title: Structural determinants of the C-terminal helix-kink-helix motif essential for protein stability and survival promoting activity of DJ-1.
abstract: 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.
authors.affiliation: Laboratory of Alzheimer's and Parkinson's Disease Research, Department of Biochemistry, Ludwig Maximilians University of Munich, 80336 Munich, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Görner
authors.forename: Eve
authors.initials: E
authors.lastname: Holtorf
authors.forename: Jens
authors.initials: J
authors.lastname: Waak
authors.forename: Thu-Trang
authors.initials: TT
authors.lastname: Pham
authors.forename: Daniela M
authors.initials: DM
authors.lastname: Vogt-Weisenhorn
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wurst
authors.forename: Christian
authors.initials: C
authors.lastname: Haass
authors.forename: Philipp J
authors.initials: PJ
authors.lastname: Kahle
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D019610:Cytoprotection / Q000187:drug effects / Q000235:genetics; D004622:Embryo, Mammalian / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D048848:MAP Kinase Kinase Kinase 5 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D015513:Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics; D016877:Oxidants / Q000494:pharmacology; D016716:PC12 Cells; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism; D054464:Peroxiredoxins; D000071617:Protein Deglycase DJ-1; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D015513:Oncogene Proteins; D016877:Oxidants; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins; D051057:Proto-Oncogene Proteins c-akt; D048848:MAP Kinase Kinase Kinase 5; C105131:PARK7 protein, human; C503466:PARK7 protein, mouse; D000071617:Protein Deglycase DJ-1