Mineralization of the matrix in disease and health

描述(申请人提供)：从海洋向陆地过渡的生命的一个古老遗产是骨骼和肾脏在调节矿化和Ca/PO4动态平衡方面的作用。骨肾轴中的关键角色是一组相关的细胞外基质蛋白(兄弟姐妹)。这些蛋白质都定位在染色体4Q上的一个紧密聚集的区域。一种独特的蛋白PHEX，间接或直接调节至少两个兄弟蛋白(MEPE和DMP1)。PHEX是一种寻找底物的锌金属内肽酶，也可能作为孤儿配体或受体发挥作用。PHEX功能的丧失会导致X连锁低磷血症性软骨病，而一种新的细胞因子FGF23(一种磷酸素)在磷酸盐表型中起主要作用。我们的数据证实了PHEX与MEPE的直接结合，MEPE是一种在骨骼、牙齿和肾脏近端曲管中表达的兄弟蛋白。这些蛋白质之间的结合发生在PHEX锌结合基序和MEPE的一个称为ASARM基序的短COOH末端区域之间。这个短小的区域对蛋白质分解具有极强的抵抗力，并且在物种间是保守的。从生物学上讲，释放的ASARM多肽是一种有效的矿化抑制剂，抑制肾脏磷酸盐的处理，并与羟基磷灰石结合。PHEX与MEPE的结合可能有助于调节ASARM多肽的释放，从而局部控制矿化。PHEX还与游离ASARM多肽高亲和力结合，并通过水解使多肽失活。在HYP中，除了丧失PHEX功能外，MEPE和成骨细胞蛋白水解酶活性也有明显上调。这会导致MEPE和DMP1的降解，并从这些蛋白和其他兄弟蛋白(DSPP、骨桥蛋白BSP、釉质蛋白)中释放ASARM-肽。这些抗蛋白酶的ASARM多肽积聚在肾脏、骨骼、尿液和循环中。为了研究兄弟姐妹、PHEX和ASARM多肽的作用，我们建立了在骨骼和肾脏分别高表达MEPE蛋白50X和7X的转基因小鼠(MEPE-TRG)。我们的具体目标是：1.鉴定MEPE-TRG小鼠的骨骼和肾脏PO4表型；2.确定MEPE-TRG、HYP和NPT2a-/-小鼠的MEPE和ASARM多肽的加工形式对骨和肾脏的钙化作用；3.设计PHEX合成肽作为工具，探索ASARM多肽和Phe在健康和疾病中的作用。与公共健康相关：最近发现的一类新的骨齿和肾脏蛋白(兄弟姐妹)在维护骨骼健康和调节磷酸盐方面发挥了重要作用。来自这些蛋白质的生物活性加工肽在疾病和健康中影响矿化、骨形成、骨生长和软组织钙化。对这些新蛋白质的研究将有助于开发治疗矿物质丢失疾病(遗传性和肿瘤获得性)、肾性骨营养不良、肾移植、异位动脉钙化、肾脏钙化和骨质疏松症的方法。

DESCRIPTION (provided by applicant): An ancient legacy of life transitioning from oceans to land is the role of the skeleton and kidney in regulating mineralization and Ca/PO4 homeostasis. Key players in this bone kidney axis are a group of related extracellular matrix proteins (SIBLINGs). These proteins all map to a tightly clustered region on chromosome 4q. A unique protein PHEX, regulates at least two SIBLING proteins (MEPE and DMP1), indirectly or directly. PHEX is a zn-metalloendopeptidase in search of a substrate and may also play a role as an orphan ligand or receptor. Loss of PHEX function results in X-linked hypophosphatemic rickets and a novel cytokine FGF23 (a phosphatonin), plays a major role in the phosphate phenotype. Our data confirms direct binding of PHEX to MEPE, a SIBLING protein expressed in bone, teeth and in the proximal convoluted tubules of the kidney. The binding between these proteins occurs between the PHEX Zn-binding motif and a short COOH-terminal region of MEPE called the ASARM-motif. This short region is exquisitely resistant to proteolysis and conserved across species. Biologically, the released ASARM-peptide is a potent inhibitor of mineralization, inhibits renal phosphate handling and binds to hydroxyapatite. Binding of PHEX to MEPE may serve to regulate release of the ASARM peptide and thus locally control mineralization. PHEX also binds with high affinity to free ASARM- peptide and also inactivates the peptide by hydrolysis. In HYP, as well as a loss of PHEX function, there is a marked up regulation of MEPE and osteoblastic protease activity. This results in degradation of MEPE and DMP1 and release of ASARM-peptides from both these proteins and perhaps other SIBLING proteins (DSPP, osteopontin BSP, enamelin). These protease resistant ASARM-peptides accumulate in kidney, bone, urine and in the circulation. To study the role of SIBLINGs, PHEX and ASARM- peptides we have made transgenic mice (MEPE-TRG) that overexpress MEPE protein 50X in bone and 7X in kidney. Our specific aims are: 1. Characterize bone and renal PO4 phenotypes of MEPE-TRG mice, 2. Determine the bone and renal- calcification roles of processed forms of MEPE and ASARM-peptides in MEPE-TRG, HYP and NPT2a-/- mice, 3. Design PHEX synthetic peptides as tools to probe the role of ASARM peptides and PHEX in health and disease. PUBLIC HEALTH RELEVANCE: A new class of recently discovered bone-teeth and renal proteins (SIBLINGs) play a major role in upholding the health of the skeleton and regulating phosphate. Bioactive processed peptides from these proteins impact on mineralization, bone formation, bone growth and soft tissue calcification in disease and health. The study of these new proteins will help develop therapies for mineral loss disorders (inherited and tumor-acquired), renal osteodystrophy, renal transplantation, ectopic arterial-calcification, renal calcification and osteoporosis.