Protease inhibitors regulate various biological processes and prevent host tissue/organ damage. Specific inhibition/regulation of proteases is clinically valuable for treating several diseases. Psoriasis affects the skin in the limbs and scalp of the body, and the contribution of cysteine and serine proteases to the development of skin inflammation is well documented. Cysteine protease inhibitors from ticks have high specificity, selectivity, and affinity to their target proteases and are efficient immunomodulators. However, their potential therapeutic effect on psoriasis pathogenesis remains to be determined. Therefore, we tested four tick cystatins (Sialostatin L, Sialostatin L2, Iristatin, and Mialostatin) in the recently developed, innate immunity-dependent mannan-induced psoriasis model. We explored the effects of protease inhibitors on clinical symptoms and histological features. In addition, the number and percentage of immune cells (dendritic cells, neutrophils, macrophages, and γδT cells) by flow cytometry, immunofluorescence/immunohistochemistry and, the expression of pro-inflammatory cytokines (TNF-a, IL-6, IL-22, IL-23, and IL-17 family) by qPCR were analyzed using skin, spleen, and lymph node samples. Tick protease inhibitors have significantly decreased psoriasis symptoms and disease manifestations but had differential effects on inflammatory responses and immune cell populations, suggesting different modes of action of these inhibitors on psoriasis-like inflammation. Thus, our study demonstrates, for the first time, the usefulness of tick-derived protease inhibitors for treating skin inflammation in patients.

• Klíčová slova
• autoimmune disease, immune responses, protease inhibitors, psoriasis, tick,
• MeSH
• dermatitida * MeSH
• endopeptidasy MeSH
• inhibitory cysteinových proteinas MeSH
• inhibitory proteas MeSH
• lidé MeSH
• mannany MeSH
• přirozená imunita MeSH
• proteasy MeSH
• psoriáza * chemicky indukované   farmakoterapie   MeSH
• zánět farmakoterapie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endopeptidasy MeSH
• inhibitory cysteinových proteinas MeSH
• inhibitory proteas MeSH
• mannany MeSH
• proteasy 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

Cysteine protease is a superfamily of widespread proteolytic enzymes and plays a major role in larval invasion, migration, exsheathing, survival and immune evasion in parasites. In the present study, the gene coding cysteine proteinase of the nematode Trichinella spiralis (Owen, 1835) was cloned into pQE-80L and subsequently expressed in E. coli JM109. The rTsCP was purified and its antigenicity was identified by Western blot and ELISA. Using anti-rTsCP serum the native TsCP was identified in muscle larval crude proteins. The results of quantitative real-time PCR and immunofluorescence test demonstrated that the TsCP was expressed in all stages of T. spiralis and located mainly in cuticle, stichosome and reproductive organs. The immunisation of mice with rTsCP elicited Th2-predominant immune responses. Anti-rTsCP antibodies could partially inhibit the in vitro larval invasion of intestinal epithelial cells and kill the newborn larvae by an antibody-dependent cell-mediated dose-dependent cytotoxicity. The vaccinated mice exhibited a 54% reduction of adults and a 33% reduction of muscle larvae following challenge infection. The results suggested that the TsCP might be an indispensable protein in Trichinella invasion, development and survival of T. spiralis in hosts, and could be a potential vaccine target against infection.

• Klíčová slova
• Trichinellosis, cysteine protease, development, invasion,
• MeSH
• cysteinové proteasy genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu MeSH
• geneticky modifikované mikroorganismy genetika   MeSH
• klonování DNA MeSH
• larva enzymologie   genetika   růst a vývoj   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• organismy bez specifických patogenů MeSH
• proteiny červů genetika   metabolismus   MeSH
• sekvenční analýza DNA veterinární   MeSH
• Trichinella spiralis enzymologie   genetika   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cysteinové proteasy MeSH
• proteiny červů MeSH

We investigated activities of the cysteine protease cathepsin B (CB; EC 3.4.22.1), the levels of reduced glutathione (GSH) and cysteine and the activity of gamma-glutamyltransferase (gamma-GT; EC 2.3.2.2) in squamous-cell lung carcinoma (SQCLC) and the lung parenchyma specimens from surgically treated patients. The basal CB activity, assayed in tissue extracts in the absence of exogenous activators, was significantly higher in SQCLC compared to the lung. The residual CB activity, remaining in tissue extracts after preincubation at 37 degrees C, was not any longer significantly different in SQCLC and the lungs. The inhibited CB activity, calculated as the difference between the basal and residual CB activities, was significantly higher in SQCLC compared to the lung. In the case of the cysteine protease cathepsin C (CC; EC 3.4.14.1), neither the basal nor the residual nor the inhibited CC activities in SQCLC and the lung were significantly different. Compared to CC, the powerfulness of endogenous cysteine protease inhibitors to inhibit CB was much higher in both SQCLC and the lung. The cysteine protease inhibitors from SQCLC and the lung which effectively inhibited CB could be related to the inhibitors with an apparent M(r) ranging from 10,000 to 30,000. Isoelectric focusing studies indicated significant differences in the progress of inhibition of the activity of CB isoforms in SQCLC and lung parenchyma extracts. The levels of both GSH and Cys were significantly higher in SQCLC compared to the lung and the level of GSH was significantly higher in Stage III tumors compared to Stage I tumors. The activity of gamma-GT was not significantly different in SQCLC and the lung but it was significantly higher in Stage I tumors compared to Stage III tumors and showed a significant negative correlation with GSH level in SQCLC. Dithiothreitol did not increase the basal activity of CB from SQCLC and the lung which indicates that reversibly oxidized forms of CB do not accumulate in the tumors and the lungs. The basal activity of CB from SQCLC and the lung was competitively inhibited by Cys. Moreover, increasing Cys concentrations had a modulatory effect on the basal activity of CB from SQCLC and the lung which was featured by Cys-induced inhibition of CB activity and by subsequent Cys-effected recovery of CB activity from its previous inhibition by Cys.

• MeSH
• časové faktory MeSH
• cystein metabolismus   farmakologie   MeSH
• dithiothreitol farmakologie   MeSH
• dospělí MeSH
• gama-glutamyltransferasa metabolismus   MeSH
• glutathion metabolismus   MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• isoelektrická fokusace MeSH
• kathepsin B metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádory plic enzymologie   MeSH
• senioři MeSH
• spinocelulární karcinom enzymologie   MeSH
• sulfhydrylové sloučeniny farmakologie   MeSH
• teplota MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• dithiothreitol MeSH
• gama-glutamyltransferasa MeSH
• glutathion MeSH
• inhibitory cysteinových proteinas MeSH
• kathepsin B MeSH
• sulfhydrylové sloučeniny MeSH

Cysteine protease from grapevine (Vitis vinifera) belongs to those resistant proteins, which survive the process of vinification and can therefore be detected as wine components. Its amino acid sequence shows a homology to other members of the papain family, but the enzyme has only partially been explored so far. In order to get more biochemical information with the help of mass spectrometry (MS), wine proteins were collected by ultrafiltration and separated by gel permeation chromatography. The purified enzyme surprisingly displayed a high molecular mass value of around 200 kDa, indicating a possible oligomeric status and aggregation, as it entered only negligibly the separating 10% gel during polyacrylamide gel electrophoresis. The isoelectric point (pI) value of 3.6 was determined by chromatofocusing. Matrix-assisted laser desorption/ionization (MALDI)-MS was employed to evaluate the cleavage specificity and usefulness of the isolated cysteine protease in protein and peptide research. A potential applicability could be anticipated from the efficient digestion performance in volatile ammonium formate buffers at pH 3. Common peptides were digested and the resulting products analyzed by MS/MS sequencing. Then, mixtures of protein standards and extracted barley nuclear proteins were processed in the same way. Grape cysteine protease is nonspecific but shows a certain preference for Arg, Lys, and also Leu residues. Compared with papain, it seems not to require fully the presence of a large hydrophobic residue adjacent to that at the cleavage site. The enzyme is suitable for protein research as it produces peptides of a reasonable length in acidic pH.

• Klíčová slova
• RD21, cysteine protease, digestion, liquid chromatography, papain, peptide,
• MeSH
• cysteinové proteasy chemie   izolace a purifikace   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• ovoce enzymologie   MeSH
• peptidy analýza   metabolismus   MeSH
• proteiny analýza   metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• substrátová specifita MeSH
• tandemová hmotnostní spektrometrie MeSH
• víno analýza   MeSH
• Vitis enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cysteinové proteasy MeSH
• peptidy MeSH
• proteiny MeSH

Rhodesain is the major cysteine protease of the protozoan parasite Trypanosoma brucei and a therapeutic target for sleeping sickness, a fatal neglected tropical disease. We designed, synthesized and characterized a bimodal activity-based probe that binds to and inactivates rhodesain. This probe exhibited an irreversible mode of action and extraordinary potency for the target protease with a kinac /Ki value of 37,000 M-1 s-1 . Two reporter tags, a fluorescent coumarin moiety and a biotin affinity label, were incorporated into the probe and enabled highly sensitive detection of rhodesain in a complex proteome by in-gel fluorescence and on-blot chemiluminescence. Furthermore, the probe was employed for microseparation and quantification of rhodesain and for inhibitor screening using a competition assay. The developed bimodal rhodesain probe represents a new proteomic tool for studying Trypanosoma pathobiochemistry and antitrypanosomal drug discovery.

• Klíčová slova
• activity-based probes, affinity chromatography, cysteine proteases, imaging agents, peptidomimetics, rhodesain, size exclusion chromatography,
• MeSH
• biotin MeSH
• cysteinové proteasy * MeSH
• fluorescence MeSH
• proteomika MeSH
• Trypanosoma brucei brucei * MeSH
• Trypanosoma * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biotin MeSH
• cysteinové proteasy * MeSH
• rhodesain MeSH Prohlížeč

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

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 μM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 μM. There was no toxicity to any of the host cell lines at 10-100 μM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.

• MeSH
• antivirové látky farmakologie   MeSH
• Cercopithecus aethiops MeSH
• fenylalanin farmakologie   MeSH
• glykoprotein S, koronavirus chemie   metabolismus   MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• internalizace viru účinky léků   MeSH
• kathepsin L antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• nádorové buněčné linie MeSH
• piperaziny farmakologie   MeSH
• proteinové domény MeSH
• proteolýza MeSH
• SARS-CoV-2 účinky léků   MeSH
• tosylové sloučeniny farmakologie   MeSH
• Vero buňky MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• antivirové látky MeSH
• fenylalanin MeSH
• glykoprotein S, koronavirus MeSH
• inhibitory cysteinových proteinas MeSH
• kathepsin L MeSH
• N-pip-phenylalanine-homophenylalanine-vinyl sulfone phenyl MeSH Prohlížeč
• piperaziny MeSH
• spike protein, SARS-CoV-2 MeSH Prohlížeč
• tosylové sloučeniny 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

• 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