Prolyl endopeptidase (PEP) is a proline-specific oligopeptidase which, in vitro, is be able to rapidly degrade several neuropeptides, including Substance P and arginine-vasopressin, by limited proteolysis. Furthermore PEP inhibitors work as cognitive enhancers in different animal models. It has been hypothesized that these effects are due to modulation of neuropeptide bioactivity by PEP. But the interaction between the neuropeptides and their receptors takes place on the cell surface. The aim of this work was to investigate the molecular basis of the reported positive effects of PEP inhibitors on learning and memory as well as on neurotoxicity. The first part of this work includes the investigation of the subcellular localization of PEP in human neuronal and glial cell lines. The second part deals with the influence of PEP inhibitors on signal transduction processes. In the present work we could shown that PEP is exclusively localized in the cytosol of human neuronal and glial cell lines. Subcellular localization studies by using a newly established anti-PEP monoclonal antibody and a EGFP-fusion technique showed that PEP is mainly localized in the perinuclear space, more than in the cell periphery. Colocalization studies have been shown that PEP almost completely colocalized with tubulin cytoskeleton structures. Using antisense cell lines with reduced PEP expression as well as specific inhibitors, we were able to show an inverse correlation between Ins(1,4,5)P3 concentration and PEP activity. Furthermore, antisense cell lines and cells treated with a PEP inhibitor showed an amplified Ins(1,4,5)P3 signal after Substance P stimulation. All data presented, strongly suggest an indirect involvement of PEP in second-messenger pathways by intracellular interactions with potential cross-talk to signal transduction mediated by neuropeptides. |