Metal Homeostatic Mechanisms in Yeast

酵母中的金属稳态机制

• 批准号: 7935576
• 负责人: Dennis R. Winge
• 金额: $ 14.04万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7935576
• 关键词: ATP phosphohydrolase; Abbreviations; Address; Affect; Aminopeptidase P; Arabidopsis; Ataxia; Binding; Bioavailable; Biogenesis; Cardiomyopathies; Cell Nucleus; Cell physiology; Cells; Chelating Agents; Complex; Copper; Coupled; Cuprozinc Superoxide Dismutase; Cytoplasm; Disease; Enzymes; Etiology; Eukaryota; Evaluation; Exhibits; Familial Amyotrophic Lateral Sclerosis; Family; Fibroblasts; Functional disorder; Genes; Genetic Screening; Golgi Apparatus; Grant; High Pressure Liquid Chromatography; Homeostasis; Homologous Gene; Human; Immunoprecipitation; In Vitro; Inner mitochondrial membrane; Ions; Iron; Lead; Letters; Ligands; Link; Liver; Manganese Superoxide Dismutase; Mediating; Membrane; Metabolic Diseases; Metals; Mitochondria; Mitochondrial Matrix; Modeling; Mus; Neonatal; Nomenclature; Nuclear; Orthologous Gene; Outer Mitochondrial Membrane; Pathway interactions; Phase; Physiology; Printing; Process; Proteins; Reaction; Reading; Respiratory distress; Role; Saccharomyces cerevisiae; Sideroblastic Anemia; Signal Pathway; Signal Transduction; Source; Specificity; Structure; Sulfur; Transcription Coactivator; Transcriptional Activation; Vesicle; Yeasts; bathocuproine sulfonate; bathophenanthroline; cofactor; cytochrome c oxidase; frataxin; glutaredoxin; human SOD2 protein; mutant; novel; public health relevance; respiratory; structural biology; superoxide dismutase 1; trafficking; transcription factor

DESCRIPTION (provided by applicant): This proposal addresses novel mitochondrial-cytoplasm signaling pathways in Saccharomyces cerevisiae as a model eukaryote. In the first mitochondrial-cytoplasm pathway, we discovered a new paradigm in copper trafficking distinct from metallochaperone copper shuttling. This pathway involves the candidate translocation of a copper-ligand (CuL) complex used in metallation of cytochrome c oxidase (CcO) and superoxide dismutase-1 (Sod1) within the mitochondria. In addition to yeast, the CuL complex is a significant source of mitochondrial copper in human fibroblasts, mouse liver and Arabidopsis. We postulate that translocation of the CuL complex into the mitochondria occurs through a yet unidentified mitochondrial transporter after Cu(I) is bound to an abundant free ligand in the cytoplasm. We aim to elucidate the structure of the CuL complex, verify its translocation into the mitochondrial matrix and identify the transporters as three major objectives. Studies in these objectives will involve analytical and structural biology approaches to deduce the ligand structure and a combination of cellular and in vitro mitochondrial import studies to characterize the translocation pathway. We will attempt to identify the transporters through a genetic screen and the evaluation of a bank of respiratory deficient mutants. A second mitochondrial-cytoplasm pathway relates to iron sensing by the Aft1 transcriptional activator. Transcriptional activation by Aft1 is inhibited in iron-replete cells by a signal emanating from the mitochondrial exporter Atm1. Aft1 function is coupled to the mitochondrial iron status, as a number of yeast mutants exhibiting mitochondrial iron accumulation show constitutively active Aft1. We aim to determine the mechanism by which iron-inhibition of Aft1 is blocked in these mutants. Iron sensing by Aft1 is dependent on two glutaredoxins and two newly identified proteins Fra1 and Fra2. We aim to elucidate how these proteins mediate Fe-inhibition of Aft1. Human cells have homologs to Atm1 and Grx3/4 with the human ortholog of Atm1, ABCB7, having a clear role in iron homeostasis. Our recent studies suggest that an expandable matrix iron pool exists. This iron pool is expanded in yeast mutants impaired in iron-sulfur cluster biogenesis and can lead to mis-metallation of superoxide dismutase-2 (Sod2) and constitutive activation of the iron-responsive transcriptional activator Aft1. The unifying themes in this grant are the presence of bioavailable pools of copper and iron within the matrix and mitochondrial-cytoplasm cross talk as a key component in copper and iron homeostasis in eukaryotes including humans. Cross talk between mitochondria and the nucleus has wide implications in a number of metabolic diseases and disorders. PUBLIC HEALTH RELEVANCE: Copper metallation of cytochrome oxidase and superoxide dismutase-1 by the copper- ligand complex within the mitochondria is relevant in human physiology. Deficiencies in cytochrome oxidase assembly lead to respiratory distress in humans presenting as neonatal cardiomyopathies. Mutants of the mitochondrial superoxide dismutase-1 are implicated in the etiology of familial amyotrophic lateral sclerosis. Dysfunction in the human ABCB7 mitochondrial iron exporter is known to cause X-linked sideroblastic anemia with ataxia.

描述（由申请人提供）：该提案涉及酿酒酵母作为真核生物模型中的新型胞浆-细胞质信号传导途径。在第一个细胞质-细胞质途径中，我们发现了一个不同于金属伴侣铜穿梭的新的铜转运模式。该途径涉及用于线粒体内细胞色素c氧化酶（CcO）和超氧化物歧化酶-1（Sod 1）代谢的铜配体（CuL）复合物的候选易位。除了酵母，CuL复合物是人成纤维细胞、小鼠肝脏和拟南芥中线粒体铜的重要来源。我们推测，易位的CuL复合物进入线粒体发生通过一个尚未确定的线粒体转运后，Cu（I）结合到细胞质中的丰富的游离配体。我们的目的是阐明的CuL复合物的结构，验证其易位到线粒体基质，并确定为三个主要目标的转运。这些目标的研究将涉及分析和结构生物学方法来推断配体结构，以及细胞和体外线粒体输入研究的组合来表征易位途径。我们将尝试通过遗传筛选和评估一个呼吸缺陷突变体库来确定转运蛋白。第二个线粒体-细胞质途径与Aft 1转录激活因子的铁传感有关。在铁充足的细胞中，通过线粒体出口商Atm 1发出的信号抑制Aft 1的转录激活。Aft 1功能与线粒体铁状态相关联，因为许多表现出线粒体铁积累的酵母突变体显示出组成型活性Aft 1。我们的目的是确定铁抑制Aft 1在这些突变体中被阻断的机制。Aft 1的铁传感依赖于两种谷氧还蛋白和两种新鉴定的蛋白Fra 1和Fra 2。我们的目的是阐明这些蛋白质如何介导铁抑制Aft 1。人类细胞具有与Atm 1和Grx 3/4的同源物，其中Atm 1的人类直系同源物ABCB 7在铁稳态中具有明确的作用。我们最近的研究表明，存在一个可扩展的基质铁池。这种铁池在铁硫簇生物发生受损的酵母突变体中扩大，并可能导致超氧化物歧化酶-2（Sod 2）的误代谢和铁响应转录激活因子Aft 1的组成性激活。在这个补助金的统一主题是存在的生物可利用池的铜和铁的矩阵和细胞质的细胞质串扰作为一个关键组成部分，在铜和铁稳态的真核生物，包括人类。线粒体和细胞核之间的相互作用在许多代谢疾病和紊乱中具有广泛的意义。公共卫生关系：通过线粒体内的铜-配体复合物对细胞色素氧化酶和超氧化物歧化酶-1的铜代谢与人体生理学相关。细胞色素氧化酶组装缺陷导致人类呼吸窘迫，表现为新生儿心肌病。线粒体超氧化物歧化酶-1的突变体与家族性肌萎缩侧索硬化症的病因有关已知人类ABCB 7线粒体铁输出蛋白功能障碍会导致X连锁铁粒幼细胞性贫血伴共济失调。