In vitro AMPylation/Adenylylation of Alpha-synuclein by HYPE/FICD

• Published in: Bio-protocol 2020-09, Vol.10 (18), p.e3760-e3760
• Main Authors: Camara, Ali, Sanyal, Anwesha, Dutta, Sayan, Rochet, Jean-Christophe, Mattoo, Seema
• Format: Article
• Language: English
• Subjects: Biology; Methods
• ISSN: 2331-8325; 2331-8325
• 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.