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1.  Overexpression of the Natural Inhibitor of Cysteine Peptidases in Leishmania mexicana Leads to Reduced Virulence and a Th1 Response▿  
Infection and Immunity  2009;77(7):2971-2978.
Leishmania mexicana cysteine peptidases (CPs) have been identified as important parasite virulence factors. More recently, a natural inhibitor of CPs (ICP) from L. mexicana has been characterized, and ICP mutants have been created. Infection of BALB/c mice with ICP null mutants or ICP reexpressing mutants resulted in nonhealing, progressively growing lesions albeit slightly attenuated compared with the growth of lesions produced by wild-type parasites. In contrast, BALB/c mice infected with mutants overexpressing ICP were able to significantly control lesion growth or heal. While BALB/c mice infected with wild-type parasites, ICP null mutants, or ICP reexpressing mutants produced significant antibody responses, including immunoglobulin E (IgE), no Th1 response, as indicated by antigen-induced splenocyte gamma interferon (IFN-γ) production, could be demonstrated. In contrast, BALB/c mice infected with mutants overexpressing ICP produced significantly less antibody, particularly IgE, as well as significantly reduced splenocyte interleukin-4 and enhanced IFN-γ production. BALB/c mice were able to resolve infection following infection with one ICP overexpressing clone, which was subsequently used for vaccination studies with BALB/c mice. However, no protection was afforded these mice when they were challenged with wild-type parasites. Nevertheless, two other mouse strains susceptible to L. mexicana, C3H and C57BL/6, vaccinated with overexpressing ICP mutants were able to control challenge infection associated with an enhanced Th1 response. This study confirms that L. mexicana CPs are virulence factors and that ICPs have therapeutic potential.
doi:10.1128/IAI.00558-08
PMCID: PMC2708542  PMID: 19433541
2.  Role of the Trypanosoma brucei natural cysteine peptidase inhibitor ICP in differentiation and virulence 
Molecular Microbiology  2007;66(4):991-1002.
ICP is a chagasin-family natural tight binding inhibitor of Clan CA, family C1 cysteine peptidases (CPs). We investigated the role of ICP in Trypanosoma brucei by generating bloodstream form ICP-deficient mutants (Δicp). A threefold increase in CP activity was detected in lysates of Δicp, which was restored to the levels in wild type parasites by re-expression of the gene in the null mutant. Δicp displayed slower growth in culture and increased resistance to a trypanocidal synthetic CP inhibitor. More efficient exchange of the variant surface glycoprotein (VSG) to procyclin during differentiation from bloodstream to procyclic form was observed in Δicp, a phenotype that was reversed in the presence of synthetic CP inhibitors. Furthermore, we showed that degradation of anti-VSG IgG is abolished when parasites are pretreated with synthetic CP inhibitors, and that parasites lacking ICP degrade IgG more efficiently than wild type. In addition, Δicp reached higher parasitemia than wild type parasites in infected mice, suggesting that ICP modulates parasite infectivity. Taken together, these data suggest that CPs of T. brucei bloodstream form play a role in surface coat exchange during differentiation, in the degradation of internalized IgG and in parasite infectivity, and that their function is regulated by ICP.
doi:10.1111/j.1365-2958.2007.05970.x
PMCID: PMC2680270  PMID: 17944830
3.  A potential role for ICP, a leishmanial inhibitor of cysteine peptidases, in the interaction between host and parasite 
Molecular microbiology  2004;54(5):1224-1236.
Summary
The biological role of a natural inhibitor of cysteine peptidases (designated ICP) of Leishmania has been investigated by genetic manipulation of the parasite. Null mutants grew normally in vitro, were as infective to macrophages in vitro as wild-type parasites, but had reduced infectivity to mice. Mutants re-expressing ICP from a single gene gave partial restoration of virulence in vivo, whereas mutants over-expressing ICP secreted the inhibitor and showed markedly reduced virulence in mice. Promastigotes of the null mutants had similar cysteine peptidase activities as the wild-type parasites, suggesting that ICP is not required for the expression or processing of the enzymes. The only proteins found to bind to ICP in promastigote cell lysates were fully processed forms of CPA and CPB, showing that ICP does not bind in abundance either to zymogens of the cysteine peptidases or other leishmanial proteins. However, only a small proportion of ICP co-localised with CPA and CPB in the promastigote (in the endoplasmic reticulum and Golgi) and the majority of ICP resided in vesicles that are apparently distinct from endosomes and the multivesicular tubule (MVT)-lysosome. These data suggest that ICP has a role other than modulation of the activity of the parasite's own cysteine peptidases and their normal trafficking to the MVT-lysosome via the flagellar pocket. The finding that ICP partially co-localised with an endocytosed cysteine peptidase leads us to postulate that ICP has a role in protection of the parasite against the hydrolytic environment of the sandfly gut and/or the parasitophorous vacuole of host macrophages.
doi:10.1111/j.1365-2958.2004.04355.x
PMCID: PMC2244714  PMID: 15554964
cysteine peptidase inhibitor; ICP; chagasin; lysosomes; endosomes; Leishmania
4.  Cross-Talk between Malarial Cysteine Proteases and Falstatin: The BC Loop as a Hot-Spot Target 
PLoS ONE  2014;9(4):e93008.
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.
doi:10.1371/journal.pone.0093008
PMCID: PMC3974720  PMID: 24699522
5.  Leishmania mexicana metacaspase is a negative regulator of amastigote proliferation in mammalian cells 
Cell Death & Disease  2012;3(9):e385-.
Metacaspases (MCAs) are caspase family cysteine peptidases that have been implicated in cell death processes in plants, fungi and protozoa. MCAs have also been suggested to be involved in cell cycle control, differentiation and clearance of aggregates; they are virulence factors. Dissecting the function of MCAs has been complicated by the presence in many organisms of multiple MCA genes or limitations on genetic manipulation. We describe here the creation of a MCA gene-deletion mutant (Δmca) in the protozoan parasite Leishmania mexicana, which has allowed us to dissect the role of the parasite's single MCA gene in cell growth and cell death. Δmca parasites are viable as promastigotes, and differentiate normally to the amastigote form both in in vitro macrophages infection and in mice. Δmca promastigotes respond to cell death inducers such as the drug miltefosine and H2O2 similarly to wild-type (WT) promastigotes, suggesting that MCAs do not have a caspase-like role in execution of L. mexicana cell death. Δmca amastigotes replicated significantly faster than WT amastigotes in macrophages and in mice, but not as axenic culture in vitro. We propose that the Leishmania MCA acts as a negative regulator of amastigote proliferation, thereby acting to balance cell growth and cell death.
doi:10.1038/cddis.2012.113
PMCID: PMC3461358  PMID: 22951982
metacaspase; caspase; cysteine peptidase; programmed cell death
6.  Identification of Semicarbazones, Thiosemicarbazones and Triazine Nitriles as Inhibitors of Leishmania mexicana Cysteine Protease CPB 
PLoS ONE  2013;8(10):e77460.
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.
doi:10.1371/journal.pone.0077460
PMCID: PMC3797739  PMID: 24146999
7.  IrAE – an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus 
Ticks are ectoparasitic blood-feeders and important vectors for pathogens including arboviruses, rickettsiae, spirochetes and protozoa. As obligate blood-feeders, one possible strategy to retard disease transmission is disruption of the parasite’s ability to digest host proteins. However, the constituent peptidases in the parasite gut and their potential interplay in the digestion of the blood meal are poorly understood. We have characterized a novel asparaginyl endopeptidase (legumain) from the hard tick Ixodes ricinus (termed IrAE), which is the first such characterization of a clan CD family C13 cysteine peptidase (protease) in arthropods. By RT-PCR of different tissues, IrAE mRNA was only expressed in the tick gut. Indirect immunofluorescence and electron microscopy localized IrAE in the digestive vesicles of gut cells and within the peritrophic matrix. IrAE was functionally expressed in Pichia pastoris and reacted with a specific peptidyl fluorogenic substrate, and acyloxymethyl ketone and aza-asparagine Michael acceptor inhibitors. IrAE activity was unstable at pH ≥ 6.0 and was shown to have a strict specificity for asparagine at P1 using a positional scanning synthetic combinatorial library. The enzyme hydrolyzed protein substrates with a pH optimum of 4.5, consistent with the pH of gut cell digestive vesicles. Thus, IrAE cleaved the major protein of the blood meal, hemoglobin, to a predominant peptide of 4 kDa. Also, IrAE trans-processed and activated the zymogen form of Schistosoma mansoni cathepsin B1 – an enzyme contributing to hemoglobin digestion in the gut of that bloodfluke. The possible functions of IrAE in the gut digestive processes of I. ricinus are compared with those suggested for other hematophagous parasites.
doi:10.1016/j.ijpara.2006.12.020
PMCID: PMC2587490  PMID: 17336985
Asparaginyl endopeptidase; Legumain; protease; Tick; Midgut; Hemoglobin digestion; Ixodes ricinus
8.  Trypanosoma brucei Metacaspase 4 Is a Pseudopeptidase and a Virulence Factor* 
The Journal of Biological Chemistry  2011;286(46):39914-39925.
Background: Metacaspases are multifunctional cysteine peptidases.
Results: Trypanosoma brucei metacaspase 4 is a catalytically inactive metacaspase homologue required for parasite virulence, which interacts with an active parasite metacaspase during release from the cell.
Conclusion: Metacaspase 4 is a pseudopeptidase virulence factor.
Significance: Extracellular release and proteolytic processing provide novel insights into metacaspase function.
Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five metacaspases (MCA1–MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure. MCA4 is expressed primarily in the bloodstream form of the parasite and associates with the flagellar membrane via dual myristoylation/palmitoylation. Loss of function phenotyping revealed critical roles for MCA4; rapid depletion by RNAi caused lethal disruption to the parasite's cell cycle, yet the generation of MCA4 null mutant parasites (Δmca4) was possible. Δmca4 had normal growth in axenic culture but markedly reduced virulence in mice. Further analysis revealed that MCA4 is released from the parasite and is specifically processed by MCA3, the only metacaspase that is both palmitoylated and enzymatically active. Accordingly, we have identified that the multiple metacaspases in T. brucei form a membrane-associated proteolytic cascade to generate a pseudopeptidase virulence factor.
doi:10.1074/jbc.M111.292334
PMCID: PMC3220528  PMID: 21949125
Caspase; Cysteine Protease; Enzyme Mutation; Parasite; Secretion; Trypanosoma brucei; Pseudopeptidase; Virulence Factor; Cysteine Peptidase; Caspase Family
9.  Expression of Multiple CPB Genes Encoding Cysteine Proteases Is Required for Leishmania mexicana Virulence In Vivo  
Infection and Immunity  2003;71(6):3190-3195.
Leishmania mexicana mutants deficient in the multicopy CPB gene array have reduced virulence, demonstrated by poor lesion growth in BALB/c mice and induction of a protective Th1 response. Reinsertion of the amastigote-specific CPB2.8 or metacyclic stage-specific CPB2 gene into a CPB-deficient mutant L. mexicana failed to restore either a Th2 response or sustained virulence. However, reexpression of multiple CPB genes from a cosmid significantly restored virulence. This was characterized by increased lesion and parasite growth and the acquisition of a Th2 response, as determined by measuring interleukin-4 production and immunoglobulin G1 (IgG1) and IgE levels. These studies confirm that L. mexicana cysteine proteases are important virulence factors and provide an explanation for the presence in L. mexicana of a multicopy tandem array of CPB genes.
doi:10.1128/IAI.71.6.3190-3195.2003
PMCID: PMC155739  PMID: 12761098
10.  In silico predicted epitopes from the COOH-terminal extension of cysteine proteinase B inducing distinct immune responses during Leishmania (Leishmania) amazonensis experimental murine infection 
BMC Immunology  2011;12:44.
Background
Leishmania parasites have been reported to interfere and even subvert their host immune responses to enhance their chances of survival and proliferation. Experimental Leishmania infection in mice has been widely used in the identification of specific parasite virulence factors involved in the interaction with the host immune system. Cysteine-proteinase B (CPB) is an important virulence factor in parasites from the Leishmania (Leishmania) mexicana complex: it inhibits lymphocytes Th1 and/or promotes Th2 responses either through proteolytic activity or through epitopes derived from its COOH-terminal extension. In the present study we analyzed the effects of Leishmania (Leishmania) amazonensis CPB COOH-terminal extension-derived peptides on cell cultures from murine strains with distinct levels of susceptibility to infection: BALB/c, highly susceptible, and CBA, mildly resistant.
Results
Predicted epitopes, obtained by in silico mapping, displayed the ability to induce cell proliferation and expression of cytokines related to Th1 and Th2 responses. Furthermore, we applied in silico simulations to investigate how the MHC/epitopes interactions could be related to the immunomodulatory effects on cytokines, finding evidence that specific interaction patterns can be related to in vitro activities.
Conclusions
Based on our results, we consider that some peptides from the CPB COOH-terminal extension may influence host immune responses in the murine infection, thus helping Leishmania survival.
doi:10.1186/1471-2172-12-44
PMCID: PMC3167762  PMID: 21824434
11.  Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes 
PLoS Pathogens  2010;6(3):e1000825.
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.
Author Summary
Plasmodium sporozoites are transmitted by Anopheles mosquitoes to the vertebrate host. They migrate through the skin before entering blood vessels and being transported with the bloodstream to liver sinusoids. There the sporozoites transmigrate through Kupffer cells and several hepatocytes before they invade a final hepatocyte and develop into thousands of merozoites. These daughter parasites are transported inside host cell-derived vesicles (merosomes) back to the bloodstream where they are finally released and infect red blood cells. Most of these processes depend on the activity of proteases, which must be tightly controlled to avoid proteolytic destruction of the parasite. We have identified a potent cysteine protease inhibitor of the rodent parasite Plasmodium berghei, which is expressed throughout the life cycle of the parasite. The inhibitor appears to play a 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.
doi:10.1371/journal.ppat.1000825
PMCID: PMC2845656  PMID: 20361051
12.  Molecular cloning and expression analysis of peptidase genes in the fish-pathogenic scuticociliate Miamiensis avidus 
Background
Parasite peptidases have been actively studied as vaccine candidates or drug targets for prevention or treatment of parasitic diseases because of their important roles for survival and/or invasion in the host. Like other parasites, the facultative histophagous ciliate Miamiensis avidus would possess peptidases that are closely associated with the invasion into the host tissue and survival in the host.
Results
The 17 genes encoding peptidases, including seven cathepsin-like cysteine peptidases, four serine carboxypeptidases, a eukaryotic aspartyl protease family protein, an ATP-dependent metalloprotease FtsH family protein, three leishmanolysin family proteins and a peptidase family M49 protein were identified from a Miamiensis avidus cDNA library by BLAST X search. Expression of genes encoding two cysteine peptidases, three leishmanolysin-like peptidases and a peptidase family M49 protein was up-regulated in the cell-fed ciliates compared to the starved ciliates. Especially, one cysteine peptidase (MaPro 4) and one leishmanolysin-like peptidase (MaPro 14) were transcribed more than 100-folds in the cell-fed ciliates.
Conclusions
The genetic information and transcriptional characteristics of the peptidases in the present results would be helpful to elucidate the role of peptidases in the invasion of scuticociliates into their hosts.
doi:10.1186/1746-6148-9-10
PMCID: PMC3599101  PMID: 23311870
Scuticociliates; Miamiensis avidus; Peptidases; RT-PCR
13.  MEROPS: the peptidase database 
Nucleic Acids Research  2007;36(Database issue):D320-D325.
Peptidases (proteolytic enzymes or proteases), their substrates and inhibitors are of great relevance to biology, medicine and biotechnology. The MEROPS database (http://merops.sanger.ac.uk) aims to fulfil the need for an integrated source of information about these. The organizational principle of the database is a hierarchical classification in which homologous sets of peptidases and protein inhibitors are grouped into protein species, which are grouped into families and in turn grouped into clans. Important additions to the database include newly written, concise text annotations for peptidase clans and the small molecule inhibitors that are outside the scope of the standard classification; displays to show peptidase specificity compiled from our collection of known substrate cleavages; tables of peptidase–inhibitor interactions; and dynamically generated alignments of representatives of each protein species at the family level. New ways to compare peptidase and inhibitor complements between any two organisms whose genomes have been completely sequenced, or between different strains or subspecies of the same organism, have been devised.
doi:10.1093/nar/gkm954
PMCID: PMC2238837  PMID: 17991683
14.  Inhibition profile of Leishmania mexicana arginase reveals differences with human arginase I 
Arginase (ARG), the enzyme that catalyzes the conversion of arginine to ornithine and urea, is the first and committed step in polyamine biosynthesis in Leishmania. The creation of a conditionally lethal Δarg null mutant in Leishmania mexicana has established that ARG is an essential enzyme for the promastigote form of the parasite and that the enzyme provides an important defense mechanism for parasite survival in the eukaryotic host. Furthermore, human ARGI (HsARGI) has also been implicated as a key factor in parasite proliferation. Thus, inhibitors of ARG offer a rational paradigm for drug design. To initiate a search for inhibitors of the L. mexicana ARG (LmARG), recombinant LmARG and HsARGI enzymes were purified from Escherichia coli. Both LmARG and HsARGI were specific for L-arginine and exhibited no activity with either D-arginine or agmatine as possible substrates. LmARG exhibited a Km of 25 ± 4 mM for L-arginine, a pH optimum ~9.0, and was dependent upon the presence of a divalent cation, preferentially manganese. A Km of 13.5 ± 2 mM for L-arginine was calculated for the HsARGI. A collection of 37 compounds was evaluated against both enzymes. Twelve of these compounds were identified as being either strong inhibitors of both LmARG and HsARGI or differential inhibitors between the two enzymes. Of the 12 compounds, six were selected for further analysis and the type and extent of inhibition determined.
doi:10.1016/j.ijpara.2010.12.006
PMCID: PMC3062745  PMID: 21232540
Leishmania; Arginase; Polyamines; Inhibitors; Amino acids
15.  α-Ketoheterocycles as inhibitors of Leishmania mexicana cysteine protease CPB 
ChemMedChem  2010;5(10):1734-1748.
Cysteine proteases of the papain superfamily are present in nearly all eukaryotes and also play pivotal roles in the biology of parasites. Inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Inspired by the in vivo antiparasitic activity of the vinyl sulfone based cysteine protease inhibitors (CPIs), a series of α-ketoheterocycles 1-15 has been developed as reversible inhibitors of a recombinant L. mexicana cysteine protease CPB2.8. The isoxazoles 1-3 and especially the oxadiazole 15 are potent reversible inhibitors of CPB2.8, however, in vitro whole-organism screening against a panel of protozoan parasites did not fully correlate with the observed inhibition of the cysteine protease.
doi:10.1002/cmdc.201000265
PMCID: PMC3245848  PMID: 20799311
cysteine proteases; inhibitors; ketoheterocycle; parasite CPB; Trypanosoma
16.  Effects of steroidal allenic phosphonic acid derivatives on the parasitic protists Leishmania donovani, Leishmania mexicana mexicana, and Pneumocystis carinii carinii. 
Several pathogenic fungi and protozoa are known to have sterols distinct from those of their mammalian hosts. Of particular interest as targets for drug development are the biosyntheses of the sterols of important parasites such as the kinetoplastid flagellates and the AIDS-associated opportunistic protist Pneumocystis carinii. These pathogens synthesize sterols with an alkyl group at C-24, and some have a double bond at C-22 of the side chain. Humans and other mammalian hosts are incapable of C-24 alkylation and C-22 desaturation. In the present study, three steroidal compounds with side chains substituted by phosphonyl-linked groups were synthesized and tested for their effects on Leishmania donovani and L. mexicana mexicana culture growth. The compounds inhibited organism proliferation at concentrations in micrograms per milliliter. The most potent inhibitors of this group of compounds were characterized by two ethyl groups at the phosphate function. Leishmania organisms treated with 17-[2-(diethylphosphonato) ethylidienyl]3-methoxy-19-norpregna-1,3,5-triene exhibited reduced growth after transfer into inhibitor-free medium. Because there are currently no axenic methods available for the continuous subcultivation of P. carinii, the effects of these drugs on this organism were evaluated by two alternative screening methods. The same two diethyl phosphonosteroid compounds that inhibited Leishmania proliferation were also the most active against P. carinii as determined by the potent effect they had on reducing cellular ATP content. Cystic as well as trophic forms responded to the drug treatments, as evaluated by a dual fluorescent staining live-dead assay. Other modifications of steroidal phosphonates may lead to the development of related drugs with increased activity and specificity for the pathogens.
PMCID: PMC163678  PMID: 8980773
17.  The crystal structure and functional characterization of an immunomodulatory salivary cystatin from the soft tick Ornithodoros moubata 
The Biochemical journal  2010;429(1):103-112.
Synopsis
The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help overcome the host’s defense during host-parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease-vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 Å and used to describe the structure-inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the proinflammatory cytokines TNF-α and IL-12, and proliferation of antigen-specific CD4+ T cells. This suggests that OmC2 may suppress the host’s adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.
doi:10.1042/BJ20100280
PMCID: PMC3523712  PMID: 20545626
cathepsin; cystatin; immune cells; structure-activity relationship; parasite; peptidase inhibitor
18.  Inhibitor of Cysteine Proteases Is Critical for Motility and Infectivity of Plasmodium Sporozoites 
mBio  2013;4(6):e00874-13.
ABSTRACT
Malaria is transmitted when motile sporozoites are injected into the dermis by an infected female Anopheles mosquito. Inside the mosquito vector, sporozoites egress from midgut-associated oocysts and eventually penetrate the acinar cells of salivary glands. Parasite-encoded factors with exclusive vital roles in the insect vector can be studied by classical reverse genetics. Here, we characterized the in vivo roles of Plasmodium berghei falstatin/ICP (inhibitor of cysteine proteases). This protein was previously suggested to act as a protease inhibitor during erythrocyte invasion. We show by targeted gene disruption that loss of ICP function does not affect growth inside the mammalian host but causes a complete defect in sporozoite transmission. Sporogony occurred normally in icp(−) parasites, but hemocoel sporozoites showed a defect in continuous gliding motility and infectivity for salivary glands, which are prerequisites for sporozoite transmission to the mammalian host. Absence of ICP correlates with enhanced cleavage of circumsporozoite protein, in agreement with a role as a protease regulator. We conclude that ICP is essential for only the final stages of sporozoite maturation inside the mosquito vector. This study is the first genetic evidence that an ICP is necessary for the productive motility of a eukaryotic parasitic cell.
IMPORTANCE
Cysteine proteases and their inhibitors are considered ideal drug targets for the treatment of a wide range of diseases, including cancer and parasitic infections. In protozoan parasites, including Leishmania, Trypanosoma, and Plasmodium, cysteine proteases play important roles in life cycle progression. A mouse malaria model provides an unprecedented opportunity to study the roles of a parasite-encoded inhibitor of cysteine proteases (ICP) over the entire parasite life cycle. By precise gene deletion, we found no evidence that ICP influences disease progression or parasite virulence. Instead, we discovered that this factor is necessary for parasite movement and malaria transmission from mosquitoes to mammals. This finding in a fast-moving unicellular protozoan has important implications for malaria intervention strategies and the roles of ICPs in the regulation of eukaryotic cell migration.
doi:10.1128/mBio.00874-13
PMCID: PMC3870247  PMID: 24281719
19.  MEROPS: the peptidase database 
Nucleic Acids Research  2004;32(Database issue):D160-D164.
Peptidases (proteolytic enzymes) are of great relevance to biology, medicine and biotechnology. This practical importance creates a need for an integrated source of information about them, and also about their natural inhibitors. The MEROPS database (http://merops.sanger.ac.uk) aims to fill this need. The organizational principle of the database is a hierarchical classification in which homologous sets of the proteins of interest are grouped in families and the homologous families are grouped in clans. Each peptidase, family and clan has a unique identifier. The database has recently been expanded to include the protein inhibitors of peptidases, and these are classified in much the same way as the peptidases. Forms of information recently added include new links to other databases, summary alignments for peptidase clans, displays to show the distribution of peptidases and inhibitors among organisms, substrate cleavage sites and indexes for expressed sequence tag libraries containing peptidases. A new way of making hyperlinks to the database has been devised and a BlastP search of our library of peptidase and inhibitor sequences has been added.
doi:10.1093/nar/gkh071
PMCID: PMC308805  PMID: 14681384
20.  PI3K-γ inhibitors in the therapeutic intervention of diseases caused by obligate intracellular pathogens 
Our increased understanding of host pathogen interactions shows that pathogens could capitalize on host cell pathways to favor entry and disease establishment. One such pathway used by Leishmania mexicana to enter into neutrophils and macrophages is the PI3Kγ signaling pathway. We recently showed that the use of the PI3Kγ inhibitor AS-605240 for the treatment of experimental L. mexicana infection in mice resulted in significantly lower parasite burdens and lesion sizes than WT untreated mice. Further, AS-605240 was found to be as effective as Sodium Stibogluconate, the drug of choice for treatment of L. mexicana infection, in reducing parasite burdens in mice. Here, we provide potential mechanisms of PI3Kγ blockade in promoting resistance to L. mexicana infection in mice. As a proof of principle, we propose that targeting host cell signaling pathways used in the establishment of infection could be a possible therapeutic option in the management of obligate intracellular pathogens.
doi:10.4161/cib.23360
PMCID: PMC3609848  PMID: 23749323
Leishmania; AS-605240; PI3Kγ; cell migration; macrophages; neutrophils; phagocytosis
21.  Are metacaspases caspases? 
The Journal of Cell Biology  2007;179(3):375-380.
The identification of caspases as major regulators of apoptotic cell death in animals initiated a quest for homologous peptidases in other kingdoms. With the discovery of metacaspases in plants, fungi, and protozoa, this search had apparently reached its goal. However, there is compelling evidence that metacaspases lack caspase activity and that they are not responsible for the caspaselike activities detected during plant and fungal cell death. In this paper, we attempt to broaden the discussion of these peptidases to biological functions beyond apoptosis and cell death. We further suggest that metacaspases and paracaspases, although sharing structural and mechanistic features with the metazoan caspases, form a distinct family of clan CD cysteine peptidases.
doi:10.1083/jcb.200705193
PMCID: PMC2064784  PMID: 17967946
22.  Leishmania Trypanothione Synthetase-Amidase Structure Reveals a Basis for Regulation of Conflicting Synthetic and Hydrolytic Activities*S⃞ 
The Journal of Biological Chemistry  2008;283(25):17672-17680.
The bifunctional trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania.
doi:10.1074/jbc.M801850200
PMCID: PMC2427367  PMID: 18420578
23.  MEROPS: the database of proteolytic enzymes, their substrates and inhibitors 
Nucleic Acids Research  2013;42(D1):D503-D509.
Peptidases, their substrates and inhibitors are of great relevance to biology, medicine and biotechnology. The MEROPS database (http://merops.sanger.ac.uk) aims to fulfill the need for an integrated source of information about these. The database has hierarchical classifications in which homologous sets of peptidases and protein inhibitors are grouped into protein species, which are grouped into families, which are in turn grouped into clans. Recent developments include the following. A community annotation project has been instigated in which acknowledged experts are invited to contribute summaries for peptidases. Software has been written to provide an Internet-based data entry form. Contributors are acknowledged on the relevant web page. A new display showing the intron/exon structures of eukaryote peptidase genes and the phasing of the junctions has been implemented. It is now possible to filter the list of peptidases from a completely sequenced bacterial genome for a particular strain of the organism. The MEROPS filing pipeline has been altered to circumvent the restrictions imposed on non-interactive blastp searches, and a HMMER search using specially generated alignments to maximize the distribution of organisms returned in the search results has been added.
doi:10.1093/nar/gkt953
PMCID: PMC3964991  PMID: 24157837
24.  Head-to-Tail Intramolecular Interaction of Herpes Simplex Virus Type 1 Regulatory Protein ICP27 Is Important for Its Interaction with Cellular mRNA Export Receptor TAP/NXF1 
mBio  2010;1(5):e00268-10.
Herpes simplex virus type 1 (HSV-1) protein ICP27 has many important functions during infection that are achieved through interactions with a number of cellular proteins. In its role as a viral RNA export protein, ICP27 interacts with TAP/NXF1, the cellular mRNA export receptor, and both the N and C termini of ICP27 must be intact for this interaction to take place. Here we show by bimolecular fluorescence complementation (BiFC) that ICP27 interacts directly with TAP/NXF1 during infection, and this interaction failed to occur with an ICP27 mutant bearing substitutions of serines for cysteines at positions 483 and 488 in the C-terminal zinc finger. Recently, we showed that ICP27 undergoes a head-to-tail intramolecular interaction, which could make the N- and C-terminal regions accessible for binding to TAP/NXF1. To determine the importance of intramolecular association of ICP27 to its interaction with TAP/NXF1, we performed BiFC-based fluorescence resonance energy transfer (FRET) by acceptor photobleaching. BiFC-based FRET showed that the interaction between ICP27 and TAP/NXF1 occurred in living cells upon head-to-tail intramolecular association of ICP27, further establishing that TAP/NXF1 interacts with both the N and C termini of ICP27.
IMPORTANCE
ICP27 is a key regulatory protein during herpes simplex virus type 1 (HSV-1) infection. ICP27 interacts with a number of cellular proteins, and an important question asks how these interactions are regulated during infection. We showed previously that ICP27 undergoes a head-to-tail intramolecular interaction, and here we show that the cellular mRNA export receptor protein TAP/NXF1 interacts with ICP27 after its head-to-tail association. Several proteins that interact with ICP27 require that the N and C termini of ICP27 be intact. These results demonstrate that the head-to-tail interaction of ICP27 may regulate some of its protein interactions perhaps through alternating between open and closed configurations.
doi:10.1128/mBio.00268-10
PMCID: PMC2975367  PMID: 21060739
25.  MEROPS: the peptidase database 
Nucleic Acids Research  2009;38(Database issue):D227-D233.
Peptidases, their substrates and inhibitors are of great relevance to biology, medicine and biotechnology. The MEROPS database (http://merops.sanger.ac.uk) aims to fulfil the need for an integrated source of information about these. The database has a hierarchical classification in which homologous sets of peptidases and protein inhibitors are grouped into protein species, which are grouped into families, which are in turn grouped into clans. The classification framework is used for attaching information at each level. An important focus of the database has become distinguishing one peptidase from another through identifying the specificity of the peptidase in terms of where it will cleave substrates and with which inhibitors it will interact. We have collected over 39 000 known cleavage sites in proteins, peptides and synthetic substrates. These allow us to display peptidase specificity and alignments of protein substrates to give an indication of how well a cleavage site is conserved, and thus its probable physiological relevance. While the number of new peptidase families and clans has only grown slowly the number of complete genomes has greatly increased. This has allowed us to add an analysis tool to the relevant species pages to show significant gains and losses of peptidase genes relative to related species.
doi:10.1093/nar/gkp971
PMCID: PMC2808883  PMID: 19892822

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