RESUMO
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and results in the activation of the unfolded protein response (UPR), which aims at restoring ER homeostasis. However, when the stress is too severe the UPR switches from being a pro-survival response to a pro-death one, and the molecular mechanisms underlying ER stress-mediated death have remained incompletely understood. In this study, we identified receptor interacting protein kinase 1 (RIPK1)-a kinase at the crossroad between life and death downstream of various receptors-as a new regulator of ER stress-induced death. We found that Ripk1-deficient MEFs are protected from apoptosis induced by ER stressors, which is reflected by reduced caspase activation and PARP processing. Interestingly, the pro-apoptotic role of Ripk1 is independent of its kinase activity, is not regulated by its cIAP1/2-mediated ubiquitylation, and does not rely on the direct regulation of JNK or CHOP, two reportedly main players in ER stress-induced death. Instead, we found that ER stress-induced apoptosis in these cells relies on death receptor-independent activation of caspase-8, and identified Ripk1 upstream of caspase-8. However, in contrast to RIPK1-dependent apoptosis downstream of TNFR1, we did not find Ripk1 associated with caspase-8 in a death-inducing complex upon unresolved ER stress. Our data rather suggest that RIPK1 indirectly regulates caspase-8 activation, in part via interaction with the ER stress sensor inositol-requiring protein 1 (IRE1).
Assuntos
Apoptose/genética , Caspase 8/genética , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Doxiciclina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/genética , Fibroblastos , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genéticaRESUMO
Autophagy and apoptosis are two important and interconnected stress-response mechanisms. However, the molecular interplay between these two pathways is not fully understood. To study the fate and function of autophagic proteins at the onset of apoptosis, we used a cellular model system in which autophagy precedes apoptosis. IL-3 depletion of Ba/F3 cells caused caspase (casp)-mediated cleavage of Beclin-1 and PI3KC3, two crucial components of the autophagy-inducing complex. We identified two casp cleavage sites in Beclin-1, TDVD(133) and DQLD(149), cleavage at which yields fragments lacking the autophagy-inducing capacity. Noteworthy, the C-terminal fragment, Beclin-1-C, localized predominantly at the mitochondria and sensitized the cells to apoptosis. Moreover, on isolated mitochondria, recombinant Beclin-1-C was able to induce the release of proapoptotic factors. These findings point to a mechanism by which casp-dependent generation of Beclin-1-C creates an amplifying loop enhancing apoptosis upon growth factor withdrawal.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Autofagia , Caspases/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/análise , Proteínas Reguladoras de Apoptose/genética , Proteína Beclina-1 , Linhagem Celular , Humanos , Interleucina-3/genética , Interleucina-3/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Caspase 1 is a cysteinyl aspartate-specific proteinase involved in the maturation of inflammatory cytokines such as pro-IL-1beta (interleukin-1beta) and pro-IL-18. Caspase 1 clusters phylogenetically together with human caspases 4, 5 and 12 and murine caspases 11 and 12, and forms the group of the so-called inflammatory caspases. Caspase 1 consists of an N-terminal CARD (caspase recruitment domain) and a proteolytic domain containing the catalytic residues. The CARD-containing prodomain is involved in the formation of the protease-activating inflammasome complex. We have also found that the prodomain is necessary and sufficient for the activation of NF-kappaB (nuclear factor kappaB). The human genome also contains three caspase-1-related CARD-only decoy proteins [COP (CARD-only protein), INCA (inhibitory CARD) and ICEBERG], which are located near the caspase 1 locus. In this mini-review, we focus on the evolutionary aspects of the inflammatory caspase locus in the human, chimpanzee, Rhesus monkey, mouse and rat. Furthermore, we discuss the functional characteristics of the caspase-1-related CARD-only proteins in relation to caspase-1-mediated IL-1beta maturation and NF-kappaB activation.