Nutritional Copper Signaling and Homeostasis

说明(申请人提供)：铜是一种基本的微量营养素，因为它在催化氧化还原反应的酶中发挥作用。在人类中，获得性铜缺乏是罕见的，因为铜的日需求量很低，但铜缺乏可伴随营养不良，与孟克斯综合征等遗传性疾病有关，或因过量摄入锌补充剂而引起。而边际缺铜，心血管疾病的一个促成因素，可能并不少见。该项目的长期目标是：i)在特定的铜酶途径的背景下，发现铜缺乏细胞的适应性生化变化；ii)剖析实现这些变化的潜在调控机制；以及iii)了解适应性修饰的生理基础。莱茵衣藻是这项研究的模式生物，因为定义明确的合成生长介质有助于微量元素动态平衡的研究，并且经典生化和遗传学方法的全部力量可以与最先进的基因组和蛋白质组方法相结合。先前对衣藻缺铜反应的研究确立了铜依赖酶的概念，这种酶在铜细胞中表达，以补偿铜酶功能的丧失(例如，细胞色素C6作为质蓝蛋白的备份，CRD2作为多铜铁氧酶的备份)，并导致了CRRL的发现，CRRL是一种新的DNA结合蛋白，是所有已知的铜缺乏反应所必需的，也参与维持铜完整细胞中铜的动态平衡。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(铁氧合酶)和Ftr1(铁渗透酶)功能相关的生化功能，并监测其在铜和铁营养条件下的表达和亚细胞定位；2)通过突变和体内、体外功能分析(包括固化结合、金属结合和氧化还原状态)，剖析了铜调节蛋白CRRL的3个结构域--DNA结合的SBP结构域、锚蛋白重复序列和富含半胱氨酸的C末端，确定了铜依赖的CRRL调节和定位的模式，为其作为铜营养和缺氧调节因子的作用建立了模型；以及3)通过对-铜与+铜细胞和ErR I与野生型细胞的专业寡核苷酸芯片分析，以及基于亚蛋白质组凝胶和多维层析的富铜与缺铜细胞的分析，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.