Nutritional Copper Signaling and Homeostasis

营养铜信号传导和体内平衡

基本信息

批准号：
8842133
负责人：
SABEEHA MERCHANT
金额：
$ 38.98万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-06-01 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8842133
关键词：
ATP phosphohydrolase Acclimatization Accounting Aerobic Alleles Animal Model Bacteria Binding Sites Bioavailable Biochemical Biochemical Genetics Cell physiology Cells Chemistry Chlamydomonas Copper DNA Binding Domain Defect Disease model Down-Regulation Elements Ensure Enzymes Family Flavins Fluorescent Dyes Fractionation Gene Expression Profile Gene Targeting Genes Genetic Screening Genome Goals Hepatolenticular Degeneration Homeostasis Host Defense Ions Knock-out Life Location Malnutrition Mammalian Cell Membrane Messenger RNA Metabolic Metabolic Pathway Metabolism Metals Methods Microscopy Minerals Modeling Molecular Chaperones Monoamine Oxidase Movement Mutation Nutrient Nutritional Organism Oxidases Oxidation-Reduction Pathway interactions Pattern Peptide Hydrolases Phenotype Plastids Plastocyanin Prosthesis Proteins Reaction Recycling Regulation Regulon Resolution Response Elements Signal Transduction Site Structure System Tertiary Protein Structure Testing Thylakoid Membranes Thylakoids Trace Elements Up-Regulation Work Zinc Zinc deficiency amine oxidase copper-binding protein design fungus gain of function genetic analysis genetic approach high throughput screening hypocupremia knock-down loss of function microbial mouse model mutant novel nutrition operation promoter research study reverse genetics success transcription factor transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): Cu is an essential nutrient for nearly all forms of life because it serves as an enzyme prosthetic group for catalyzing redox reactions or reactions involving O2 chemistry. These reactions are central to aerobic life. Our long term goal is to understand the metabolism of copper, especially when it is a limiting nutrient or in a situation of metabolic defect. Two decades ago, we discovered the paradigm of Cu sparing in the model organism, Chlamydomonas, where a Cu-independent protein can, in a situation of Cu-deficiency, replace an otherwise abundant Cu protein. The wide-spread occurrence of this mechanism in many microbial systems for various trace mineral nutrients (Fe, Zn, Mo, to list a few) is now well-established. Metal sparing mechanisms are likely to be important for the success of certain infectious bacteria and fungi in evading host defense systems, where metal sequestration is used as a defensive strategy. A new paradigm, discovered in the previous project period, is Cu "salvage", in which Cu is removed from a non-essential protein so that it can be recycled and used for the synthesis of an essential cuproprotein. In Chlamydomonas, replacement and salvage mechanisms are turned on in copper-deficient cells by a copper-sensing transcription factor, CRR1. Its target genes are associated with copper response elements, which serve as binding sites for the DNA binding domain of CRR1. We have identified all the CRR1 target genes in the Chlamydomonas genome by next gen transcriptome profiling (RNA-Seq). In Specific Aim 1, we will undertake biochemical and reverse genetic analysis of select target genes, IRT2 (encoding a ZIP family FeII transporter), AOF1 (encoding a flavin amine oxidase), 142634 (encoding a down-regulated plastid-targeted metallochaperone) to further elaborate the copper sparing pathway, and of RSEP1 (encoding a thylakoid lumen protease) and CTR3 (encoding a soluble copper binding protein) to assess their function in the salvage pathway. In zinc-deficient Chlamydomonas cells, copper is hyper-accumulated in bio-inaccessible compartments, resulting in functional copper-deficiency. This phenotype has been observed recently in mammalian cells with disruptions in Cu homeostasis factors. Hyper- accumulation of Cu in Chlamydomonas requires CRR1. In Aim 2, we will undertake biochemical characterization of these copper-loaded compartments and we will use high throughput screening methods for a classical genetic approach for the discovery of factors involved in loading and unloading Cu into these compartments. CRR1 is also required in Chlamydomonas for zinc homeostasis. Besides ZIP family transporters, two novel proteins, 123019 and 117548, with COG0523 domains (conserved in all kingdoms of life) are highly up-regulated in zinc-deficiency. In Aim 3, we will distinguish the mechanism of regulation of the corresponding genes, identify zinc response elements, and use gain-of-function mutants (by over-expression) to deduce whether they are zinc chaperones.

描述（由申请人提供）：Cu 是几乎所有生命形式的必需营养素，因为它充当催化氧化还原反应或涉及 O2 化学的反应的酶辅基。这些反应是有氧生活的核心。我们的长期目标是了解铜的代谢，特别是当它是一种限制性营养素或处于代谢缺陷的情况下。二十年前，我们在模型生物衣藻中发现了铜节约的范例，在铜缺乏的情况下，不依赖铜的蛋白质可以替代原本丰富的铜蛋白质。这种机制在许多微生物系统中广泛存在于各种微量矿物质营养素（铁、锌、钼等）中，现已得到证实。金属节约机制可能对于某些传染性细菌和真菌成功逃避宿主防御系统很重要，其中金属隔离被用作防御策略。上一个项目期间发现的一个新范例是铜“回收”，其中铜从非必需蛋白质中去除，以便可以回收并用于合成必需铜蛋白。在衣藻中，缺铜细胞中的替代和挽救机制是由铜感应转录因子 CRR1 启动的。其靶基因与铜反应元件相关，铜反应元件充当 CRR1 DNA 结合域的结合位点。我们通过下一代转录组分析 (RNA-Seq) 鉴定了衣藻基因组中的所有 CRR1 靶基因。在具体目标 1 中，我们将对选定的目标基因 IRT2（编码 ZIP 家族 FeII 转运蛋白）、AOF1（编码黄素胺氧化酶）、142634（编码下调的质体靶向金属伴侣）进行生化和反向遗传分析，以进一步阐述铜节约途径以及 RSEP1（编码类囊体腔蛋白酶）和 CTR3 （编码可溶性铜结合蛋白）以评估其在补救途径中的功能。在缺锌的衣藻细胞中，铜在生物无法进入的隔室中过度积累，导致功能性铜缺乏。最近在铜稳态因子破坏的哺乳动物细胞中观察到了这种表型。衣藻中铜的过度积累需要 CRR1。在目标 2 中，我们将对这些载铜区室进行生化表征，并将使用经典遗传方法的高通量筛选方法，以发现将铜加载和卸载到这些区室中所涉及的因素。衣藻中也需要 CRR1 来维持锌稳态。除了 ZIP 家族转运蛋白外，两种具有 COG0523 结构域（在所有生命王国中保守）的新型蛋白 123019 和 117548 在缺锌时高度上调。在目标3中，我们将区分相应基因的调控机制，识别锌反应元件，并使用功能获得突变体（通过过度表达）来推断它们是否是锌伴侣。