PROTEOMICS OF REGULATED EXOCYTOSIS IN OSTEOCLASTS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 骨吸收是由一种机制介导的，其中破骨细胞-骨界面的局部酸化取决于与CLC-7氯离子通道电荷偶联的空泡型H+ ATP酶。酸化的微环境首先动员骨的矿物质相，然后通过溶酶体半胱氨酸蛋白酶组织蛋白酶K消化有机基质。虽然H+ ATP酶、CLC-7和组织蛋白酶K对骨吸收至关重要，但对它们如何靶向细胞的极化皱褶膜知之甚少。令人信服的证据表明，这三个骨吸收部分位于非吸收破骨细胞的溶酶体中，并在细胞活化时被转运到皱褶边缘。因此，表征破骨细胞中溶酶体靶向分泌的分子机制将促进我们对它们如何降解骨的理解。考虑到这一点，我们已经从成熟的破骨细胞中分离出了溶酶体样囊泡组分，其中含有组织蛋白酶K和LAMP 2（Igp 110），一种溶酶体相关的膜蛋白。另外，我们发现，有针对性的破坏在小鼠的突触结合蛋白VII（SYT VII），一种蛋白质，介导的溶酶体内容物在成纤维细胞和神经内分泌细胞的分泌，抑制组织蛋白酶K的分泌，破骨细胞和骨吸收在体外和体内。鉴于1）组织蛋白酶K分泌到破骨细胞的吸收微环境中，并且LAMP 2定位于吸收破骨细胞的皱褶边缘，以及2）syt VII调节成纤维细胞和神经内分泌细胞中的溶酶体分泌，我们假设骨细胞的骨吸收是通过涉及syt VII的机制由溶酶体样囊泡的胞吐作用介导的。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。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.