Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity.

We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes.

Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from Entamoeba histolytica.

EhCP-B9, a cysteine protease (CP) involved in Entamoeba histolytica virulence, is a potential target for disease diagnosis and drug design. After purification from inclusion bodies produced in Escherichia coli, the recombinant EhCP-B9 precursor (ppEhCP-B9) can be refolded using detergents as artificial chaperones. However, the conformational changes that occur during ppEhCP-B9 refolding remain unknown. Here, we comprehensively describe conformational changes of ppEhCP-B9 that are induced by various chemical detergents acting as chaperones, including non-ionic, zwitterionic, cationic and anionic surfactants. We monitored the effect of detergent concentration and incubation time on the secondary and tertiary structures of ppEhCP-B9 using fluorescence and circular dichroism (CD) spectroscopy. In the presence of non-ionic and zwitterionic detergents, ppEhCP-B9 adopted a ß-enriched structure (ppEhCP-B9(ß1)) without proteolytic activity at all detergent concentrations and incubation times evaluated. ppEhCP-B9 also exhibits a ß-rich structure in low concentrations of ionic detergents, but at concentrations above the critical micelle concentration (CMC), the protein acquires an α+ß structure, similar to that of papain but without proteolytic activity (ppEhCP-B9(α+ß1)). Interestingly, only within a narrow range of experimental conditions in which SDS concentrations were below the CMC, ppEhCP-B9 refolded into a ß-sheet rich structure (ppEhCP-B9(ß2)) that slowly transforms into a different type of α+ß conformation that exhibited proteolytic activity (ppEhCP-B9(α+ß2)) suggesting that enzymatic activity is gained as slow transformation occurs.