SUMO化修饰对人源胸腺嘧啶DNA糖基化酶的结构影响及活性调控

目的· 研究SUMO 化修饰对胸腺嘧啶DNA 糖基化酶（thymine DNA glycosylase，TDG）蛋白的结构、稳定性及活性的影响。方法· 建立体外表达纯化体系，获取可用于晶体筛选及活性检测的SUMO-1-TDG 蛋白。通过晶体筛选、衍射数据收集及结构解析，分析SUMO-1-TDG 的分子结构。利用蛋白热稳定性实验检测在SUMO 化修饰前后TDG 稳定性的变化。建立TDG 活性测试体系，探讨SUMO 化修饰对TDG 活性产生的影响。结果· 通过结构解析得到SUMO-1-TDG 分子结构。稳定性、活性实验检测发现TDG 蛋白熔解温度（Tm）值提高约16℃，催化活性提升约9.70 %。...

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Bibliographic Details
Published in上海交通大学学报（医学版） Vol. 38; no. 1; pp. 24 - 29
Main Author 付天然;张良
Format Journal Article
LanguageChinese
Published 上海交通大学基础医学院药理学教研室,上海,200025 2018
Subjects
SUMO化
晶体结构
活性检测
稳定性实验
胸腺嘧啶DNA糖基化酶
晶体结构
sumoylation
稳定性实验
activity assay
crystal structure
胸腺嘧啶DNA糖基化酶
活性检测
SUMO化
thermal shift assay
thymine DNA glycosylase
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Summary:目的· 研究SUMO 化修饰对胸腺嘧啶DNA 糖基化酶（thymine DNA glycosylase，TDG）蛋白的结构、稳定性及活性的影响。方法· 建立体外表达纯化体系，获取可用于晶体筛选及活性检测的SUMO-1-TDG 蛋白。通过晶体筛选、衍射数据收集及结构解析，分析SUMO-1-TDG 的分子结构。利用蛋白热稳定性实验检测在SUMO 化修饰前后TDG 稳定性的变化。建立TDG 活性测试体系，探讨SUMO 化修饰对TDG 活性产生的影响。结果· 通过结构解析得到SUMO-1-TDG 分子结构。稳定性、活性实验检测发现TDG 蛋白熔解温度（Tm）值提高约16℃，催化活性提升约9.70 %。结论· SUMO-1 分子结合TDG 对其进行修饰使得结合位点附近氨基酸形成分子间相互作用，并进一步参与调控TDG 蛋白的稳定性及催化活性。
Bibliography:31-2045/R
thymine DNA glycosylase; sumoylation; crystal structure; thermal shift assay; activity assay
FU Tian-ran, ZHANG Liang（ Department of Pharmacology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China）
Objective To study the effect of sumoylation on the structure, stability and activity of human thymine DNA glycosylase （TDG）. Methods. Expression and purification systems were established for obtaining SUMO-1-TDG protein with high purity which can be used for crystal screening and activity detection. Structure of SUMO-1-TDG was solved after crystal screening, diffraction data collection and structure analysis. The change of TDG stability led by sumoylation was detected through a protein thermal shift assay. In addition, an activity assay was applied to investigate the effect of sumoylation on the activity of TDG. Results A high-resolution structure of SUMO-1-TDG which could clearly describe the interaction between TDG and SUMO-1 was solved. The melting temperature （Tm） v
ISSN:1674-8115
DOI:10.3969/j.issn.1674-8115.2018.01.005