The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP) - Colocalization of KPI/APP and PTP in secretory vesicles

Proteolytic processing of proenkephalin and proneuropeptides is required fo r the production of active neurotransmitters and peptide hormones. Variatio ns in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nex in 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of th e amyloid precursor protein (APP), potently inhibited the proenkephalin pro cessing enzyme known as prohormone thiol protease (PTP), with a K-i,K-app o f 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typica l of kunitz protease inhibitor interactions with target proteases, In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles P N2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met )enkephalin in secretary vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles th at contain PTP, In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these comp onents in functional secretory vesicles. These results suggest a role for K PI/APP inhibition of PTP in regulated secretory vesicles. In addition, thes e results are the first to identify an endogenous protease target of KPI/AP P, which is developmentally regulated in aging and Alzheimer's disease.