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，细胞内ADP- 核糖基化是维持基因组完整性所必需的。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。ARH 3特异性逆转poly（ADP- 核糖）和丝氨酸处的单ADP核糖基化，丝氨酸是DNA损伤后修饰的主要位点，而 ARH 1在精氨酸处切割单ADP核糖基化。然而，在理解上存在着根本性的差距， 底物的选择性，催化作用，和功能的比活性的ARHs，这在很大程度上是由于缺乏 定量和方便的工具以及关于底物结合的活性形式的结构信息不足。的 本申请的目的是开发新的定量分析，其选择性地测量逆转的 残基特异性ADP核糖基化并阐明底物选择性的机制及其各自的作用 ARH 3的酶活性。为了支持这一目标，我们制定了一个高度敏感的，定量的， 方便的基于荧光的测定，其特异性地监测聚（ADP-核糖）或丝氨酸 通过ARH 3的单ADP核糖基化。我们还确定了ARH 3的初始结构， 印刷受体.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 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 和高效催化。在目标1中，我们将全面开发基于荧光的定量分析， 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 用于特定底物识别和ARH切割的碱基。在目标3中，我们将确定ARH 3分离- 功能突变体，以确定ARH 3的每种酶活性的作用。我们的研究将提供新的 研究自然界中各种ADP核糖基化代谢酶的定量工具， 了解ARH的机制和功能是维持生命的基本过程。

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.