Cysteine proteinases are key virulence factors of the protozoan parasiteEntamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of theE. histolyticagenome project has revealed more than 40 genes encoding cysteine proteinases. We have focused onE. histolyticacysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasiveE. histolyticaand is highly expressed and released. Recombinant EhCP1 was expressed inEscherichia coliand refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI inhibitors.

Infantile hemangiomas (IH) are the most common benign vascular tumors of childhood, with an incidence of 5–10% during the first year of age. Propranolol is considered the first-line treatment for this condition. Potentially there is a high probability of negative results to therapy, because in many countries there are no treatment protocols or propranolol formulations appropriate for the pediatric population. The objective of the present study was to evaluate the impact of pharmacist interventions such as detecting, analyzing, and solving problems presented during treatment with propranolol in patients with IH.

Protein disulfide isomerases (PDI) are eukaryotic oxidoreductases that catalyze the formation and rearrangement of disulfide bonds during folding of substrate proteins. Structurally, PDI enzymes share as a common feature the presence of at least one active thioredoxin-like domain. PDI enzymes are also involved in holding, refolding, and degradation of unfolded or misfolded proteins during stressful conditions. TheEhPDI enzyme (a 38 kDa polypeptide with two active thioredoxin-like domains) has been used as a model to gain insights into protein folding and disulfide bond formation inE. histolytica. Here, we performed a functional complementation assay, using a ΔdsbC mutant ofE. coli, to test whetherEhPDI exhibits isomerase activityin vivo. Our preliminary results showed thatEhPDI exhibits isomerase activity; however, further mutagenic analysis revealed significant differences in the functional role of each thioredoxin-like domain. Additional studies confirmed thatEhPDI protects heat-labile enzymes against thermal inactivation, extending our knowledge about its chaperone-like activity. The characterization ofEhPDI, as an oxidative folding catalyst with chaperone-like function, represents the initial step to dissect the molecular mechanisms involved in protein folding inE. histolytica.