The superior salinity tolerance of bread wheat cultivar Shanrong No. 3 is unlikely to be caused by elevated Ta-sro1 poly-(ADP-ribose) polymerase activity

Structural and biochemical analyses demonstrate that the elevated salinity tolerance of bread wheat cultivar Shanrong No. 3 is unlikely to be caused by elevated Ta-sro1 poly(ADP-ribose) polymerase activity

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

Characterization of the mono-ADP-ribosylation by ARTD10 : substrates, consequences and reversibility

Posttranslational modifications are involved in basically all cellular processes. Some of them have been studied quite extensively, such as phosphorylation and ubiquitination. Others, mono-ADP-ribosylation for example, have currently barely been investigated. Mono-ADP-ribosylating enzymes transfer an ADP-ribose moiety from the cofactor NAD+ onto a target substrate. ARTD10 has been demonstrated to be an enzyme catalyzing the transfer of mono-ADP-ribose, but has not been investigated in more detail. Here a protein microarray-based substrate-screen is presented, not only for ARTD10 substrates but also for ARTD8 substrates. The results are validated and analyzed. To characterize the functional consequences of mono-ADP-ribosylation, GSK3beta is used as prototype substrate. We could show that mono-ADP-ribosylation of GSK3beta inhibits kinase activity in vitro as well as in cells. Moreover, we identified MDO2 as ADP-ribosylhydrolase capable of removing ADP-ribose from both ARTD10 itself and GSK3beta, which suffices to restore kinase activity. The interaction between ARTD10 and GSK3beta was addressed by bioinformatical modeling studies and GSK3beta was identified as kinase of ARTD10 in vitro. Lastly, we investigated methods to identify ADP-ribosylation sites by mass spectrometry and by peptide arrays, for which the currently employed methods are summarized in the introduction. This study implies that mono-ADP-ribosylating enzymes are highly specific, as only a small percentage of the 8000 proteins tested on the protein microarrays were modified. Moreover, the functional consequence of mono-ADP-ribosylation for a substrate protein described here has not been shown before for any eukaryotic intracellular mono-ADP-ribosylating enzyme and adds a new dimension to the known regulatory mechanisms of GSK3beta. The hydrolyzing activities of MDO2 are also a novelty unlike any published before for the removal of mono-ADP-ribosylation and indicate that mono-ADP-ribosylation is a dynamic posttranslational modification. Together, these findings provide a basis for future research addressing the physiological relevance of mono-ADP-ribosylation in eukaryotic cells

Characterization of the mono-ADP-ribosylation by ARTD10 : substrates, consequences and reversibility

Posttranslational modifications are involved in basically all cellular processes. Some of them have been studied quite extensively, such as phosphorylation and ubiquitination. Others, mono-ADP-ribosylation for example, have currently barely been investigated. Mono-ADP-ribosylating enzymes transfer an ADP-ribose moiety from the cofactor NAD+ onto a target substrate. ARTD10 has been demonstrated to be an enzyme catalyzing the transfer of mono-ADP-ribose, but has not been investigated in more detail. Here a protein microarray-based substrate-screen is presented, not only for ARTD10 substrates but also for ARTD8 substrates. The results are validated and analyzed. To characterize the functional consequences of mono-ADP-ribosylation, GSK3beta is used as prototype substrate. We could show that mono-ADP-ribosylation of GSK3beta inhibits kinase activity in vitro as well as in cells. Moreover, we identified MDO2 as ADP-ribosylhydrolase capable of removing ADP-ribose from both ARTD10 itself and GSK3beta, which suffices to restore kinase activity. The interaction between ARTD10 and GSK3beta was addressed by bioinformatical modeling studies and GSK3beta was identified as kinase of ARTD10 in vitro. Lastly, we investigated methods to identify ADP-ribosylation sites by mass spectrometry and by peptide arrays, for which the currently employed methods are summarized in the introduction. This study implies that mono-ADP-ribosylating enzymes are highly specific, as only a small percentage of the 8000 proteins tested on the protein microarrays were modified. Moreover, the functional consequence of mono-ADP-ribosylation for a substrate protein described here has not been shown before for any eukaryotic intracellular mono-ADP-ribosylating enzyme and adds a new dimension to the known regulatory mechanisms of GSK3beta. The hydrolyzing activities of MDO2 are also a novelty unlike any published before for the removal of mono-ADP-ribosylation and indicate that mono-ADP-ribosylation is a dynamic posttranslational modification. Together, these findings provide a basis for future research addressing the physiological relevance of mono-ADP-ribosylation in eukaryotic cells

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

Regulation of NF-κB signalling by the mono-ADP-ribosyltransferase ARTD10

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-AD-Pribosyltransferases is ARTD10/PARP10. It possesses two ubiquitin-interaction motifs, a unique feature among ARTD/PARP enzymes. Here, we find that the ARTD10 ubiquitin-interaction motifs bind to K63-linked poly-ubiquitin, a modification that is essential for NF-kappa B 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-1 beta 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-ADP-ribosylation