RESUMO
RIPK1 is a multi-functional kinase that regulates cell death and inflammation and has been implicated in the pathogenesis of inflammatory diseases. RIPK1 acts in a kinase-dependent and kinase-independent manner to promote or suppress apoptosis and necroptosis, but the underlying mechanisms remain poorly understood. Here, we show that a mutation (R588E) disrupting the RIPK1 death domain (DD) caused perinatal lethality induced by ZBP1-mediated necroptosis. Additionally, these mice developed postnatal inflammatory pathology, which was mediated by necroptosis-independent TNFR1, TRADD, and TRIF signaling, partially requiring RIPK3. Our biochemical mechanistic studies revealed that ZBP1- and TRIF-mediated activation of RIPK3 required RIPK1 kinase activity in wild-type cells but not in Ripk1R588E/R588E cells, suggesting that DD-dependent oligomerization of RIPK1 and its interaction with FADD determine the mechanisms of RIPK3 activation by ZBP1 and TRIF. Collectively, these findings revealed a critical physiological role of DD-dependent RIPK1 signaling that is important for the regulation of tissue homeostasis and inflammation.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Inflamação , Necroptose , Proteínas de Ligação a RNA , Proteína Serina-Treonina Quinases de Interação com Receptores , Transdução de Sinais , Animais , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Camundongos , Inflamação/metabolismo , Inflamação/imunologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/genética , Morte Celular , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Domínios Proteicos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Apoptose , Mutação , Proteína de Domínio de Morte Associada a Receptor de TNFRESUMO
Digested dietary fats are taken up by enterocytes where they are assembled into pre-chylomicrons in the endoplasmic reticulum followed by transport to the Golgi for maturation and subsequent secretion to the circulation1. The role of mitochondria in dietary lipid processing is unclear. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with specific ablation of the mitochondrial aspartyl-tRNA synthetase DARS2 (ref. 2), the respiratory chain subunit SDHA3 or the assembly factor COX10 (ref. 4) in intestinal epithelial cells showed accumulation of large lipid droplets (LDs) in enterocytes of the proximal small intestine and failed to thrive. Feeding a fat-free diet suppressed the build-up of LDs in DARS2-deficient enterocytes, which shows that the accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed an impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in intestinal epithelial cells. DARS2 deficiency caused a distinct lack of mature chylomicrons concomitant with a progressive dispersal of the Golgi apparatus in proximal enterocytes. This finding suggests that mitochondrial dysfunction results in impaired trafficking of chylomicrons from the endoplasmic reticulum to the Golgi, which in turn leads to storage of dietary lipids in large cytoplasmic LDs. Taken together, these results reveal a role for mitochondria in dietary lipid transport in enterocytes, which might be relevant for understanding the intestinal defects observed in patients with mitochondrial disorders5.
Assuntos
Gorduras na Dieta , Enterócitos , Metabolismo dos Lipídeos , Mitocôndrias , Animais , Camundongos , Aspartato-tRNA Ligase/metabolismo , Quilomícrons/metabolismo , Gorduras na Dieta/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Retículo Endoplasmático/metabolismo , Enterócitos/metabolismo , Enterócitos/patologia , Células Epiteliais/metabolismo , Complexo de Golgi/metabolismo , Intestinos , Gotículas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologiaRESUMO
Pathways controlling intestinal epithelial cell (IEC) death regulate gut immune homeostasis and contribute to the pathogenesis of inflammatory bowel diseases. Here, we show that caspase-8 and its adapter FADD act in IECs to regulate intestinal inflammation downstream of Z-DNA binding protein 1 (ZBP1)- and tumor necrosis factor receptor-1 (TNFR1)-mediated receptor interacting protein kinase 1 (RIPK1) and RIPK3 signaling. Mice with IEC-specific FADD or caspase-8 deficiency developed colitis dependent on mixed lineage kinase-like (MLKL)-mediated epithelial cell necroptosis. However, MLKL deficiency fully prevented ileitis caused by epithelial caspase-8 ablation, but only partially ameliorated ileitis in mice lacking FADD in IECs. Our genetic studies revealed that caspase-8 and gasdermin-D (GSDMD) were both required for the development of MLKL-independent ileitis in mice with epithelial FADD deficiency. Therefore, FADD prevents intestinal inflammation downstream of ZBP1 and TNFR1 by inhibiting both MLKL-induced necroptosis and caspase-8-GSDMD-dependent pyroptosis-like death of epithelial cells.
Assuntos
Caspase 8/genética , Proteína de Domínio de Morte Associada a Fas/genética , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Quinases/metabolismo , Animais , Apoptose/genética , Caspase 8/metabolismo , Morte Celular/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Células Epiteliais/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Perfilação da Expressão Gênica , Homeostase/genética , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Proteínas Quinases/genéticaRESUMO
Epithelial barrier defects are implicated in the pathogenesis of inflammatory bowel disease (IBD); however, the role of microbiome dysbiosis and the cytokine networks orchestrating chronic intestinal inflammation in response to barrier impairment remain poorly understood. Here, we showed that altered Schaedler flora (ASF), a benign minimal microbiota, was sufficient to trigger colitis in a mouse model of intestinal barrier impairment. Colitis development required myeloid-cell-specific adaptor protein MyD88 signaling and was orchestrated by the cytokines IL-12, IL-23, and IFN-γ. Colon inflammation was driven by IL-12 during the early stages of the disease, but as the mice aged, the pathology shifted toward an IL-23-dependent inflammatory response driving disease chronicity. These findings reveal that IL-12 and IL-23 act in a temporally distinct, biphasic manner to induce microbiota-driven chronic intestinal inflammation. Similar mechanisms might contribute to the pathogenesis of IBD particularly in patients with underlying intestinal barrier defects.
Assuntos
Colite/imunologia , Doenças Inflamatórias Intestinais/imunologia , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Mucosa Intestinal/patologia , Microbiota/imunologia , Animais , Doença Crônica , Modelos Animais de Doenças , Humanos , Inflamação , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-12/genética , Interleucina-23/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Quimeras de TransplanteRESUMO
Mutations of the ADAR1 gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including Aicardi-Goutières syndrome and bilateral striatal necrosis1-3. The IFN-inducible p150 isoform of ADAR1 contains a Zα domain that recognizes RNA with an alternative left-handed double-helix structure, termed Z-RNA4,5. Hemizygous ADAR1 mutations in the Zα domain cause type I IFN-mediated pathologies in humans2,3 and mice6-8; however, it remains unclear how the interaction of ADAR1 with Z-RNA prevents IFN activation. Here we show that Z-DNA-binding protein 1 (ZBP1), the only other protein in mammals known to harbour Zα domains9, promotes type I IFN activation and fatal pathology in mice with impaired ADAR1 function. ZBP1 deficiency or mutation of its Zα domains reduced the expression of IFN-stimulated genes and largely prevented early postnatal lethality in mice with hemizygous expression of ADAR1 with mutated Zα domain (Adar1mZα/- mice). Adar1mZα/- mice showed upregulation and impaired editing of endogenous retroelement-derived complementary RNA reads, which represent a likely source of Z-RNAs activating ZBP1. Notably, ZBP1 promoted IFN activation and severe pathology in Adar1mZα/- mice in a manner independent of RIPK1, RIPK3, MLKL-mediated necroptosis and caspase-8-dependent apoptosis, suggesting a novel mechanism of action. Thus, ADAR1 prevents endogenous Z-RNA-dependent activation of pathogenic type I IFN responses by ZBP1, suggesting that ZBP1 could contribute to type I interferonopathies caused by ADAR1 mutations.
Assuntos
Adenosina Desaminase , Interferon Tipo I , Proteínas de Ligação a RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Apoptose , Caspase 8/metabolismo , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/imunologia , Camundongos , Mutação , Necroptose , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Cellular inhibitor of apoptosis proteins (cIAPs) are RING-containing E3 ubiquitin ligases that ubiquitylate receptor-interacting protein kinase 1 (RIPK1) to regulate TNF signalling. Here, we established mice simultaneously expressing enzymatically inactive cIAP1/2 variants, bearing mutations in the RING domains of cIAP1/2 (cIAP1/2 mutant RING, cIAP1/2MutR ). cIap1/2MutR/MutR mice died during embryonic development due to RIPK1-mediated apoptosis. While expression of kinase-inactive RIPK1D138N rescued embryonic development, Ripk1D138N/D138N /cIap1/2MutR/MutR mice developed systemic inflammation and died postweaning. Cells expressing cIAP1/2MutR and RIPK1D138N were still susceptible to TNF-induced apoptosis and necroptosis, implying additional kinase-independent RIPK1 activities in regulating TNF signalling. Although further ablation of Ripk3 did not lead to any phenotypic improvement, Tnfr1 gene knock-out prevented early onset of systemic inflammation and premature mortality, indicating that cIAPs control TNFR1-mediated toxicity independent of RIPK1 and RIPK3. Beyond providing novel molecular insights into TNF-signalling, the mouse model established in this study can serve as a useful tool to further evaluate ongoing therapeutic protocols using inhibitors of TNF, cIAPs and RIPK1.
Assuntos
Proteínas Inibidoras de Apoptose , Receptores Tipo I de Fatores de Necrose Tumoral , Animais , Camundongos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Morte Celular , Apoptose , Inflamação/genética , Inflamação/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.
Assuntos
Caspase 8/metabolismo , Instabilidade Cromossômica , Neoplasias do Colo/enzimologia , Fibroblastos/enzimologia , Mitose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Aneuploidia , Animais , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Fibroblastos/patologia , Células HT29 , Humanos , Inflamação/enzimologia , Inflamação/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais , Quinase 1 Polo-LikeRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The biological function of Z-DNA and Z-RNA, nucleic acid structures with a left-handed double helix, is poorly understood1-3. Z-DNA-binding protein 1 (ZBP1; also known as DAI or DLM-1) is a nucleic acid sensor that contains two Zα domains that bind Z-DNA4,5 and Z-RNA6-8. ZBP1 mediates host defence against some viruses6,7,9-14 by sensing viral nucleic acids6,7,10. RIPK1 deficiency, or mutation of its RIP homotypic interaction motif (RHIM), triggers ZBP1-dependent necroptosis and inflammation in mice15,16. However, the mechanisms that induce ZBP1 activation in the absence of viral infection remain unknown. Here we show that Zα-dependent sensing of endogenous ligands induces ZBP1-mediated perinatal lethality in mice expressing RIPK1 with mutated RHIM (Ripk1mR/mR), skin inflammation in mice with epidermis-specific RIPK1 deficiency (RIPK1E-KO) and colitis in mice with intestinal epithelial-specific FADD deficiency (FADDIEC-KO). Consistently, functional Zα domains were required for ZBP1-induced necroptosis in fibroblasts that were treated with caspase inhibitors or express RIPK1 with mutated RHIM. Inhibition of nuclear export triggered the Zα-dependent activation of RIPK3 in the nucleus resulting in cell death, which suggests that ZBP1 may recognize nuclear Z-form nucleic acids. We found that ZBP1 constitutively bound cellular double-stranded RNA in a Zα-dependent manner. Complementary reads derived from endogenous retroelements were detected in epidermal RNA, which suggests that double-stranded RNA derived from these retroelements may act as a Zα-domain ligand that triggers the activation of ZBP1. Collectively, our results provide evidence that the sensing of endogenous Z-form nucleic acids by ZBP1 triggers RIPK3-dependent necroptosis and inflammation, which could underlie the development of chronic inflammatory conditions-particularly in individuals with mutations in RIPK1 and CASP817-20.
Assuntos
Inflamação/metabolismo , Necroptose , Proteínas de Ligação a RNA/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Caspase 8/metabolismo , Feminino , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Nucleicos/metabolismo , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Dermatopatias/genética , Dermatopatias/metabolismo , Dermatopatias/patologiaRESUMO
RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.
Assuntos
Caspase 8/metabolismo , Doenças Hereditárias Autoinflamatórias/metabolismo , Mutação , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Caspase 3/metabolismo , Feminino , Doenças Hereditárias Autoinflamatórias/genética , Doenças Hereditárias Autoinflamatórias/patologia , Humanos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linhagem , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor κB (NF-κB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-κB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.
Assuntos
Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Celulas de Paneth/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose/genética , Células Cultivadas , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelB/genéticaRESUMO
Macrophages are a crucial component of the innate immune system in sensing pathogens and promoting local and systemic inflammation. RIPK1 and RIPK3 are homologous kinases, previously linked to activation of necroptotic death. In this study, we have described roles for these kinases as master regulators of pro-inflammatory gene expression induced by lipopolysaccharide, independent of their well-documented cell death functions. In primary macrophages, this regulation was elicited in the absence of caspase-8 activity, required the adaptor molecule TRIF, and proceeded in a cell autonomous manner. RIPK1 and RIPK3 kinases promoted sustained activation of Erk, cFos, and NF-κB, which were required for inflammatory changes. Utilizing genetic and pharmacologic tools, we showed that RIPK1 and RIPK3 account for acute inflammatory responses induced by lipopolysaccharide in vivo; notably, this regulation did not require exogenous manipulation of caspases. These findings identified a new pharmacologically accessible pathway that may be relevant to inflammatory pathologies.
Assuntos
Imunidade Inata , Inflamação/imunologia , Macrófagos/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Caspase 8/genética , Caspase 8/metabolismo , Células Cultivadas , Feminino , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais , TranscriptomaRESUMO
Caspase-8 is the initiator caspase of extrinsic apoptosis1,2 and inhibits necroptosis mediated by RIPK3 and MLKL. Accordingly, caspase-8 deficiency in mice causes embryonic lethality3, which can be rescued by deletion of either Ripk3 or Mlkl4-6. Here we show that the expression of enzymatically inactive CASP8(C362S) causes embryonic lethality in mice by inducing necroptosis and pyroptosis. Similar to Casp8-/- mice3,7, Casp8C362S/C362S mouse embryos died after endothelial cell necroptosis leading to cardiovascular defects. MLKL deficiency rescued the cardiovascular phenotype but unexpectedly caused perinatal lethality in Casp8C362S/C362S mice, indicating that CASP8(C362S) causes necroptosis-independent death at later stages of embryonic development. Specific loss of the catalytic activity of caspase-8 in intestinal epithelial cells induced intestinal inflammation similar to intestinal epithelial cell-specific Casp8 knockout mice8. Inhibition of necroptosis by additional deletion of Mlkl severely aggravated intestinal inflammation and caused premature lethality in Mlkl knockout mice with specific loss of caspase-8 catalytic activity in intestinal epithelial cells. Expression of CASP8(C362S) triggered the formation of ASC specks, activation of caspase-1 and secretion of IL-1ß. Both embryonic lethality and premature death were completely rescued in Casp8C362S/C362SMlkl-/-Asc-/- or Casp8C362S/C362SMlkl-/-Casp1-/- mice, indicating that the activation of the inflammasome promotes CASP8(C362S)-mediated tissue pathology when necroptosis is blocked. Therefore, caspase-8 represents the molecular switch that controls apoptosis, necroptosis and pyroptosis, and prevents tissue damage during embryonic development and adulthood.
Assuntos
Apoptose/genética , Caspase 8/genética , Caspase 8/metabolismo , Necroptose/genética , Piroptose/genética , Animais , Linhagem Celular , Células Cultivadas , Ativação Enzimática/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Inflamassomos/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/enzimologia , Queratinócitos/citologia , Queratinócitos/patologia , Camundongos , Mutação , Receptor TIE-2/genética , Receptor TIE-2/metabolismoRESUMO
Reprogramming of mitochondria provides cells with the metabolic flexibility required to adapt to various developmental transitions such as stem cell activation or immune cell reprogramming, and to respond to environmental challenges such as those encountered under hypoxic conditions or during tumorigenesis1-3. Here we show that the i-AAA protease YME1L rewires the proteome of pre-existing mitochondria in response to hypoxia or nutrient starvation. Inhibition of mTORC1 induces a lipid signalling cascade via the phosphatidic acid phosphatase LIPIN1, which decreases phosphatidylethanolamine levels in mitochondrial membranes and promotes proteolysis. YME1L degrades mitochondrial protein translocases, lipid transfer proteins and metabolic enzymes to acutely limit mitochondrial biogenesis and support cell growth. YME1L-mediated mitochondrial reshaping supports the growth of pancreatic ductal adenocarcinoma (PDAC) cells as spheroids or xenografts. Similar changes to the mitochondrial proteome occur in the tumour tissues of patients with PDAC, suggesting that YME1L is relevant to the pathophysiology of these tumours. Our results identify the mTORC1-LIPIN1-YME1L axis as a post-translational regulator of mitochondrial proteostasis at the interface between metabolism and mitochondrial dynamics.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Metabolismo dos Lipídeos , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Hipóxia Celular , Linhagem Celular , Proliferação de Células , Humanos , Lipídeos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metaloendopeptidases/genética , Proteínas Mitocondriais/genética , ProteóliseRESUMO
TNF is an inflammatory cytokine that upon binding to its receptor, TNFR1, can drive cytokine production, cell survival, or cell death. TNFR1 stimulation causes activation of NF-κB, p38α, and its downstream effector kinase MK2, thereby promoting transcription, mRNA stabilization, and translation of target genes. Here we show that TNF-induced activation of MK2 results in global RIPK1 phosphorylation. MK2 directly phosphorylates RIPK1 at residue S321, which inhibits its ability to bind FADD/caspase-8 and induce RIPK1-kinase-dependent apoptosis and necroptosis. Consistently, a phospho-mimetic S321D RIPK1 mutation limits TNF-induced death. Mechanistically, we find that phosphorylation of S321 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Thus, MK2-mediated phosphorylation of RIPK1 serves as a checkpoint within the TNF signaling pathway that integrates cell survival and cytokine production.
Assuntos
Apoptose/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Caspase 8/metabolismo , Relação Dose-Resposta a Droga , Proteína de Domínio de Morte Associada a Fas/metabolismo , Células HT29 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos , NF-kappa B/metabolismo , Necrose , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
The maintenance of immune homeostasis requires regulatory T cells (Treg cells). Here we found that Treg cellspecific ablation of Ubc13, a Lys63 (K63)-specific ubiquitin-conjugating enzyme, caused aberrant T cell activation and autoimmunity. Although Ubc13 deficiency did not affect the survival of Treg cells or expression of the transcription factor Foxp3, it impaired the in vivo suppressive function of Treg cells and rendered them sensitive to the acquisition of T helper type 1 (TH1) cell and interleukin 17 (IL-17)-producing helper T (TH17) celllike effector phenotypes. This function of Ubc13 involved its downstream target, the kinase IKK. The Ubc13-IKK signaling axis controlled the expression of specific Treg cell effector molecules, including IL-10 and SOCS1. Collectively, our findings suggest that the Ubc13-IKK signaling axis regulates the molecular program that maintains Treg cell function and prevents Treg cells from acquiring inflammatory phenotypes.
Assuntos
Autoimunidade/imunologia , Diferenciação Celular/imunologia , Quinase I-kappa B/metabolismo , Linfócitos T Reguladores/imunologia , Enzimas de Conjugação de Ubiquitina/imunologia , Animais , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Quinase I-kappa B/deficiência , Quinase I-kappa B/imunologia , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais/imunologia , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/imunologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Linfócitos T Reguladores/citologia , Células Th1/citologia , Células Th1/imunologia , Células Th17/citologia , Células Th17/imunologia , Enzimas de Conjugação de Ubiquitina/deficiência , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Multiple lung pathogens such as chemical agents, H5N1 avian flu, or SARS cause high lethality due to acute respiratory distress syndrome. Here we report that Toll-like receptor 4 (TLR4) mutant mice display natural resistance to acid-induced acute lung injury (ALI). We show that TLR4-TRIF-TRAF6 signaling is a key disease pathway that controls the severity of ALI. The oxidized phospholipid (OxPL) OxPAPC was identified to induce lung injury and cytokine production by lung macrophages via TLR4-TRIF. We observed OxPL production in the lungs of humans and animals infected with SARS, Anthrax, or H5N1. Pulmonary challenge with an inactivated H5N1 avian influenza virus rapidly induces ALI and OxPL formation in mice. Loss of TLR4 or TRIF expression protects mice from H5N1-induced ALI. Moreover, deletion of ncf1, which controls ROS production, improves the severity of H5N1-mediated ALI. Our data identify oxidative stress and innate immunity as key lung injury pathways that control the severity of ALI.
Assuntos
Estresse Oxidativo , Síndrome do Desconforto Respiratório/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Humanos , Influenza Humana/metabolismo , Interleucina-6/metabolismo , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Fosfolipídeos/metabolismo , Síndrome Respiratória Aguda Grave/metabolismo , Transdução de SinaisRESUMO
Apoptosis and necroptosis are two regulated cell death mechanisms; however, the interaction between these cell death pathways in vivo is unclear. Here we used cerebral ischemia/reperfusion as a model to investigate the interaction between apoptosis and necroptosis. We show that the activation of RIPK1 sequentially promotes necroptosis followed by apoptosis in a temporally specific manner. Cerebral ischemia/reperfusion insult rapidly activates necroptosis to promote cerebral hemorrhage and neuroinflammation. Ripk3 deficiency reduces cerebral hemorrhage and delays the onset of neural damage mediated by inflammation. Reduced cerebral perfusion resulting from arterial occlusion promotes the degradation of TAK1, a suppressor of RIPK1, and the transition from necroptosis to apoptosis. Conditional knockout of TAK1 in microglial/infiltrated macrophages and neuronal lineages sensitizes to ischemic infarction by promoting apoptosis. Taken together, our results demonstrate the critical role of necroptosis in mediating neurovascular damage and hypoperfusion-induced TAK1 loss, which subsequently promotes apoptosis and cerebral pathology in stroke and neurodegeneration.
Assuntos
Apoptose/fisiologia , Necroptose/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Lesões Encefálicas/metabolismo , Morte Celular , Inflamação/patologia , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Acidente Vascular Cerebral/patologiaRESUMO
Psoriasis is a common chronic inflammatory skin disease with a prevalence of about 2% in the Caucasian population. Tumor necrosis factor (TNF) plays an essential role in the pathogenesis of psoriasis, but its mechanism of action remains poorly understood. Here we report that the development of psoriasis-like skin inflammation in mice with epidermis-specific inhibition of the transcription factor NF-κB was triggered by TNF receptor 1 (TNFR1)-dependent upregulation of interleukin-24 (IL-24) and activation of signal transducer and activator of transcription 3 (STAT3) signaling in keratinocytes. IL-24 was strongly expressed in human psoriatic epidermis, and pharmacological inhibition of NF-κB increased IL-24 expression in TNF-stimulated human primary keratinocytes, suggesting that this mechanism is relevant for human psoriasis. Therefore, our results expand current views on psoriasis pathogenesis by revealing a new keratinocyte-intrinsic mechanism that links TNFR1, NF-κB, ERK, IL-24, IL-22R1, and STAT3 signaling to disease initiation.
Assuntos
Citocinas/fisiologia , Queratinócitos/patologia , Psoríase/etiologia , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Células Cultivadas , Cruzamentos Genéticos , Citocinas/biossíntese , Citocinas/genética , Modelos Animais de Doenças , Epiderme/patologia , Regulação da Expressão Gênica/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Humanos , Quinase I-kappa B/deficiência , Quinase I-kappa B/fisiologia , Interleucinas/fisiologia , Queratinócitos/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/metabolismo , Psoríase/patologia , Psoríase/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Fator de Transcrição STAT3/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Receptor-interacting protein kinase 1 (RIPK1) regulates cell death and inflammation through kinase-dependent and -independent functions. RIPK1 kinase activity induces caspase-8-dependent apoptosis and RIPK3 and mixed lineage kinase like (MLKL)-dependent necroptosis. In addition, RIPK1 inhibits apoptosis and necroptosis through kinase-independent functions, which are important for late embryonic development and the prevention of inflammation in epithelial barriers. The mechanism by which RIPK1 counteracts RIPK3-MLKL-mediated necroptosis has remained unknown. Here we show that RIPK1 prevents skin inflammation by inhibiting activation of RIPK3-MLKL-dependent necroptosis mediated by Z-DNA binding protein 1 (ZBP1, also known as DAI or DLM1). ZBP1 deficiency inhibited keratinocyte necroptosis and skin inflammation in mice with epidermis-specific RIPK1 knockout. Moreover, mutation of the conserved RIP homotypic interaction motif (RHIM) of endogenous mouse RIPK1 (RIPK1mRHIM) caused perinatal lethality that was prevented by RIPK3, MLKL or ZBP1 deficiency. Furthermore, mice expressing only RIPK1mRHIM in keratinocytes developed skin inflammation that was abrogated by MLKL or ZBP1 deficiency. Mechanistically, ZBP1 interacted strongly with phosphorylated RIPK3 in cells expressing RIPK1mRHIM, suggesting that the RIPK1 RHIM prevents ZBP1 from binding and activating RIPK3. Collectively, these results show that RIPK1 prevents perinatal death as well as skin inflammation in adult mice by inhibiting ZBP1-induced necroptosis. Furthermore, these findings identify ZBP1 as a critical mediator of inflammation beyond its previously known role in antiviral defence and suggest that ZBP1 might be implicated in the pathogenesis of necroptosis-associated inflammatory diseases.