The PINK1 p.I368N Mutation Affects Protein Stability and Kinase Activity with Its Structural Change

Background : Mutations in the recessive genes PINK1 and PARKIN are the most common causes of early-onset Parkinsonʼs disease (PD). The mitochondrial ubiquitin (Ub) kinase PINK1 mediates, together with the cytosolic E3 Ub ligase PARKIN, mitochondrial quality control. Thereby, damaged mitochondria are identified to prevent their accumulation and eventually cell death. A detailed understanding of PINK1 mutations will help to further our understanding of PD. Objective : The aim of this study was to examine the exact molecular pathogenic mechanisms of PINK1 p.I368N. Methods : We investigated molecular mechanisms on the structural and functional level in patientsʼ fibroblasts and in cell-based, biochemical assays. Results : Under endogenous conditions, PINK1 p.I368N is expressed, imported in healthy mitochondria similar to PINK1 wild type. Upon mitochondrial damage, however, full-length PINK1 p.I368N is unstable on the outer mitochondrial membrane and consequently mitochondrial quality control declines. We found that stress-induced interaction between PINK1 p.I368N and TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity. We further confirmed experimentally that the kinase activity of the PINK1 p.I368N mutant is abolished. Conclusions : We revealed two mechanisms that lead to loss of function of PINK1 upon mutation.

[1]  David S. Park,et al.  Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment , 2012, EMBO reports.

[2]  F. Fiesel,et al.  Disease relevance of phosphorylated ubiquitin (p-S65-Ub) , 2015, Autophagy.

[3]  H. Lorenz,et al.  Intramembrane protease PARL defines a negative regulator of PINK1- and PARK2/Parkin-dependent mitophagy , 2015, Autophagy.

[4]  Heung-Chin Cheng,et al.  Analysis of the regulatory and catalytic domains of PTEN‐induced kinase‐1 (PINK1) , 2012, Human mutation.

[5]  S. Endo,et al.  L347P PINK1 mutant that fails to bind to Hsp90/Cdc37 chaperones is rapidly degraded in a proteasome-dependent manner , 2008, Neuroscience Research.

[6]  S. Gygi,et al.  Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. , 2014, Molecular cell.

[7]  Roland L. Dunbrack,et al.  Assignment of protonation states in proteins and ligands: combining pKa prediction with hydrogen bonding network optimization. , 2012, Methods in molecular biology.

[8]  Batsal Devkota,et al.  Motion of transfer RNA from the A/T state into the A‐site using docking and simulations , 2012, Proteins.

[9]  Atsushi Tanaka,et al.  PINK1 Is Selectively Stabilized on Impaired Mitochondria to Activate Parkin , 2010, PLoS biology.

[10]  S. Przedborski,et al.  Cytosolic cleaved PINK1 represses Parkin translocation to mitochondria and mitophagy , 2014, EMBO reports.

[11]  Jeffrey Skolnick,et al.  Template‐based protein structure modeling using TASSERVMT , 2012, Proteins.

[12]  A. Burlingame,et al.  A Neo-Substrate that Amplifies Catalytic Activity of Parkinson’s-Disease-Related Kinase PINK1 , 2013, Cell.

[13]  B. de Strooper,et al.  PINK1 Kinase Catalytic Activity Is Regulated by Phosphorylation on Serines 228 and 402* , 2014, The Journal of Biological Chemistry.

[14]  Scott E. Martin,et al.  High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy , 2013, Nature.

[15]  David W. Miller,et al.  Mutations in PTEN-induced putative kinase 1 associated with recessive parkinsonism have differential effects on protein stability. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[16]  H. Dopazo,et al.  Phylogenetic and in silico structural analysis of the Parkinson disease‐related kinase PINK1 , 2011, Human Mutation.

[17]  R. King,et al.  Identification and application of the concepts important for accurate and reliable protein secondary structure prediction , 1996, Protein science : a publication of the Protein Society.

[18]  E. Valente,et al.  Mitochondrial import and enzymatic activity of PINK1 mutants associated to recessive parkinsonism. , 2005, Human molecular genetics.

[19]  Fabienne C. Fiesel,et al.  PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1 , 2010, Nature Cell Biology.

[20]  Soojay Banerjee,et al.  PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity , 2014, The Journal of cell biology.

[21]  Jeffrey Skolnick,et al.  Improving threading algorithms for remote homology modeling by combining fragment and template comparisons , 2010, Proteins.

[22]  A. Lees,et al.  Altered cleavage and localization of PINK1 to aggresomes in the presence of proteasomal stress , 2006, Journal of neurochemistry.

[23]  J. Burman,et al.  The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy , 2015, Nature.

[24]  T. Lithgow,et al.  Tom40, the import channel of the mitochondrial outer membrane, plays an active role in sorting imported proteins , 2003, The EMBO journal.

[25]  P. Janik,et al.  Incidence of mutations in the PARK2, PINK1, PARK7 genes in Polish early-onset Parkinson disease patients. , 2013, Neurologia i neurochirurgia polska.

[26]  S. Minoshima,et al.  Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism , 1998, Nature.

[27]  M. Beal,et al.  PINK1 Defect Causes Mitochondrial Dysfunction, Proteasomal Deficit and α-Synuclein Aggregation in Cell Culture Models of Parkinson's Disease , 2009, PloS one.

[28]  William Lin,et al.  Characterization of PINK1 processing, stability, and subcellular localization , 2008, Journal of neurochemistry.

[29]  C. Sander,et al.  The PDBFINDER database: a summary of PDB, DSSP and HSSP information with added value , 1996, Comput. Appl. Biosci..

[30]  T. Caulfield,et al.  PINK1, Parkin, and Mitochondrial Quality Control: What can we Learn about Parkinson’s Disease Pathobiology? , 2016, Journal of Parkinson's disease.

[31]  R. Youle,et al.  Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin. , 2012, Developmental cell.

[32]  Ron Elber,et al.  SSALN: An alignment algorithm using structure‐dependent substitution matrices and gap penalties learned from structurally aligned protein pairs , 2005, Proteins.

[33]  Elisabeth L. Moussaud-Lamodière,et al.  (Patho‐)physiological relevance of PINK1‐dependent ubiquitin phosphorylation , 2015, EMBO reports.

[34]  M. Farrer,et al.  Heterozygous PINK1 p.G411S increases risk of Parkinson’s disease via a dominant-negative mechanism , 2016, Brain : a journal of neurology.

[35]  R. Youle,et al.  PINK1 is degraded through the N-end rule pathway , 2013, Autophagy.

[36]  R. Nussbaum,et al.  Hereditary Early-Onset Parkinson's Disease Caused by Mutations in PINK1 , 2004, Science.

[37]  Jeffrey Skolnick,et al.  Protein structure prediction by pro-Sp3-TASSER. , 2009, Biophysical journal.

[38]  José L. Medina-Franco,et al.  Molecular dynamics simulations of human DNA methyltransferase 3B with selective inhibitor nanaomycin A. , 2011, Journal of structural biology.

[39]  Elisabeth L. Moussaud-Lamodière,et al.  Early-onset Parkinson's disease due to PINK1 p.Q456X mutation--clinical and functional study. , 2014, Parkinsonism & related disorders.

[40]  Miratul M. K. Muqit,et al.  PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65 , 2012, Open Biology.

[41]  Keehyoung Joo,et al.  Improving physical realism, stereochemistry, and side‐chain accuracy in homology modeling: Four approaches that performed well in CASP8 , 2009, Proteins.

[42]  J. Skolnick,et al.  Ab initio protein structure prediction using chunk-TASSER. , 2007, Biophysical journal.

[43]  Z. Xiang,et al.  On the role of the crystal environment in determining protein side-chain conformations. , 2002, Journal of molecular biology.

[44]  A. Brice,et al.  What genetics tells us about the causes and mechanisms of Parkinson's disease. , 2011, Physiological reviews.

[45]  D. Selkoe,et al.  Pink1 Parkinson mutations, the Cdc37/Hsp90 chaperones and Parkin all influence the maturation or subcellular distribution of Pink1. , 2008, Human molecular genetics.

[46]  T. Caulfield,et al.  Inter-ring rotation of apolipoprotein A-I protein monomers for the double-belt model using biased molecular dynamics. , 2011, Journal of molecular graphics & modelling.

[47]  Seung Yup Lee,et al.  Analysis of TASSER‐based CASP7 protein structure prediction results , 2007, Proteins.

[48]  T. Hirokawa,et al.  Ubiquitin is phosphorylated by PINK1 to activate parkin , 2014, Nature.

[49]  Keiji Tanaka,et al.  A Dimeric PINK1-containing Complex on Depolarized Mitochondria Stimulates Parkin Recruitment* , 2013, The Journal of Biological Chemistry.

[50]  C. Sander,et al.  Errors in protein structures , 1996, Nature.

[51]  S F Altschul,et al.  Iterated profile searches with PSI-BLAST--a tool for discovery in protein databases. , 1998, Trends in biochemical sciences.

[52]  J. Harper,et al.  The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy. , 2015, Molecular cell.

[53]  Y. Saeki,et al.  Phosphorylated ubiquitin chain is the genuine Parkin receptor , 2015, The Journal of cell biology.

[54]  William Lin,et al.  Structural determinants of PINK1 topology and dual subcellular distribution , 2010, BMC Cell Biology.

[55]  S. Weber,et al.  The PINK1/Parkin-mediated mitophagy is compromised by PD-associated mutations , 2010, Autophagy.

[56]  A. Whitworth,et al.  PINK1 cleavage at position A103 by the mitochondrial protease PARL , 2010, Human molecular genetics.

[57]  N. Hattori,et al.  PINK1 autophosphorylation upon membrane potential dissipation is essential for Parkin recruitment to damaged mitochondria , 2012, Nature Communications.

[58]  S. Przedborski,et al.  Pink1 Kinase and Its Membrane Potential (Δψ)-dependent Cleavage Product Both Localize to Outer Mitochondrial Membrane by Unique Targeting Mode* , 2012, The Journal of Biological Chemistry.