RESUMO
Cellular defects which prevent apoptotic cell death can result in the generation of hyperproliferative disorders and can prevent the effective treatment of such diseases. The majority of cellular defects which result in apoptosis resistance lie upstream of caspase activation. We have described chimeric caspase molecules consisting of the prodomain of caspase-2 fused to the amino terminus of caspase-3, and which are tagged at the carboxyl terminus with green fluorescent protein (GFP) to allow direct visualisation of transfected cells. Here we show that these chimeric caspase molecules possess potent, rapid cell-killing activity in cell lines which display a range of defects resulting in apoptosis resistance.
Assuntos
Apoptose , Caspases/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia de Células T/metabolismo , Osteossarcoma/metabolismo , Proteínas Recombinantes de Fusão/toxicidade , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Animais , Células COS/efeitos dos fármacos , Células COS/metabolismo , Caspase 2 , Caspase 3 , Sobrevivência Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fluorescência Verde , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia de Células T/genética , Proteínas Luminescentes/genética , Osteossarcoma/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
RAIDD, a caspase recruitment domain (CARD) containing molecule, interacts with procaspase-2 in a CARD-dependent manner. This interaction has been suggested to mediate the recruitment of caspase-2 to the tumour necrosis factor receptor 1 (TNFR1). In this paper we have studied the subcellular localization of RAIDD and its interaction with caspase-2. We demonstrate that endogenous RAIDD is mostly localized in the cytoplasm and to some extent in the nucleus. RAIDD localization is not affected by TNF-treatment of HeLa cells, but in cells ectopically expressing caspase-2, a fraction of RAIDD is recruited to the nucleus. In transfected cells, coexpression of RAIDD and caspase-2 leads to CARD-dependent colocalization of the two proteins to discrete subcellular structures. We further show that overexpression of the RAIDD-CARD results in the formation of filamentous structures due to CARD-mediated oligomerization. These structures were similar to death effector filaments (DEFs) formed by FADD and FLICE death effector domains (DEDs), and partially colocalized with DEFs. Our results suggest that similar to the DED, the RAIDD-CARD has the ability to form higher order complexes, believed to be important in apoptotic execution. We also present evidence that RAIDD-CARD oligomerization may be regulated by intramolecular folding of the RAIDD molecule.
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Caspase 2 , Caspases/metabolismo , Dimerização , Células HeLa , Humanos , Dobramento de Proteína , Receptores do Fator de Necrose Tumoral/metabolismoRESUMO
Caspases are a family of evolutionarily conserved cysteine proteases that constitute the effector arm of the apoptotic machinery. Studies in Caenorhabditis elegans, Drosophila melanogaster, and mouse point to evolutionarily conserved caspase function in developmentally programmed cell death in metazoans. Whereas in the nematode all developmental cell death is mediated by a single caspase, in Drosophila and the mouse some caspases appear to regulate cell death in a spatio-temporally restricted manner. This article reviews what we currently know about the roles of various caspases in the execution of developmentally programmed cell death and what may be expected from future research in this field.
Assuntos
Apoptose/fisiologia , Caspases/fisiologia , Animais , Apoptose/genética , Caenorhabditis elegans/citologia , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Caspases/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Genes de Helmintos , Genes de Insetos , Camundongos , MutaçãoRESUMO
Caspases are cysteine proteases that play an essential role in apoptosis. Initial activation of caspases defines the key step in apoptotic execution. Based on primary structure, caspases can be divided into two groups, those with long amino-terminal prodomains (class I), and those with relatively short prodomains (class II). On overexpression in mammalian cells, class I caspases can induce cell death that is dependent on their autocatalytic activity. Recent studies suggest that the long prodomains in some class I caspases are able to mediate dimerization of procaspase molecules, thereby promoting autoprocessing. In this communication, we demonstrate that fusion of the prodomain of a class I caspase (Nedd2/caspase-2) with procaspase-3 greatly augments autocatalysis and apoptosis induction by the chimeric caspase-3 molecule. The chimeric caspase-3 molecules were able to form homodimers in Saccharomyces cerevisiae and were efficiently processed in transfected mammalian cells. These results provide direct evidence for a role of a class I caspase prodomain in caspase autoactivation and processing and establish a basis for functional hierarchy among the two classes of caspases.
