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

摘要 残基特异的可逆蛋白质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，细胞内ADP的动态平衡- 核糖化对于维持基因组的完整性是必不可少的。腺苷二磷酸核糖受体水解酶(ARHS)是一种 金属酶家族，调节特定部位的ADP-核糖基化。ARH3特异性逆转聚(ADP- 核糖)和丝氨酸上的单-ADP-核糖化，这是DNA损伤后的主要修饰部位，而 ARH1在精氨酸上裂解单-ADP-核糖化。然而，在认识上存在着根本的差距 底物选择性、催化作用和ARHS的特定活性的功能，这在很大程度上是由于缺乏 定量和方便的工具和关于底物结合活性形式的结构信息不足。这个 这项应用的目的是开发新的定量分析方法，选择性地测量细胞的逆转 残基特异的ADP-核糖基化，并阐明底物选择性的机制和每种作用 ARH3的酶活性。为了支持这一目标，我们开发了一种高度敏感、定量和 简便的基于荧光的分析方法，可特异性地监测聚(ADP-核糖)或丝氨酸的逆转 ARH3的单-ADP-核糖基化。我们还确定了ARH3结合完整的初始结构 底物。在这些强劲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 Arhs的金属配位状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分析方法，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 ARHS识别和切割特定底物的基础。在目标3中，我们将确定ARH3分离- 功能缺失突变体以确定ARH3的每一种酶活性的作用。我们的研究将提供新的 定量工具研究自然界中不同的ADP核糖化代谢酶并促进我们的 了解ARHs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.