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1.
Nogueira de Melo AC d'Avila-Levy CM Dias FA Armada JL Silva HD Lopes AH Santos AL Branquinha MH Vermelho AB 《International journal for parasitology》2006,36(4):415-422
The cell-associated and extracellular peptidases of Herpetomonas megaseliae grown in brain-heart infusion and in modified Roitman's complex media were analyzed by measuring peptidase activity on gelatin, casein and hemoglobin in zymograms. Casein was the best proteinaceous substrate for the peptidase detection on both growth conditions. However, no proteolytic activity was detected when hemoglobin was used. Our results showed that cellular cysteine peptidase (115-100, 40 and 35 kDa) and metallopeptidase (70 and 60 kDa) activities were detected on both media in casein and gelatin zymograms. Additionally, the use of casein in the gel revealed a distinct acidic metallopeptidase of 50 kDa when the parasite was cultured in the modified Roitman's complex medium. Irrespective of the culture medium composition, H. megaseliae released metallopeptidases exclusively in the extracellular environment. The presence of gp63-like molecules on the H. megaseliae surface was shown by flow cytometry using anti-gp63 antibody raised against recombinant gp63 from Leishmania mexicana. The pre-treatment of parasites with phospholipase C reduced the number of gp63-positive cells, suggesting that these molecules were glycosylphosphatidylinositol-anchored to the surface. Additionally, the supernatant obtained from phospholipase C-treated cells and probed with anti-cross-reacting determinant confirmed that at least a 52 kDa gp63-like molecule is glycosylphosphatidylinositol-anchored. Furthermore, we assessed a possible function for the gp63-like molecules in H. megaseliae on the interaction with explanted guts of its original host, Megaselia scalaris, and with an experimental model employing Aedes aegypti. Parasites pre-treated with either anti-gp63 antibody or phospholipase C showed a significant reduction in the adhesion to M. scalaris and A. aegypti guts. Similarly, the pre-treatment of the explanted guts with purified gp63 diminished the interaction process. Collectively, these results corroborate the ubiquitous existence of gp63 homologues in insect trypanosomatids and the potential adhesion of these molecules to invertebrate host tissues. 相似文献
2.
Vanina E. AlvarezGabriela T. Niemirowicz Juan J. Cazzulo 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(1):195-206
Trypanosoma cruzi, the agent of the American Trypanosomiasis, Chagas disease, contains cysteine, serine, threonine, aspartyl and metallo peptidases. The most abundant among these enzymes is cruzipain, a cysteine proteinase expressed as a mixture of isoforms, some of them membrane-bound. The enzyme is an immunodominant antigen in human chronic Chagas disease and seems to be important in the host/parasite relationship. Inhibitors of cruzipain kill the parasite and cure infected mice, thus validating the enzyme as a very promising target for the development of new drugs against the disease. In addition, a 30 kDa cathepsin B-like enzyme, two metacaspases and two autophagins have been described. Serine peptidases described in the parasite include oligopeptidase B, a member of the prolyl oligopeptidase family involved in Ca2+-signaling during mammalian cell invasion; a prolyl endopeptidase (Tc80), against which inhibitors are being developed, and a lysosomal serine carboxypeptidase. Metallopeptidases homologous to the gp63 of Leishmania spp. are present, as well as two metallocarboxypeptidases belonging to the M32 family, previously found only in prokaryotes. The proteasome has properties similar to those of other eukaryotes, and its inhibition by lactacystin blocks some differentiation steps in the life cycle of the parasite. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. 相似文献
3.
Tatiana A. Semashko Elena A. Vorotnikova Valeriya F. Sharikova Konstantin S. Vinokurov Yulia A. Smirnova Yakov E. Dunaevsky Mikhail A. Belozersky Brenda Oppert Elena N. Elpidina Irina Y. Filippova 《Analytical biochemistry》2014
This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp = pyroglutamyl; Xaa = Phe or Val; and Y = pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes. 相似文献
4.
Elias CG Pereira FM Dias FA Silva TL Lopes AH d'Avila-Levy CM Branquinha MH Santos AL 《Experimental parasitology》2008,120(4):343-352
We have characterized the cysteine peptidase production by Phytomonas serpens, a tomato trypanosomatid. The parasites were cultivated in four distinct media, since growth conditions could modulate the synthesis of bioactive molecules. The proteolytic profile has not changed qualitatively regardless the media, showing two peptidases of 38 and 40 kDa; however, few quantitative changes were observed including a drastic reduction (around 70%) on the 40 and 38 kDa peptidase activities when parasites were grown in yeast extract and liver infusion trypticase medium, respectively, in comparison with parasites cultured in Warren medium. The time-span of growth did not significantly alter the protein and peptidase expression. The proteolytic activities were blocked by classical cysteine peptidase inhibitors (E-64, leupeptin, and cystatin), being more active at pH 5.0 and showing complete dependence to reducing agents (dithiothreitol and l-cysteine) for full activity. The cysteine peptidases were able to hydrolyze several proteinaceous substrates, including salivary gland proteins from Oncopeltus fasciatus, suggesting broad substrate utilization. By means of agglutination, fluorescence microscopy, flow cytometry and Western blotting analyses we showed that both cysteine peptidases produced by P. serpens share common epitopes with cruzipain, the major cysteine peptidase of Trypanosoma cruzi. Moreover, our data suggest that the 40 kDa cysteine peptidase was located at the P. serpens cell surface, attached to membrane domains via a glycosylphosphatidylinositol anchor. The 40 kDa peptidase was also detected in the cell-free culture supernatant, in an active form, which suggests secretion of this peptidase to the extracellular environment. 相似文献
5.
Leandro S. Sangenito Rubem F. S. Menna-Barreto Claudia M. d′Avila-Levy André L. S. Santos Marta H. Branquinha 《PloS one》2014,9(12)
Background
Aspartic peptidase inhibitors have shown antimicrobial action against distinct microorganisms. Due to an increase in the occurrence of Chagas'' disease/AIDS co-infection, we decided to explore the effects of HIV aspartic peptidase inhibitors (HIV-PIs) on Trypanosoma cruzi, the etiologic agent of Chagas'' disease.Methodology and Principal Findings
HIV-PIs presented an anti-proliferative action on epimastigotes of T. cruzi clone Dm28c, with IC50 values ranging from 0.6 to 14 µM. The most effective inhibitors, ritonavir, lopinavir and nelfinavir, also had an anti-proliferative effect against different phylogenetic T. cruzi strains. The HIV-PIs induced some morphological alterations in clone Dm28c epimastigotes, as reduced cell size and swollen of the cellular body. Transmission electron microscopy revealed that the flagellar membrane, mitochondrion and reservosomes are the main targets of HIV-PIs in T. cruzi epimastigotes. Curiously, an increase in the epimastigote-into-trypomastigote differentiation process of clone Dm28c was observed, with many of these parasites presenting morphological alterations including the detachment of flagellum from the cell body. The pre-treatment with the most effective HIV-PIs drastically reduced the interaction process between epimastigotes and the invertebrate vector Rhodnius prolixus. It was also noted that HIV-PIs induced an increase in the expression of gp63-like and calpain-related molecules, and decreased the cruzipain expression in epimastigotes as judged by flow cytometry and immunoblotting assays. The hydrolysis of a cathepsin D fluorogenic substrate was inhibited by all HIV-PIs in a dose-dependent manner, showing that the aspartic peptidase could be a possible target to these drugs. Additionally, we verified that ritonavir, lopinavir and nelfinavir reduced drastically the viability of clone Dm28c trypomastigotes, causing many morphological damages.Conclusions and Significance
The results contribute to understand the possible role of aspartic peptidases in T. cruzi physiology, adding new in vitro insights into the possibility of exploiting the use of HIV-PIs in the clinically relevant forms of the parasite. 相似文献6.
Saraiva VB Wengert M Gomes-Quintana E Heise N Caruso-Neves C 《Archives of biochemistry and biophysics》2009,481(1):65-351
Miltefosine has been shown to be a very active compound against Trypanosoma cruzi. Here, we evaluated the effects of miltefosine on the activity of the Na+-ATPase and protein kinase C (PKC) present in the plasma membrane of T. cruzi. Furosemide (2 mM), a specific inhibitor of Na+-ATPase, abolished the growth of T. cruzi showing a crucial role of this enzyme to parasite growth. Miltefosine inhibited the Na+-ATPase activity with IC50 = 18 ± 5 μg mL−1. This effect was shown to be reversible, dependent on the pH and Ca2+. The inhibition was not observed when the membranes were solubilized with 0.1% deoxycholate, suggesting that the interaction between the enzyme and membrane phospholipids might be important for the drug effect. Miltefosine also inhibited the parasite PKC activity, but through a Na+-ATPase-independent way. Altogether the results indicate that miltefosine inhibits T. cruzi growth through, at least in part, the inhibition of both Na+-ATPase and PKC activities. 相似文献
7.
d'Avila-Levy CM Santos LO Marinho FA Dias FA Lopes AH Santos AL Branquinha MH 《Current microbiology》2006,52(6):439-444
In this study, we demonstrated that metallopeptidase inhibitors (EDTA, EGTA, and 1,10-phenanthroline) were able to arrest
Phytomonas serpens growth in distinct patterns. This parasite released exclusively metallopeptidases to the extracellular environment, whereas
in cellular extracts only cysteine peptidases were detected. In addition, an extracellular polypeptide of 60 kDa reacted in
Western blotting probed with polyclonal antibody raised against gp63 of Leishmania amazonensis. In the cellular parasite extract, this antibody recognized bands migrating at 63 and 52 kDa, which partitioned on both aqueous
and membrane-rich fractions. Flow cytometry and fluorescence microscopy analyses showed that the gp63-like molecules have
a surface location. Moreover, phospholipase C (PLC)-treated parasites reduced the number of gp63-positive cells. The anti-cross-reacting
determinant (CRD) and anti-gp63 antibodies recognized the 60-kDa band in the supernatant from PLC-treated cells, suggesting
that this protein is glycosylphosphatidylinositol-anchored to the plasma membrane. This is the first report on the presence
of gp63-like molecules in members of the Phytomonas genus. The pretreatment of the parasites with anti-gp63 antibody significantly diminished their adhesion index to explanted
salivary glands of the phytophagous insect Oncopeltus fasciatus, suggesting a potential involvement of the gp63-like molecules in the adhesive process of this plant trypanosomatid. 相似文献
8.
9.
Erika Gracielle Pinto Daniel C. Pimenta Marta Maria Antoniazzi Carlos Jared Andre Gustavo Tempone 《Experimental parasitology》2013
Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC50 values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC50 value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC50 = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies. 相似文献
10.
11.
Daniel B. Liarte Silvane M.F. Murta Alvaro J. Romanha 《Experimental parasitology》2009,123(4):283-291
A multiplex PCR was developed for simultaneous detection of Trypanosoma cruzi DNA and classification of the parasite strain into groups I and II. As little as 10 fg of T. cruzi DNA could be detected by multiplex PCR. The technique was shown to be specific for T. cruzi DNA, since no PCR amplification products were obtained with DNA from other tripanosomatid species. Multiplex PCR was validated by assaying genomic DNA from 34 strains of T. cruzi that had been previously characterized; 24 blood samples from experimentally-infected mice and non-infected controls; 20 buffy coat samples from patients in the acute phase of Chagas disease and non-infected individuals, and 15 samples of feces from naturally-infected Triatoma infestans. T. cruzi samples from patients and from Y strain-infected mice were classified by multiplex PCR as T. cruzi II and samples from T. infestans and Colombiana strain-infected mice as T. cruzi I. 相似文献
12.
Four positional isomers of Thiastearate (TS) and Isoxyl (Thiocarlide) were assayed as fatty acid desaturase inhibitors in Trypanosoma cruzi epimastigotes. 9-TS did not exert a significant effect on growth of T. cruzi, nor on the fatty acid profile of the parasite cells. One hundred micromolars of 10-TS totally inhibited growth, with an effective concentration for 50% growth inhibition (EC50) of 3.0 ± 0.2 μM. Growth inhibition was reverted by supplementing the culture media with oleate. The fatty acid profile of treated cells revealed that conversion of stearate to oleate and palmitate to palmitoleate were drastically reduced and, as a consequence, the total level of unsaturated fatty acids decreased from 60% to 32%. Isoxyl, a known inhibitor of stearoyl-CoA Δ9 desaturase in mycobacteria, had similar effects on T. cruzi growth (EC50 2.0 ± 0.3 μM) and fatty acid content, indicating that Δ9 desaturase was the target of both drugs. 12- and 13-TS were inhibitors of growth with EC50 values of 50 ± 2 and 10 ± 3 μM, respectively, but oleate or linoleate were unable to revert the effect. Both drugs increased the percentage of oleate and palmitate in the cell membrane and drastically reduced the content of linoleate from 38% to 16% and 12%, respectively, which is in agreement with a specific inhibition of oleate Δ12 desaturase. The absence of corresponding enzyme activity in mammalian cells and the significant structural differences between trypanosome and mammalian Δ9 desaturases, together with our results, highlight these enzymes as promising targets for selective chemotherapeutic intervention. 相似文献
13.
14.
Two aspartyl proteases activities were identified and isolated from Trypanosoma cruzi epimastigotes: cruzipsin-I (CZP-I) and cruzipsin-II (CZP-II). One was isolated from a soluble fraction (CZP-II) and the other was solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CZP-I). The molecular mass of both proteases was estimated to be 120 kDa by HPLC gel filtration and the activity of the enzymes was detected in a doublet of bands (56 and 48 kDa) by substrate-sodium dodecyl sulphate-polyacrylamide-gelatin gel electrophoresis. Substrate specificity studies indicated that the enzymes consistently hydrolyze the cathepsin D substrate Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu-O4MP but failed to hydrolyze serine and other protease substrates. Both proteases activities were strongly inhibited by the classic inhibitor pepstatin-A (?68%) and the aspartic active site labeling agent, 1,2-epoxy-3-(phenyl-nitrophenoxy) propane (?80%). These findings show that both proteases are novel T. cruzi acidic proteases. The physiological function of these enzymes in T. cruzi has under investigation. 相似文献
15.
Nucleoside diphosphate kinases (NDPKs) are multifunctional enzymes involved mainly in the conservation of nucleotides and deoxynucleotides at intracellular levels. Here we report the characterization of two NDPKs from the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease. TcNDPK1 and TcNDPK2 were biochemically characterized presenting different kinetic parameters and regulation mechanisms. NDPK activity was mainly detected in soluble fractions according to the digitonin extraction technique; however 20% of the activity remains insoluble at digitonin concentrations up to 5 mg ml−1. TcNDPK1 is a short enzyme isoform, whereas TcNDPK2 is a long one containing a DM10 motif. In addition, two other putative NDPK genes (TcNPDK3 and TcNDPK4) were detected by data mining at the T. cruzi genome database. The large number and diversity of NDPK isoforms are in agreement with those previously observed for other T. cruzi phosphotransferases, such as adenylate kinases. 相似文献
16.
Santos AL d'Avila-Levy CM Dias FA Ribeiro RO Pereira FM Elias CG Souto-Padrón T Lopes AH Alviano CS Branquinha MH Soares RM 《International journal for parasitology》2006,36(1):47-56
In this study, we report the ultrastructural and growth alterations caused by cysteine peptidase inhibitors on the plant trypanosomatid Phytomonas serpens. We showed that the cysteine peptidase inhibitors at 10 microM were able to arrest cellular growth as well as promote alterations in the cell morphology, including the parasites becoming short and round. Additionally, iodoacetamide induced ultrastructural alterations, such as disintegration of cytoplasmic organelles, swelling of the nucleus and kinetoplast-mitochondrion complex, which culminated in parasite death. Leupeptin and antipain induced the appearance of microvillar extensions and blebs on the cytoplasmic membrane, resembling a shedding process. A 40 kDa cysteine peptidase was detected in hydrophobic and hydrophilic phases of P. serpens cells after Triton X-114 extraction. Additionally, we have shown through immunoblotting that anti-cruzipain polyclonal antibodies recognised two major polypeptides in P. serpens, including a 40 kDa component. Flow cytometry analysis confirmed that this cruzipain-like protein has a location on the cell surface. Ultrastructural immunocytochemical analysis demonstrated the presence of the cruzipain-like protein on the surface and in small membrane fragments released from leupeptin-treated parasites. Furthermore, the involvement of cysteine peptidases of P. serpens in the interaction with explanted salivary glands of the phytophagous insect Oncopeltus fasciatus was also investigated. When P. serpens cells were pre-treated with either cysteine peptidase inhibitors or anti-cruzipain antibody, a significant reduction of the interaction process was observed. Collectively, these results suggest that cysteine peptidases participate in several biological processes in P. serpens including cell growth and interaction with the invertebrate vector. 相似文献
17.
Catherine X. Moss Elaine Brown Alana Hamilton Pieter Van der Veken Koen Augustyns Jeremy C. Mottram 《PloS one》2015,10(3)
The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei survival. All S9 serine peptidases were dispensable for parasite survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on parasite survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1). This gene was essential for parasite survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei survival. 相似文献
18.
Gradia DF Rau K Umaki AC de Souza FS Probst CM Correa A Holetz FB Avila AR Krieger MA Goldenberg S Fragoso SP 《International journal for parasitology》2009,39(1):49-58
We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185 bp long and encodes a 44.3 kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes. 相似文献
19.
Alexandra Schwarz Jeremy M. Sternberg Nora Medrano-Mercado Jen C.C. Hume Günter A. Schaub 《International journal for parasitology》2009,39(9):1021-1029
Hematophagous arthropods such as Triatomainfestans, the vector of Trypanosomacruzi, elicit host-immune responses during feeding. Characterization of antibody responses to salivary antigens offers the potential to develop immunologically based monitoring techniques for exposure to re-emergent triatomine bug populations in peridomestic animals. IgG-antibody responses to the salivary antigens of T.infestans have been detected in chickens as soon as 2 days after the first exposure to five adult bugs. Chickens and guinea pigs regularly exposed to this number of triatomines showed a significantly lower anti-saliva antibody titre than animals exposed to 25 adults and fifth instars of four different T.infestans strains originating from Bolivia and from Northern Chile. Highly immunogenic salivary antigens of 14 and 21 kDa were recognised by all chicken sera and of 79 kDa by all guinea pig sera. Cross-reactivity studies using saliva or salivary gland extracts from different hematophagous species, e.g. different triatomines, bed bugs, mosquitoes, sand flies and ticks, as well as chicken sera exposed to triatomines and mosquitoes, demonstrated that the 14 and 21 kDa salivary antigens were only found in triatomines. Sera from peridomestic chickens and guinea pigs in sites of known T.infestans challenge in Bolivia also recognised the 14 and 21 kDa antigens. These represent promising epidemiological markers for the detection of small numbers of feeding bugs and hence may be a new tool for vector surveillance in Chagas disease control programs. 相似文献
20.
Lívia Almeida Uehara Otacílio C. Moreira Ana Carolina Oliveira Patrícia Azambuja Ana Paula Cabral Araujo Lima Constan?a Britto André Luis Souza dos Santos Marta Helena Branquinha Claudia Masini d'Avila-Levy 《PLoS neglected tropical diseases》2012,6(12)