Frataxin, Iron Storage, and Neurodegeneration

描述（由申请人提供）：拟议研究的主要重点是负责维持氧化磷酸化（OXPHOS）和保护免受氧化损伤的机制。我们假设线粒体蛋白frataxin代表了一个关键的机制，以处理氧化还原活性铁安全内的线粒体。缺乏酵母frataxin已被证明会导致线粒体铁积累，线粒体DNA完整性的丧失，以及铁硫簇维持的全面缺乏。在人类中，共济失调蛋白缺陷导致神经退行性疾病，弗里德赖希共济失调。这些发现表明，共济失调蛋白参与铁稳态，并且该功能的丧失导致氧化损伤，从而导致神经元细胞死亡。初步结果表明，人共济失调蛋白可以与自身组装，并以稳定和生物可利用的形式结合铁。该提案的直接目标是阐明人类共济失调蛋白的作用机制。本研究的主要目的是：（1）对人共济失调蛋白的组装、铁结合和抗氧化活性等关键氨基酸残基进行定点突变，（2）在大肠杆菌中表达突变蛋白。（3）在真核生物模型中，研究严重突变体对OXPHOS维持和氧化应激敏感性的影响，并对S.啤酒。这项研究将为Friedreich共济失调的发病机制和氧化损伤在神经退行性疾病中的作用提供新的信息。

DESCRIPTION (provided by applicant): The main focus of the proposed research are the mechanisms responsible for maintenance of oxidative phosphorylation (OXPHOS) and protection from oxidative damage. We have hypothesized that the mitochondrial protein frataxin represents a critical mechanism to handle redox active iron safely within the mitochondrion. Lack of yeast frataxin has been shown to result in mitochondrial iron accumulation, loss of mitochondrial DNA integrity, and a global deficiency in the maintenance of iron-sulfur clusters. In humans, frataxin defects lead to a neurodegenerative disease, Friedreich ataxia. These findings indicate that frataxin is involved in iron homeostasis and that loss of this function results in oxidative damage leading to neuronal cell death. Preliminary results indicate that human frataxin can assemble with itself and bind iron in stable and bioavailable form. The immediate goal of this proposal is to elucidate the mechanism of action of human frataxin. The following specific aims are proposed: (1) To carry out site directed mutagenesis of conserved amino acid residues predicted to be critical for the assembly, iron binding, and anti-oxidant properties of human frataxin; (2) To express mutant proteins in E. coli and characterize them by biophysical and biochemical methods; (3) To test the effects of severe mutants on OXPHOS maintenance and sensitivity to oxidative stress in the eukaryotic model, S. cerevisiae. This research should provide new information relevant to the pathogenesis of Friedreich ataxia and the role of oxidative damage in neurodegenerative disorders.