A desthiobiotin labelled NAD+ analogue to uncover Poly(ADP‐ribose) polymerase 1 protein targets

Abstract

AbstractADP‐ribosylation is a post‐translational modification catalyzed by the enzyme family of polyadenosine diphosphate (ADP)‐ribose) polymerases (PARPs). This enzymatic process involves the transfer of single or multiple ADP‐ribose molecules onto proteins, utilizing nicotinamide adenine dinucleotide (NAD+) as a substrate. It, thus, plays a pivotal role in regulating various biological processes. Unveiling PARP‐selective protein targets is crucial for a better understanding of their biological functions. Nonetheless, this task proves challenging due to overlapping targets shared among PARP family members. Therefore, we applied the “bump‐and‐hole” strategy to modify the nicotinamide binding site of PARP1 by introducing a hydrophobic pocket (“hole”). This PARP1‐mutant binds an orthogonal NAD+ (Et‐DTB‐NAD+) containing an ethyl group (“bump”) at the nicotinamide moiety. Furthermore, we added a desthiobiotin (DTB) tag directly to the adenosine moiety, enabling affinity enrichment of ADP‐ribosylated proteins. Employing this approach, we successfully identified protein targets modified by PARP1 in cell lysate. This strategy expands the arsenal of chemically modified NAD+ analogs available for studying ADP‐ribosylation, providing a powerful tool to study these critical post‐translational modifications.