中华神经科杂志
中華神經科雜誌
중화신경과잡지
Chinese Journal of Neurology
2010年
11期
795-800
,共6页
汪克建%龙志敏%高宝兵%赵蕾%陆蔚天%甘胜伟%贺桂琼
汪剋建%龍誌敏%高寶兵%趙蕾%陸蔚天%甘勝偉%賀桂瓊
왕극건%룡지민%고보병%조뢰%륙위천%감성위%하계경
神经元%膜蛋白质类%蛋白酶抑制药
神經元%膜蛋白質類%蛋白酶抑製藥
신경원%막단백질류%단백매억제약
Neurons%Membrane proteins%Cysteine proteinase inhibitors
目的 探讨神经细胞内前咽缺陷蛋白-1(Aph-1)的蛋白降解是经蛋白酶体途径还是经溶酶体途径介导.方法 在人神经母细胞瘤细胞(SH-SY5Y)建立稳定表达Aph-1细胞株的基础上,应用蛋白酶体和溶酶体酶抑制剂分别处理Aph-1细胞株,并结合Western blotting、放射性同位素脉冲示踪技术(Pulse-chase)、免疫荧光双标等技术,检测细胞内Aph-1的蛋白表达变化.结果 Western blotting结果显示,特异性蛋白酶体抑制剂能显著提高神经细胞内源性和外源性Aph-1的蛋白表达水平,且高度特异的蛋白酶体抑制剂乳胞素(Lactacystin)对Aph-1表达的增强效应呈剂量依赖性和时间依赖性;而非蛋白酶体蛋白酶抑制剂ALLM和溶酶体酶抑制剂对Aph-1的蛋白表达无影响;Pulsechase结果显示,蛋白酶体抑制剂可增强细胞内新合成Aph-1的表达水平,其增强作用是通过阻止35S-Aph-1的蛋白降解实现;免疫荧光双标结果显示,Aph-1与泛素在细胞内共存.结论 神经细胞内Aph-1的蛋白降解由蛋白酶体途径介导,与溶酶体途径无关;Aph-1在降解之前经泛素化修饰.
目的 探討神經細胞內前嚥缺陷蛋白-1(Aph-1)的蛋白降解是經蛋白酶體途徑還是經溶酶體途徑介導.方法 在人神經母細胞瘤細胞(SH-SY5Y)建立穩定錶達Aph-1細胞株的基礎上,應用蛋白酶體和溶酶體酶抑製劑分彆處理Aph-1細胞株,併結閤Western blotting、放射性同位素脈遲示蹤技術(Pulse-chase)、免疫熒光雙標等技術,檢測細胞內Aph-1的蛋白錶達變化.結果 Western blotting結果顯示,特異性蛋白酶體抑製劑能顯著提高神經細胞內源性和外源性Aph-1的蛋白錶達水平,且高度特異的蛋白酶體抑製劑乳胞素(Lactacystin)對Aph-1錶達的增彊效應呈劑量依賴性和時間依賴性;而非蛋白酶體蛋白酶抑製劑ALLM和溶酶體酶抑製劑對Aph-1的蛋白錶達無影響;Pulsechase結果顯示,蛋白酶體抑製劑可增彊細胞內新閤成Aph-1的錶達水平,其增彊作用是通過阻止35S-Aph-1的蛋白降解實現;免疫熒光雙標結果顯示,Aph-1與汎素在細胞內共存.結論 神經細胞內Aph-1的蛋白降解由蛋白酶體途徑介導,與溶酶體途徑無關;Aph-1在降解之前經汎素化脩飾.
목적 탐토신경세포내전인결함단백-1(Aph-1)적단백강해시경단백매체도경환시경용매체도경개도.방법 재인신경모세포류세포(SH-SY5Y)건립은정표체Aph-1세포주적기출상,응용단백매체화용매체매억제제분별처리Aph-1세포주,병결합Western blotting、방사성동위소맥충시종기술(Pulse-chase)、면역형광쌍표등기술,검측세포내Aph-1적단백표체변화.결과 Western blotting결과현시,특이성단백매체억제제능현저제고신경세포내원성화외원성Aph-1적단백표체수평,차고도특이적단백매체억제제유포소(Lactacystin)대Aph-1표체적증강효응정제량의뢰성화시간의뢰성;이비단백매체단백매억제제ALLM화용매체매억제제대Aph-1적단백표체무영향;Pulsechase결과현시,단백매체억제제가증강세포내신합성Aph-1적표체수평,기증강작용시통과조지35S-Aph-1적단백강해실현;면역형광쌍표결과현시,Aph-1여범소재세포내공존.결론 신경세포내Aph-1적단백강해유단백매체도경개도,여용매체도경무관;Aph-1재강해지전경범소화수식.
Objectives To investigate whether degradation of anterior pharynx decfective-1(Aph-1) goes through proteasomal pathway or lysosomal pathway.Methods Various methods such as cell culture,Western blotting,pulse-chase metabolic labeling technique,double immunofluoresecnt staining,combined with proteasomal and lysosomal inhibition were used to check Aph-1 expression level in stable Aph-1-transfected or non-transfected neuronal(SH-SY5Y)cell line.Results Using Western blotting,treating the neuronal cells with proteasome specific inhibitors significantly increased the expression of both endogenous and exogenous Aph-1.The effect of the proteasome inhibitors on Aph-1 expression was dose-and time-dependent Lysosomal pathway was not involved in Aph-1 degradation. Pulse-chase metabolic labeling experiment showed that the turnover of newly-synthesized radiolabeled Aph-1 protein was blocked by Lactacystin.Double immunofluorescent staining revealed colocalization of Aph-1 and ubiquitin in the same cells.Conclusion Degradation of Aph-1 protein is mediated by proteasomal pathway in neuronal cells,and is not related to lysosomal pathway.Aph-1 protein is ubiquitinated before degradation.
