Osteoclast activation in uremic bone disease

描述(摘自申请人的摘要)：空泡V-ATPase(V-ATPase)在真核生物的内吞途径中起着重要的作用 细胞。在某些类型的细胞中，如破骨细胞和某些肾上皮细胞， V-ATPase在高水平表达，是特化的 细胞的功能。在破骨细胞和肾上皮细胞中，V-ATPase 储存在细胞质中的囊泡或小管中，直到细胞遇到 激活信号。然后，V-ATPase被运输到 质膜。尽管这种传输是V-ATPase 都受到监管，直到最近，人们对潜在的机制知之甚少。 V-ATPase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 离子泵与微丝之间的相互作用。这笔赠款将考验 假设V-ATPase和F-肌动蛋白之间的相互作用解释了 V-ATPase在破骨细胞和其他细胞中的运输。结合的相互作用 V-ATPase和F-肌动蛋白之间是由V-ATPase的B亚基介导的； 因此，B亚基代表了肌动蛋白家族中一个新的和独特的成员 结合蛋白。 肌动蛋白和V-ATPase之间的相互作用可以用 代表N-末端结构域的细菌表达的融合蛋白 B亚基。这笔赠款将继续对装订进行定性 V-ATPase与肌动蛋白相互作用的分子和生化研究 技术，目标是了解大范围内的绑定相互作用 细节。有了这些信息，B亚基的突变分子结构 将创建能够与其他VATPase亚基集成以 支持质子泵活动，但缺乏结合肌动蛋白的能力。 这些结构将在破骨细胞培养模型中表达以进行测试 V-ATPase与V-ATPase结合的生理学意义 F-肌动蛋白。具体目标是： I.获取有关F-肌动蛋白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。 II.研究V-ATPase-F-肌动蛋白结合对F-肌动蛋白组织和功能的影响 V-ATPase的酶功能。 测试结合作用在破骨细胞培养模型中的重要性。 因为V-ATPase是所有细胞生命的关键酶，并且一直是 与临床疾病有关，包括骨质疏松症和肾小管 酸中毒，了解它们的调节将对两者都非常重要 基础科学和临床科学。

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.