Dissection of the Molecular Details of Fe Deficiency Acclimation

缺铁驯化的分子细节剖析

• 批准号: 8433584
• 负责人: Crysten Elizabeth Blaby
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8433584
• 关键词: Acclimatization; Acetates; Aerobic; Bioenergetics; Biological Assay; Carbon; Carbon Dioxide; Carboxypeptidase; Cell physiology; Cells; Chlamydomonas; Chlamydomonas reinhardtii; Chloroplasts; DNA Binding; DNA biosynthesis; Detection; Dissection; Ensure; Enzymes; Eukaryota; Event; Ferritin; Future; Gene Expression Profile; Gene Proteins; Genes; Genetic Screening; Genome; Goals; Green Algae; Growth; Health; Homeostasis; Human; Individual; Infection; Iron; Knowledge; Laboratories; Lead; Libraries; Life; Location; Mammals; Membrane; Messenger RNA; Metabolic; Metabolism; Methodology; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Molecular Biology; Nutrient; Organelles; Organism; Outcome; Pathway interactions; Peptide Hydrolases; Phenotype; Photosynthesis; Population; Predisposition; Process; Protein Kinase; Proteins; Protocols documentation; Reaction; Recycling; Regulation; Respiration; Role; Signal Pathway; Signal Transduction; Source; Stress; Techniques; Tertiary Protein Structure; Testing; Time; Training; Ubiquitination; Vacuole; Work; Yeasts; base; cofactor; cytotoxic; design; extracellular; human disease; interest; iron deficiency; literature survey; member; multicatalytic endopeptidase complex; mutant; novel; nutrition; oxidation; positional cloning; programs; protein degradation; response; screening; trafficking; transcription factor; transcriptome sequencing

DESCRIPTION (provided by applicant): Iron (Fe) is an abundant protein cofactor required for the activity of a myriad of proteins and consequently is essential for numerous cellular functions ranging from DNA synthesis to respiration. Therefore, the cell must ensure that a sufficient supply of Fe is available to these Fe-dependent proteins but, at the same time, excess Fe in the cell, which can lead to cytotoxic reactions, must be avoided. Although our understanding of Fe homeostasis has benefited from over half a century of study in numerous organisms, the molecular details concerning intracellular Fe trafficking are lacking. The Merchant group has developed Chlamydomonas reinhardtii as a reference organism for studying Fe metabolism in the context of poor Fe nutrition. As it is estimated that roughly one-third of the world's population suffers from symptomatic Fe deficiency and multiple human diseases are caused by mis-regulation of Fe homeostasis, understanding these mechanisms is crucial. One under-characterized response is the recycling of Fe from dispensable proteins when extracellular Fe is unavailable. How this process is regulated at the molecular level is not known. The goal of this project is to identify the trafficking pathways of Fe within the cell during Fe-limitation and discover proteins responsible for accomplishing and regulating Fe recycling. The specific aims of this project are three-fold. First, cells will be biochemically fractionated and distribution of Fe between Fe-utilizing and Fe-storage compartments in Fe-replete vs. -deficient conditions will be determined, especially in the context of carbon source utilization (respiration vs. photosynthesis). Second, reverse genetics will be employed to characterize the involvement of Fe-regulated genes predicted to be involved in Fe recycling. Characterization will be aided by determining the subcellular location of these proteins and how these genes are regulated by Fe. Third, a classical genetic screen will be implemented to discover novel components of Fe homeostasis that may not be regulated at the gene or mRNA level and, therefore, have escaped the notice of transcriptome studies. These aims were chosen to set a precedent for understanding the mechanism of regulated Fe trafficking in the cell, characterize genes known to be induced by Fe-limitation and discover novel genes involved in acclimating to Fe-limitation. In addition, this project has been designed to provide training with a wide-range of techniques and to gain expertise in working with a reference eukaryotic organism.

描述（由申请人提供）：铁（Fe）是多种蛋白质活性所需的丰富蛋白质辅因子，因此对于从DNA合成到呼吸的许多细胞功能至关重要。因此，细胞必须确保这些依赖性蛋白质有足够的Fe供应，但必须避免避免细胞中的过量Fe（可能导致细胞毒性反应）。尽管我们对FE稳态的理解受益于半个多世纪的许多生物研究，但缺乏有关细胞内Fe贩运的分子细节。商人群体已经开发了雷目氏菌作为参考生物体，用于在不良的Fe营养中研究FE代谢。据估计，大约三分之一的世界人口患有症状的FE缺乏和多种人类疾病是由于FE稳态的错误调节引起的，因此了解这些机制至关重要。一个特征较低的反应是当不可用的细胞外Fe时，可抵达蛋白的Fe回收。该过程在分子水平上的调节方式尚不清楚。该项目的目的是确定FE限制期间FE在细胞内的贩运途径，并发现负责完成和调节FE回收的蛋白质。该项目的具体目的是三倍。首先，将在Fe-Repleplete和Fe-Stemage室之间进行生物化学分馏，而Fe储存室之间的Fe分布将确定，而缺乏 - 缺陷条件将被确定，尤其是在碳源利用率的情况下（呼吸与光合作用）。其次，将采用反向遗传学来表征预测参与Fe回收的Fe调节基因的参与。通过确定这些蛋白质的亚细胞位置以及如何通过Fe调节这些基因，将有助于表征。第三，将实施一个经典的遗传筛查，以发现可能在基因或mRNA水平不受调节的Fe稳态组成部分，因此已经逃脱了转录组研究的通知。选择这些目标是为了理解细胞中受调节的运输机制的先例，其特征是通过Fe限制诱导的基因，并发现涉及适应Fe限制的新基因。此外，该项目旨在为培训提供广泛的技术，并在使用参考真核生物方面获得专业知识。