Substantial progress has been made in deciphering the enzymatic mechanisms responsible for opioid 3eptide synthesis within the past decade. Opioid precursors are now known to be cleaved by prohormone convertases 1 and 2 (PC1 and PC2), subtilisin-like proteases restricted to neuroendocrine tissues, to produce small bioactive opioid peptides. Our work during the past two cycles of this grant has focused on the expression and biochemical characterization of PC1 and PC2. We have produced both enzymes in recombinant form, and have defined their specificity on the model opioid peptide precursor proenkephalin, both in vitro and in vivo, and have identified potent inhibitors of both enzymes through the use of combinatorial peptide library screening. Most recently, in collaboration with the crystallography group of Wolfram Bode, we have obtained the first crystal structure of this family of enzymes, the related enzyme furin. In this renewal application, we propose to apply the structural information on convertases gained thus far to perform site-directed mutagenesis of PC1 and PC2; this will help us to understand the molecular basis for the specific biochemical properties of each enzymes. We will continue our collaboration with the Bode group to obtain the crystal structures of PC1, PC2 and proPC2. We will also continue our successful collaboration with the Houghten group in identifying specific and potent non-peptide convertase inhibitors. Lastly, in collaboration with Melvin Simon, we will combine the reagents generated through previous funding of this grant, recombinant proenkephalin and recombinant convertases, with other recombinant substrates and processing enzymes to produce a robust in vitro system for the synthesis of natural peptides from recombinant precursors for functional studies. Taken together these studies will provide us with novel information on the biochemical mechanisms of opioid peptide formation and may provide reagents useful for he therapeutic inhibition of convertases. ? ? ?
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| Ramos-Molina, B; Martin, M G; Lindberg, I (2016) PCSK1 Variants and Human Obesity. Prog Mol Biol Transl Sci 140:47-74 |
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| Liew, Chong Wee; Assmann, Anke; Templin, Andrew T et al. (2014) Insulin regulates carboxypeptidase E by modulating translation initiation scaffolding protein eIF4G1 in pancreatic ? cells. Proc Natl Acad Sci U S A 111:E2319-28 |
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