MITOCHONDRIAL IRON METABOLISM IN AGING

衰老过程中的线粒体铁代谢

• 批准号: 6207160
• 负责人: KATHLEEN S CROWLEY
• 金额: $ 3.92万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6207160
• 关键词: adenosinetriphosphatase; aging; cell cycle; cell death; cell line; cell senescence; enzyme activity; flow cytometry; fluorimetry; free radical oxygen; immunologic assay /test; iron metabolism; laboratory rabbit; mitochondria; nutrition of aging; nutrition related tag; oxidative stress; protein localization; transfection; yeasts

The long-term goals of this work is to understand the role of mitochondrial iron metabolism in regulating life span and cell death. Iron is an essential nutrient, however, in the presence of naturally produced reactive oxygen intermediates, iron augments free radical formation. Free radicals are suspected of causing cellular damage, for example, DNA fragmentation associated with aging, and high cellular iron concentration and sensitivity to the oxidizing potential of iron is associated with cell death observed in post- ischemic reperfusion injury and Friedreich's ataxia. In the cell, mitochondria are the major site of iron utilization and the major site of reactive oxygen intermediate formation implying that the ability of the mitochondria to manage iron may be extremely important to cell viability. Disruption of the gene for a previously undefined yeast traffic ATPase (mtATPase) that is suspected of regulating mitochondrial iron uptake and/or metabolism based on preliminary results, was shown to cause morphological defects similar to those observed during cell senescence. The overall aim of the proposed work is to confirm that this traffic ATPase is a mitochondrial iron uptake protein and determine if this traffick ATPase affects the overall cell cycle, as suspected based on preliminary data. Specifically the aims are: 1) to verify that the traffick ATPase is disrupted in a previously constructed mutant cell line and prove that a decrease in mtATPase expression causes the mutant phenotype by introducing an ATPase expression vector into mutant cells, 2) to prove that mtATPase is involved in mitochondrial iron metabolism by determining iron uptake and iron management activity of mtATPase disruption mutants and mtATPase over-expressing strains, and to verify that mtATPase is a mitochondrial protein by localizing the protein in situ using immunoassays, and 3) to prove that the mtATPase-1 cell line has an altered mitochondrial content and an altered cell cycle and life span by cytometry, fluorometry, and gene expression analysis. Determining the underlying mechanisms for mitochondrial iron handling is essential for future studies aimed at defining the role of iron aging, and for better understanding diseases associated with free radical damage and maladaptive iron metabolism. Since preliminary data indicates that a mammalian mtATPase homologue exists, a long term consequence of this work may be discovery of more effective treatment for iron related disorders and diseases.

这项工作的长期目标是了解线粒体铁代谢在调节寿命和细胞死亡中的作用。 铁是一种必需的营养素，然而，在天然产生的活性氧中间体存在的情况下，铁会增加自由基的形成。 自由基被怀疑会引起细胞损伤，例如，与衰老相关的DNA断裂，以及高细胞铁浓度和对铁氧化电位的敏感性与在缺血性再灌注损伤和弗里德赖希共济失调中观察到的细胞死亡相关。 在细胞中，线粒体是铁利用的主要部位和活性氧中间体形成的主要部位，这意味着线粒体管理铁的能力可能对细胞活力极其重要。 根据初步结果，先前未定义的酵母运输 ATP 酶 (mtATP 酶) 基因的破坏被怀疑可调节线粒体铁的吸收和/或代谢，结果表明会导致与细胞衰老期间观察到的形态缺陷相似的形态缺陷。 拟议工作的总体目标是确认这种运输 ATP 酶是一种线粒体铁摄取蛋白，并确定这种运输 ATP 酶是否影响整个细胞周期，正如根据初步数据所怀疑的那样。 具体目标是：1) 验证先前构建的突变细胞系中的运输 ATP 酶是否被破坏，并通过将 ATP 酶表达载体引入突变细胞来证明 mtATP 酶表达的减少会导致突变表型，2) 证明 mtATP 酶通过测定 mtATPase 破坏突变体和 mtATPase 过表达菌株的铁摄取和铁管理活性来参与线粒体铁代谢，并通过以下方式验证 mtATPase 是线粒体蛋白使用免疫测定法对蛋白质进行原位定位，3) 通过细胞计数、荧光测定和基因表达分析证明 mtATPase-1 细胞系的线粒体含量发生了改变，细胞周期和寿命也发生了改变。 确定线粒体铁处理的潜在机制对于未来旨在确定铁老化作用的研究以及更好地了解与自由基损伤和适应不良铁代谢相关的疾病至关重要。 由于初步数据表明存在哺乳动物 mtATP 酶同源物，因此这项工作的长期结果可能是发现针对铁相关病症和疾病的更有效治疗方法。