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Biochem Biophys Res Commun. 2019 Nov 27. pii: S0006-291X(19)32270-3. doi: 10.1016/j.bbrc.2019.11.142. [Epub ahead of print]

Preserved proteinase K-resistant core after amplification of alpha-synuclein aggregates: Implication to disease-related structural study.

Author information

1
Laboratory of Structural Neuropathology, Doshisha University Graduate School of Brain Science, 1-3 Miyakodanitatara, Kyotanabe-shi, Kyoto, 610-0394, Japan.
2
Laboratory of Structural Neuropathology, Doshisha University Graduate School of Brain Science, 1-3 Miyakodanitatara, Kyotanabe-shi, Kyoto, 610-0394, Japan. Electronic address: toyamana@mail.doshisha.ac.jp.
3
Laboratory of Structural Neuropathology, Doshisha University Graduate School of Brain Science, 1-3 Miyakodanitatara, Kyotanabe-shi, Kyoto, 610-0394, Japan; Department of Neurology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
4
Department of Neurology and Neuropathology (the Brain Bank for Aging Research), Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, 35-2, Sakae-chou, Itabashi-ku, Tokyo, 173-0015, Japan.
5
Laboratory of Structural Neuropathology, Doshisha University Graduate School of Brain Science, 1-3 Miyakodanitatara, Kyotanabe-shi, Kyoto, 610-0394, Japan. Electronic address: nnukina@mail.doshisha.ac.jp.

Abstract

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.

KEYWORDS:

Alpha-synuclein; Mass spectrometry; Protein misfolding cyclic amplification; Seeding reaction; Synucleinopathy

PMID:
31785806
DOI:
10.1016/j.bbrc.2019.11.142

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