【作者】 姚凌雁；

【导师】 唐北沙；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 背景：帕金森病(Parkinson’s disease, PD)是临床上常见的神经退行性疾病之一。PD的临床表现主要为静止性震颤、运动迟缓、步态异常和肌强直,PD的病因与发病机制尚无确切定论,主流研究认为这是一种与衰老、环境及遗传因素密切相关的疾病。PINK1基因(PTEN-induced putative kinase1)是在2004年被克隆的PARK6致病基因,属于常染色体隐性遗传早发性帕金森综合征的致病基因之一,该基因编码产生一种由581个氨基酸组成有激酶活性的线粒体蛋白,包括一个由34个氨基酸组成的线粒体定位结构域和一个由354个氨基酸组成的激酶结构域,此激酶结构域与Ca2+/钙调蛋白家族的丝氨酸/苏氨酸激酶高度同源。PINK1蛋白首先于2003年被证明它的激酶区域在体外实验中有自磷酸化活性,PINK1蛋白能通过自身磷酸化后,调节其自身的激酶活性；PINK1蛋白也能参与体外磷酸化,如磷酸化组蛋白H1或酪蛋白。有研究发现PINK1基因的致病突变影响了PINK1蛋白的磷酸化。但未有PINK1蛋白自身磷酸化位点体外实验的鉴定研究报道,及PINK1蛋白的自身磷酸化修饰位点改变对其体外激酶活性影响研究报道。在前期工作中我们发现了两种PINK1基因新的纯合致病突变(T313M, R492X),两者均位于激酶结构域中,从而导致其激酶功能改变,也可能对稳定PINK1蛋白的结构起重要作用,这两种致病突变的致病机理仍有待探讨。目的：探讨PINK1蛋白自磷酸化修饰功能及磷酸化底物功能参与帕金森病致病的机制方法：1、应用谷胱甘肽亲和层析法纯化法、体外磷酸化和质谱技术鉴定PINK1蛋白的自身磷酸化修饰位点；2、应用体外磷酸化和放射自显影技术明确PINK1蛋白的自磷酸化修饰位点；3、应用体外磷酸化和放射自显影技术检钡(?)PINK1的自磷酸化修饰位点突变、T313M、R492X突变对PINK1蛋白的激酶活性的影响；4、应用脂质体过表达、细胞免疫荧光化学染色的方法检测自磷酸化修饰位点对PINK1在真核细胞中的亚细胞定位的影响；5、应用脂质体过表达、chase-time技术研究白磷酸化位点突变对PINK1自身降解的影响：6、应用脂质体过表达、细胞免疫荧光化学染色的方法检钡(?)PINK1自磷酸化修饰位点突变、T313M、R492X突变PINK1对Parkin亚细胞定位的影响。结果：1、质谱结果显示一个PINK1蛋白可能的自身磷酸化位点：PINK1第465位丝氨酸残基-Ser465,通过生物信息比对可知,该氨基酸残基在绝大多数种属中高度保守。2、放射自显影结果示：在体外磷酸化条件下,野生型PINK1蛋白能够被自身磷酸化,而S465A突变型PINK1蛋白自身磷酸化水平较野生型降低,两者的差异具有统计学意义(P<0.05)。3、放射自显影结果示：在体外磷酸化条件下,野生型PINK1蛋白能够磷酸化酪蛋白(casein),而T313M、R492X、S465A突变型PINK1蛋白磷酸化底物水平较野生型降低,三种突变型PINK1蛋白分别与野生型PINK1蛋白进行两两比较的差异均具有统计学意义(P<0.05)。4、HEK293细胞免疫荧光化学染色结果显示：野生型PINK1蛋白分布于细胞胞质,呈典型的点状分布的线粒体定位；而S465A、S465D突变型PINK1蛋白的亚细胞定位没有明显变化,仍呈胞浆中线粒体定位。5、Chase-time实验结果显示：S465A、S465D突变型PINK1蛋白降解的半衰期较野生型延长,降解减慢。6、HEK293细胞免疫荧光化学染色结果显示：野生型PINK1蛋白和S465D突变型PINK1蛋白均促进Parkin蛋白转移到聚集的线粒体上,S465A. T313M. R492X突变型PINK1蛋白促进此过程的效率明显下降。结论：1、PINK1蛋白第465位丝氨酸残基Ser465为其体外自磷酸化的位点之一；2、PINK1蛋白第465位丝氨酸残基Ser465突变、PINK1致病突变T313M、R492X均能引起PINK1体外磷酸化激酶活性降低；3、PINK1蛋白第465位丝氨酸残基Ser465对PINK1蛋白真核细胞内亚细胞定位无影响；4、PINK1蛋白第465位丝氨酸残基Ser465与PINK1蛋白在真核细胞内自身稳定性相关；5、PINK1蛋白第465位丝氨酸残基Ser465(?)调节真核细胞内PINK1蛋白与Parkin蛋白的线粒体共定位；6、PINK1致病突变T313M、R492X均能引起真核细胞内PINK1蛋白与Parkin蛋白的线粒体共定位的效率降低。更多还原

【Abstract】 Background:Parkinson’s disease (PD) is one of the most common neurodegenerative disorders clinically. The clinical manifestations of PD are characterized by resting tremor, bradykinesia, gait abnormalities and muscle rigidity. The etiology and pathogenesis of PD are. not thoroughly understood, and the mainstream studies suggest that aging, environmental and genetic factors are closely related to this disease. PINKl (PTEN-induced putative kinase1) gene was cloned in2004as a PARK6-related gene, which is responsible for autosomal recessive early-onset Parkinson’s syndrome. It encodes a mitochondrial kinase consisting of581amino acids. PINK1gene includes a mitochondrial targeting domain consisting of34amino acids and a kinase domain consisting of354amino acids. The kinase domain is highly homologous with serine/threonine kinase of Ca2+/calmodulin protein family.The kinase domain of PINK1protein acts autophosphorylation activity in vitro, which was described in2003for the first time. The autophosphorylation of PINK1protein may regulate the phosphorylation kinase activity itself. PINK1protein also participates in phosphorylating substrates in vitro, such as histone HI or casein. Moreover, many studies show that disease-causing mutations in PINK1gene can affect the phosphorylation of PINK1protein. However, there are no reports on identification of PINK1protein autophosphorylation sites in vitro and how the PINK1protein autophosphorylation activity affects its kinase activity in vitro. In previous work, we found two PINK1gene homozygous disease-causing mutations (T313M, R492X), both of which are located in the kinase domain, resulting in the change in its kinase function and the stability of PINK1protein probably. The role of these two pathogenic mutations in the pathogenesis of PD remains an open question.Objective:To explore how the autophosphorylation and phosphorylation activity of PINK1protein participates in the pathogenic mechanisms involved in Parkinson’s diseaseMethods:1、Glutathione affinity chromatography purification method, the method of phosphorylation in vitro and mass spectrometry are utilized for the identification of autophosphorylation modification sites of PINK1protein;2、The method of Phosphorylation in vitro and autoradiography are utilized for detecting autophosphorylation activity of autophosphorylation site-mutant PINK1protein;3、The method of Phosphorylation In vitro and autoradiography are utilized for detecting kinase activity of autophosphorylation site-mutant PINKland T313M, R492X-mutant PINK1;4、Plasmid transfection and immunofluorescence staining method are utilized for detecting subcellular localization of autophosphorylation site-mutant PINK1in eukaryotic cells;5、Plasmid transfection and Chase-time technology are utilized for detecting the degradation of autophosphorylation site-mutant PINK1.6、Plasmid transfection and immunofluorescence staining method are utilized for detecting how autophosphorylation site-mutant PINK1and T313M, R492X mutant PINK1affect subcellular localization of Parkin in eukaryotic cells.Results:1、Mass spectrometry results show that the PINK1465serine residues-Ser465may be an autophosphorylation site. The biological information shows that this amino acid residues is highly conserved in the vast majority of species;2、Autoradiograph shows:In vitro, wild-type PINK1protein acts autophosphorylation activity, and the autophosphorylation level of S465A mutant PINK1protein reduced compared with wild-type, the difference was statistically significant (P<0.05);3、Autoradiograph showed:in vitro, the wild-type PINK1protein can phosphorylate casein (casein), the phosphorylating substrate level of T313M, R492X, S465A mutant PINK1protein reduced compared with the wild-type. As the three mutant PINK1protein mentioned above compare with the wild-type PINK1protein, differences in comparison were statistically significant (P<0.05);4、HEK293cells by immunofluorescence staining showed that: wild-type PINK1protein located in the cytoplasm of the cell, which was typical mitochondrial localization of punctate distribution; subcellular localization of S465A, S465D mutant PINK1protein did not change significantly;5、Chase-time experimental results show that:the degradation half-life of S465A mutant PINK1protein prolongs compared to wild-type PINK1protein, degradation half-life of S465D mutant PINK1protein is basically the same with S465A mutant PINK1protein.6、Immunofluorescence staining in HEK293cells showed that:the wild-type PINK1protein and S465D mutant PINK1protein can promote the transfer and aggregation of Parkin protein in mitochondria, S465A, T313M, R492X mutant PINK1protein can not promote this process.Conclusion:1、PINK1protein465serine residues Ser465is one of the autophosphorylation sites in vitro;2、Mutantion of PINK1protein465serine residues Ser465, PINK1pathogenic mutation T313M, R492X can reduce the kinase activity of PINK1in vitro;3、PINK1protein465serine residues Ser465may not influence the subcellular localization of PINK1in eukaryotic cells;4、PINK1protein465serine residues Ser465can influence the degradation pathway of PINK1protein in eukaryotic cells;5、PINK1protein465serine residues Ser465can regulate co-localization of PINK1and Parkin in mitochondria in eukaryotic cells.6、PINK1pathogenic mutation T313M, R492X can reduce co-localization of PINK1and Parkin in mitochondria in eukaryotic cells.更多还原