RESUMO
ABSTRACT: Regulation of lineage biases in hematopoietic stem and progenitor cells (HSPCs) is pivotal for balanced hematopoietic output. However, little is known about the mechanism behind lineage choice in HSPCs. Here, we show that messenger RNA (mRNA) decay factors regnase-1 (Reg1; Zc3h12a) and regnase-3 (Reg3; Zc3h12c) are essential for determining lymphoid fate and restricting myeloid differentiation in HSPCs. Loss of Reg1 and Reg3 resulted in severe impairment of lymphopoiesis and a mild increase in myelopoiesis in the bone marrow. Single-cell RNA sequencing analysis revealed that Reg1 and Reg3 regulate lineage directions in HSPCs via the control of a set of myeloid-related genes. Reg1- and Reg3-mediated control of mRNA encoding Nfkbiz, a transcriptional and epigenetic regulator, was essential for balancing lymphoid/myeloid lineage output in HSPCs in vivo. Furthermore, single-cell assay for transposase-accessible chromatin sequencing analysis revealed that Reg1 and Reg3 control the epigenetic landscape on myeloid-related gene loci in early stage HSPCs via Nfkbiz. Consistently, an antisense oligonucleotide designed to inhibit Reg1- and Reg3-mediated Nfkbiz mRNA degradation primed hematopoietic stem cells toward myeloid lineages by enhancing Nfkbiz expression. Collectively, the collaboration between posttranscriptional control and chromatin remodeling by the Reg1/Reg3-Nfkbiz axis governs HSPC lineage biases, ultimately dictating the fate of lymphoid vs myeloid differentiation.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Linhagem da Célula/genética , Células-Tronco Hematopoéticas/metabolismo , Medula Óssea/metabolismo , Hematopoese/genética , RNA Mensageiro/metabolismo , Diferenciação Celular/genéticaRESUMO
Thiol-reactive reagents designed for the chemical modification of proteins cannot, in general, be used directly for the modification of intracellular targets because the presence of millimolar concentrations of glutathione inside cells effectively outcompetes reaction with target thiols. Here we report an equilibrium, entropic strategy for achieving target selectivity using a cyanoacrylate-based thiol-reactive cross-linker (BCNA) with two reactive sites. This compound exhibits â³200-fold selectivity for reaction with target peptides and proteins containing appropriately spaced pairs of thiols, versus reaction with mono-thiols. Photo-isomerization of the azobenzene moiety of the cross-linker can be used to affect the conformation of the target peptide or protein. This approach suggests a general strategy for the chemical modification of intracellular peptide and protein targets.
Assuntos
Compostos Azo , Proteínas , Reagentes de Ligações Cruzadas/química , Compostos Azo/química , Peptídeos/química , Compostos de Sulfidrila/químicaRESUMO
BACKGROUND: Pulmonary arterial hypertension (PAH) is a type of pulmonary hypertension (PH) characterized by obliterative pulmonary vascular remodeling, resulting in right-sided heart failure. Although the pathogenesis of PAH is not fully understood, inflammatory responses and cytokines have been shown to be associated with PAH, in particular, with connective tissue disease-PAH. In this sense, Regnase-1, an RNase that regulates mRNAs encoding genes related to immune reactions, was investigated in relation to the pathogenesis of PH. METHODS: We first examined the expression levels of ZC3H12A (encoding Regnase-1) in peripheral blood mononuclear cells from patients with PH classified under various types of PH, searching for an association between the ZC3H12A expression and clinical features. We then generated mice lacking Regnase-1 in myeloid cells, including alveolar macrophages, and examined right ventricular systolic pressures and histological changes in the lung. We further performed a comprehensive analysis of the transcriptome of alveolar macrophages and pulmonary arteries to identify genes regulated by Regnase-1 in alveolar macrophages. RESULTS: ZC3H12A expression in peripheral blood mononuclear cells was inversely correlated with the prognosis and severity of disease in patients with PH, in particular, in connective tissue disease-PAH. The critical role of Regnase-1 in controlling PAH was also reinforced by the analysis of mice lacking Regnase-1 in alveolar macrophages. These mice spontaneously developed severe PAH, characterized by the elevated right ventricular systolic pressures and irreversible pulmonary vascular remodeling, which recapitulated the pathology of patients with PAH. Transcriptomic analysis of alveolar macrophages and pulmonary arteries of these PAH mice revealed that Il6, Il1b, and Pdgfa/b are potential targets of Regnase-1 in alveolar macrophages in the regulation of PAH. The inhibition of IL-6 (interleukin-6) by an anti-IL-6 receptor antibody or platelet-derived growth factor by imatinib but not IL-1ß (interleukin-1ß) by anakinra, ameliorated the pathogenesis of PAH. CONCLUSIONS: Regnase-1 maintains lung innate immune homeostasis through the control of IL-6 and platelet-derived growth factor in alveolar macrophages, thereby suppressing the development of PAH in mice. Furthermore, the decreased expression of Regnase-1 in various types of PH implies its involvement in PH pathogenesis and may serve as a disease biomarker, and a therapeutic target for PH as well.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Animais , Biomarcadores , Citocinas , Hipertensão Pulmonar Primária Familiar , Hipertensão Pulmonar/metabolismo , Mesilato de Imatinib , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1beta , Interleucina-6/genética , Interleucina-6/metabolismo , Leucócitos Mononucleares/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Camundongos , Fator de Crescimento Derivado de Plaquetas , Artéria Pulmonar , Estabilidade de RNA , Ribonucleases/genética , Ribonucleases/metabolismo , Remodelação VascularRESUMO
Toll-like receptor (TLR) stimulation induces glycolysis and the production of mitochondrial reactive oxygen species (ROS), both of which are critical for inflammatory responses in macrophages. Here, we demonstrated that cyclin J, a TLR-inducible member of the cyclin family, reduced cytokine production in macrophages by coordinately controlling glycolysis and mitochondrial functions. Cyclin J interacted with cyclin-dependent kinases (CDKs), which increased the phosphorylation of a subset of CDK substrates, including the transcription factor FoxK1 and the GTPase Drp1. Cyclin J-dependent phosphorylation of FoxK1 decreased the transcription of glycolytic genes and Hif-1α activation, whereas hyperactivation of Drp1 by cyclin J-dependent phosphorylation promoted mitochondrial fragmentation and impaired the production of mitochondrial ROS. In mice, cyclin J in macrophages limited the growth of tumor xenografts and protected against LPS-induced shock but increased the susceptibility to bacterial infection. Collectively, our findings indicate that cyclin J-CDK signaling promotes antitumor immunity and the resolution of inflammation by opposing the metabolic changes that drive inflammatory responses in macrophages.
Assuntos
Imunidade Inata , Macrófagos , Animais , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Macrófagos/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
RNA in extracellular vesicles (EVs) are uptaken by cells, where they regulate fundamental cellular functions. EV-derived mRNA in recipient cells can be translated. However, it is still elusive whether "naked nonvesicular extracellular mRNA" (nex-mRNA) that are not packed in EVs can be uptaken by cells and, if so, whether they have any functions in recipient cells. Here, we show the entrance of nex-mRNA in the nucleus, where they exert a translation-independent function. Human nex-interleukin-1ß (IL1ß)-mRNA outside cells proved to be captured by RNA-binding zinc finger CCCH domain containing protein 12D (ZC3H12D)-expressing human natural killer (NK) cells. ZC3H12D recruited to the cell membrane binds to the 3'-untranslated region of nex-IL1ß-mRNA and transports it to the nucleus. The nex-IL1ß-mRNA in the NK cell nucleus upregulates antiapoptotic gene expression, migration activity, and interferon-γ production, leading to the killing of cancer cells and antimetastasis in mice. These results implicate the diverse actions of mRNA.
Assuntos
Núcleo Celular/metabolismo , Espaço Extracelular/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Animais , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Meios de Cultivo Condicionados/metabolismo , Endorribonucleases/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Células Matadoras Naturais/metabolismo , Camundongos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/farmacologia , Proteínas de Ligação a RNA/metabolismoRESUMO
BACKGROUND Brugada syndrome is a potentially fatal cardiac arrhythmia characterized by incomplete right bundle-branch block (RBB) and characteristic ST-segment elevation in the anterior electrocardiogram (ECG) leads. This report is of a case of type 2 Brugada syndrome, and discusses the importance of preoperative history and ECG evaluation. CASE REPORT A 32-year-old man was scheduled for tympanoplasty. His preoperative ECG revealed saddleback-type J waves in V2 (>2 mm) and ST increase (>1 mm) detected 1 week before elective surgery, but the ECG 1 year before showed normal. He had no notable past history. Anesthesia was induced with remifentanil and propofol, and maintained with sevoflurane in combination with remifentanil. Routine monitoring of vital signs was supplemented with V2 monitoring on the ECG. The heart rate was maintained at above 60 beats/min using ephedrine. The course of the operation was uneventful. CONCLUSIONS We managed anesthesia for a patient with a type 2 Brugada syndrome ECG without events, probably because he had no notable past history such as syncope. Type 2 and type 3 Brugada syndrome ECGs are difficult to recognize, and patients with them are considered to be less risky than a patient with a type I ECG. However, as Brugada syndrome ECG is dynamic and changeable, a type 2 or 3 Brugada syndrome ECG can change to a type I ECG under some conditions, and thus should not be overlooked, and the patient's past history or symptoms, such as syncope, should be carefully investigated.
Assuntos
Síndrome de Brugada , Adulto , Arritmias Cardíacas , Síndrome de Brugada/diagnóstico , Bloqueio de Ramo , Eletrocardiografia , Humanos , Masculino , TimpanoplastiaRESUMO
BACKGROUND: Severe aortic stenosis (AS) is increasing in the aging society and is a serious condition for anesthetic management. However, approximately one-third of patients with severe AS are asymptomatic. Echocardiography is the most reliable method to detect AS, but it takes time and is costly. METHODS: Data were obtained retrospectively from patients who underwent surgery and preoperative transthoracic echocardiography (TTE). LVH on ECG was determined by voltage criteria (Sv1 + Rv5 or 6 ≥3.5 mV) and/or the strain pattern in V5 and V6. Severe AS was defined as a mean transaortic pressure gradient ≥40 mmHg or aortic valve area ≤1.0 cm2 by TTE. RESULTS: Data for 470 patients aged 28-94 years old were obtained. One hundred and twenty-six patients had severe AS. LVH on ECG by voltage criteria alone was detected in 182 patients, LVH by strain pattern alone was detected in 80 patients and LVH by both was detected in 55 patients. Multivariable logistic analysis revealed that LVH by the strain pattern or voltage criteria, diabetes mellitus, and age were significantly associated with severe AS. The AUC for the ROC curve for LVH by voltage criteria alone was 0.675 and the cut-off value was 3.84 mm V, and the AUC for the ROC for age was 0.675 and the cut-off value was 74 years old. CONCLUSION: Our study suggests that patients who are 74 years old or over with LVH on ECG, especially those with DM, should undergo preoperative TTE in order to detect severe AS.
Assuntos
Estenose da Valva Aórtica/diagnóstico , Doenças Assintomáticas , Eletrocardiografia , Hipertrofia Ventricular Esquerda/diagnóstico , Cuidados Pré-Operatórios/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Anestesia/efeitos adversos , Estenose da Valva Aórtica/complicações , Feminino , Humanos , Hipertrofia Ventricular Esquerda/etiologia , Japão , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controle , Curva ROC , Estudos Retrospectivos , Fatores de Risco , Índice de Gravidade de Doença , Procedimentos Cirúrgicos Operatórios/efeitos adversosRESUMO
BACKGROUND: In a patient with very long-chain acyl-Coenzyme A dehydrogenase (VLCAD) deficiency, metabolism of fatty acids is impaired and a supply of alternative energy is limited when glucose level is insufficient on starvation. CASE PRESENTATION: A 37-year-old woman with VLCAD deficiency was diagnosed with an ovarian cyst and was scheduled for laparoscopic ovarian cystectomy. Glucose was administered intravenously with the start of fasting. Anesthesia was induced with remifentanil, midazolam, and thiamylal, maintained with desflurane and remifentanil. Body temperature was maintained at 36.2-36.7 °C. During anesthesia, hypoglycemia did not occur, creatine kinase levels were in the normal range, and myoglobinuria was not detected. No shivering was observed after extubation. CONCLUSIONS: Glucose was administered to avoid perioperative hypoglycemia. Body temperature was controlled to avoid shivering, which would otherwise increase skeletal muscle energy needs. Blood creatine kinase did not increase, and myoglobinuria was not detected; thus, rhabdomyolysis was unlikely to develop.
RESUMO
Chaetomium globosum is a filamentous fungus from which we have previously isolated a series of interesting natural products. Here, we isolated a previously unknown natural product from the culture of C. globosum. Through spectroscopic and crystallographic characterization, we determined the compound to be a new dimerized azaphilone-type product which we termed cochliodone J (1). Furthermore, our investigation into the biological activity of the natural product determined that 1 was cytotoxic to human cervix carcinoma HeLa cells with an IC50 of 17.3 µM. Lastly, a plausible biosynthetic mechanism for 1 is suggested based on our previous study on the biosynthesis of a closely related compound, cochliodone A (2).
Assuntos
Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Chaetomium/química , Pigmentos Biológicos/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Benzopiranos/química , Benzopiranos/isolamento & purificação , Feminino , Células HeLa , Humanos , Concentração Inibidora 50 , Pigmentos Biológicos/química , Pigmentos Biológicos/isolamento & purificaçãoRESUMO
Regnase-1-mediated mRNA decay (RMD), in which inflammatory mRNAs harboring specific stem-loop structures are degraded, is a critical part of proper immune homeostasis. Prior to initial translation, Regnase-1 associates with target stem-loops but does not carry out endoribonucleolytic cleavage. Single molecule imaging revealed that UPF1 is required to first unwind the stem-loops, thus licensing Regnase-1 to proceed with RNA degradation. Following translation, Regnase-1 physically associates with UPF1 using two distinct points of interaction: The Regnase-1 RNase domain binds to SMG1-phosphorylated residue T28 in UPF1; in addition, an intrinsically disordered segment in Regnase-1 binds to the UPF1 RecA domain, enhancing the helicase activity of UPF1. The SMG1-UPF1-Regnase-1 axis targets pioneer rounds of translation and is critical for rapid resolution of inflammation through restriction of the number of proteins translated by a given mRNA. Furthermore, small-molecule inhibition of SMG1 prevents RNA unwinding in dendritic cells, allowing post-transcriptional control of innate immune responses.
Assuntos
Macrófagos Peritoneais/imunologia , Degradação do RNAm Mediada por Códon sem Sentido/imunologia , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Ribonucleases/genética , Transativadores/genética , Animais , Fibroblastos/citologia , Fibroblastos/imunologia , Células HEK293 , Células HeLa , Homeostase/genética , Homeostase/imunologia , Humanos , Imunidade Inata , Inflamação , Sequências Repetidas Invertidas , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Knockout , Mutação , Cultura Primária de Células , Ligação Proteica , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/imunologia , RNA Mensageiro/metabolismo , Ribonucleases/deficiência , Ribonucleases/imunologia , Imagem Individual de Molécula , Transativadores/imunologiaRESUMO
RNA-modulating factors not only regulate multiple steps of cellular RNA metabolism, but also emerge as key effectors of the immune response against invading viral pathogens including human immunodeficiency virus type-1 (HIV-1). However, the cellular RNA-binding proteins involved in the establishment and maintenance of latent HIV-1 reservoirs have not been extensively studied. Here, we screened a panel of 62 cellular RNA-binding proteins and identified NEDD4-binding protein 1 (N4BP1) as a potent interferon-inducible inhibitor of HIV-1 in primary T cells and macrophages. N4BP1 harbours a prototypical PilT N terminus-like RNase domain and inhibits HIV-1 replication by interacting with and degrading viral mRNA species. Following activation of CD4+ T cells, however, N4BP1 undergoes rapid cleavage at Arg 509 by the paracaspase named mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1). Mutational analyses and knockout studies revealed that MALT1-mediated inactivation of N4BP1 facilitates the reactivation of latent HIV-1 proviruses. Taken together, our findings demonstrate that the RNase N4BP1 is an efficient restriction factor of HIV-1 and suggest that inactivation of N4BP1 by induction of MALT1 activation might facilitate elimination of latent HIV-1 reservoirs.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ativação Viral/genética , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Infecções por HIV/metabolismo , Humanos , Interferon-alfa/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Domínios Proteicos , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Latência ViralRESUMO
Postoperative cognitive dysfunction (POCD) is a serious complication of anesthesia and surgery, and the major risk factor of POCD is aging. Although the exact pathophysiology of POCD remains unknown, two possible and reliable mechanisms have been proposed: neuroinflammation and neurodegeneration, i.e., amyloid ß accumulation and/or tau protein phosphorylation, by surgery and/or general anesthetics. White matter lesions (WML) are produced by chronic cerebral hypoperfusion, frequently observed in elderly people, and closely related to cognitive decline. As recent studies have revealed that WML are a significant risk factor for POCD in humans, and we previously also demonstrated that persistent hypocapnea or hypotension caused neuronal damage in the caudoputamen or the hippocampus in a rat model of chronic cerebral hypoperfusion, which features global cerebral WML without neuronal damage and is recognized as a good model of human vascular dementia especially in elderly people, we hypothesize that in addition to those two previously proposed mechanisms, perioperative vital sign changes that cause reductions in cerebral blood flow might contribute to POCD in patients with WML, whose cerebral blood flow is already considerably decreased.
Assuntos
Disfunção Cognitiva/fisiopatologia , Complicações Cognitivas Pós-Operatórias/fisiopatologia , Substância Branca/patologia , Idoso , Envelhecimento , Animais , Isquemia Encefálica/patologia , Circulação Cerebrovascular , Hipocampo/patologia , Humanos , Neurônios/patologia , Ratos , Fatores de RiscoRESUMO
Inhaled pathogens including Pseudomonas aeruginosa initially encounter airway epithelial cells (AECs), which are poised to evoke cell-intrinsic innate defense, affecting second tier of hematopoietic cell-mediated immune reaction. However, it is largely unknown how pulmonary immune responses mediated by a variety of immune cells are coordinated. Here we show that Regnase-1, an endoribonuclease expressed in AECs and immune cells, plays an essential role in coordinating innate responses and adaptive immunity against P. aeruginosa infection. Intratracheal treatment of mice with heat-killed P. aeruginosa resulted in prolonged disappearance of Regnase-1 consistent with sustained expression of Regnase-1 target inflammatory genes, whereas the transcription factor NF-κB was only transiently activated. AEC-specific deletion of Regnase-1 not only augmented innate defenses against P. aeruginosa but also enhanced secretion of Pseudomonas-specific IgA and Th17 accumulation in the lung, culminating in conferring significant resistance against P. aeruginosa re-infection in vivo. Although Regnase-1 directly controls distinct sets of genes in each of AECs and T cells, degradation of Regnase-1 in both cell types is beneficial for maximizing acquired immune responses. Collectively, these results demonstrate that Regnase-1 orchestrates AEC-mediated and immune cell-mediated host defense against pulmonary bacterial infection.
Assuntos
Pulmão/imunologia , Pneumonia Bacteriana/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/fisiologia , Mucosa Respiratória/metabolismo , Ribonucleases/metabolismo , Células Th17/imunologia , Imunidade Adaptativa , Animais , Anticorpos Antibacterianos/metabolismo , Imunidade Inata , Imunoglobulina A/metabolismo , Pulmão/microbiologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Ribonucleases/genética , Transdução de SinaisRESUMO
Iron metabolism is regulated by transcriptional and post-transcriptional mechanisms. The mRNA of the iron-controlling gene, transferrin receptor 1 (TfR1), has long been believed to be negatively regulated by a yet-unidentified endonuclease. Here, we show that the endonuclease Regnase-1 is critical for the degradation of mRNAs involved in iron metabolism in vivo. First, we demonstrate that Regnase-1 promotes TfR1 mRNA decay. Next, we show that Regnase-1-/- mice suffer from severe iron deficiency anemia, although hepcidin expression is downregulated. The iron deficiency anemia is induced by a defect in duodenal iron uptake. We reveal that duodenal Regnase-1 controls the expression of PHD3, which impairs duodenal iron uptake via HIF2α suppression. Finally, we show that Regnase-1 is a HIF2α-inducible gene and thus provides a positive feedback loop for HIF2α activation via PHD3. Collectively, these results demonstrate that Regnase-1-mediated regulation of iron-related transcripts is essential for the maintenance of iron homeostasis.
Assuntos
Antígenos CD/metabolismo , Homeostase , Ferro/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Estabilidade de RNA , Receptores da Transferrina/metabolismo , Ribonucleases/metabolismo , Anemia/metabolismo , Anemia/patologia , Animais , Antígenos CD/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Duodeno/metabolismo , Ferritinas/metabolismo , Camundongos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Transferrina/genética , Elementos de Resposta/genética , Ribonucleases/deficiência , Transcrição GênicaRESUMO
Akirin2, an evolutionarily conserved nuclear protein, is an important factor regulating inflammatory gene transcription in mammalian innate immune cells by bridging the NF-κB and SWI/SNF complexes. Although Akirin is critical for Drosophila immune responses, which totally rely on innate immunity, the mammalian NF-κB system is critical not only for the innate but also for the acquired immune system. Therefore, we investigated the role of mouse Akirin2 in acquired immune cells by ablating Akirin2 function in B lymphocytes. B cell-specific Akirin2-deficient (Cd19(Cre/+)Akirin2(fl/fl)) mice showed profound decrease in the splenic follicular (FO) and peritoneal B-1, but not splenic marginal zone (MZ), B cell numbers. However, both Akirin2-deficient FO and MZ B cells showed severe proliferation defect and are prone to undergo apoptosis in response to TLR ligands, CD40, and BCR stimulation. Furthermore, B cell cycling was defective in the absence of Akirin2 owing to impaired expression of genes encoding cyclin D and c-Myc. Additionally, Brg1 recruitment to the Myc and Ccnd2 promoter was severely impaired in Akirin2-deficient B cells. Cd19(Cre/+)Akirin2(fl/fl) mice showed impaired in vivo immune responses to T-dependent and -independent Ags. Collectively, these results demonstrate that Akirin2 is critical for the mitogen-induced B cell cycle progression and humoral immune responses by controlling the SWI/SNF complex, further emphasizing the significant function of Akirin2 not only in the innate, but also in adaptive immune cells.
Assuntos
Linfócitos B/imunologia , Linhagem da Célula/imunologia , Imunidade Humoral , Ativação Linfocitária , Proteínas Repressoras/imunologia , Animais , Antígenos CD19/genética , Antígenos CD19/imunologia , Apoptose , Linfócitos B/citologia , Antígenos CD40/genética , Antígenos CD40/imunologia , Proliferação de Células , Ciclina D/genética , Ciclina D/imunologia , Ciclina D2/genética , Ciclina D2/imunologia , DNA Helicases/genética , DNA Helicases/imunologia , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Regiões Promotoras Genéticas , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
Transcription of inflammatory genes in innate immune cells is coordinately regulated by transcription factors, including NF-κB, and chromatin modifiers. However, it remains unclear how microbial sensing initiates chromatin remodeling. Here, we show that Akirin2, an evolutionarily conserved nuclear protein, bridges NF-κB and the chromatin remodeling SWI/SNF complex by interacting with BRG1-Associated Factor 60 (BAF60) proteins as well as IκB-ζ, which forms a complex with the NF-κB p50 subunit. These interactions are essential for Toll-like receptor-, RIG-I-, and Listeria-mediated expression of proinflammatory genes including Il6 and Il12b in macrophages. Consistently, effective clearance of Listeria infection required Akirin2. Furthermore, Akirin2 and IκB-ζ recruitment to the Il6 promoter depend upon the presence of IκB-ζ and Akirin2, respectively, for regulation of chromatin remodeling. BAF60 proteins were also essential for the induction of Il6 in response to LPS stimulation. Collectively, the IκB-ζ-Akirin2-BAF60 complex physically links the NF-κB and SWI/SNF complexes in innate immune cell activation. By recruiting SWI/SNF chromatin remodellers to IκB-ζ, transcriptional coactivator for NF-κB, the conserved nuclear protein Akirin2 stimulates pro-inflammatory gene promoters in mouse macrophages during innate immune responses to viral or bacterial infection.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica , Imunidade Inata , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Citocinas/metabolismo , Feminino , Humanos , Listeria monocytogenes/fisiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Repressoras/genética , Deleção de Sequência , Ativação TranscricionalRESUMO
Previously, we reported that an artificial zinc-finger protein (AZP)-staphylococcal nuclease (SNase) hybrid (designated AZP-SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP-SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP-SNase to the single-chain FokI dimer (scFokI) that we had developed previously. We demonstrated that transfection with a plasmid expressing the resulting hybrid nuclease (designated AZP-scFokI) inhibited HPV-18 DNA replication in transient replication assays using mammalian cells more efficiently than AZP-SNase. Then, by linker-mediated PCR analysis, we confirmed that AZP-scFokI cleaved an HPV-18 ori plasmid around its binding site in mammalian cells. Finally, a modified MTT assay revealed that AZP-scFokI did not show any significant cytotoxicity. Thus, the newly developed AZP-scFokI hybrid is expected to serve as a novel antiviral reagent for the neutralization of human DNA viruses with less fewer potential side effects.
Assuntos
Replicação do DNA/genética , DNA Viral/genética , DNA Viral/metabolismo , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/metabolismo , Sítios de Ligação/genética , Biotecnologia , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/toxicidade , Feminino , Células HEK293 , Papillomavirus Humano 18/patogenicidade , Humanos , Nuclease do Micrococo/genética , Nuclease do Micrococo/toxicidade , Reação em Cadeia da Polimerase , Especificidade por Substrato , Transfecção , Dedos de Zinco/genéticaRESUMO
Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase that destabilizes a set of mRNAs, including Il6 and Il12b, through cleavage of their 3' UTRs. Although Regnase-1 inactivation leads to development of an autoimmune disease characterized by T cell activation and hyperimmunoglobulinemia in mice, the mechanism of Regnase-1-mediated immune regulation has remained unclear. We show that Regnase-1 is essential for preventing aberrant effector CD4(+) T cell generation cell autonomously. Moreover, in T cells, Regnase-1 regulates the mRNAs of a set of genes, including c-Rel, Ox40, and Il2, through cleavage of their 3' UTRs. Interestingly, T cell receptor (TCR) stimulation leads to cleavage of Regnase-1 at R111 by Malt1/paracaspase, freeing T cells from Regnase-1-mediated suppression. Furthermore, Malt1 protease activity is critical for controlling the mRNA stability of T cell effector genes. Collectively, these results indicate that dynamic control of Regnase-1 expression in T cells is critical for controlling T cell activation.
Assuntos
Caspases/metabolismo , Ativação Linfocitária , Proteínas de Neoplasias/metabolismo , Ribonucleases/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Doenças Autoimunes/imunologia , Humanos , Interleucina-2/genética , Células Jurkat , Glicoproteínas de Membrana/genética , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Ligante OX40 , Proteínas Proto-Oncogênicas c-rel/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Fatores de Necrose Tumoral/genéticaRESUMO
Posttranscriptional regulation of IL-6 has been largely uncharacterized, with the exception of the ribonuclease Regnase-1, which prevents autoimmunity by destabilizing IL-6 mRNA. Here, we identified AT-rich interactive domain-containing protein 5A (Arid5a) as a unique RNA binding protein, which stabilizes IL-6 but not TNF-α mRNA through binding to the 3' untranslated region of IL-6 mRNA. Arid5a was enhanced in macrophages in response to LPS, IL-1ß, and IL-6. Arid5a deficiency inhibited elevation of IL-6 serum level in LPS-treated mice and suppressed IL-6 levels and the development of T(H)17 cells in experimental autoimmune encephalomyelitis. Importantly, Arid5a inhibited the destabilizing effect of Regnase-1 on IL-6 mRNA. These results indicate that Arid5a plays an important role in promotion of inflammatory processes and autoimmune diseases.
Assuntos
Proteínas de Ligação a DNA/imunologia , Encefalomielite Autoimune Experimental/imunologia , Interleucina-6/imunologia , Estabilidade de RNA/imunologia , Proteínas de Ligação a RNA/imunologia , Fatores de Transcrição/imunologia , Regiões 3' não Traduzidas/genética , Animais , Técnicas de Cultura de Células , Proteínas de Ligação a DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Interleucina-6/sangue , Lipopolissacarídeos , Luciferases , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
TNFR-associated factor family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1) is critical for the activation of IFN regulatory factor 3 and type I IFN production upon virus infection. A set of TBK1-binding proteins, 5-azacytidine-induced gene 2 (AZI2; also known as NAP1), TANK, and TBK1-binding protein 1 (TBKBP1), have also been implicated in the production of type I IFNs. Among them, TANK was found to be dispensable for the responses against virus infection. However, physiological roles of AZI2 and TBKBP1 have yet to be clarified. In this study, we found that none of these TBK1-binding proteins is critical for type I IFN production in mice. In contrast, AZI2, but not TBKBP1, is critical for the differentiation of conventional dendritic cells (cDCs) from bone marrow cells in response to GM-CSF. AZI2 controls GM-CSF-induced cell cycling of bone marrow cells via TBK1. GM-CSF-derived DCs from AZI2-deficient mice show severe defects in cytokine production and T cell activation both in vitro and in vivo. Reciprocally, overexpression of AZI2 results in efficient generation of cDCs, and the cells show enhanced T cell activation in response to Ag stimulation. Taken together, AZI2 expression is critical for the generation of cDCs by GM-CSF and can potentially be used to increase the efficiency of immunization by cDCs.