RESUMO
A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and pro-inflammatory cytokine levels.
Assuntos
Proteínas de Transporte/biossíntese , Caspase 8/biossíntese , Inflamassomos/metabolismo , Mitocôndrias/metabolismo , Apoptose/genética , Autofagia/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Proteínas de Transporte/genética , Caspase 8/genética , Células Cultivadas , Peptidil-Prolil Isomerase F , Ciclofilinas/antagonistas & inibidores , Ciclofilinas/genética , Humanos , Interleucina-1beta/biossíntese , Mitocôndrias/patologia , Mitofagia/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR , Receptores Toll-Like/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Host innate immune responses to DNA viruses involve members of the nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein (NLRP) family, which form "inflammasomes" that activate caspase-1, resulting in proteolytic activation of cytokines interleukin (IL)-1ß and IL-18. We hypothesized that DNA viruses would target inflammasomes to overcome host defense. A Vaccinia virus (VACV) B-cell CLL/lymphoma 2 (Bcl-2) homolog, F1L, was demonstrated to bind and inhibit the NLR family member NLRP1 in vitro. Moreover, infection of macrophages in culture with virus lacking F1L (ΔF1L) caused increased caspase-1 activation and IL-1ß secretion compared with wild-type virus. Virulence of ΔF1L virus was attenuated in vivo, causing altered febrile responses, increased proteolytic processing of caspase-1, and more rapid inflammation in lungs of infected mice without affecting cell death or virus replication. Furthermore, we found that a hexapeptide from F1L is necessary and sufficient for inhibiting the NLRP1 inflammasome in vitro, thus identifying a peptidyl motif required for binding and inhibiting NLRP1. The functional importance of this NLRP1-binding motif was further confirmed by studies of recombinant ΔF1L viruses reconstituted either with the wild-type F1L or a F1L mutant that fails to bind NLRP1. Cellular infection with wild-type F1L reconstituted virus-suppressed IL-1ß production, whereas mutant F1L did not. In contrast, both wild-type and mutant versions of F1L equally suppressed apoptosis. In vivo, the NLR nonbinding F1L mutant virus exhibited an attenuated phenotype similar to ΔF1L virus, thus confirming the importance of F1L interactions with NLRP1 for viral pathogenicity in mice. Altogether, these findings reveal a unique viral mechanism for evading host innate immune responses.
Assuntos
Regulação Viral da Expressão Gênica , Imunidade Inata , Inflamassomos/metabolismo , Vaccinia virus/metabolismo , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Caspases/metabolismo , Chlorocebus aethiops , Citocinas/metabolismo , Células HEK293 , Células HeLa , Humanos , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fenótipo , Proteínas Recombinantes/metabolismo , Células Vero , VirulênciaRESUMO
Inflammasomes are cytosolic innate immune sensors of pathogen infection and cellular damage that induce caspase-1-mediated inflammation upon activation. Although inflammation is protective, uncontrolled excessive inflammation can cause inflammatory diseases and can be detrimental, such as in coronavirus disease (COVID-19). However, the underlying mechanisms that control inflammasome activation are incompletely understood. Here we report that the leucine-rich repeat (LRR) protein ribonuclease inhibitor (RNH1), which shares homology with LRRs of NLRP (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing) proteins, attenuates inflammasome activation. Deletion of RNH1 in macrophages increases interleukin (IL)-1ß production and caspase-1 activation in response to inflammasome stimulation. Mechanistically, RNH1 decreases pro-IL-1ß expression and induces proteasome-mediated caspase-1 degradation. Corroborating this, mouse models of monosodium urate (MSU)-induced peritonitis and lipopolysaccharide (LPS)-induced endotoxemia, which are dependent on caspase-1, respectively, show increased neutrophil infiltration and lethality in Rnh1 -/- mice compared with wild-type mice. Furthermore, RNH1 protein levels were negatively related with disease severity and inflammation in hospitalized COVID-19 patients. We propose that RNH1 is a new inflammasome regulator with relevance to COVID-19 severity.
Assuntos
COVID-19/patologia , Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Proteínas de Repetições Ricas em Leucina/metabolismo , Animais , COVID-19/imunologia , Caspase 1/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Gravidade do Paciente , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond-Blackfan anemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor 1 (RNH1) is a ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here, we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 and E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knockdown in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.
Assuntos
Embrião de Mamíferos/metabolismo , Eritropoese , Fator de Transcrição GATA1/biossíntese , Células-Tronco Hematopoéticas/metabolismo , Biossíntese de Proteínas , Proteínas/metabolismo , Animais , Embrião de Mamíferos/citologia , Fator de Transcrição GATA1/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Células K562 , Camundongos , Camundongos Knockout , Proteínas/genética , Subunidades Ribossômicas Maiores/genética , Subunidades Ribossômicas Maiores/metabolismoRESUMO
The 14 kDa homodimeric N1L protein is a potent vaccinia and variola (smallpox) virulence factor. It is not essential for viral replication, but it causes a strong attenuation of viral production in culture when deleted. The N1L protein is predicted to contain the BH3-like binding domain characteristic of Bcl-2 family proteins, and it is able to bind the BH3 peptides. Its overexpression has been reported to prevent infected cells from committing apoptosis. Therefore, interfering with the N1L apoptotic blockade may be a legitimate therapeutic strategy affecting the viral growth. By using in silico ligand docking and an array of in vitro assays, we have identified submicromolar (600 nM) N1L antagonists belonging to the family of polyphenols. Their affinity is comparable to that of the BH3 peptides (70-1000 nM). We have also identified the natural polyphenol resveratrol as a moderate N1L inhibitor. Finally, we show that our ligands efficiently inhibit growth of vaccinia virus.