A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils.
A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils.
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DOI：
10.1371/journal.pone.0241161
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Lindberg I
中科院分区：
文献类型：
--
作者：
Jarvela TS;Chaplot K;Lindberg I
α-突触核蛋白预形成原纤维 (PFF) 代表了一种有前途的模型系统，用于研究 α-突触核蛋白蛋白病聚集体细胞间传递的细胞过程。然而，由于 PFF 粘附在细胞表面的倾向，区分内化 PFF 和保留在细胞外环境中的 PFF 命运的能力仍然有限。去除 PFF 需要重复洗涤和/或特异性猝灭细胞外荧光 PFF 信号。在本文中，我们提出了一种分析内化α-突触核蛋白命运的新方法。我们在 α-突触核蛋白和绿色荧光蛋白之间插入了烟草蚀纹病毒 (TEV) 蛋白酶切割位点，并在与标记的 PFF 孵育后用 TEV 蛋白酶对细胞进行短暂处理。由于 TEV 蛋白酶具有高度特异性、无毒且在生理条件下具有活性，因此可利用免受 TEV 裂解的保护来区分内化的 PFF 和仍附着在细胞表面的 PFF。使用这种实验范例，可以通过活细胞或固定细胞显微镜以及蛋白质印迹分析下游细胞内事件。我们认为这种方法将有助于了解 PFF 在各种实验操作下内吞作用后的命运。
Alpha-synuclein pre-formed fibrils (PFFs) represent a promising model system for the study of cellular processes underlying cell-to-cell transmission of alpha-synuclein proteopathic aggregates. However, the ability to differentiate the fate of internalized PFFs from those which remain in the extracellular environment remains limited due to the propensity for PFFs to adhere to the cell surface. Removal of PFFs requires repeated washing and/or specific quenching of extracellular fluorescent PFF signals. In this paper we present a new method for analyzing the fate of internalized alpha-synuclein. We inserted a tobacco etch virus (TEV) protease cleavage site between alpha-synuclein and green fluorescent protein and subjected cells to brief treatment with TEV protease after incubation with tagged PFFs. As the TEV protease is highly specific, non-toxic, and active under physiological conditions, protection from TEV cleavage can be used to distinguish internalized PFFs from those which remain attached to the cell surface. Using this experimental paradigm, downstream intracellular events can be analyzed via live or fixed cell microscopy as well as by Western blotting. We suggest that this method will be useful for understanding the fate of PFFs after endocytosis under various experimental manipulations.
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影响因子：
1.2
作者：
Patterson, Joseph R.;Polinski, Nicole K.;Sortwell, Caryl E.
通讯作者：
Sortwell, Caryl E.
影响因子：
3.3
作者：
Volpicelli-Daley LA;Gamble KL;Schultheiss CE;Riddle DM;West AB;Lee VM
通讯作者：
Lee VM
DOI：
10.1126/science.1227157
发表时间：
2012-11-16
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Luk KC;Kehm V;Carroll J;Zhang B;O'Brien P;Trojanowski JQ;Lee VM
通讯作者：
Lee VM
影响因子：
64.8
作者：
Peelaerts, W.;Bousset, L.;Baekelandt, V.
通讯作者：
Baekelandt, V.
影响因子：
8
作者：
Wintgens, Jan P.;Wichert, Sven P.;Wehr, Michael C.
通讯作者：
Wehr, Michael C.