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.
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