Four day inhibition of prolyl oligopeptidase causes significant changes in the peptidome of rat brain, liver and kidney

Abstract

Prolyl oligopeptidase (PREP) cleaves short peptides at the C-side of proline. Although several proline containing neuropeptides have been shown to be efficiently cleaved by PREP in vitro, the actual physiological substrates of this peptidase are still a matter of controversy. The aim of this study was to evaluate the changes in the peptidome of rat tissues caused by a repeated 4-day administration of the potent and specific PREP inhibitor KYP-2047, using our recently developed iTRAQ-based technique. We found tissue-dependent changes in the levels of specific subsets of peptides mainly derived from cytosolic proteins. Particularly in the kidney, where the levels of cytochrome c oxidase were found decreased, many of the altered peptides originated from mitochondrial proteins being involved in energy metabolism. However, in the hypothalamus, we found significant changes in peptides derived from hormone precursors. We could not confirm a role of PREP as the metabolising enzyme for β-endorphin, galanin, octadecaneuropeptide, neuropeptide-glutamic acid-isoleucine, substance P, somatostatin, enkephalin and neuropeptide Y. Furthermore, changes in the degradation patterns of some of these neuropeptides, and also most of those derived from other larger proteins, did not follow specificity to proline. After a 4-day treatment, we found a significant amount of peptides, all derived from secreted pro-proteins, being cleaved with pair of basic residue specificity. In vitro experiments indicated that PREP modifies the endogenous dibasic residue specific proteolysis, in a KYP-2047 sensitive way. These findings suggest that PREP may act indirectly within the routes leading to the specific peptide changes that we observed. The data reported here suggest a wider tissue specific physiological role of PREP rather than the mere metabolism of proline containing active peptides and hormones.