Cu Homeostasis and Cu-ATPases in Polarized Epithelia

极化上皮细胞中的 Cu 稳态和 Cu-ATP 酶

• 批准号: 7133531
• 负责人: Ann L Hubbard
• 金额: $ 31.37万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7133531
• 关键词: adenosinetriphosphatase; anions; apical membrane; biological signal transduction; cell population study; cellular polarity; chloride channels; copper; enzyme activity; epithelium; gene expression; immunoelectron microscopy; intestinal mucosa; laboratory rat; liver cells; metal metabolism; molecular dynamics; mutant; protein protein interaction; protein structure function; proteomics

Our long-term goal is to elucidate the molecular mechanisms of regulated Cu efflux by two mammalian Cu- ATPases in polarized epithelial cells. ATP7A in intestinal epithelial cells delivers Cu to the circulation (basolateral environment) and ATP7B in hepatic cells delivers Cu to the bile (apical environment). Mutations in either protein cause human disease, emphasizing their importance in Cu-homeostasis. Based on extensive preliminary data in vivo and in vitro, we propose to test three hypotheses. In AIM 1 we hypothesize that in Cu-loaded hepatic and intestinal cells, Cu-ATPases pump Cu into unique vesicles that fuse with the basolateral (ATP7A) or apical (ATP7B) plasma membrane (PM) to release "stored" Cu. We will identify these compartments using immuno-EM and immuno-isolation/proteomic approaches. In AIM 2 we hypothesize that the chloride channel, CIC4, is an anion shunt for both Cu-ATPases in their secretory and efflux function/locations. In basal Cu, We will determine if Cu loading of ceruloplasmin in WIF-B cells by ATP7B and hephaestin in Caco-2 cells by ATP7A occurs in chloride-free conditions and after knock-down of CIC4. We will also examine the role of CIC4 in Cu efflux from the cells.In Aim 3, we hypothesize that when Cu levels are elevated, unique structural signals in the N-terminus of ATP7B regulate its trafficking/apical efflux function. Our test includes biochemically isolating and identifying new protein interactors, elucidating the role of a newly-identified modulator of Cu efflux (Murr1) on the dynamics/function of wt and selected N-terminal mutants of exogenous ATP7B, and using the LEG rat model of Wilson Disease to confirm and extend in vitro findings. Approaches will include RNAi and overexpression of wt modulators, as well as yeast two hybrid. Also in AIM 2 we hypothesize that when Cu levels are lowered, ATP7A and ATP7B are retrieved from their distinct compartments through recognition of structural signals in their C-termini. We will use yeast two hybrid and affinity isolation of putative protein interactors from extracts of relevant tissue.

我们的长期目标是阐明两种哺乳动物Cu-调节的Cu外排的分子机制 极化上皮细胞中的ATPases。肠上皮细胞中的ATP7A将Cu传递到循环 （基底外侧环境）和肝细胞中的ATP7B将CU提供给胆汁（顶部环境）。突变 在任何一种蛋白质中，都会引起人类疾病，强调它们在Cu Homeostasis中的重要性。基于 我们建议在体内和体外进行广泛的初步数据，以检验三个假设。在目标1中我们 假设在加载的Cu肝和肠细胞中，Cu-Atpases将Cu泵入独特的囊泡中 与基底外侧（ATP7A）或顶端（ATP7B）质膜（PM）融合以释放“存储” Cu。我们将 使用免疫EM和免疫异常/蛋白质组学方法识别这些隔室。在目标2中我们 假设氯化物通道CIC4是它们分泌中两个Cu-atpase的阴离子分流，并且 外排功能/位置。在基底Cu中，我们将确定是否通过 通过ATP7A在CACO-2细胞中的ATP7B和Hephaestin发生在无氯化物条件下以及在敲击之后 CIC4。我们还将检查CIC4在细胞中的Cu 4中的作用。在AIM 3中，我们假设当 CU水平升高，在ATP7B的N末端的独特结构信号调节其贩运/顶端 外排功能。我们的测试包括生化隔离和识别新的蛋白质相互作用，并阐明 Cu外排（MURR1）新识别的调节剂在WT和选定的N末端的动力学/功能上的作用 外源ATP7B的突变体，并使用威尔逊疾病的腿大鼠模型确认和 扩展体外发现。方法将包括RNAi和WT调节剂的过表达以及酵母 两个混合动力。同样在AIM 2中，我们假设当Cu级别降低时，检索ATP7A和ATP7B 从它们的独特隔室通过识别C-termini中的结构信号。我们将使用酵母 从相关组织提取物提取的两个杂种和亲和力分离。