Recognition of mono-ADP ribosylated ARTD10 substrates by ARTD8 macrodomains and acetylation of ARTD10
Year: 2014
Type: dissertation
Abstract: ADP-ribosylation is an ancient posttranslational modification occurring throughout the kingdom of life. Poly-ADP-ribosylation mediated by members of the ARTD (PARP) family plays a pivotal role in the regulation of cellular processes including DNA repair, apoptosis, and signaling. Four different types of domains, e.g. certain macrodomains, have been reported to function as readers for poly-ADP-ribosylation and recruit further effector proteins. Beyond, most of these are also capable of binding to free ADP-ribose but none of them was shown to recognize mono-ADP-ribose attached to proteins. On the other hand several members of the ARTD (formerly PARP) family are unable to poly-ADP-ribosylate substrates but they transfer single units of ADP-ribose onto substrates. A representative and also the best-described member of this ARTD subfamily is ARTD10 (formerly PARP10). In this study two protein domains interacting with mono-ADP-ribosylated ARTD10 and substrates of ARTD10 were identified and analyzed. Initially, a co-localization between ARTD10 and ARTD8, another mono-ADP-ribosyltransferase of the ARTD family, was observed in cells. Since co-localization might suggest interactions between two proteins, the three macrodomains and the WWE domain of ARTD8 were examined for mediating this interaction. It was found that macrodomains 2 and 3 of the three macrodomains of ARTD8 are capable of binding to mono-ADP-ribosylated ARTD10, the recognition being strictly dependent on the mono-ADP-ribosylation. Poly-ADP-ribosylated proteins like activated ARTD1 could not associate with these two domains, distinguishing them from other macrodomains that are described as poly-ADP-ribosylation reader modules. Solving the crystal structures of these macrodomains confirmed their capacity to bind free ADP-ribose. Furthermore, macrodomains 2 and 3 were demonstrated to recognize transiently expressed as well as endogenous ARTD10 but not its catalytically inactive mutant in cells. Additionally it was found that this recognition is not restricted to modified ARTD10 itself but the three tested substrates glycogen synthase kinase 3beta (GSK3beta), NF-kappaB essential modulator (NEMO) and the small G protein Ran-GTP could all be established as interaction partners of macrodomains of ARTD8 after mono-ADP-ribosylation in vitro and Ran-GTP also in cells. Thus this study contributes to the investigation of mono-ADP-ribosylation mediated by enzymes of the ARTD family as a so far poorly characterized posttranslational modification. First evidence of an intracellular mono-ADP-ribosylation activity by ARTD10 is provided by this data. The discovery of two reader domains for mono-ADP-ribosylated ARTD10 substrates provides a tool, which will assist by the characterization of intracellular ARTD10 substrates and investigation of the biological effects caused by mono-ADP-ribosylation in the future. Insights from studies of ARTD1 (formerly PARP1), the founding member of the ARTD family, stress the importance of posttranslational modification and regulation by phosphorylation and acetylation for the enzymatic activity. For ARTD10 a phosphorylation site at threonine T101 by CyclinE/CDK2 has been reported in the literature so far. This phosphorylation stimulates the enzymatic activity. In this work it was found that GCN5 acetylates ARTD10 and lysine K274 was mapped as the targeted site. This site is directly neighboring the glycine-rich region and located between this region and the RNA recognition motif of ARTD10. It remains to be investigated in the future, whether this acetylation site has an effect on the function of these two motifs in ARTD10.
Source: RWTH Publications (RWTH Aachen)
Authors: Alexandra Forst, Bernhard Lüscher
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1041126220