Biochemical characterization of the core structure of alpha-synuclein filaments.
Intracellular filamentous aggregates comprised of alpha-synuclein such as Lewy bodies and glial cytoplasmic inclusions are the defining hallmarks of a subset of neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. We have analyzed biochemical and structural properties of alpha-synuclein filaments assembled in vitro or extracted from brains of patients with multiple system atrophy and found that both types of filaments are insoluble to detergents and partially resistant to proteinase K digestion. Immunoelectron microscopy and immunoblot analysis showed that both amino and carboxyl termini of alpha-synuclein in in vitro assembled filaments were degraded by proteinase K treatment, whereas the central portion of alpha-synuclein is resistant to proteinase K and retains filamentous structures. Protein sequencing and mass spectrometric analyses of the proteinase K-resistant, minimal fragment of 7 kDa revealed that amino acid residues 31-109 of alpha-synuclein constitute the core unit of the filaments. These observations suggest that the central half of the alpha-synuclein polypeptide, containing five tandem repeats as well as a part of the carboxyl-terminal acidic region, forms the core structure of alpha-synuclein filaments, which is coated by the amino- and carboxyl-terminal portions at the periphery.
Miake
Hirotomo
H
Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Mizusawa
Hidehiro
H
Iwatsubo
Takeshi
T
Hasegawa
Masato
M
D000595:Amino Acid Sequence; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D019636:Neurodegenerative Diseases / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051843:Synucleins; D051844:alpha-Synuclein
D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C497604:SNCA protein, human; D051843:Synucleins; D051844:alpha-Synuclein; D019286:Endopeptidase K