ADP-ribosyltransferases (ARTs) catalyze the transfer of ADP-ribose from NAD(+) onto substrates. Some ARTs generate in an iterative process ADP-ribose polymers that serve as adaptors for distinct protein domains. Other ARTs, exemplified by ARTD10, function as mono-ADP-ribosyltransferases, but it has been unclear whether this modification occurs in cells and how it is read. We observed that ARTD10 colocalized with ARTD8 and defined its macrodomains 2 and 3 as readers of mono-ADP-ribosylation both in vitro and in cells. The crystal structures of these two ARTD8 macrodomains and isothermal titration calorimetry confirmed their interaction with ADP-ribose. These macrodomains recognized mono-ADP-ribosylated ARTD10, but not poly-ADP-ribosylated ARTD1. This distinguished them from the macrodomain of macroH2A1.1, which interacted with poly- but not mono-ADP-ribosylated substrates. Moreover, Ran, an ARTD10 substrate, was also read by ARTD8 macrodomains. This identifies readers of mono-ADP-ribosylated proteins, defines their structures, and demonstrates the presence of this modification in cells.

Adenosine diphosphate-ribosylation is a post-translational modification mediated by intracellular and membrane-associated extracellular enzymes and many bacterial toxins. The intracellular enzymes modify their substrates either by poly-ADP-ribosylation, exemplified by ARTD1/PARP1, or by mono-ADP-ribosylation. The latter has been discovered only recently, and little is known about its physiological relevance. The founding member of mono-ADPribosyltransferases is ARTD10/PARP10. It possesses two ubiquitin-interaction motifs, a unique feature among ARTD/PARP enzymes. Here, we find that the ARTD10 ubiquitininteraction motifs bind to K63-linked poly-ubiquitin, a modification that is essential for NF-kB signalling. We therefore studied the role of ARTD10 in this pathway. ARTD10 inhibits the activation of NF-kB and downstream target genes in response to interleukin-1b and tumour necrosis factor-a, dependent on catalytic activity and poly-ubiquitin binding of ARTD10. Mechanistically ARTD10 interferes with poly-ubiquitination of NEMO, which interacts with and is a substrate of ARTD10. Our findings identify a novel regulator of NF-kB signalling and provide evidence for cross-talk between K63-linked poly-ubiquitination and mono-ADPribosylation.

BackgroundAlthough ADP-ribosylation has been described five decades ago, only recently a distinction has been made between eukaryotic intracellular poly- and mono-ADP-ribosylating enzymes. Poly-ADP-ribosylation by ARTD1 (formerly PARP1) is best known for its role in DNA damage repair. Other polymer forming enzymes are ARTD2 (formerly PARP2), ARTD3 (formerly PARP3) and ARTD5/6 (formerly Tankyrase 1/2), the latter being involved in Wnt signaling and regulation of 3BP2. Thus several different functions of poly-ADP-ribosylation have been well described whereas intracellular mono-ADP-ribosylation is currently largely undefined. It is for example not known which proteins function as substrate for the different mono-ARTDs. This is partially due to lack of suitable reagents to study mono-ADP-ribosylation, which limits the current understanding of this post-translational modification.ResultsWe have optimized a novel screening method employing protein microarrays, ProtoArrays®, applied here for the identification of substrates of ARTD10 (formerly PARP10) and ARTD8 (formerly PARP14). The results of this substrate screen were validated using in vitro ADP-ribosylation assays with recombinant proteins. Further analysis of the novel ARTD10 substrate GSK3β revealed mono-ADP-ribosylation as a regulatory mechanism of kinase activity by non-competitive inhibition in vitro. Additionally, manipulation of the ARTD10 levels in cells accordingly influenced GSK3β activity. Together these data provide the first evidence for a role of endogenous mono-ADP-ribosylation in intracellular signaling.ConclusionsOur findings indicate that substrates of ADP-ribosyltransferases can be identified using protein microarrays. The discovered substrates of ARTD10 and ARTD8 provide the first sets of proteins that are modified by mono-ADP-ribosyltransferases in vitro. By studying one of the ARTD10 substrates more closely, the kinase GSK3β, we identified mono-ADP-ribosylation as a negative regulator of kinase activity.

BackgroundADP-ribosylation is a posttranslational modification catalyzed in cells by ADP-ribosyltransferases (ARTD or PARP enzymes). The ARTD family consists of 17 members. Some ARTDs modify their substrates by adding ADP-ribose in an iterative process, thereby synthesizing ADP-ribose polymers, the best-studied example being ARTD1/PARP1. Other ARTDs appear to mono-ADP-ribosylate their substrates and are unable to form polymers. The founding member of this latter subclass is ARTD10/PARP10, which we identified as an interaction partner of the nuclear oncoprotein MYC. Biochemically ARTD10 uses substrate-assisted catalysis to modify its substrates. Our previous studies indicated that ARTD10 may shuttle between the nuclear and cytoplasmic compartments. We have now addressed this in more detail.ResultsWe have characterized the subcellular localization of ARTD10 using live-cell imaging techniques. ARTD10 shuttles between the cytoplasmic and nuclear compartments. When nuclear, ARTD10 can interact with MYC as measured by bimolecular fluorescence complementation. The shuttling is controlled by a Crm1-dependent nuclear export sequence and a central ARTD10 region that promotes nuclear localization. The latter lacks a classical nuclear localization sequence and does not promote full nuclear localization. Rather this non-conventional nuclear localization sequence results in an equal distribution of ARTD10 between the cytoplasmic and the nuclear compartments. ARTD10 forms discrete and dynamic bodies primarily in the cytoplasm but also in the nucleus. These contain poly-ubiquitin and co-localize in part with structures containing the poly-ubiquitin receptor p62/SQSTM1. The co-localization depends on the ubiquitin-associated domain of p62, which mediates interaction with poly-ubiquitin.ConclusionsOur findings demonstrate that ARTD10 is a highly dynamic protein. It shuttles between the nuclear and cytosolic compartments dependent on a classical nuclear export sequence and a domain that mediates nuclear uptake. Moreover ARTD10 forms discrete bodies that exchange subunits rapidly. These bodies associate at least in part with the poly-ubiquitin receptor p62. Because this protein is involved in the uptake of cargo into autophagosomes, our results suggest a link between the formation of ARTD10 bodies and autophagy.Lay abstractPost-translational modifications refer to changes in the chemical appearance of proteins and occur, as the name implies, after proteins have been synthesized. These modifications frequently affect the behavior of proteins, including alterations in their activity or their subcellular localization. One of these modifications is the addition of ADP-ribose to a substrate from the cofactor NAD+. The enzymes responsible for this reaction are ADP-ribosyltransferases (ARTDs or previously named PARPs). Presently we know very little about the role of mono-ADP-ribosylation of proteins that occurs in cells. We identified ARTD10, a mono-ADP-ribosyltransferase, as an interaction partner of the oncoprotein MYC. In this study we ...

is a post-translational modification that regulates various physiological processes, including DNA damage repair, gene transcription and signal transduction. Intracellular ADP-ribosyltransferases (ARTDs or PARPs) modify their substrates either by poly-or mono-ADP-ribosylation. Previously we identified ARTD10 (formerly PARP10) as a mono-ADP-ribosyltransferase, and observed that exogenous ARTD10 but not ARTD10-G888W, a catalytically inactive mutant, interferes with cell proliferation. To expand on this observation, we established cell lines with inducible ARTD10 or ARTD10-G888W. Consistent with our previous findings, induction of the wild-type protein but not the mutant inhibited cell proliferation, primarily by inducing apoptosis. During apoptosis, ARTD10 itself was targeted by caspases. We mapped the major cleavage site at EIAMD406↓S, a sequence that was preferentially recognized by caspase-6. Caspase-dependent cleavage inhibited the pro-apoptotic activity of ARTD10, as ARTD10(1-406) and ARTD10(407-1025), either alone or together, were unable to induce apoptosis, despite catalytic activity of the latter. Deletion of the N-terminal RNA recognition motif in ARTD10(257-1025) also resulted in loss of pro-apoptotic activity. Thus our findings indicate that the RNA recognition motif contributes to the pro-apoptotic effect, together with the catalytic domain. We suggest that these two domains must be physically linked to stimulate apoptosis, possibly targeting ARTD10 through the RNA recognition motif to specific substrates that control cell death. Moreover, we established that knockdown of ARTD10 reduced apoptosis in response to DNA-damaging agents. Together, these findings indicate that ARTD10 is involved in the regulation of apoptosis, and that, once apoptosis is activated, ARTD10 is cleaved as part of negative feedback regulation. Structured digital abstractCaspase-8 cleaves ARTD10 by protease assay (View interaction) Caspase-6 cleaves ARTD10 by protease assay (View interaction) Abbreviations ART, ADP-ribosyltransferase; ARTD, ADP-ribosyltransferase diphtheria toxin-like; FACS, fluorescence-activated cell sorting; HA, hemagglutinin; PI, propidium iodide; RRM, RNA recognition motif.