Bone matrix and bone resorption

描述（由申请人提供）：本项目研究破骨细胞（OC）生物学，以及正常和病理性骨重建的机制，有两个特定的目的。SA 1将继续研究我们发现的一种名为Plekhm 1的新的骨硬化症基因。SA 2，将在最后一个未定位的大鼠骨硬化品系中定位遗传病变，op. Aim 1来自我们以前的工作，该工作发现了新基因Plekhm 1中骨硬化切牙缺失（ia）大鼠的致病突变。我们还发现PLEKHM 1移码突变导致人类骨硬化症。IA大鼠和人类患者都具有TRAP+，非再吸收破骨细胞，具有缺陷性皱褶边缘（RB）和缺陷性分泌。我们的同事最近还发现了一个显性点突变（R714 C）在PLEKHM 1在低骨量和局灶性硬化症的患者，我们提出这是一个功能获得性突变。该基因编码一个大的（>1000 aa）蛋白质，具有几个结构域，表明模块化功能。Plekhm 1以依赖于GTP结合Rab 7的方式定位于内体晚期。Rab 7+囊泡通常向细胞核移动，但在OC中，它们移动到RB。Plekhm 1具有RUN结构域、2个普列克底物蛋白同源（PH）结构域和C-1结构域。IA大鼠蛋白具有完整的RUN结构域，缺乏蛋白的其余部分，并且不能与Rab 7囊泡一起定位。我们将调查的本地化的plekhm 1吸收与非吸收OC，并将筛选候选蛋白质的潜在作用，在一个复杂的需要易位OC分泌囊泡的RB和分泌到吸收陷窝的内容，影响骨吸收。在SA1.1下，我们将通过共聚焦和免疫电子显微镜、IP和下拉测定、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plekhm 1和Rab 7的蛋白质复合物。我们将比较再吸收和非再吸收OC以及野生型与IA RA OC。在SA1.2下，我们将研究PH结构域结合特定磷脂的能力，并通过酶抑制试验确定plekhm 1活性是否与许多OC功能一样受PI 3激酶调节。我们还将研究位于第二PH结构域的R714 C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，以确定它是否受甘油二酯的调节，并需要锌离子的活性。我们还将研究候选氨基酸的点突变的影响，以确定PH和C-1结构域中的特定aa是否影响其重要的生物结合活性。在SA 2下，我们将继续绘制大鼠中最后一个未绘制的骨硬化突变，op大鼠具有大的TRAP+无功能OC。它的图谱位置和测序结果表明，它不是在以前已知的骨硬化症基因。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，并对含有突变的染色体间隔中的基因进行测序。我们目前正在进行F11代育种，并正在测试F12育种者。当我们确定了突变，它无疑将导致更多的调查基因及其在破骨细胞生物学的作用。公共卫生相关性： 包括骨质疏松症、关节炎、牙周骨疾病、关节置换松动和肿瘤骨转移在内的广泛的骨疾病都具有超过骨形成的共同骨丢失。骨丢失是由称为破骨细胞的细胞进行的，我们发现的每一个影响破骨细胞活性的新因素都是治疗的潜在目标。该提案旨在研究调节破骨细胞形成和活性的新基因，以更好地了解骨骼生物学，并可能找到治疗这些广泛的骨骼疾病的新方法。

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.