【作者】 牟英峰；

【导师】 唐北沙；

【作者基本信息】 中南大学 ， 神经病学， 2009， 博士

【摘要】 帕金森病(Parkinson’s disease,PD)是一种常见于老年人的神经退行性疾病。目前其发病机制尚不清楚,但是众多研究显示,线粒体功能障碍在PD发病机制中起了重要作用:线粒体毒素(如MPTP)可以在人类或动物模型引起PD样表现;PD患者的线粒体功能障碍常见;PD致病基因之一的编码产物PINK1蛋白在线粒体上发挥其生物学功能。研究显示,PINK1蛋白定位于线粒体,对线粒体具有保护功能。了解PINK1蛋白的功能对于理解PD的发病机制以及治疗具有重要意义。虽然已经对PINK1蛋白的功能进行了许多研究,目前对其如何发挥保护线粒体功能并不清楚。许多蛋白质通过相互作用参与生物学过程而发挥其功能,因此寻找PINK1蛋白的相互作用蛋白是理解PINK1蛋白功能的一个重要途径。为了寻找可能与PINK1蛋白存在相互作用的蛋白,我们从如下5个方面进行了研究:1.首先应用基因重组技术,以本研究小组前期工作中构建的pcDNA3.1-PINK1为模板,成功构建了野生型PINK1蛋白酵母表达质粒pGBKT7-PINK1。经测序证实PINK1基因阅读框正确,载体构建正确。然后将pGBKT7-PINK1转化到酵母细胞AH109中,应用Western blot技术,检测PINK1蛋白在酵母菌株AH109中的表达情况。结果发现PINK1蛋白能够在酵母菌株AH109中正确表达。2.将27μl MATCHMAKER Human Fetal Brain GAL4 cDNA &genomic libraries原始菌液铺到300个150mm皿中,在30℃温箱中培养24-36小时至菌落融合,刮取菌落,共收集菌液1000ml。使用QIAGEN Tip2500试剂盒抽提1000ml菌液的cDNA质粒,共获得2.593mg文库cDNA质粒。3.采用大规模酵母质粒转化技术,将所抽提的cDNA文库质粒转化至酵母细胞AH109/pGBKT7-PINK1,进行人胎脑cDNA文库筛选。按照文库说明书要求,共进行了4次文库筛选,共筛选了6.65×10~6个克隆,筛选得到77个His~+克隆,继续进行Ade~+/Mell~+表型检测,我们总共获得了61个Ade~+/His~+/Mell~+克隆。4.61个Ade~+/His~+/Mell~+克隆经过划线、PCR及酶谱分析消除重复克隆,我们获得了49个Ade~+/His~+/Mell~+克隆。通过自激活验证消除能够自激活的克隆,最终我们筛选获得了2个真阳性克隆。经过测序和在NCBI数据库中进行BLAST,这两个真阳性克隆都是已知的蛋白质,它们是KIF1A(kinesin family member 1A)和BAG5(BCL2-associated athanogene 5)。这说明PINK1蛋白可能与KIF1A和BAG5分别存在相互作用。5.由于我们获得的KIF1A序列位于其mRNA的不翻译区,因此认为KIF1A不能作为PINK1蛋白互作蛋白的候选蛋白。而我们筛选到的BAG5序列位于其氨基端编码区,故继续进行了BAG5与PINK1蛋白是否存在互作的验证。收集HEK293A细胞中过表达的HA-PINK1蛋白,与JM109表达的GST-BAG5相孵育,在G4B(Glutathione 4 B)匀浆中进行pull down结合实验,Western blot中应用抗HA抗体作免疫印迹,结果发现在Input及HA-PINK1+GST-BAG5中均见一条63kDa大小的全长型和一条52kDa大小的成熟型PINK1条带;将PKH3-HA-PINK1和pEGFP-N3-BAG5质粒共转染HEK293A细胞,用抗GFP抗体免疫沉淀BAG5蛋白,分别用抗HA抗体和抗GFP抗体做免疫印迹,结果发现在经抗GFP抗体沉淀的蛋白复合物中检测到GFP和GFP-BAG5蛋白,说明免疫沉淀成功;用抗HA抗体做免疫印迹,结果发现共转染细胞组中存在一条63kDa大小的全长型PINK1条带及一条52kDa大小的成熟型PINK1条带。这些结果说明PINK1蛋白与BAG5在细胞体内、外均存在相互作用。继续进行体外结合实验证实PINK1蛋白与BAG5之间的相互作用是直接相互作用。我们首次采用酵母双杂交技术筛选PINK1蛋白的相互作用蛋白,首次发现PINK1蛋白可能与BAG5和KIF1A分别存在相互作用,首次证实PINK1蛋白与BAG5存在直接相互作用。本研究结果不但为进一步深入研究PINK1基因的功能奠定了基础,而且对于我们理解PINK1基因在PD发病机制中的作用、PD的治疗药物的开发以及线粒体功能障碍与PD的关系,甚至是线粒体与衰老的关系均具有重要意义。更多还原

【Abstract】 Parkinson’s disease(PD) is a common neurodegenerative disorder. The cause of the disease is still unknown,although substantial evidence suggests that mitochondrial dysfunction is a major contributor:Several mitochondrial toxins induce PD-like symptoms in humans and animal models;systemic mitochondrial dysfunction appears to be a feature of a large proportion of PD sufferers;and several genes involved in rare heritable forms of Parkinsonism have been implicated in mitochondrial biology,including the PTEN-induced kinase 1(PINK1).PINK1 protein has been shown to localize in the mitochondria.The mechanism by which PINK1 protects cells remains unknown.Studying on the function of PINK1 is important to understand the neuropathology of PD.Many,if not all,essential biological processes require selective interactions between proteins.Therefore,an important step in the analysis of PINK1 function is identification of the interaction partners of PINK1. The objectives of the present study are to identify proteins which may interact with PINK1 on the following five aspects:1.The full-length code domain sequence of PINK1 was amplified from pcDNA3.1-P1NK1 which was constructed in our previous experiments and was cloned to pGBKT7 vector.The integrity of the constructs was confirmed by sequencing.Yeast strain AH109 cells were transiently transformed with pGBKT7-PINK1.We prepared Western blots from the transformants and confirmed that PINK1 protein could be normally expressed in Saccharomyces cerevisiae(strain AH109). 2.In order to obtain enough plasmids for library screening in yeast, we must amplify the premaded MATCHMAKER Human Fetal Brain GAL4 cDNA & genomic libraries.Plate 27μl library directly on 300 150-mm plates,and incubate at 30℃for 24-36 hours,the resulting colonies were nearly confluent.Then we scraped colonies,and obtained 1000 ml yields.Then prepare eDNA plasmid with QIAGEN Tip2500 kit. We obtained 2.593mg library plasmid in total.3.By using large-scale transformation,AH109/pGBKT7-PINK1 cells were transformed with library plasmid.According to the manual of the library,we screened for MATCHMAKER Human Fetal Brain GAL4 cDNA & genomie libraries four times.We screened 6.65×10~6 independent clones in total,and acquired 77 His~+ clones.After retesting Ade~+/Mel1~+ phenotypes,61 Ade~+/His~+/Mel1~+ clones were acquired.4.By restreaking Ade~+/His~+/Mel1~+ clones,amplifying library inserts by PCR and characterizing PCR products by digesting with HaeⅢ,we sorted clones to eliminate duplicates.We acquired 49 Ade~+/His~+/Mel1~+ clones.By eliminating self-activating clones,we acquired 2 true positive clones.By sequencing and BLAST in NCBI,we know the two positive inserts are part of KIF1A(kinesin family member 1A) and BAG5 (BCL2-associated athanogene 5).PINK1 protein may interact with KIF1A and BAG5 respectively.5.KIF1A is not supposed to be a candidate protein interacting with PINK1 because the acquired sequence is located on untranslated region of KIF1A.The acquired sequence of BAG5 is located on its N-terminal coding region,so we continued experiments on confirming interaction between PINK1 and BAG5.Incubate GST-BAG5 fusion protein from JM109 and HA-PINK1 fusion protein from HEK293A cells together with G4B(Glutathione 4B),which were then collected and washed.Collected complexes were resolved by SDS-PAGE and processed for further analysis by Western blotting,63kDa and 52kDa PINK1 protein bands in both input and HA-PINK1+GST-BAG5 were found.Collect cell extracts from HEK293A cells cotransfected with PKH3-HA-PINK1 and pEGFP-N3-BAGS,immunoprecipitate the cell extracts with anti-GFP antibody,then perform Western blotting by using anti-HA antibody, 63kDa and 52kDa PINK1 protein bands in cotransfected cells were found, both the full-length and the processed forms of endogenous PINK1 co-immunoprecipitated with BAGS.These results suggest that PINK1 interacts with BAG5 in vitro and in vivo.The following in vitro binding assay showed us that PINK1 directly interacts with BAG5.For the first time,we used yeast two-hybrid system to identify PINK1 interacting proteins and acquired two proteins,which were BAG5 and KIF1A,either of them may interact with PINK1.And we confirmed that PINK1 directly interacts with BAG5.These data not only establish the basis for further investigating PINK1 gene function,but also suggest a novel PINK1 related mitochondrial pathway in PD pathogenesis,even in understanding the relationship between mitochondrial and aging.更多还原