【作者】 王雪晶；

【导师】 唐北沙；

【作者基本信息】 中南大学 ， 神经病学， 2010， 博士

【摘要】 背景：帕金森病(Parkinson’s disease, PD)是常见的神经退行性疾病之一,以静止性震颤、肌强直、运动减少和姿势平衡障碍等症状为特征。PD在65岁以上人群患病率高达1.7%,85岁以上人群患病率高达4.8%，严重威胁着中老年人的身体健康。随着PD相关易感基因的发现,遗传因素在PD发病机制中的作用越来越受到关注。自1997年以来已定位了16个遗传性PD的基因位点,并克隆了11个遗传性PD致病基因,其中parkin、PINK1、DJ-1、ATP13A2、PLA2G6和FBX07与常染色体隐性遗传早发性帕金森综合征(autosomal recessive early-onset parkinsonism, AREP)发病相关。Parkin基因是1998年克隆的第一个AREP的致病基因。Parkin蛋白是一个含465个氨基酸的蛋白质,分子量约为52kDa,在中脑黑质等脑部各区域广泛表达。Parkin蛋白的功能迄今未完全阐明,相关研究证实Parkin作为E3泛素蛋白连接酶参与蛋白质泛素化过程,并具有线粒体保护作用。PINK1 (PTEN-induced putative kinase 1)基因是继parkin基因后第二常见的AREP致病基因。该基因定位于1p36区域,共跨越1.8Kb，包含8个外显子,编码一个由581个氨基酸组成的蛋白质。研究表明PINK1蛋白主要参与调节线粒体的生理功能,可能通过减轻应激状态下线粒体功能障碍和细胞凋亡发挥其保护神经元的作用。目前研究发现PINK1蛋白与其他蛋白之间的相互作用可能对于PD的发病具有极其重要的意义。BAG (BCL2-associated athanogene, BAG)家族蛋白由于与抗凋亡蛋白bcl-2相互作用而被发现。BAG5 (BCL2-associated athanogene5)是该蛋白家族中唯一包含多个BAG结构域的蛋白,BAG5蛋白可通过4个BAG结构域与相关的蛋白质发生相互作用。动物实验研究发现BAG5在小鼠的大脑皮质、海马、红核和黑质致密部均有表达,在黑质致密部的多巴胺能神经元表达水平较高。目前BAG5的功能仍不十分明确,有研究发现BAG5能够促进PD动物模型多巴胺能神经元退行性变,抑制了Hsp70分子伴侣的活性；但也有研究发现BAG5蛋白能够促进Hsp70的分子伴侣活性。前期工作中,我们已成功运用酵母双杂交系统进行了PINK1相互作用蛋白的筛选,并成功地筛选到BAG5蛋白,且相互作用编码区位于BAG5蛋白CDS编码区的91-425bp(31-142氨基酸之间)处；此后构建了PINK1与BAG5的真核及原核表达质粒,应用免疫共沉淀技术证实PINK1蛋白与BAG5蛋白在真核细胞内能够相互结合,进一步应用体外结合实验证实PINK1蛋白与BAG5蛋白存在直接相互结合。本研究在前期研究的基础上进一步探讨BAG5、PINK1及Parkin蛋白三者的相互作用机制如何参与了PD的发病过程。目的：探讨BAG5、PINK1及Parkin蛋白的相互作用参与PD致病过程的机制。方法：1.应用免疫荧光共定位技术,研究野生型PINK1蛋白及BAG5的蛋白亚细胞分布、亚细胞共定位及二者共同转染后亚细胞分布的改变。2.应用体外蛋白结合实验技术,研究PINK1蛋白与BAG5蛋白的相互结合结构域。3.应用体外磷酸化与放射自显影技术,研究PINK1蛋白能否直接磷酸化修饰BAG5蛋白。4.应用chase-time技术,研究BAG5蛋白是否调节了野生型及突变型PINK1蛋白的降解半衰期。5.应用脂质体过表达、RNA干扰、免疫共沉淀技术,研究BAG5蛋白对野生型及R492X突变型PINK1蛋白泛素化降解的调节。6.应用脂质体过表达、RNA干扰、免疫共沉淀技术,研究BAG5蛋白及Parkin蛋白对PINK1蛋白泛素化降解水平的共同调节。7.应用脂质体过表达、免疫共沉淀技术,研究野生型、突变型Parkin蛋白对于PINK1蛋白泛素化降解水平的调节。8.应用免疫组织化学、western-blot技术,研究MPTP诱导的小鼠PD模型黑质多巴胺能神经元中BAG5、PINK1及Parkin蛋白的变化。结果：1.应用细胞免疫荧光化学技术证实PINK1及BAG5蛋白存在亚细胞共定位,且二者共转染后BAG5蛋白部分呈现线粒体聚集。2.应用体外蛋白结合实验证实BAG5蛋白的4个结构域：BAG5(9-86)(第一个BAG结构域)、BAG5(182-260)(第二个BAG结构域)、BAG5(365-442)(第四个BAG结构域)及BAG5(87-181)(第一第二BAG结构域之间区域)均可与野生型PINK1蛋白直接结合。PINK1蛋白的PINK1(156-509)(PINK1蛋白激酶结构域)可与野生型BAG5直接结合。3.应用GST Pull-down技术与放射自显影技术,通过γ-32P标记的ATP进行体外磷酸化实验证明：PINK1蛋白不能体外磷酸化修饰BAG5蛋白。4.应用chase-time技术证实BAG5蛋白延长了野生型PINK1蛋白的降解半衰期,起到了稳定野生型PINK1蛋白的作用；同时缩短了R492X突变型PINK1蛋白的降解半衰期,起到了促进R492X突变型PINK1蛋白降解的作用。5.应用脂质体过表达、RNA干扰、免疫共沉淀技术研究证实BAG5蛋白能够对野生型及突变型PINK1蛋白泛素化水平进行调节。BAG5蛋白能够减少野生型PINK1蛋白的多聚泛素化水平,延缓野生型PINK1蛋白的泛素化降解；同时BAG5蛋白能够增加R492X突变型PINK1蛋白的多聚泛素化水平,从而促进R492X突变型PINK1蛋白的泛素化降解。6.应用脂质体过表达、RNA干扰、免疫共沉淀技术研究证实BAG5与Parkin蛋白通过不同的途径调节PINK1蛋白的泛素化降解,二者相对独立的对PINK1蛋白的泛素化降解过程进行调节。7.应用脂质体过表达、免疫共沉淀技术研究证实野生型Parkin蛋白减少PINK1蛋白的泛素化降解,突变型Parkin蛋白增加PINK1蛋白的泛素化降解。8.应用免疫组织化学、western-blot技术研究发现MPTP诱导的小鼠PD模型黑质多巴胺能神经元中BAG5及Parkin蛋白的表达上调而PINK1蛋白表达下调。结论：1.PINK1蛋白与BAG5蛋白存在细胞内共定位。2.PINK1蛋白与BAG5蛋白的结合结构域为PINK1蛋白的激酶结构域；BAG5蛋白与PINK1蛋白的结合结构域为BAG5蛋白的第一、第二、第四个BAG结构域及第一第二BAG结构域之间区域。3. BAG5蛋白不是PINK1蛋白的直接磷酸化底物。4.BAG5蛋白抑制了野生型PINK1蛋白的泛素化降解,促进了R492X突变型PINK1蛋白的泛素化降解,BAG5蛋白能够稳定野生型PINK1蛋白,清除R492X突变型PINK1蛋白。5. BAG5蛋白及Parkin蛋白均可通过泛素蛋白酶体通路独立的调节PINK1蛋白的泛素化降解。6. Parkin蛋白通过促进PINK1蛋白E3连接酶的降解而抑制了PINK1蛋白的泛素化降解,稳定PINK1蛋白水平。7. BAG5、PINK1及Parkin蛋白均参与了MPTP诱导的PD的病理过程。更多还原

【Abstract】 Background:Parkinson’s disease (PD) is a common neurodegenerative disorder which is characterized by resting tremor, muscle rigidity, balance and posture to reduce obstacles to movement. Prevalence rate of PD over 65 years old is up to 1.7 percent, and the people over 85 years old are reach to 4.8 percent, and PD is a serious threat to the health of the elderly. The molecular genetic studies have led to the identification of different monogenic forms of PD. Genetic factors in the pathogenesis of Parkinson’s disease is becoming increasingly important. From 1999,16 loci for autosomal recessive Parkinsonism have been mapped, and 11 genes have been cloned, they are parkin、PINK1、DJ-1、ATP13A2、PLA2G6 and FBXO7, which are related to autosomal recessive early-onset parkinsonism.Parkin was a protein which in humans was encoded by the PARK2 gene in 1998, and expressed 465 amino acid product. The precise function of this protein is unknown; however, the protein is a component of a multiprotein E3 ubiquitin ligase complex which in turn is part of the ubiquitin-proteasome system that mediates the targeting of substrate proteins for proteasomal degradation, also play an important role in protecting the mitochondria’s function.In 2004, Valente found that PINK1 gene (PTEN-induced putative kinase 1) was the second common virulence gene besides Parkin. The PINK1 gene comprises eight exons, which encode a ubiquitously expressed 581 amino acid product. The protein contains an N-terminal mitochondrial targeting domain and a highly conserved kinase domain that sequence comparisons suggest is a serine/threonine protein kinase structurally belonging to the calmodulin-dependent kinase class of protein kinases. To date, functional studies have shown that PINK1 protein may play an important role in protecting the mitochondria’s function, wild-type PINK1 protects cells against proteasomal inhibition as well as staurosporine-induced apoptosis. The study suggested that the interaction between PINK1 protein and other protein many play an important role in the pathogenesis of PD.Bcl-2-associated athanogene (BAG)-family proteins were originally identified by their ability to associate with the anti-apoptotic protein, Bcl-2. BAG-family proteins contain a single BAG domain, except for BAG-5 which has four BAG repeats. BAG-5 expresses in the brain cortex, hippocampus, red nucleus and substantia nigra pars compacta, and mainly gathered in the substantia nigra pars compacta. Little is known about the role of BAG-5 in cells other than its ability to bind Hsp70, and to inhibit both parkin E3 ligase and Hsp70 chaperone activity enhancing dopaminergic neuron degeneration.We have used yeast two-hybrid system to identify PINKI interacting protein and aquired the protein BAG5. We confirmed that PINKI directly interacts with BAG5. We confirmed the interaction in vivo by co-immunoprecipitation and in vitro by pull-down, These data not only establish the basis for further investigating PINK1 protein function, but also suggest the interaction between BAG5 protein, PINK1 protein and Parkin protein many play an important role in the pathogenesis of PD.Objective:To research the possible mechanism of the interaction between the PINK1 protein, Parkin protein and BAG protein in the Parkinson’s disease.Methods:1) By using the electrophysiological method, we studied Intracellular distribution of wild-type PINK1 protein and wild-type BAG5 protein、subcellular distribution of PINK1 and BAG5 after cotransformation.2) We generated different deletion of BAG5 and PINK1 to identificated of the domains in BAG5 interaction with PNK1 and the domains in PINK1 interaction with BAG5 by GST pull-down assay.3) To further identify whether BAG5 can be phosphorylated by PINK1 by using autoradiography and phosphorylation in vitro. 4) Chase-time experiment was undertaken to investigate the effect of BAG5 protein on the half-lives of wild-type PINK1 and R492X mutation of PINK1.5) Plasmid transfection, RNA interference technique and co-immunoprecipitation were utilized to analyze co-regulation of BAG5 and PINK1 on the ubiquitination of wild-type PINK1.6) Plasmid transfection, RNA interference technique and co-immunoprecipitation were utilized to analyze co-regulation of BAG5 and PINK1 on the ubiquitination of wild-type PINK1.7) Plasmid transfection and co-immunoprecipitation were utilized to analyze the effects of wild-type Parkin and the mutations of Parkin on the ubiquitination of wild-type PINK1.8) Immunohistochemistry and western-blot were utilized to analyze the level of BAG5、PINK1 and Parkin in the MPTP-induced PD model of dopaminergic neuron in substantia nigra.Results:1) The subcellular co-localization of PINK1 protein and BAG5 protein were detected by immunocytochemistry labeling, and after cotransformating in HEK 293 cell the subcellular localization of BAG5 protein changed and showed aggregation in mitochondrion.2) Using GST pull-down assay to detect the 5 domains:BAG5(9-86) (the first BAG domain)、BAG5(182-260) (the second BAG domain)、BAG5(365-442) (the forth BAG domain) and BAG5(87-181) (the domain between the first BAG domain and the second BAG domain) all can directly interact with PINK1. PINK1(156-509)(the serine/ threonine protein kinase domain) can directly interact with BAG5.3) Using GST pull-down assay and autoradiography we found BAG5 can not be phosphorylated by PINK1 in vitro.4) We found BAG5 protein prolonged the half-lives of wild-type PINK1, but shortened the half-lives of R492X mutation of PINK1. BAG5 protein stabilized the wild-type PINK1 and promoted the degradation of R492X mutation of PINK1.5) We found BAG5 protein can regulate the ubiquitination of wild-type PINK1 and the R492X mutation of PINK1 by using Plasmid transfection, RNA interference technique and co-immunoprecipitation. BAG5 protein can inhibit the ubiquitination of wild-type PINK1, and promote the ubiquitination of of R492X mutation of PINK1.6) By using Plasmid transfection, RNA interference technique and co-immunoprecipitation, we found BAG5 protein and Parkin protein through different pathway to regulate the ubiquitination of wild-type PINK1 and they regulate the ubiquitination of wild-type PINK1 independently.7) We found wild-type Parkin inhibit the ubiquitination of wild-type PINK1, and promote the ubiquitination of R492X mutation of PINK 1, by using Plasmid transfection, RNA interference technique and co-immunoprecipitation.8) By using immunohistochemistry and western-blot technique, we found the protein expression level of BAG5 and Parkin increased, however, the level of PINK1 protein decreased in the MPTP-induced PD model of dopaminergic neuron in substantia nigra.Conclusion:1) There are co-localization of PINK 1 protein and BAG5 protein in the cell.2) The first BAG domain, the second BAG domain, the forth BAG domain and the domain between the first BAG domain and the second BAG domain all can directly interact with PINK1. The serine /threonine protein kinase domain of PINK1 can directly interact with BAG5.3) BAG5 can not be directly phosphorylated by PINK1 in vitro.4) BAG5 protein can regulate the degradation of the wild-type PINK1 and the R492X mutation of PINK1; BAG5 protein can inhibit the ubiquitination of wild-type PINK1, and promote the ubiquitination of R492X mutation of PINK1.5) BAG5 protein and Parkin protein through different pathway to regulate the ubiquitination of wild-type PINK1 and they regulate the ubiquitination of wild-type PINK1 independently.6) The E3 ligase of PINK1 is the substrate of Parkin, Parkin promote the degradation of E3 ligase of PINK1, and inhibits the ubiquitination of wild-type PINK1.7) BAG5、PINK1 and Parkin proteins involved in the pathological process of MPTP-induced PD.更多还原