文章摘要
黄颡鱼STAT3乙酰化位点验证及其与SIRTs去乙酰化酶的关系研究
Validation of STAT3 acetylation site and its relationship with SIRTs deacetylase in yellow catfish Pelteobagrus fulvidracoZHENG Hua, XU Yichuang, ZHAO Tao, LV Wuhong, YU Angen, HE Yang,
投稿时间:2020-12-25  修订日期:2021-02-01
DOI:
中文关键词: 黄颡鱼  STAT3  乙酰化  SIRTs  过表达
英文关键词: Pelteobagrus fulvidraco  STAT3  Acetylation  SIRTs  Overexpression
基金项目:国家自然科学基金;国家重点研发计划项目
作者单位邮编
郑华 华中农业大学水产学院 430070
徐一闯 华中农业大学水产学院 
赵涛 华中农业大学水产学院 
吕武宏 华中农业大学水产学院 
余岸艮 华中农业大学水产学院 
何杨 华中农业大学水产学院 
谭肖英 华中农业大学水产学院 
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中文摘要:
      本实验主要研究黄颡鱼STAT3乙酰化位点及其与SIRTs去乙酰化酶家族之间的关系。首先,构建带Flag标签的STAT3过表达质粒及带HA、GFP标签的SIRTs过表达质粒,并对STAT3可能发生乙酰化的位点进行突变体构建。其次,转染STAT3及突变体和STAT3、 SIRTs共转染于HEK 293T细胞,利用免疫印迹、免疫沉淀、免疫荧光技术等技术检测结果。结果表明,STAT3是一种胞浆蛋白,与野生型STAT3相比,突变体K685R的乙酰化水平明显降低,突变体K680R和K712R次之。而突变体K49R、K87R、K714R的乙酰化水平与野生型STAT3无显著性差异。这表明K685是STAT3的关键乙酰化位点。免疫沉淀和免疫荧光结果显示,SIRT2、SIRT7与STAT3发生蛋白互作并催化STAT3的去乙酰化,而SIRT5不与STAT3发生蛋白互作,对其乙酰化水平无明显变化。本研究揭示了黄颡鱼STAT3的乙酰化位点及STAT3和SIRTs之间的关系,为探讨STAT3蛋白乙酰化修饰在黄颡鱼营养代谢中的作用奠定基础。
英文摘要:
      The present study was conducted to study the acetylation site of STAT3 and its relationship with SIRTs deacetylase family in yellow catfish. The STAT3 overexpression plasmids containing Flag tag and the SIRTs overexpression plasmids containing HA and GFP tags were constructed, and mutants were constructed for the possible acetylated sites of STAT3. Then, STAT3 was transfected and the mutant was co-transfected with STAT3 and SIRTs into HEK 293T cells, and Western blotting, immunoprecipitation and immunofluorescence techniques were used. The results showed that STAT3 is a cytoplasmic protein, and the acetylation level of its mutant K685R was significantly lower than that of wild-type STAT3, followed by mutant K712R and K680R. The acetylation levels of mutants K49R, K87R and K714R have no significant changes compared with wild-type STAT3. The immunoprecipitation and immunofluorescence showed that SIRT2 and SIRT7 and interacted with STAT3 and catalyzed the deacetylation of STAT3. However, SIRT5 does not interact with STAT3 and accordingly does not changes its acetylation levels of STAT3. The study revealed, for the first time, the acetylation site of STAT3 and the relationship between STAT3 and SIRTs in yellow catfish, and laid the foundation for exploring the role of STAT3 acetylation modification in the nutrient metabolism of yellow catfish.
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