RESUMO
PTEN-induced putative kinase 1 (Pink1) is a recently identified gene linked to a recessive form of familial Parkinson's disease (PD). The kinase contains a mitochondrial localization sequence and is reported to reside, at least in part, in mitochondria. However, neither the manner by which the loss of Pink1 contributes to dopamine neuron loss nor its impact on mitochondrial function and relevance to death is clear. Here, we report that depletion of Pink1 by RNAi increased neuronal toxicity induced by MPP(+). Moreover, wild-type Pink1, but not the G309D mutant linked to familial PD or an engineered kinase-dead mutant K219M, protects neurons against MPTP both in vitro and in vivo. Intriguingly, a mutant that contains a deletion of the putative mitochondrial-targeting motif was targeted to the cytoplasm but still provided protection against 1-methyl-4-phenylpyridine (MPP(+))/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity. In addition, we also show that endogenous Pink1 is localized to cytosolic as well as mitochondrial fractions. Thus, our findings indicate that Pink1 plays a functional role in the survival of neurons and that cytoplasmic targets, in addition to its other actions in the mitochondria, may be important for this protective effect.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/antagonistas & inibidores , Neurônios/enzimologia , Neurotoxinas/antagonistas & inibidores , Doença de Parkinson/enzimologia , Proteínas Quinases/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Sobrevivência Celular/genética , Citoplasma/enzimologia , Dopaminérgicos/toxicidade , Camundongos , Camundongos Endogâmicos , Mitocôndrias/enzimologia , Mutação , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Doença de Parkinson/genética , Proteínas Quinases/análise , Proteínas Quinases/genética , Interferência de RNARESUMO
Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Oncogênicas/metabolismo , Transtornos Parkinsonianos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Encéfalo/metabolismo , Linhagem Celular , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Genes Mitocondriais , Genes Recessivos , Glutationa/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Estresse Oxidativo , PTEN Fosfo-Hidrolase/metabolismo , Transtornos Parkinsonianos/genética , Peroxirredoxinas , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Desglicase DJ-1 , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The formation of cysteine-sulfinic acid has recently become appreciated as a modification that links protein function to cellular oxidative status. Human DJ-1, a protein associated with inherited parkinsonism, readily forms cysteine-sulfinic acid at a conserved cysteine residue (Cys106 in human DJ-1). Mutation of Cys106 causes the protein to lose its normal protective function in cell culture and model organisms. However, it is unknown whether the loss of DJ-1 protective function in these mutants is due to the absence of Cys106 oxidation or the absence of the cysteine residue itself. To address this question, we designed a series of substitutions at a proximal glutamic acid residue (Glu18) in human DJ-1 that alter the oxidative propensity of Cys106 through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys106 to be oxidized to Cys106-sulfinic acid under mild conditions. In contrast, the E18D mutation stabilizes a cysteine-sulfenic acid that is readily reduced to the thiol in solution and in vivo. We show that E18N and E18Q can both partially substitute for wild-type DJ-1 using mitochondrial fission and cell viability assays. In contrast, the oxidatively impaired E18D mutant behaves as an inactive C106A mutant and fails to protect cells. We therefore conclude that formation of Cys106-sulfinic acid is a key modification that regulates the protective function of DJ-1.
Assuntos
Cisteína/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Oncogênicas/metabolismo , Ácidos Sulfínicos/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Cisteína/genética , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas Mitocondriais/genética , Mutação , Proteínas Oncogênicas/genética , Oxirredução , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteína Desglicase DJ-1RESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial and apparently sporadic Parkinson disease. LRRK2 is a multidomain protein kinase with autophosphorylation activity. It has previously been shown that the kinase activity of LRRK2 is required for neuronal toxicity, suggesting that understanding the mechanism of kinase activation and regulation may be important for the development of specific kinase inhibitors for Parkinson disease treatment. Here, we show that LRRK2 predominantly exists as a dimer under native conditions, a state that appears to be stabilized by multiple domain-domain interactions. Furthermore, an intact C terminus, but not N terminus, is required for autophosphorylation activity. We identify two residues in the activation loop that contribute to the regulation of LRRK2 autophosphorylation. Finally, we demonstrate that LRRK2 undergoes intramolecular autophosphorylation. Together, these results provide insight into the mechanism and regulation of LRRK2 kinase activity.