Discovery of novel peptide hormones using prohormone convertase screening

Bioactive peptides play critical roles in diverse biological phenomena; the discovery of novel peptides thus has the potential to open entirely new fields of research. The successful sequencing of entire genomes of various organisms has yielded numerous novel candidate precursor genes; however, the identification of novel bioactive peptides from such genomic information has been hampered by their small size. In this study, we attempt to identify novel peptides using a combinatorial screening approach: a predictive algorithm followed by "restriction protease" screening. The algorithm was trained using various properties associated with known peptide precursors. Approximately 150 genes were identified as coding for candidate peptide precursors. In a preliminary screening, 21 genes were chosen from this list and expressed in tagged form in HEK293 cells. Proteolysis using prohormone convertases 1 and 2 and furin was performed in order to confirm cleavage of expressed proteins. Sixteen of 21 precursors were cleaved by one or more convertases; 5 proteins were not cleaved and may represent false bioinformatics hits. In situ hybridization of mouse brain with antisense cDNA from a cleaved precursor indicated limited expression within the mediobasal hypothalamus. These results demonstrate that our bioinformatics/convertase screening approach is a promising method for peptide hormone discovery. (DA05984)