Nutritional Copper Signaling and Homeostasis

营养铜信号传导和体内平衡

基本信息

批准号：
7277414
负责人：
SABEEHA MERCHANT
金额：
$ 8.82万
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 2009-03-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Copper is an essential micronutrient because of its role in enzymes that catalyze redox reactions. In humans, acquired copper-deficiency is rare because of the low daily requirement of copper, but copper-deficiency can accompany malnutrition, be associated with genetic disorders like Menkes syndrome or result from overconsumption of zinc supplements. And marginal copper deficiency, a contributing factor in cardiovascular disease, may not be uncommon. The long-term objective of this project is to i) discover the adaptive biochemical changes in copper-deficient cells in the context of specific cuproenzyme-containing pathways, ii) dissect the underlying regulatory mechanisms for achieving these changes, and iii) understand the physiological rationale for the adaptive modifications. Chlamydomonas reinhardtii is the model organism for the study because the well-defined synthetic growth medium facilitates studies of trace element homeostasis, and the full power of classical biochemical and genetic approaches can be combined with state of the art genomic and proteomic methods. The previous studies of copper-deficiency responses in Chlamydomonas established the concept of "back up" copper-independent enzymes that are expressed in -Cu cells to compensate for loss of function of cuproenzymes (e.g. cyt c6, as a back up for plastocyanin and Crd2 as a back up for the multicopper ferroxidase), and led to the discovery of Crrl, a novel DNA binding protein that is required for all known copper-deficiency responses and is involved also in maintaining copper homeostasis in a copper replete cell. In this project period, the investigators will: 1) identify CRD2, which is required for high affinity iron uptake in a copper-deficient cell, determine its biochemical function in relation to Fox1 (ferroxidase) and Ftr1 (iron permease) function, and monitor its expression and sub-cellular location in response to copper and iron nutrition; 2) dissect 3 domains in the copper regulator Crrl - the DNA-binding SBP domain, ankyrin repeats and the Cys-rich C-terminus - by mutagenesis and in vivo and in vitro functional analysis including CuRE binding, metal binding and redox state, and determine the pattern of copper-dependent Crrl regulation and location, to develop a model for its action as a regulator of copper nutrition and hypoxia; and 3) identify novel targets of copper-deficiency by specialized oligonucleotide microarray analyses of -Cu vs. +Cu cells and err I vs. wild-type cells, in parallel with sub-proteomic gel- and multi-dimensional chromatography-based analyses of copper-replete vs. copper-deficient cells.

描述（由申请人提供）：铜是必不可少的微量营养素，因为它在催化氧化还原反应的酶中的作用。在人类中，由于铜的每日需求较低，因此获得的铜缺陷很少见，但是铜缺陷可能伴随营养不良，与Menkes综合征（如Menkes综合征）或锌补充剂过度消耗导致的遗传疾病有关。边缘铜缺乏症是心血管疾病的促成因素，可能并不少见。该项目的长期目的是i）在特定的含库酶的途径的背景下，发现铜缺陷细胞的适应性生化变化，ii）剖析了实现这些变化的基本调节机制，并且iiii）了解适应性修饰的生理原理。衣原体Reinhardtii是该研究的模型生物，因为明确定义的合成培养基促进了痕量元素稳态的研究，并且可以将经典生物化学和遗传方法的全部力量与ART基因组和蛋白质组学方法的状态相结合。 The previous studies of copper-deficiency responses in Chlamydomonas established the concept of "back up" copper-independent enzymes that are expressed in -Cu cells to compensate for loss of function of cuproenzymes (e.g. cyt c6, as a back up for plastocyanin and Crd2 as a back up for the multicopper ferroxidase), and led to the discovery of Crrl, a novel DNA所有已知的铜缺陷反应所需的结合蛋白，也参与了在铜细胞中维持铜稳态。在这个项目期间，研究人员将：1）确定CRD2（在铜缺陷型细胞中获得高亲和力铁的吸收所需的CRD2，确定其与FOX1（铁氧化酶）和FTR1（Iron Permence）功能相关的生化功能，并监测其对铜和铁营养的响应的表达和亚细胞位置； 2）通过诱变，体内和体外功能分析，在铜调节剂CRRL中解剖3个结构域 - DNA结合SBP结构域，Ankyrin重复和富含Cys的C-末端 - 固化结合，包括金属结合状态，并确定copper crrl crrl规范和位置的模型，并确定copper的模型，并确定copper的模型，以确保结合结合和体外功能分析。缺氧；和3）通过与亚蛋白质组凝胶和多维色谱分析的铜铜，铜 - 重新培养物，铜铜细胞的分析，通过专门的寡核苷酸微阵列分析确定铜缺陷的新靶标。