RESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/fisiologia , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Sequência Conservada , Proteína de Domínio de Morte Associada a Fas , Humanos , Células Jurkat , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
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.
Assuntos
Callithrix/imunologia , Epitopos Imunodominantes/imunologia , Imunoterapia Ativa/métodos , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Proteína Básica da Mielina/imunologia , Proteína Proteolipídica de Mielina/imunologia , Animais , Autoanticorpos/biossíntese , Encéfalo/patologia , Citocinas/biossíntese , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Esquema de Medicação , Epitopos Imunodominantes/administração & dosagem , Imunossupressores/administração & dosagem , Imunossupressores/imunologia , Injeções Intravenosas , Ativação Linfocitária/imunologia , Imageamento por Ressonância Magnética , Masculino , Esclerose Múltipla/patologia , Proteína Básica da Mielina/administração & dosagem , Proteína Proteolipídica de Mielina/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismoRESUMO
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.
Assuntos
Apoptose , Linfócitos B/fisiologia , Proteínas de Transporte/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Receptores de Antígenos de Linfócitos B/fisiologia , Receptor fas/fisiologia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Proteínas Reguladoras de Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Feminino , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/fisiologia , Regulação para CimaRESUMO
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.
