Mitochondrial aconitase: Fe-S cluster biogenesis and interaction with mtDNA

线粒体顺乌头酸酶：Fe-S簇的生物发生及其与线粒体DNA的相互作用

• 批准号: 7797998
• 负责人: DEBKUMAR PAIN
• 金额: $ 31.68万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7797998
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Aconitate Hydratase; Active Sites; Affect; Aging; Apoproteins; Back; Biogenesis; Cells; Citrates; Citric Acid Cycle; DNA Binding; DNA Maintenance; Data; Defect; Enzymes; Ferredoxin; Genes; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Human; Hydrolysis; In Vitro; Iron; Isocitrates; Life; Maintenance; Mediating; Membrane; Mitochondria; Mitochondrial DNA; Mitochondrial Matrix; Molecular; Molecular Chaperones; NADH; NADP; Nuclear; Nucleotides; Oligonucleotides; Oxidation-Reduction; Oxidative Stress; Oxidoreductase; Pathway interactions; Phosphotransferases; Physiology; Play; Process; Production; Protein Import; Proteins; Role; Saccharomyces cerevisiae; Scaffolding Protein; Site; Source; Specificity; Staging; Sulfur; Testing; Tricarboxylic Acids; Yeasts; cysteine desulfurase; in vivo; isocitrate; mutant; oxidative damage; persulfides; public health relevance; research study; yeast protein

DESCRIPTION (provided by applicant): Mitochondrial aconitase is highly sensitive to oxidative damage during aging. It reversibly converts citrate to isocitrate in the tricarboxylic acid (TCA) cycle. The cubane [4Fe-4S]2+ cluster of aconitase is essential for its catalytic activity, but it also renders the enzyme highly vulnerable to oxidative stress. We have discovered that isolated wild-type Saccharomyces cerevisiae mitochondria contain a nucleotide (GTP, NAD(P)H and ATP)- dependent machinery for iron-sulfur cluster (ISC) biogenesis of aconitase (Aco1p). The cluster biogenesis occurs in the mitochondrial matrix by a multi-step process requiring multiple components. In Aim I, we will biochemically dissect these steps and determine the nucleotide requirements of each. In Aims II-IV, we will study proteins that we found are directly relevant to nucleotide-dependent processes in ISC biogenesis of Aco1p. These include a GTPase (Mtg1p), a NADH kinase (Pos5p), a NADPH-requiring reductase (Arh1p), and an ATPase (Ssq1p). Mitochondria lacking any of these proteins are deficient in ISC biogenesis of Aco1p and display greatly reduced aconitase activity. However, the causal defects in these mutant mitochondria must be different since these proteins have different functions. Mtg1p is a GTPase in the mitochondrial matrix, and we will test our hypothesis that Mtg1p mediates the effects of matrix GTP on ISC biogenesis of Aco1p (Aim II). Pos5p is a NADH kinase and is required for NADPH production in the matrix. We will determine if the role of Pos5p in ISC biogenesis of Aco1p is mediated by its effects on NADPH levels in the matrix (Aim III). NADPH is likely utilized by reductase(s) such as ferredoxin reductase (Arh1p), which may provide reducing equivalents for one or more steps in cluster biogenesis, and this will be tested as part of Aim III. Ssq1p is an Hsp70 chaperone with ATPase activity in the matrix. Therefore some of the effects of ATP on ISC biogenesis of Aco1p are likely mediated by Ssq1p, and this will be explored (Aim IV). In addition to Ssq1p, yeast mitochondria contain an abundant Hsp70 (Ssc1p) that is involved in protein import. In contrast, human mitochondria contain a single Hsp70 (hSSC1), and thus it may participate in both protein import and ISC biogenesis, and we will test this possibility as part of Aim IV. Aco1p is essential for mitochondrial DNA (mtDNA) stability, and this activity is independent of its enzymatic activity in the TCA cycle. Aim V is to investigate interaction of yeast and human mitochondrial aconitases with mtDNA. We will determine if human mitochondrial aconitase, like yeast Aco1p, is also bifunctional and participates in the maintenance of mtDNA. Nucleotides and/or redox status of the matrix may play a critical role in distributing aconitase between the TCA cycle and mt-nucleoids, and we will examine these possibilities. The mechanism underlying the biogenesis of ISCs of mitochondrial aconitase in yeast is likely to be very similar to that in human. Homologs of most, if not all, yeast proteins that participate in the process also exist in human. Thus, conclusions from yeast studies proposed here will be informative to human physiology. Public Health Relevance: Mitochondrial aconitase requires a 4Fe-4S cluster for its enzymatic activity in the tricarboxylic acid (TCA) cycle and is highly sensitive to oxidative damage during aging. In yeast, aconitase is also essential for the maintenance of mitochondrial DNA, and this activity is independent of its catalytic activity in the TCA cycle. This proposal seeks to investigate the molecular mechanism of Fe-S cluster biogenesis of aconitase and its interaction with mitochondrial DNA.

描述（由申请人提供）：线粒体乌头酸酶对衰老过程中的氧化损伤高度敏感。它在三羧酸 (TCA) 循环中可逆地将柠檬酸转化为异柠檬酸。乌头酸酶的立方烷 [4Fe-4S]2+ 簇对其催化活性至关重要，但它也使该酶极易受到氧化应激的影响。我们发现分离的野生型酿酒酵母线粒体含有核苷酸（GTP、NAD(P)H 和 ATP）依赖性机制，用于乌头酸酶 (Aco1p) 的铁硫簇 (ISC) 生物发生。簇生物发生通过需要多种成分的多步骤过程在线粒体基质中发生。在目标 I 中，我们将从生化角度剖析这些步骤并确定每个步骤的核苷酸需求。在目标 II-IV 中，我们将研究与 Aco1p ISC 生物发生中的核苷酸依赖性过程直接相关的蛋白质。其中包括 GTP 酶 (Mtg1p)、NADH 激酶 (Pos5p)、NADPH 需要的还原酶 (Arh1p) 和 ATP 酶 (Ssq1p)。缺乏任何这些蛋白质的线粒体在 Aco1p 的 ISC 生物合成中存在缺陷，并且表现出乌头酸酶活性大大降低。然而，这些突变线粒体的因果缺陷一定是不同的，因为这些蛋白质具有不同的功能。 Mtg1p 是线粒体基质中的 GTP 酶，我们将检验我们的假设，即 Mtg1p 介导基质 GTP 对 Aco1p 的 ISC 生物合成的影响（目标 II）。 Pos5p 是一种 NADH 激酶，是基质中 NADPH 产生所必需的。我们将确定 Pos5p 在 Aco1p ISC 生物合成中的作用是否是通过其对基质中 NADPH 水平的影响来介导的（目标 III）。 NADPH 可能被铁氧还蛋白还原酶 (Arh1p) 等还原酶利用，它可以为簇生物发生中的一个或多个步骤提供还原当量，这将作为目标 III 的一部分进行测试。 Ssq1p 是一种 Hsp70 伴侣，在基质中具有 ATP 酶活性。因此，ATP 对 Aco1p ISC 生物发生的一些影响可能是由 Ssq1p 介导的，对此将进行探索（目标 IV）。除了 Ssq1p 之外，酵母线粒体还含有丰富的 Hsp70 (Ssc1p)，参与蛋白质输入。相比之下，人类线粒体含有一个 Hsp70 (hSSC1)，因此它可能参与蛋白质输入和 ISC 生物发生，我们将作为 Aim IV 的一部分测试这种可能性。 Aco1p 对于线粒体 DNA (mtDNA) 稳定性至关重要，并且该活性与其在 TCA 循环中的酶活性无关。目标 V 是研究酵母和人类线粒体乌头酸酶与 mtDNA 的相互作用。我们将确定人类线粒体乌头酸酶（如酵母 Aco1p）是否也具有双功能并参与线粒体 DNA 的维护。核苷酸和/或基质的氧化还原状态可能在 TCA 循环和 mt 核苷之间分布乌头酸酶中发挥关键作用，我们将研究这些可能性。酵母中线粒体乌头酸酶 ISC 生物发生的机制可能与人类中的非常相似。参与该过程的大多数（如果不是全部）酵母蛋白的同源物也存在于人类中。因此，这里提出的酵母研究的结论将为人类生理学提供信息。 公共健康相关性：线粒体乌头酸酶需要 4Fe-4S 簇才能发挥其在三羧酸 (TCA) 循环中的酶活性，并且对衰老过程中的氧化损伤高度敏感。在酵母中，乌头酸酶对于维持线粒体 DNA 也至关重要，并且这种活性与其在 TCA 循环中的催化活性无关。该提案旨在研究乌头酸酶 Fe-S 簇生物合成的分子机制及其与线粒体 DNA 的相互作用。