FoxO1磷酸化对低氧下大鼠骨骼肌细胞蛋白质合成和分解的影响  

Effects of phosphorylation sites of FoxO1 on protein synthesis and proteolysis in rat skeletal muscle cells exposure to hypoxia

作　　者：贾杰 付鹏宇[2] 朱镕鑫 许春艳[2] 龚丽景 JIA Jie;FU Pengyu;ZHU Rongxin;XU Chunyan;GONG Lijing(China Institute of Sport and Health Science,Beijing Sport University,Beijing,100084;Sport Science College,Beijing Sport University,Beijing,100084;Shanghai Research Institute of Sport Sciences,Shanghai,200030,China)

机构地区：[1]北京体育大学中国运动与健康研究院,北京100084 [2]北京体育大学运动人体科学学院,北京100084 [3]上海体育科学研究所,上海200030

出　　处：《第三军医大学学报》2020年第15期1519-1530,共12页Journal of Third Military Medical University

基　　金：中央高校基本科研业务费专项资金(2019PT003);国家自然科学基金面上项目(31771317)。

摘　　要：目的探究FoxO1不同磷酸化位点在低氧暴露下大鼠L6成肌细胞蛋白质合成和分解中的调节作用。方法构建FoxO1过表达、FoxO1^T24A/D、FoxO1^S250A/D、FoxO1^S313A/D位点的质粒,使用腺病毒作为载体,分别感染分化后的大鼠L6骨骼肌细胞6 h,将细胞分别命名为rFoxO1、T24A、T24D、S250A、S250D、S313A、S313D,并对应分为7个转染组,以转染空白对照质粒作为对照组(EGFP),随后将各转染组再分为常氧组(normoxic group,C)和低氧组(hypoxic group,H)。采用荧光共聚焦显微镜观察腺病毒转染情况;采用Western blot对嘌呤霉素(puromycin)结合多肽进行分析,并对泛素(ubiquitin)标记蛋白和分解相关蛋白表达进行检测。结果与C-EGFP组相比,C-rFoxO1组puromycin结合多肽(1.38±0.06)、mTOR(1.54±0.21)和p-mTOR(1.57±0.34)表达显著增加(P<0.05)。与C-EGFP组相比,C-T24D组mTOR(1.33±0.35)和p-mTOR(1.60±0.44)表达显著上升(P<0.01);与C-T24D组相比,H-T24D组mTOR表达显著降低(P<0.01)。与C-EGFP组相比,C-S250A组蛋白质积累显著上升(P<0.01),与C-S250A组相比,H-S250A组蛋白质积累显著下降(P<0.05)。与EGFP组相比,S313A和S313D组蛋白质积累差异无统计学意义(P>0.05)。结论FoxO1通过上调mTOR活性来增加蛋白质合成,但受低氧调控;低氧下FoxO1经Thr24和Ser250两个位点磷酸化来抑制机体中蛋白质合成。Objective To investigate the effects of different phosphorylation sites of FoxO1 on protein synthesis and proteolysis in rat skeletal muscle L6 cells under the condition of hypoxia.Methods Adenoviral vectors(FoxO1,FoxO1^T24 A/D,FoxO1^S250 A/D,and FoxO1^AS313 A/D)were constructed,and then used to infect the differentiated rat skeletal muscle cells respectively with appropriate multiples of infection(MOI).These L6 cells were corresponding named to 8 groups:EGFP,rFoxO1,T24 A,T24 D,S250 A,S250 D,S313 A and S313 D,and then each transfection group was divided into normoxic group(C)and hypoxic group(H).Adenovirus transfection was observed using a fluorescence confocal microscope.Puromycin-binding peptides,ubiquitin labeling protein,synthesis and decomposition-related proteins were detected using Western blotting.Results Compared with the C-EGFP group,the expression levels of puromycin-binding polypeptide(1.38±0.06),mTOR(1.54±0.21)and p-mTOR(1.57±0.34)were significantly increased in the C-rFoxO1 group(all P<0.05).The levels of mTOR(1.33±0.35)and p-mTOR(1.60±0.44)were also elevated in the C-T24 D group than the C-EGFP group(P<0.01).The level of mTOR in the H-T24 D group was notably reduced when compared with the C-T24 D group(P<0.01).Protein accumulation was remarkably increased in the C-S250 A group than the C-EGFP group(P<0.01),and was statistically decreased in the H-S250 A group than the C-S250 A group(P<0.05).But no such differences were seen in the protein accumulation in the S313 A and S313 D groups when compared with that in the C-EGFP group(P>0.05).Conclusion FoxO1 can improve protein synthesis by enhancing the activity of mTOR,but this process is strictly regulated by hypoxia.Under hypoxic condition,FoxO1 inhibits protein synthesis via phosphorylation sites Thr24 and Ser250.

关 键 词：FOXO1 磷酸化位点 蛋白质合成 蛋白质分解 翻译表面感应 

分 类 号：R322.74[医药卫生—人体解剖和组织胚胎学] R329.26[医药卫生—基础医学]