PROTEINS REGULATING CU-ATPASES IN EPITHELIA

我们的长期目标是阐明两种哺乳动物Cu-ATP酶 调节极化上皮细胞中的铜稳态。这些Cu-ATP酶具有两种功能， 使它们以铜敏感和可逆方式在细胞内重新分布或运输成为必要。第一、 它们将铜转运到分泌途径中以金属化新合成的铜酶。他们还 流出铜肠上皮细胞中的ATP 7A将饮食Cu递送至循环（基底外侧 肝细胞中的ATP 7 B将过量的Cu递送至胆汁（顶端环境）。这个项目 主要研究调节Cu-ATP酶运输和输出功能的细胞蛋白。在目标1A中，我们 将确定过表达或敲除后极化肝WIF-B细胞中ATP 7 B的动力学 当铜离子被抑制时，动力蛋白亚基p62与ATP 7 B（而不是ATP 7A）的N端结构域结合。 升高。p62被认为指导ATP 7 B阳性囊泡沿沿着微管的顶端运动。 将采用免疫荧光和铜-64流出测定法滴定细胞铜。 目的1B集中于我们仅在ATP 7 B的N-末端发现的9个氨基酸的顶端靶向基序。我们 我将与项目3 + 4合作，以确定靶向基序是否与ATP 7 B的其他结构域相互作用 并且如果含有与基底外侧蛋白融合的该基序的嵌合体将靶向顶膜。到 为了鉴定提出结合靶向基序的蛋白（X），我们将使用2种方法：基于膜的 酵母双杂交系统;和候选蛋白质的推定结合结构域的筛选。在目标2中，我们 确定PDZ蛋白AIPP靶向/保留内源性ATP 7A至/中的机制。 极化的肠Caco-2细胞的基底外侧区域。我们将过度表达并使用 与目标1A类似的方法，并与项目3合作，以确定AIPP 1水平是否/如何影响 铜出口。在目标3中，我们将研究ATOX 1（两种Cu-ATP酶的铜分子伴侣）的作用，及其在细胞中的作用。 提出的修饰剂FKBP 52在复合杂合子小鼠（ATOX 1 +/-; 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.