Nutritional Copper Signaling and Homeostasis

营养铜信号传导和体内平衡

基本信息

批准号：
8185585
负责人：
SABEEHA MERCHANT
金额：
$ 40.5万
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/8185585
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 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 RNA 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 high throughput screening hypocupremia knock-down loss of function microbial mouse model mutant novel nutrition operation positional cloning promoter research study success transcription factor

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. PUBLIC HEALTH RELEVANCE: Mineral nutrients like Cu and Zn are required by all forms of life because the elements contribute to structures of molecules and they also provide catalytic centers; hence deficiencies, resulting from genetic defect or poor nutrition, can hinder normal metabolism. In this project, a model organism Chlamydomonas is used to discover mechanisms of Cu storage, sparing and salvage in Cu and Zn deficient cells. These mechanisms optimize the use of the mineral nutrients for the most important metabolic pathways.

描述（由申请人提供）：CU是几乎所有生活形式的必不可少的营养素，因为它是催化氧化还原反应或涉及O2化学反应的酶假体。这些反应对有氧生活至关重要。我们的长期目标是了解铜的新陈代谢，尤其是当它是一种有限的养分或新陈代谢缺陷的情况时。二十年前，我们在模型生物体中发现了cu的范式，在这种情况下，在铜缺乏症的情况下，独立于Cu的蛋白质可以取代原本丰富的Cu蛋白质。现在已经建立了许多微生物系统（Fe，Zn，Mo，列出几种）的许多微生物系统中这种机制的广泛发生。金属保留机制对于某些传染性细菌和真菌在逃避宿主防御系统中的成功可能很重要，在这种防御系统中，金属隔离被用作防御策略。在上一个项目时期发现的一个新范式是Cu“打捞”，其中将Cu从非必需的蛋白质中取出，以便可以回收并用于合成必需的蛋白质蛋白。在衣原体中，通过铜感应转录因子CRR1在缺陷铜细胞中打开替代和打捞机制。它的靶基因与铜响应元件有关，铜响应元件是CRR1的DNA结合结构域的结合位点。我们已经通过下一个基因转录组分析（RNA-Seq）确定了衣原体基因组中的所有CRR1靶基因。 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类囊体管腔蛋白酶）和Ctr3（编码可溶性铜结合蛋白）以评估其在打捞途径中的功能。在缺乏锌的衣原体细胞中，铜在生物不可接近的隔室中均超过铜，从而导致功能性铜缺陷。最近在哺乳动物细胞中观察到这种表型，在CU稳态因子中有破坏。 Cu在衣原体中的过度积累需要CRR1。在AIM 2中，我们将对这些铜隔间的生化表征进行生化表征，并将使用高吞吐量筛选方法进行经典的遗传方法，以发现与将CU卸载和将CU卸载到这些隔室中所涉及的因素。锌稳态的衣原体也需要CRR1。除了拉链家庭转运蛋白外，两种新型蛋白质，即123019和117548，带有COG0523域（在所有王国中保守）在锌缺陷方面高度上调。在AIM 3中，我们将区分相应基因调节的机理，识别锌响应元件，并使用功能获得的突变体（通过过表达）推断出它们是否是锌伴侣。公共卫生相关性：各种形式的生命都需要矿物质营养素，因为元素有助于分子的结构，并且还提供催化中心。因此，由于遗传缺陷或营养不良而导致的缺陷会阻碍正常的代谢。在该项目中，模型有机衣原体用于发现Cu和Zn缺陷细胞中Cu储存，保留和打捞的机制。这些机制优化了最重要的代谢途径的矿物营养素的使用。