食管癌细胞线粒体中MYH9蛋白的分布及与SLP-2结合的研究

MYH9 Protein Partly Distributes in Mitochondria of Esophageal Carcinoma Cell and Binds with SLP-2

目的研究非肌性肌球蛋白重链9(myosin,heavy chain 9,non-muscle,MYH9)蛋白在食管鳞癌细胞线粒体内的分布,及MYH9蛋白与线粒体相关蛋白SLP-2的结合情况。方法使用抗stomatinlike protein 2(SLP-2)抗体,在食管鳞癌细胞系KYSE150细胞的裂解液里进行免疫共沉淀,共沉淀物使用MALDI-TOF-MS蛋白质质谱进行分析,以分析与线粒体相关蛋白SLP-2结合的分子。对分析结果再继续使用免疫共沉淀及GST pull down法进行验证。使用蔗糖密度梯度离心纯化分离细胞线粒体,利用Western blot明确MYH9在食管癌细胞线粒体内的分布。结果在食管鳞癌细胞系KYSE150细胞的裂解液内,MYH9蛋白可与SLP-2蛋白共沉淀,并且MYH9蛋白也能与GST-SLP-2结合。MYH9蛋白既存在于KYSE150细胞分离纯化后的细胞质中,也存在于分离出的线粒体中。结论 MYH9在食管癌细胞内部分分布于线粒体中,并与线粒体相关蛋白SLP-2相互结合。MYH9可能影响食管癌细胞线粒体的功能。

Objective To investigate the distribution of myosin, heavy polypeptide 9(MYH9) protein in the mitochondria of esophageal squamous cell carcinoma(ESCC), and the interaction between MYH9 protein and a mitochondria-associated protein, stomatin-like protein 2(SLP-2). Methods In order to find the binding protein of a new reported mitochondria-associated protein SLP-2, co-immunoprecipitation(Co-IP) was conducted by anti-SLP-2 antibody in the lysate of ESCC cell lines KYSE150. Precipitated mixture was analyzed by matrixassisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Co-IP and glutathione S transferase(GST) pull down assay were performed to further confirm the result of MALDI-TOF-MS. To detect the distribution of MYH9 in ESCC mitochondria, sucrose density gradient centrifugation was adopted to isolate the mitochondria for Western blot. Results In the lysate of ESCC cell lines KYSE150,MYH9 protein coimmunoprecipitated with SLP-2 protein and binded with GST-SLP-2. MYH9 protein existed in both isolated cytoplasm and purified mitochondria of ESCC cell lines KYSE150. Conclusion Parts of MYH9 distributes in the mitochondria of ESCC and bind with a mitochondria-associated protein SLP-2. MYH9 may affect the function of ESCC mitochondria.