RESUMO
DJ-1 is a multifunctional protein that plays roles in transcriptional regulation and antioxidative stress, and loss of its function is thought to result in the onset of Parkinson's disease (PD). Here, we report that DJ-1 was sumoylated on a lysine residue at amino-acid number 130 (K130) by PIASxalpha or PIASy. The K130 mutation abrogated all of the functions of DJ-1, including ras-dependent transformation, cell growth promotion and anti-UV-induced apoptosis activities. Sumoylation of DJ-1 was increased after UV irradiation concomitant with a pI shift to an acidic point of DJ-1. Furthermore, L166P, a mutant DJ-1 found in PD patients, and K130RX, an artificial mutant containing four mutations in DJ-1, were improperly sumoylated, and they became insoluble, partly localized in the mitochondria and degraded by the proteasome system. Both L166P-expressing cells and DJ-1-knockdown cells were found to be highly susceptible to UV-induced cell apoptosis.
Assuntos
Proteínas Oncogênicas/metabolismo , Proteína SUMO-1/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Sequência de Bases , Linhagem Celular , DNA/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisina/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteína Desglicase DJ-1 , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Solubilidade , Transfecção , Raios UltravioletaRESUMO
Omi/HtrA2 is a mitochondrial serine protease that is released into the cytosol during apoptosis and promotes cytochrome c (Cyt c)dependent caspase activation by neutralizing inhibitor of apoptosis proteins (IAPs) via its IAP-binding motif. The protease activity of Omi/HtrA2 also contributes to the progression of both apoptosis and caspase-independent cell death. In this study, we found that wild-type Omi/HtrA2 is more effective at caspase activation than a catalytically inactive mutant of Omi/HtrA2 in response to apoptotic stimuli, such as UV irradiation or tumor necrosis factor. Although similar levels of Omi/HtrA2 expression, XIAP-binding activity, and Omi/HtrA2 mitochondrial release were observed among cells transfected with catalytically inactive and wild-type Omi/HtrA2 protein, XIAP protein expression after UV irradiation was significantly reduced in cells transfected with wild-type Omi/HtrA2. Recombinant Omi/HtrA2 was observed to catalytically cleave IAPs and to inactivate XIAP in vitro, suggesting that the protease activity of Omi/HtrA2 might be responsible for its IAP-inhibiting activity. Extramitochondrial expression of Omi/HtrA2 indirectly induced permeabilization of the outer mitochondrial membrane and subsequent Cyt c-dependent caspase activation in HeLa cells. These results indicate that protease activity of Omi/HtrA2 promotes caspase activation through multiple pathways.