p62敲除对细胞自噬的影响及其对泛素化修饰蛋白募集的作用

Effect of p62 knockout on cellular autophagy and its role in recruitment of ubiquitination-modified proteins

目的探讨p62敲除对细胞自噬及泛素化蛋白募集的影响。方法将野生型(Wild Type,WT)和p62基因敲除(p62 knockout,p62-KO)的大鼠肾上皮细胞(NRK)分别分为野生型阴性对照组(WT CT组)和野生型饥饿4 h组(WT Sta4 h组)、p62基因敲除阴性对照组(p62-KO CT组)和p62基因敲除饥饿4 h组(p62-KO Sta4 h组)。WT CT组和p62-KO CT组中加入普通培养基,对细胞进行空白对照处理;WT Sta4 h组和p62-KO Sta4 h组中加入饥饿培养基,对细胞进行饥饿4 h处理。免疫印迹法鉴定和检测p62基因的敲除及自噬体标志蛋白微管相关蛋白1轻链3(LC3)、泛素化修饰蛋白(Ub)的表达;电子显微镜观察自噬体形成;激光共聚焦显微镜法观察LC3蛋白阳性点状结构的数量。结果鉴定p62基因成功被敲除。饥饿处理4 h后,WT和p62-KO细胞中均能观察到自噬结构(包括自噬体和自噬溶酶体)。与WT CT组比较,WT Sta4 h组中LC3蛋白酯化和Ub蛋白表达水平升高(P<0.05),LC3蛋白阳性点状结构数量显著增加(P<0.01);与p62-KO CT组比较,p62-KO Sta4 h组中LC3蛋白酯化和Ub蛋白表达水平升高(P<0.05),LC3蛋白阳性点状结构数量显著增加(P<0.01)。结论p62参与细胞自噬并募集泛素化修饰蛋白,最终影响选择性自噬体底物的凝聚和降解。

Objective To explore the effects of p62 knockout on cellular autophagy and ubiquitinated protein recruitment.Methods Wild type(WT)and p62 knockout(p62-KO)rat kidney epithelial cells(NRK)were divided into wild type negative control group(WT CT group)and wild type starvation 4 h group(WT Sta4 h group),p62 knockout negative control group(p62-KO CT group)and p62 knockout starvation 4 h group(p62-KO Sta4 h group).Normal medium was added to the WT CT group and p62-KO CT group,and the cells were treated with blank control,starvation medium was added to the WT Sta4 h group and p62-KO Sta4 h group,and the cells were treated with starvation 4 h.Immunoblotting method was used to identify and detect the knockout of p62 gene and the expression of autophagosome marker proteins microtubule-associated protein 1 light chain 3(LC3)and ubiquitination-modified protein(Ub),electron microscopy was used to observe the formation of autophagosomes,and the number of LC3 protein-positive punctate structures was observed by laser confocal microscopy.Results The p62 gene was identified to be successfully knocked out.After 4 h of starvation treatment,autophagic structures(including autophagosomes and autophagolysosomes)were observed in both WT and p62-KO cells.Compared with the WT CT group,the levels of LC3 protein esterification and Ub protein expression were increased in the WT Sta4 h group(P<0.05),and the number of LC3 protein-positive punctate structures were significantly increased(P<0.01),compared with the p62-KO CT group,the levels of LC3 protein esterification and Ub protein expression were increased in the p62-KO Sta4 h group(P<0.05),and the LC3 protein positive punctate structures were significantly increased(P<0.01).Concl-usion p62 is involved in cellular autophagy and recruits ubiquitination-modified proteins,which ultimately affect the cohesion and degradation of selective autophagosome substrates.