RESUMO
The CD95 (Fas/APO-1) death-inducing signaling complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC.
Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Transdução de Sinais , Receptor fas/química , Apoptose , Caspase 10/metabolismo , Caspase 8/metabolismo , Dimerização , Proteína de Domínio de Morte Associada a Fas/metabolismo , Células HeLa , Humanos , Espectrometria de Massas/métodos , Microscopia de Fluorescência/métodos , Modelos Biológicos , Modelos Teóricos , Receptor fas/metabolismoRESUMO
Cellular FADD-like interleukin-1beta-converting enzyme inhibitory proteins (c-FLIPs; isoforms c-FLIP long [c-FLIP(L)], c-FLIP short [c-FLIP(S)], and c-FLIP Raji [c-FLIP(R)]) regulate caspase-8 activation and death receptor (DR)-induced apoptosis. In this study, using a combination of mathematical modeling, imaging, and quantitative Western blots, we present a new mathematical model describing caspase-8 activation in quantitative terms, which highlights the influence of c-FLIP proteins on this process directly at the CD95 death-inducing signaling complex. We quantitatively define how the stoichiometry of c-FLIP proteins determines sensitivity toward CD95-induced apoptosis. We show that c-FLIP(L) has a proapoptotic role only upon moderate expression in combination with strong receptor stimulation or in the presence of high amounts of one of the short c-FLIP isoforms, c-FLIP(S) or c-FLIP(R). Our findings resolve the present controversial discussion on the function of c-FLIP(L) as a pro- or antiapoptotic protein in DR-mediated apoptosis and are important for understanding the regulation of CD95-induced apoptosis, where subtle differences in c-FLIP concentrations determine life or death of the cells.
Assuntos
Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Modelos Biológicos , Transdução de Sinais , Receptor fas/metabolismo , Algoritmos , Western Blotting , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Caspase 8/metabolismo , Simulação por Computador , Células HeLa , Humanos , Isoformas de Proteínas/metabolismo , Receptor fas/genéticaRESUMO
This study explores the dilemma in cellular signaling that triggering of CD95 (Fas/APO-1) in some situations results in cell death and in others leads to the activation of NF-kappaB. We established an integrated kinetic mathematical model for CD95-mediated apoptotic and NF-kappaB signaling. Systematic model reduction resulted in a surprisingly simple model well approximating experimentally observed dynamics. The model postulates a new link between c-FLIP(L) cleavage in the death-inducing signaling complex (DISC) and the NF-kappaB pathway. We validated experimentally that CD95 stimulation resulted in an interaction of p43-FLIP with the IKK complex followed by its activation. Furthermore, we showed that the apoptotic and NF-kappaB pathways diverge already at the DISC. Model and experimental analysis of DISC formation showed that a subtle balance of c-FLIP(L) and procaspase-8 determines life/death decisions in a nonlinear manner. We present an integrated model describing the complex dynamics of CD95-mediated apoptosis and NF-kappaB signaling.
Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Transdução de Sinais , Receptor fas/metabolismo , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspases/metabolismo , Morte Celular , Linhagem da Célula , Sobrevivência Celular , Ativação Enzimática , Células HeLa , Humanos , Quinase I-kappa B/metabolismo , Cinética , Modelos Biológicos , NF-kappa B/metabolismo , Ligação Proteica , Receptor fas/genéticaRESUMO
Apoptosis is a process common to all multicellular organisms. Apoptosis leads to the elimination of cells via a complex but highly defined cellular programme. Defects in the regulation of apoptosis result in serious diseases such as cancer, autoimmunity, AIDS and neurodegeneration. Recently, a substantial step forward in understanding the complex apoptotic pathways has been made by utilising systems biology approaches. Systems biology combines rigorous mathematical modelling with experimental approaches in a closed loop cycle for advancing our knowledge about complex biological processes. In this review we describe the contemporary systems biology studies devoted to apoptotic signalling and focus on the question of how systems biology helps to understand life/death decisions made in the cell and to develop new approaches to rational treatment strategies.