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Acetylation dependent functions of Rab22a-NeoF1 Fusion Protein in Osteosarcoma.
Theranostics ( IF 12.4 ) Pub Date : 2020-6-19 , DOI: 10.7150/thno.46082
Xiaoting Liang 1 , Xin Wang 1 , Yaohui He 2 , Yuanzhong Wu 1 , Li Zhong 1 , Wen Liu 2 , Dan Liao 1 , Tiebang Kang 1
Affiliation  

Background: Rab22a-NeoF1 fusion gene containing the 1-38aa of Rab22a (Rab22a1-38) plays a decisive role in driving tumor metastasis by activating RhoA via binding to SmgGDS607. However, its intercellular regulation remains unknown./nMethods: 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./nResults: 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 vivo, which are also diminished by p300/CBP inhibitor C646./nConclusion: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 Rab22a1-38.

中文翻译:

Rab22a-NeoF1融合蛋白在骨肉瘤中的乙酰化依赖功能。

背景:包含Rab22a的1-38aa的Rab22a-NeoF1融合基因(Rab22a 1-38)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,它的细胞间监管依然是unknown./n方法:Rab22a-NeoF1的Lys7(K7)乙酰化最初是通过质谱鉴定的。共转染,免疫沉淀和蛋白质印迹被用来表征负责Rab22a-NeoF1的K7乙酰化的乙酰基转移酶和脱乙酰基酶,并定义蛋白质之间的相互作用。Rab22a-NeoF1的K7乙酰化的特异性由其特异性抗K7ac-Rab22a-NeoF1抗体及其K7R突变体确定。RhoA-GTP通过RhoA激活测定法测量。分别通过不带基质胶基质和带基质胶基质的Tr​​answell试验评估迁移和侵袭。体内原位骨肉瘤转移模型用于监测稳定表达U2OS / MTX300-Luc载体,Rab22a-NeoF1或其K7R突变体在有或没有C646(p300 / CBP的相对特异性抑制剂)时的肺转移。不成对的学生被用于统计significance./n测试结果: RAB22A-NeoF1的K7通过乙酰化的p300 / CBP而由两个HDAC6和SIRT1脱乙酰化。RAB22A-NeoF1的K7R突变体缺乏其结合SmgGDS607并随后失去了它的促进功能,诸如的RhoA的激活,细胞迁移,侵入和肺转移瘤在体外在VIV O,其也由P300 / CBP抑制剂减少C646./n结论:Rab22a-NeoF1的促进功能取决于其在骨肉瘤中的K7乙酰化作用,靶向这种乙酰化作用(例如C646)可能有益于Rab22a 1-38阳性的癌症患者,尤其是骨肉瘤患者。
更新日期:2020-06-23
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