Bone matrix and bone resorption

描述(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，以及正常和病理性骨重建的机制。SA 1将继续我们发现的一种名为Plekhm1的新的骨化病基因的研究。Sa 2，将定位最后一个未定位的大鼠骨质疏松株OP的遗传损伤。目的1从我们以前的工作中发现了一个新的基因Plekhm1在骨化门牙缺失(Ia)大鼠中发生了致病突变。我们还发现，PLEKHM1移码突变会导致人类的骨化病。IA大鼠和人类患者都有TRAP+、不吸收的破骨细胞，具有有缺陷的褶皱边界(RB)和有缺陷的分泌物。我们的同事最近还在一名低骨量和局灶性硬化症患者中发现了PLEKHM1的一个显性点突变(R714C)，我们认为这是一种功能获得突变。该基因编码一个大的(&gt；1000aa)蛋白，有几个结构域表明具有模块化的功能。Plekhm1以一种依赖于GTP结合的Rab7的方式定位到内体的晚期。Rab7+囊泡通常向核移动，但在OCS中，它们向Rb移动。Plekhm1有一个Run结构域、2个Pleckstrin同源(PH)结构域和一个C-1结构域。IA大鼠蛋白具有完整的Run结构域，缺乏其余蛋白，并且无法与Rab7囊泡定位。我们将研究plekhm1在吸收和非吸收OCS中的定位，并将筛选候选蛋白，以寻找将OC分泌囊泡转移到RB并将其内容分泌到吸收陷窝中以影响骨吸收的复合体中的潜在角色。在SA1.1下，我们将通过共聚焦和免疫电子显微镜、IP和下拉试验、微管破裂、蛋白质交联和高灵敏度质谱分析相结合的方法来研究包括plekhm1和Rab7在内的蛋白质复合体。我们将比较捕食和非捕食的OCS，以及野生型和ia ra OCS。根据SA1.2，我们将研究PH结构域与特定磷脂结合的能力，并通过酶抑制试验确定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R714C突变，位于第二个PH结构域，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甘油调节，并需要锌离子才能发挥其活性。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结构域的特定AA是否影响其重要的生物结合活性。在SA2下，我们将继续定位大鼠OP中最后一个未被定位的成骨突变。OP大鼠有大的、TRAP+的、不起作用的OCS。到目前为止，它的图谱位置和测序结果表明它不在先前已知的骨化病基因中。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，并对包含突变的染色体区间中的基因进行测序。我们目前是F11代，正在为F12繁殖者进行测试。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： 广泛存在的骨病包括骨质疏松症、关节炎、牙周骨病、关节置换松动、肿瘤骨转移等，这些疾病的共同特点是骨丢失超过骨形成。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动的新因素都是治疗的潜在靶点。这项提议旨在研究调控破骨细胞形成和活动的新基因，以更好地了解一般的骨生物学，并有可能找到治疗这些广泛存在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.