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.     

Identification of Semicarbazones,Thiosemicarbazones and Triazine Nitriles as Inhibitors of Leishmania mexicana Cysteine Protease CPB

Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.     

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.     

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 .     

Endosome sorting and autophagy are essential for differentiation and virulence of Leishmania major

Cellular remodeling during differentiation is essential for life-cycle progression of many unicellular eukaryotic pathogens such as Leishmania, but the mechanisms involved are largely uncharacterized. The role of endosomal sorting in differentiation was analyzed in Leishmania major by overexpression of a dominant-negative ATPase, VPS4. VPS4(E235Q) accumulated in vesicles from the endocytic pathway, and the mutant L. major was deficient in endosome sorting. Mutant parasites failed to differentiate to the obligate infective metacyclic promastigote form. Furthermore, the autophagy pathway, monitored via the expression of autophagosome marker GFP-ATG8, and shown to normally peak during initiation of metacyclogenesis, was disrupted in the mutants. The defect in late endosome-autophagosome function in the VPS4(E235Q) parasites made them less able to withstand starvation than wild-type L. major. In addition, a L. major ATG4-deficient mutant was found also to be defective in the ability to differentiate. This finding, that transformation to the infective metacyclic form is dependent on late endosome function and, more directly, autophagy, makes L. major a good model for studying the roles of these processes in differentiation.     

Natural sesquiterpene lactones are active against Leishmania mexicana

In this paper, the effects of 3 natural sesquiterpene lactones, i.e., helenalin (Hln), mexicanin (Mxc), and dehydroleucodine (DhL), were evaluated using cultured Leishmania mexicana promastigotes. It was observed that the compounds inhibited the in vitro growth of the parasites at relatively low concentrations. The effect was rapid and irreversible with an estimated IC50 of 2-4 microM, while all the lactones were more effective than ketoconazole. Moreover, these compounds exhibited low cytotoxicity for mammalian cells. Hln induced strong vacuolization of the parasite cytoplasm, although pericellular microtubules were preserved. The 3 lactones induced DNA fragmentation as judged by the high labeling with the fluorescent TUNEL method, which was confirmed by electrophoresis on agarose gels. The ability of the parasites to invade Vero cells was also decreased by exposure to low concentrations of the compounds. We conclude that these compounds can affect the parasite's life cycle, possibly through multiple mechanisms. Identification of the molecular targets of these natural products and their effects on amastigotes should be determined to evaluate the possible therapeutic use of the compounds against leishmaniasis.     

Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes.

The CPB genes of the protozoan parasite Leishmania mexicana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1-S3 and S1'-S3' subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3' by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3 to S3' subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P2' position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.     

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.     

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.     

Inhibition of growth of Leishmania mexicana mexicana by Leishmania mexicana amazonensis during "in vitro" co-cultivation

Inhibition of one Leishmania subspecies by exometabolites of another subspecies, a phenomenon not previously reported, is suggested by our recent observations in cell cloning experiments with Leishmania mexicana mexicana and Leishmania mexicana amazonensis. Clones were identified using the technique of schizodeme analysis. The phenomenon observed is clearly relevant to studies of parasite isolation, leishmanial metabolism, cross-immunity and chemotherapy.     

Leishmania tropica and Leishmania mexicana: cross-immunity in mice

The effect of a previous or concurrent Leishmania tropica major infection on a L. mexicana infection was studied. Mice which were recovering from or had recovered from a L. tropica infection were found to be totally resistant to L. mexicana. Infection of mice already carrying a L. mexicana infection with L. tropica resulted in subsequent ulceration and eventual healing of the lesions caused by both Leishmania species. Mice infected with L. mexicana were found normally to be no more susceptible to L. tropica than untreated mice: Only when L. tropica infections were located in the region of a draining lymph node already serving a L. mexicana infection did lesions of the former parasite persist.     

Subcellular localisation of purine-metabolising enzymes in Leishmania mexicana mexicana

Leishmania mexicana mexicana cultured promastigotes were fractionated by isopycnic centrifugation on linear sucrose gradients. Guanine, hypoxanthine and xanthine phosphoribosyltransferase activities were found to be associated with glycosomes, whereas adenine phosphoribosyltransferase was cytosolic. 3'- and 5'-nucleotidases and IMP dehydrogenase were shown to be particulate, the former two possibly being associated with the plasma membrane, IMP dehydrogenase with the endoplasmic reticulum. Nucleosidases and deaminases were found to be cytosolic. The results demonstrate that intracellular separation of enzymes could play a part in the regulation of the parasite's purine metabolism.     

Stable ornithine decarboxylase in promastigotes of Leishmania mexicana mexicana

Studies on the decarboxylation of ornithine in Leishmania mexicana have shown that this activity corresponds to a true ornithine decarboxylase rather than to an oxidative decarboxylation or aminotransferase reaction, both of which also give rise to the release of CO2. The stoichiometric relationship between substrate and products has indicated that extracts of L. mexicana were able to catalyse the formation of an unknown compound besides putrescine and CO2. The addition of cycloheximide to cultures of L. mexicana allowed us to demonstrate that ornithine decarboxylase degradation in vivo was extremely slow in this parasite. This remarkable stability of the enzyme is only comparable to that found in Trypanosoma brucei and contrasts with the high turnover rate of ornithine decarboxylases of different mammalian cells.     

Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana.

The cell surface of the parasitic protozoan Leishmania mexicana is coated by glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids. To investigate whether the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for dolichol-phosphate-mannose synthase (DPMS) and attempted to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of dolichol-phosphate mannose, the sugar donor for all mannose additions in the biosynthesis of both the anchor and free GPIs, except for a alpha1-3-linked mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for dolichol-phosphate-mannose in other glycosylation pathways in L.mexicana is minimal. Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes were first transfected with an episomal copy of lmdpms, indicating that lmdpms, and possibly GPI biosynthesis, is essential for parasite growth. As evidence presented in this and previous studies indicates that neither GPI-anchored glycoproteins nor lipophosphoglycan are required for growth of cultured parasites, it is possible that the abundant and functionally uncharacterized free GPIs are essential membrane components.     

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.     

Sphingolipids are essential for differentiation but not growth in Leishmania

Sphingolipids (SLs) play critical roles in eukaryotic cells in the formation of lipid rafts, membrane trafficking, and signal transduction. Here we created a SL null mutant in the protozoan parasite Leishmania major through targeted deletion of the key de novo biosynthetic enzyme serine palmitoyltransferase subunit 2 (SPT2). Although SLs are typically essential, spt2- Leishmania were viable, yet were completely deficient in de novo sphingolipid synthesis, and lacked inositol phosphorylceramides and other SLs. Remarkably, spt2- parasites maintained 'lipid rafts' as defined by Triton X-100 detergent resistant membrane formation. Upon entry to stationary phase spt2- failed to differentiate to infective metacyclic parasites and died instead. Death occurred not by apoptosis or changes in metacyclic gene expression, but from catastrophic problems leading to accumulation of small vesicles characteristic of the multivesicular body/multivesicular tubule network. Stage specificity may reflect changes in membrane structure as well as elevated demands in vesicular trafficking required for parasite remodeling during differentiation. We suggest that SL-deficient Leishmania provide a useful biological setting for tests of essential SL enzymes in other organisms where SL perturbation is lethal.     

Cysteine peptidases and their inhibitors in breast and genital cancer

Cysteine proteinases and their inhibitors probably play the main role in carcinogenesis and metastasis. The metastasis process need external proteolytic activities that pass several barriers which are membranous structures of the connective tissue which includes, the basement membrane of blood vessels. Activities of the proteinases are regulated by endogenous inhibitors and activators. The imbalance between cysteine proteinases and cystatins seems to be associated with an increase in metastatic potential in some tumors. It has also been reported that proteinase inhibitors, specific antibodies for these enzymes and inhibition of the urokinase receptor may prevent cancer cell invasion. Some proteinase inhibitor could serve as agents for cancer treatment.