An Li, Gao Hong, Zhong Yi, Liu Yanqiu, Cao Ying, Yi Jing, Huang Xiang, Wen Chunlei, Tong Rui, Pan Zhijun, Yan Xu, Liu Meiyan, Wang Shengzhao, Bai Xue, Wu Hao, Hu Tingju
Guizhou Medical University, Guiyang, 550004 Guizhou People's Republic of China.
Department of Anesthesiology, The Affiliated Hospital of Guizhou Medical University, No. 28 Guiyi St, Yunyan District, Guiyang, 550004 Guizhou People's Republic of China.
Cytotechnology. 2023 Jun;75(3):207-217. doi: 10.1007/s10616-023-00570-6. Epub 2023 Feb 3.
To investigate the involvement of stress induced phosphoprotein 1 (STIP1), heat shock protein (HSP) 70, and HSP90 in ubiquitination of connexin 43 (Cx43) in rat H9c2 cardiomyocytes. Co-immunoprecipitation was used to detect protein-protein interactions and Cx43 ubiquitination. Immunofluorescence was used for protein co-localization. The protein binding, Cx43 protein expression, and Cx43 ubiquitination were reanalyzed in H9c2 cells with modified STIP1 and/or HSP90 expression. STIP1 bound to HSP70 and HSP90, and Cx43 bound to HSP40, HSP70, and HSP90 in normal H9c2 cardiomyocytes. Overexpression of STIP1 promoted the transition of Cx43-HSP70 to Cx43-HSP90 and inhibited Cx43 ubiquitination; knockdown of STIP1 resulted in the opposite effects. Inhibition of HSP90 counteracted the inhibitory effect of STIP1 overexpression on Cx43 ubiquitination. STIP1 suppresses Cx43 ubiquitination in H9c2 cardiomyocytes by promoting the transition of Cx43-HSP70 to Cx43-HSP90.
研究应激诱导磷蛋白1（STIP1）、热休克蛋白（HSP）70和HSP90在大鼠H9c2心肌细胞中对连接蛋白43（Cx43）泛素化的影响。采用免疫共沉淀法检测蛋白质-蛋白质相互作用及Cx43泛素化。采用免疫荧光法进行蛋白质共定位。在STIP1和/或HSP90表达改变的H9c2细胞中重新分析蛋白质结合、Cx43蛋白表达及Cx43泛素化情况。在正常H9c2心肌细胞中，STIP1与HSP70和HSP90结合，Cx43与HSP40、HSP70和HSP90结合。STIP1过表达促进Cx43-HSP70向Cx43-HSP90转变并抑制Cx43泛素化；敲低STIP1则产生相反作用。抑制HSP90可抵消STIP1过表达对Cx43泛素化的抑制作用。STIP1通过促进Cx43-HSP70向Cx43-HSP90转变来抑制H9c2心肌细胞中Cx43的泛素化。
