Evidence for the existence of a proteasome inToxoplasma gondii: intracellular localization and specific peptidase activities

Abstract

Summary :The proteasome is a large intracellular protein complex whose main function is proteolytic removal of damaged proteins. It has recently been shown that the proteasome has a crucial role in the pathogenesis of protozoan parasites. We attempted to characterize the proteasome of T. gondii (RH strain). In immunoblot experiments, we showed that MCP231 monoclonal antibody, directed against the human 20S proteasome, labelled homologous proteins in T. gondii with a pattern similar to that observed in mammalian cells. The study of in vitro proteolytic activities showed that chymotrypsin-like activity (the only activity obtained with archaebacteria) was present in Toxoplasma, with K m and specific activity values close to those observed with eukaryotic cells. Immunofluorescence studies showed that the Toxoplasma proteasome predominated in the cytosol. KEY WORDS :Toxoplasma gondii, proteasome, immunoblotting, chymotrypsinlike, trypsin-like, fluorescent antibody. Résumé : MISE EN ÉVIDENCE DU PROTÉASOME DE TOXOPLASMA GONDII-. LOCALISATION INTRACELLULAIRE ET ACTIVITÉS SPÉCIFIQUES PEPTIDASIQUES

In vitro effects of gliotoxin, a natural proteasome inhibitor, on the infectivity and proteolytic activity of Toxoplasma gondii

“…The proteasome has several peptidase activities, of which the most extensively investigated is ''chymotrypsin-like'', as it cleaves substrates on the carboxyl side of a hydrophobic residue. In a previous study (Paugam et al 2001), we obtained similar K m and specific activity values for Toxoplasma and HeLa proteasomes with Suc-LLVY-AMC (0.06 mM, 0.08 mM; 16.7 pmol, 32 pmol min -1 mg -1 , respectively). No published data are available on gliotoxin inhibition of protozoan parasites.…”

“…It has recently been shown that progression through the life-cycle of various protozoan parasites like Entamoeba (Gonzalez et al 1999), Leishmania (Robertson 1999), and Trypanosoma (de Diego et al 2001) requires a functional proteasome. We have previously demonstrated the presence of a proteasome in T. gondii by intracellular localization and detection of specific peptidase activity (Paugam et al 2001); and proteasome inhibitors have been tested for the treatment of malaria in an experimental model (Gantt et al 1998). Following the recent demonstration that fungal gliotoxin efficiently inhibits the proteasome (Kroll et al 1999), we studied the effect of gliotoxin on the proteolytic activity of the Toxoplasma proteasome and on Toxoplasma infectivity in vitro.…”

Critical role for the immunoproteasome subunit LMP7 in the resistance of mice to Toxoplasma gondii infection

“…2D). As T. gondii is known to have proteasomes 23, 24, there was a possibility that the enhanced activity was due to increased activity of T. gondii proteasomes. Therefore, we collected T. gondii from each preparation but could not detect proteolytic activity in the parasites (Fig.…”

The Ubiquitin Proteome of Toxoplasma gondii Reveals Roles for Protein Ubiquitination in Cell-Cycle Transitions

“…The T. gondii proteasome contains homologues of many components found in other eukaryotes, and additional proteins unique to T. gondii (Paugam et al, 2003; Paugam et al, 2001). The T. gondii genome possesses a conserved and functional repertoire of ERAD proteins (Agrawal et al, 2009), a large number of candidate ubiquitin ligases and deubiquitinases, as well as ubiquitin and ubiquitin-like modifiers (Ponder and Bogyo, 2007; Ponts et al, 2008).…”