Structural determinants of the C-terminal helix-kink-helix motif essential for protein stability and survival promoting activity of DJ-1.
Mutations in the PARK7 gene encoding DJ-1 cause autosomal recessive Parkinson disease. The most deleterious point mutation is the L166P substitution, which resides in a structure motif comprising two alpha-helices (G and H) separated by a kink. Here we subjected the C-terminal helix-kink-helix motif to systematic site-directed mutagenesis, introducing helix-incompatible ... proline residues as well as conservative substitutions into the helical interface. Furthermore, we generated deletion mutants lacking the H-helix, the kink, and the entire C terminus. When transfected into neural and nonneural cell lines, steady-state levels of G-helix breaking and kink deletion mutants were dramatically lower than wild-type DJ-1. The effects of H-helix breakers were comparably smaller, and the non-helix breaking mutants only slightly destabilized DJ-1. The decreased steady-state levels were due to accelerated protein degradation involving in part the proteasome. G-helix breaking DJ-1 mutations abolished dimer formation. These structural perturbations had functional consequences on the cytoprotective activities of DJ-1. The destabilizing mutations conferred reduced cytoprotection against H(2)O(2) in transiently retransfected DJ-1 knock-out mouse embryonic fibroblasts. The loss of survival promoting activity of the DJ-1 mutants with destabilizing C-terminal mutations correlated with impaired anti-apoptotic signaling. We found that wild-type, but not mutant DJ-1 facilitated the Akt pathway and simultaneously blocked the apoptosis signal-regulating kinase 1, with which DJ-1 interacted in a redox-dependent manner. Thus, the G-helix and kink are critical determinants of the C-terminal helix-kink-helix motif, which is absolutely required for stability and the regulation of survival-promoting redox signaling of the Parkinson disease-associated protein DJ-1.
Mesh Terms:
Amino Acid Motifs, Animals, Apoptosis, Cell Survival, Cytoprotection, Embryo, Mammalian, Fibroblasts, Humans, Hydrogen Peroxide, Intracellular Signaling Peptides and Proteins, MAP Kinase Kinase Kinase 5, Mice, Mice, Knockout, Mutagenesis, Site-Directed, Mutation, Missense, Oncogene Proteins, Oxidants, PC12 Cells, Parkinson Disease, Protein Folding, Protein Processing, Post-Translational, Protein Structure, Tertiary, Proto-Oncogene Proteins c-akt, Rats, Signal Transduction, Structure-Activity Relationship
Amino Acid Motifs, Animals, Apoptosis, Cell Survival, Cytoprotection, Embryo, Mammalian, Fibroblasts, Humans, Hydrogen Peroxide, Intracellular Signaling Peptides and Proteins, MAP Kinase Kinase Kinase 5, Mice, Mice, Knockout, Mutagenesis, Site-Directed, Mutation, Missense, Oncogene Proteins, Oxidants, PC12 Cells, Parkinson Disease, Protein Folding, Protein Processing, Post-Translational, Protein Structure, Tertiary, Proto-Oncogene Proteins c-akt, Rats, Signal Transduction, Structure-Activity Relationship
J. Biol. Chem.
Date: May. 04, 2007
PubMed ID: 17331951
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