Nutritional Copper Signaling and Homeostasis

描述（由申请人提供）：铜是一种必需的微量营养素，因为它在催化氧化还原反应的酶中起作用。在人类中，由于铜的每日需求量较低，获得性缺铜是罕见的，但缺铜可能伴随营养不良，与Menkes综合征等遗传性疾病有关，或由锌补充剂过度消耗引起。边缘性铜缺乏是心血管疾病的一个促成因素，可能并不罕见。该项目的长期目标是i）在特定的含铜酶途径的背景下发现铜缺乏细胞中的适应性生化变化，ii）剖析实现这些变化的潜在调控机制，以及iii）理解适应性修饰的生理学原理。莱茵衣藻是该研究的模式生物，因为定义明确的合成生长培养基有利于微量元素稳态的研究，并且经典生物化学和遗传方法的全部功能可以与最先进的基因组学和蛋白质组学方法相结合。以往对衣原体铜缺乏反应的研究建立了“备份”铜非依赖性酶的概念，这些酶在-Cu细胞中表达以补偿铜酶功能的丧失（例如cyt c6，作为质体蓝素的备份，Crd 2作为多铜铁氧化酶的备份），并导致Crrl的发现，一种新的DNA结合蛋白，是所有已知的铜缺乏反应所需的，也参与维持铜充足细胞中的铜稳态。在本项目期间，研究者将：1）鉴定CRD 2，其是在铜缺乏细胞中高亲和力铁摄取所必需的，确定其与Fox 1相关的生化功能（铁氧化酶）和Ftr 1（铁通透酶）功能，并监测其响应铜和铁营养的表达和亚细胞定位; 2）通过诱变和包括CuRE结合、金属结合和氧化还原状态在内的体内和体外功能分析，解剖铜调节剂Crrl中的3个结构域-DNA结合SBP结构域、锚蛋白重复序列和富含Cys的C末端，并确定铜依赖性Crrl调节和定位的模式，以开发其作为铜营养和缺氧调节剂的作用模型;和3）通过-Cu与+Cu细胞和err I与野生型细胞的专门寡核苷酸微阵列分析鉴定铜缺乏的新靶点，与基于亚蛋白质组学凝胶和多维色谱的分析的铜-充足与铜-缺乏细胞平行。

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.