Preserved proteinase K-resistant core after amplification of alpha-synuclein aggregates: Implication to disease-related structural study
Preserved proteinase K-resistant core after amplification of alpha-synuclein aggregates: Implication to disease-related structural study
复制标题
α-突触核蛋白聚集体扩增后保留的蛋白酶 K 抗性核心：对疾病相关结构研究的意义
DOI：
10.1016/j.bbrc.2019.11.142
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发表时间：
2020
影响因子：
3.1
通讯作者：
Nukina Nobuyuki
中科院分区：
文献类型：
--
作者：
Yoshinaga Saki;Yamanaka Tomoyuki;Miyazaki Haruko;Okuzumi Ayami;Hiyama Akiko;Murayama Shigeo;Nukina Nobuyuki
许多与神经退行性疾病相关的病理蛋白在发病过程中错误折叠，聚集形成淀粉样纤维。α-突触核蛋白(α-SYN)是一种病理蛋白，在帕金森病(PD)、路易体痴呆(DLB)和多系统萎缩症(MSA)的大脑中积聚，被称为突触核病。最近，人们建议用异常堆积蛋白的结构特征来确定疾病的表型。然而，这些积累的蛋白质的生化和结构特征仍然知之甚少。我们先前报道了α-SYN的序列和种子结构相关的多态纤维，并通过质谱仪(MS)分析确定了该多态。在本研究中，我们将该方法应用于α-SYN聚集体的分析。为了对蛋白酶K抗性核心进行MS分析，我们首先通过种子反应和蛋白质误折叠循环扩增(PmCA)扩增α-SYN聚集体，以获得足够数量的聚集体。利用病脑的十二烷基硫酸钠不溶性部分，我们成功地扩增了足够多的α-SYN聚集体，用于MS分析。通过对扩增前后α-SYN聚合体的蛋白酶K抗性核心的MS分析，我们区分了小鼠和人的DNA-SYN聚合体。结果表明，α-SYN扩增后纤维的结构特征被保留下来，这些方法可用于研究神经退行性疾病的病理蛋白。
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.