摘要： 目的·挖掘F-box蛋白22（F-box only protein 22，FBXO22）的底物并明确其作用机制，进一步探讨FBXO22在肿瘤细胞凋亡中的作用。方法·利用免疫沉淀联合质谱分析寻找并验证FBXO22的相互作用蛋白凋亡诱导因子（apoptosis-inducing factor，AIF）。采用变性条件下泛素化修饰实验检测FBXO22对AIF泛素化修饰的影响。通过短发夹RNA敲低FBXO22、过表达FBXO22检测AIF的表达水平。利用流式细胞仪检测FBXO22敲低或过表达对结肠癌细胞凋亡的影响。结果·免疫沉淀联合质谱分析显示FBXO22可与AIF结合，变性条件下泛素化修饰实验显示FBXO22可介导AIF的泛素化修饰。敲低FBXO22可上调AIF水平，过表达FBXO22可下调AIF水平。流式细胞术结果显示，敲低FBXO22可促进由甲基硝基亚硝基胍（N-methyl-N
'-nitro-N-nitrosoguanidine，MNNG）等诱导的结肠癌细胞凋亡，过表达FBXO22则可抑制MNNG等诱导的结肠癌细胞凋亡。结论·FBXO22通过泛素化修饰降解AIF进而调控由AIF介导的结肠癌细胞凋亡。
关键词: F-box蛋白22, 凋亡诱导因子, 泛素化修饰, 细胞凋亡 Abstract: Objective·To explore the substrate of F-box only protein 22 (FBXO22) and its mechanism, and further study the role of FBXO22 in cancer cell apoptosis.
Methods·The interaction protein of FBXO22, namely apoptosis-inducing factor (AIF), was searched and identified by immunoprecipitation (IP)-coupled liquid chromatograph mass spectrometer/mass spectrometer (LC-MS/MS) technology. The effect of FBXO22 on ubiquitination of AIF was detected by ubiquitin modification assay under denatured conditions. Short hairpin RNA (shRNA) knockdown FBXO22 and overexpression FBXO22 were used to detect the expression of AIF. The effect of FBXO22 knockdown or overexpression on apoptosis of colon cancer cells was detected by flow cytometry.
Results·IP-coupled LC-MS/MS technology showed that FBXO22 could interact with AIF, and ubiquitin modification assay under denaturing conditions showed that FBXO22 could mediate ubiquitination of AIF. Knockdown of FBXO22 could up-regulate the level of AIF, while overexpression of FBXO22 could down-regulate the level of AIF. The results of flow cytometry showed that knockdown of FBXO22 could promote the apoptosis of colon cancer cells induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), while overexpression of FBXO22 could inhibit the apoptosis of colon cancer cells induced by MNNG.
Conclusion· FBXO22 regulates AIF-mediated apoptosis of colon cancer cells by ubiquitination and degradation of AIF.
Key words: F-box only protein 22 (FBOX22), apoptosis-inducing factor (AIF), ubiquitination modification, cell apoptosis PDF全文下载地址:
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