Peptide neurotransmitters are synthesized as protein precursors that require proteolytic processing to form the active neuropeptides. The goal of this proposal is to obtain biochemical and molecular characterization of two components -- a novel 'prohormone thiol protease' (PTP) and its endogenous alpha1-antichymotrypsin-like (ACT-like) protease inhibitor -- that may be involved in enkephalin and tachykinin precursor processing. Only the mature processed enkephalin and substance P peptides, and not the precursors, function as neurotransmitters. Therefore, investigation of PTP as a major processing enzyme and its regulation by ACT-like protease inhibitor is crucial for understanding molecular mechanisms of neurotransmission. Biochemical assessment of PTP's precursor selectivity, cleavage site specificity, and in vitro processing of recombinant proenkephalin (PE) and beta-protachykinin (beta-PT) will be conducted. High level expression of PE and beta-PT in E. coli provides adequate quantities of precursors needed for in vitro PTP kinetic studies with PE and beta-PT near their in vivo concentrations. Importantly, recombinant precursors allow identification of processing products by peptide microsequencing analyses. Molecular cloning will utilize PTP's partial NH2-terminal amino acid sequence in multiple cloning approaches: (a) RT-PCR and RACE PCR (polymerase chain reaction) with complementary degenerate oligonucleotides, combined with nested PCR, to generate a partial PTP cDNA for screening cDNA libraries, (b) use of complementary oligonucleotides for cDNA library screening, (c) expression cloning using an antibody that recognizes PTP. The PTP cDNA will allow comparison of the primary structure of PTP with other proteases. In the second part of this project, inhibition of PTP by alpha1-ACT-like protein will be investigated with respect to inhibitory potency, interactions with PTP, and microsequencing. Colocalization of PTP and ACT-like inhibitor in neuroendocrine cells will be assessed by immunofluorescence and immunoelectron microscopy. In the last part of this project, studies will assess the functional importance of PTP in cellular PE processing by inhibiting PTP with a potent cysteine protease inhibitor, and by inhibiting PTP expression with antisense oligonucleotides. These studies will provide significant advances in understanding how PTP and ACT-like inhibitor(s) are involved in peptide neurotransmitter production. Knowledge obtained will provide insight into new therapeutic strategies for normal and diseased brains.
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