RESUMO
Inositol-requiring enzyme 1 (IRE1) is an evolutionarily conserved sensor of endoplasmic reticulum (ER) stress and mediates a key branch of the unfolded protein response in eukaryotic cells. It is an ER-resident transmembrane protein that possesses Ser/Thr protein kinase and endoribonuclease (RNase) activities in its cytoplasmic region. IRE1 is activated through dimerization/oligomerization and autophosphorylation at multiple sites, acting through its RNase activity to restore the functional capacity of the ER. However, it remains poorly defined in vivo how the autophosphorylation events of endogenous IRE1 govern its dynamic activation and functional output. Here, we generated a mouse model harboring a S724A knock-in mutation (Ern1S724A/S724A) and investigated the importance of phosphorylation at Ser724 within the kinase activation loop of murine IRE1α. We found that in mouse embryonic fibroblast cells and in primary hepatocytes, S724A mutation resulted in markedly reduced IRE1α autophosphorylation in parallel with blunted activation of its RNase activity to catalyze X-box binding protein 1 (Xbp1) mRNA splicing. Furthermore, ablation of IRE1α phosphorylation at Ser724 exacerbated ER stress-induced hepatic steatosis in tunicamycin-treated Ern1S724A/S724A mice. This was accompanied by significantly decreased hepatic production of spliced XBP1 protein but increased CCAAT-enhancer-binding protein homologous protein (CHOP) level, along with suppressed expression of key metabolic regulators of fatty acid ß-oxidation and lipid secretion. These results demonstrate a critical role of phosphorylation at Ser724 of IRE1α in dynamically controlling its kinase activity, and thus its autophosphorylation state, which is coupled to activation of its RNase activity in counteracting hepatic steatosis under ER stress conditions.
Assuntos
Estresse do Retículo Endoplasmático , Endorribonucleases , Proteínas Serina-Treonina Quinases , Animais , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Ativação Enzimática , Fígado Gorduroso/genética , Fibroblastos/metabolismo , Camundongos , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
The unfolded protein response (UPR) in plants is elicited by endoplasmic reticulum stress, which can be brought about by adverse environmental conditions. The response is mediated by a conserved signalling network composed of two branches - one branch involving inositol requiring enzyme1- basic leucine zipper60 (IRE1-bZIP60) signalling pathway and another branch involving the membrane transcription factors, bZIP17 and -28. The UPR has been reported in Chlamydomonas reinhardtii, a unicellular green alga, which lacks some canonical UPR signalling components found in vascular plants, raising the question whether C. reinhardtii uses other means such as oxidative signalling or Regulated IRE1-Dependent Decay to activate the UPR. In vascular plants, IRE1 splices bZIP60 mRNA in response to endoplasmic reticulum stress by cutting at a site in the RNA that is highly conserved in structure and sequence. Monocots have a single IRE1 gene required for viability in rice, while dicots have two IRE1 genes, IRE1a and -b. Brassicas have a third IRE1 gene, IRE1c, which lacks a lumenal domain, but is required in combination with IRE1b for gametogenesis. Vascular and non-vascular plants upregulate a similar set of genes in response to endoplasmic reticulum stress despite differences in the complexity of their UPR signalling networks.
Assuntos
Proteínas de Plantas/metabolismo , Plantas/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Chlamydomonas reinhardtii/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Plantas/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismoRESUMO
Inositol-requiring enzyme 1 (IRE1) is an endoplasmic reticulum (ER)-resident transmembrane protein that senses ER stress and is evolutionarily conserved from yeast to humans. IRE1 possesses both Ser/Thr protein kinase and endoribonuclease (RNase) activities within its cytoplasmic domain and is activated through autophosphorylation and dimerization/oligomerization. It mediates a critical arm of the unfolded protein response to manage ER stress provoked by lumenal overload of unfolded/misfolded proteins. Emerging lines of evidence have revealed that in mammals, IRE1α functions as a multifunctional signal transducer that responds to metabolic cues and nutrient stress conditions, exerting profound and broad effects on metabolic homeostasis. In this review, we cover recent advances in our understanding of how IRE1α integrates a variety of metabolic and stress signals and highlight its tissue-specific or context-dependent metabolic activities. We also discuss how dysregulation of this metabolic stress sensor during handling of excessive nutrients in cells contributes to the progression of obesity and metabolic disorders.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologia , Proteína 1 de Ligação a X-Box/genéticaRESUMO
Mycobacterium avium, a slow-growing nontuberculous mycobacterium, causes fever, diarrhoea, loss of appetite, and weight loss in immunocompromised people. We have proposed that endoplasmic reticulum (ER) stress-mediated apoptosis plays a critical role in removing intracellular mycobacteria. In the present study, we investigated the role of the regulated IRE1-dependent decay (RIDD) pathway in macrophages during M. avium infection based on its role in the regulation of gene expression. The inositol-requiring enzyme 1 (IRE1)/apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) signalling pathway was activated in macrophages after infection with M. avium. The expression of RIDD-associated genes, such as Bloc1s1 and St3gal5, was decreased in M. avium-infected macrophages. Interestingly, M. avium-induced apoptosis was significantly suppressed by pretreatment with irestatin (inhibitor of IRE1α) and 4µ8c (RIDD blocker). Macrophages pretreated with N-acetyl cysteine (NAC) showed decreased levels of reactive oxygen species (ROS), IRE1α, and apoptosis after M. avium infection. The expression of Bloc1s1 and St3gal5 was increased in NAC-pretreated macrophages following infection with M. avium. Growth of M. avium was significantly increased in irestatin-, 4µ8c-, and NAC-treated macrophages compared with the control. The data indicate that the ROS-mediated ER stress response induces apoptosis of M. avium-infected macrophages by activating IRE1α-RIDD. Thus, activation of IRE1α suppresses the intracellular survival of M. avium in macrophages.
Assuntos
Apoptose/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Camundongos , Mycobacterium avium/patogenicidade , Células RAW 264.7 , Tuberculose Aviária/metabolismo , Tuberculose Aviária/microbiologiaRESUMO
Cell viability requires the maintenance of intracellular homeostasis through the unfolded protein response mediated by receptors localized on the endoplasmic reticulum (ER) membrane. The receptor IRE1 mediates not only various adaptive outputs but also programmed cell death (PCD) under varying stress levels. However, little is known about the mechanism by which the same receptors trigger different responses in plants. Arabidopsis Golgi anti-apoptotic protein 1 (GAAP1) and GAAP3 resist PCD upon ER stress and negatively modulate the adaptive response of the IRE1-bZIP60 pathway through IRE1 association. To elucidate the mechanism underlying the anti-PCD activity of GAAPs, we attempted to isolate interactors of GAAPs by yeast two-hybrid screening. Membrane-associated progesterone receptor 3 (MAPR3) was isolated as one of the factors interacting with GAAP. Mutations in GAAP1/GAAP3 and/or MAPR3 enhanced the sensitivity of seedlings to ER stress. Whole-transcriptome analysis combined with quantitative reverse transcription-PCR and cellular analysis showed that regulated IRE1-dependent decay (RIDD) and autophagy were impaired in mutants mapr3, gaap1mapr3, and gaap3mapr3. MAPR3, GAAP1, and GAAP3 interacted with IRE1B as determined by protein interaction assays. These data suggest that the interaction of GAAP1/GAAP3 with MAPR3 mitigates ER stress to some extent through regulating IRE10-mediated RIDD and autophagy.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Estresse do Retículo Endoplasmático , Transdução de Sinais , Proteínas de Arabidopsis/genética , Autofagia/genética , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação/genética , Ligação Proteica , Estabilidade de RNA/genéticaRESUMO
Inositol-requiring enzyme 1 (IRE1) is the most conserved transducer of the unfolded protein response (UPR), a homeostatic response that preserves proteostasis. Intriguingly, via its endoribonuclease activity, IRE1 produces either adaptive or death signals. This occurs through both unconventional splicing of XBP1 mRNA and regulated IRE1-dependent decay of mRNA (RIDD). Whereas XBP1 mRNA splicing is cytoprotective in response to endoplasmic reticulum (ER) stress, RIDD has revealed many unexpected features. For instance, RIDD cleaves RNA at an XBP1-like consensus site but with an activity divergent from XBP1 mRNA splicing and can either preserve ER homeostasis or induce cell death. Here we review recent findings on RIDD and propose a model of how IRE1 RNase activity might control cell fate decisions.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Animais , Endorribonucleases/metabolismo , Humanos , Ribonucleases/metabolismoRESUMO
RNA splicing patterns in antibody-secreting cells are shaped by endoplasmic reticulum stress, ELL2 (eleven-nineteen lysine-rich leukemia gene 2) induction, and changes in the levels of snRNAs. Endoplasmic reticulum stress induces the unfolded protein response comprising a highly conserved set of genes crucial for cell survival; among these is Ire1, whose auto-phosphorylation drives it to acquire a regulated mRNA decay activity. The mRNA-modifying function of phosphorylated Ire1 non-canonically splices Xbp1 mRNA and yet degrades other cellular mRNAs with related motifs. Naïve splenic B cells will activate Ire1 phosphorylation early on after lipopolysaccharide (LPS) stimulation, within 18 h; large-scale changes in mRNA content and splicing patterns result. Inhibition of the mRNA-degradation function of Ire1 is correlated with further differences in the splicing patterns and a reduction in the mRNA factors for snRNA transcription. Some of the >4000 splicing changes seen at 18 h after LPS stimulation persist into the late stages of antibody secretion, up to 72 h. Meanwhile some early splicing changes are supplanted by new splicing changes introduced by the up-regulation of ELL2, a transcription elongation factor. ELL2 is necessary for immunoglobulin secretion and does this by changing mRNA processing patterns of immunoglobulin heavy chain and >5000 other genes.
Assuntos
Processamento Alternativo/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Processamento Alternativo/genética , Animais , Linfócitos B/metabolismo , Estresse do Retículo Endoplasmático/genética , Humanos , Splicing de RNA/genética , RNA Mensageiro/metabolismo , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
Compromised protein folding capacity in the endoplasmic reticulum (ER) leads to a protein traffic jam that produces a toxic environment called ER stress. However, the ER smartly handles such a critical situation by activating a cascade of proteins responsible for sensing and responding to the noxious stimuli of accumulated proteins. The ER protein load is higher in secretory cells, such as liver hepatocytes, which are thus prone to stress-mediated toxicity and various diseases, including alcohol-induced liver injury, fatty liver disease, and viral hepatitis. Therefore, we discuss the molecular cues that connect ER stress to hepatic diseases. Moreover, we review the literature on ER stress-regulated miRNA in the pathogenesis of liver diseases to give a comprehensive overview of mechanistic insights connecting ER stress and miRNA in the context of liver diseases. We also discuss currently discovered regulated IRE1 dependent decay in regulation of hepatic diseases.
Assuntos
Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Endorribonucleases/metabolismo , Hepatopatias/etiologia , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Dobramento de Proteína , Transporte ProteicoRESUMO
Antibody secretion is executed by plasma cells that are generated in the periphery and migrate to the bone marrow to establish a long lived pool. The terminal differentiation of B lymphocytes into plasma cells is executed by a network of transcription factors that cross-regulate each other in order to irreversibly promote this transition. While major progress has been made in the understanding the transcriptional activity of the underlying master regulators, much less is known on the metabolic regulation of plasma cell differentiation that is required to support antibody synthesis, folding and secretion at high levels and allow their long-lasting survival. In this review we will address the known cross talks between the transcription and metabolic control of plasma cells and elaborate on the gaps of knowledge in the field.
Assuntos
Diferenciação Celular , Metabolismo Energético , Plasmócitos/citologia , Plasmócitos/metabolismo , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Plasmócitos/imunologia , Transdução de Sinais , Estresse Fisiológico/genética , Estresse Fisiológico/imunologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Inositol-requiring enzyme 1 (IRE1) is a kinase and ribonuclease that executes the splicing of X box binding protein 1 (XBP-1) mRNA in response to the accumulation of unfolded protein in the ER, a signal cascade termed the unfolded protein response. Recently, IRE1 has been implicated in mRNA and miRNA cleavage and degradation, a pathway termed regulated IRE1-dependent decay (RIDD). Deletion of XBP-1 in the liver and pancreas strongly enhances RIDD by upregulating IRE1 protein levels and enhancing its ribo-nuclease activity. Because XBP-1 is essential for generating plasma cells with developed secretory capacity, we sought to evaluate the contribution of RIDD to this regulation. Mice were conditionally deleted for XBP-1 and/or IRE1 in their B-cell lineage. Similarly to the liver, deletion of XBP-1 induces IRE1 expression in LPS-treated B cells. In vitro, IRE1 cleaves the mRNA of secretory µ chains, which explains the reduction in secretory µ mRNA and its synthesis in XBP-1 KO plasma cells. In accordance, the IgM response is partially restored in XBP-1/IRE1 double KO mice relative to XBP-1 KO mice. Interestingly, the IgG1 response is reduced to a similar level in XBP-1 KO, IRE1 KO, and their double knockout animals. Our data demonstrate a specific contribution by RIDD in curtailing immunoglobulin synthesis and secretion.
Assuntos
Formação de Anticorpos/fisiologia , Imunoglobulinas/biossíntese , Proteínas de Membrana/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Plasmócitos/ultraestrutura , Proteínas Serina-Treonina Quinases/genética , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição de Fator Regulador X , Linfócitos T/imunologia , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
As they commit to plasma cell differentiation, B lymphocytes must swiftly gear up to produce and secrete huge amounts of antibodies. To develop their secretory capacity, B cells exploit a signaling pathway that is employed by all eukaryotic cells in response to endoplasmic reticulum stress. An article by Benhamron et al. in this issue of the European Journal of Immunology, [Eur. J. Immunol. 2014. 44: 867-876] sheds new light on why an intact IRE1/XBP-1 signaling relay is central to orchestrate the full-blown expansion of the secretory machinery needed for massive antibody production.
Assuntos
Formação de Anticorpos/fisiologia , Imunoglobulinas/biossíntese , Proteínas de Membrana/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , AnimaisRESUMO
MicroRNAs (miRNA) are generally described as negative regulators of gene expression. However, some evidence suggests that they may also play positive roles. As such, we reported that miR-1291 leads to a GPC3 mRNA expression increase in hepatoma cells through a 3' untranslated region (UTR)-dependent mechanism. In the absence of any direct interaction between miR-1291 and GPC3 mRNA, we hypothesized that miR-1291 could act by silencing a negative regulator of GPC3 mRNA expression. Based on in silico predictions and experimental validation, we demonstrate herein that miR-1291 represses the expression of the mRNA encoding the endoplasmic reticulum (ER)-resident stress sensor IRE1α by interacting with a specific site located in the 5' UTR. Moreover, we show, in vitro and in cultured cells, that IRE1α cleaves GPC3 mRNA at a 3' UTR consensus site independently of ER stress, thereby prompting GPC3 mRNA degradation. Finally, we show that the expression of a miR-1291-resistant form of IRE1α abrogates the positive effects of miR-1291 on GPC3 mRNA expression. Collectively, our data demonstrate that miR-1291 is a biologically relevant regulator of GPC3 expression in hepatoma cells and acts through silencing of the ER stress sensor IRE1α.
Assuntos
Endorribonucleases/metabolismo , Inativação Gênica , Glipicanas/metabolismo , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Regiões 3' não Traduzidas , Linhagem Celular Tumoral , Biologia Computacional/métodos , Estresse do Retículo Endoplasmático , Endorribonucleases/genética , Glipicanas/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/genética , Clivagem do RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Transfecção , TransgenesRESUMO
The endoplasmic-reticulum (ER) stress response constitutes a cellular process that is triggered by a variety of conditions that disturb folding of proteins in the ER. Eukaryotic cells have developed an evolutionarily conserved adaptive mechanism, the unfolded protein response (UPR), which aims to clear unfolded proteins and restore ER homeostasis. In cases where ER stress cannot be reversed, cellular functions deteriorate, often leading to cell death. Accumulating evidence implicates ER stress-induced cellular dysfunction and cell death as major contributors to many diseases, making modulators of ER stress pathways potentially attractive targets for therapeutics discovery. Here, we summarize recent advances in understanding the diversity of molecular mechanisms that govern ER stress signaling in health and disease. This article is part of a Special Section entitled: Cell Death Pathways.
Assuntos
Estresse do Retículo Endoplasmático , Animais , Morte Celular , Doença , Humanos , Modelos Biológicos , Transdução de Sinais , Resposta a Proteínas não DobradasRESUMO
The unfolded protein response (UPR) is a cellular mechanism that protects cells during stress conditions in which there is an accumulation of misfolded proteins in the endoplasmic reticulum (ER). UPR activates three signaling pathways that function to alleviate stress conditions and promote cellular homeostasis and cell survival. During unmitigated stress conditions, however, UPR activation signaling changes to promote cell death through apoptosis. Interestingly, cancer cells take advantage of this pathway to facilitate survival and avoid apoptosis even during prolonged cell stress conditions. Here, we discuss different signaling pathways associated with UPR and focus specifically on one of the ER signaling pathways activated during UPR, inositol-requiring enzyme 1α (IRE1). The rationale is that the IRE1 pathway is associated with cell fate decisions and recognized as a promising target for cancer therapeutics. Here we discuss IRE1 inhibitors and how they might prove to be an effective cancer therapeutic.
RESUMO
Angelica gigas NAKAI (AG) is a popular traditional medicinal herb widely used to treat dyslipidemia owing to its antioxidant activity. Vascular disease is intimately linked to obesity-induced metabolic syndrome, and AG extract (AGE) shows beneficial effects on obesity-associated vascular dysfunction. However, the effectiveness of AGE against obesity and its underlying mechanisms have not yet been extensively investigated. In this study, 40 high fat diet (HFD) rats were supplemented with 100-300 mg/kg/day of AGE to determine its efficacy in regulating vascular dysfunction. The vascular relaxation responses to acetylcholine were impaired in HFD rats, while the administration of AGE restored the diminished relaxation pattern. Endothelial dysfunction, including increased plaque area, accumulated reactive oxygen species, and decreased nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) Ser1177 phosphorylation, were observed in HFD rats, whereas AGE reversed endothelial dysfunction and its associated biochemical signaling. Furthermore, AGE regulated endoplasmic reticulum (ER) stress and IRE1α sulfonation and its subsequent sirt1 RNA decay through controlling regulated IRE1α-dependent decay (RIDD) signaling, ultimately promoting NO bioavailability via the SIRT1-eNOS axis in aorta and endothelial cells. Independently, AGE enhanced AMPK phosphorylation, additionally stimulating SIRT1 and eNOS deacetylation and its associated NO bioavailability. Decursin, a prominent constituent of AGE, exhibited a similar effect in alleviating endothelial dysfunctions. These data suggest that AGE regulates dyslipidemia-associated vascular dysfunction by controlling ROS-associated ER stress responses, especially IRE1α-RIDD/sirt1 decay and the AMPK-SIRT1 axis.
Assuntos
Dislipidemias , Sirtuína 1 , Ratos , Animais , Sirtuína 1/metabolismo , Endorribonucleases/genética , Endotélio Vascular/metabolismo , Células Endoteliais/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Acetilação , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Obesidade/metabolismo , Óxido Nítrico/metabolismoRESUMO
Neuronal cells are highly functioning but also extremely stress-sensitive cells. By defending the neuronal cells against pathogenic insults, microglial cells, a unique cell type, act as the frontline cavalry in the central nervous system (CNS). Their remarkable and unique ability to self-renew independently after their creation is crucial for maintaining normal brain function and neuroprotection. They have a wide range of molecular sensors that help maintain CNS homeostasis during development and adulthood. Despite being the protector of the CNS, studies have revealed that persistent microglial activation may be the root cause of innumerable neurodegenerative illnesses, including Alzheimer's disease (AD), Parkinson's disease (PD), and Amyotrophic Lateral Sclerosis (ALS). From our vigorous review, we state that there is a possible interlinking between pathways of Endoplasmic reticulum (ER) stress response, inflammation, and oxidative stress resulting in dysregulation of the microglial population, directly influencing the accumulation of pro-inflammatory cytokines, complement factors, free radicals, and nitric oxides leading to cell death via apoptosis. Recent research uses the suppression of these three pathways as a therapeutic approach to prevent neuronal death. Hence, in this review, we have spotlighted the advancement in microglial studies, which focus on their molecular defenses against multiple stresses, and current therapeutic strategies indirectly targeting glial cells for neurodevelopmental diseases.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Doença de Parkinson , Humanos , Microglia/metabolismo , Doenças Neurodegenerativas/metabolismo , Sistema Nervoso Central/metabolismo , Doença de Alzheimer/metabolismo , Doença de Parkinson/metabolismoRESUMO
BACKGROUND: IRE1 is an unfolded protein response (UPR) sensor with kinase and endonuclease activity. It plays a central role in the endoplasmic reticulum (ER) stress response through unconventional splicing of XBP1 mRNA and regulated IRE1-dependent decay (RIDD). Multiple myeloma (MM) cells are known to exhibit an elevated level of baseline ER stress due to immunoglobulin production, however RIDD activity has not been well studied in this disease. In this study, we aimed to investigate the potential of RNA-sequencing in the identification of novel RIDD targets in MM cells and to analyze the role of these targets in MM cells. METHODS: In vitro IRE1-cleavage assay was combined with RNA sequencing. The expression level of RIDD targets in MM cell lines was measured by real-time RT-PCR and Western blot. RESULTS: Bioinformatic analysis revealed hundreds of putative IRE1 substrates in the in vitro assay, 32 of which were chosen for further validation. Looking into the secondary structure of IRE1 substrates, we found that the consensus sequences of IRF4, PRDM1, IKZF1, KLF13, NOTCH1, ATR, DICER, RICTOR, CDK12, FAM168B, and CENPF mRNAs were accompanied by a stem-loop structure essential for IRE1-mediated cleavage. In fact, we show that mRNA and protein levels corresponding to these targets were attenuated in an IRE1-dependent manner by treatment with ER-stress-inducing agents. In addition, a synergistic effect between IMiDs and ER-stress inducers was found. CONCLUSION: This study, using RNA sequencing, shows that IRE1 RNase has a broad range of mRNA substrates in myeloma cells and demonstrates for the first time that IRE1 is a key regulator of several proteins of importance in MM survival and proliferation.
RESUMO
IRE1α is one of the three ER transmembrane transducers of the Unfolded Protein Response (UPR) activated under endoplasmic reticulum (ER) stress. IRE1α activation has a dual role in cancer as it may be either pro- or anti-tumoral depending on the studied models. Here, we describe the discovery that exogenous expression of IRE1α, resulting in IRE1α auto-activation, did not affect cancer cell proliferation in vitro but resulted in a tumor-suppressive phenotype in syngeneic immunocompetent mice. We found that exogenous expression of IRE1α in murine colorectal and Lewis lung carcinoma cells impaired tumor growth when syngeneic tumor cells were subcutaneously implanted in immunocompetent mice but not in immunodeficient mice. Mechanistically, the in vivo tumor-suppressive effect of overexpressing IRE1α in tumor cells was associated with IRE1α RNAse activity driving both XBP1 mRNA splicing and regulated IRE1-dependent decay of RNA (RIDD). We showed that the tumor-suppressive phenotype upon IRE1α overexpression was characterized by the induction of apoptosis in tumor cells along with an enhanced adaptive anti-cancer immunosurveillance. Hence, our work indicates that IRE1α overexpression and/or activation in tumor cells can limit tumor growth in immunocompetent mice. This finding might point toward the need of adjusting the use of IRE1α inhibitors in cancer treatments based on the predominant outcome of the RNAse activity of IRE1α.
Assuntos
Endorribonucleases , Neoplasias , Animais , Endorribonucleases/genética , Endorribonucleases/metabolismo , Imunidade , Camundongos , Processos Neoplásicos , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella, the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1α-dependent decay (RIDD) of Bloc1s1 mRNA encoding BLOS1, a protein that promotes endosome-lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1α were resistant to infection. Inactivation of the Bloc1s1 gene impaired the ability to assemble BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella-containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Bloc1s1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus murine hepatitis virus also subverted the RIDD-BLOS1 axis to promote intracellular replication. Our work establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.
Assuntos
Brucella , Brucelose , Animais , Brucelose/metabolismo , Brucelose/microbiologia , Endorribonucleases/metabolismo , Endossomos/metabolismo , Camundongos , Proteínas Serina-Treonina QuinasesRESUMO
BACKGROUND: Genetically modified dendritic cells (DCs) modulate the alloimmunity of T lymphocytes by regulating antigen presentation. METHODS: We generated mice with specific deletion of the X-box-binding protein 1 (XBP1) allele in bone marrow cells and cultured bone marrow-derived DCs (Xbp1-/- BMDCs) from these animals. We then tested the phenotype of Xbp1-/- BMDCs, evaluated their capability to activate allogeneic T cells and investigated their mechanistic actions. We developed a mouse model of allogeneic heart transplantation in which recipients received PBS, Xbp1-/- BMDCs, a suboptimal dose of cyclosporine A (CsA), or Xbp1-/- BMDCs combined with a suboptimal dose of CsA to evaluate the effects of Xbp1-/- BMDC transfusion on alloimmunity and on the survival of heart allografts. RESULTS: The deletion of XBP1 in BMDCs exploited the IRE1-dependent decay of TAPBP mRNA to reduce the expression of MHC-I on the cell surface, altered the capability of BMDCs to activate CD8+ T cells, and ultimately suppressed CD8+ T-cell-mediated allogeneic rejection. The adoptive transfer of Xbp1-/- BMDCs inhibited CD8+ T-cell-mediated rejection. In addition, XBP1-deficient BMDCs were weak stimulators of allogeneic CD4+ T cells despite expressing high levels of MHC-II and costimulatory molecules on their cell surface. Moreover, the adoptive transfer of Xbp1-/- BMDCs inhibited the production of circulating donor-specific IgG. The combination of Xbp1-/- BMDCs and CsA treatment significantly prolonged the survival of allografts compared to CsA alone. CONCLUSIONS: The deletion of XBP1 induces immunosuppressive BMDCs, and treatment with these immunosuppressive BMDCs prevents alloimmune rejection and improves the outcomes of heart transplantation. This finding provides a promising therapeutic target in combating transplant rejection and expands knowledge of inducing therapeutic DCs.