| In vitro AMPylation/Adenylylation of Alpha-synuclein by HYPE/FICD
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Author:
Date:
2020-09-20
[Abstract] One of the major histopathological hallmarks of Parkinson’s disease are Lewy bodies (LBs) –cytoplasmic inclusions, enriched with fibrillar forms of the presynaptic protein alpha-synuclein (α-syn). Progressive deposition of α-syn into LBs is enabled by its propensity to fibrillize into insoluble aggregates. We recently described a marked reduction in α-syn fibrillation in vitro upon posttranslational modification (PTM) by the Fic (Filamentation induced by cAMP) family adenylyltransferase HYPE/FICD (Huntingtin yeast-interacting protein E/FICD). Specifically, HYPE utilizes ATP to covalently decorate key threonine residues in α-syn’s N-terminal and NAC (non-amyloid-β component) regions with AMP (adenosine monophosphate), in a PTM termed AMPylation or adenylylation. Status quo in ...
[摘要] [摘要 ] 帕金森氏病的主要组织病理学标志之一是路易体（LB） –细胞质内含物，富含纤维状形式的突触前蛋白α-突触核蛋白（α-syn）。由于α-syn易于原纤维化成不溶性聚集体，因此可以逐步沉积到LBs中。我们最近描述了Fic（由cAMP诱导的细丝化）家族腺苷酸转移酶HYPE / FICD（与亨廷顿酵母相互作用的蛋白E / FICD）在翻译后修饰（PTM）后体外α-syn纤颤的明显减少。具体而言，HYPE利用ATP在称为AMPylation 或adenylylation 的PTM中，以AMP（单磷酸腺苷）共价修饰α-syn's N末端和NAC（非淀粉样β成分）区域中的关键苏氨酸残基。HYPE底物（例如α-syn）的体外AMPyl化反应现状使用多种ATP类似物，包括放射性标记的α - 32 P-ATP 或α - 33 P-ATP，荧光ATP类似物，生物素化ATP类似物（N6- [6-六甲基] -ATP-生物素），以及基于点击化学的烷基-ATP方法检测基于凝胶的AMPylation 。当前描述HYPE介导AMPylation 的分步方案的文献依赖于α - 33 P-ATP核苷酸，而不是更常见的α - 32 P-ATP。尽管有效，但前一种方法需要长时间且危险的DMSO-PPO（二甲基亚砜- 聚苯基恶唑）沉淀。因此，我们提供了基于α - 33 ...
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| Generation and Testing of Fluorescent Adaptable Simple Theranostic (FAST) Proteins
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Author:
Date:
2020-07-05
[Abstract] This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens new possibilities that are not achievable with proteins produced in bacteria or yeast, such as direct use of the fluorescent protein-secreting cells in live co-culture assays. The Fluorescent Adaptable Simple Theranostic (FAST) protein system includes a histidine purification tag and a tobacco etch virus (TEV) cleavage site, allowing the purification tag and fluorescent protein to be removed for therapeutic use. This protocol is split into five ...
[摘要] [摘要] 该方案提供了一种逐步建立重组荧光融合蛋白的方法，可从哺乳动物细胞系分泌。这项技术建立在许多其他重组蛋白和荧光蛋白技术的基础上，但它是第一个利用荧光融合蛋白直接分泌到细胞培养上清液中的技术之一。这为细菌或酵母产生的蛋白质开辟了新的可能性，例如直接使用荧光蛋白分泌细胞进行活体共培养实验。该荧光适应性简单热（FAST）蛋白系统包括组氨酸纯化标签和烟草蚀刻病毒（TEV）裂解位点，允许去除纯化标签和荧光蛋白用于治疗用途。该方案分为五个部分：（A）感兴趣基因（GOI）和感兴趣蛋白（POI）的电子表征；（B）表达载体的设计；（C）表达载体的组装；（D）用表达载体转染真核细胞系；（E）检测重组蛋白。这种广泛的方案只需聚合酶链反应（PCR）和细胞培养训练即可完成。另外，协议的每个部分都可以独立使用。
[背景] ...
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