摘要
目的通过构建铜缺乏的细胞模型探究是否存在铜依赖的酶参与铜缺乏导致的心肌肥厚的发生。方法使用CRISPR/Cas9技术,构建Ctr1基因敲除的大鼠心肌细胞株H9c2(Ctr1^(-/-));收集野生型(WT)、Ctr1^(-/-)、铜螯合剂TTM处理的H9c2细胞中的蛋白质,通过铜亲和树脂蛋白质结合实验联合质谱分析筛选铜依赖的酶;通过Western blot检测缺铜细胞中该酶的下游信号通路。结果成功构建Ctr1基因敲除的H9c2细胞株,该细胞株中铜含量显著降低(P<0.05);质谱分析筛选出能与铜相互作用的去泛素化酶OTUB1;铜缺乏的H9c2细胞中泛素化水平升高;铜缺乏条件下H9c2细胞中OTUB1靶蛋白DEPTOR水平下降;mTOR信号通路激活。结论去泛素化酶OTUB1参与铜缺乏的细胞应答,提示其可能参与铜缺乏引起的心肌肥厚的发生。
Objective By establishing a copper deficiency cell model to explore whether there are copper-dependent enzymes involved in cardiac hypertrophy caused by copper deficiency.Methods The Ctr1 knockout H9c2 cell strain was established by CRISPR/Cas9 technology;The proteins in wildtype(WT),Ctr1^(-/-),and copper chelator TTM-treated H9c2 cells were collected,and the protein was screened by copper affinity resin pull down experiment combined with mass spectrometry analysis to find out an enzyme which could interact with copper;The downstream signaling pathway of this enzyme in copper-deficient cells was detected by Western blot.Results The H9c2 cell strain with knockout of Ctr1 was successfully established,and the copper level in this cell strain was significantly reduced(P<0.05).Mass spectrometry analysis screened out the deubiquitinase OTUB1 that could interact with copper;The level of ubiquitination in copper-deficient H9c2 cells was increased;Under the condition of copper deficiency,the level of OTUB1 target protein DEPTOR decreased,and the mTOR signaling pathway was activated in H9c2 cells.Conclusions The deubiquitinase OTUB1 is involved in the cellular response to copper deficiency,suggesting that it may be involved in the development of copper deficiency-induced cardiac hypertrophy.
作者
张宇飞
朱基彦
瞿思遥
张祝琴
刘德培
ZHANG Yu-fei;ZHU Ji-yan;QU Si-yao;ZHANG Zhu-qin;LIU De-pei(Department of Molecular Biology and Biochemistry, State Key Laboratory of Medical Molecular Biology,Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC,Beijing 100005,China)
出处
《基础医学与临床》
2022年第7期1020-1025,共6页
Basic and Clinical Medicine
基金
国家重点研究与发展计划(2021YFA0804903)
中国医学科学院医学科学创新基金(CIFMS,2021-I2M-1-016)。