Metal Homeostatic Mechanisms in Yeast

酵母中的金属稳态机制

• 批准号: 7826959
• 负责人: Dennis R. Winge
• 金额: $ 26.07万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-24 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7826959
• 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)：本提案将酿酒酵母中新的线粒体-细胞质信号通路作为真核生物的模型。在第一个线粒体-细胞质途径中，我们发现了不同于金属配位蛋白铜穿梭的铜运输的新范式。这一途径涉及线粒体内用于细胞色素c氧化酶(CcO)和超氧化物歧化酶-1(SOD1)金属化的铜-配体(CuL)络合物的候选易位。除了酵母，CuL复合体也是人类成纤维细胞、小鼠肝脏和拟南芥中线粒体铜的重要来源。我们推测，在铜(I)与细胞质中丰富的自由配体结合后，CuL络合物通过一种尚未确定的线粒体转运蛋白进入线粒体。我们的目标是阐明CuL复合体的结构，验证其移位到线粒体基质中，并将转运蛋白确定为三个主要目标。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，并结合细胞和体外线粒体导入研究来表征易位途径。我们将尝试通过基因筛查和对呼吸缺陷突变体库的评估来鉴定转运蛋白。第二条线粒体-细胞质途径与Aft1转录激活子对铁的感知有关。在铁充足的细胞中，Aft1的转录激活受到线粒体出口蛋白Atm1发出的信号的抑制。Aft1功能与线粒体铁状态有关，因为许多表现出线粒体铁积累的酵母突变株表现出结构性的活性Aft1。我们的目标是确定在这些突变体中阻断Aft1铁抑制的机制。Aft1对铁的感知依赖于两个谷氧还蛋白和两个新发现的蛋白质Fra1和Fra2。我们的目的是阐明这些蛋白如何介导铁抑制Aft1。人类细胞具有Atm1和Grx3/4的同源基因，与人类的Atm1和ABCB7同源，在铁稳态中具有明显的作用。我们最近的研究表明，存在一个可膨胀的基质铁池。这个铁库在铁-硫簇生物发生受损的酵母突变株中扩大，可能导致超氧化物歧化酶-2(Sod2)的错误金属化和铁响应转录激活因子Aft1的结构性激活。这笔赠款的统一主题是在基质中存在生物可利用的铜和铁池，以及线粒体-细胞质串扰，作为包括人类在内的真核生物中铜和铁稳态的关键成分。线粒体和细胞核之间的串扰在许多代谢性疾病和紊乱中具有广泛的意义。与公共健康相关：线粒体内铜配体复合体对细胞色素氧化酶和超氧化物歧化酶-1的铜金属化与人体生理学有关。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突变与家族性肌萎缩侧索硬化症的病因有关。已知人类ABCB7线粒体铁输出器功能障碍会导致X连锁铁粒母细胞贫血并共济失调。