丝氨酰tRNA合成酶(SerRS)结合并激活去乙酰化酶SIRT2的机制  

The mechanism of seryl-tRNA synthetase(SerRS)binding and activating the deacetylase SIRT2

作　　者：秦懿偲 周德健 李超群[1] 余巍 QIN Yi-cai;ZHOU De-jian;LI Chao-qun;YU Wei(Department of Microbiology,School of Life Sciences,Fudan University,Shanghai 200438,China)

机构地区：[1]复旦大学生命科学学院微生物系,上海200438

出　　处：《复旦学报（医学版）》2020年第4期506-512,530,共8页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金(91749120,31771545)。

摘　　要：目的探究丝氨酰tRNA合成酶(seryl-tRNA synthetase,SerRS)结合并激活去乙酰化酶SIRT2的机制。方法构建SerRS与SIRT2质粒,在293T细胞中进行转染。免疫共沉淀方法检测SerRS与SIRT2、SIRT2突变型之间的相互作用。通过原核纯化蛋白质,体外荧光方法检测SerRS对SIRT2酶活影响。通过结构模拟探究SIRT2的H187位点调控机制。通过Western blot方法检测SerRS的乙酰化修饰。结果SerRS能与SIRT2相互作用,并增强其去乙酰化酶活性。SIRT2 H187Y突变使SIRT2失活,同时也阻断了SIRT2与其SerRS的结合。进一步通过结构模拟发现,SIRT2上的H187Y突变与Q267位点在空间上发生冲突,可能改变SIRT2的构象,进而影响与SerRS的结合界面,且SIRT2能够去乙酰化SerRS。结论SIRT2第187位组氨酸能够调控SerRS与SIRT2的结合,为靶向SIRT2的药物研发提供了新的思路。Objective To investigate the mechanism of seryl-t RNA synthetase(Ser RS)binding and activating the deacetylase SIRT2.Methods The plasmids of Ser RS and SIRT2 were constructed and transfected in 293 T cells.Co-immunoprecipitation was used to detect the interactions between Ser RS and SIRT2 or SIRT2 mutations.Proteins were purified in prokaryotes,and the effect of Ser RS on the deacetylation activity of SIRT2 in vitro was detected by monitoring the fluorescence intensity.Structural simulation was used to investigate the regulation of H187 site on SIRT2.Western blot was used to detect the acetylation level of Ser RS.Results Ser RS interacted with SIRT2 and enhanced its deacetylation activity.SIRT2 H187 Y mutation inhibited SIRT2 activity,which also abrogated its interaction with Ser RS.Through analyzing structural modeling,we found that H187 Y mutation had spatial conflict with Q267,which possibly changed the conformation of SIRT2,and thereby affected the binding interface with Ser RS.SIRT2 could also decrease Ser RS acetylation.Conclusion Histidine 187 in SIRT2 could regulate the binding with Ser RS,which provided a new direction for drug discovery targeting SIRT2.

关 键 词：乙酰化 丝氨酰tRNA合成酶(SerRS) SIRT2 血管生成 

分 类 号：Q559[生物学—生物化学] Q343