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个哺乳动物Cu-atpase的分子机制调节极化上皮细胞中的铜稳态。这些Cu-atpases具有两个功能需要以铜敏感和可逆的方式进行细胞内再分配或贩运。第一的，他们将铜运输到分泌途径，以金属合成的库酶。他们也是外排铜。肠上皮细胞中的ATP7A将饮食CU提供给循环（基底外侧）环境），肝细胞中的ATP7B为胆汁（顶环境）提供过多的Cu。这个项目专注于调节Cu-Atpases运输和输出功能的细胞蛋白。在AIM 1 A中，我们过度表达或敲击后，将确定偏振肝WIF-B细胞中ATP7B的动力学沿Dynactin亚基p62，该铜的N末端结构域（非ATP7A）在铜为高架。提出了p62来指导ATP7B阳性囊泡沿微管的顶端运动。将进行免疫荧光和铜-64外排测定的细胞铜的滴定。 AIM 1B专注于我们仅在ATP7B的N末端发现的9个氨基酸顶靶向基序。我们将与项目3 + 4合作，以确定目标图案是否与ATP7B的其他域相互作用如果包含该基序融合到基底外侧蛋白的嵌合体将靶向根尖膜。到确定提议结合靶向基序的蛋白质（x），我们将使用两种方法：一种基于膜的方法酵母 - 两种混合动力系统；以及候选蛋白的假定结合结构域的屏幕。在AIM 2中，我们将确定PDZ蛋白AIPP靶向/将内源性ATP7A靶向/中的机制极化肠Caco-2细胞的基底外侧区域。我们将过表达并使用与AIM 1A中类似的方法并与项目3合作，以确定AIPP1级别是否影响/如何影响 CU出口。在AIM 3中，我们将研究ATOX1的作用，Atox1是Cu-atpases的铜伴侣伴侣及其的作用提议的修饰符FKBP52，在化合物杂合子小鼠的体内体内稳态中（ATOX1 +/-; FKBP +/-）。我们将与项目2和3合作研究这些动物的肝脏，肠和肾脏。