黑色素瘤相关抗原MAGEA1的定位及其相互作用蛋白质的鉴定  

Localization of Melanoma-associated Antigen MAGEA1 and Identification of Its Interacting Proteins

作　　者：李瑜颖 钟子琳 陈建军 LI Yu-Ying;ZHONG Zi-Lin;CHEN Jian-Jun(Pediatrics Department of Shanghai Fourth People’s Hospital Affiliated to Tongji University,Shanghai 200434,China)

机构地区：[1]同济大学附属上海市第四人民医院儿科,上海200434

出　　处：《中国生物化学与分子生物学报》2021年第6期765-771,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.81371062);国家重点研发计划项目(No.2020YFA0112500)资助。

摘　　要：黑色素瘤相关抗原家族A1蛋白(melanoma associated antigen family A1,MAGEA1)在生殖细胞和多种组织学来源的肿瘤中有表达,但其作用机制尚不清楚。本研究通过构建带Flag和GFP标签的MAGEA1真核表达重组质粒,将其转染至HeLa、HEK293T细胞内。利用Western印迹、免疫细胞荧光法、免疫共沉淀、细胞核蛋白质与细胞质蛋白质分离和线粒体提取等技术,检测其在细胞内的表达与定位以及与其他蛋白质的相互作用情况。免疫细胞化学和蛋白质印迹结果显示,过表达的MAGEA1主要定位于细胞质,并部分与线粒体共定位。通过免疫共沉淀验证了MAGEA1与TRIM31、SNW1、HDAC1之间存在相互作用,并且发现MAGEA1可能主要与位于细胞质中的HDAC1相互作用。以上研究表明,MAGEA1可能参与不同的细胞内调节途径,部分与线粒体共定位;与TRIM31、SNW1、HDAC1相互作用,且可能主要与位于细胞质中的HDAC1蛋白相互作用,推测其可能参与到蛋白质泛素化和Notch信号通路。本研究的结果为后续深入研究MAGEA1的作用机制奠定了实验基础。Melanoma associated antigen family A1(MAGEA1)is expressed in germ cells and tumors of various histological origins,but its mechanism is still unclear.In this study,the eukaryotic recombinant MAGEA1 expression plasmids with Flag or GFP tags were constructed and transfected into HeLa and HEK293T cells.Western blotting,immunocytochemistry,co-immunoprecipitation,nuclear protein and cytoplasmic protein separation,and mitochondrial isolation were used to detect the expression and location of MAGEA1 and its interaction with other proteins in cells.The results of immunocytochemistry(ICC)and Western blotting showed that the overexpressed MAGEA1 was mainly localized in the cytoplasm and partially co-localized with mitochondria.Co-immunoprecipitation experiments verified the interactions between MAGEA1 and TRIM31,SNW1,HDAC1,and found that MAGEA1 may mainly interact with HDAC1 in the cytoplasm.The studies above indicate that MAGEA1 may be involved in different cellular biological processes and co-localize with mitochondria.It interacts with TRIM31,SNW1 and HDAC1,while MAGEA1 may mainly interact with HDAC1 in the cytoplasm.We propose that it may be involved in protein ubiquitination and the Notch signaling pathway.The results of this study laid an experimental foundation for the subsequent in-depth study of the mechanism of MAGEA1.

关 键 词：黑色素瘤相关抗原家族A1 亚细胞定位 线粒体 E3泛素连接酶TRIM31 组蛋白去乙酰酶1 

分 类 号：R34[医药卫生—基础医学]