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

Saki Yoshinaga, Tomoyuki Yamanaka, Haruko Miyazaki, Ayami Okuzumi, Akiko Hiyama, Shigeo Murayama, Nobuyuki Nukina

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)655-661
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume522
Issue number3
DOIs
Publication statusPublished - Feb 12 2020
Externally publishedYes

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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