The Trypanosoma cruzi Virulence Factor Oligopeptidase B (OPBTc) Assembles into an Active and Stable Dimer

Oligopeptidase B, a processing enzyme of the prolyl oligopeptidase family, is considered as an important virulence factor in trypanosomiasis. Trypanosoma cruzi oligopeptidase B (OPBTc) is involved in host cell invasion by generating a Ca2+-agonist necessary for recruitment and fusion of host lysosomes at the site of parasite attachment. The underlying mechanism remains unknown and further structural and functional characterization of OPBTc may help clarify its physiological function and lead to the development of new therapeutic molecules to treat Chagas disease. In the present work, size exclusion chromatography and analytical ultracentrifugation experiments demonstrate that OPBTc is a dimer in solution, an association salt and pH-resistant and independent of intermolecular disulfide bonds. The enzyme retains its dimeric structure and is fully active up to 42°C. OPBTc is inactivated and its tertiary, but not secondary, structure is disrupted at higher temperatures, as monitored by circular dichroism and fluorescence spectroscopy. It has a highly stable secondary structure over a broad range of pH, undergoes subtle tertiary structure changes at low pH and is less stable under moderate ionic strength conditions. These results bring new insights into the structural properties of OPBTc, contributing to future studies on the rational design of OPBTc inhibitors as a promising strategy for Chagas disease chemotherapy

Localisation of a multicatalytic protease complex in Trypanosoma brucei brucei

Trypanosoma brucei brucei are tsetse-transmitted, extracellular protozoan parasite that causes Nagana, a cattle disease similar to sleeping sickness caused in humans by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Proteases of these parasites have come under increasing scrutiny because of their importance in such aspects as disease pathogenesis and as potential drug targets. Recently, a multicatalytic protease complex, the proteasome, has also become of interest because of its potential involvement in cell-cycle development. The proteasome is the central protease of the non lysosomal ubiquitin dependent pathway of protein degradation. Here we report the subcellular localization of 20S proteasome of Trypanosoma brucei brucei, using subcellular fractionation and enzymatic assays. Homogenate prepared from bloodstream form of T. brucei was fractionated by differential centrifugation into nuclear [NU] fraction, a large-granule [LG] fraction, small-granule [SG] fraction, microsomal [MI] and soluble cytosolic [CY] fractions. Each of these subcellular fractions was enriched in a particular organelle. The identity of these organelles was evaluated by assaying for the activity of marker enzymes. The proteasome activity was measured in the fractions using Z-Gly-Gly-Leu-AMC as substrate, peptide aldehyde ZLeu-Leu-Leu-H as specific inhibitor. The enzymatic activity was detected mainly in the cytosolic fraction (6.7-fold enrichment relative to the whole homogenate), but also in the nuclear fraction (2.8-fold). Our findings indicate that the internal location of the trypanosomal proteasome may not be restricted to the cytosol and nuclei as has previously been demonstrated in the eukaryotic cells.Key words: Trypanosoma brucei, 20S proteasome. subcellular localizatio

Characterisation of the antitrypanosomal activity of peptidyl alpha-aminoalkyl phosphonate diphenyl esters.

Two groups of irreversible serine peptidase inhibitors, peptidyl chloromethyl ketones and peptidyl phosphonate diphenyl esters, were examined for antitrypanosomal activity against the bloodstream form of Trypanosoma brucei brucei. Both peptidyl chloromethyl ketones and peptidyl phosphonate diphenyl esters inhibited trypsin-like peptidases of the parasites and exhibited antitrypanosomal activity at micromolar concentrations. In live T. b. brucei, labelled analogues of both of these groups of inhibitors primarily targeted an 80-kDa peptidase, possibly a serine oligopeptidase known as oligopeptidase B. In an in vivo mouse model of infection, one of these inhibitors, carbobenzyloxyglycyl-4-amidinophenylglycine phosphonate diphenyl ester, was curative at 5 mg kg(-1) day(-1) but appeared toxic at higher doses. There was no significant correlation between the inhibitory potency (as evaluated against purified T. b. brucei oligopeptidase B) and the in vitro antitrypanosomal efficacy of either group of inhibitors, suggesting that these inhibitors were acting on multiple targets within the parasites, or had different cell permeability properties. These findings suggest that serine peptidases may represent novel chemotherapeutic targets in African trypanosomes