Acetylation dependent functions of Rab22a-NeoF1 Fusion Protein in Osteosarcoma
Acetylation dependent functions of Rab22a-NeoF1 Fusion Protein in Osteosarcoma
复制标题
Rab22a-NeoF1 融合蛋白在骨肉瘤中的乙酰化依赖性功能
DOI：
10.7150/thno.46082
复制
发表时间：
2020-01-01
期刊：
影响因子：
12.4
通讯作者：
Kang, Tiebang
中科院分区：
文献类型：
--
作者：
Liang, Xiaoting;Wang, Xin;Kang, Tiebang
背景：含有Rab22a第1 - 38位氨基酸（Rab22a 1 - 38 ）的Rab22a - NeoF1融合基因，通过与SmgGDS607结合激活RhoA，在驱动肿瘤转移中起决定性作用。然而，其细胞间调控机制仍不明确。
方法：首先通过质谱鉴定Rab22a - NeoF1的赖氨酸7（K7）乙酰化。采用共转染、免疫沉淀和蛋白质免疫印迹法来鉴定负责Rab22a - NeoF1 K7乙酰化的乙酰转移酶和去乙酰化酶，并明确蛋白质之间的相互作用。利用特异性的抗K7乙酰化Rab22a - NeoF1抗体及其K7R突变体来确定Rab22a - NeoF1 K7乙酰化的特异性。通过RhoA激活实验检测RhoA - GTP水平。分别采用无Matrigel基质和含Matrigel基质的Transwell实验评估细胞迁移和侵袭能力。构建原位骨肉瘤转移体内模型，以监测稳定表达载体、Rab22a - NeoF1或其K7R突变体的U2OS/MTX300 - Luc细胞在有无p300/CBP相对特异性抑制剂C646作用下的肺转移情况。采用非配对Student t检验进行统计学显著性分析。
结果：Rab22a - NeoF1的K7位点由p300/CBP进行乙酰化修饰，而HDAC6和SIRT1可使其去乙酰化。Rab22a - NeoF1的K7R突变体无法与SmgGDS607结合，进而丧失其促进功能，如在体外和体内对骨肉瘤中RhoA的激活、细胞迁移、侵袭及肺转移的促进作用，p300/CBP抑制剂C646也可削弱这些功能。
结论：在骨肉瘤中，Rab22a - NeoF1的促癌功能依赖于其K7乙酰化，针对这种乙酰化（如使用C646）可能使癌症患者，尤其是Rab22a 1 - 38阳性的骨肉瘤患者受益。
Background: Rab22a-NeoF1 fusion gene containing the 1-38aa of Rab22a (Rab22a 1-38 ) plays a decisive role in driving tumor metastasis by activating RhoA via binding to SmgGDS607. However, its intercellular regulation remains unknown.Methods: The Lys7 (K7) acetylation of Rab22a-NeoF1 was initially identified by mass spectrum. Co-transfection, immunoprecipitation and Western blotting were used to characterize the acetyltransferases and deacetylases responsible for the K7 acetylation of Rab22a-NeoF1, and to define the interaction of proteins. The specificity of K7 acetylation of Rab22a-NeoF1 was determined by its specific anti-K7ac-Rab22a-NeoF1 antibody and its K7R mutant. RhoA-GTP was measured by RhoA activation assay. The migration and invasion were assessed by Transwell assay without and with Matrigel matrix, respectively. The orthotopic osteosarcoma metastasis model in vivo was used to monitor the lung metastases of U2OS/MTX300-Luc stably expressing Vector, Rab22a-NeoF1 or its K7R mutant with or without C646, a relatively specific inhibitor of p300/CBP. The unpaired Student t test was used for the statistical significance.Results: The K7 of Rab22a-NeoF1 is acetylated by p300/CBP while is de-acetylated by both HDAC6 and SIRT1. The K7R mutant of Rab22a-NeoF1 lacks its binding to SmgGDS607 and subsequently lost its promoting functions, such as activation of RhoA, cell migration, invasion and lung metastasis in osteosarcoma in vitro and in viv o, which are also diminished by p300/CBP inhibitor C646.Conclusion: The promoting function of Rab22a-NeoF1 is dependent on its K7 acetylation in osteosarcoma, and targeting this acetylation (e.g., C646) may benefit cancer patients, in particular osteosarcoma patients, who are positive for the Rab22a 1-38 .