Cu homeostasis by the Cu-ATPase, ATPase, ATP7B in Polarized Hepatic Cells

极化肝细胞中 Cu-ATPase、ATPase、ATP7B 的 Cu 稳态

基本信息

批准号：
8543707
负责人：
Ann Hubbard
金额：
$ 36.15万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-15 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8543707
关键词：
ATP phosphohydrolase Adenoviruses Affect Apical B-Lymphocytes Behavior Bile fluid Binding Biochemical Biotinylation Blood Circulation Cells Characteristics Chimera organism Co-Immunoprecipitations Collaborations Copper Cytoplasm Cytoplasmic Tail Destinations Diet Disease Environment Epithelial Cells Epithelium Exhibits Funding Goals Golgi Apparatus Health Hepatic Hepatocyte Hepatolenticular Degeneration Homeostasis Image Instruction Intestines Ions Kidney Liver Lung Mediating Membrane Membrane Transport Proteins Metals Methods Missense Mutation Modeling Molecular Mus Mutation N-terminal Nature Neurologic Pathway interactions Phenotype Point Mutation Property Proteins Proteomics Recycling Signal Transduction Surface Symptoms Testing Tissues Vesicle Wilson disease protein Work antiporter apical membrane base copper-transporting ATPase disease-causing mutation human disease in vivo liver injury mutant protein expression protein transport research study stem trafficking

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. 1) They transport copper Into the secretory pathway to metallate newly-synthesized cuproenzymes. 2) They also export 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). In this PPG, we will focus on the hepatic Cu-ATPase, ATP7B. Like the other membrane transport proteins being studied in this PPG, ATP7B trafficks In a regulated manner to the apical region of a polarized epithelial cell, the hepatocyte. Once there, it exports the metal ion Cu(l) Into bile. But the signals/mechanism(s) mediating the copper-sensitive targeting of ATP7B, the functional nature ofthe vesicles carrying the protein and the Cu(l) export mechanisms it uses at the apical membrane are currently unknown. Based on new results from two years of PPG funding, we will continue our studies in WIF-B cells and in vivo. In Aim 1, we will identify sequences and domains in ATP7B itself that regulate its copper-dependent dynamics (TGN-retention, apical targeting and Cu(l) export). We will generate adenoviruses encoding C- and new N-terminal Wilson Disease :(WD)-causing GFP-ATP7B mutations, express them in WlF-B for protein expression and trafficking +/- Cu, and In MNK y/- cells (no ATP7A or ATP7B) for their Cu(l)-loading activity. We will determine Intramolecular interactions of ATP7B through analysis of 7B/7A chimera expression/trafficking phenotypes. We will perform FRAP studies on selected mutants. In Aim 2, we will continue Identification, isolation and functional characterization of vesicles carrying endogenous ATP7B (and Cu(l)?) to the apical region of hepatocytes of copper-loaded mice. We will also identify ATP7B protein interactors +/- Cu. We will determine if ATP7B trafficks via the well-studied Rablla apical recycling compartment. Finally, we will express selected WD-ATP7B mutants in ATP7B-/- mice to determine the mutant protein's Cu(l) export phenotype in vivo.

我们的长期目标是阐明两种哺乳动物Cu-atpases调节偏振上皮细胞中铜稳态的分子机制。这些Cu-atpass具有两个功能，需要以铜敏感和可逆的方式进行细胞内的再分配或运输。 1）他们将铜运输到分泌途径，以金属合成的库酶。 2）他们还出口铜。肠上皮细胞中的ATP7A可为循环（基底外侧环境）提供饮食中的CU，而肝细胞中的ATP7B向胆汁（顶部环境）提供了过多的Cu。在此PPG中，我们将专注于肝Cu-ATPase ATP7B。就像在该PPG中研究的其他膜转运蛋白一样，ATP7B运输的方式受到调节的方式，向极化上皮细胞的顶端区域，即肝细胞。到达那里后，它将金属离子铜（L）导出到胆汁中。但是，目前尚不清楚介导ATP7B的铜敏感靶向的信号/机制，即ATP7B的铜敏感性，携带蛋白质的囊泡的功能性质和它在顶端膜上使用的Cu（L）导出机制目前尚不清楚。基于两年PPG资金的新结果，我们将继续在Wif-B细胞和体内进行研究。在AIM 1中，我们将在ATP7B本身中确定调节其铜依赖性动力学（TGN-驱动，顶端靶向和CU（L）导出）的序列和域。我们将生成编码C-和新的N末端Wilson病的腺病毒：（WD）引起GFP-ATP7B突变，在WLF-B中表达它们以用于蛋白质表达和运输+/- CU，以及MNK Y/ - 细胞（no） ATP7A或ATP7B）用于其Cu（L）加载活动。我们将通过分析7b/7a嵌合体表达/运输表型来确定ATP7B的分子内相互作用。我们将对选定的突变体进行FRAP研究。在AIM 2中，我们将继续鉴定，分离和功能表征，这些囊泡携带内源性ATP7B（和Cu（L）？）到铜载体小鼠的肝细胞的顶端区域。我们还将识别ATP7B蛋白质相互作用+/- CU。我们将确定ATP7B的贩运是否通过研究精心的Rablla顶端回收室。最后，我们将在ATP7B - / - 小鼠中表达选定的WD-ATP7B突变体，以确定体内突变蛋白的Cu（L）导出表型。