Cu Homeostasis and Cu-ATPases in Polarized Epithelia

我们的长期目标是阐明两种哺乳动物Cu-2受体调控Cu外排的分子机制。 极化上皮细胞中的ATP酶。肠上皮细胞中的ATP 7A将Cu输送到循环中 肝细胞中的ATP 7 B将Cu递送至胆汁（基底外侧环境），而肝细胞中的ATP 7 B将Cu递送至胆汁（顶端环境）。突变 在任何蛋白质引起人类疾病，强调其在铜稳态的重要性。基于 在体内和体外的广泛的初步数据，我们建议测试三个假设。在AIM 1中， 假设在负载Cu肝和肠细胞中，Cu-ATP酶将Cu泵入独特的囊泡中， 与基底外侧（ATP 7A）或顶端（ATP 7 B）质膜（PM）融合以释放“储存的”Cu。我们将 使用免疫EM和免疫分离/蛋白质组学方法鉴定这些隔室。在AIM 2中， 假设氯离子通道CIC 4是分泌型和分泌型Cu-ATP酶的阴离子旁路， 外排功能/位置。在基础Cu中，我们将通过以下方法确定WIF-B细胞中铜蓝蛋白的Cu负载： 在无氯条件下，ATP 7A敲低Caco-2细胞中的ATP 7 B和hephaestin， CIC4.我们还将研究CIC 4在铜流出细胞中的作用。 Cu水平升高，ATP 7 B N-末端的独特结构信号调节其运输/顶端 外排功能我们的测试包括生物化学分离和识别新的蛋白质相互作用，阐明 新鉴定的Cu流出调节剂（Murr 1）对wt和选择的N-末端的动力学/功能的作用 外源性ATP 7 B突变体，并使用Wilson病的LEG大鼠模型来证实和 扩展体外研究结果。方法将包括RNAi和wt调节剂的过表达，以及酵母表达。 两种混合动力。同样在AIM 2中，我们假设当Cu水平降低时，ATP 7A和ATP 7 B被回收 通过识别其C-末端的结构信号从其不同的隔室中分离。我们将使用酵母 从相关组织的提取物中进行推定的蛋白质相互作用物的双杂交和亲和分离。

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.