ADP-ribosylation and NAD+ Utilizing Enzymes

Mareike Bütepage, Sarah Krieg, Laura Eckei, Jinyu Li, Giulia Rossetti, Patricia Verheugd, Bernhard Lüscher, Bütepage, Mareike, Krieg, Sarah, Eckei, Laura, Li, Jinyu, Rossetti, Giulia, Verheugd, Patricia, Lüscher, Bernhard

Mono-ADP-ribosylation is a posttranslational modification, which is catalyzed in cells by certain members of the ADP-ribosyltransferase diphtheria toxin-like family (ARTD) of ADP-ribosyltransferases (aka PARP enzymes). It involves the transfer of a single residue of ADP-ribose (ADPr) from the cofactor NAD+ onto substrate proteins. Although 12 of the 17 members of the ARTD family have been defined as mono-ARTDs in in vitro assays, relatively little is known about their exact cellular functions. A major challenge is the detection of mono-ADP-ribosylated (MARylated) proteins in cells as no antibodies are available that detect exclusively MARylated proteins. As an alternative to classical antibodies, the MAR-specific binding domains macro2 and macro3 of Artd8 can be utilized alone or in combination, to demonstrate intracellular auto-modification levels of ARTD10 in cells in both co-immunoprecipitation and co-localization experiments. Here we demonstrate that different macrodomain constructs of human ARTD8 and murine Artd8, alone or in combination, exert differences with regard to their interaction with ARTD10 in cells. Precisely, while the macrodomains of murine Artd8 interacted with ARTD10 in cells in a MARylation-dependent manner, the macrodomains of human ARTD8 interacted with ARTD10 independent of its catalytic activity. Moreover, we show that a combination of macro2 and macro3 of murine Artd8 was recruited more efficiently to ARTD10 during co-localization experiments compared to the single domains. Therefore, murine Artd8 macrodomain constructs can serve as a tool to evaluate intracellular ARTD10 auto-modification levels using the described methods, while the human ARTD8 macrodomains are less suited because of ADPr-independent binding to ARTD10. Protocols for co-immunoprecipitation and co-localization experiments are described in detail.