Osteoclast activation in uremic bone disease

尿毒症骨病中的破骨细胞活化

• 批准号: 6915600
• 负责人: LEXIE Shannon HOLLIDAY
• 金额: $ 24.02万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-17 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6915600
• 关键词: actins; adenosinetriphosphatase; bone metabolism; endocytosis; enzyme activity; enzyme mechanism; hydrogen transport; intracellular transport; laboratory mouse; microfilaments; osteoclasts; protein binding; protein protein interaction; protein transport; site directed mutagenesis; tissue /cell culture

DESCRIPTION (Adapted from the Applicant's Abstract): Vacuolar V-ATPases (V-ATPases) have an essential role in the endocytic pathways of eukaryotic cells. In some cell types, like osteoclasts and certain renal epithelial cells, V-ATPases are expressed at high levels and are required for the specialized functions of the cells. In osteoclasts and renal epithelial cells, V-ATPases are stored in vesicles or tubules in the cytoplasm until the cell encounters an activating signal. V-ATPases are then transported to specialized domains of the plasma membrane. Although this transport is a crucial means by which V-ATPases are regulated, until recently little was known about the underlying mechanisms. A specific interaction between V-ATPases and filamentous actin (F-actin) has been identified by our lab. This represents the first example of a direct interaction between an ion pump and microfilaments. This grant will test the hypothesis that interaction between V-ATPase and F-actin accounts for the transport of V-ATPases in osteoclasts and other cells. The binding interaction between V-ATPase and F-actin is mediated by the B-subunit of V-ATPase; the B-subunit thus represents a new and unique member of the family of actin binding proteins. The interaction between actin and V-ATPase can be reconstituted using bacterially-expressed fusion proteins representing the N-terminal domains of B-subunits. This grant will continue characterization of the binding interaction between V-ATPase and actin using molecular and biochemical techniques with the goal of understanding the binding interaction in great detail. With this information, mutated molecular constructs of the B-subunit will be created which are able to integrate with other VATPase subunits to support proton pumping activity, but which lack the capacity to bind actin. These constructs will be expressed in an osteoclast cell culture model to test the physiologic importance of the binding interaction between V-ATPase and F-actin. The specific aims are: I. To obtain detailed molecular information about the F-actin binding site on the B-subunit, and generate mutant B subunits that lack actin binding but retain catalytic activity. II. To study effects of V-ATPase-F-actin binding on F-actin organization and V-ATPase enzymatic function. III. To test the importance of the binding interaction in osteoclast cell culture models. Because V-ATPases are crucial enzymes to the lives of all cells, and have been implicated in clinical disorders including osteoporosis and renal tubular acidosis, understanding their regulation will be of great importance for both basic and clinical science.

说明书（改编自申请人的摘要）：极性V-ATP酶 （V-ATP酶）在真核生物的内吞途径中具有重要作用。 细胞在某些细胞类型中，如破骨细胞和某些肾上皮细胞， V-ATP酶以高水平表达，并且是专门的细胞所必需的。 细胞的功能。在破骨细胞和肾上皮细胞中， 储存在细胞质中的小泡或小管中，直到细胞遇到 激活信号V-ATP酶然后被运输到细胞的专门结构域， 质膜尽管这种转运是V-ATP酶 是受调控的，直到最近，人们对潜在的机制知之甚少。 V-ATP酶和丝状肌动蛋白（F-肌动蛋白）之间的特异性相互作用， 已经被我们的实验室确认了这是第一个直接 离子泵和微丝之间的相互作用。这一举措将考验 假设V-ATP酶和F-肌动蛋白之间的相互作用解释了 V-ATP酶在破骨细胞和其它细胞中的转运。的结合相互作用 V-ATP酶与F-actin之间的相互作用是由V-ATP酶的B亚基介导的， 因此，B亚基是肌动蛋白家族中一个新的独特成员 结合蛋白 肌动蛋白和V-ATP酶之间的相互作用可以用 细菌表达的融合蛋白，其代表 B亚单位这项赠款将继续表征的约束力 V-ATP酶与肌动蛋白相互作用分子生物学研究 技术的目标是了解绑定的相互作用， 详细有了这些信息，B亚基的突变分子结构 能够与其他VATP酶亚基整合， 支持质子泵活性，但缺乏结合肌动蛋白的能力。 这些构建体将在破骨细胞培养模型中表达，以测试 V-ATP酶和 肌动蛋白具体目标是： I.为了获得关于F-actin结合位点的详细分子信息， B亚基，并产生缺乏肌动蛋白结合但 保持催化活性。 二.研究V-ATP酶-F-actin结合对F-actin组织结构的影响， V-ATPase酶功能。 三.在破骨细胞培养模型中测试结合相互作用的重要性。 因为V-ATP酶是所有细胞生命的关键酶， 与包括骨质疏松症和肾小管 酸中毒，了解他们的调节将是非常重要的， 基础和临床科学。

项目成果

Vacuolar H+-ATPases (V-ATPases) as therapeutic targets: a brief review and recent developments.

• DOI: 10.21037/biotarget.2017.12.01
• 发表时间: 2017-12-01
• 期刊: Biotarget
• 作者: Holliday, L Shannon

Regulation of vacuolar H(+)-ATPase in microglia by RANKL.

• DOI: 10.1016/j.bbrc.2009.08.122
• 发表时间: 2009-11-06
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Serrano, Eric M.;Ricofort, Ryan D.;Zuo, Jian;Ochotny, Noelle;Manolson, Morris F.;Holliday, L. Shannon
• 通讯作者: Holliday, L. Shannon