Bone matrix and bone resorption

描述（由申请人提供）：此申请是对NOT-OD-09-058的回应，“NIH宣布为竞争性修订申请提供恢复法案资金。”这是一项竞争性修订申请RO1 DE 007444，“骨基质和骨吸收”，我们正在研究我们发现的一种名为plekhm1的蛋白质的破骨细胞生物学。有证据表明，plekhm1是破骨细胞正常转运、分泌和骨吸收所必需的。在大鼠模型和人类患者中，截断plekhm1蛋白的突变会导致骨质疏松症和破骨细胞失活。相反，我们的合作者最近在一名人类患者中发现了一个点突变R714C，它似乎会导致过度的破骨细胞活性。这导致破骨细胞分泌活性增加和骨骼骨质减少。目前的项目正在研究与plekhm1相互作用的结合伙伴，以控制破骨细胞中的囊泡运动和分泌，以及生物活性脂质对其的调节。这个建议将给我们正在进行的项目增加一个新的具体目标。我们假设plekhm1-R714C突变是导致过度骨质流失的显性功能获得突变。我们将在新的特定目标下通过生成小鼠模型来验证这一假设，在该模型中，内源性的野生型plekhm1被plekhm1- r714c基因敲入取代。采用DEXA、pQCT和microCT对小鼠骨骼表型进行评价。还将测量血清TRAP水平，以确定小鼠系表型与点突变杂合的人类患者的程度。透射电镜和免疫电镜研究将检查突变的破骨细胞。来自敲入小鼠的细胞将用于研究点突变引起的破骨细胞生物学和plekhm1结合和调节的变化。新聘博士后1名，为期2年。我们也在申请资金购买一台荧光显微镜，用它我们既可以对细胞培养中的标记蛋白表达进行常规筛选，也可以进行反卷积共聚焦成像实验，以筛选与plekhm1共定位的候选蛋白，plekhm1是破骨细胞正常囊泡运输、分泌和骨吸收所需的复合体的一部分。

DESCRIPTION (provided by applicant): This application is in response to NOT-OD-09-058, "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications." It is a competitive revision request for grant RO1 DE 007444, "Bone matrix and bone resorption," under which we are investigating the osteoclast cell biology of a protein we discovered called plekhm1. Evidence indicates that plekhm1 is required for normal trafficking of vesicles, secretion, and bone resorption by osteoclasts. Mutations that truncate the plekhm1 protein cause osteopetrosis and inactive osteoclasts in a rat model and in human patients. In contrast, a point mutation, R714C, was recently discovered by our collaborators in a human patient, which appears to cause excessive osteoclast activity. This leads to increased secretory activity by osteoclasts and an osteopenic skeleton. The current project is examining binding partners that interact with plekhm1 to control vesicle movement and secretion in osteoclasts, as well as its regulation by bioactive lipids. This proposal would add a new specific aim to our ongoing project. We hypothesize that the plekhm1-R714C mutation is a dominant, gain-of-function mutation that leads to excessive bone loss. We will test this hypothesis under the new specific aim by generating a mouse model in which the endogenous, wild type plekhm1 is replaced by gene knock-in with plekhm1-R714C. The skeletal phenotype of the mice will be evaluated by DEXA, pQCT and microCT. Serum TRAP levels will also be measured to determine the extent to which the mouse line phenocopies the human patient who is heterozygous for the point mutation. TEM and immune-EM studies will examine mutant osteoclasts. Cells derived from the knock-in mice will be used to investigate the alterations in osteoclast biology and plekhm1 binding and regulation caused by the point mutation. A new post-doctoral fellow will be hired to carry out this project, which will last for 2 years. We are also requesting funds to purchase a fluorescence microscope with which we can both perform routine screening for tagged protein expression in cell cultures and perform deconvolution confocal imaging experiments to screen candidate proteins for co-localization with plekhm1, as part of a complex required for normal vesicle trafficking, secretion, and bone resorption by osteoclasts. PUBLIC HEALTH RELEVANCE: Project narrative: Widespread bone disorders, including osteoporosis, arthritis, periodontal bone disease, joint replacement loosening, and tumor metastasis to bone, all have in common bone loss that exceeds bone formation. Bone loss is carried out by cells called osteoclasts, and the more deeply we understand how osteoclasts work, the more opportunities will arise for potential therapeutics to control their activity. This proposal aims to investigate a new gene we discovered which causes bone disease in animal models and in human patients. Deeper understanding of this gene and how it controls the bone resorbing activity of osteoclasts will give a better knowledge of bone biology in general, and potentially will lead to new ways to treat these all-too-common bone disorders.