Background

Deposition of the small (140 amino acids), intrinsically disordered protein alpha-synuclein (α-syn) into Lewy bodies (LBs) is a major hallmark of Parkinson’s disease (PD) (Weinreb et al., 1996; Spillantini et al., 1997). On its way to forming the insoluble aggregates found in LBs, monomeric α-syn adopts a β-sheet-rich, amyloid-like conformation, leading to progressive fibrillation and concomitant insolubility (Iwai et al., 1995). Multiple lines of research describe a compelling correlation between post-translational modifications (PTMs) of α-syn and its oligomeric state. Phosphorylation at Ser 129, for example, is prevalent on aggregated α-syn found within LBs (Chen and Feany, 2005). Tyrosine nitrations of α-syn have been linked to formation of stable oligomers and reduced fibril formation (Yamin et al., 2003), while O-GlcNAcylation of threonine residues reduces α-syn aggregation (Levine et al., 2019). Recently, we described a novel α-syn modification–adenylylation or AMPylation–which reduces α-syn fibrillation in vitro (Sanyal et al., 2019). Moreover, AMPylated α-syn displayed diminished disease-linked phenotypes, such as membrane permeabilization (Ysselstein et al., 2017; Sanyal et al., 2019).

We demonstrated that HYPE, the sole Fic protein in humans, carried out AMPylation of α-syn (Worby et al., 2009; Sanyal et al., 2019). Like other members of the conserved Fic domain protein family, HYPE’s AMPylation activity is intrinsically regulated by an inhibitory helix called α_inh (Engel et al., 2012). Mutation of HYPE’s conserved Glu234 to Gly within the α_inh renders HYPE constitutively active for its adenylyltransferase activity (Engel et al., 2012; Sanyal et al., 2015). HYPE then covalently attaches the AMP portion of an ATP co-substrate onto target hydroxyl side-chain residues (e.g., serine or threonine) (Ham et al., 2014; Sanyal et al., 2015; Truttmann et al., 2016; Preissler et al., 2017). Indeed, tandem mass spectrometric analysis of purified, recombinant α-syn mapped AMPylation sites to three threonines–T33, T54, and T75–located within regions hypothesized to be crucial for oligomerization (Sanyal et al., 2019).

Several biochemical assays monitoring Fic-mediated AMPylation have been reported (Worby et al., 2009; Mattoo et al., 2011; Yarbrough et al., 2009; Lewallen et al., 2012; Truttmann et al., 2016; Dedic et al., 2016). Most common amongst these are assays using a fluorescent (Fl-ATP) or radioactive (α-³²P-ATP or α-³³P-ATP) ATP analogs. While Fl-ATP has served as a useful research tool, its large, bulky fluorescent moiety is known to stunt kinetic efficiencies (Lewallen et al., 2012). Moreover, no step-by-step protocol for Fl-ATP AMPylation has been documented. A detailed procedure for α-³³P-ATP, however, is available (Truttmann and Ploegh, 2017). This method entails incubation of HYPE and its protein target with radiolabelled α-³³P-ATP, followed by separation via SDS-PAGE, DMSO-PPO precipitation, and then imaging of radiolabeled proteins on autoradiography film. Though effective in detecting AMPylation, this procedure suffers from additional time-consuming steps that are expensive and require hazardous chemicals (e.g., DMSO-PPO and autoradiography exposure and development). Herein, we present an optimized method for monitoring AMPylation in vitro, using α-syn as a target and α-³²P-ATP as a nucleotide source. α-³²P-ATP preserves signal sensitivity, while eliminating DMSO-PPO precipitation. Our protocol also replaces the expensive single-use autoradiography film with a reusable Phosphor screen, which allows more sensitive detection of radioactive phosphate-containing proteins under ambient light and at shorter exposure times. Given the broad specificities of Fic proteins and other enzymes for co-substrates including GTP, CTP, phosphocholine, and even ATP used for phosphorylation, this protocol can be adapted to assess other PTMs in vitro (Mattoo et al., 2011; Veyron et al., 2018).