RESUMEN
Death receptors in the TNF receptor superfamily signal for apoptosis via the ordered recruitment of FADD and caspase-8 to a death-inducing signaling complex (DISC). However, the nature of the protein-protein interactions in the signaling complex is not well defined. Here we show that FADD self-associates through a conserved RXDLL motif in the death effector domain (DED). Despite exhibiting similar binding to both Fas and caspase-8 and preserved overall secondary structure, FADD RDXLL motif mutants cannot reconstitute FasL- or TRAIL-induced apoptosis and fail to recruit caspase-8 into the DISC of reconstituted FADD-deficient cells. Abolishing self-association can transform FADD into a dominant-negative mutant that interferes with Fas-induced apoptosis and formation of microscopically visible receptor oligomers. These findings suggest that lateral interactions among adapter molecules are required for death receptor apoptosis signaling and implicate self-association into oligomeric assemblies as a key function of death receptor adapter proteins in initiating apoptosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/fisiología , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Secuencia Conservada , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Células Jurkat , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Mature T cells initially respond to Ag by activation and expansion, but high and repeated doses of Ag cause programmed cell death and can suppress T cell-mediated diseases in rodents. We evaluated repeated systemic Ag administration in a marmoset model of experimental allergic encephalomyelitis that closely resembles the human disease multiple sclerosis. We found that treatment with MP4, a chimeric, recombinant polypeptide containing human myelin basic protein and human proteolipid protein epitopes, prevented clinical symptoms and did not exacerbate disease. CNS lesions were also reduced as assessed in vivo by magnetic resonance imaging. Thus, specific Ag-directed therapy can be effective and nontoxic in primates.
Asunto(s)
Callithrix/inmunología , Epítopos Inmunodominantes/inmunología , Inmunoterapia Activa/métodos , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Proteína Básica de Mielina/inmunología , Proteína Proteolipídica de la Mielina/inmunología , Animales , Autoanticuerpos/biosíntesis , Encéfalo/patología , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Relación Dosis-Respuesta Inmunológica , Esquema de Medicación , Epítopos Inmunodominantes/administración & dosificación , Inmunosupresores/administración & dosificación , Inmunosupresores/inmunología , Inyecciones Intravenosas , Activación de Linfocitos/inmunología , Imagen por Resonancia Magnética , Masculino , Esclerosis Múltiple/patología , Proteína Básica de Mielina/administración & dosificación , Proteína Proteolipídica de la Mielina/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Cross-linking of the B cell antigen receptor (BCR) induces resistance to Fas (APO-1 / CD95)-dependent apoptosis and thereby regulates one mechanism of B cell selection during antigen stimulation. To investigate the molecular mechanism by which BCR signaling regulates the Fas pathway, we examined the expression of constituents of the death-inducing signaling complex (DISC), including Fas, FADD, caspase-8 and cellular FLICE-inhibitory protein (c-FLIP). No significant changes in the cellular levels of Fas, FADD or caspase-8 were observed after BCR cross-linking. By contrast, the long isoform of c-FLIP (c-FLIP(L)) was significantly up-regulated by BCR cross-linking in primary B cells and in two B cell lines, A20 and WEHI-279. Moreover, transfection of c-FLIP(L) into A20 cells inhibited Fas-dependent apoptosis and suppressed recruitment of caspase-8 to the DISC. BCR cross-linking or FLIP overexpression also protects B cells from TRAIL-induced apoptosis. Thus, BCR signaling up-regulates c-FLIP(L) and suppresses the Fas- and TRAIL-receptor apoptosis pathways which could be important for tolerance and selection of antigen-specific B cells.
Asunto(s)
Apoptosis , Linfocitos B/fisiología , Proteínas Portadoras/fisiología , Péptidos y Proteínas de Señalización Intracelular , Receptores de Antígenos de Linfocitos B/fisiología , Receptor fas/fisiología , Animales , Anticuerpos Antiidiotipos/inmunología , Proteínas Reguladoras de la Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Femenino , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia ArribaRESUMEN
Definition of the immune process that causes demyelination in multiple sclerosis is essential to determine the feasibility of Ag-directed immunotherapy. Using the nonhuman primate, Callithrix jacchus jacchus (common marmoset), we show that immunization with myelin basic protein and proteolipid protein determinants results in clinical disease with significant demyelination. Demyelination was associated with spreading to myelin oligodendrocyte glycoprotein (MOG) determinants that generated anti-MOG serum Abs and Ig deposition in central nervous system white matter lesions. These data associate intermolecular "determinant spreading" with clinical autoimmune disease in primates and raise important issues for the pathogenesis and treatment of multiple sclerosis.