title: Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation, Membrane Association, and Internalization.
abstract: Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.
authors.affiliation: From the Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario M5T 2S8, Canada.
authors.forename: Filsy
authors.initials: F
authors.lastname: Samuel
authors.affiliation: the Integrative Cell Biology Program.
authors.forename: William P
authors.initials: WP
authors.lastname: Flavin
authors.affiliation: From the Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario M5T 2S8, Canada.
authors.forename: Sobia
authors.initials: S
authors.lastname: Iqbal
authors.affiliation: the Departments of Pharmacology and Neurosciences, Central Nervous System Research Group (GRSNC), Faculty of Medicine, Université de Montréal, Québec H4T 1J4, Canada, and.
authors.forename: Consiglia
authors.initials: C
authors.lastname: Pacelli
authors.affiliation: From the Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario M5T 2S8, Canada.
authors.forename: Sri Dushyaanthan
authors.initials: SD
authors.lastname: Sri Renganathan
authors.affiliation: the Departments of Pharmacology and Neurosciences, Central Nervous System Research Group (GRSNC), Faculty of Medicine, Université de Montréal, Québec H4T 1J4, Canada, and.
authors.forename: Louis-Eric
authors.initials: LE
authors.lastname: Trudeau
authors.affiliation: the Integrative Cell Biology Program, Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Campbell
authors.affiliation: From the Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario M5T 2S8, Canada, the Departments of Medical Biophysics and.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Fraser
authors.affiliation: From the Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario M5T 2S8, Canada, Medicine, University of Toronto, Toronto, Ontario M5T 2S8, Canada a.tandon@utoronto.ca.
authors.forename: Anurag
authors.initials: A
authors.lastname: Tandon
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D002478:Cells, Cultured; D059290:Dopaminergic Neurons / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D004705:Endocytosis; D006801:Humans; D008636:Mesencephalon / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D051379:Mice; D009154:Mutation; D010300:Parkinson Disease / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D066263:Protein Aggregation, Pathological / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism* / Q000473:pathology; D051844:alpha-Synuclein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C497604:SNCA protein, human; D051844:alpha-Synuclein; D012694:Serine; C496126:PLK2 protein, human; D017346:Protein Serine-Threonine Kinases
keywords: Parkinson disease; endocytosis; fibril; post-translational modification (PTM); protein kinase; protein misfolding; protein self-assembly; vesicles