IDENTIFICATION OF INHIBITORS AND SUBSTANCE PHEX

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 磷酸盐动态平衡对健康和疾病都很重要。在肾功能衰竭患者中，磷酸盐水平升高与心血管疾病死亡增加有关。此外，还有导致低磷血症的遗传性疾病和以矿化受损为特征的骨骼疾病。PHEX是一种在骨骼中表达的细胞表面金属蛋白水解酶，调节磷酸盐稳态和骨矿化。PHEX突变增加了FGF23的表达，FGF23是一种调节肾脏磷酸盐重吸收和1，25(OH)2D3产生的成纤维细胞生长因子。然而，PHEX突变是如何导致FGF23升高的尚不清楚。 这项研究的目标是确定苯丙氨酸底物和抑制剂。我们正在使用噬菌体展示和基于我们对FGF23的PHEX调节的观察结果的候选方法来识别与PHEX结合并抑制其功能的多肽。这些多肽将为进一步研究PHEX的功能和开发治疗高磷血症的药物奠定基础。生理上相关的PHEX底物的鉴定也可能揭示一种新的激素途径，将PHEX缺乏与肾脏磷酸盐消耗联系起来。 为了做到这一点，我们首先建立了一种内部猝灭的荧光肽底物分析来测量PHEX的活性。我们已经证明了苯丙氨酸具有剂量依赖性地切割合成底物的能力。然后，我们检测了与FGF23调节相关的牙本质基质蛋白-1(DMP1)和基质细胞外磷酸糖蛋白(MEPE)，以确定它们对FGF23的影响是否可能是通过直接调节PHEX来实现的。我们发现DMP1和MEPE都能抑制PHEX的活性。为了进一步评估MEPE的效果，我们检测了MEPE的切割产物ASARM肽，它也与DMP1同源。我们发现，磷酸化的ASARM多肽抑制了PHEX酶的活性。这些抑制物调节FGF23表达的能力的体外和体内研究正在我们实验室进行。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phosphate homeostasis is important in health and disease. In patients with renal failure, elevated phosphate levels are associated with increased death from cardiovascular disease. Additionally, there are inherited disorders that lead to hypophosphatemia and bone diseases characterized by impaired mineralization. Phex is a cell surface metalloprotease expressed in bone which regulates phosphate homeostasis and bone mineralization. Phex mutations increase expression of FGF23, a fibroblast growth factor regulating kidney phosphate reabsorption and 1,25(OH)2D3 production. However, how Phex mutations cause elevated FGF23 is unclear. The goal of this study is to identify Phex substrates and inhibitors. We are using both phage display and a candidate approach based on our observations regarding Phex-regulation of FGF23 to identify peptides that bind to Phex and inhibit its function. These peptides will be useful for further study of Phex function and potential development of drug therapies to treat hyperphosphatemic disorders. The identification of physiologically relevant Phex substrates will also likely uncover a novel hormonal pathway linking Phex deficiency to renal phosphate wasting. To accomplish this, we first set up an internally quenched fluorogenic peptide substrate assay to measure PHEX activity. We have shown the ability of Phex to dose-dependently cleave the synthetic substrate. Then, we tested dentin matrix protein-1 (DMP1) and matrix extracellular phosphoglycoprotein (MEPE), which are associated with regulation of FGF23, to see if their effect on FGF23 may be mediated by direct regulation of Phex. We found that both DMP1 and MEPE inhibit Phex activity. To further evaluate the effect of MEPE, we examined the ASARM peptide, a cleavage product of MEPE, which is also homologous to DMP1. We found that the phosphorylated ASARM peptide inhibited Phex enzyme activity. The in vitro and in vivo studies of the ability of these inhibitors to regulate FGF23 expression are ongoing in our lab.