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）是一种丰富的蛋白质辅助因子，是无数蛋白质活性所必需的，因此是从DNA合成到呼吸等许多细胞功能所必需的。因此，细胞必须确保这些依赖铁的蛋白质有足够的铁供应，但与此同时，必须避免细胞中过量的铁，这可能导致细胞毒性反应。尽管我们对铁稳态的理解受益于半个多世纪以来对许多生物的研究，但关于细胞内铁运输的分子细节还缺乏。Merchant小组开发了莱茵衣藻，作为研究铁营养不良情况下铁代谢的参考生物。据估计，世界上大约三分之一的人口患有症状性缺铁，多种人类疾病是由铁稳态失调引起的，因此了解这些机制至关重要。一种未被充分描述的反应是，当细胞外铁不可用时，从必要的蛋白质中回收铁。这个过程是如何在分子水平上调控的尚不清楚。本项目的目标是确定铁在细胞内的运输途径，并发现负责完成和调节铁循环的蛋白质。该项目的具体目标有三个方面。首先，细胞将被生化分解，并确定在富铁与缺铁条件下，铁在利用和储存间的分布，特别是在碳源利用的背景下（呼吸作用与光合作用）。其次，反向遗传学将用于表征铁调控基因参与铁循环的预测。表征将通过确定这些蛋白质的亚细胞位置以及这些基因如何被铁调节来辅助。第三，将实施经典的遗传筛选来发现铁稳态的新成分，这些成分可能不受基因或mRNA水平的调节，因此逃过了转录组研究的注意。选择这些目标是为了了解细胞中受调节的铁运输机制，表征已知的受铁限制诱导的基因，并发现参与适应铁限制的新基因，从而开创先例。此外，这个项目的目的是提供广泛的技术培训，并获得与参考真核生物一起工作的专门知识。