Reversal of residue-specific ADP-ribosylations by ADP-ribosyl-acceptor hydrolases

摘要 残基特异性、可逆的蛋白质 ADP 核糖基化调节广泛的生物过程，包括 DNA 损伤反应、基因表达和细胞死亡。因此，细胞 ADP- 的稳态 核糖基化对于维持基因组完整性至关重要。 ADP-核糖基受体水解酶 (ARH) 是一种 调节位点特异性 ADP-核糖基化的金属酶家族。 ARH3 特异性逆转聚 (ADP- 核糖）和丝氨酸处的单 ADP 核糖基化，这是 DNA 损伤后修饰的主要位点，而 ARH1 裂解精氨酸处的单 ADP 核糖基化。然而，人们在认识上存在着根本性的差距。 ARHs 的底物选择性、催化作用和特定活性的功能，这很大程度上是由于缺乏 定量和方便的工具以及底物结合活性形式的结构信息不足。的 该应用的目的是开发新颖的定量分析方法，选择性地测量 残基特异性 ADP-核糖基化，并阐明底物选择性的机制和每个的作用 ARH3 的酶活性。为了支持这一目标，我们开发了一个高度敏感、定量和 方便的基于荧光的测定，专门监测聚（ADP-核糖）或丝氨酸的逆转 ARH3 的单 ADP 核糖基化。我们还确定了与完整的 ARH3 结合的初始结构 基材。在这些强有力的初步数据的指导下，我们将追求三个具体目标来检验我们的假设 ARH 的金属配位态和独特的结构可塑性与底物选择性有关 和高效的催化作用。在目标 1 中，我们将全面开发基于荧光的定量检测方法，选择性地 监测残基特异性单 ADP 核糖基化的逆转。在目标 2 中，我们将确定结构 ARH 特异性底物识别和切割的碱基。在目标 3 中，我们将确定 ARH3 分离 - 功能突变体来定义 ARH3 每种酶活性的作用。我们的研究将提供新的 研究自然界中多种 ADP-核糖基化代谢酶并推进我们的研究的定量工具 了解 ARH 作为维持生命的基本过程的机制和功能。

Abstract Residue-specific, reversible protein ADP-ribosylations regulate a broad range of biological processes, including DNA damage responses, gene expression, and cell death. Therefore, homeostasis of cellular ADP- ribosylations is essential for maintaining genomic integrity. ADP-ribosyl-acceptor hydrolases (ARHs) are a family of metalloenzymes that regulate site-specific ADP-ribosylations. ARH3 specifically reverses poly(ADP- ribose) and mono-ADP-ribosylation at serine, a major site for modification following DNA damage, whereas ARH1 cleaves mono-ADP-ribosylation at arginine. However, there is a fundamental gap in understanding of substrate selectivity, catalysis, and function of specific activities of ARHs, which is largely due to the lack of quantitative and convenient tools and insufficient structural information on substrate-bound active forms. The objective of this application is to develop novel quantitative assays that selectively measure the reversal of residue-specific ADP-ribosylations and to elucidate the mechanism of substrate selectivity and the role of each enzymatic activity of ARH3. In support of this objective, we have developed a highly sensitive, quantitative, and convenient fluorescence-based assays that specifically monitor the reversal of poly(ADP-ribose) or serine mono-ADP-ribosylation by ARH3. We have also determined initial structures of ARH3 bound to intact substrates. Guided by these strong preliminary data, we will pursue three specific aims to test our hypothesis that the metal-coordination states and unique structural plasticity of ARHs are linked to substrate selectivity and efficient catalysis. In Aim 1, we will fully develop quantitative fluorescence-based assays that selectively monitor the reversal of residue-specific mono-ADP-ribosylations. In Aim 2, we will determine the structural bases for the specific substrate recognition and cleavage by ARHs. In Aim 3, we will identify ARH3 separation- of-function mutants to define the role of each enzymatic activity of ARH3. Our studies will provide new quantitative tools to study diverse ADP-ribosylation-metabolizing enzymes in nature and advance our understanding of the mechanisms and functions of ARHs as essential processes for maintaining life.