alpha-Synuclein Inhibition of Mitochondrial Protein Import

 描述（由申请人提供）：帕金森病（PD）是一种常见的神经退行性疾病，可导致运动障碍、认知和精神症状以及自主神经功能障碍。遗传和环境因素与PD发病机制有关，并且似乎线粒体缺陷和突触蛋白α-突触核蛋白的积累在大多数疾病形式中是常见的。此外，有证据表明线粒体功能障碍和α-突触核蛋白蓄积之间存在双向相互作用。线粒体复合物I的抑制导致α-突触核蛋白的增加和寡聚化，并且α-突触核蛋白水平的增加导致线粒体损伤和活性氧（ROS）的产生。α-突触核蛋白与线粒体之间相互作用的性质仍然不清楚，目前尚不清楚未经修饰的单体α-突触核蛋白是否对这些效应负责，或者是否与发病机制有关的翻译后修饰，例如寡聚化、多巴胺修饰、磷酸化或硝化作用很重要。线粒体包含自己的基因组，但它只编码13种蛋白质，因此它们必须输入它们所包含的>1000种蛋白质中的约99%。线粒体蛋白质输入通过复杂且高度调节的系统发生，其中最好的理解是识别用于输入的蛋白质上的N-末端线粒体靶向信号（MTS）。虽然没有共有序列，但MTS特征性地形成由受体蛋白TOM 20识别的两亲性螺旋，该受体蛋白TOM 20是线粒体外膜（TOM）的移位酶复合物的亚基。虽然单体α-突触核蛋白在溶液中是一种本质上无序的蛋白质，但它与膜中的阴离子脂质结合，形成两亲性螺旋 类似于已知的MTS基序。在这种情况下，我们有强有力的证据表明，某些形式的后修饰的α-突触核蛋白特异性地结合到TOM 20上，并干扰靶向蛋白质的输入。本研究拟从以下几个方面研究α-synuclein与线粒体蛋白质输入机制的相互作用：（1）确定抑制线粒体蛋白质输入的α-synuclein的具体形式;（2）确定关键的结合配偶体和结合参数;（3）确定α-synuclein诱导的线粒体蛋白质输入障碍的功能后果;（4）研究α-synuclein与线粒体蛋白质输入机制的相互作用。（4）研究阻断线粒体蛋白输入是否会引起类似于α-突触核蛋白过表达的黑质纹状体神经变性;和（5）检查线粒体输入的遗传操作以防止黑质中AAV 2介导的α-突触核蛋白过表达的潜力。

 DESCRIPTION (provided by applicant): Parkinson disease (PD) is a common neurodegenerative disorder resulting in motor impairment, cognitive and psychiatric symptoms and autonomic dysfunction. Genetic and environmental factors have been implicated in PD pathogenesis, and it appears that mitochondrial defects and accumulation of the synaptic protein, α- synuclein, are common to most forms of the disease. Moreover, there is evidence of a bidirectional interaction between mitochondrial dysfunction and α-synuclein accumulation. Inhibition of mitochondrial complex I leads to accretion and oligomerization of α-synuclein, and increased levels of α-synuclein cause mitochondrial impairment and production of reactive oxygen species (ROS). The nature of the interaction between α- synuclein and mitochondria remains obscure, and it is unclear whether unmodified monomeric α-synuclein is responsible for these effects, or whether posttranslational modifications which have been implicated in pathogenesis, such as oligomerization, dopamine modification, phosphorylation or nitration are important. Mitochondria contain their own genome, but it encodes only 13 proteins, so they must import about 99% of the >1000 proteins they contain. Mitochondrial protein import occurs through complex and highly regulated systems, the best understood of which recognizes N-terminal mitochondrial targeting signals (MTS) on proteins destined for import. While there is no consensus sequence, MTS characteristically form an amphipathic helix that is recognized by a receptor protein, TOM20, which is a subunit of the translocase complex of the outer mitochondrial membrane (TOM). Although monomeric α-synuclein is an intrinsically disordered protein in solution, in association with anionic lipids in membranes, it forms an amphipathic helix similar to known MTS motifs. In this context, we have strong evidence that certain forms of posttranslationally-modified α-synuclein bind specifically to TOM20 and interfere with import of mitochondrially-targeted proteins. We propose to study the interaction of α-synuclein with mitochondrial import machinery in the following aims: (1) Define the specific forms of α-synuclein that inhibit mitochondrial protein import; (2) Define key binding partners and binding parameters; (3) Determine functional consequences of α-synuclein-induced impairment of mitochondrial protein import; (4) Investigate whether blockade of mitochondrial protein import causes nigrostriatal neurodegeneration similar to α-synuclein overexpression; and (5) Examine the potential for genetic manipulation of mitochondrial import to protect against AAV2-mediated α-synuclein overexpression in substantia nigra.