Cercariae of the bird schistosome Trichobilharzia regenti and of the human schistosome Schistosoma mansoni employ proteases to invade the skin of their definitive hosts. To investigate whether a similar proteolytic mechanism is used by both species, cercarial extracts of T. regenti and S. mansoni were biochemically characterized, with the primary focus on cysteine peptidases. A similar pattern of cysteine peptidase activities was detected by zymography of cercarial extracts and their chromatographic fractions from T. regenti and S. mansoni. The greatest peptidase activity was recorded in both species against the fluorogenic peptide substrate Z-Phe-Arg-AMC, commonly used to detect cathepsins B and L, and was markedly inhibited (> 96%) by Z-Phe-Ala-CHN2 at pH 4.5. Cysteine peptidases of 33 kDa and 33-34 kDa were identified in extracts of T. regenti and S. mansoni cercariae employing a biotinylated Clan CA cysteine peptidase-specific inhibitor (DCG-04). Finally, cercarial extracts from both T. regenti and S. mansoni were able to degrade native substrates present in skin (collagen II and IV, keratin) at physiological pH suggesting that cysteine peptidases are important in the pentration of host skin.

• MeSH
• cysteinové endopeptidasy účinky léků   metabolismus   MeSH
• diazomethan analogy a deriváty   farmakologie   MeSH
• gelová chromatografie MeSH
• inhibitory proteas farmakologie   MeSH
• keratiny metabolismus   MeSH
• kolagen metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• leucin analogy a deriváty   metabolismus   MeSH
• Schistosoma mansoni enzymologie   MeSH
• Schistosomatidae enzymologie   MeSH
• vazebná místa MeSH
• želatina metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• benzyloxycarbonylphenylalanylalanine diazomethyl ketone MeSH Prohlížeč
• cysteinové endopeptidasy MeSH
• DCG 04 MeSH Prohlížeč
• diazomethan MeSH
• inhibitory proteas MeSH
• keratiny MeSH
• kolagen MeSH
• leucin MeSH
• želatina MeSH

The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.

• Klíčová slova
• Trichomonas vaginalis, cysteine peptidase, glycosylation, mannose 6-phosphate receptor, phagolysosome, proteome,
• MeSH
• cystein metabolismus   MeSH
• cysteinové proteasy * metabolismus   MeSH
• fagozomy metabolismus   MeSH
• lidé MeSH
• lyzozomy metabolismus   MeSH
• proteasy metabolismus   MeSH
• proteomika MeSH
• Trichomonas vaginalis * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• cysteinové proteasy * MeSH
• proteasy MeSH

It has been proposed that the natural cysteine peptidase inhibitor ICP of Leishmania mexicana protects the protozoan parasite from insect host proteolytic enzymes, thereby promoting survival. To test this hypothesis, L. mexicana mutants deficient in ICP were evaluated for their ability to develop in the sand fly Lutzomyia longipalpis. No significant differences were found between the wild-type parasites, two independently derived ICP-deficient mutants, or mutants overexpressing ICP; all lines developed similarly in the sand fly midgut and produced heavy late-stage infections. In addition, recombinant L. mexicana ICP did not inhibit peptidase activity of the midgut extracts in vitro. We conclude that ICP has no major role in promoting survival of L. mexicana in the vectorial part of its life cycle in L. longipalpis.

• MeSH
• cysteinové endopeptidasy fyziologie   MeSH
• hmyzí proteiny fyziologie   MeSH
• inhibitory cysteinových proteinas genetika   fyziologie   MeSH
• interakce hostitele a parazita MeSH
• Leishmania mexicana genetika   patogenita   MeSH
• protozoální proteiny genetika   fyziologie   MeSH
• Psychodidae enzymologie   parazitologie   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cysteinové endopeptidasy MeSH
• hmyzí proteiny MeSH
• inhibitory cysteinových proteinas MeSH
• protozoální proteiny MeSH

The gills of the common carp, whose mucosal surface belongs to the key defence mechanisms of piscine immunity, can be infested with both the larval and adult stage of Eudiplozoon nipponicum (Monogenea). Although on their own, monogeneans do not considerably compromise their hosts' health status, fish with epithelial barriers damaged in parasite feeding and attachment sites are at an increased risk of bacterial challenge with possible harmful consequences. Several studies suggest that helminth parasites of teleost fish evade and manipulate host immune system via their excretory-secretory products, but our knowledge of these processes in the monogeneans is limited. Cysteine peptidase inhibitors (CPI), which are found in the secretions of numerous parasites, often induce immunosuppression by subverting Th1 mechanisms and drawing the immune system towards a Th2/Treg response. We employed the qPCR to test the effect of recently characterised CPI of E. nipponicum (rEnStef) on the mRNA expression of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 produced by porcine macrophages in vitro. After an initial preincubation with rEnStef, we stimulated the macrophages using LPS. By inducing a Th1 pro-inflammatory response, we imitated the immune reaction during a bacterial challenge in tissue damaged by the feeding and attachment of E. nipponicum. We observed a significant dose-dependent downregulation of the expression of TNF-α and IL-10 cytokines. The observed suppression of TNF-alpha expression by rEnStef could result in decreased pathogen control, which might in turn lead to increased rates of secondary bacterial infections in fish infected by E. nipponicum.

• Klíčová slova
• Cystatin, Cytokines, IL-10, Immunomodulation, Monogenea, TNF-α,
• MeSH
• cytokiny * účinky léků   metabolismus   MeSH
• imunomodulace MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• interleukin-10 metabolismus   MeSH
• kapři parazitologie   MeSH
• makrofágy * účinky léků   metabolismus   MeSH
• prasata MeSH
• rekombinantní proteiny farmakologie   MeSH
• TNF-alfa účinky léků   metabolismus   MeSH
• Trematoda imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny * MeSH
• inhibitory cysteinových proteinas MeSH
• interleukin-10 MeSH
• rekombinantní proteiny MeSH
• TNF-alfa MeSH

The quantum mechanics (QM)-based scoring function that we previously developed for the description of noncovalent binding in protein-ligand complexes has been modified and extended to treat covalent binding of inhibitory ligands. The enhancements are (i) the description of the covalent bond breakage and formation using hybrid QM/semiempirical QM (QM/SQM) restrained optimizations and (ii) the addition of the new ΔG(cov)' term to the noncovalent score, describing the "free" energy difference between the covalent and noncovalent complexes. This enhanced QM-based scoring function is applied to a series of 20 vinyl sulfone-based inhibitory compounds inactivating the cysteine peptidase cathepsin B1 of the Schistosoma mansoni parasite (SmCB1). The available X-ray structure of the SmCB1 in complex with a potent vinyl sulfone inhibitor K11017 is used as a template to build the other covalently bound complexes and to model the derived noncovalent complexes. We present the correlation of the covalent score and its constituents with the experimental binding data. Four outliers are identified. They contain bulky R1' substituents structurally divergent from the template, which might induce larger protein rearrangements than could be accurately modeled. In summary, we propose a new computational approach and an optimal protocol for the rapid evaluation and prospective design of covalent inhibitors with a conserved binding mode.

• MeSH
• cysteinové proteasy chemie   metabolismus   MeSH
• inhibitory cysteinových proteinas chemie   farmakologie   MeSH
• krystalografie rentgenová MeSH
• kvantová teorie * MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• Schistosoma mansoni enzymologie   MeSH
• sulfony chemie   farmakologie   MeSH
• vinylové sloučeniny chemie   farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cysteinové proteasy MeSH
• inhibitory cysteinových proteinas MeSH
• sulfony MeSH
• vinylové sloučeniny MeSH

In this study we investigated the levels of two lysosomal cysteine protease proteins cathepsin B (CB) and cathepsin L (CL) and the levels of three cysteine protease inhibitor proteins stefin A (SFA), stefin B (SFB) and cystatin C (CNC) in squamous-cell lung carcinoma (SQCLC) and matched lung parenchyma specimens and examined the inhibition of CB and cathepsin C (CC) activities by endogenous inhibitors in extracts from SQCLC, lung adenocarcinoma (LAC) and lung parenchyma specimens. We found that Stage I SQCLCs contained significantly increased levels of CB protein, CB activity and SFA protein as compared to matched lungs. Neither the levels of CL protein nor the levels of SFB protein nor the levels of CNC protein in Stage I SQCLCs and the lungs were significantly different, but the levels of CB and CL proteins as well as the levels of SFA and SFB proteins showed significant positive correlation in SQCLCs. In SQCLCs as well as in the lungs the level of SFB protein was significantly higher than the level of SFA protein or the level of CNC protein. In the lungs the levels of SFA protein and CNC protein revealed a weak negative correlation trend. In extracts from SQCLCs the level of SFA protein showed a weak negative correlation with the residual CB activity (i.e. the activity remaining after extract preincubation) whereas in extracts from the lungs the level of CNC protein displayed a weak negative correlation trend with the residual CB activity and with the residual CC activity. We observed that SQCLCs and LACs contained not only a significantly increased activity of CB but also a significantly higher inhibitory potential against the activity of endogenous CB as compared to matched lungs. Leupeptin, a small inhibitor of CB, was capable to protect CB in lung carcinoma and lung parenchyma extracts from preincubation-induced inhibition, revealing an active-site directed and competitive nature of CB inhibition by endogenous cystatins. Ultrafiltration passaged protein preparations of nominal Mr < or = 30,000 obtained from extracts of SQCLCs inhibited significantly higher quantities of activity of purified bovine spleen CC than did such protein preparations from matched lungs. Reaction courses of purified bovine spleen CC that had been preincubated with such protein preparations resembled those of endogenous CC from SQCLC and lung extracts showing a slow steady-state approach. These observations and the relaxation kinetics of CC from SQCLC and lung extracts suggest that CC in the extracts may be complexed with some cystatins. In conclusion, our results indicate that quantitatively different combinations of cystatins are the major constituents of the inhibitory potential against CB and CC in SQCLCs and the lungs.

• MeSH
• adenokarcinom enzymologie   metabolismus   MeSH
• cystatin A MeSH
• cystatin B MeSH
• cystatin C MeSH
• cystatiny metabolismus   MeSH
• cysteinové endopeptidasy MeSH
• dipeptidylpeptidasy a tripeptidylpeptidasy metabolismus   MeSH
• dospělí MeSH
• endopeptidasy * MeSH
• inhibitory cysteinových proteinas metabolismus   MeSH
• kathepsin B metabolismus   MeSH
• kathepsin C MeSH
• kathepsin L MeSH
• kathepsiny metabolismus   MeSH
• kinetika MeSH
• leupeptiny farmakologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lyzozomy enzymologie   MeSH
• nádory plic enzymologie   metabolismus   MeSH
• plíce enzymologie   MeSH
• senioři MeSH
• senzitivita a specificita MeSH
• skot MeSH
• spinocelulární karcinom enzymologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CST3 protein, human MeSH Prohlížeč
• CSTA protein, human MeSH Prohlížeč
• CSTB protein, human MeSH Prohlížeč
• CTSL protein, human MeSH Prohlížeč
• cystatin A MeSH
• cystatin B MeSH
• cystatin C MeSH
• cystatiny MeSH
• cysteinové endopeptidasy MeSH
• dipeptidylpeptidasy a tripeptidylpeptidasy MeSH
• endopeptidasy * MeSH
• inhibitory cysteinových proteinas MeSH
• kathepsin B MeSH
• kathepsin C MeSH
• kathepsin L MeSH
• kathepsiny MeSH
• leupeptin MeSH Prohlížeč
• leupeptiny MeSH

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.

• Klíčová slova
• Cysteine peptidases, Enzymatic peptide synthesis, Multicomponent enzyme mixtures, Selective peptide substrates, Substrates of peptidases,
• MeSH
• cysteinové proteasy izolace a purifikace   metabolismus   MeSH
• enzymatické testy metody   MeSH
• fluorescenční barviva analýza   metabolismus   MeSH
• hydrolýza MeSH
• molekulární modely MeSH
• peptidy chemie   metabolismus   MeSH
• substrátová specifita MeSH
• Tenebrio enzymologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cysteinové proteasy MeSH
• fluorescenční barviva MeSH
• peptidy MeSH

A transcriptional product of a gene encoding cathepsin B-like peptidase in the bird schistosome Trichobilharzia regenti was identified and cloned. The enzyme was named TrCB2 due to its 77% sequence similarity to cathepsin B2 from the important human parasite Schistosoma mansoni. The zymogen was expressed in the methylotropic yeast Pichia pastoris; procathepsin B2 underwent self-processing in yeast media. The peptidolytic activity of the recombinant enzyme was characterised using synthetic fluorogenic peptide substrates at optimal pH 6.0. Functional studies using different specific inhibitors proved the typical cathepsin B-like nature of the enzyme. The S(2) subsite specificity profile of recombinant TrCB2 was obtained. Using monospecific antibodies against the recombinant enzyme, the presence of cathepsin B2 was confirmed in extracts from cercariae (infective stage) and schistosomula (early post-cercarial stage) of T. regenti on Western blots. Also, cross-reactivity was observed between T. regenti and S. mansoni cathepsins B2 in extracts of cercariae, schistosomula or adults. In T. regenti, the antisera localised the enzyme to post-acetabular penetration glands of cercariae implying an important role in the penetration of host skin. The ability of recombinant TrCB2 to degrade skin, serum and nervous tissue proteins was evident. Elastinolytic activity suggests that the enzyme might functionally substitute the histolytic role of the serine class elastase known from S. mansoni and Schistosoma haematobium but not found in Schistosoma japonicum or in bird schistosomes.

• MeSH
• cystein biosyntéza   MeSH
• cysteinové proteasy biosyntéza   genetika   fyziologie   MeSH
• hlemýždi MeSH
• kachny MeSH
• kathepsin B biosyntéza   MeSH
• králíci MeSH
• krocani MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• Pichia MeSH
• Schistosoma mansoni enzymologie   MeSH
• Schistosoma enzymologie   MeSH
• sekvence aminokyselin MeSH
• skot MeSH
• substrátová specifita MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• myši MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• cysteinové proteasy MeSH
• kathepsin B MeSH

• MeSH
• inhibitory cysteinových proteinas MeSH
• inhibitory proteas analýza   MeSH
• lidé MeSH
• papain MeSH
• spektrofotometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inhibitory cysteinových proteinas MeSH
• inhibitory proteas MeSH
• papain MeSH

Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells.

• MeSH
• členovci enzymologie   MeSH
• Culicidae enzymologie   MeSH
• cysteinové proteasy metabolismus   MeSH
• klíšťata enzymologie   MeSH
• paraziti enzymologie   MeSH
• stravovací zvyklosti fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• cysteinové proteasy MeSH