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，获得性铜缺乏症很少见，因为每天对铜的需求量很低，但铜缺乏症可能伴随营养不良，可能与门克斯综合征等遗传疾病有关，也可能是过量服用锌补充剂的结果。微量缺铜是心血管疾病的一个诱因，这种情况可能并不罕见。该项目的长期目标是：1)发现特定含铜酶途径下缺铜细胞的适应性生化变化；2)剖析实现这些变化的潜在调节机制；3)了解适应性修饰的生理原理。莱茵衣藻是该研究的模式生物，因为定义良好的合成生长培养基有利于微量元素稳态的研究，并且经典生化和遗传方法的全部力量可以与最先进的基因组学和蛋白质组学方法相结合。前人对衣藻铜缺乏反应的研究建立了“备份”铜不依赖酶的概念，这些酶在-Cu细胞中表达，以补偿铜原酶的功能丧失（例如cyt c6作为质体青素的备份，Crd2作为多铜氧化铁酶的备份），并导致Crrl的发现。一种新型的DNA结合蛋白，是所有已知的缺铜反应所必需的，也参与维持缺铜细胞中的铜稳态。在本项目期间，研究人员将：1)鉴定缺铜细胞高亲和力铁摄取所需的CRD2，确定其与Fox1（铁氧化酶）和Ftr1（铁渗透酶）功能相关的生化功能，并监测其表达和亚细胞位置对铜和铁营养的响应；2)通过诱变和体内外功能分析，解剖铜调控因子Crrl中的3个结构域——dna结合的SBP结构域、锚蛋白重复序列和富含cys的c端，包括CuRE结合、金属结合和氧化还原状态，确定铜依赖性Crrl的调控模式和定位，建立其作为铜营养和缺氧调控因子的模型；3)通过专门的寡核苷酸微阵列分析-Cu与+Cu细胞和err I与野生型细胞，以及基于亚蛋白质组学凝胶和多维色谱的铜富集与缺铜细胞分析，确定铜缺乏的新靶点。

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.