title: Role of alpha-synuclein carboxy-terminus on fibril formation in vitro.
abstract: Alpha-synuclein (alpha-syn) is the major component of intracellular inclusions in several neurodegenerative diseases, and the conversion of soluble alpha-syn into filamentous aggregates may contribute to disease pathogenesis. Since mechanisms leading to the formation of alpha-syn inclusions are unclear, in vitro models of alpha-syn aggregation may yield insights into this process. To that end, we examined the consequences on the progressive deletion of the carboxy-terminus of alpha-syn in regulating fibril formation, and we show here that carboxy-terminal truncated alpha-syn proteins aggregate faster than the full-length molecule. Protease digestion and immunoelectron microscopy indicate that the alpha-syn amino- and carboxy-termini are more solvent exposed than the central core and that filaments formed from carboxy-terminal truncated alpha-syn are narrower in diameter than the full-length molecule. Moreover, seeding experiments under conditions where full-length alpha-syn did not readily aggregate revealed that carboxy-truncated alpha-syn extending from amino acids 1-102 and 1-110 but not 1-120 were efficient in seeding full-length alpha-syn aggregation over a range of concentrations. Using site-directed mutagenesis, the negatively charged residues 104, 105 and 114, 115 in the carboxy-terminus were implicated in this reduced aggregation and the lack of seeding of full-length alpha-syn fibrillogenesis by 1-120. Our data support the view that the middle region of alpha-syn forms the core of alpha-syn filaments and that negative charges in the carboxy-terminus counteract alpha-syn aggregation. Thus, the carboxy-terminus of alpha-syn may regulate aggregation of full-length alpha-syn and determine the diameter of alpha-syn filaments.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Ian V J
authors.initials: IV
authors.lastname: Murray
authors.forename: Benoit I
authors.initials: BI
authors.lastname: Giasson
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Quinn
authors.forename: Vishwanath
authors.initials: V
authors.lastname: Koppaka
authors.forename: Paul H
authors.initials: PH
authors.lastname: Axelsen
authors.forename: Harry
authors.initials: H
authors.lastname: Ischiropoulos
authors.forename: John Q
authors.initials: JQ
authors.lastname: Trojanowski
authors.forename: Virginia M-Y
authors.initials: VM
authors.lastname: Lee
mesh_terms: D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D020894:Microfibrils / Q000648:ultrastructure*; D016253:Microscopy, Immunoelectron; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence; D051843:Synucleins; D051844:alpha-Synuclein
chemical_list: D017931:DNA Primers; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C497604:SNCA protein, human; D051843:Synucleins; D051844:alpha-Synuclein