The post-translational modification (PTM) and signaling molecule poly(ADP-ribose) (PAR) has an impact on diverse biological processes. This PTM is regulated by a series of ADP-ribosyl glycohydrolases (PARG enzymes) that cleave polymers and/or liberate monomers from their protein targets. Existing methods for monitoring these hydrolases rely on detection of the natural substrate, PAR, commonly achieved via radioisotopic labeling. Here we disclose a general substrate for monitoring PARG activity, TFMU-ADPr, which directly reports on total PAR hydrolase activity via release of a fluorophore; this substrate has excellent reactivity, generality (processed by the major PARG enzymes), stability, and usability. A second substrate, TFMU-IDPr, selectively reports on PARG activity only from the enzyme ARH3. Use of these probes in whole-cell lysate experiments has revealed a mechanism by which ARH3 is inhibited by cholera toxin. TFMU-ADPr and TFMU-IDPr are versatile tools for assessing small-molecule inhibitors in vitro and probing the regulation of ADP-ribosyl catabolic enzymes.

中文翻译：

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用连续荧光底物监测聚（ADP-核糖基）糖基水解酶活性。

翻译后修饰（PTM）和信号分子聚（ADP-核糖）（PAR）对多种生物过程都有影响。此PTM受一系列ADP-核糖基糖水解酶（PARG酶）调控，该酶可将聚合物裂解和/或将单体从其蛋白质靶标中释放出来。监测这些水解酶的现有方法依赖于通常通过放射性同位素标记实现的天然底物PAR的检测。在这里，我们公开了用于监测PARG活性的通用底物TFMU-ADPr，其通过荧光团的释放直接报告总PAR水解酶活性。这种底物具有出色的反应性，通用性（由主要的PARG酶处理），稳定性和可用性。第二种底物TFMU-IDPr仅从酶ARH3选择性报告PARG活性。在全细胞裂解物实验中使用这些探针揭示了霍乱毒素可抑制ARH3的机制。TFMU-ADPr和TFMU-IDPr是用于评估体外小分子抑制剂和探索ADP-核糖基分解代谢酶调控的多功能工具。