Cu Homeostasis and Cu-ATPases in Polarized Epithelia

我们的长期目标是阐明两种哺乳动物铜流出调节的分子机制 极化上皮细胞中的 ATP 酶。肠上皮细胞中的 ATP7A 将铜输送至循环系统 （基底外侧环境）和肝细胞中的 ATP7B 将铜输送至胆汁（顶端环境）。突变 任何一种蛋白质都会导致人类疾病，强调它们在铜稳态中的重要性。基于 根据体内和体外的广泛初步数据，我们建议检验三个假设。在 AIM 1 中，我们 假设在负载 Cu 的肝细胞和肠细胞中，Cu-ATP 酶将 Cu 泵入独特的囊泡中， 与基底外侧 (ATP7A) 或顶端 (ATP7B) 质膜 (PM) 融合，释放“储存的”Cu。我们会 使用免疫电镜和免疫分离/蛋白质组学方法识别这些区室。在《AIM 2》中，我们 假设氯离子通道 CIC4 是 Cu-ATP 酶分泌和分泌过程中的阴离子分流通道。 流出功能/位置。在基础 Cu 中，我们将通过以下方式确定 WIF-B 细胞中铜蓝蛋白的 Cu 负载量： ATP7A 在 Caco-2 细胞中产生 ATP7B 和 hephaestin，发生在无氯条件下，并且在敲低 CIC4。我们还将研究 CIC4 在 Cu 从细胞流出中的作用。在目标 3 中，我们假设当 Cu 水平升高，ATP7B N 末端的独特结构信号调节其运输/顶端 流出功能。我们的测试包括生化分离和识别新的蛋白质相互作用物，阐明 新鉴定的 Cu 流出调节剂 (Murr1) 对 wt 和选定 N 末端的动力学/功能的作用 外源 ATP7B 突变体，并使用 LEG 威尔逊病大鼠模型来确认和 扩展体外研究结果。方法包括 RNAi 和 wt 调节剂以及酵母的过度表达 两个杂交种。同样在 AIM 2 中，我们假设当 Cu 水平降低时，ATP7A 和 ATP7B 会恢复 通过识别其 C 末端的结构信号，将其从不同的区室中分离出来。我们将使用酵母 从相关组织提取物中对假定的蛋白质相互作用物进行两种杂交和亲和分离。

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.