泛素连接酶SMURF1催化ADAR1的多聚泛素化修饰  

The Ubiquitin Ligase SMURF1 Catalyzes the Polyubiquitination of ADAR1

作　　者：周文淼 王红霞 刘昆梅 刘菁 屈昱良[1,2] 郭乐 ZHOU Wen-Miao;WANG Hong-Xia;LIU Kun-Mei;LIU Jing;QU Yu-Liang;GUO Le(School of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China;School of Laboratory Medicine,Ningxia Medical University,Yinchuan 750004,China;Ningxia Key Laboratory of Cerebrocranial Diseases,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学临床医学院,银川750004 [2]宁夏医科大学检验学院,银川750004 [3]宁夏医科大学颅脑疾病重点实验室,银川750004

出　　处：《中国生物化学与分子生物学报》2023年第11期1630-1637,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.82260321,No.82160497,No.32070930);宁夏自然科学基金(No.2022AAC02034);宁夏高等学校科学研究项目(No.NYG2022045)资助。

摘　　要：既往研究发现,SMAD特异性E3泛素蛋白连接酶1(SMAD specific E3 ubiquitin protein ligase 1,SMURF1)通过其E3泛素连接酶活性介导自噬进程,然而SMURF1的泛素化底物蛋白质仍有待进一步挖掘。本文利用免疫共沉淀(Co-IP)联合蛋白质谱分析捕获并鉴定THP-1细胞中SMURF1的相互作用蛋白质集合物,发现在THP-1细胞中SMURF1可与222种蛋白质物理性结合,RNA腺苷脱氨酶1(adenosine deaminase acting on RNA 1,ADAR1)具有较高的肽段结合分数。构建SMURF1过表达载体并转染到HEK-293T细胞中,Co-IP和Western印迹检测验证外源性SMURF1与内源性ADAR1存在相互作用。qRT-PCR和Western印迹检测结果显示,在HEK-293T细胞中过表达SMURF1后ADAR 1 mRNA水平差异无统计学意义、蛋白质水平明显降低(P<0.05)。用放线菌酮(CHX)分别处理正常和过表达SMURF1的HEK-293T细胞,Western印迹检测显示,过表达SMURF1后ADAR1的半衰期缩短(P<0.05)。进一步在HEK-293T细胞中共转染泛素(Ub)过表达载体和SMURF1过表达载体,通过Co-IP和Western印迹检测结果证实,过表达SMURF1后ADAR1的多聚泛素化水平显著增加(P<0.05)。在蛋白酶体抑制剂(MG132)作用后,Western印迹检测结果表明,蛋白酶体降解途径受抑制后SMURF1对ADAR1的负调控作用减弱(P<0.05)。本研究表明,SMURF1可以与ADAR1相互作用,催化ADAR1的多聚泛素化修饰并介导其蛋白酶体途径降解,为探索SMURF1通过影响ADAR1蛋白质稳定性而具备的多种生物学功能提供理论基础。It is known that SMAD specific E3 ubiquitin protein ligase 1(SMURF1)mediates autophagy through its E3 ubiquitin ligase activity,but the ubiquitinated substrates of SMURF1 need to be further explored.In this paper,the interacting proteins of SMURF1 in THP-1 cells were captured and identified by co-immunoprecipitation(Co-IP)combined with mass spectrometry.It was found that SMURF1 could physically bind to 222 proteins in THP-1 cells,and Adenosine deaminase acting on RNA 1(ADAR1)had a higher peptide binding score.SMURF1 overexpression vectors were constructed and transfected into HEK-293T cells,then Co-IP and Western blotting assays verified the interaction between exogenous SMURF1 and endogenous ADAR1.qRT-PCR and Western blotting assays were carried out after transfecting SMURF1 overexpression vectors in HEK-293T cells,which identified that overexpression of SMURF1 attenuated the protein levels of ADAR1(P<0.05).However,there was no significant difference in the mRNA level of ADAR 1.HEK-293T cells with normal and overexpressing SMURF1 were treated with cycloheximide(CHX),respectively,and Western blotting assays showed a shortened half-life of ADAR1 after overexpression of SMURF1(P<0.05).Furthermore,overexpression of SMURF1 increased the polyubiquitination level of ADAR1 as detected by Co-IP and Western blot(P<0.05).After the proteasome inhibitor(MG132)treatment,the Western blotting assay was performed to demonstrate that the negative regulatory effect of SMURF1 on ADAR1 was weakened after the proteasome degradation pathway was attenuated(P<0.05).This study shows that SMURF1 interacts with ADAR1,catalyzes the polyubiquitination of ADAR1 and mediates its degradation through the proteasome pathway,which provides a theoretical basis for exploring the various biological functions of SMURF1 by affecting the stability of ADAR1.

关 键 词：SMAD特异性E3泛素蛋白连接酶1 RNA腺苷脱氨酶1 泛素化 翻译后修饰 

分 类 号：Q7[生物学—分子生物学]