Frataxin, Iron Storage, and Neurodegeneration

描述(由申请人提供)：建议研究的主要焦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代表了在线粒体内安全处理氧化还原活性铁的关键机制。缺乏酵母Frataxin已被证明导致线粒体铁积累，线粒体DNA完整性丧失，以及在维持铁-硫簇方面的全球缺陷。在人类中，Frataxin缺陷会导致一种神经退行性疾病，Friedreich共济失调。这些发现表明，Frataxin参与了铁稳态，这一功能的丧失会导致氧化损伤，导致神经细胞死亡。初步结果表明，人的Frataxin可以自我组装，并以稳定的生物可利用形式与铁结合。这项建议的直接目标是阐明人类Frataxin的作用机制。建议的具体目标如下：(1)对预计对人Frataxin的组装、铁结合和抗氧化特性至关重要的保守氨基酸残基进行定点突变；(2)在大肠杆菌中表达突变蛋白，并用生物物理和生化方法对其进行鉴定；(3)在真核模型酿酒酵母中测试严重突变对OXPHOS维持和氧化应激敏感性的影响。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.