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

• 批准号: 8379309
• 负责人: Ann Hubbard
• 金额: $ 37.52万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8379309
• 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; Diet; Disease; Environment; Epithelial Cells; Epithelium; 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; Principal Investigator; 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; mutant; protein expression; protein transport; research study; stem; trafficking

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 targefing 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 confinue 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 mutafions, 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 interacfions 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 characterizafion 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. RELEVANCE (See instructions): Copper-transporting ATPases, ATP7A and ATP7B, are important In health and disease. Hepatic ATP7B ; normally exports excess copper from the liver. In the absence of ATP7B function, affected people develop j Wilson Disease, which is due to copper overioad and is characterized by liver damage and/or abnormal ; neurological symptoms. Thus, understanding the dynamics of hepafic ATP7B is important. ¿:

我们的长期目标是阐明两个哺乳动物Cu-atpase的分子机制 调节铜稳态在两极分子的细胞中。 需要以铜敏感和可逆的方式进行细胞内再分配或贩运。1） 他们将铜运送到分泌物中，以金属合成的鸡蛋酶2 同样出口铜。 环境），肝细胞中的ATP7B为胆汁（顶端环境）提供了过多的CU。 我们将重点放在肝cu-atpase上，例如其他膜运输蛋白。 在此PPG中，ATP7B以受调节方式运输到两极分化的上皮层的顶端区域 肝细胞。 ATP7B的铜敏感性TARGEFING 它在顶端Membne使用的出口机制目前是基于两个的新结果。 多年的PPG资金，我们将在Wif-B细胞和AIM 1中进行研究。 ATP7B本身中调节其铜偏侧动力学的序列和域 靶向（L）出口）。 ：（ WD） - 导致GFP-ATP7B Mutafions，在WLF-B中表达蛋白质表达和运输+/- CU， 在MNK y/-cell中（无ATP7A或ATP7B），我们将确定分子内分子。 通过分析7b/7a嵌合体表达/运输表型，ATP7B的间隔 对AIM 2中选定突变体的FRAP研究，我们将继续识别，隔离和功能 速度狂欢的内源性ATP7B（和Cu（l）？）的表征到肝细胞的顶端区域 加载的小鼠。 通过精心研究的Rablla Apill Express选择的贩运。 ATP7B - / - 小鼠中的WD-ATP7B突变体确定体内突变蛋白的Cu（L）导出表型。 相关性（请参阅教学）： 铜转运ATPases，ATP7A和ATP7B在健康和疾病中很重要 通常，出口在没有ATP7B功能的情况下从肝脏中退出 J Wilson病，该病是由于铜过量的，其特征是肝损伤和/或异常 因此，神经症状。 挥之不去：