Substrate specificity profiling of M32 metallocarboxypeptidases from Trypanosoma cruzi and Trypanosoma brucei.

Metallocarboxypeptidases (MCPs) of the M32 family, while broadly distributed among prokaryotic organisms, have so far been only found in a few eukaryotes including trypanosomatids. Among these organisms are human and animal pathogens of medical relevance such as Trypanosoma brucei and Trypanosoma cruzi, the respective causative agents of sleeping sickness and Chagas disease. The M32 MCP orthologues found in these parasites share 72% protein sequence identity. They also present a cytosolic localization, a similar pattern of expression and a marked preference for Arg/Lys residues at P1'. To further explore MCPs substrate specificity beyond the S1' subsite, we employed four positional scanning synthetic combinatorial libraries (PS-SC) of fluorescence resonance energy transfer (FRET) peptides. Our results indicated that the T. brucei enzyme has a restricted selectivity for Phe in P1 position compared to T. cruzi MCP-1, which presented a wider range of substrate acceptance. The S2, S3 and S4 subsites, on the other hand, could accommodate a broad range of residues. On the basis of these results, we synthesized for each enzyme a series of FRET substrates which contained the most favourable residues in every position. In particular, for both MCPs acting on FRET pentapeptide substrates, catalytic efficiencies were ∼100 times higher compared with previously described chromogenic substrates. In fact, the fluorogenic peptide Abz-LLKFK(Dnp)-OH (Abz = ortho-aminobenzoic acid; Dnp = 2, 4-dinitrophenyl) described here can be used to monitor accurately TbMCP-1 activity in parasite cell-free extracts. These results provide valuable insights to develop selective substrates and inhibitors, to further understand the mechanisms and functions of M32 MCPs.

Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: differences and similarities with its orthologue in Trypanosoma cruzi.

Metallocarboxypeptidases (MCP) of the M32 family of peptidases have been identified in a number of prokaryotic organisms but they are absent from eukaryotic genomes with the remarkable exception of those of trypanosomatids. The genome of Trypanosoma brucei, the causative agent of Sleeping Sickness, encodes one such MCP which displays 72% identity to the characterized TcMCP-1 from Trypanosoma cruzi. As its orthologue, TcMCP-1, Trypanosoma brucei MCP is a cytosolic enzyme expressed in both major stages of the parasite. Purified recombinant TbMCP-1 exhibits a significant hydrolytic activity against the carboxypeptidase B substrate FA (furylacryloil)-Ala-Lys at pH 7.0-7.8 resembling the T. cruzi enzyme. Several divalent cations had little effect on TbMCP-1 activity but increasing amounts of Co(2+) inhibited the enzyme. Despite having similar tertiary structure, both protozoan MCPs display different substrate specificity with respect to P1 position. Thus, TcMCP-1 enzyme cleaved Abz-FVK-(Dnp)-OH substrate (where Abz: o-aminobenzoic acid and Dnp: 2,4-dinitrophenyl) whereas TbMCP-1 had no activity on this substrate. Comparative homology models and sequence alignments using TcMCP-1 as a template led us to map several residues that could explain this difference. To verify this hypothesis, site-directed mutagenesis was undertaken replacing the TbMCP-1 residues by those present in TcMCP-1. We found that the substitution A414M led TbMCP-1 to gain activity on Abz-FVK-(Dnp)-OH, thus showing that this residue is involved in specificity determination, probably being part of the S1 sub-site. Moreover, the activity of both protozoan MCPs was explored on two vasoactive compounds such as bradykinin and angiotensin I resulting in two different hydrolysis patterns.

Angiotensin-(1-7) upregulates central nitric oxide synthase in spontaneously hypertensive rats.

Increased blood pressure in hypertension is hypothesized to be caused by high sympathetic nervous system (SNS) activity. Since Ang (1-7) exerts an inhibitory neuromodulatory effect on the SNS through a NO-mediated mechanism, we tested the hypothesis that Ang (1-7) alters centrally nitric oxide synthase (NOS) activity and expression in spontaneously hypertensive rats (SHR). Since NOS activity is altered in relation to the development of hypertension in rats, we evaluated the effect of Ang-(1-7) on hypothalamic NOS activity in two different ages in SHR, corresponding to a prehypertensive phase (3-4 weeks) and a established hypertension (13-14 weeks) and compared with age-matched Wistar-Kyoto (WKY) rats. NOS activity was measured by the conversion of [³H]L-arginine to citrulline. Ang-(1-7) caused an impairment in NOS activity in prehypertensive SHR (26 ± 4% reduction), while it induced an increase in NOS activity at established hypertension (48 ± 9% increase). In contrast, Ang-(1-7) did not modify NOS activity in age-matched WKY rats. In another set of experiments, Ang-(1-7) was injected into the anterior hypothalamic area, mean arterial blood pressure (MAP) was registered and after 30, 60 and 180 min nNOS expression was evaluated by Western-blot. Ang-(1-7) decreased MAP after 10 min of injection and this effect was blocked by a NOS inhibitor. nNOS expression increased after 180 min of Ang-(1-7) intrahypothalamic injection in both WKY and SHR (WKY: 3.6-fold increase above basal; SHR: 1.85-fold increase above basal). Our results suggest that Ang-(1-7) upregulates hypothalamic NOS in a hypertensive state as a compensatory and protective mechanism to combat hypertension.