RESUMO
DJ-1 was recently identified as a gene product responsible for a subset of familial Parkinson's disease (PD). The mechanisms by which mutations in DJ-1 alter its function and account for PD-related pathology remained largely unknown. We show that DJ-1 is processed by caspase-6 and that the caspase-6-derived C-terminal fragment of DJ-1 fully accounts for associated p53-dependent cell death. In line with the above data, we show that a recently described early-onset PD-associated mutation (D149A) renders DJ-1 resistant to caspase-6 proteolysis and abolishes its protective phenotype. Unlike the D149A mutation, the L166P mutation that prevents DJ-1 dimerization does not impair its proteolysis by caspase-6 although it also abolishes DJ-1 antiapoptotic function. Therefore, we show here that DJ-1 loss of function could be due to impaired caspase-6 proteolysis and we document the fact that various DJ-1 mutations could lead to PD pathology through distinct molecular mechanisms.
Assuntos
Caspase 6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Proteínas Oncogênicas/genética , Doença de Parkinson/genética , Substituição de Aminoácidos , Animais , Apoptose , Encéfalo/metabolismo , Células Cultivadas , Dimerização , Regulação para Baixo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas/metabolismo , Doença de Parkinson/metabolismo , Proteína Desglicase DJ-1 , Proteína Supressora de Tumor p53/metabolismoRESUMO
The present study analyses, by two-dimensional polyacrylamide gel electrophoresis, the protease SP220K isolated from renal cell carcinoma. The pure molecule is separated using either immobilized pH gradient or isoelectric focusing in conventional carrier ampholyte in the first dimension. Some interactions with the acrylamide matrix in isoelectric focusing are discussed. The results demonstrate that two-dimensional gel electrophoresis performed with enriched media such as basolateral membranes, allows the detection of the protease. In addition, the non detection of the molecule up to now by this methodology can be explained by the high tendency of oligomerization of SP220K. Effectively the high molecular weight form of the molecule of 220 kDa is favoured in two-dimensional gel electrophoresis over monomeric forms which are better detected in SDS PAGE. This was confirmed by immunostaining performed with an antiserum to SP220K produced by nitrocellulose-bound antigen.