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外流的分子机制。 极化上皮细胞中的ATPase。肠上皮细胞中的ATP7A将铜输送到循环中 肝细胞中的ATP7B将铜输送到胆汁中(尖端环境)。突变 这两种蛋白质中的任何一种都会导致人类疾病，强调它们在铜稳态中的重要性。基于 大量的体内和体外初步数据，我们建议检验三个假说。在目标1中我们 假设在铜负荷的肝脏和肠道细胞中，铜-三磷酸腺苷酶将铜泵入独特的囊泡中， 与基底外侧(ATP7A)或根尖(ATP7B)质膜(PM)融合，释放“储存的”铜。我们会 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，我们 假设氯离子通道CIC4是铜-三磷酸腺苷酶分泌和分泌过程中的阴离子分流通道。 外流功能/位置。在基础铜中，我们将通过以下方法确定铜蓝蛋白在WIF-B细胞中的载量 ATP7A诱导的Caco-2细胞中的ATP7B和Hephestn在无氯条件下发生，并在敲除 CIC4.我们还将研究CIC4在细胞铜外流中的作用。 铜水平升高，ATP7B N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鉴定新的蛋白质相互作用因子，阐明 新发现的铜外流调节剂(Murr1)对wt和选择性N-末端动力学/功能的影响 外源ATP7B的突变体，并用肝豆状核变性大鼠模型验证和 扩大体外研究结果。方法将包括RNAi和wt调节子的过度表达，以及酵母 两个混血儿。同样在目标2中，我们假设当铜水平降低时，ATP7A和ATP7B被恢复 通过识别其C-末端的结构信号，从它们不同的隔间中分离出来。我们将使用酵母 从相关组织的提取物中分离出两种杂交性和亲和性可能的蛋白质相互作用因子。

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.