PARP10 Multi-Site Auto- and Histone MARylation Visualized by Acid-Urea Gel Electrophoresis

Abstract

Poly-ADP-ribose polymerase (PARP)-family ADP-ribosyltransferases function in various signaling pathways, predominantly in the nucleus and cytosol. Although PARP inhibitors are in clinical practice for cancer therapy, the enzymatic activities of individual PARP family members are yet insufficiently understood. We studied PARP10, a mono-ADP-ribosyltransferase and potential drug target. Using acid-urea gel electrophoresis, we found that the isolated catalytic domain of PARP10 auto-ADP-ribosylates (MARylates) at eight or more acceptor residues. We isolated individual species with either singular or several modifications and then analyzed them by mass spectrometry. The results confirmed multi-site MARylation in a random order and identified four acceptor residues. The mutagenesis of singular acceptor residues had a minor impact on the overall auto-MARylation level and no effect on the MARylation of histone H3.1. Together, our results suggest that PARP10 automodification may have functions in the regulation of intramolecular or partner binding events, rather than of its enzymatic catalysis. This contributes to a better understanding of PARP10 functions, and, in the long run, to gauging the consequences of PARP inhibitor actions.

Systematic analysis of ADP-ribose detection reagents and optimisation of sample preparation to detect ADP-ribosylationin vitroand in cells

“…We could furthermore confirm that ARH3 reverses both PAR and serine-linked MARylation ( Figure 1c ), but also appears to have some activity towards PARP10 ( Figure 1b ),. This was hinted at in earlier work studying reversal of ADP-ribosylation by PARP10 (6) and would be expected if PARP10 also automodifies on serine as reported (51). ARH1 reverses the arginine modification introduced by mART2.2 very efficiently, but has no activity towards other modified amino acids ( Figure 1d ).…”

“…We purified the known erasers of ADP-ribosylation from bacteria: MACROD1, MACROD2, TARG1, PARG, ARH1 and ARH3 (Figure 1a). The majority of described activities we could confirm, for example MACROD1, MACROD2 and TARG1 reverse the modification introduced by PARP10 although not fully, as has been seen before (Figure 1b and Supplementary Figure 2) (6,51). We could furthermore confirm that ARH3 reverses both PAR and serine-linked MARylation (Figure 1c), but also appears to have some activity towards PARP10 (Figure 1b),.…”

“…Partial reversal of PARP10 automodification by PARG and ARH3 was observed before, which may be reversal of oligomers that PARP10 was reported to generate (6). A more recent study using recombinant proteins also hinted at PARP10 promiscuity, as the modification of serine, arginine and glutamate by PARP10 was detected using mass spectrometry (51). We incubated the protein with different hydrolases and combinations thereof.…”

Rapid Analysis of ADP-Ribosylation Dynamics and Site-Specificity Using TLC-MALDI

“…García-Saura and co-workers recently described an acid-urea gel system that aids visualization of multisite ADP-ribose modifications; their findings highlight the value of additional technologies to study multisite ADP-ribosylation—especially those that allow for quantification of multisite labeling. 23 …”

Are PARPs promiscuous?

“…If indeed the macrodomain-containing hydrolases MACROD1, MACROD2 and TARG1 reverse modification of acidic residues, the question remains what the residual signal may be. In a mass-spectrometry based analysis of PARP10 automodification, glutamate, arginine and serines were identified as modification sites [ 59 ], which forms a putative explanation for the remaining signal after hydrolase incubation. However, sequential incubation of PARP10 with first MACROD1 and then ARH1, ARH3, PARG or combinations thereof did not lead to a further reduction of the signal [ 60 ].…”