Solution structure of the inhibitor of cysteine proteases 1 from Entamoeba histolytica reveals a possible auto regulatory mechanism

The genome of Entamoeba histolytica encodes approximately 50 Cysteine Proteases (CPs) whose activity is regulated by two Inhibitors of Cysteine Proteases (ICPs), EhICP1 and EhICP2. The main difference between both EhICPs is the acquisition of a 17 N-terminal targeting signal in EhICP2 and three exposed cysteine residues in EhICP1. The three exposed cysteines in EhICP1 potentiate the formation of cross-linking species that drive heterogeneity. Here we solved the NMR structure of EhICP1 using a mutant protein without accessible cysteines. Our structural data shows that EhICP1 adopts an immunoglobulin fold composed of seven β-strands, and three solvent exposed loops that resemble the structures of EhICP2 and chagasin. EhICP1 and EhICP2 are able to inhibit the archetypical cysteine protease papain by intercalating their BC loops into the protease active site independently of the character of the residue (serine or threonine) responsible to interact with the active site of papain. EhICP1 and EhICP2 present signals of functional divergence as they clustered in different clades. Two of the three exposed cysteines in EhICP1 are located at the DE loop that intercalates into the CP substrate-binding cleft. We propose that the solvent exposed cysteines of EhICP1 play a role in regulating its inhibitory activity and that in oxidative conditions, the cysteines of EhICP1 react to form intra and intermolecular disulfide bonds that render an inactive inhibitor. EhICP2 is not subject to redox regulation, as this inhibitor does not contain a single cysteine residue. This proposed redox regulation may be related to the differential cellular localization between EhICP1 and EhICP2.     

The cysteine protease inhibitors EhICP1 and EhICP2 perform different tasks in the regulation of endogenous protease activity in trophozoites of Entamoeba histolytica

Trophozoites of E. histolytica are equipped with two chagasin-like cysteine protease inhibitors, EhICP1 and EhICP2, also known as amoebiasin 1 and 2. Expression studies using E. invadens as model organism showed that corresponding mRNAs were detectable in both life stages of the parasite, cyst and trophozoite state. Unlike EhICP1 known to act in the cytosol, EhICP2 co-localized with cysteine protease EhCP-A1 in lysosome-like vesicles, as demonstrated by immunofluorescence microscopy. Silencing or overexpressing of the two inhibitors did not show any effect on morphology and viability of the trophozoites. Overexpression of the EhICPs, however, although dramatically dampening the proteolytic activity of cell extracts from the corresponding cell lines, did not influence expression rate or localization of the major amoebic cysteine proteases as well as phagocytosis and digestion of erythrocytes. Activity gels of cell extracts from strains overexpressing ehicp1 showed a drastically reduced activity of EhCP-A1 suggesting a high affinity of EhICP1 towards this protease. From these data, we propose that EhCP-A1 accidentally released into the cytosol is the main target of EhICP1, whereas EhICP2, beside its role in house-keeping processes, may control the proteolytic processing of other hydrolases or fulfils other tasks different from protease inhibition.     

Two cysteine protease inhibitors, EhICP1 and 2, localized in distinct compartments, negatively regulate secretion in Entamoeba histolytica

The enteric protozoan parasite Entamoeba histolytica uniquely possesses two isotypes of ICPs, a novel class of inhibitors for cysteine proteases. These two EhICPs showed a remarkable difference in the ability to inhibit cysteine protease (CP) 5, a well-established virulence determinant, whereas they equally inhibited CP1 and CP2. Immunofluorescence imaging and cellular fractionation showed that EhICP1 and EhICP2 are localized to distinct compartments. While EhICP1 is localized to the soluble cytosolic fraction, EhICP2 is targeted from lysosomes to phagosomes upon erythrocyte engulfment. Overexpression of either EhICP1 or EhICP2 caused reduction of intracellular CP activity, but not the amount of CP, and decrease in the secretion of all major CPs, suggesting that both EhICPs are involved in the trafficking and/or interference with the major CP activity. These data indicate that the two EhICPs, present in distinct subcellular compartments, negatively regulate CP secretion, and, thus, the virulence of this parasite.     

Identification of EhICP1, a chagasin-like cysteine protease inhibitor of Entamoeba histolytica

Based on the Entamoeba histolytica genome project (www.sanger.ac.uk/Project/E_histolytical/) we have identified a cysteine protease inhibitor, EhICP1 (amoebiasin 1), with significant homology to chagasin. Recombinant EhICP1 inhibited the protease activity of papain and that of a trophozoite lysate with Ki's in the picomolar range. By immunocytology, we localized the endogenous approximately 13 kDa EhICP1 in a finely dotted subcellular distribution discrete from the vesicles containing the amoebic cysteine protease, EhCP1 (amoebapain). In an overlay assay, we observed binding of recombinant EhICP1 to EhCP1. As a heptapeptide (GNPTTGF) corresponding to the second conserved chagasin motif inhibited the protease activity of both papain (K) 1.5 microM) and trophozoite extract (Ki in sub-mM range), it may be a candidate for the rational development of anti-amoebiasis drugs.     

Purification, refolding and autoactivation of the recombinant cysteine proteinase EhCP112 from Entamoeba histolytica

The cysteine proteinase EhCP112 and the adhesin EhADH112 assemble to form the EhCPADH complex involved in Entamoeba histolytica virulence. To further characterize this cysteine proteinase, the recombinant full-length EhCP112 enzyme was expressed and purified under denaturing conditions. After a refolding step under reductive conditions, the inactive precursor (ppEhCP112) was processed to a 35.5 kDa mature and active enzyme (EhCP112). The thiol specific inhibitor E-64, but not serine or aspartic proteinase inhibitors arrested this activation process. The activation step of the proenzyme followed by the mature enzyme suggests an autocatalytic process during EhCP112 maturation. The experimentally determined processing sites observed during EhCP112 activation lie close to processing sites of other cysteine proteinases from parasites. The kinetic parameters of the mature EhCP112 were determined using hemoglobin and azocasein as substrates. The proteinase activity of EhCP112 was completely inhibited by thiol inhibitors, E-64, TLCK, and chymostatin, but not by general proteinase inhibitors. Since EhCP112 is a proteinase involved in the virulence of E. histolytica, a reliable source of active EhCP112 is a key step for its biochemical characterization and to carry out future protein structure-function studies.     

Mimicking a p53‐MDM2 interaction based on a stable immunoglobulin‐like domain scaffold

Antibodies recognize protein targets with great affinity and specificity. However, posttranslational modifications and the presence of intrinsic disulfide‐bonds pose difficulties for their industrial use. The immunoglobulin fold is one of the most ubiquitous folds in nature and it is found in many proteins besides antibodies. An example of a protein family with an immunoglobulin‐like fold is the Cysteine Protease Inhibitors (ICP) family I42 of the MEROPs database for protease and protease inhibitors. Members of this protein family are thermostable and do not present internal disulfide bonds. Crystal structures of several ICPs indicate that they resemble the Ig‐like domain of the human T cell co‐receptor CD8α As ICPs present 2 flexible recognition loops that vary accordingly to their targeted protease, we hypothesize that members of this protein family would be ideal to design peptide aptamers that mimic protein‐protein interactions. Herein, we use an ICP variant from Entamoeba histolytica (EhICP1) to mimic the interaction between p53 and MDM2. We found that a 13 amino‐acid peptide derived from p53 can be introduced in 2 variable loops (DE, FG) but not the third (BC). Chimeric EhICP1‐p53 form a stable complex with MDM2 at a micromolar range. Crystal structure of the EhICP1‐p53(FG)‐loop variant in complex with MDM2 reveals a swapping subdomain between 2 chimeric molecules, however, the p53 peptide interacts with MDM2 as in previous crystal structures. The structural details of the EhICP1‐p53(FG) interaction with MDM2 resemble the interaction between an antibody and MDM2.     

Crystal structure of chagasin, the endogenous cysteine-protease inhibitor from Trypanosoma cruzi

Trypanosoma cruzi chagasin belongs to a recently discovered family of cysteine protease inhibitors found in lower eukaryotes and prokaryotes but not in mammals. Chagasin binds tightly to cruzain, the major lysosomal T. cruzi cysteine protease, involved with infectivity and survival of the parasite in mammalian host cells. In the scope of a project to characterize proteins diferentially expressed during T. cruzi metacyclogenesis, we have determined the crystal structure of chagasin, which is now the first X-ray structure of a chagasin-like cysteine protease inhibitor to be reported. The structure was solved by the SIRAS method and refined at 1.7A resolution and a comparison with the two NMR structures available revealed some differences in the loops involved in binding to cysteine proteases. The highly flexible loop 4 could be entirely modeled and residues 29-33 from loop 2 form a 3(10)-helix structure that may be important to stabilize the loop conformation. Chagasin crystal structure was docked to the highest resolution structure available of cruzain and a model of chagasin-cruzain interaction was analyzed. The knowledge of the chagasin crystal structure may contribute to the elucidation of the molecular mechanism involved in the inhibition of cruzain and other T. cruzi cysteine proteases.     

Overexpression of cysteine proteinase 2 in Entamoeba histolytica or Entamoeba dispar increases amoeba-induced monolayer destruction in vitro but does not augment amoebic liver abscess formation in gerbils

To study the role of cysteine proteinases in the pathogenicity of Entamoeba histolytica , we have attempted to overexpress the three main cysteine proteinases (EhCP1, EhCP2, EhCP5) of this parasite in trophozoites of E. histolytica as well as in non-pathogenic Entamoeba dispar by episomal transfection. Although each of the corresponding coding sequences were cloned in identical expression plasmids, we were unable to overexpress EhCP1 and EhCP5, respectively, but could substantially induce expression of EhCP2 in both amoeba species by sevenfold, leading to a threefold increase in total cysteine proteinase activity. Overexpression of EhCP2 did not influence expression of other cysteine proteinases and could be attributed to an increase of a single 35 kDa activity band in substrate gel electrophoresis. In contrast to previous findings, which indicated that amoeba cysteine proteinases are involved in erythrophagocytosis and liver abscess formation, cells overexpressing EhCP2 showed no difference in erythrophagocytosis or liver abscess formation compared with respective controls. However, overexpression of EhCP2 in both amoeba species resulted in a marked increase of in vitro monolayer destruction.     

Cross-Talk between Malarial Cysteine Proteases and Falstatin: The BC Loop as a Hot-Spot Target

Cysteine proteases play a crucial role in the development of the human malaria parasites Plasmodium falciparum and Plasmodium vivax. Our earlier studies demonstrated that these enzymes are equipped with specific domains for defined functions and further suggested the mechanism of activation of cysteine proteases. The activities of these proteases are regulated by a new class of endogenous inhibitors of cysteine proteases (ICPs). Structural studies of the ICPs of Trypanosoma cruzi (chagasin) and Plasmodium berghei (PbICP) indicated that three loops (termed BC, DE, and FG) are crucial for binding to target proteases. Falstatin, an ICP of P. falciparum, appears to play a crucial role in invasion of erythrocytes and hepatocytes. However, the mechanism of inhibition of cysteine proteases by falstatin has not been established. Our study suggests that falstatin is the first known ICP to function as a multimeric protein. Using site-directed mutagenesis, hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops, inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds. These results suggest that the BC loop of falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases. This finding suggests new strategies for the development of anti-malarial agents based on protease-inhibitor interactions.     

Cloning, expression and evaluation of the efficacy of a recombinant Entamoeba histolytica cysteine proteinase (EhCP4) antigen in minipig

Cysteine proteinases 4 (EhCP4) of Entamoeba histolytica are considered important for ameba pathogenicity. The recombinant gene was obtained by cloning and expression of the EhCP4 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in minipig against challenge infection in a minipig-E. histolytica model. There was a 53.16% reduction (P<0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-EhCP4 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P<0.001). This is a first report demonstrating that a recombinant form of EhCP4 generated in E. coli, to immunize a minipig model of E. histolytica, and there is significant protection. This study may help to understand the EhCP4 for human in the future.     

Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.     

Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model

Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba 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 the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and 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.     

The structure of chagasin in complex with a cysteine protease clarifies the binding mode and evolution of an inhibitor family

Protein inhibitors of proteolytic enzymes regulate proteolysis and prevent the pathological effects of excess endogenous or exogenous proteases. Cysteine proteases are a large family of enzymes found throughout the plant and animal kingdoms. Disturbance of the equilibrium between cysteine proteases and natural inhibitors is a key event in the pathogenesis of cancer, rheumatoid arthritis, osteoporosis, and emphysema. A family (I42) of cysteine protease inhibitors (http://merops.sanger.ac.uk) was discovered in protozoan parasites and recently found widely distributed in prokaryotes and eukaryotes. We report the 2.2 A crystal structure of the signature member of the I42 family, chagasin, in complex with a cysteine protease. Chagasin has a unique variant of the immunoglobulin fold with homology to human CD8alpha. Interactions of chagasin with a target protease are reminiscent of the cystatin family inhibitors. Protein inhibitors of cysteine proteases may have evolved more than once on nonhomologous scaffolds.     

Structural basis for the regulation of cysteine-protease activity by a new class of protease inhibitors in Plasmodium

Plasmodium cysteine proteases are essential for host-cell invasion and egress, hemoglobin degradation, and intracellular development of the parasite. The temporal, site-specific regulation of cysteine-protease activity is a prerequisite for survival and propagation of Plasmodium. Recently, a new family of inhibitors of cysteine proteases (ICPs) with homologs in at least eight Plasmodium species has been identified. Here, we report the 2.6?? X-ray crystal structure of the C-terminal, inhibitory domain of ICP from P. berghei (PbICP-C) in a 1:1 complex with falcipain-2, an important hemoglobinase of Plasmodium. The structure establishes Plasmodium ICP as a member of the I42 class of chagasin-like protease inhibitors but with large insertions and differences in the binding mode relative to other family members. Furthermore, the PbICP-C structure explains why host-cell cathepsin B-like proteases and, most likely, also the protease-like domain of Plasmodium SERA5 (serine-repeat antigen 5) are no targets for ICP.     

The structure of Leishmania mexicana ICP provides evidence for convergent evolution of cysteine peptidase inhibitors

Clan CA, family C1 cysteine peptidases (CPs) are important virulence factors and drug targets in parasites that cause neglected diseases. Natural CP inhibitors of the I42 family, known as ICP, occur in some protozoa and bacterial pathogens but are absent from metazoa. They are active against both parasite and mammalian CPs, despite having no sequence similarity with other classes of CP inhibitor. Recent data suggest that Leishmania mexicana ICP plays an important role in host-parasite interactions. We have now solved the structure of ICP from L. mexicana by NMR and shown that it adopts a type of immunoglobulin-like fold not previously reported in lower eukaryotes or bacteria. The structure places three loops containing highly conserved residues at one end of the molecule, one loop being highly mobile. Interaction studies with CPs confirm the importance of these loops for the interaction between ICP and CPs and suggest the mechanism of inhibition. Structure-guided mutagenesis of ICP has revealed that residues in the mobile loop are critical for CP inhibition. Data-driven docking models support the importance of the loops in the ICP-CP interaction. This study provides structural evidence for the convergent evolution from an immunoglobulin fold of CP inhibitors with a cystatin-like mechanism.     

Evaluation of the efficacy of a recombinant Entamoeba histolytica cysteine proteinase (EhCP112) antigen in minipig

Cysteine proteinases 112 (EhCP112) of Entamoeba histolytica are considered important for ameba pathogenicity. The recombinant gene was obtained by cloning and expression of the EhCP112 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in minipig against challenge infection in a minipig-E. histolytica model. There was a 46.29% reduction (P<0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-EhCP112 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P<0.001). This is a first report demonstrating that a recombinant form of EhCP112 generated in E. coli, to immunize a minipig model of E. histolytica, and there is significant protection. This study may help to understand the EhCP112 for human in the future.     

Resistance of Entamoeba histolytica to the cysteine proteinase inhibitor E64 is associated with secretion of pro-enzymes and reduced pathogenicity

Cysteine proteinases (CPs) have been considered suitable targets for the development of antiparasitic drugs. To assess the importance of CPs for the growth and pathogenicity of the protozoan parasite Entamoeba histolytica we have cultured amoebae in the presence of various cysteine proteinase inhibitors (CPIs). It was found that broad range CPIs, which are membrane permeable and rapidly enter the cell, are highly toxic at micromolar concentrations, and all attempts to generate E. histolytica mutants resistant to these CPIs were unsuccessful. In contrast, the broad range CPI E64, which does not permeate membranes as well, was deleterious at much higher concentrations, and amoebae rapidly developed resistance to this inhibitor. Compared with sensitive wild-type cells, E64-resistant E. histolytica were substantially reduced in the expression of various CP genes and were able to secrete unprocessed enzyme into the culture medium. Moreover, E64 resistance was associated with a significant reduction in virulence, because these cells were greatly impaired in the ability to generate liver abscesses in experimentally infected gerbils.