Cysteine peptidases CPA and CPB are vital for autophagy and differentiation in Leishmania mexicana

In the past, ultrastructural investigations of Leishmania mexicana amastigotes revealed structures that were tentatively identified as autophagosomes. This study has now provided definitive data that autophagy occurs in the parasite during differentiation both to metacyclic promastigotes and to amastigotes, autophagosomes being particularly numerous during metacyclic to amastigote form transformation. Moreover, the results demonstrate that inhibiting two major lysosomal cysteine peptidases (CPA and CPB) or removing their genes not only interferes with the autophagy pathway but also prevents metacyclogenesis and transformation to amastigotes, thus adding support to the hypothesis that autophagy is required for cell differentiation. The study suggests that L. mexicana CPA and CPB perform similar roles to the aspartic peptidase PEP4 and the serine peptidase PRB1 in Saccharomyces cerevisiae. The results also provide an explanation for why L. mexicana CPA/CPB-deficient mutants transform to amastigotes very poorly and lack virulence in macrophages and mice.     

Heparin Modulates the Endopeptidase Activity of Leishmania mexicana Cysteine Protease Cathepsin L-Like rCPB2.8

Background

Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity.

Methodology/Principal Findings

The data analysis revealed that the presence of heparin affects all steps of the enzyme reaction: (i) it decreases 3.5-fold the k ₁ and 4.0-fold the k ₋₁, (ii) it affects the acyl-enzyme accumulation with pronounced decrease in k ₂ (2.7-fold), and also decrease in k ₃ (3.5-fold). The large values of ΔG  =  12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys²⁵)-S⁻/(His¹⁶³)-Im⁺ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme.

Conclusions/Significance

Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface.     

Expression of Cysteine Proteinases by Metacyclic Promastigotes of Leishmania mexicana

ABSTRACT. The expression of cysteine proteinases by metacyclic promastigotes of Leishmania mexicana was investigated using gelatin polyacrylamide gel electrophoresis. Two prominent bands were detected which distinguished metacyclics from multiplicative promastigotes, lacking detectable cysteine proteinase activity, and amastigotes, with a distinct banding pattern composed of multiple enzymes. A correlation between relative activity of the metacyclic-specific bands and the prevalence of metacyclics was found both during the growth cycle in vitro as metacyclogenesis occurred, and by comparison of stationary phase populations from consecutive subpassages in vitro. Irreversible inhibition of the metacyclic activities using N-benzyloxycarbonyl-phenylalanyl-alanyl diazomethane did not inhibit metacyclic to amastigote transformation in vitro. These activities provide a useful biochemical marker for the metacyclic promastigotes of L. mexicana .     

In Search of Selective Inhibitors of Cysteine Protease,Cathepsin K

Two potential azapeptide inhibitors of cathepsin K were designed and synthesized. To analyze in detail interactions between these azainhibitors and the investigated cysteine protease, molecular dynamics simulations were performed. For the obtained compounds the equilibrium constants for dissociation of inhibitor – enzyme complex, K_i, were determined. The examined azapeptides proved to be not as potent inhibitors of cathepsin K as they were expected to be according to the results of simulations. However, these calculations provide valuable information about probable structures of this type of peptidomimetics in the catalytic pocket of cathepsin K, which could be useful in designing of more selective inhibitors of this cysteine protease.     

半胱氨酸蛋白酶拟肽抑制剂设计新进展

半胱氨酸蛋白酶包括多种酶,这些酶在广泛的生命过程中发挥作用。人类正常的半胱氨酸蛋白酶表达失调,寄生虫、病毒的半胱氨酸蛋白酶表达与多种病理情况相关。对于这类疾病,抑制半胱氨酸蛋白酶是一个可行的药物治疗策略。当前这类药物设计的目标是3种结构不同的半胱氨酸蛋白酶,即木瓜蛋白酶家族、半胱氨酸-天冬氨基特异性蛋白酶家族(caspases)和小核糖核酸病毒科半胱氨酸蛋白酶抑制剂家族。本文综述了近年来有关半胱氨酸蛋白酶抑制剂的设计思路。     

Cysteine protease B of Leishmania mexicana inhibits host Th1 responses and protective immunity

C3H mice infected with Leishmania mexicana fail to develop a protective Th1 response, and are unable to cure. In this study, we show that L. mexicana cysteine proteases suppress the antileishmanial immune response. Previous studies demonstrated that deletion of the entire multicopy cysteine protease B (CPB) gene array in L. mexicana is associated with decreased parasite virulence, potentially attributable to factors related to parasite fitness rather than to direct effects on the host immune response. We now show that C3H mice infected with the L. mexicana deletion mutant (Deltacpb) initially develop lesions that grow at rates comparable to those of wild-type L. mexicana-infected mice. However, in contrast to controls, Deltacpb-induced lesions heal with an accompanying Th1 immune response. Lesion resolution was Th1 dependent, as Deltacpb-infected IL-12p40(-/-) and STAT4(-/-) mice developed high parasite burdens and progressive disease. Moreover, when L. major was transfected with a cosmid expressing multiple L. mexicana CPB genes, this parasite induced a significantly lower IFN-gamma response compared with wild-type L. major. These data indicate that cysteine proteases of L. mexicana are critical in suppressing protective immune responses and that inhibition of CPB may prove to be a valuable immunomodulatory strategy for chronic forms of leishmaniasis.     

Identification of mitogen-activated protein kinase homologues from Leishmania mexicana

Mitogen-activated protein kinases are key-regulatory elements in the differentiation, proliferation, apoptosis and stress response of eukaryotic cells. Our recent identification of a mitogen-activated protein kinase homologue in Leishmania mexicana which is essential for the proliferation of the amastigote stage of the parasite living in the parasitophorous vacuole of the infected macrophage prompted us to screen the genome of L. mexicana for additional mitogen-activated protein kinase homologues using degenerate oligonucleotide primers in a polymerase chain reaction amplification approach. We cloned and sequenced the genes for eight new mitogen-activated protein kinase homologues which were subsequently shown to be present in one copy per haploid genome. The mRNA levels of the kinases varied significantly in pro- and amastigote life stages of the parasite. We used the structural information of the p38 stress-activated protein kinase, which belongs to the family of mitogen-activated protein kinases, for the alignment of the deduced proteins and the verification of the predicted secondary structure elements. All new mitogen-activated protein kinases reveal the typical 12 subdomain primary structure, the conserved residues characterising serine/threonine protein kinases and the characteristic TXY motif in the phosphorylation lip. Typical features of some of the molecules are amino acid insertions between the subdomains and long carboxy-terminal amino acid extensions carrying putative src-homology 3-binding motifs.     

Design,Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors

Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K_i and IC₅₀ values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC₅₀ values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a).     

Carboxydipeptidase activities of recombinant cysteine peptidases. Cruzain of Trypanosoma cruzi and CPB of Leishmania mexicana.

The recombinant cysteine peptidases, cruzain from Trypanosoma cruzi and CPB2.8DeltaCTE from Leishmania mexicana, are cathepsin L-like and characteristically endopeptidases. In this study, we characterized the carboxydipeptidase activities of these enzymes and compared them with those of human recombinant cathepsin B and cathepsin L. The analysis used the internally quenched fluorescent peptide Abz-FRFK*-OH and some of its analogues, where Abz is ortho-aminobenzoic acid and K* is (2,4-dinitrophenyl)-epsilon-NH2-lysine. These peptides were demonstrated to be very sensitive substrates, due to the strong quenching effect of K* on the fluorescence of the Abz group. The carboxydipeptidase activity of cruzain was shown to be very similar to that of cathepsin B, while that of CPB2.8DeltaCTE is closer to the carboxydipeptidase activity of cathepsin L. The S2 subsite architecture of cruzain and the nature of the amino acid at the P2 position of the substrates determine its carboxydipeptidase activity and gives further and direct support to the notion that the carboxydipeptidase activity of the papain family cysteine peptidases rely on the S2-P2 interaction [N?gler D. K., Tam, W., Storer, A.C., Krupa, J.C., Mort, J.S. & Menard, R. (1999) Biochemistry38, 4868-4874]. Cruzain and CPB2.8DeltaCTE presented a broad pH-range for both the endo- and exo-peptidase activities, although the later is approximately one order of magnitude lower. This feature, that is not common in related mammalian cysteine peptidases, is consistent with the enzymes being exposed to different environmental conditions and having different locations during parasite development.     

Cysteine peptidases as virulence factors of Leishmania

Leishmania mexicana amastigotes are particularly rich in cysteine peptidases (CPs), which play important roles in facilitating the survival and growth of the parasites in mammals. The importance of the CPs as virulence factors and their potential as drug targets and vaccine candidates has been investigated extensively. Recent years, however, have heralded advances in our knowledge and understanding of leishmanial CPs on two fronts. Firstly, genome analysis has revealed the great diversity of CPs, and, secondly, the ways in which the most widely studied CPs, designated CPB, influence the interaction between parasite and mammalian host have been elucidated. These topics are the focus of this review.     

Di-O-alkylglycerol, mono-O-alkylglycerol and ceramide inositol phosphates of Leishmania mexicana mexicana promastigotes

Three acidic unsaponifiable lipid fractions were isolated by chromatographic methods from sandfly vector stages (promastigotes) of a protozoan parasite of man, Leishmania mexicana mexicana, cultured in vitro. Fast atom bombardment mass spectrometry, fast atom bombardment collision induced tandem mass spectrometry and metabolic labeling were used to characterize these lipids as di-O-alkylphosphatidyl-inositols, lyso-1-O-alkylphosphatidylinositols and inositol phosphosphingolipids. Molecular species of the dialkyl forms, new to natural product biochemistry, had a 20:0 substituent and either 17:1 or 18:1. The monoalkyl forms had either 17:0 or 18:0. The predominant ceramide had the 16:1 base and the lesser component the 16:0 base. In both, the N-acyl group was 18:0.     

Molecular Design,Synthesis and Trypanocidal Activity of Dipeptidyl Nitriles as Cruzain Inhibitors

A series of compounds based on the dipeptidyl nitrile scaffold were synthesized and assayed for their inhibitory activity against the T. cruzi cysteine protease cruzain. Structure activity relationships (SARs) were established using three, eleven and twelve variations respectively at the P1, P2 and P3 positions. A K_i value of 16 nM was observed for the most potent of these inhibitors which reflects a degree of non-additivity in the SAR. An X-ray crystal structure was determined for the ligand-protein complex for the structural prototype for the series. Twenty three inhibitors were also evaluated for their anti-trypanosomal effects and an EC₅₀ value of 28 μM was observed for the most potent of these. Although there remains scope for further optimization, the knowledge gained from this study is also transferable to the design of cruzain inhibitors based on warheads other than nitrile as well as alternative scaffolds.     

Purine phosphoribosyltransferases of Leishmania mexicana mexicana and other flagellate protozoa

Amastigotes and cultured promastigotes of Leishmania mexicana mexicana and L. m. amazonensis, cultured promastigotes of L. donovani and L. tarentolae, and the culture forms of Crithidia fasciculata, Herpetomonas muscarum muscarum and H. m. ingenoplastis all possessed four phosphoribosyltransferase (PRTase) activities: adenine PRTase, hypoxanthine PRTase, guanine PRTase and xanthine PRTase. The enzymes of L. m. mexicana required divalent cations for activity; Mn2+ or Co2+ produced maximal activity in most cases. Hypoxanthine PRTase, guanine PRTase and xanthine PRTase from all organisms were sedimentable in part, suggesting that they may occur within glycosomes. The enzymes of L. m. mexicana cultured promastigotes were inhibited by a range of purine analogues.     

Identification of Protease Inhibitors by a Fast Fluorimetric Assay

Anomalous protease activities are associated with many diseases. Great efforts are paid for selecting specific protease modulators for therapeutic approaches. We have selected new modulators of enzyme activity by an homogeneous assay based on a doubly labeled small peptide used as substrate of trypsin. The substrate incorporates the fluorophore 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS) at one end and an EDANS-quenching moiety (Dabcyl, (4-(4-dimethylaminophenylazo)-benzoic acid)) on the other end. Following cleavage by trypsin, the peptide-EDANS product is released interrupting the fluorescence resonance energy transfer effect and yielding bright fluorescence, which can be detected using excitation wavelengths at 335–345 nm and emission wavelengths at 485–510 nm. The method optimized, tested by detecting the strong inhibiting effect of α1-antitrypsin on trypsin activity, has been developed on 384 multi-well plates in a volume of 10 μL, using an automated platform. From the screening of a chemical library, four compounds that inhibit trypsin activity with IC₅₀s in the micromolar range have been identified. Interestingly, the most active compound (M4) shows a chemical structure recapitulating that of other more potent inhibitors with thiourea and halogenated centers. Molecular docking studies show that M4 is a competitive inhibitor recognizing most residues within or nearby the catalytic pocket.     

Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana

The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.     

Purification and characterization of proteolytic enzymes of Leishmania mexicana mexicana amastigotes and promastigotes

Leishmania mexicana mexicana amastigote and promastigote soluble proteinases were purified using gel filtration and ion exchange chromatography. For the amastigotes, two main proteinase activity peaks were separated with both methods. These accounted for approximately 10% and 90% of the total activity. Characterization of the two activities for substrate specificity and sensitivity to inhibitors indicated that the major peak from both column methods contained enzymes with the characteristics of cysteine proteinases. SDS-polyacrylamide gel electrophoresis of the enzyme from the major peak purified by gel filtration revealed one polypeptide with a molecular weight in the region of 31 000. In contrast, the activity of the minor peak eluted from the columns was of higher molecular weight (67 000) and was similar to metalloproteinases. Purification of the soluble proteinases in the promastigote of L. m. mexicana produced only one activity peak from both column techniques. This activity (mol. wt 67 000) corresponded to the high molecular weight proteinase of the amastigote. The purified proteinases were active on 4-nitroanilide and 7-amino-4-methylcoumarin derivatives of various small peptides. The high molecular weight proteinases of both amastigotes and promastigotes were similarly active against most of the peptides, suggesting a low specificity of the enzymes. In contrast, the low molecular weight amastigote proteinases were particularly active against two of the substrates, namely BZ-Pro-Phe-Arg-Nan and Z-Phe-Arg-MCA. These results indicate that a highly active, substrate-specific, soluble proteinase, with characteristics of a cysteine proteinase, is produced upon transformation of the L. m. mexicana promastigote to amastigote. The discovery and characterization of this enzyme offers opportunities for the development of new antileishmanial agents.