Department of Biosciences and Nutrition, Karolinska Institutet, 14157 Huddinge, Sweden.
Center for Molecular Protein Science, Department of Chemistry, Lund University, 22100 Lund, Sweden.
Cells. 2021 Mar 15;10(3):654. doi: 10.3390/cells10030654.
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.
多聚 ADP-核糖聚合酶 (PARP)-家族 ADP-核糖基转移酶在各种信号通路中发挥作用，主要在核和细胞质中。尽管 PARP 抑制剂已在癌症治疗的临床实践中应用，但个别 PARP 家族成员的酶活性仍未得到充分理解。我们研究了 PARP10，一种单 ADP-核糖基转移酶和潜在的药物靶点。使用酸脲凝胶电泳，我们发现 PARP10 的分离催化结构域自身进行 ADP-核糖基化 (MARylation)，在 8 个或更多的受体残基上。我们分离出具有单个或多个修饰的单个物种，然后通过质谱进行分析。结果证实了随机顺序的多部位 MARylation，并鉴定了四个受体残基。单个受体残基的突变对整体自身 MARylation 水平的影响较小，对组蛋白 H3.1 的 MARylation 没有影响。总之，我们的结果表明，PARP10 的自动修饰可能在调节分子内或伴侣结合事件中发挥作用，而不是在其酶催化作用中发挥作用。这有助于更好地理解 PARP10 的功能，并且从长远来看，有助于评估 PARP 抑制剂作用的后果。
