Bone matrix and bone resorption

描述（由申请人提供）：本项目在两个特定目标下研究破骨细胞（OC）生物学以及正常和病理骨重塑的机制。SA 1将继续研究我们发现的一种名为Plekhm1的新型骨质疏松基因。sa2将绘制最后一个未绘制的大鼠骨质疏松菌株（op. Aim 1）的遗传病变。Aim 1来自我们之前的工作，该工作发现在骨质疏松门牙缺失（ia）的大鼠中存在一个新基因Plekhm1的致病突变。我们还发现PLEKHM1移码突变导致人类骨质疏松症。ia大鼠和人类患者均有TRAP+、非吸收性破骨细胞，伴有皱褶边界缺陷和分泌缺陷。我们的同事最近也在一例低骨量局灶性硬化症患者中发现了PLEKHM1的显性点突变（R714C），我们认为这是一种功能获得突变。该基因编码一个大的（>1000 aa）蛋白，具有多个结构域，表明其功能模块化。Plekhm1以依赖于gtp结合的Rab7的方式定位于核内体的晚期。Rab7+囊泡通常向细胞核移动，但在OCs中，它们向RB移动。Plekhm1有一个RUN结构域、2个pleckstrin同源结构域（PH）和一个C-1结构域。ia大鼠蛋白有一个完整的RUN结构域，缺乏其余的蛋白，不能与Rab7小泡定位。我们将研究plekhm1在骨吸收与非骨吸收OC中的定位，并筛选候选蛋白，以确定其在将OC分泌囊泡转移到RB并将其内容物分泌到骨吸收腔隙中以影响骨吸收所需的复合物中的潜在作用。在SA1.1下，我们将通过共聚焦和免疫电镜、IP和下拉试验、微管破坏、蛋白质交联和高灵敏度质谱法的组合来研究包括plekhm1和Rab7的蛋白质复合物。我们将比较吸收和非吸收oc以及野生型oc和野生型oc。在SA1.2下，我们将研究PH结构域结合特定磷脂的能力，并通过酶抑制实验确定plekhm1的活性是否像许多OC功能一样受到pi3激酶的调节。我们还将研究位于第二PH结构域的R714C突变，以确定其对脂质结合特异性和强度的影响。我们将研究C-1结构域，以确定它是否受二酰基甘油调节，并需要锌离子来维持其活性。我们还将研究候选氨基酸点突变的影响，以确定PH和C-1结构域的特异性aa是否影响其重要的生物结合活性。在SA2下，我们将继续绘制大鼠op中最后一个未绘制的骨质疏松突变。op大鼠具有较大的TRAP+无功能oc。迄今为止，它的图谱位置和测序结果表明它不在先前已知的骨质疏松基因中。我们正在维持一个正在进行的异交育种项目，以缩小候选区域，并在包含突变的染色体间隔内对基因进行测序。我们目前在F11代，正在测试F12育种者。当我们确定突变时，无疑将导致对该基因及其在破骨细胞生物学中的作用的进一步研究。公共卫生相关性：

DESCRIPTION (provided by applicant): This project investigates osteoclast (OC) biology, and mechanisms of normal and pathological bone remodeling under 2 Specific Aims. SA 1 will continue our investigations of a novel osteopetrosis gene we discovered called Plekhm1. SA 2, will map the genetic lesion in the last unmapped rat osteopetrotic strain, op. Aim 1 grows from our previous work which found the causative mutation in the osteopetrotic incisors absent (ia) rat in a novel gene, Plekhm1. We also showed that a PLEKHM1 frameshift mutation causes osteopetrosis in humans. Both ia rats and the human patient have TRAP+, non-resorbing osteoclasts with defective ruffled borders (RB) and defective secretion. Our colleagues also recently discovered a dominant point mutation (R714C) in PLEKHM1 in a patient with low bone mass and focal sclerosis which we propose is a gain-of- function mutation. The gene encodes a large (>1000 aa) protein with several domains suggesting modularized function. Plekhm1 localizes late to endosomes in a manner dependent on GTP-bound Rab7. Rab7+ vesicles normally move toward the nucleus, but in OCs they move to the RB. Plekhm1 has a RUN domain, 2 pleckstrin homology (PH) domains, and a C-1 domain. The ia rat protein has an intact RUN domain, lacks the rest of the protein, and fails to localize with Rab7 vesicles. We will investigate the localization of plekhm1 in resorbing vs. non-resorbing OCs and will screen candidate proteins for potential roles in a complex needed to translocate OC secretory vesicles to the RB and secrete their contents into the resorption lacuna to effect bone resorption. Under SA1.1, we will investigate the protein complex that includes plekhm1 and Rab7 by a combination of confocal and immunoelectron microscopy, IP and pull-down assays, microtubule disruption, protein crosslinking and high-sensitivity mass spectrophotometry. We will compare resorbing and non-resorbing OCs and wild type vs. ia ra OCs. Under SA1.2, we will study the ability of the PH domains to bind specific phospholipids and determine by enzyme inhibition assays if plekhm1 activity, like many OC functions, is regulated by PI3-kinase. We will also study the R714C mutation, located in the 2nd PH domain, to determine its impact on lipid binding specificity and strength. We will investigate the C-1 domain to determine if it is regulated by diacylglycerol and requires zinc ions for its activity. We will also study the effects of point mutations of candidate amino acids to determine if specific aa in the PH and C-1 domains impact their important biological binding activities. Under SA2, we will continue to map the last remaining unmapped osteopetrotic mutation in the rat, op. The op rat has large, TRAP+, non-functioning OCs. Its map position and sequencing results to date indicate it is not in a previously known osteopetrosis gene. We are maintaining an ongoing out-crossed breeding project to narrow the candidate region and are sequencing genes in the chromosome interval containing the mutation. We are currently at generation F11 and are testing for F12 breeders. When we identify the mutation, it will undoubtedly lead to additional investigations of the gene and its role in osteoclast biology. PUBLIC HEALTH RELEVANCE: 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 every new factor we discover that affects osteoclast activity is a potential target for therapeutics. This proposal aims to investigate new genes that regulate the formation and activity of osteoclasts to better understand bone biology in general, and potentially to find new ways to treat these widespread bone disorders.