Leung, A. K. L. | Search bioRxiv

bioRxiv2019

Alphavirus nsP3 ADP-ribosylhydrolase Activity Disrupts Stress Granule Formation

Formation of stress granules (SGs), cytoplasmic condensates of stalled translation initiation complexes, is regulated by post-translational protein modification. Alphaviruses interfere with SG formation in response to inhibition of host protein synthesis through the activities of nonstructural protein 3 (nsP3). nsP3 has a conserved N-terminal macrodomain that binds and can remove ADP-ribose from ADP-ribosylated proteins and a C-terminal hypervariable domain that binds essential SG component G3BP1. We showed that the hydrolase activity of chikungunya virus nsP3 macrodomain removed ADP-ribosylation of G3BP1 and suppressed SG formation. ADP-ribosylhydrolase-deficient nsP3 mutants allowed stress-induced cytoplasmic condensation of translation initiation factors. nsP3 also disassembled SG-like aggregates enriched with translation initiation factors that are induced by the expression of FUS mutant R495X linked to amyotrophic lateral sclerosis. Therefore, our data indicate that regulation of ADP-ribosylation controls the localization of translation initiation factors during virus infection and other pathological conditions.

cell biology↗

bioRxiv2019

An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation

ADP-ribosylation is integral to a diverse range of cellular processes such as DNA repair, chromatin regulation and RNA processing. However, proteome-wide investigation of its cellular functions has been limited due to numerous technical challenges including the complexity of the poly(ADP-ribose) (PAR) chains, low abundance of the modification and lack of sensitive enrichment methods. To facilitate live-cell profiling of ADP-ribosylated proteins, we show that 2-alkyne-adenosine (2YnAd) is metabolically incorporated in mammalian cells and enables fluorescent detection and robust affinity enrichment of the modified proteins. We then present an integrated chemical biology approach that involves simultaneous metabolic incorporation of 2YnAd and the previously reported 6-alkyne-adenosine (6YnAd) in live cells followed by click chemistry with a capture reagent to facilitate highly sensitive and comprehensive enrichment of the modified proteins. By combining this dual metabolic labelling strategy with the tandem mass tag (TMT) isobaric mass spectrometry, we have quantified the responses of thousands of endogenous proteins to clinical PARP inhibitors Olaparib and Rucaparib. Our study provides insight into the wider scope of PARP targets and will help to advance further characterisation of their functional roles.

biochemistry↗