RESUMO
The activation of T cells by the T cell antigen receptor (TCR) results in the formation of signaling protein complexes (signalosomes), the composition of which has not been analyzed at a systems level. Here, we isolated primary CD4+ T cells from 15 gene-targeted mice, each expressing one tagged form of a canonical protein of the TCR-signaling pathway. Using affinity purification coupled with mass spectrometry, we analyzed the composition and dynamics of the signalosomes assembling around each of the tagged proteins over 600 s of TCR engagement. We showed that the TCR signal-transduction network comprises at least 277 unique proteins involved in 366 high-confidence interactions, and that TCR signals diversify extensively at the level of the plasma membrane. Integrating the cellular abundance of the interacting proteins and their interaction stoichiometry provided a quantitative and contextual view of each documented interaction, permitting anticipation of whether ablation of a single interacting protein can impinge on the whole TCR signal-transduction network.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Mapas de Interação de Proteínas/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Camundongos , Camundongos Transgênicos , Cultura Primária de Células , Mapeamento de Interação de Proteínas/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/genéticaRESUMO
Members of the family of NOD-like receptors (NLRs) play essential roles in innate immunity by detecting intracellular 'pathogen-associated molecular patterns' (PAMPs) and 'danger-associated molecular patterns' (DAMPs). These molecules reveal the presence of pathogenic infection, abiotic stress, environmental insults, cellular damage, and cell death. NLR family members can be divided in two functional groups. One group consists of intracellular receptors, such as NLRP1, NLRP3, NLRP6 and NLRC4, which mediate the assembly of inflammasome complexes leading to the activation of procaspase-1. The second group includes members such as NOD1 and NOD2, and mediates the assembly of complexes that activate MAPK and NF-κB signaling pathways. We review the roles of NLR family members in health and disease, with emphasis on the signaling mechanisms in cell death and inflammation.
Assuntos
Proteínas de Transporte/imunologia , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Animais , Caspase 1/imunologia , Humanos , Inflamassomos/imunologia , NF-kappa B/imunologia , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologiaRESUMO
Protein interaction domains belonging to the death domain-fold superfamily are six-helix bundles that mediate the assembly of large protein complexes involved in apoptotic and inflammatory signaling. Typically, death domains (DDs), a subfamily of the death domain-fold superfamily, harbor six delineated interaction patches on their surfaces that mediate three distinct and conserved types of interaction designated as types I, II, and III. Here, we show that caspase recruitment domains (CARDs), another subfamily of the death domain-fold superfamily, multimerize by employing at least two of the three reported interaction types that were identified in DDs. On the one hand, the CARD of procaspase-1 binds the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) through a type I interaction that involves a patch surrounding residue Asp-27. On the other hand, the CARD of procaspase-1 auto-oligomerizes through a type III interaction involving a patch surrounding residue Arg-45. This oligomerization allows binding of receptor-interacting protein 2 (RIP2). In addition, we show that a 1:1 interaction between ASC and procaspase-1 is sufficient for procaspase-1 to gain proteolytic activity, whereas the formation of a higher order CARD complex involving ASC, procaspase-1, and RIP2 is required for effective procaspase-1-mediated NF-κB activation. These findings indicate that the CARD of procaspase-1 is differently involved in the formation of procaspase-1 activating platforms and procaspase-1-mediated, RIP2-dependent NF-κB activation.
Assuntos
Caspase 1/metabolismo , Complexos Multienzimáticos/metabolismo , NF-kappa B/metabolismo , Multimerização Proteica/fisiologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais/fisiologia , Caspase 1/genética , Ativação Enzimática/fisiologia , Células HEK293 , Humanos , Complexos Multienzimáticos/genética , NF-kappa B/genética , Estrutura Terciária de Proteína , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genéticaRESUMO
The death-fold superfamily encompasses four structurally homologous subfamilies that engage in homotypic, subfamily-restricted interactions. The Death Domains (DDs), the Death Effector Domains (DEDs), the CAspase Recruitment Domains (CARDs) and the PYrin Domains (PYDs) constitute key building blocks involved in the assembly of multimeric complexes implicated in signaling cascades leading to inflammation and cell death. We review the molecular basis of these homotypic domain-domain interactions in light of their structure, function and evolution. In addition, we elaborate on three distinct types of asymmetric interactions that were recently identified from the crystal structures of three multimeric, death-fold complexes: the MyDDosome, the PIDDosome and the Fas/FADD-DISC. Insights into the mechanisms of interaction of death-fold domains will be useful to design strategies for specific modulation of complex formation and might lead to novel therapeutic applications.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Evolução Molecular , Humanos , Mediadores da Inflamação/química , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Filogenia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Estrutura Secundária de ProteínaRESUMO
Interleukin-33 (IL-33) is a member of the IL-1 family and is involved in polarization of T cells toward a T helper 2 (Th2) cell phenotype. IL-33 is thought to be activated via caspase-1-dependent proteolysis, similar to the proinflammatory cytokines IL-1 beta and IL-18, but this remains unproven. Here we showed that IL-33 was processed by caspases activated during apoptosis (caspase-3 and -7) but was not a physiological substrate for caspases associated with inflammation (caspase-1, -4, and -5). Furthermore, caspase-dependent processing of IL-33 was not required for ST2 receptor binding or ST2-dependent activation of the NF-kappaB transcription factor. Indeed, caspase-dependent proteolysis of IL-33 dramatically attenuated IL-33 bioactivity in vitro and in vivo. These data suggest that IL-33 does not require proteolysis for activation, but rather, that IL-33 bioactivity is diminished through caspase-dependent proteolysis within apoptotic cells. Thus, caspase-mediated proteolysis acts as a switch to dampen the proinflammatory properties of IL-33.
Assuntos
Caspase 1/imunologia , Caspase 3/imunologia , Caspase 7/imunologia , Interleucinas/imunologia , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Animais , Apoptose/imunologia , Caspase 1/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/genética , Interleucinas/metabolismo , Linfócitos/enzimologia , Linfócitos/imunologia , Linfócitos/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia , NF-kappa B/metabolismo , Receptores de InterleucinaRESUMO
This review provides an overview of the biochemistry and activation of inflammatory caspases, and focuses on their therapeutic potential as disease targets in pathologies such as sepsis, Crohn's disease, rheumatoid arthritis, traumatic brain injury and amyotrophic lateral sclerosis (ALS). We summarize the proof-of-principal evidence obtained by studies in several corresponding experimental disease models confirming the validity of strategies targeting inflammatory caspases. We discuss the use of inflammatory caspase inhibitors, such as VX-740 (Pralnacasan) and VX-765, in clinical studies for rheumatoid arthritis and osteoarthritis. Finally, we point out recent approaches identifying novel peptidomimetic or non-peptide caspase inhibitors with suitable clinical profiles.