PROTEOMICS OF REGULATED EXOCYTOSIS IN OSTEOCLASTS

破骨细胞中调控胞吐作用的蛋白质组学

• 批准号: 7139402
• 负责人: F. Patrick ROSS
• 金额: $ 20.13万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7139402
• 关键词: bone; cysteine endopeptidases; lysosomes; osteoclasts; secretion

DESCRIPTION (provided by applicant): Bone resorption is mediated by a mechanism in which local acidification of the osteoclast-bone interface depends on a vacuolar type H+ATPase charged coupled to the CLC-7 chloride channel. The acidified microenvironment first mobilizes the mineral phase of bone followed by organic matrix digestion via the lysosomal cysteine protease, Cathepsin K. Although the H+ATPase, CLC-7 and cathepsin K are critical to bone resorption, little is known about how they are targeted to the cell's polarized ruffled membrane. Compelling evidence indicates that these three bone resorptive moieties are localized in lysosomes in non-resorbing osteoclasts and are transported to the ruffled border when the cells are activated. Thus, characterizing the molecular mechanisms of the targeted secretion of lysosomes in osteoclasts will promote our understanding of how they degrade bone. With this in mind, we have isolated a lysosome-like vesicular fraction from mature osteoclasts, which contains cathepsin K and LAMP2 (Igp110), a lysosome-associated membrane protein. Separately, we find that targeted disruption in mice of synaptotagmin VII (syt VII), a protein that mediates secretion of lysosomal contents in fibroblasts and the neuro-endocrine cells , inhibits cathepsin K secretion by osteoclasts and bone resorption in vitro and in vivo. Given that 1) cathepsin K is secreted into the resorptive microenvironment of osteoclasts and LAMP2 is localized at the ruffled border of resorbing osteoclasts and 2) syt VII regulates lysosome secretion in fibroblasts and neuroendocrine cells, we hypothesize that osteoclastic bone resorption is mediated by exocytosis of lysosomal-like vesicles, via a mechanism involving sytVII. Thus, our specific aims are to: 1) identify the molecular machinery regulating lysosome trafficking and secretion by detailed proteomic analysis of osteoclast lysosomes and 2) define the mechanisms by which syt VII regulates lysosome secretion and osteoclast function by identifying its binding proteins in osteoclasts.

说明书(申请人提供)：骨吸收是由破骨细胞-骨界面的局部酸化依赖于连接到ClC-7氯通道的空泡型H+ATPase的机制来调节的。酸化的微环境首先动员骨的矿物相，然后通过溶酶体半胱氨酸蛋白酶组织蛋白酶K进行有机基质消化。尽管H+ATPase、ClC-7和组织蛋白酶K对骨吸收至关重要，但人们对它们如何靶向细胞的极化褶皱膜知之甚少。令人信服的证据表明，这三个骨吸收部分定位在非吸收破骨细胞的溶酶体中，当细胞被激活时，它们被运输到褶皱的边缘。因此，研究破骨细胞中溶酶体靶向分泌的分子机制将有助于我们理解破骨细胞如何降解骨。考虑到这一点，我们从成熟的破骨细胞中分离出一种溶酶体样囊泡部分，其中包含组织蛋白酶K和LAMP2(Igp110)，LAMP2是一种溶酶体相关的膜蛋白。另外，我们在小鼠中发现定向干扰突触凝集素VII(Syt VII)，一种介导成纤维细胞和神经内分泌细胞溶酶体内容分泌的蛋白质，在体外和体内抑制破骨细胞分泌组织蛋白酶K和骨吸收。鉴于1)组织蛋白酶K被分泌到破骨细胞的吸收微环境中，LAMP2定位于吸收破骨细胞的褶皱边缘，2)syt VII调节成纤维细胞和神经内分泌细胞中溶酶体的分泌，我们推测破骨细胞性骨吸收是通过sytVII参与的溶酶体样囊泡排出介导的。因此，我们的具体目标是：1)通过对破骨细胞溶酶体的详细蛋白质组学分析，确定调节溶酶体运输和分泌的分子机制；2)通过鉴定其在破骨细胞中的结合蛋白，确定SyT VII调节溶酶体分泌和破骨细胞功能的机制。