【作者】 应征；

【导师】 王光辉；

【作者基本信息】 中国科学技术大学 ， 神经生物学， 2009， 博士

【摘要】 许多神经退行性疾病如老年性痴呆、帕金森病、肌萎缩性侧索硬化症、多聚谷胺酰胺病、朊蛋白病和家族性神经抑丝酶包涵体脑病的核心病理特征是由错误折叠的致病蛋白异常积聚而导致的聚集,致病蛋白突变后因蛋白构像的改变而导致错误折叠。这些蛋白的聚集体常与泛素、分子伴侣和蛋白酶体的亚单位结合在一起。肌萎缩侧索硬化症(ALS)的发病与铜锌过氧化物歧化酶SOD1的基因突变有关,而脊髓小脑共济失调3型(SCA3)/马查多-约瑟夫病(MJD)的致病蛋白为ataxin-3。这两种致病蛋白对神经元有毒性作用,在病人脑中都是错误折叠且形成聚集体。近期有文献报道这两个疾病相关蛋白都与内质网有关联,参与调控内质网相关降解通路。它们在内质网上的异常积聚可导致内质网应激。内质网关联降解通路是真核细胞中极为重要的一个蛋白质量控制系统,负责清除错误折叠的内质网内蛋白。许多未正确装配或错误折叠的蛋白都会在内质网内大量沉积,于是被选择成为内质网关联降解通路的底物由内质网经泛素蛋白酶体系统降解。底物蛋白的泛素化是这条通路中必不可少的环节,而泛素化的实现依赖于泛素连接酶(E3)。至今已有至少五种E3s被发现与内质网关联降解通路有关,其中两个研究得最多的是:gp78和Hrd1。和其它的内质网相关E3相似,gp78是一个内质网跨膜蛋白,含有RING finger型的活性结构域,此结构域暴露在细胞质环境中,可催化底物蛋白的泛素化。本论文主要探索了内质网关联降解通路对ALS及SCA3/MJD这两种疾病的相关蛋白的调控。首先,我们发现了gp78可以与SOD1和ataxin-3这两个蛋白结合,过表达gp78可以促进这两个蛋白的泛素化及降解,而沉默gp78的表达可以稳定这两个蛋白。其次,我们的结果表明gp78在突变体SOD1及ataxin-3的转染细胞中,以及在ALS发病鼠中,都有表达的上调。最后,我们观察到gp78通过这种对SOD1的降解调控,可以减缓突变体SOD1的聚集并保护细胞免受这种由SOD1聚集体引起的细胞死亡。由此可以看出,gp78可以调控SOD1和ataxin-3经内质网关联降解通路降解,动物实验结果提示这种降解调控作用可能参与了ALS和SCA3/MJD的发病过程。更多还原

【Abstract】 The hallmark of many neurodegenerative disorders,such as Alzheimer’s disease (AD),Parkinson’s disease(PD),amyotrophic lateral sclerosis(ALS),polyglutamine diseases and prion diseases and familial encephalopathy with neuroserpin inclusion bodies(FENIB),is the intracellular aggregates formed by the accumulation of aberrant misfolded disease proteins.Mutations of these proteins may alter the conformation and result in their misfolding.Furthermore,these misfolded proteins can form aggregates which are highly ubiquitinated,along with molecular chaperones and proteasome subunits.Copper-zinc superoxide dismutase(SOD1) is an antioxidant enzyme.Mutations of SOD1 are associated with ALS.Ataxin-3 is another neurodegenerative disease protein in association with Machado-Joseph disease(also known as spinocerebellar ataxia type 3,SCA3).These two disease proteins are misfolded,aggregated and neurotoxic in diseased brains.It was recently reported that these two proteins are associated with ER(endoplasmic reticulum) and both of them are involved in ERAD (ER-associated degradation).Abnormal accumulation of them in the ER induces ER stress.ERAD is a protein quality control system in eukaryotes to degrade misfolded proteins in the ER.In ERAD process,misfolded or unassembled target proteins are selected as substrates and finally degraded by the cytoplasmic 26S proteasome through ubiquitin-proteasome system(UPS).Many unassembled or misfolded proteins are accumulated in the ER and degraded through ERAD.Ubiquitin ligase (E3)-mediated ubiquitination of proteins is an important process for protein degradation by ERAD system.Up to date,al least five E3s,including gp78 and Hrd1, have been identified to be involved in mammalian ERAD.Similar to other ERAD E3s, mammalian gp78 is an ER membrane-spanning protein with the RING finger-type ubiquitin ligase activity site that is localized on the ER surface towards the cytoplasmic side.In this thesis,we mainly explored the role of ERAD in ALS and sCA3/MJD process.We show that gp78 interacts with both SOD1 and ataxin-3.Overexpression of gp78 promotes the ubiquitination and degradation of these two proteins,whereas knockdown of gp78 stabilizes them.Moreover,gp78 is increased in cells transfected with these two mutant proteins as well as in ALS mice.Furthermore,gp78 represses aggregate formation of mutant SOD1 and protect cells against mutant SOD1-induced cell death.Thus,our results suggest that gp78 functions in the regulation of SOD1 and ataxin-3 to target them for ERAD.Potentially,this regulation may underlie the pathogenesis of ALS and SCA3/MJD.更多还原