PROTEINS REGULATING CU-ATPASES IN EPITHELIA

调节上皮细胞中铜ATP酶的蛋白质

基本信息

批准号：
7690603
负责人：
Ann L Hubbard
金额：
$ 29.58万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-15 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7690603
关键词：
ATP phosphohydrolase Affect Amino Acids Animals Apical B-Lymphocytes Bile fluid Binding Binding Sites Biochemical Biology Biotinylation Blood Circulation Breeding C-terminal Caco-2 Cells Cell membrane Cell surface Cells Chimera organism Copper Cytoplasmic Protein Cytoplasmic Vesicles Environment Epithelial Cells Epithelium Excision Goals Hepatic Hepatocyte Heterozygote Homeostasis Homo Human Hybrids Immunofluorescence Immunologic In Vitro Intestines Ions Kidney Knock-out Length Letters Liver MDCK cell Mediating Membrane Metals Methods Microtubules Molecular Molecular Chaperones Molecular Conformation Movement Mus N-terminal PDZ protein Pathway interactions Play Protein Binding Proteins Regulation Role Route Signal Transduction Sorting - Cell Movement Tacrolimus Binding Proteins Tail Testing Tissues Titrations Tyrosine Vesicle Wilson disease protein Work Yeasts apical membrane base bathocuproine disulfonate dimer dynactin human disease in vivo kidney cell knock-down mutant novel overexpression polarized cell protein protein interaction tacrolimus binding protein 4 trafficking trans-Golgi Network yeast two hybrid system

项目摘要

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.

我们的长期目标是阐明两种哺乳动物 Cu-ATP 酶的分子机制调节极化上皮细胞中的铜稳态。这些 Cu-ATP 酶有两个功能：需要它们以铜敏感且可逆的方式在细胞内重新分布或运输。第一的，它们将铜转运到分泌途径中，使新合成的铜酶金属化。他们还流出铜。肠上皮细胞中的 ATP7A 将饮食中的铜输送到循环系统（基底外侧环境），肝细胞中的 ATP7B 将过量的铜输送到胆汁（顶端环境）。这个项目专注于调节 Cu-ATP 酶运输和输出功能的细胞蛋白。在目标 1 A 中，我们将确定极化肝 WIF-B 细胞中 ATP7B 过表达或敲除后的动态当铜存在时，它会结合 ATP7B（不是 ATP7A）的 N 端结构域。升高。 p62 被认为可以指导 ATP7B 阳性囊泡沿微管的顶端运动。将使用免疫荧光滴定细胞铜并测定铜 64 流出量。 Aim 1B 重点关注我们仅在 ATP7B 的 N 末端发现的 9 个氨基酸顶端靶向基序。我们将与项目 3 + 4 合作，确定靶向基序是否与 ATP7B 的其他结构域相互作用如果含有与基底外侧蛋白融合的基序的嵌合体将靶向顶膜。到识别提议结合靶向基序的蛋白质 (X)，我们将使用 2 种方法：基于膜的方法酵母-二杂交系统；以及候选蛋白质的推定结合域的筛选。在目标 2 中，我们将确定 PDZ 蛋白 AIPP 靶向/保留内源 ATP7A 的机制极化肠道 Caco-2 细胞的基底外侧区域。我们将使用以下方法过度表达并敲低 AIPP1 与目标 1A 中的方法类似，并与项目 3 合作确定 AIPP1 水平是否/如何影响铜出口。在目标 3 中，我们将研究 ATOX1（两种 Cu-ATP 酶的铜伴侣）及其作用提出的修饰剂 FKBP52 在复合杂合子小鼠体内铜稳态中的作用 (ATOX1+/-; FKBP+/-)。我们将与项目 2 和 3 合作研究这些动物的肝脏、肠道和肾脏。