Nucleolar-nucleoplasmic shuttling of TARG1 and its control by DNA damage-induced poly-ADP-ribosylation and by nucleolar transcription

Macrodomains are conserved protein folds associated with ADP-ribose binding and turnover. ADP-ribosylation is a posttranslational modification catalyzed primarily by ARTD (aka PARP) enzymes in cells. ARTDs transfer either single or multiple ADP-ribose units to substrates, resulting in mono- or poly-ADP-ribosylation. TARG1/C6orf130 is a macrodomain protein that hydrolyzes mono-ADP-ribosylation and interacts with poly-ADP-ribose chains. Interactome analyses revealed that TARG1 binds strongly to ribosomes and proteins associated with rRNA processing and ribosomal assembly factors. TARG1 localized to transcriptionally active nucleoli, which occurred independently of ADP-ribose binding. TARG1 shuttled continuously between nucleoli and nucleoplasm. In response to DNA damage, which activates ARTD1/2 (PARP1/2) and promotes synthesis of poly-ADP-ribose chains, TARG1 re-localized to the nucleoplasm. This was dependent on the ability of TARG1 to bind to poly-ADP-ribose. These findings are consistent with the observed ability of TARG1 to competitively interact with RNA and PAR chains. We propose a nucleolar role of TARG1 in ribosome assembly or quality control that is stalled when TARG1 is re-located to sites of DNA damage

Mono-ADP-ribosylation by ARTD10 a new modification in NF-kappaB signaling

ADP ribosylation is a post translational modification implicated in many physiological processes. It was originally identified as the enzymatic mechanism associated with several bacterial toxins. Subsequently cellular enzymes have been defined that ADP ribosylate substrates, most relevant here are the intracellular ADP ribosyltransferases of the diphtheria toxin like subfamily (ARTDs). Beside enzymes that catalyze poly ADP ribosylation, exemplified by ARTD1, recently enzymes restricted to mono ADP ribosylation were identified with ARTD10 being the founding member. ARTD10 possesses a unique domain structure due to two ubiquitin interaction motifs (UIMs). In this study biological functions of ARTD10 associated with the UIMs have been investigated. The characterization of the UIMs of ARTD10 demonstrated their ability to specifically interact with K63 linked polyubiquitin. This form of ubiquitination is an essential post translational modification in NF kappa B signaling. Therefore we addressed a potential role for ARTD10 in this pathway. We found that ARTD10 inhibited NF-kappaB activation, p65 translocation into the nucleus and expression of downstream target genes in response to the cytokines IL-1beta and TNF alpha. Catalytic activity of and polyubiquitin binding by ARTD10 were required to execute this function. We present evidence that ARTD10 interferes with polyubiquitination of NEMO, which interacts with ARTD10 and was identified as substrate of ARTD10 dependent mono ADP ribosylation. Together these results define ARTD10 as a novel regulator of NF-kappaB signaling and provide evidence for crosstalk between mono ADP ribosylation and K63 linked polyubiquitination. Furthermore we identified ARTD10 as substrate of IKKepsilon. ARTD10 was phosphorylated by IKKepsilon in vitro as well as in cells. Phosphorylation by IKKepsilon had no apparent impact on catalytic activity but influenced the subcellular localization of ARTD10, which normally shuttles between the nucleus and the cytoplasm. In response to catalytically active IKKepsilon, but not an inactive mutant, ARTD10 accumulated in the nucleus. This finding supports a possible nuclear function of ARTD10 and may contribute to the interaction with MYC and modification of additional nuclear targets such as histones, thereby contributing to the regulation of chromatin

Insight into the Mechanism of Intramolecular Inhibition of the Catalytic Activity of Sirtuin 2 (SIRT2)

Sirtuin 2 (SIRT2) is a NAD+-dependent deacetylase that has been associated with neurodegeneration and cancer. SIRT2 is composed of a central catalytic domain, the structure of which has been solved, and N- and C-terminal extensions that are thought to control SIRT2 function. However structural information of these N- and C-terminal regions is missing. Here, we provide the first full-length molecular models of SIRT2 in the absence and presence of NAD+. We also predict the structural alterations associated with phosphorylation of SIRT2 at S331, a modification that inhibits catalytic activity. Bioinformatics tools and molecular dynamics simulations, complemented by in vitro deacetylation assays, provide a consistent picture based on which the C-terminal region of SIRT2 is suggested to function as an autoinhibitory region. This has the capacity to partially occlude the NAD+ binding pocket or stabilize the NAD+ in a non-productive state. Furthermore, our simulations suggest that the phosphorylation at S331 causes large conformational changes in the C-terminal region that enhance the autoinhibitory activity, consistent with our previous findings that phosphorylation of S331 by cyclin-dependent kinases inhibits SIRT2 catalytic activity. The molecular insight into the role of the C-terminal region in controlling SIRT2 function described in this study may be useful for future design of selective inhibitors targeting SIRT2 for therapeutic applications

ARTD10 substrate identification on protein microarrays: regulation of GSK3β by mono-ADP-ribosylation

BACKGROUND: Although 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. RESULTS: We 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. CONCLUSIONS: Our 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