【作者】 王洪枫；

【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】 脊髓小脑共济失调三型(SCA3)即马查多-约瑟夫病(MJD),是一种常染色体显性遗传的神经退行性疾病。在各种脊髓小脑共济失调疾病中,SCA3是最常见的亚型.SCA3的主要症状是小脑变性,锥体和外锥体病变,进行性眼外肌麻痹和面部抽搐等。国际报道SCA3病人的平均发病年龄为37.4±14.1岁,国内基因诊断的SCA-3/MJD患者平均发病年龄为33.9±9.5岁。它的发病主要是由于致病基因MJD1产物ataxin-3的C末端发生多聚谷氨酰胺数目的异常扩增引起的.Ataxin-3在人体内广泛存在,与其它多聚谷氨酰胺疾病一样,当polyQ扩增时,蛋白会形成错误折叠。SCA3的显著特征是在脑组织细胞中形成大量泛素化的聚集,这些由多聚谷氨酰胺蛋白异常折叠所引起的聚集常常和分子伴侣,转录因子以及蛋白酶体共定位在一起,这些现象提示SCA3的发病机理可能与改变或损伤上述蛋白功能有关。泛素蛋白酶体通路(UPP)是细胞内重要的降解途径,短寿命蛋白,异常折叠蛋白或者损伤多肽等都通过UPP降解。底物蛋白先是经过泛素激活酶(E1),泛素结合酶(E2),泛素连接酶(E3)等多种酶的多步反应后被标记上泛素链,然后被转运蛋白带到蛋白酶体降解。19S蛋白酶体是催化亚基,是由多个亚单位组成,其中有六个具有ATP酶活性的蛋白。这六个ATP酶具有一定的底物特异性,通过单个的突变可以找到各自不同的特异底物。P45是其中的一个ATP酶,它可以结合Sp1和vitamin D受体并促进后两者的降解。Ataxin-3包含有2个或3个UIM,通过UIM区域可以结合泛素化底物并通过ataxin-3的Josephin domain来行使去泛素酶的功能.Ataxin-3还可以结合HHR23和VCP,它们具有将泛素化底物运送到蛋白酶体的功能,这提示ataxin-3和蛋白酶体有着紧密的联系。另外研究表明ataxin-3是泛素蛋白酶体底物,但是明确的降解机制还不清楚。我们的实验显示ataxin-3与蛋白酶体有直接的联系。在体外和细胞内野生型(正常长度polyQ)和突变(异常扩增polyQ)ataxin-3与19S蛋白酶体上具有ATP酶活性的亚基p45直接结合。细胞内共转染实验表明p45可以调控的ataxin-3的降解,体外降解系统中我们得到同样的结论。这种调控作用可以被蛋白酶体抑制剂MG-132所阻断。在N2a或者HEK293细胞系中,过表达的p45可以显著地促进ataxin-3的降解,而另外两种神经退行性疾病相关蛋白,SOD1和alDhasynuclein却不被p45调控,这说明p45对ataxin-3的调控是特异的。另外p45的ATP酶活性对于它的功能也十分重要,实验表明丧失ATP酶活性的p45不再促进ataxin-3的降解.我们确认了ataxin-3与p45结合的位点在N端,另一组实验则证明p45对ataxin-3的调控作用依赖于ataxin-3的泛素结合区(UIM),但是仅有UIM区又不足以造成p45对它的降解调控。p45与蛋白酶体上其他几个具有ATP酶活性的蛋白同源性很高,我们发现只有p45对ataxin-3具有调节降解的作用,这也说明了此促进降解的作用是特异的。综上我们得出结论:蛋白酶体上具有ATP酶活性的蛋白p45可以特异性的促进ataxin-3通过蛋白酶降解,这种作用是通过与ataxin-3 N端的直接结合并且依赖于UIM功能区域的。这一发现可能为我们更好的了解SCA3的发病机制,寻求治疗SCA3的方法提供一些帮助。更多还原

【Abstract】 Spinocerebellar ataxia(SCA3),also called Machado-Joseph disease(MJD),is an autosomal dominant neurodegenerative disorder.It is the most common subtype in spinocerebellar ataxia.which is characterized clinically by cerebellar ataxia,pyramidal and extrapyramidal signs,progressive extraocular muscle paralysis and facial tic.It’s reported that the mean age at onset is 37.4+14.1 years in the whole world,and in China the mean age is about 33.9+9.5 years.It is caused by an expansion of the polyglutamine tract near the C-terminus of the MJD-1 gene product,ataxin-3.Ataxin-3 is ubiquitously expressed in the body.The hallmark of MJD,as well as other polyglutamine diseases,is the formation of high ubiquitinated nuclear inclusions in affected brain,resulting from an abnormal accumulation of misfolded polyglutamine protein,along with molecular chaperones,transcription factors or co-activators and the components of proteasome.The presence of above proteins to inclusions formed by expanded ataxin-3 suggests that a functional alteration or impairment of those proteins may occur,thus contributing to MJD pathogenesis.The ubiquitin proteasome pathway(UPP) is a proteolysis route for most non-lysosomal degradation proteins,including both short-lived regulatory proteins and constitutively stable cellular proteins with long half-lives,as well as misfolded and damaged polypeptides.Usually,the proteins are polyubiquitinated by a multistep process,involving at least three types of enzyme called a ubiquitin-activating enzyme (E1),a ubiquitin-conjugating enzyme(E2),and a ubiquitin-protein ligase(E3) before they are degraded by the proteasome.The 19S subcomplex is composed of several non- ATPase subunits and six ATPase subunits to function in selective degradation of proteins and regulation of the activity of the 20S proteasome.The functional specificity of different proteasomal ATPase subunits remains unclear,but mutations in individual ATPase subunits cause different phenotypes and reduce the degradation of some substrates,while not affecting others.Within the six ATPase subunits,p45 has been shown to interact with Sp1 and vitamin D receptor(VDR) and to enhance the degradation of Sp1 and VDR specifically by the proteasome both in vitro and in vivo.Ataxin-3 contains two or three ubiquitin-interacting motifs(UIMs) based on its splice variant,functioning in modulating the de-ubiqutination of its substrates through its Joseph domain,a ubiquitin protease(UBP).Ataxin-3 also interacts with proteins functionally associated with UPP,such as HHR23 proteins and valosin-containing protein(VCP/p97),suggesting that there are tight links between ataxin-3 and the proteasome.Studies have shown that ataxin-3 is degraded by the proteasome, however,the precise mechanism of the degradation of ataxin-3 is still unknown.Here we show a direct link between ataxin-3 and the proteasome.Using in vitro and in cells studies,p45,an ATPase subunit of the 26S proteasome,interacts with normal ataxin-3 as well as expanded ataxin-3 directly.In reconstituted degradation assay,p45 stimulates the degradation of ataxin-3 by the proteasome.The regulation can be blocked by MG-132.In N2a cells or 293 cells,overexpression of p45 strikingly enhances the clearance of both normal and expanded ataxin-3,but not alpha synuclein or SOD1,implying a functional specificity of p45 in this proteolytic process. In contrast,an ATPase mutant of p45,which is still able to bind ataxin-3,loses the ability to induce the degradation of ataxin-3.Furthermore,we confirmed that the N-terminus of ataxin-3 interacts with p45 and demonstrated that the degradation of ataxin-3 driven by p45 depends on the presence of its UIMs,but the UIMs are not sufficient for this regulation.As p45 is an ATPase of the 19S proteasome,we wonder whether other ATPase of the 19S proteasome also promotes the degradation of ataxin-3.The data suggest that p45 specifically drives the process.Thus our findings suggest that the ATPase subunit of the 19S proteasome p45 can specifically regulated the degradation of polyglutamine protein ataxin-3,and this effect is through the proteasome pathway.The UIMs of ataxin-3 is not sufficient but important.The binding sites are the N-termins.The findings here will be of help to expand our knowledge of ataxin-3 functions,as well as its possible role in SCA3.更多还原