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Mammalian iron-sulfur cluster biogenesis

哺乳动物铁硫簇生物发生

基本信息

批准号：
10915317
负责人：
TRACEY A. ROUAULT
金额：
$ 159.43万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10915317
关键词：
2019-nCoV Aconitate Hydratase Apoproteins Attenuated Binding Binding Sites Biogenesis Biological Assay Biological Availability Biological Markers Carrier Proteins Cell Nucleus Cells Citric Acid Cycle Collaborations Common Cold Complex Cytosol Data Development Disease Electron Transport Complex III Enzymes Exoribonucleases FGF21 gene Ferredoxin Fibroblasts Friedreich Ataxia Genes Genetic Transcription Goals Hamsters Homeostasis Human Cell Line In Vitro Inductively Coupled Plasma Mass Spectrometry Infection Informatics Iron Iron Overload Iron-Sulfur Proteins Lactic Acidosis Ligation Mammalian Cell Mass Spectrum Analysis Mediating Metabolic Pathway Mitochondria Mitochondrial Diseases Mitochondrial Matrix Modeling Molecular Chaperones Mutagenesis Mutation Myopathy Neonatal Nonstructural Protein Nuclear Oral Oxidants Oxidoreductase Pathogenesis Pathogenicity Pathway interactions Patients Peptides Phenotype Physiological Plaque Assay Porphobilinogen Synthase Primer Extension Process Prosthesis Protein Isoforms Proteins Publishing RNA RNA Binding RNA Viruses RNA-Binding Proteins Regulation Research Respiratory Chain Respiratory physiology Role SARS-CoV-2 infection Scaffolding Protein Scanning Sideroblastic Anemia Skeletal Muscle Specificity Structure Study models Subgroup Succinate Dehydrogenase Sulfur Sulofenur System Thioctic Acid Tissues Translations Viral Viral Physiology Virus Work Zinc candidate identification cofactor coronavirus disease cysteine desulfurase fibroblast growth factor 21 frataxin gene product helicase heme biosynthesis human disease iron deficiency lipoic acid synthase lymphoblast metabolic abnormality assessment overexpression oxidation protein function rare genetic disorder replicase respiratory scaffold skeletal muscle metabolism stem tempol tissue/cell culture

项目摘要

IRP1 is an iron-sulfur protein related to mitochondrial aconitase, a citric acid cycle enzyme, and it functions as a cytosolic aconitase in cells that are iron replete. In iron-depleted cells, IRP1 loses its iron sulfur cofactor, and the apoprotein switches to become an RNA binding protein. IRP1 involves a transition from a form of IRP1 in which a 4Fe-4S cluster is bound, to a form that loses both iron and aconitase activity. The 4Fe-4S containing protein does not bind RNA stem-loops known asIREs. Controlled degradation of the iron-sulfur cluster and mutagenesis reveals that the physiologically relevant form of the RNA binding protein in iron-depleted cells is apoprotein. The status of the cluster appears to determine whether IRP1 will bind RNA. Over the past decade, we have identified mammalian enzymes of iron-sulfur cluster assembly that are homologous to the NifS, ISCU and Nif U, ferredoxin and ferredoxin reductase genes implicated in bacterial iron-sulfur cluster assembly, and we have shown that these gene products facilitate assembly of the iron- sulfur cluster of IRP1. We have discovered that frataxin transcription is iron-dependently regulated and frataxin expression decreases when there is cytosolic iron deficiency in wild-type and in fibroblasts and lymphoblasts from Friedreich ataxia patients. We discovered that a mutation in the scaffold protein, ISCU, causes a rare myopathy. In both Friedreich ataxia and ISCU myopathy, our data indicate that mitochondrial iron overload occurs in conjunction with cytosolic iron depletion. In collaboration, we discovered that mutations in NFU1 and BOLA3 mutations cause a human disease characterized by lactic acidosis and lipoic acid deficiency. We predicted that other rare genetic diseases characterized by mitochondrial compromise were caused by mutations in the genes responsible for iron-sulfur cluster biogenesis, and we collaborated to discover that mutations of NFS1 cause neonatal mitochondrial disease. We are characterizing the steps that chaperone transfer of nascent iron-sulfur clusters from their association with the initial assembly apparatus to proteins that require iron-sulfur clusters for function. We have extensively studied the metabolic remodeling of skeletal muscle metabolism in ISCU myopathy and discovered several compensatory pathways that help to maintain energy homeostasis. We have also discovered multiple reasons that limit the phenotype of ISU myopathy to skeletal muscles, while largely sparing other tissues. We are developing antisense treatment therapy for ISCU myopathy, and we recently demonstrated that FGF21 is a good biomarker for muscle disease in ISCU myopathy. We are also actively working to discover how SDHB acquires its three Fe-S clusters, and we have demonstrated that HSC20 cochaperone mediated iron sulfur cluster delivery is critical for iron sulfur acquisition of respiratory chains I-III. We are evaluating many more candidate recipients of iron sulfur clusters, and we expect our studies will greatly increase the number of known mammalian iron sulfur proteins. We established that Fe-S biogenesis occurs de novo in the cytosol, and that the chaperone HSC20 connects initial cytosolic biogenesis with the CIAO1-dependent Fe-S delivery platform in the cytosol by binding to a LYR motif in CIAO1. Thus Fe-S biogenesis occurs in parallel both in the mitochondrial matrix and in the cytosol of mammalian cells. Our work challenges the paradigm that initial Fe-S biogenesis occurs only in the mitochondrial matrix, and ABCB7 exports a component critical to Fe-S synthesis in mammalian cytosol. We are working to clarify the molecular interactions that promote transfer of Fe-S clusters to recipients using the HSC20 cochaperone system, followed in some cases by use of secondary scaffold proteins that confer specificity to subgroups of Fe-S recipients. Using informatics looking for iterations of the LYR motif, followed by overexpression and ICP-MS, we are in the process of identifying previously unrecognized Fe-S proteins, which we believe are common and represented in multiple key metabolic pathways of mammalian cells. We discovered that SARS-CoV-2 coopts the mammalian iron sulfur biogenesis machinery to supply iron sulfur cofactors for the viral nsp-12 replicase. The replicase incorporates two cubane iron sulfur cofactors, and they are needed for replicase function and for binding the associated helicase, nsp 13. The helicase, nsp13, ligates an iron sulfur cluster that is required for full function. Iron sulfur cofactors are vulnerable to oxidation. The stable nitroxide, Tempol, is an oxidant that degrades the iron sulfur cofactor of the replicase in vitro in primer extension assays. When tissue culture cells are infected with SARS-CoV-2, the addition of Tempol works as an antiviral by disabling the replicase. When Tempol was given to hamsters infected with SARS-CoV-2, Tempol attenuated the pathogenic effects of infection. We are working to develop Tempol as an oral antiviral for use against Covid infection. We are pursuing the hypothesis that many viruses utilize iron sulfur cofactors for function. Iron sulfur cofactors facilitate use of cellular reducing equivalents that the virus may utilize to support its energy requirements. Several other SARs-CoV-2 proteins are candidate iron sulfur proteins. We are establishing conditions to grow and analyze OC43, a viral cause of the common cold that is related to SARs-CoV-2. OC43 has a different mechanism for entering host cells, but the replicase, helicase, exoribonuclease and other non-structural proteins are highly conserved, making it a useful model for studies of coronaviral diseases. We aim to define the role of iron sulfur cofactors in coronaviral infections and to analyze structures and mechanisms of replication and translation through collaborations. Our ability to discover iron sulfur cofactors is due to our development of a system for predicting candidate proteins, over-expressing in human cell lines, and purification under anaerobic conditions that protect the iron sulfur cofactor from disassembly by oxidation. Numerous pathways in mammalian cells likely depend on function of proteins that are not yet recognized to depend on iron sulfur cofactors. Through publishing our research, we aim to galvanize discovery of iron sulfur proteins in multiple critical metabolic pathways in mammalian cells.

IRP1是一种与线粒体乌头酶（一种柠檬酸循环酶）相关的铁硫蛋白，它在富含铁的细胞中作为胞质乌头酶发挥作用。在缺铁细胞中，IRP1失去其铁硫辅助因子，载脂蛋白转换为RNA结合蛋白。IRP1涉及从一种结合4Fe-4S簇的IRP1转变为一种既失去铁又失去乌头酶活性的IRP1。含有4Fe-4S的蛋白不结合被称为asres的RNA茎环。铁硫簇的受控降解和诱变表明，缺铁细胞中RNA结合蛋白的生理相关形式是载脂蛋白。簇的状态似乎决定了IRP1是否会结合RNA。在过去的十年中，我们已经确定了铁硫簇组装的哺乳动物酶，这些酶与细菌铁硫簇组装相关的NifS， ISCU和Nif U，铁氧还蛋白和铁氧还蛋白还原酶基因同源，我们已经证明这些基因产物促进IRP1的铁硫簇组装。我们发现frataxin的转录是铁依赖性调节的，在野生型和弗里德赖希共济失调患者的成纤维细胞和淋巴细胞中，当细胞内缺铁时，frataxin的表达减少。我们发现支架蛋白ISCU的突变导致了一种罕见的肌病。在弗里德里希共济失调和ISCU肌病中，我们的数据表明，线粒体铁过载与细胞质铁耗尽同时发生。在合作中，我们发现NFU1和BOLA3突变导致一种以乳酸酸中毒和硫辛酸缺乏症为特征的人类疾病。我们预测其他以线粒体受损为特征的罕见遗传疾病是由负责铁硫簇生物发生的基因突变引起的，我们合作发现NFS1突变导致新生儿线粒体疾病。我们描述了从与初始组装装置的关联到需要铁硫团簇功能的蛋白质的伴侣转移的步骤。我们对ISCU肌病骨骼肌代谢的代谢重塑进行了广泛的研究，发现了几种有助于维持能量稳态的代偿途径。我们还发现了多种原因，将ISU肌病的表型限制在骨骼肌，而在很大程度上保留了其他组织。我们正在开发ISCU肌病的反义治疗方法，我们最近证明FGF21是ISCU肌病肌肉疾病的良好生物标志物。我们也在积极探索SDHB如何获得其三个Fe-S簇，并且我们已经证明HSC20共伴侣蛋白介导的铁硫簇递送对于呼吸链I-III的铁硫获取至关重要。我们正在评估更多铁硫簇的候选受体，我们期望我们的研究将大大增加已知哺乳动物铁硫蛋白的数量。我们发现Fe-S的生物发生在细胞质中是从头开始的，并且伴侣HSC20通过结合CIAO1中的LYR基序，将细胞质中初始的Fe-S生物发生与依赖CIAO1的细胞质中Fe-S递送平台连接起来。因此，Fe-S在哺乳动物细胞的线粒体基质和细胞质中同时发生。我们的工作挑战了最初的Fe-S生物生成仅发生在线粒体基质中的范式，ABCB7输出对哺乳动物细胞质中Fe-S合成至关重要的成分。我们正在努力阐明使用HSC20合作伙伴系统促进Fe-S簇向受体转移的分子相互作用，随后在某些情况下使用二级支架蛋白赋予Fe-S受体亚群特异性。利用信息学寻找LYR基序的迭代，然后是过表达和ICP-MS，我们正在鉴定以前未被识别的Fe-S蛋白，我们认为这些蛋白在哺乳动物细胞的多个关键代谢途径中很常见。

项目成果

期刊论文数量（20）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Insertion mutants in Drosophila melanogaster Hsc20 halt larval growth and lead to reduced iron-sulfur cluster enzyme activities and impaired iron homeostasis.

DOI：
10.1007/s00775-013-0988-2
发表时间：
2013-04
期刊：
JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY
影响因子：
3
作者：
Uhrigshardt, Helge;Rouault, Tracey A.;Missirlis, Fanis
通讯作者：
Missirlis, Fanis

Cochaperone binding to LYR motifs confers specificity of iron sulfur cluster delivery.

DOI：
10.1016/j.cmet.2014.01.015
发表时间：
2014-03-04
期刊：
Cell metabolism
影响因子：
29
作者：
Maio N;Singh A;Uhrigshardt H;Saxena N;Tong WH;Rouault TA
通讯作者：
Rouault TA

Novel frataxin isoforms may contribute to the pathological mechanism of Friedreich ataxia.

新型的Frataxin同工型可能有助于弗里德里希共济失调的病理机理。

DOI：
10.1371/journal.pone.0047847
发表时间：
2012
期刊：
PloS one
影响因子：
3.7
作者：
Xia H;Cao Y;Dai X;Marelja Z;Zhou D;Mo R;Al-Mahdawi S;Pook MA;Leimkühler S;Rouault TA;Li K
通讯作者：
Li K

Mammalian iron sulfur cluster biogenesis and human diseases.