The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series Abz-GFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X=Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr605 and Ala607 in TOP and at Tyr606 and Gly608 in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. The kinetic parameters for the hydrolysis of substrates with systematic variations at position P1 showed that Tyr605 and Tyr606 of TOP and NEL respectively, played a role in subsite S1. Ala607 of TOP and Gly608 of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Tyr605 was an important anchor for its interaction with wild-type TOP. The hydroxy groups of Tyr605 and Tyr606 did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-kcat/Km dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.

The consequences of sleep deprivation on memory, cognition, nociception, stress, and endocrine function are related to the balance of neuropeptides, with peptidases being particularly essential. Thimet oligopeptidase (THOP1) is a metallopeptidase implicated in the metabolism of many sleep-related peptides, including angiotensin I, gonadotropin releasing hormone (GnRH), neurotensin, and opioid peptides. In the present study, we evaluated the effect of sleep deprivation and sleep recovery in male rats on THOP1 expression and specific activity in the central nervous system. In the striatum and hypothalamus, THOP1 activity decreased following sleep deprivation and a recovery period. Meanwhile, THOP1 activity and immunoexpression increased in the hippocampal dentate gyrus during the sleep recovery period. Changes in THOP1 expression after sleep deprivation and during sleep recovery can potentially alter the processing of neuropeptides. In particular, processing of opioid peptides may be related to the known increase in pain sensitivity in this model. These results suggest that THOP1 may be an important player in the effects of sleep deprivation.

Some central effects of cocaine administration seem to be related to angiotensin II (Ang II) or its metabolites. Nonetheless, it is still an open question whether or not the levels of angiotensin I-converting enzyme (ACE), the main Ang II generating enzyme, are modified by cocaine administration. To evaluate the effect of acute and subchronic cocaine administration on ACE activity and mRNA expression, male rats were randomly assigned to saline or cocaine group. Acute and subchronic cocaine administration induced a significant increase in ACE activity and mRNA expression in the frontal cortex and striatum but not in the hippocampus. These results suggest that some of the Ang II related effects of cocaine upon the central nervous system can be mediated by changes on the expression and activity of ACE in the striatum and frontal cortex.

The effect of sleep deprivation on proteolytic activity in the central nervous system has not been frequently addressed in the literature. We have started to study the changes on peptidase activities in animal models of paradoxical sleep deprivation (PSD), mainly focusing on metallopeptidases like thimet oligopeptidase (EP24.15) known to process neuropeptides. In the present study, we demonstrated that PSD in rats induces changes both in the expression and activity of EP24.15 in the male rat hypothalamus and striatum tissue extracts. In the hypothalamus, the significant decrease in activity and mRNA levels, after PSD, was only totally reversed after 96h of sleep recovery. In the striatum, albeit significative, changes in mRNA and activity do not parallel changes on protein content. Neurotensin, GnRH, dynorphins, enkephalins, orphanin/nociceptin are among the many neuropeptides whose balance can be modified by the modifications on EP24.15 activity and can be, at least partially, related with many of the physiological changes observed in PSD and during the sleep recovery periods. Grant Funding Source: Supported by FAPESP, CNPq and AFIP

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