科研之友
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摘要
The accumulation of alpha-synuclein (alpha-syn) fibrils in neuronal inclusions is the defining pathological process in Parkinson's disease (PD). A pathogenic role for alpha-syn fibril accumulation is supported by the identification of dominantly inherited alpha-syn (SNCA) gene mutations in rare cases of familial PD. Fibril formation involves a spontaneous nucleation event in which soluble alpha-syn monomers associate to form seeds, followed by fibril growth during which monomeric alpha-syn molecules sequentially associate with existing seeds. To better investigate this process, we developed sensitive assays that use the fluorescein arsenical dye FlAsH (fluorescein arsenical hairpin binder) to detect soluble oligomers and mature fibrils formed from recombinant alpha-syn protein containing an N-terminal bicysteine tag (C2--alpha-syn). Using seed growth by monomer association (SeGMA) assays to measure fibril growth over 3 h in the presence of C2-alpha-syn monomer, we observed that some familial PD-associated alpha-syn mutations (i.e. H50Q and A53T) greatly increased growth rates, whereas others (E46K, A30P, and G51D) decreased growth rates. Experiments with wild-type seeds extended by mutant monomer and vice versa revealed that single-amino acid differences between seed and monomer proteins consistently decreased growth rates. These results demonstrate that alpha-syn monomer association during fibril growth is a highly ordered process that can be disrupted by misalignment of individual amino acids and that only a subset of familial-PD mutations causes fibril accumulation through increased fibril growth rates. The SeGMA assays reported herein can be utilized to further elucidate structural requirements of alpha-syn fibril growth and to identify growth inhibitors as a potential therapeutic approach in PD.
- 出版日期2017-6-2
