RESUMO
Dysfunction of proteasomal protein degradation is involved in neurodegeneration in Parkinson's disease (PD). Recently we identified the regulatory proteasomal subunit S6 ATPase as a novel interactor of synphilin-1, which is a substrate of the ubiquitin-ligase Parkin (PARK2) and an interacting protein of alpha-synuclein (PARK1). To further investigate a potential role in the pathogenesis of PD, we performed a detailed mutation analysis of the S6 ATPase gene in a large sample of 486 German sporadic and familial PD patients. Direct sequencing revealed two novel intronic variants. An insertion/deletion variant in intron 5 of the S6 ATPase gene was more frequent in patients compared to controls. Moreover, this variant was significantly more frequent in early-onset compared to late-onset PD patients. The identification of a genetic link between a regulatory proteasomal subunit and PD further underscores the relevance of disturbed protein degradation in PD.
Assuntos
Adenosina Trifosfatases/genética , Doença de Parkinson/epidemiologia , Doença de Parkinson/genética , Complexo de Endopeptidases do Proteassoma/genética , ATPases Associadas a Diversas Atividades Celulares , Idoso , Alelos , Proteínas de Transporte/genética , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , DNA Complementar/biossíntese , DNA Complementar/genética , Feminino , Genótipo , Alemanha/epidemiologia , Humanos , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Polimorfismo de Fragmento de Restrição , Desnaturação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Synphilin-1 is linked to Parkinson's disease (PD), based on its role as an alpha-synuclein (PARK1)-interacting protein and substrate of the ubiquitin E3 ligase Parkin (PARK2) and because of its presence in Lewy bodies (LB) in brains of PD patients. We found that overexpression of synphilin-1 in cells leads to the formation of ubiquitinated cytoplasmic inclusions supporting a derangement of the ubiquitin-proteasome system in PD. We report here a novel specific interaction of synphilin-1 with the regulatory proteasomal protein S6 ATPase (tbp7). Functional characterization of this interaction on a cellular level revealed colocalization of S6 and synphilin-1 in aggresome-like intracytoplasmic inclusions. Overexpression of synphilin-1 and S6 in cells caused reduced proteasomal activity associated with a significant increase in inclusion formation compared to cells expressing synphilin-1 alone. Steady-state levels of synphilin-1 in cells were not altered after cotransfection of S6 and colocalization of synphilin-1-positive inclusions with lysosomal markers suggests the presence of an alternative lysosomal degradation pathway. Subsequent immunohistochemical studies in brains of PD patients identified S6 ATPase as a component of LB. This is the first study investigating the physiological role of synphilin-1 in the ubiquitin proteasome system. Our data suggest a direct interaction of synphilin-1 with the regulatory complex of the proteasome modulating proteasomal function.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/etiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Encéfalo/patologia , Humanos , Corpos de Inclusão/metabolismo , Corpos de Lewy , Lisossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologiaRESUMO
Recently targeted disruption of Omi/HtrA2 has been found to cause neurodegeneration and a parkinsonian phenotype in mice. Using a candidate gene approach, we performed a mutation screening of the Omi/HtrA2 gene in German Parkinson's disease (PD) patients. In four patients, we identified a novel heterozygous G399S mutation, which was absent in healthy controls. Moreover, we identified a novel A141S polymorphism that was associated with PD (P<0.05). Both mutations resulted in defective activation of the protease activity of Omi/HtrA2. Immunohistochemistry and functional analysis in stably transfected cells revealed that S399 mutant Omi/HtrA2 and to a lesser extent, the risk allele of the A141S polymorphism induced mitochondrial dysfunction associated with altered mitochondrial morphology. Cells overexpressing S399 mutant Omi/HtrA2 were more susceptible to stress-induced cell death than wild-type. On the basis of functional genomics, our results provide a novel link between mitochondrial dysfunction and neurodegeneration in PD.
