Tools for investigating ADP-ribosylation of specific PARP members

Abstract

ADP-ribosylation is a post-translational modification that plays a critical role in several cellular processes, including DNA damage repair. It is catalyzed by the family of poly(ADP-ribose) polymerases (PARPs), by transferring single or multiple ADP-ribose molecules to proteins using nicotinamide adenine dinucleotide (NAD+) as a substrate. For a better understanding of their biological function, it is crucial to study the ADP-ribosylation of individual members of the PARP family. However, due to the overlapping functions of PARP members and the fact that they all use NAD+ as a substrate, this remains a challenge. Within this thesis novel approaches were developed to investigate ADP-ribosylation of individual PARP members. To this end, the “bump-hole strategy” was used, in which the nicotinamide binding site of a specific PARP member is modified by introducing a hydrophobic pocket (“hole”’) so that it can bind an orthogonal NAD+ containing a small alkyl or benzyl moiety (“bump”) on the nicotinamide moiety. In addition, a fluorescent or affinity tag was introduced directly onto the C2 position of the adenine moiety of the NAD+, allowing a direct read out of the ADP-ribosylation signal or enrichment of ADP-ribosylated proteins. To apply this strategy, it was first necessary to determine whether modifications at the C2 position of the adenine, such as the affinity tag d-desthiobiotin (DTB) are tolerated by the PARP enzymes. Therefore, the DTB labeled NAD+ analog DTB-NAD+ was used and its acceptance as a substrate of various PARP members was investigated in an auto-ADP ribosylation assay. Using this assay, it was shown that all PARPs tested (PARP1, 2, 3, 5a, 5b, 6, 10, and 14) accept DTB-NAD+ as a substrate. Based on this result orthogonal NAD+ analogs were synthesized that can no longer be used as a substrate by wild-type (wt)-PARPs and thus can be used in the bump-hole strategy together with a specific PARP-mutant. The nicotinamide site of the NAD+ analogs was modified with either a methyl (Me), ethyl (Et), or benzyl (Bn) group and the adenine moiety with a fluorophore 5’’-carboxy-tetramethyl rhodamine (TMR) or an affinity label DTB, resulting in the new NAD+ analogs Et-TMR-NAD+, Me-DTB-NAD+, Et-DTB-NAD+, and Bn-DTB-NAD+. These molecules were then evaluated in the bump-hole strategy for PARP1. For this purpose, the acceptance of all four NAD+s by the mutant KA-PARP1, which has a hydrophobic pocket (“hole”) in the nicotinamide binding site, was tested in vitro in auto-ADP ribosylation assays and compared with several wt-PARPs. Et-DTB-NAD+ showed the best results, since it was used as a substrate by KA-PARP1, but not or only to a very low extent by the tested wt-PARPs (PARP1, 2, 3, 5a, 5b). This new enzyme-substrate pair (KA-PARP1 and Et-DTB-NAD+) was then successfully used in affinity enrichment experiments in combination with LC-MS/MS proteomics. This led to the identification of 45 potential PARP1-specific protein targets. In the final section of this work, the method was extended to investigate ADP-ribosylation directly in living cells. To achieve this, the NAD+ analogs were transfected using the transfection reagent DOTAP. In addition to the KA-PARP1 and Et-DTB-NAD+ enzyme-substrate pair, the IG-PARP7 and Bn-DTB-NAD+ enzyme-substrate pair was employed. The attempt to ADP-ribosylate proteins using KA-PARP1 in cells was unsuccessful, likely due to its low activity. However, ADP-ribosylation of proteins was successful when using the PARP7 mutant IG-PARP7 together with its substrate Bn-DTB-NAD+. Thus, a novel enzyme-substrate pair was found potentially allowing the investigation of PARP7-specific ADP-ribosylation in cells.