Proteome-wide identification of the endogenous ADP-ribosylome of mammalian cells and tissue.

Rita Martello,Michael L Nielsen,Clifford Young,Mario Leutert,Vera Bilan,Stephanie Jungmichel,Michael O Hottiger,Sara C Larsen

doi:10.1038/ncomms12917

Rita Martello, Michael L Nielsen + Show 6 more

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https://doi.org/10.1038/ncomms12917

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Abstract

Although protein ADP-ribosylation is involved in diverse biological processes, it has remained a challenge to identify ADP-ribose acceptor sites. Here, we present an experimental workflow for sensitive and unbiased analysis of endogenous ADP-ribosylation sites, capable of detecting more than 900 modification sites in mammalian cells and mouse liver. In cells, we demonstrate that Lys residues, besides Glu, Asp and Arg residues, are the dominant in vivo targets of ADP-ribosylation during oxidative stress. In normal liver tissue, we find Arg residues to be the predominant modification site. The cellular distribution and biological processes that involve ADP-ribosylated proteins are different in cultured cells and liver tissue, in the latter of which the majority of sites were found to be in cytosolic and mitochondrial protein networks primarily associated with metabolism. Collectively, we describe a robust methodology for the assessment of the role of ADP-ribosylation and ADP-ribosyltransferases in physiological and pathological states.

Highlights

Protein ADP-ribosylation is involved in diverse biological processes, it has remained a challenge to identify ADP-ribose acceptor sites
We have developed a technology for sensitive analysis of endogenous ADP-ribosylation sites in both cells and tissues that overcomes several of the above-mentioned limitations of current approaches
We treat the cellular peptide digest with PAR glycohydrolase (PARG), thereby converting all PARylated amino acids to their MARylated counterparts[19]

Summary

Introduction

Protein ADP-ribosylation is involved in diverse biological processes, it has remained a challenge to identify ADP-ribose acceptor sites. Protein ADP-ribosylation refers to the process where an ADP-ribose moiety is transferred from NAD þ to the amino acid side-chains of target proteins (as mono-ADPribose, MAR) or to an already protein bound ADP-ribose to form poly-ADP-ribose (PAR). These modifications are primarily catalysed by a class of enzymes known as ADP-ribosyltransferases (ARTs), with certain Sirtuin deacetylases being able to catalyse ADP-ribosylation[1]. Protein ADP-ribosylation is a low-abundant PTM that is rapidly degraded To overcome this challenge cellular PARG knockdowns (siPARG) or knockouts have been developed[9,12]. While ADP-ribosylation has been known for more than 50 years, the cellular stoichiometry of the modification has remained elusive, primarily due to the lack of methodologies that can elucidate such information[17]

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