Biochemical and functional characterization of prolyl oligopeptidase

Resumen

Prolyl oligopeptidase (POP) is a serine protease that in vitro cleaves small peptides at the carboxyl side of an internal proline residue. Substance P, arginine-vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this peptidase. POP has been implicated in a variety of brain processes, including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension, and psychiatric disorders. POP has been considered to be a soluble cytoplasmic enzyme. However, due the lack of co-localization of the enzyme and its substrates, the role of POP in the metabolism of neuropeptides has been criticized. Low levels of POP-like activity have been detected in membranes and in extracellular fluids such as serum, cerebrospinal fluid, seminal fluid, and urine, suggesting the existence of noncytoplasmic forms. Furthermore, a closely associated membrane prolyl endopeptidase (PE) activity has been previously detected in synaptosomes and shown to be different from the cytoplasmic POP activity. For a better understanding of the neuropeptide metabolism through PEs, we have isolated, purified and characterized this membrane-bound PE, herein referred to as mPOP. Although, when attached to membranes, mPOP presents certain features that distinguish it from the classical POP, our results indicate that this protein has the same amino acid sequence as POP except for the possible addition of a hydrophobic membrane anchor. In order to step further, we have also evaluated the neuropeptides levels in different brain areas from rats after POP inhibitor treatment to this and, we have developed a new analytical method combining (a) sample snap-freezing and boiling buffer extraction, to limit protein degradation and reduce sample complexity; (b) pH two-dimensional liquid reverse-phase chromatography to enhance resolution; and (c) iTRAQ isobaric labelling to identify the rat brain peptides whose levels were differentially changed due to in vivo POP inhibition. The method has shown to be sensitive enough to detect differences between treated and controls animals. However, none of the in vitro substrates have been found significantly altered during the experiment, suggesting that they could not be in vivo substrates. Finally as a complementary work, we have evaluated the POP activity levels in plasmas from multiple sclerosis (MS) patients. Previous unpublished results from our laboratory have shown that plasmas from MS patients have higher inhibitory capacity on POP than those from controls. Our results show that MS patients have a decreased POP activity that is correlated with the disease severity. Altogether and considering the recent publications supporting the idea of POP as a modulator of the inflammatory response, our data could lead to the identification of new therapeutic targets or diagnostic applications.