Preserved proteinase K-resistant core after amplification of alpha-synuclein aggregates: Implication to disease-related structural study.
Many pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils during pathogenesis. One of the pathological proteins, alpha-synuclein (α-syn), accumulates in the brains of Parkinson disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are designated as synucleinopathies. Recently, structural properties of abnormal accumulated proteins are suggested to determine the disease phenotype. However, the biochemical and structural characteristics of those accumulated proteins are still poorly understood. We previously reported the sequence and seed-structure-dependent polymorphic fibrils of α-syn and the polymorphism was identified by proteinase K-resistant cores determined by mass spectrometry (MS) analysis. In this study, we applied this method to analyze α-syn aggregates of MSA and DLB. To perform MS analysis on proteinase K-resistant cores, we first performed amplification of α-syn aggregates by seeding reaction and protein misfolding cyclic amplification (PMCA) to obtain a sufficient amount of aggregates. Using SDS insoluble fraction of the disease brain, we successfully amplified enough α-syn aggregates for MS analysis. We differentiated between mouse and human α-syn aggregates by MS analysis on proteinase K-resistant cores of the aggregates before and after amplification. The results suggest that structural properties of amplified α-syn fibrils are preserved after PMCA and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders.
['Yoshinaga|Saki|S|', 'Yamanaka|Tomoyuki|T|', 'Miyazaki|Haruko|H|', 'Okuzumi|Ayami|A|', 'Hiyama|Akiko|A|', 'Murayama|Shigeo|S|', 'Nukina|Nobuyuki|N|']
D000368:Aged D000818:Animals D001921:Brain D019286:Endopeptidase K D005260:Female D006801:Humans D008297:Male D051379:Mice D008875:Middle Aged D066329:Protein Aggregates D066263:Protein Aggregation, Pathological D000080874:Synucleinopathies D051844:alpha-Synuclein
Alpha-synuclein Synucleinopathy Mass spectrometry Seeding reaction Protein misfolding cyclic amplification