Methods to Study TCDD-Inducible Poly-ADP-Ribose Polymerase (TIPARP) Mono-ADP-Ribosyltransferase Activity

Abstract

TCDD-inducible poly-ADP-ribose polymerase (TIPARP; also known as PARP7 and ARTD14) is a mono-ADP-ribosyltransferase that has emerged as an important regulator of innate immunity, stem cell pluripotency, and transcription factor regulation. Characterizing TIPARP's catalytic activity and identifying its target proteins are critical to understanding its cellular function. Here we describe methods that we use to characterize TIPARP catalytic activity and its mono-ADP-ribosylation of its target proteins.

Loss of Tiparp Results in Aberrant Layering of the Cerebral Cortex

“…Mono-ADP-ribosylation (MAR) of α-tubulin was verified by immunoprecipitation as previously described (Hutin et al, 2018a). In brief, mNSCs were harvested and the cell pellet was lysed in cell lysis buffer mNSCs (200 mM NaCl, 1% NP40, and 20 mM HEPES at pH 7.4, supplemented with protease inhibitor cocktail; Sigma).…”

“…However, it is activated by numerous endogenous ligands and plays important roles in immune function, inflammation, stem cell differentiation, neurologic development and behavior (Stockinger et al, 2014). It has previously been reported that TIPARP functions as part of a negative feedback loop regulating AHR activity and that Tiparp –/– mice exhibit an increased sensitivity to dioxin-induced toxicities primarily due to increased AHR activity (Hutin et al, 2018).…”

Structure-Guided Design and In-Cell Target Profiling of a Cell-Active Target Engagement Probe for PARP Inhibitors

“…11 Finally, recombinant glutathione S-transferase (GST)-tagged PARP6 was incubated with a biotinylated analogue of NAD + and increasing concentrations of PARPYnD. The reactions were separated by gel electrophoresis, transferred to nitrocellulose, and immunoblotted with NeutrAvidin-HRP (Figure 2C); 22 dose-dependent inhibition of PARP6 auto-MARylation was observed, indicating that PARPYnD binds to PARP6 and inhibits its catalytic activity. These data support the proposal that PARPYnD is an effective tool to interrogate the biomolecular profile of AZ9482 and AZ0108, and therefore, the molecule was taken forward to preliminary live cell profiling studies.…”

“… Finally, recombinant glutathione S -transferase (GST)-tagged PARP6 was incubated with a biotinylated analogue of NAD + and increasing concentrations of PARPYnD . The reactions were separated by gel electrophoresis, transferred to nitrocellulose, and immunoblotted with NeutrAvidin-HRP (Figure C); dose-dependent inhibition of PARP6 auto-MARylation was observed, indicating that PARPYnD binds to PARP6 and inhibits its catalytic activity.…”