ADP-ribosylation (ADPr) is a biologically and clinically important post-translational modification, but little is known about the amino acids it targets on cellular proteins. Here we present a proteomic approach for direct in vivo identification and quantification of ADPr sites on histones.We have identified 12 unique ADPr sites in human osteosarcoma cells and report serine ADPr as a new type of histone mark that responds to DNA damage.

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that synthesise ADP-ribosylation (ADPr), a reversible modification of proteins that regulates many different cellular processes. Several mammalian PARPs are known to regulate the DNA damage response, but it is not clear which amino acids in proteins are the primary ADPr targets. Previously, we reported that ARH3 reverses the newly discovered type of ADPr (ADPr on serine residues; Ser-ADPr) and developed tools to analyse this modification (Fontana et al., 2017). Here, we show that Ser-ADPr represents the major fraction of ADPr synthesised after DNA damage in mammalian cells and that globally Ser-ADPr is dependent on HPF1, PARP1 and ARH3. In the absence of HPF1, glutamate/aspartate becomes the main target residues for ADPr. Furthermore, we describe a method for site-specific validation of serine ADP-ribosylated substrates in cells. Our study establishes serine as the primary form of ADPr in DNA damage signalling.

ADP-ribosylation is a post-translational modification (PTM) of proteins found in organisms from all kingdoms of life which regulates many important biological functions including DNA repair, chromatin structure, unfolded protein response and apoptosis. Several cellular enzymes, such as macrodomain containing proteins PARG [poly(ADP-ribose) glycohydrolase] and TARG1 [terminal ADP-ribose (ADPr) protein glycohydrolase], reverse protein ADP-ribosylation. In the present study, we show that human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (hNUDT16) represents a new enzyme class that can process protein ADP-ribosylation in vitro, converting it into ribose-5'-phosphate (R5P) tags covalently attached to the modified proteins. Furthermore, our data show that hNUDT16 enzymatic activity can be used to trim ADP-ribosylation on proteins in order to facilitate analysis of ADP-ribosylation sites on proteins by MS.

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Modification of proteins with ubiquitin and ubiquitin-like molecules is involved in the regulation of almost every biological process. Historically, each conjugation pathway has its unique set of E1, E2 and E3 enzymes that lead to activation and conjugation of their cognate molecules. Here, we present the unexpected finding that under stress conditions, the ubiquitin E1 enzyme Ube1 mediates conjugation of the ubiquitin-like molecule NEDD8. Inhibition of the 26S proteasome, heat shock and oxidative stress cause a global increase in NEDDylation. Surprisingly, this does not depend on the NEDD8 E1-activating enzyme, but rather on Ube1. A common event in the tested stress conditions is the depletion of "free" ubiquitin. A decrease in "free" ubiquitin levels in the absence of additional stress is sufficient to stimulate NEDDylation through Ube1. Further analysis on the NEDD8 proteome shows that the modified NEDDylated proteins are simultaneously ubiquitinated. Mass spectrometry on the complex proteome under stress reveals the existence of mixed chains between NEDD8 and ubiquitin. We further show that NEDDylation of the p53 tumor suppressor upon stress is mediated mainly through Ube1. Our studies reveal an unprecedented interplay between NEDD8 and ubiquitin pathways operating in diverse cellular stress conditions.