PROTEINS REGULATING CU-ATPASES IN EPITHELIA

我们的长期目标是阐明两种哺乳动物铜-ATPase的分子机制 调节极化上皮细胞铜的动态平衡。这些铜-ATPase有两个功能 有必要以对铜敏感和可逆的方式在细胞内重新分配或贩运它们。第一, 它们将铜运输到分泌途径中，以金属化新合成的铜酶。他们也 喷出的铜。肠上皮细胞中的ATP7A将饮食中的铜输送到循环中(基侧 肝细胞中的ATP7B将过量的铜输送到胆汁中(尖端环境)。这个项目 重点是调节铜-三磷酸腺苷酶的运输和出口功能的细胞蛋白质。在目标1 A中，我们 将确定ATP7B在极化的肝脏WIF-B细胞中过度表达或敲击后的动力学 下调动力蛋白亚基p62，当铜被激活时，它与ATP7B(而不是ATP7A)的N-末端结构域结合 抬高了。P62被认为指导ATP7B阳性小泡沿微管的顶端运动。 将进行细胞铜的免疫荧光滴定和铜-外排的测定。 目的1B是我们在ATP7B的N端发现的一个9个氨基酸的顶端靶向基序。我们 我将与项目3和4合作，以确定目标基序是否与ATP7B的其他域相互作用 如果包含这个基序的嵌合体融合到基侧蛋白，就会靶向顶膜。至 鉴定蛋白(X)建议结合靶向基序，我们将使用两种方法：一种基于膜的 酵母-双杂交系统；以及候选蛋白可能结合结构域的筛选。在目标2中，我们将 确定PDZ蛋白AIPP靶向/保留内源性ATP7A到/中的机制 极化的肠道Caco-2细胞的基底外侧区。我们将过度表达并拆分AIPP1 方法与目标1A中的方法类似，并与项目3协作，以确定AIPP1水平是否/如何影响 铜出口。在目标3中，我们将研究ATOX1的作用，ATOX1是两种铜-ATPase的铜伴侣，以及它的 建议的修改剂，FKBP52，体内铜稳态复合杂合子小鼠(ATOX1/-； FKBP/-)。我们将与项目2和项目3合作，研究这些动物的肝脏、肠道和肾脏。

Our long-term goal is to elucidate the molecular mechanisms by which two mammalian Cu-ATPases regulate copper homeostasis in polarized epithelial cells. These Cu-ATPases have two functions that necessitate their intracellular redistribution, or trafficking, in a copper-sensitive and reversible manner. First, they transport copper into the secretory pathway to metallate newly-synthesized cuproenzymes. They also efflux copper. ATP7A in intestinal epithelial cells delivers dietary Cu to the circulation (basolateral environment), and ATP7B in hepatic cells delivers excess Cu to the bile (apical environment). This project focuses on cellular proteins that modulate the Cu-ATPases' trafficking and export functions. In Aim 1 A, we will determine the dynamics of ATP7B in the polarized hepatic WIF-B cells, after over-expressing or knocking down the dynactin subunit, p62, which binds the N-terminal domain of ATP7B (not ATP7A) when copper is elevated. p62 is proposed to direct the apical movement of ATP7B-positive vesicles along microtubules. Titration of cellular copper with immunofluorescence and copper-64 efflux determinations will be performed. Aim 1B focuses on a 9 amino acid apical targeting motif we discovered in the N-terminus of only ATP7B. We will collaborate with Projects 3 + 4 to determine if the targeting motif interacts with other domains of ATP7B and if a chimera containing this motif fused to a basolateral protein will target to the apical membrane. To identify the protein (X) proposed to bind the targeting motif, we will use 2 approaches: a membrane-based yeast-two hybrid system; and a screen of putative binding domains of candidate proteins. In Aim 2, we will determine the mechanism by which a PDZ protein, AIPP, targets/retains endogenous ATP7A to/in the basolateral region of polarized intestinal Caco-2 cells. We will over-express and knock down AIPP1 using approaches similar to those in Aim 1A and collaborate with Project 3 to determine if/how AIPP1 levels affect Cu export. In Aim 3, we will study the role of ATOX1, the copper chaperone for both Cu-ATPases, and its proposed modifier, FKBP52, in copper homeostasis in vivo in compound heterozygote mice (ATOX1+/-; FKBP+/-). We will collaborate with Projects 2 and 3 to study the liver, intestine and kidney in these animals.