(from abstract) Many neuroendocrine peptides are synthesized as precursor proteins; post-translational processing of these precursors is a key step in the production of biologically active peptides. The enzymes involved in this processing are important regulators of these intercellular messengers. A variety of endopeptidases and exopeptidases have been proposed to be involved in neuropeptide processing. In this grant application the applicants describe studies to examine the contribution of various processing enzymes in the generation of opioid peptides using transgenic animals and cell lines. The first specific aim is to examine dynorphin processing in animal models that lack specific peptide processing enzymes. They will assess the extent of involvement of carboxypeptidase E (CPE) in dynorphin processing by comparing the profiles of various dynorphin peptides in fat/fat mice (lacking active CPE) with those in control mice. In addition, they will assess the extent of involvement of prohormone convertase 2 (PC2) in dynorphin processing by comparing the profiles in PC2 K/O mice (that lack PC2) with those in control mice. The second specific aim is to examine the contribution of various peptide processing enzymes to dynorphin biosynthesis using a cell culture system that inefficiently processes dynorphin. The investigators will examine whether a dynorphin processing defect in NIT-3 cells (that lack CPE) can be overcome by endocrine carboxypeptidases or by prohormone convertases. These studies will provide information about the interactions of various processing enzymes that could modulate their function. The studies proposed in this grant application will address the physiological role of the processing enzymes in a neuropeptide biosynthesis. The information gained through these studies will provide insights into the multiple regulatory steps (including cross-talk between enzymes) that govern neuropeptide processing which cold modulate the levels of neuropeptides in normal an dysfunctional cellular events such as those seen in several neurological disorders.
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