RESUMEN
Cellular FLICE-like inhibitory protein (c-FLIP) regulates extrinsic apoptosis by controlling procaspase-8 activation through its tandem N-terminal death effector domains (DEDs). Here, we present the expression and purification of c-FLIP tandem DEDs (tDED) from Escherichia coli. We observed that the c-FLIPtDED maintains monomeric form under near-physiological pH condition in vitro. Our results also reveal a significant correlation between the pH conditions and the structure of c-FLIPtDED (F114A). The described methods and results would be helpful for follow-up study on the structural and functional of c-FLIP.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Dominio Efector de Muerte , Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Estudios de SeguimientoRESUMEN
The formation of death-inducing signaling complex (DISC) and death effector domain (DED) filament initiates extrinsic apoptosis. Recruitment and activation of procaspase-8 at the DISC are regulated by c-FLIP. The interaction between c-FLIP and procaspase-8 is mediated by their tandem DEDs (tDED). However, the structure of c-FLIPtDED and how c-FLIP interferes with procaspase-8 activation at the DISC remain elusive. Here, we solved the monomeric structure of c-FLIPtDED (F114G) at near physiological pH by solution nuclear magnetic resonance (NMR). Structural superimposition reveals c-FLIPtDED (F114G) adopts a structural topology similar to that of procaspase-8tDED. Our results provide a structural basis for understanding how c-FLIP interacts with procaspase-8 and the molecular mechanisms of c-FLIP in regulating cell death.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Dominio Efector de Muerte , Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/metabolismo , Transducción de SeñalRESUMEN
Apoptosis is a process of programmed cell death which has an important role in tissue homeostasis and in the control of organism development. Here, we focus on information concerning the role of the extrinsic apoptotic pathway in the control of human erythropoiesis. We discuss the role of tumor necrosis factor α (TNFα), tumor necrosis factor ligand superfamily member 6 (FasL), tumor necrosis factor-related apoptosis-inducing (TRAIL) and caspases in normal erythroid maturation. We also attempt to initiate a discussion on the observations that mature erythrocytes contain most components of the receptor-dependent apoptotic pathway. Finally, we point to the role of the extrinsic apoptotic pathway in ineffective erythropoiesis of different types of ß-thalassemia.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/genética , Eritropoyesis/genética , Transducción de Señal/genética , Talasemia beta/sangre , Caspasas/metabolismo , Dominio Efector de Muerte/genética , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Eritropoyesis/efectos de los fármacos , Proteína Ligando Fas/metabolismo , Humanos , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Talasemia beta/genéticaRESUMEN
The assembly of the death-inducing signaling complex (DISC) and death effector domain (DED) filaments at CD95/Fas initiates extrinsic apoptosis. Procaspase-8 activation at the DED filaments is controlled by short and long c-FLIP isoforms. Despite apparent progress in understanding the assembly of CD95-activated platforms and DED filaments, the detailed molecular mechanism of c-FLIP action remains elusive. Here, we further addressed the mechanisms of c-FLIP action at the DISC using biochemical assays, quantitative mass spectrometry, and structural modeling. Our data strongly indicate that c-FLIP can bind to both FADD and procaspase-8 at the DED filament. Moreover, the constructed in silico model shows that c-FLIP proteins can lead to the formation of the DISCs comprising short DED filaments as well as serve as bridging motifs for building a cooperative DISC network, in which adjacent CD95 DISCs are connected by DED filaments. This network is based on selective interactions of FADD with both c-FLIP and procaspase-8. Hence, c-FLIP proteins at the DISC control initiation, elongation, and composition of DED filaments, playing the role of control checkpoints. These findings provide new insights into DISC and DED filament regulation and open innovative possibilities for targeting the extrinsic apoptosis pathway.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Dominio Efector de Muerte , Secuencia de Aminoácidos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/química , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Células HeLa , Humanos , Células Jurkat , Modelos Moleculares , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Receptor fas/metabolismoRESUMEN
Caspase-8 is an apoptotic protease that is activated by a proximity-induced dimerization mechanism within the death-inducing signaling complex (DISC). The death effector domain (DED) of caspase-8 is involved in protein-protein interactions and is essential for the activation. Here, we report two crystal structures of the dimeric DEDs of the F122A mutant of caspase-8, both of which illustrate a novel domain-swapped dimerization, while differ in the relative orientation of the two subunits and the solvent exposure of the conserved hydrophobic patch Phe122/Leu123. We demonstrate that mutations disrupting the dimerization of the DEDs abrogate the formation of cellular death effector filaments (DEFs) and the induced apoptosis by overexpressed DEDs. Furthermore, such dimerization-disrupting mutations also impair the activation of the full-length caspase-8 and the downstream apoptosis cascade. The structures provide new insights into understanding the mechanism underlying the activation of procaspase-8 within the DISC and DEFs.
Asunto(s)
Caspasa 8/química , Caspasa 8/genética , Dominio Efector de Muerte , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación Puntual , Multimerización de Proteína , Caspasa 8/metabolismo , Cristalografía por Rayos X , Estabilidad de Enzimas , Células HeLa , Humanos , Células Jurkat , Modelos Moleculares , Proteínas Mutantes/metabolismo , Estructura Cuaternaria de Proteína , Solubilidad , Receptor fas/metabolismoRESUMEN
Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.