Solution structure of the inhibitor of cysteine proteases 1 from Entamoeba histolytica reveals a possible auto regulatory mechanism (original) (raw)
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Gene, 2011
Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereas EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight β-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1999
Papain from Carica papaya, an easily available cysteine protease, is the best-studied representative of this family of enzymes. The three dimensional structure of papain is very similar to that of other cysteine proteases of either plant (actinidin, caricain, papaya protease IV) or animal (cathepsins B, K, L, H) origin. As abnormalities in the activities of mammalian cysteine proteases accompany a variety of diseases, there has been a long-lasting interest in the development of potent and selective inhibitors for these enzymes. A covalent inhibitor of cysteine proteases, designed as a combination of epoxysuccinyl and peptide moieties, has been modeled in the catalytic pocket of papain. A number of its configurations have been generated and relaxed by constrained simulated annealing-molecular dynamics in water. A clear conformational variability of this inhibitor is discussed in the context of a conspicuous conformational diversity observed earlier in several solid-state structures of other complexes between cysteine proteases and covalent inhibitors. The catalytic pockets S2 and even more so S3, as defined by the pioneering studies on the papain^ZPACK, papain^E64c and papain^leupeptin complexes, appear elusive in view of the evident flexibility of the present inhibitor and in confrontation with the obvious conformational scatter seen in other examples. This predicts limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit the minute differences in the catalytic pockets of various members of this family. A simultaneous comparison of the three published proenzyme structures suggests the enzyme's prosegment binding loopp rosegment interface as a new potential target for selective inhibitors of papain-related thiol proteases. ß 1999 Elsevier Science B.V. All rights reserved.
Identification of EhICP1, a chagasin-like cysteine protease inhibitor of Entamoeba histolytica
FEBS Letters, 2005
Based on the Entamoeba histolytica genome project (www.sanger.ac.uk/Projects/E\_histolytica/) 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 K i Õs in the picomolar range. By immunocytology, we localized the endogenous $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 i 1.5 lM) and trophozoite extract (K i in sub-mM range), it may be a candidate for the rational development of anti-amoebiasis drugs.
Journal of Biological Chemistry, 2010
Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design. Entamoeba histolytica is the causative protozoan parasite of amebic colitis and liver abscesses and the second leading cause of death from parasitic diseases (1). Invasive E. histolytica trophozoites secrete 10-1,000-fold more cysteine proteinases than non-invasive Entamoeba dispar (2), whereas EhCP 2-deficient trophozoites demonstrate reduced virulence in animal models (3). During amebic invasion, EhCPs degrade the mucus barrier and destroy the extracellular matrix (4) underlying the epithelium, thereby causing epithelial detachment and ulceration (4, 5). Amebic penetration through ulcers facilitates entry into the mucosa and subsequently spread to the liver (3-5). Furthermore, EhCPs interrupt the function of the host immune system by cleaving immune molecules, including IgA and IgG (6, 7), processing complement C3 (2) and inactivating complement C3a and C5a (8). They also generate mature IL-1 from pro-IL-1 (9) but inactivate pro-IL-18 (10). These alternations of the immune response contribute to ineffective host defenses and thereby excessive inflammation and tissue damage. Genome-wide homology searches have identified 50 E. histolytica genes encoding cysteine proteases (11, 12), most of which belong to the C1 papain superfamily. These enzymes are expressed as zymogens with a hydrophobic pre-domain of 12-20 residues, a pro-domain of 55-148 residues, a catalytic domain of 190-488 residues, and six cysteine residues that form the disulfide bridges to stabilize the tertiary structure (13, 14). The EhCPs that have been characterized to date include EhCP1 (EhCP-A1) (15, 16), EhCP2 (EhCP-A2) (14), EhCP3 (EhCP-A3) (10, 14), EhCP5 (EhCP-A5) (15, 17), and EhCP112 (EhCP-B9) (18). These enzymes all undergo autocatalytic activation in which the inhibitory pro-domain is cleaved from the zymogen. They also have a cathepsin L-like structure, but like cathepsin B, these enzymes can also accommodate Arg at P2 (14). Among the remaining EhCPs,
FEBS Letters, 1988
High hydrophobicity of the second amino acid N-terminal to the scissile bond (P, residue) is generally considered to be the major factor in the specificity of the substrates for cysteine proteases of the papain family. To examine the catalytic contribution of the S,P, hydrogen bond apparent from X-ray crystallographic studies, the kinetics of Z-Phe-Gly-OEt and its thiono derivative were compared. The thiono compound contains a sulfur atom in place of the carbonyl oxygen of the phenylalanine residue. It was found that the specificity rate constants for the reactions of the thiono substrate with various cysteine proteases are lower by 2-3 orders of magnitude as compared to the corresponding rate constants for the 0x0 substrate. This remarkable effect is not expected in the light of previous studies indicating that the change from oxygen to sulfur in the P, residue was without an appreciable effect. The results are interpreted in terms of a distorted binding of the thiono substrate.
FEBS Letters, 2006
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.
PLoS ONE, 2013
The cysteine biosynthetic pathway is essential for survival of the protist pathogen Entamoeba histolytica, and functions by producing cysteine for countering oxidative attack during infection in human hosts. Serine acetyltransferase (SAT) and Oacetylserine sulfhydrylase (OASS) are involved in cysteine biosynthesis and are present in three isoforms each. While EhSAT1 and EhSAT2 are feedback inhibited by end product cysteine, EhSAT3 is nearly insensitive to such inhibition. The active site residues of EhSAT1 and of EhSAT3 are identical except for position 208, which is a histidine residue in EhSAT1 and a serine residue in EhSAT3. A combination of comparative modeling, multiple molecular dynamics simulations and free energy calculation studies showed a difference in binding energies of native EhSAT3 and of a S208H-EhSAT3 mutant for cysteine. Mutants have also been generated in vitro, replacing serine with histidine at position 208 in EhSAT3 and replacing histidine 208 with serine in EhSAT1. These mutants showed decreased affinity for substrate serine, as indicated by K m , compared to the native enzymes. Inhibition kinetics in the presence of physiological concentrations of serine show that IC50 of EhSAT1 increases by about 18 folds from 9.59 mM for native to 169.88 mM for H208S-EhSAT1 mutant. Similar measurements with EhSAT3 confirm it to be insensitive to cysteine inhibition while its mutant (S208H-EhSAT3) shows a gain of cysteine inhibition by 36% and the IC50 of 3.5 mM. Histidine 208 appears to be one of the important residues that distinguish the serine substrate from the cysteine inhibitor.
Structural studies of cysteine proteases and their inhibitors
actabp.pl
Cysteine proteases (CPs) are responsible for many biochemical processes occurring in living organisms and they have been implicated in the development and progression of several diseases that involve abnormal protein turnover. The activity of CPs is regulated among others by their specific inhibitors: cystatins. The main aim of this review is to discuss the structure-activity relationships of cysteine proteases and cystatins, as well as of some synthetic inhibitors of cysteine proteases structurally based on the binding fragments of cystatins.
Journal of Biological Chemistry, 2004
Human Ero1␣ is an endoplasmic reticulum (ER)-resident protein responsible for protein disulfide isomerase (PDI) oxidation. To clarify the molecular mechanisms underlying its function, we generated a panel of cysteine replacement mutants and analyzed their capability of: 1) complementing a temperature-sensitive yeast Ero1 mutant, 2) favoring oxidative folding in mammalian cells, 3) forming mixed disulfides with PDI and ERp44, and 4) adopting characteristic redox-dependent conformations. Our results reveal that two essential cysteine triads (Cys 85 -Cys 94 -Cys 99 and Cys 391 -Cys 394 -Cys 397 ) cooperate in electron transfer, with Cys 94 likely forming mixed disulfides with PDI. Dominant negative phenotypes arise when critical residues within the triads are mutated (Cys 394 , Cys 397 , and to a lesser extent Cys 99 ). Replacing the first cysteine in either triad (Cys 85 or Cys 391 ) generates mutants with weaker activity. In addition, mutating either Cys 85 or Cys 391 , but not Cys 397 , reverts the dominant negative phenotype of the C394A mutant. These findings suggest that interactions between the two triads, dependent on Cys 85 and Cys 391 , are important for Ero1␣ function, possibly stabilizing a platform for efficient PDI oxidation.