帕金森病相关蛋白Parkin在β-Synuclein P123H突触核蛋白病理变化过程中的作用研究

发布时间：2018-03-05 08:02

本文选题：Parkin　切入点：突变体　出处：《河南大学》2014年硕士论文　论文类型：学位论文

【摘要】：Parkin（PARK2）与常染色体隐性遗传的家族性帕金森病相关，Parkin蛋白分子量为52kD，主要分布在细胞质中，是一种E3泛素连接酶，可以保护对细胞毒性的各种应激，并且在帕金森病的保护中起重要作用。β-synP123H突变体能够在细胞中形成溶酶体样包涵体，从而导致自噬性细胞退化。国内外对Parkin介导的线粒体自噬有诸多报道，但是对于Parkin在β-synP123H诱导的突触核蛋白病理变化过程中对蛋白的降解、细胞的自噬和存活所起到的作用和机制并不清楚。这项研究中，我们探讨Parkin及其突变体在β-synP123H诱导的突触核蛋白病理变化过程中的作用，有助于澄清帕金森病突触核蛋白病理变化过程的发病机制，为帕金森病的治疗和预防提供新的理论依据。本实验首先通过PCR构建带有HA标签的Parkin野生型（ParkinWT）和两步PCR构建带有HA标签的Parkin突变体R42P、T240R、R275W、C441R的真核表达质粒，通过双酶切和测序确定序列的正确性，通过Western Blot的方法确定质粒能够在真核细胞上表达；通过细胞爬片和免疫荧光技术了解野生型和突变型Parkin在细胞内的分布特征；通过WST-1和LDH的测定来了解和分析野生型和突变型的Parkin本身对于细胞的存活的影响以及对细胞产生的毒性。发现野生型Parkin弥散分布于细胞胞浆内，而突变型Parkin在细胞的胞浆内细胞核周围呈小颗粒状的包涵体存在，Parkin突变体具有细胞毒性。其次，构建并筛选出稳定表达Parkin野生型和突变型R42P、T240R、R275W、C441R的Hela细胞株，在稳定表达Parkin的细胞株上，运用免疫荧光双标记的方法，研究Parkin及其突变体对线粒体的影响。我们发现突变型Parkin导致线粒体的聚集和片段化，FCCP处理细胞导致线粒体去极化，ParkinWT能够被募集到去极化的线粒体上，但是Parkin突变体丧失了这一功能。最后，，在稳定表达Parkin野生型和突变型T240R的Hela细胞株上，瞬时转染pCEP4，β-syn野生型（β-synWT）和β-synP123H，分别通过Parkin和β-syn，Parkin和LC3以及β-syn和p62的免疫荧光双标记实验，LC3和β-syn的Western Blot实验，研究ParkinWT和ParkinT240R对自噬和β-synP123H的作用，发现ParkinWT能够促进β-synP123H通过自噬溶酶体途径降解，减缓β-synP123H在细胞内的累积，起到保护细胞的作用，Parkin突变体的作用与ParkinWT的作用相反。在稳定表达β-synP123H的293T细胞上瞬时转染pTRE2hyg、Parkin野生型和四种突变体后，检测Atg5、Beclin-1的水平和mTOR通路上的mTOR、p70S6K、4E-BP1的磷酸化水平也验证了这一点。结论：野生型和突变型Parkin在细胞内的分布特征不同，Parkin突变体具有细胞毒性，能够使线粒体片段化和聚集，而且不能被募集到去极化的线粒体上，诱导细胞退化；野生型Parkin通过调控mTOR通路调节自噬，促使β-synP123H进入自噬小体和溶酶体内降解，起到保护细胞的作用，Parkin突变体抑制β-synP123H蛋白降解，促进β-synP123H蛋白的累积，增强路易体痴呆相关的神经退化。
[Abstract]:Parkin (PARK2) associated with autosomal recessive familial Parkinson disease, Parkin protein molecular weight of 52kD, mainly distributed in the cytoplasm, an E3 ubiquitin ligase that can protect all the stress on the cell toxicity, and play an important role in the protection of Parkinson's disease. Beta -synP123H mutant can form a lysosome like inclusion in body cells, leading to autophagic cell degeneration.
There are many reports about Parkin mediated mitochondrial autophagy at home and abroad, but it is not clear about the role and mechanism of Parkin in protein degradation, cell autophagy and survival in the pathological change of beta -synP123H induced synuclein.
In this study, we investigate the synuclein pathological changes in Parkin and its mutant beta induced -synP123H, help to clarify the pathogenesis of Parkinson's disease synuclein pathology process, for the treatment and prevention of Parkinson's disease and provide new theoretical basis.
This experiment firstly constructed by PCR with HA tag Parkin wild type (ParkinWT) and the two step construction of PCR Parkin mutant R42P, HA tagged T240R, R275W, eukaryotic expression plasmid C441R, to determine the correct sequence by double enzyme digestion and sequencing, the Western Blot method to determine the plasmid expression in it nuclear cells; the cell slide and immunofluorescence technique of wild type and mutant Parkin distribution in cells was determined by WST-1 and LDH; to understand and analyze the wild type and mutant Parkin itself for cell survival and toxicity on the cells. It was found that the wild type Parkin dispersed in in the cytoplasm and around the mutant Parkin in the cytoplasm of cells within the nucleus is small granular inclusion bodies, Parkin mutants with cell toxicity.
Secondly, constructed and screened stable expression of wild type Parkin and mutant R42P, T240R, R275W, Hela, C441R cells, the stable expression of Parkin cell line, by using the method of immunofluorescence double labeling, effects of Parkin and its mutants of mitochondria. We found that the mutant Parkin leads to aggregation and fragmentation of mitochondria FCCP treatment, cells leading to mitochondrial depolarization, ParkinWT can be recruited to the depolarization of mitochondria, but Parkin mutant lost this function.
Finally, the stable expression of Parkin wild-type and mutant T240R in Hela cell line, transient transfection of pCEP4 and wild-type -syn beta (beta -synWT) and 3 -synP123H, respectively by Parkin and Parkin and beta -syn, LC3 and beta -syn and p62 immunofluorescence double labeling experiment, Western Blot experiment LC3 and beta -syn and the effects of ParkinWT and ParkinT240R on autophagy and beta -synP123H, ParkinWT beta can promote the degradation of -synP123H through the lysosomal pathway, the accumulation of slow beta -synP123H in cells and plays an important role in protecting cells, and the opposite effect. The effect of ParkinWT Parkin mutant in stable -synP123H expression on 293T cells transiently transfected into pTRE2hyg. Parkin wild type and four mutants, Atg5 detection, and mTOR pathway of Beclin-1 on mTOR, p70S6K, 4E-BP1 phosphorylation also proved this point.
Conclusion: the distribution of wild type and mutant Parkin in the cells of different Parkin mutants with cell toxicity, can cause mitochondrial fragmentation and aggregation, and can not be raised to the depolarization of mitochondria, induce cell degeneration; wild type Parkin regulates mTOR pathway regulates autophagy, promote beta -synP123H into autophagosomes and solution enzyme degradation in vivo, plays an important role in protecting cells, inhibition of Parkin mutant beta -synP123H protein degradation, promote the accumulation of beta -synP123H protein, enhance the neural correlates of dementia Louis degradation.

【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

【共引文献】