RESUMO
Acidic noncaspase proteases-like cathepsins have been introduced as novel mediators of apoptosis. A clear role for these proteases and the acidic endolysosomal compartment in apoptotic signalling is not yet defined. To understand the role and significance of noncaspases in promoting and mediating cell death, it is important to determine whether an intersection of these proteases and the caspase pathway exists. We recently identified the endolysosomal aspartate protease cathepsin D (CTSD) as a target for the proapoptotic lipid ceramide. Here, we show that tumor necrosis factor (TNF)-induced CTSD activation depends on functional acid sphingomyelinase (A-SMase) expression. Ectopic expression of CTSD in CTSD-deficient fibroblasts results in an enhanced TNF-mediated apoptotic response. Intracellular colocalization of CTSD with the proapoptotic bcl-2 protein family member Bid in HeLa cells, and the ability of CTSD to cleave directly Bid in vitro as well as the lack of Bid activation in cathepsin-deficient fibroblasts indicate that Bid represents a direct downstream target of CTSD. Costaining of CTSD and Bid with Rab5 suggests that the endosomal compartments are the common 'meeting point'. Caspase-9 and -3 activation also was in part dependent on A-SMase and CTSD expression as revealed in the respective deficiency models. Our results link as novel endosomal intermediates the A-SMase and the acid aspartate protease CTSD to the mitochondrial apoptotic TNF pathway.
Assuntos
Proteínas de Transporte/metabolismo , Caspases/metabolismo , Catepsina D/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Caspase 3 , Caspase 9 , Células Cultivadas , Ceramidas/metabolismo , Ativação Enzimática/fisiologia , Feminino , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Esfingomielina Fosfodiesterase/metabolismoAssuntos
Azirinas , Proteínas de Transporte/metabolismo , Ceramidas/metabolismo , Proteínas de Membrana/metabolismo , Marcadores de Afinidade , Animais , Proteínas de Transporte/isolamento & purificação , Cromatografia de Afinidade/métodos , Indicadores e Reagentes , Radioisótopos do Iodo , Proteínas de Membrana/isolamento & purificaçãoRESUMO
We have identified the aspartic protease cathepsin D as a novel intracellular target protein for the lipid second messenger ceramide. Ceramide specifically binds to and induces CTSD proteolytic activity. A-SMase deficient cells derived from Niemann-Pick patients show decreased CTSD activity that was reconstituted by transfection with A-SMase cDNA. Ceramide accumulation in cells derived from A-ceramidase defective Farber patients correlates with enhanced CTSD activity. These findings suggest that A-SMase-derived ceramide targets endolysosomal CTSD.
Assuntos
Catepsina D/metabolismo , Ceramidas/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Esfingomielina Fosfodiesterase/fisiologia , Amidoidrolases/deficiência , Amidoidrolases/genética , Animais , Apoptose , Catepsina D/deficiência , Catepsina D/genética , Compartimento Celular , Linhagem Celular Transformada , Ceramidases , Ceramidas/farmacologia , Citocinas/fisiologia , DNA Complementar/genética , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/patologia , Células HeLa/metabolismo , Humanos , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/metabolismo , Metabolismo dos Lipídeos , Linfócitos/enzimologia , Lisossomos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças de Niemann-Pick/enzimologia , Doenças de Niemann-Pick/genética , Doenças de Niemann-Pick/patologia , Proteínas Recombinantes de Fusão/metabolismo , Esfingomielina Fosfodiesterase/deficiência , Esfingomielina Fosfodiesterase/genética , Células Tumorais Cultivadas , Células U937/metabolismoRESUMO
Ceramide has been recognized as a common intracellular second messenger for various cytokines, growth factors and other stimuli, such as CD95, chemotherapeutic drugs and stress factors. To understand the role of ceramide during apoptosis and other cellular responses, it is critically important to characterize direct targets of ceramide action. In this paper, we show that ceramide specifically binds to and activates the endosomal acidic aspartate protease cathepsin D. Direct interaction of ceramide with cathepsin D results in autocatalytic proteolysis of the 52 kDa pre-pro cathepsin D to form the enzymatically active 48/32 kDa isoforms of cathepsin D. Acid sphingomyelinase (A-SMase)-deficient cells show decreased cathepsin D activity, which could be reconstituted by transfection with A-SMase cDNA. The results of our study identify cathepsin D as the first endosomal ceramide target that colocalizes with and may mediate downstream signaling effects of A-SMase.
Assuntos
Catepsina D/metabolismo , Ceramidas/metabolismo , Esfingomielina Fosfodiesterase/química , Animais , Sequência de Bases , Ceramidas/química , Primers do DNA , Ativação Enzimática , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Processamento Pós-Transcricional do RNA , Especificidade por SubstratoRESUMO
The 55-kDa receptor for tumor necrosis factor (TR55) triggers multiple signaling cascades initiated by adapter proteins like TRADD and FAN. By use of the primary amine monodansylcadaverine (MDC), we addressed the functional role of tumor necrosis factor (TNF) receptor internalization for intracellular signal distribution. We show that MDC does not prevent the interaction of the p55 TNF receptor (TR55) with FAN and TRADD. Furthermore, the activation of plasmamembrane-associated neutral sphingomyelinase activation as well as the stimulation of proline-directed protein kinases were not affected in MDC-treated cells. In contrast, activation of signaling enzymes that are linked to the "death domain" of TR55, like acid sphingomyelinase and c-Jun-N-terminal protein kinase as well as TNF signaling of apoptosis in U937 and L929 cells, are blocked in the presence of MDC. The results of our study suggest a role of TR55 internalization for the activation of select TR55 death domain signaling pathways including those leading to apoptosis.
