LOCALIZING PEX IN LIVING CELLS WITH GREEN FLUORESCENT PROTEIN

编码一种新型金属内肽酶（PEX）的基因发生了突变

Mutations sin a gene encoding a novel metalloendopeptidase, (PEX), are found in X-linked dominant hypophosphatemic rickets and its animal models, Hyp and Gy mice. The disorder is manifest by renal phosphate wasting, and this defect is mediated by an unidentified circulating factor. Whether the bone disease is caused by the resultant hypophosphatemia, or whether there are independent mediators is unknown. The PEX protease is a member of the neutral endopeptidase family, and transcripts encoding the protein product are found in low abundance in various human and murine tissues; interestingly bone appears to e the tissue in which expression is greatest, and no kidney expression has been detected. Little is known regarding the tissue distribution of the protein itself, subcellular localization of the protein, or its precise function. The goal of this pilot and feasibility project is to further our understanding of PEX function by determining its subcellular localization and trafficking characteristics within living cells. In order to achieve this goal, we will perform the following preliminary experiments: 1) construct of fusion proteins using PEX cDNA and a plasmid vector for the reporter molecule, Green Fluorescent Protein (GFP). A N-terminal tagged PEX protein and a C-terminal tagged PEX will be constructed; preservation of correct reading frame, and directionality will be confirmed by sequencing the constructs; 2) of these constructs into a variety of cell types, and in particular bone cell lines; and (3) observation of these subcellular localization of the fusion protein by standard epifluorescent microscopy. These studies should provide exciting preliminary data for extension of the work to an independently funded larger-scale project, directed toward employing transgenic expression of the fusion protein, and classic rescue experiments addressing physiologic function of the protein. The approach offers a novel means of examining a perplexing biological problem, and holds promise as a window promise as a window in which to further investigate the pathophysiology of a poorly understood and inadequately treated skeletal disease. Core support will be critical to the performance of the proposed studies.