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

ADP-核糖基受体水解酶逆转残基特异性 ADP-核糖基化

• 批准号: 10794697
• 负责人: In-Kwon Kim
• 金额: $ 4.1万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-03-01
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10794697
• 关键词: ADP ribosylation; Arginine; Binding; Biological Assay; Biological Process; Catalysis; Cell Death; DNA Damage; Data; Disease; Enzymes; Family; Fluorescence; Gene Expression; Genomic Instability; Homeostasis; Hydrolase; Life; Link; Measurement; Measures; Metals; Modification; Monitor; Nature; Process; Proteins; Regulation; Role; Serine; Site; Structure; Testing; base; genome integrity; metalloenzyme; mutant; novel; response; tool

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.

摘要

项目成果

ATP enhances the error-prone ribonucleotide incorporation by the SARS-CoV-2 RNA polymerase.

• DOI: 10.1016/j.bbrc.2022.07.087
• 发表时间: 2022-10-15
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Pourfarjam, Yasin;Ma, Zhijun;Kim, In-Kwon
• 通讯作者: Kim, In-Kwon

Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes.

• DOI: 10.1371/journal.pone.0254022
• 发表时间: 2021
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Kasson S;Dharmapriya N;Kim IK
• 通讯作者: Kim IK

Enzymatic Coupling of a Classical Luciferase Assay for the Quantitative Monitoring of the Protein-Free ADP-ribose.

用于定量监测无蛋白质 ADP-核糖的经典荧光素酶测定的酶促偶联。

• 发表时间: 2022
• 期刊: FASEB journal : official publication of the Federation of American Societies for Experimental Biology
• 作者: Kasson,SamuelP;Kim,In-Kwon
• 通讯作者: Kim,In-Kwon