【作者】 费尔康；

【导师】 王光辉；

【作者基本信息】 中国科学技术大学 ， 神经生物学， 2008， 博士

【摘要】 神经退行性疾病是一类由脑中特定区域神经元发生退变而引起的慢性进行性神经系统疾病,通常可分为家族遗传型和散发型,在家族遗传型中,主要由于致病基因的突变而引起发病。大部分神经退行性疾病的一个共同的病理特征是致病蛋白会由于降解异常而在胞质、胞核或者胞外形成异常的积聚而形成聚集。约有20%的家族遗传型肌肉萎缩侧索硬化症（FALS）是由于编码铜-锌过氧化物歧化酶（SOD1）的基因发生突变而引起的,从而造成脑和脊髓运动神经元的选择性丧失引起发病。突变的SOD1通常在发病患者的脑和脊髓中通过聚集而形成包涵体,但其致病机制以及突变蛋白的聚集机制至今尚未能明确阐述。另一种神经退行性疾病脊髓小脑共济失调Ⅲ型（SCA3）/马查多-约瑟夫病（MJD）是由于编码谷氨酰胺的密码子CAG的数量在MJD1基因中非正常扩增而引起的一种常染色体显性遗传的多聚谷氨酰胺病。该病的主要病理特征为突变的ataxin-3（MJD1编码蛋白）在患者易感脑区内的神经元胞核内聚集形成包涵体,但其聚集机制以及致病机制至今也不清楚。神经退行性疾病中聚集的形成与这些致病蛋白的翻译后修饰作用有很大的关系。蛋白质的翻译后修饰包括磷酸化、乙酰化、甲基化、泛素化和SUMO化等,它们可以通过调节底物蛋白的活性、亚细胞器定位、稳定性或者与其它蛋白质的相互作用而达到调节蛋白功能的作用。很多神经退行性疾病相关蛋白如huntingtin、ataxin-1、α-synuclein、DJ-1和tau等由于SUMO化修饰而对蛋白功能以及疾病发病过程产生影响,也有很多研究表明多聚谷氨酰胺疾病蛋白如huntingtin,ataxin-1和DRPLA的磷酸化对于蛋白的功能以及疾病发生有很重要的影响。因此,蛋白质的翻译后修饰作用与神经退行性疾病之间存在着很大的联系。本论文主要从家族型肌萎缩侧索硬化症（FALS）的致病蛋白SOD1的SUMO化和脊髓小脑共济失调Ⅲ型（SCA3）/马查多-约瑟夫病（MJD）致病蛋白ataxin-3的磷酸化这两方面来探讨蛋白质翻译后修饰对这些神经退行性疾病致病蛋白特性的影响,主要研究结果如下:1.我们运用免疫共沉淀方法和体外SUMO化反应,发现SOD1能够被SUMO-1修饰,但是却不能被SUMO-2和SUMO-3修饰。将SOD1中第75位赖氨酸这个保守的SUMO修饰位点突变为精氨酸后,SOD1则完全不能够被SUMO-1修饰。通过进一步的细胞共转染技术,并用荧光显微镜观察发现经SUMO-1修饰后,野生型和突变型SOD1所形成的胞浆内聚集均会增加,而且SUMO-1能够被招募至SOD1所形成的聚集上,与SOD1在细胞内聚集体上有共定位现象。更进一步通过加入放线菌酮（CHX）以抑制新生蛋白的合成来检测已合成蛋白的降解速度,证实SUMO-1可以稳定野生型和突变型SOD1而不易降解。这些结果提示,SUMO-1对SOD1在第75位赖氨酸的修饰可能参与调控SOD1的稳定性以及聚集的形成,进而可能对由SOD1突变所引起的FALS的发病产生影响。2.我们运用GST Pull-down和免疫共沉淀实验证实激酶GSK 3β与ataxin-3能够结合,并且通过γ-³²P标记的ATP进行体外磷酸化实验证明正常和扩展的ataxin-3均能被激酶GSK 3β磷酸化。通过磷酸化位点的点突变实验,我们发现磷酸化的位点是在ataxin-3的第256位丝氨酸上。我们进一步构建了第256位丝氨酸丧失磷酸化的突变体S256A以及模拟磷酸化的突变体S256D,采用细胞转染和免疫印迹检测发现,扩展的ataxin-3的S256A突变体会形成高分子量的多聚体形成聚集,而没有突变的扩展的ataxin-3和其S256D突变体则只是单体。更进一步与分子伴侣Hsp40或Hsp70分别共转染后发现,Hsp70能够抑制扩展的ataxin-3的S256A突变体形成聚集。这些结果表明激酶GSK3β对ataxin-3第256位丝氨酸的磷酸化能够调节ataxin-3的聚集,该特性可能对SCA3/MJD的发病产生影响。通过以上两个方面的研究结果可以看出,蛋白质翻译后修饰作用能够调节神经退行性疾病致病蛋白的特性,影响蛋白的聚集,从而可能对于疾病的发病产生影响。更多还原

【Abstract】 Neurodegenerative diseases are chronic disorders caused by the deterioration of certain neurons.Changes in these neurons cause a function disorder,eventually lead to neuronal cell death.Neurodegenerative diseases occur either in familial or sporadic forms.Familial neurodegenerative diseases are caused by mutations in association with diseasegenes.Mutated disease proteins form the intracellular,intranuclear or extracellular aggregates,which are the common pathologic hallmarks of these diseases.The mutations in the gene encoding copper-zinc superoxide dismutase (SOD1)causes approximately 20%cases of familial amyotrophic lateral sclerosis (FALS),characterized by selective loss of motor neurons.Mutant SOD1 forms inclusions in tissues from FALS patients.However,the precise mechanism of the accumulation of mutant SOD1 remains unclear.Spinocerebellar ataxia typeⅢ(SCA3)/Machado-Joseph disease(MJD)is a dominant neurodegenerative disorder caused by an expansion of the polyglutamine tract in MJD-1 gene product,ataxin-3. The expanded ataxin-3 forms nuclear inclusions in MJD brain.The precise mechanism of inclusion formation is also not well illuminated.It was reported that post-translational modifications of desease proteins play an important role in the aggregation process.Post-translational modifications of proteins, such as phophorylation,acetylation,methylation,ubiquitination and SUMOylation, are important for the regulation of protein functions,activities or localizations after their synthesis has been completed.Recent studies have revealed that some neurodegenerative disease proteins,such as huntingtin,ataxin-1,α-synuclein,DJ-1 and tau,are modified by SUMO,implicating that SUMO modification of these disease proteins may participate in regulation of these protens’ functions and associate with their pathogenesis.And some studies show that phosphorylation of some polyglutamine disease proteins,such as huntingtin,ataxin-1 and DRPLA,plays an important role in regulating pathogenesis.Thus,post-translational modifications have critical roles in neurodegenerative diseases.In this thesis,we mainly focus on the SUMOylation of SOD1 and phosphorylation of ataxin-3.From these two aspects,we investigated the role that post-tranlational modifications act in neurodegenerative diseases.The main results are below: 1.We show that human SOD1 is a substrate modified by SUMO-1.A conversion of lysine 75 to an arginine within a SUMO consensus sequence in SOD1 completely abolishes SOD1 sumoylation.We further show that SUMO-1 modification,on both wild type and mutant SOD1,increases SOD1 steady state level and aggregation.Moreover,SUMO-1 co-localizes onto the aggregates formed by SOD1.These findings imply that SUMO-1 modification on lysine 75 may participate in regulating SOD1 stability and its aggregation process.Thus,our results suggest that sumoylation of SOD1 may be involved in the pathogenesis of FALS associated with mutant SOD1.2.We show that S256 site in ataxin-3 is phosphorylated by GSK 3β.Moreover, S256A mutant of expanded ataxin-3 forms high molecular weight protein aggregation,whereas S256D mutant and expanded ataxin-3 without mutation at this site are monomeric.The molecular chaperone Hsp70 represses the aggregation of S256A mutant.Our results imply that phosphorylation of ataxin-3 by GSK 3βat serine 256 regulates the aggregation of ataxin-3.Taking together,post-translational modifications regulate the specificities of neurodegenerative disease proteins,influence the aggregate formation and play a role in the pathogenesis of neurodegenerative diseses.更多还原