response (UPR) upregulates Gdf15 expression. Genetic and pharmacological inactivation of IRE1α is sufficient to ameliorate chemotherapy-induced anorexia and body weight loss. These results identify hepatic IRE1α as a molecular driver of GDF15-mediated anorexia and suggest that blocking IRE1α RNase activity offers a therapeutic strategy to alleviate the adverse anorexia effects in chemotherapy.

Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins.

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ in situ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.

Effect of Calcium Supplementation and TMEM16A Inhibition on Endoplasmic Reticulum Stress Induced by Dental Fluorosis in Mice.

BACKGROUND: Dental fluorosis is a discoloration of the teeth caused by the excessive consumption of fluoride. It represents a distinct manifestation of chronic fluorosis in dental tissues, exerting adverse effects on the human body, particularly on teeth. The transmembrane protein 16a (TMEM16A) is expressed at the junction of the endoplasmic reticulum and the plasma membrane. Alterations in its channel activity can disrupt endoplasmic reticulum calcium homeostasis and intracellular calcium ion concentration, thereby inducing endoplasmic reticulum stress (ERS). This study aims to investigate the influence of calcium supplements and TMEM16A on ERS in dental fluorosis. METHODS: C57BL/6 mice exhibiting dental fluorosis were subjected to an eight-week treatment with varying calcium concentrations: low (0.071%), medium (0.79%), and high (6.61%). Various assays, including Hematoxylin and Eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction (qPCR), and Western blot, were employed to assess the impact of calcium supplements on fluoride content, ameloblast morphology, TMEM16A expression, and endoplasmic reticulum stress-related proteins (calreticulin (CRT), glucose-regulated protein 78 (GRP78), inositol requiring kinase 1α (IRE1α), PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)) in the incisors of mice affected by dental fluorosis. Furthermore, mice with dental fluorosis were treated with the TMEM16A inhibitor T16Ainh-A01 along with a medium-dose calcium to investigate the influence of TMEM16A on fluoride content, ameloblast morphology, and endoplasmic reticulum stress-related proteins in the context of mouse incisor fluorosis. RESULTS: In comparison to the model mice, the fluoride content in incisors significantly decreased following calcium supplements (p < 0.01). Moreover, the expression of TMEM16A, CRT, GRP78, IRE1α, PERK, and ATF6 were also exhibited a substantial reduction (p < 0.01), with the most pronounced effect observed in the medium-dose calcium group. Additionally, the fluoride content (p < 0.05) and the expression of CRT, GRP78, IRE1α, PERK, and ATF6 (p < 0.01) were further diminished following concurrent treatment with the TMEM16A inhibitor T16Ainh-A01 and a medium dose of calcium. CONCLUSIONS: The supplementation of calcium or the inhibition of TMEM16A expression appears to mitigate the detrimental effects of fluorosis by suppressing endoplasmic reticulum stress. These findings hold implications for identifying potential therapeutic targets in addressing dental fluorosis.

SARS-CoV-2 nsp15 preferentially degrades AU-rich dsRNA via its dsRNA nickase activity.

It has been proposed that coronavirus nsp15 mediates evasion of host cell double-stranded (ds) RNA sensors via its uracil-specific endoribonuclease activity. However, how nsp15 processes viral dsRNA, commonly considered as a genome replication intermediate, remains elusive. Previous research has mainly focused on short single-stranded RNA as substrates, and whether nsp15 prefers single-stranded or double-stranded RNA for cleavage is controversial. In the present work, we prepared numerous RNA substrates, including both long substrates mimicking the viral genome and short defined RNA, to clarify the substrate preference and cleavage pattern of SARS-CoV-2 nsp15. We demonstrated that SARS-CoV-2 nsp15 preferentially cleaved pyrimidine nucleotides located in less thermodynamically stable areas in dsRNA, such as AU-rich areas and mismatch-containing areas, in a nicking manner. Because coronavirus genomes generally have a high AU content, our work supported the mechanism that coronaviruses evade the antiviral response mediated by host cell dsRNA sensors by using nsp15 dsRNA nickase to directly cleave dsRNA intermediates formed during genome replication and transcription.

mRNA Decapping Activator Pat1 Is Required for Efficient Yeast Adaptive Transcriptional Responses via the Cell Wall Integrity MAPK Pathway.

Cellular mRNA levels, particularly under stress conditions, can be finely regulated by the coordinated action of transcription and degradation processes. Elements of the 5'-3' mRNA degradation pathway, functionally associated with the exonuclease Xrn1, can bind to nuclear chromatin and modulate gene transcription. Within this group are the so-called decapping activators, including Pat1, Dhh1, and Lsm1. In this work, we have investigated the role of Pat1 in the yeast adaptive transcriptional response to cell wall stress. Thus, we demonstrated that in the absence of Pat1, the transcriptional induction of genes regulated by the Cell Wall Integrity MAPK pathway was significantly affected, with no effect on the stability of these transcripts. Furthermore, under cell wall stress conditions, Pat1 is recruited to Cell Wall Integrity-responsive genes in parallel with the RNA Pol II complex, participating both in pre-initiation complex assembly and transcriptional elongation. Indeed, strains lacking Pat1 showed lower recruitment of the transcription factor Rlm1, less histone H3 displacement at Cell Wall Integrity gene promoters, and impaired recruitment and progression of RNA Pol II. Moreover, Pat1 and the MAPK Slt2 occupied the coding regions interdependently. Our results support the idea that Pat1 and presumably other decay factors behave as transcriptional regulators of Cell Wall Integrity-responsive genes under cell wall stress conditions.

Hfq-Antisense RNA I Binding Regulates RNase E-Dependent RNA Stability and ColE1 Plasmid Copy Number.

The mechanisms and consequences of gene regulation by Hfq on trans-encoded small RNAs (sRNAs) have been well studied and documented. Recent employment of Genomic SELEX to search for Hfq-binding motifs has indicated that Hfq might frequently regulate gene expression controlled by cis-antisense RNAs. Here, we use the classic ColE1 plasmid antisense RNA-based regulation model (i.e., RNA I) to study the role of Hfq in controlling antisense regulatory functions. We show that Hfq exhibits a high binding affinity for RNA I and that binding limits RNase E cleavage, thereby stabilizing RNA I and reducing the plasmid copy number. Full-length RNA I displays a binding affinity for Hfq in the sub-micromolar range. In vivo overexpression of Hfq prolongs RNA I stability and reduces the ColE1 plasmid copy number, whereas deletion of hfq reduces RNA I stability and increases the plasmid copy number. RNA I predominantly binds to the proximal face of Hfq and exhibits competitive ability against a chromosome-borne proximal face-bound sRNA (DsrA) for Hfq binding. Through its strong promoter and high gene dosage features, plasmid-encoded antisense RNA I results in high RNA I expression, so it may antagonize the effects of trans-encoded RNAs in controlling target gene expression.

MazF-rolling circle amplification combined MALDI-TOF MS for site-specific detection of N6-methyladenosine RNA.

N6-methyladenosine (m6A) is one of the most abundant chemical modifications in RNA and has vital significance in cellular processes and tumor development. However, the accurate analysis of site-specific m6A modification remains a challenge. In this work, a MazF endoribonuclease activated rolling circle amplification (MazF-RCA) combined MALDI-TOF MS assay is developed for the detection of site-specific m6A-RNA. MazF endoribonuclease can specifically cleave the ACA motif, leaving methylated (m6A)CA motif intact. The intact methylated RNA can then be amplified through rolling circle amplification, and the generated reporter oligonucleotides are detected by MALDI-TOF MS. The assay exhibits good quantification ability, presenting a wide linear range (100 fM to 10 nM) with the limit-of-detection lower than 100 fM. Additionally, the assay can accurately detect methylated RNA in the presence of large amount of non-methylated RNA with a relative abundance of methylated RNA down to 0.5%. The developed assay was further applied to detect m6A-RNA spiked in MCF-7 cell RNA extracts, with the recovery rates in the range of 90.64-106.93%. The present assay provides a novel platform for the analysis of site-specific m6A-RNA at high specificity and sensitivity, which can promote the study of RNA methylation in clinical and biomedical research.

IRE1α regulates macrophage polarization in type 2 diabetic periodontitis through promoting endoplasmic reticulum stress.

OBJECTIVES: The aim of this study was to investigate the effect of 4µ8c, an inhibitor targeting the endoplasmic reticulum stress-associated factor IRE1α, on macrophage polarization in an experimental model of diabetic periodontitis through ex vivo experiments. MATERIALS AND METHODS: Local alveolar bone parameters were evaluated using Micro-CT following intraperitoneal administration of 4µ8c in mice with experimental diabetic periodontitis. Surface markers indicating macrophage polarization were identified using immunofluorescence. In vitro experiments were performed employing bone marrow-derived macrophages and gingival fibroblasts. Macrophage polarization was determined using flow cytometry. Principal impacted signaling pathways were identified through Western blot analysis. RESULTS: Results from both in vitro and in vivo experiments demonstrated that 4µ8c mitigated alveolar bone resorption and inflammation in mice with diabetic periodontitis. Furthermore, it modulated macrophage polarization towards the M2 phenotype and augmented M2 macrophage polarization through the MAPK signaling pathway. CONCLUSIONS: These findings suggest that inhibiting IRE1α can modulate macrophage polarization and alleviate ligature-induced diabetic periodontitis via the MAPK signaling pathway. This unveils a novel mechanism, offering a scientific foundation for the treatment of experimental diabetic periodontitis.

The IRE1α-XBP1 arm of the unfolded protein response is a host factor activated in SARS-CoV-2 infection.

SARS-CoV-2 infection can cause severe pneumonia, wherein exacerbated inflammation plays a major role. This is reminiscent of the process commonly termed cytokine storm, a condition dependent on a disproportionated production of cytokines. This state involves the activation of the innate immune response by viral patterns and coincides with the biosynthesis of the biomass required for viral replication, which may overwhelm the capacity of the endoplasmic reticulum and drive the unfolded protein response (UPR). The UPR is a signal transduction pathway composed of three branches that is initiated by a set of sensors: inositol-requiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6). These sensors control adaptive processes, including the transcriptional regulation of proinflammatory cytokines. Based on this background, the role of the UPR in SARS-CoV-2 replication and the ensuing inflammatory response was investigated using in vivo and in vitro models of infection. Mice and Syrian hamsters infected with SARS-CoV-2 showed a sole activation of the Ire1α-Xbp1 arm of the UPR associated with a robust production of proinflammatory cytokines. Human lung epithelial cells showed the dependence of viral replication on the expression of UPR-target proteins branching on the IRE1α-XBP1 arm and to a lower extent on the PERK route. Likewise, activation of the IRE1α-XBP1 branch by Spike (S) proteins from different variants of concern was a uniform finding. These results show that the IRE1α-XBP1 system enhances viral replication and cytokine expression and may represent a potential therapeutic target in SARS-CoV-2 severe pneumonia.

The inositol-requiring enzyme 1 (IRE1) endoplasmic reticulum stress pathway promotes MDA-MB-231 cell survival and renewal in response to the aryl-ureido fatty acid CTU.

Current treatment options for triple-negative breast cancer (TNBC) are limited to toxic drug combinations of low efficacy. We recently identified an aryl-substituted fatty acid analogue, termed CTU, that effectively killed TNBC cells in vitro and in mouse xenograft models in vivo without producing toxicity. However, there was a residual cell population that survived treatment. The present study evaluated the mechanisms that underlie survival and renewal in CTU-treated MDA-MB-231 TNBC cells. RNA-seq profiling identified several pro-inflammatory signaling pathways that were activated in treated cells. Increased expression of cyclooxygenase-2 and the cytokines IL-6, IL-8 and GM-CSF was confirmed by real-time RT-PCR, ELISA and Western blot analysis. Increased self-renewal was confirmed using the non-adherent, in vitro colony-forming mammosphere assay. Neutralizing antibodies to IL-6, IL-8 and GM-CSF, as well as cyclooxygenase-2 inhibition suppressed the self-renewal of MDA-MB-231 cells post-CTU treatment. IPA network analysis identified major NF-κB and XBP1 gene networks that were activated by CTU; chemical inhibitors of these pathways and esiRNA knock-down decreased the production of pro-inflammatory mediators. NF-κB and XBP1 signaling was in turn activated by the endoplasmic reticulum (ER)-stress sensor inositol-requiring enzyme 1 (IRE1), which mediates the unfolded protein response. Co-treatment with an inhibitor of IRE1 kinase and RNase activities, decreased phospho-NF-κB and XBP1s expression and the production of pro-inflammatory mediators. Further, IRE1 inhibition also enhanced apoptotic cell death and prevented the activation of self-renewal by CTU. Taken together, the present findings indicate that the IRE1 ER-stress pathway is activated by the anti-cancer lipid analogue CTU, which then activates secondary self-renewal in TNBC cells.