Cu Homeostasis and Cu-ATPases in Polarized Epithelia
极化上皮细胞中的 Cu 稳态和 Cu-ATP 酶
基本信息
- 批准号:7133531
- 负责人:
- 金额:$ 31.37万
- 依托单位:
- 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 2009-08-31
- 项目状态:已结题
- 来源:
- 关键词:adenosinetriphosphataseanionsapical membranebiological signal transductioncell population studycellular polaritychloride channelscopperenzyme activityepitheliumgene expressionimmunoelectron microscopyintestinal mucosalaboratory ratliver cellsmetal metabolismmolecular dynamicsmutantprotein protein interactionprotein structure functionproteomics
项目摘要
Our long-term goal is to elucidate the molecular mechanisms of regulated Cu efflux by two mammalian Cu-
ATPases in polarized epithelial cells. ATP7A in intestinal epithelial cells delivers Cu to the circulation
(basolateral environment) and ATP7B in hepatic cells delivers Cu to the bile (apical environment). Mutations
in either protein cause human disease, emphasizing their importance in Cu-homeostasis. Based on
extensive preliminary data in vivo and in vitro, we propose to test three hypotheses. In AIM 1 we
hypothesize that in Cu-loaded hepatic and intestinal cells, Cu-ATPases pump Cu into unique vesicles that
fuse with the basolateral (ATP7A) or apical (ATP7B) plasma membrane (PM) to release "stored" Cu. We will
identify these compartments using immuno-EM and immuno-isolation/proteomic approaches. In AIM 2 we
hypothesize that the chloride channel, CIC4, is an anion shunt for both Cu-ATPases in their secretory and
efflux function/locations. In basal Cu, We will determine if Cu loading of ceruloplasmin in WIF-B cells by
ATP7B and hephaestin in Caco-2 cells by ATP7A occurs in chloride-free conditions and after knock-down of
CIC4. We will also examine the role of CIC4 in Cu efflux from the cells.In Aim 3, we hypothesize that when
Cu levels are elevated, unique structural signals in the N-terminus of ATP7B regulate its trafficking/apical
efflux function. Our test includes biochemically isolating and identifying new protein interactors, elucidating
the role of a newly-identified modulator of Cu efflux (Murr1) on the dynamics/function of wt and selected N-terminal
mutants of exogenous ATP7B, and using the LEG rat model of Wilson Disease to confirm and
extend in vitro findings. Approaches will include RNAi and overexpression of wt modulators, as well as yeast
two hybrid. Also in AIM 2 we hypothesize that when Cu levels are lowered, ATP7A and ATP7B are retrieved
from their distinct compartments through recognition of structural signals in their C-termini. We will use yeast
two hybrid and affinity isolation of putative protein interactors from extracts of relevant tissue.
我们的长期目标是阐明两种哺乳动物铜流出调节的分子机制
极化上皮细胞中的 ATP 酶。肠上皮细胞中的 ATP7A 将铜输送至循环系统
(基底外侧环境)和肝细胞中的 ATP7B 将铜输送至胆汁(顶端环境)。突变
任何一种蛋白质都会导致人类疾病,强调它们在铜稳态中的重要性。基于
根据体内和体外的广泛初步数据,我们建议检验三个假设。在 AIM 1 中,我们
假设在负载 Cu 的肝细胞和肠细胞中,Cu-ATP 酶将 Cu 泵入独特的囊泡中,
与基底外侧 (ATP7A) 或顶端 (ATP7B) 质膜 (PM) 融合,释放“储存的”Cu。我们将
使用免疫电镜和免疫分离/蛋白质组学方法识别这些区室。在《AIM 2》中,我们
假设氯离子通道 CIC4 是 Cu-ATP 酶分泌和分泌过程中的阴离子分流通道。
流出功能/位置。在基础 Cu 中,我们将通过以下方式确定 WIF-B 细胞中铜蓝蛋白的 Cu 负载量:
ATP7A 在 Caco-2 细胞中产生 ATP7B 和 hephaestin,发生在无氯条件下,并且在敲低
CIC4。我们还将研究 CIC4 在 Cu 从细胞流出中的作用。在目标 3 中,我们假设当
Cu 水平升高,ATP7B N 末端的独特结构信号调节其运输/顶端
流出功能。我们的测试包括生化分离和识别新的蛋白质相互作用物,阐明
新鉴定的 Cu 流出调节剂 (Murr1) 对 wt 和选定 N 末端的动力学/功能的作用
外源 ATP7B 突变体,并使用 LEG 威尔逊病大鼠模型来确认和
扩展体外研究结果。方法包括 RNAi 和 wt 调节剂以及酵母的过度表达
两个杂交种。同样在 AIM 2 中,我们假设当 Cu 水平降低时,ATP7A 和 ATP7B 会恢复
通过识别其 C 末端的结构信号,将其从不同的区室中分离出来。我们将使用酵母
从相关组织提取物中对假定的蛋白质相互作用物进行两种杂交和亲和分离。
项目成果
期刊论文数量(0)
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Ann L Hubbard其他文献
Genetic, metabolic and cellular factors influencing intracellular localization of the Wilson disease protein, ATP7B
- DOI:
10.1186/1755-8166-7-s1-p68 - 发表时间:
2014-01-21 - 期刊:
- 影响因子:1.400
- 作者:
Arnab Gupta;Ashima Bhattacharjee;Nesrin Hasan;Lita Braiterman;Svetlana Lutsenko;Ann L Hubbard - 通讯作者:
Ann L Hubbard
Ann L Hubbard的其他文献
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{{ truncateString('Ann L Hubbard', 18)}}的其他基金
Cu Homeostasis and Cu-ATPases in Polarized Epithelia
极化上皮细胞中的 Cu 稳态和 Cu-ATP 酶
- 批准号:
7487972 - 财政年份:2007
- 资助金额:
$ 31.37万 - 项目类别:
AMT DUAL CAMERA SYST: PARASITES: PLASMODIUM, C FASCICULATA, T BRUCEI
AMT 双摄像头系统:寄生虫:疟原虫、束状疟原虫、布氏疟原虫
- 批准号:
7166513 - 财政年份:2005
- 资助金额:
$ 31.37万 - 项目类别:
AMT DUAL CAMERA: WILSON DIS PROTEIN, CORONAVIRUS & INFECTIOUS BRONCHITIS VIRUS
AMT 双摄像头:Wilson DIS 蛋白质、冠状病毒
- 批准号:
7166511 - 财政年份:2005
- 资助金额:
$ 31.37万 - 项目类别:
AMT DUAL CAMERA: ID GENE & PROTEINS IN BRAIN DVMT & RETINAL PIGMENTED EPITHELIUM
AMT 双摄像头:ID GENE
- 批准号:
7166512 - 财政年份:2005
- 资助金额:
$ 31.37万 - 项目类别:
AMT DUAL CAMERA SYST: BACTERIOLOGY: MYXOCOCCUS XANTHUS, YERSINA ENTEROCOLITICA
AMT 双摄像头系统:细菌学:黄色粘球菌、小肠结肠炎耶尔森氏菌
- 批准号:
7166510 - 财政年份:2005
- 资助金额:
$ 31.37万 - 项目类别:
AMT DUAL CAMERA SYST: GENOMICS & PROTEOMICS, WOUND RE-EPITHELIALIZATION
AMT 双摄像头系统:基因组学
- 批准号:
7166509 - 财政年份:2005
- 资助金额:
$ 31.37万 - 项目类别:
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