The Promise of Proteomics for the Study of ADP-Ribosylation

Abstract

ADP-ribosylation is a post-translational modification where single units (mono-ADP-ribosylation) or polymeric chains (poly-ADP-ribosylation) of ADP-ribose are conjugated to proteins by ADP-ribosyltransferases. This post-translational modification and the ADP-ribosyltransferases (also known as PARPs) responsible for its synthesis have been found to play a role in nearly all major cellular processes, including DNA repair, transcription, translation, cell signaling and cell death. Furthermore, dysregulation of ADP-ribosylation has been linked to diseases including cancers, diabetes, neurodegenerative disorders and heart failure, leading to the development of therapeutic PARP inhibitors, many of which are currently in clinical trials. The study of this therapeutically important modification has recently been bolstered by the application of mass spectrometry-based proteomics, arguably the most powerful tool for the unbiased analysis of protein modifications. Unfortunately, progress has been hampered by the inherent challenges that stem from the physicochemical properties of ADP-ribose which as a post-translational modification is highly charged, heterogeneous (linear or branched polymers, as well as monomers), labile, and found on a wide range of amino acid acceptors. In this perspective, we discuss the progress that has been made in addressing these challenges, including the recent breakthroughs in proteomics techniques to identify ADP-ribosylation sites, and future developments to provide a proteome-wide view of the many cellular processes regulated by ADP-ribosylation.

PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes

“…Moreover, due to the presence of multiple active PARPs in a cell at any given time and their compensatory nature, determining the contribution made by one specific PARP to the ADP-ribosyl proteome has proven to be difficult. Recently, a number of novel MS-based techniques have been developed to identify proteins that are ADPribosylated (protein or peptide identification) as well as the specific sites of ADP-ribosylation (Daniels et al 2015a). These methods rely on approaches to (1) degrade PAR chains down to a single ADP-ribosyl moiety and (2) cleave the ADP-ribosyl moiety to produce an identifiable mass shift in the mass spectrometer.…”

“…These studies have begun to reveal the promising ways in which PARPs may be targeted therapeutically for the treatment of disease. In this review, we discuss these topics and relate them to the future directions of the field.ADP-ribosylation is a reversible post-translational modification (PTM) of proteins resulting in the covalent attachment of a single ADP-ribose unit [i.e., mono(ADP-ribose) (MAR)] or polymers of ADP-ribose units [i.e., poly(ADP-ribose) (PAR)] on a variety of amino acid residues on target proteins (Gibson and Kraus 2012;Daniels et al 2015a). This modification is mediated by a diverse group of ADPribosyl transferase (ADPRT) enzymes that use ADP-ribose units derived from β-NAD + to catalyze the ADP-ribosylation reaction.…”

At the Interface of Three Nucleic Acids: The Role of RNA-Binding Proteins and Poly(ADP-ribose) in DNA Repair

“…This finding provides insight into why PARP is the preferred target for poly(ADP-ribosyl)ation [134]. The PAR acceptor amino acid residues identified in PARP1 and other poly(ADP-ribosyl)ation targets to date are multivarious: Lys, Arg, Glu, Asp, Cys, Ser, Thr, Sep (through the phosphate group) and Asn, although charged amino acid residues are typically responsible for this function [146-149]. Bearing in mind that the rate of PAR biosynthesis is limited to NAD + breakdown, it is tempting to suggest that the binding of ADP-ribose to a target protein in the presence of activated PARP1 occurs via any amino acid residue exposed on the protein surface [125].…”

ADP-Ribosylation Goes Normal: Serine as the Major Site of the Modification

“…However, it is the intracellular poly-ADP-ribosylation (PARylation) that appears to be essential for the maintenance of genome integrity (Ryu et al, 2015) and is at the core of the article we preview here. In the course of poly-ADP-ribosylation, a number of adenosine diphosphate ribose residues are added sequentially to a nucleophilic side chain of an amino acid (Daniels et al, 2015). At the ADPr-protein junction, this modification structurally represents protein glycosylation, despite the presence of pyrophosphates and nucleosides further in the poly-ADPr chain, which seemingly relates ADP-ribosylation to protein phosphorylation or nucleotidylation.…”

“…The most studied members of the ARTD family are PARP1 (ARTD1) and PARP2 (ARTD2), which are thought to be capable of building the PAR-chain and modifying the side-chain of amino acids to attach the first ADPrmoiety to the protein. There is a broad consensus in the literature that poly-ADP-ribosylation occurs at the side chains Glu, Asp, Lys, or Arg (Daniels et al, 2015), which make this modification disparate from the regular protein glycosylation that happens at Ser (O-glycosylation) or at Asn (N-glycosylation) (Moremen et al, 2012). It is also somewhat enigmatic how the same transferase is capable of catalyzing different chemical transformations.…”