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是一种与线粒体刺激酶，一种柠檬酸循环酶有关的铁硫蛋白，它在铁蛋白的细胞中充当胞质刺激酶。在铁缺乏的细胞中，IRP1失去其铁硫辅因子，而载脂蛋白转换为RNA结合蛋白。 IRP1涉及从IRP1的形式的过渡，其中4FE-4S簇被结合到失去铁和aconitase活性的形式。含有蛋白质的4FE-4S不结合已知Asires的RNA干循环。铁硫簇的受控降解和诱变表明，铁缺失细胞中RNA结合蛋白的生理相关形式是载脂蛋白。群集的状态似乎确定IRP1是否会结合RNA。在过去的十年中，我们已经确定了与NIFS，ISCU和NIF U，非氧蛋白和铁氧蛋白还原酶还原酶基因相关的铁硫簇组件的哺乳动物酶促进IRP1铁硫簇的组装。我们已经发现，当野生型和成纤维细胞和弗里德里希共济失调患者的成纤维细胞和成纤维细胞和淋巴母细胞中，Frataxin转录是铁依赖性调节的，而Frataxin的表达降低。我们发现脚手架蛋白ISCU中的突变会引起罕见的肌病。在Friedreich共济失调和ISCU肌病中，我们的数据表明线粒体铁超负荷与胞质铁耗竭结合发生。在合作的情况下，我们发现NFU1和BOLA3突变中的突变引起的人类疾病，其特征是乳酸性酸中毒和脂酸缺乏。我们预测，其他由线粒体妥协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的突变会引起新生儿线粒体疾病。我们正在表征将新生铁硫簇从其与初始组装设备的关联转移到需要铁硫簇以进行功能的步骤。我们已经广泛研究了ISCU肌病中骨骼肌代谢的代谢重塑，并发现了几种补偿性途径，有助于维持能量稳态。我们还发现了多种原因，这些原因将ISU肌病的表型限制在骨骼肌肉上，同时很大程度上保留了其他组织。我们正在为ISCU肌病开发反义治疗疗法，最近我们证明，FGF21是ISCU肌病中肌肉疾病的良好生物标志物。我们还在积极地努力发现SDHB如何获得其三个Fe-S簇，并且我们已经证明HSC20辅助酮介导的铁硫簇的递送对于对铁硫的呼吸链I-III至关重要。我们正在评估更多的铁硫簇的候选者，我们预计我们的研究将大大增加已知的哺乳动物铁硫蛋白的数量。我们确定Fe-S生物发生发生在细胞质中，并且伴侣HSC20通过与CIAO1中的Lyl基序结合，将初始的细胞质生物发生与CIAO1依赖性Fe-S递送平台联系起来。因此，Fe-S生物发生在线粒体基质和哺乳动物细胞的胞质溶胶中平行发生。我们的工作挑战了初始Fe-S生物发生仅发生在线粒体基质中的范式，而ABCB7导出了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辅助系统促进Fe-S簇向受体转移的分子相互作用，在某些情况下，通过使用二级支架蛋白来赋予Fe-S子群的特异性。使用寻求Lyr序列迭代的信息学，其次是过表达和ICP-MS，我们正在识别先前未识别的Fe-S蛋白，我们认为这是常见的，并且在哺乳动物细胞的多个关键代谢途径中表示。我们发现SARS-COV-2可以使用哺乳动物的铁硫生物发生机制为病毒NSP-12复制酶提供铁硫辅因子。复制酶结合了两个古巴铁硫辅助因子，并且需要复制酶功能并结合相关的解旋酶NSP13。Helicase，NSP13，它与完整功能所需的铁硫簇相连。铁硫辅因子容易受到氧化的影响。稳定的氮氧化物Tempol是一种氧化剂，可在底漆扩展测定中在体外降解复制酶的铁硫辅因子。当组织培养细胞被SARS-COV-2感染时，tempol的添加通过禁用复制酶作为抗病毒药物。当给感染SARS-COV-2感染的仓鼠时，tempol会减弱感染的致病作用。我们正在努力开发tempol作为一种口服抗病毒，以抗病感染。我们正在提出这样的假设，即许多病毒利用铁硫辅助因子来起作用。铁硫辅助因子促进了病毒可用于支持其能量需求的细胞还原等效物。其他几种SARS-COV-2蛋白是候选铁硫蛋白。我们正在建立生长和分析OC43的条件，OC43是与SARS-COV-2有关的常见感冒病毒原因。 OC43具有进入宿主细胞的不同机制，但是复制酶，解旋酶，远高核酸酶和其他非结构性蛋白质具有高度保守，使其成为冠状病毒疾病研究的有用模型。我们旨在定义铁硫辅因子在冠状病毒感染中的作用，并通过协作分析复制和翻译的结构和机制。我们发现铁硫辅助因子的能力是由于我们开发了一种用于预测候选蛋白，在人细胞系中过表达的系统，以及在厌氧条件下纯化，从而保护铁硫辅助因子免受氧化的脱隔化。哺乳动物细胞中的许多途径可能取决于蛋白质的功能，而蛋白质的功能尚未依赖于铁硫辅助因子。通过发表我们的研究，我们旨在在哺乳动物细胞中多种关键代谢途径中发现发现铁硫蛋白的发现。

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.

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.