Mineralization of the matrix in disease and health

描述(由申请人提供)： 遗传性和肿瘤获得性骨矿障碍提供了强大的模型，帮助理解了一些负责维持健康、动态、矿化骨骼的分子机制。X连锁低磷血症性软骨病(HYP)就是这样一种疾病，它与严重的矿化、肾脏磷酸盐转运和维生素D代谢缺陷有关。骨基质蛋白MEPE和成骨细胞蛋白水解酶在HYP中显著升高。此外，PHEX是一种成骨细胞表达的质膜锚定的锌金属肽酶，是导致这种疾病的主要缺陷。我们有大量的初步数据支持这样的假设，即MEPE和PHEX之间的蛋白质-蛋白质相互作用调节矿化。在体外，PHEX保护MEPE免受蛋白水解酶(尤其是组织蛋白酶B)的切割，而成骨细胞蛋白水解酶在Hyp中升高。在没有PHEX的情况下，MEPE被切割，释放出一个磷酸化的、酸性的、富含丝氨酸的天冬氨酸C末端片段(ASARM-肽)。ASARM多肽(2 KDa)对大量的蛋白酶具有显著的抵抗力，导致Hyp水平升高。ASARM-肽抑制矿化，ASARM-肽升高可能是矿化缺陷的全部或部分原因。这一应用将集中在MEPE和ASARM-肽在矿化中的作用。本研究的具体目的是：1.体外和体内正常和高血压成骨细胞及血清中MEPE-ASARM-肽(S)的结构表征和定量。2.测定MEPE ASARM多肽的体内外矿化作用。3.阐明了MEPE-PHEX蛋白-蛋白质之间的相互作用。4.升高的蛋白水解酶(S)在成骨细胞异常矿化和MEPE-PHEX相互作用中的作用。ASARM基序存在于许多牙齿-骨基质和唾液蛋白中。这些基因包括MEPE、DMP1、骨桥蛋白、Statherin、DSPP，它们都位于染色体4q21上。因此，阐明其在矿化中的作用对于了解骨成矿机制具有重要意义。重要的是，Motif扮演的角色将与治疗和理解骨折风险、骨质疏松、肿瘤引起的骨软化(OHO)、高血压、牙周病、牙齿矿化障碍、骨、肾结石和动脉异位钙化以及肾性骨营养不良有关。

DESCRIPTION (provided by applicant): Inherited and tumor acquired bone-mineral disorders have provided powerful models that have helped understand some of the molecular-mechanisms responsible for maintaining a healthy, dynamic, mineralized-skeleton. X-linked hypophosphatemic rickets (HYP) is one such disease and is associated with severe defects in mineralization, renal phosphate handling, and vitamin D metabolism. MEPE, (a bone-matrix protein) and osteoblastic-proteases are elevated markedly in HYP. Also, PHEX, an osteoblast-expressed, plasma-membrane anchored Zn-metallopeptidase is the primary defect responsible for the disease. We have substantive preliminary data that supports the hypothesis that a protein-protein interaction between MEPE and PHEX regulates mineralization. PHEX protects MEPE from proteolytic cleavage by proteases (notably cathepsin B) in-vitro and osteoblastic-proteases are elevated in Hyp. In the absence of PHEX, MEPE is cleaved, releasing a phosphorylated, acidic, serine-aspartate rich, C-terminal fragment (ASARM-peptide). The ASARM-peptide (2 kDa), is remarkably resistant to a vast array of proteases resulting in increased levels in Hyp. The ASARM-peptide inhibits mineralization and elevated ASARM-peptide is proposed to be wholly or in part responsible for the mineralization defects. This application will focus on the role of MEPE and ASARM-peptides in mineralization. Our specific aims are: 1. Structural characterization and quantification of MEPE ASARM-peptide(s) in-vitro and in-vivo in normal and Hyp osteoblasts and serum. 2. Determination of MEPE ASARM-peptide effects on mineralization in-vitro and in-vivo. 3. Elucidation of MEPE-PHEX protein-protein interactions. 4. The role of elevated protease(s) in Hyp-osteoblasts in relation to abnormal mineralization and MEPE-PHEX interactions. The ASARM-motif is present in a number of tooth-bone matrix and salivary proteins. These include MEPE, DMP1, osteopontin, statherin, DSPP that all map to chromosome 4q21. Thus, the elucidation of its role in mineralization is of prime importance for understanding bone mineralization mechanisms. Importantly, the part the motif plays will have relevance to the treatment and understanding of bone-fracture risk, osteoporosis, tumor-induced osteomalacia (OHO), HYP, periodontal disease, disorders of mineralization in teeth, bone, renal-stones and ectopic calcifications in arteries and renal-osteodystrophy.