Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes.
Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes.
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DOI：
10.1371/journal.pone.0254022
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Kim IK
中科院分区：
文献类型：
--
作者：
Kasson S;Dharmapriya N;Kim IK
ADP-核糖基化是调节多种细胞应激反应的关键翻译后修饰。 ADP-核糖基化循环由写入器和擦除器维持。例如，聚（ADP-核糖基）化循环由两种主要酶组成：聚（ADP-核糖）聚合酶（PARP）和聚（ADP-核糖）糖水解酶（PARG）。然而，历史上，ADP-核糖基化擦除机制尚未得到充分研究，主要是由于缺乏选择性监测不同 ADP-核糖基化逆转酶的特定活性的定量工具。在这里，我们开发了一种新的 NUDT5 偶联 AMP-Glo (NCAG) 测定法，专门监测 ADP-核糖基化逆转酶释放的无蛋白质 ADP-核糖。我们发现 NUDT5 选择性切割不含蛋白质的 ADP-核糖，但不切割蛋白质结合的多聚和单 ADP-核糖基化、不含蛋白质的聚 (ADP-核糖) 链或 NAD+。作为概念验证，我们成功测量了 PARG（释放单体 ADP-核糖）外切糖基水解酶活性的动力学参数，并监测了位点特异性单 ADP-核糖基受体水解酶（例如 ARH3 和 TARG1）的活性。该 NCAG 测定可用作研究多种 ADP-核糖基化逆转酶的机制的通用平台，这些酶释放无蛋白质的 ADP-核糖作为产物。此外，该测定提供了一个有用的工具来识别靶向 ADP-核糖基化代谢的小分子探针并量化细胞中的 ADP-核糖浓度。
ADP-ribosylation is a key post-translational modification that regulates a wide variety of cellular stress responses. The ADP-ribosylation cycle is maintained by writers and erasers. For example, poly(ADP-ribosyl)ation cycles consist of two predominant enzymes, poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolase (PARG). However, historically, mechanisms of erasers of ADP-ribosylations have been understudied, primarily due to the lack of quantitative tools to selectively monitor specific activities of different ADP-ribosylation reversal enzymes. Here, we developed a new NUDT5-coupled AMP-Glo (NCAG) assay to specifically monitor the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes. We found that NUDT5 selectively cleaves protein-free ADP-ribose, but not protein-bound poly- and mono-ADP-ribosylations, protein-free poly(ADP-ribose) chains, or NAD+. As a proof-of-concept, we successfully measured the kinetic parameters for the exo-glycohydrolase activity of PARG, which releases monomeric ADP-ribose, and monitored activities of site-specific mono-ADP-ribosyl-acceptor hydrolases, such as ARH3 and TARG1. This NCAG assay can be used as a general platform to study the mechanisms of diverse ADP-ribosylation reversal enzymes that release protein-free ADP-ribose as a product. Furthermore, this assay provides a useful tool to identify small-molecule probes targeting ADP-ribosylation metabolism and to quantify ADP-ribose concentrations in cells.
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DOI：
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发表时间：
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影响因子：
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作者：
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DOI：
10.2741/3297
发表时间：
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Frontiers in bioscience (Landmark edition)
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