Camara Ali, Sanyal Anwesha, Dutta Sayan, Rochet Jean-Christophe, Mattoo Seema
Department of Biological Sciences, Purdue University, West Lafayette, USA.
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, USA.
Bio Protoc. 2020 Sep 20;10(18):e3760. doi: 10.21769/BioProtoc.3760.
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 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 AMPylation reactions of HYPE substrates, such as α-syn, use a variety of ATP analogs, including radiolabeled α-P-ATP or α-P-ATP, fluorescent ATP analogs, biotinylated-ATP analogs (N6-[6-hexamethyl]-ATP-Biotin), as well as click-chemistry-based alkyl-ATP methods for gel-based detection of AMPylation. Current literature describing a step-by-step protocol of HYPE-mediated AMPylation relies on an α-P-ATP nucleotide instead of the more commonly available α-P-ATP. Though effective, this former procedure requires a lengthy and hazardous DMSO-PPO (dimethyl sulfoxide-polyphenyloxazole) precipitation. Thus, we provide a streamlined alternative to the α-P-ATP-based method, which obviates the DMSO-PPO precipitation step. Described here is a detailed procedure for HYPE mediated AMPylation of α-syn using α-P-ATP as a nucleotide source. Moreover, our use of a reusable Phosphor screen for AMPylation detection, in lieu of the standard, single-use autoradiography film, provides a faster, more sensitive and cost-effective alternative.
帕金森病的主要组织病理学特征之一是路易小体（LBs）——富含突触前蛋白α-突触核蛋白（α-syn）纤维状形式的细胞质内含物。α-syn向路易小体的渐进性沉积是由其形成不溶性聚集体的纤维化倾向所导致的。我们最近发现，Fic（由cAMP诱导的丝状化）家族腺苷酸转移酶HYPE/FICD（亨廷顿蛋白酵母相互作用蛋白E/FICD）对α-syn进行翻译后修饰（PTM）后，α-syn的纤维化显著减少。具体而言，HYPE利用ATP将AMP（单磷酸腺苷）共价修饰到α-syn的N端和NAC（非淀粉样β成分）区域的关键苏氨酸残基上，这种PTM称为AMP化或腺苷酸化。目前HYPE底物（如α-syn）的AMP化反应使用多种ATP类似物，包括放射性标记的α-P-ATP或α-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方法用于基于凝胶的AMP化检测。目前描述HYPE介导的AMP化分步方案的文献依赖于α-P-ATP核苷酸，而不是更常用的α-P-ATP。虽然这种方法有效，但前一种方法需要冗长且危险的DMSO-PPO（二甲基亚砜-聚苯基恶唑）沉淀。因此，我们提供了一种基于α-P-ATP方法的简化替代方案，该方案避免了DMSO-PPO沉淀步骤。这里描述的是使用α-P-ATP作为核苷酸来源对α-syn进行HYPE介导的AMP化的详细程序。此外，我们使用可重复使用的磷屏进行AMP化检测，而不是标准的一次性放射自显影片，提供了一种更快、更灵敏且更具成本效益的替代方案。
