(Former:) Protein inhibitors of the propeptide/prohormone convertases.
(Former:) The propeptide convertases (PCs) are endopeptidases that are responsible for proteolytic cleavage of biosynthetic precursors for peptide hormones, growth factors, receptors, envelope glycoproteins of viral pathogens, and toxins. As most propeptides or prohormones exhibit minimal biological activity, the PCs play an essential role in generating bioactive product peptides from these precursors. The PCs are co-synthesized as preproenzymes along with their propeptide substrates and are targeted into the secretory pathway. The mature enzymes function in the Golgi and in secretory granules to cleave bioactive peptides from their precursors. Even though the PCs have been studied extensively, much remains to be learned about their function. One means of examining enzyme function is to employ specific inhibitors. Studies in progress are designed to develop means of selectively blocking the activity of PCs with novel peptide or protein inhibitors. The first goal was to identify new synthetic peptide and/or naturally occurring protein inhibitors of the PCs. The active site regions of all PCs have sequence homology with the active sites of the bacterial subtilisins. A group of Streptomyces subtilisin inhibitors (SSI) has been characterized, many of which display a single basic amino acid linked to a non-basic amino acid in their reactive sites. This sequence mimics that found in mutant forms of human proinsulin. The mutation prevents PC processing at the cleavage site which normally consists of a pair of basic amino acids. Based on the similarity between subtilisin and PC active site sequences, it was postulated that SSIs may be effective PC inhibitors. To test this hypotheses, synthetic peptides containing SSI reactive site sequences and full length SSIs have been examined for their ability to inhibit recombinant PC activity in vitro using both fluorometric and prohormone cleavage assays. Effective inhibitors have been identified and their mechanism of action is being determined. An additional objective is to develop procedures for expression and targeting of potent inhibitors into the secretory pathway of intact cells. cDNAs for inhibitors which have been determined to be effective in vitro are incorporated into mammalian expression vectors which provide a mechanism for targeting inhibitors to the intracellular sites where PC-mediated precursor cleavage occurs. Transfected cells then synthesize an endogenous supply of PC inhibitor that can markedly reduce or completely block bioactive peptide cleavage from precursors. Inhibition of PC activity in vivo provides an experimental methodology that can be employed to assess PC specificity and function in intact cells. Such an approach could have other applications as well. Several of the PCs have been implicated in the processing of the HIV gp160 surface envelope protein in HIV infected cells. If gp160 cleavage is blocked, HIV virion replication is prevented. If HIV target cells were transfected with a vector that mediates inducible expression of a peptide PC inhibitor, these cells could potentially reduce the viral load in HIV infected individuals.
