肌萎缩侧索硬化症相关基因CREST在蛋白质降解系统中起关键作用  

The ALS-related Gene CREST Plays Critical Roles in Protein Degradation Systems

作　　者：曲扬 陈晨 赵琳琳[1] 丁婧 郑静[3] 程诚 张惊宇[1] QU Yang;CHEN Chen;ZHAO Lin-Lin;DING Jing;ZHENG Jing;CHENG Cheng;ZHANG Jing-Yu(Department of Neurology,The Fourth Hospital of Harbin Medical University,Harbin 150001,China;Shanghai Key Laboratory of New Drug Design and Research and Development,School of Bioengineering,East China University of Science and Technology,Shanghai 200237,China;Shanghai Key Laboratory of New Drug Design and Research and Development,School of Pharmacy,East China University of Science and Technology,Shanghai 200237,China)

机构地区：[1]哈尔滨医科大学第四医院神经内科,哈尔滨150001 [2]华东理工大学生物工程学院,上海市新药设计与研发重点实验室,上海200237 [3]华东理工大学药学院,上海市新药设计与研发重点实验室,上海200237

出　　处：《中国生物化学与分子生物学报》2020年第9期1064-1070,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：Supported by Fundamental Research Funds for the Central Universities(No.JKY012016001)。

摘　　要：蛋白质降解系统的紊乱可能是神经退行性疾病的关键致病机制之一。肌萎缩侧索硬化症(ALS)就是一类严重的神经退行性疾病。本研究旨在探索我们新报道的ALS相关基因CREST是否会影响蛋白质降解系统。我们构建了人源CREST的表达质粒或鼠源CREST shRNA的质粒,并将其转染到293细胞或从胚胎期C57BL/6小鼠分离培养的原代皮层神经元中,以此制备实验样品。使用分子生物学和生物化学方法,分析了CREST对蛋白质降解系统的影响,包括泛素蛋白酶体系统(UPS)和自噬流。利用UPS的GFP指示剂,发现过表达CREST会导致293细胞中GFP阳性信号显著降低。但CREST的过表达却不影响293细胞或培养的皮层神经元中的自噬流。然而,无论在是否使用影响自噬流药物的情况下,降低CREST的蛋白水平都能抑制培养神经元中LC3-Ⅰ向LC3-Ⅱ的转化(P<0.05)。不仅如此,LC3-Ⅱ与Ⅰ的比值在表达CREST的ALS相关突变(CREST-Q388X)的皮层神经元中显著上升(P<0.05)。上述结果表明,CREST可能在蛋白质降解系统中发挥关键作用,包括促进UPS以及维持自噬的正常功能,而在自噬调控中获得新功能的CREST-Q388X突变也将涉及一些新的致病机制。The disturbance of protein degradation systems may be a critical pathogenic mechanism underlying devastating neurodegenerative diseases,including amyotrophic lateral sclerosis(ALS).This study aims to investigate whether the new ALS-related gene CREST that we have reported affects protein degradation systems.We constructed plasmids expressing human wild-type CREST,the CREST-Q388X mutant or mouse CREST shRNA and transfected them into 293 cells or cultured primary cortical neurons isolated from embryotic C57BL/6 mice as experiment samples.Using molecular biological and biochemical tools,we identified the effects of CREST on protein degradation systems,including the ubiquitin proteasome system(UPS)and autophagic flux.Using a GFP-indicator of UPS,we found that overexpression of wild-type CREST led to a significant decrease of GFP-positive signals in 293 cells,but did not affect autophagic flux in 293 cells or cultured cortical neurons.However,decreased CREST protein levels could inhibit the conversion of LC3-Ⅰto LC3-Ⅱin cultured neurons with or without the manipulation of drugs that affected autophagic flux(P<0.05).Moreover,the LC3-Ⅱ/Ⅰratios were increased in cortical neurons expressing the ALS-associated mutant(CREST-Q388X)(P<0.05).Our results suggest that CREST may plays critical roles in protein degradation systems,including promotion of UPS and the maintenance of functional autophagic activity,and some new mechanisms may be involved in the gain-of-function of CREST-Q388X mutant in autophagy regulation.

关 键 词：肌萎缩侧索硬化症 CREST 自噬 泛素蛋白酶体系统 

分 类 号：Q7[生物学—分子生物学]