The interaction between pathogens and vectors' physiology can impact parasite transmission. Studying this interaction at the molecular level can help in developing control strategies. We study leishmaniases, diseases caused by Leishmania parasites transmitted by sand fly vectors, posing a significant global public health concern. Lipophosphoglycan (LPG), the major surface glycoconjugate of Leishmania, has been described to have several roles throughout the parasite's life cycle, both in the insect and vertebrate hosts. In addition, the sand fly midgut possesses a rich microbiota expressing lipopolysaccharides (LPS). However, the effect of LPG and LPS on the gene expression of sand fly midgut proteins or immunity effectors has not yet been documented. We experimentally fed Lutzomyia longipalpis and Phlebotomus papatasi sand flies with blood containing purified LPG from Leishmania infantum, Leishmania major, or LPS from Escherichia coli. The effect on the expression of genes encoding gut proteins galectin and mucin, digestive enzymes trypsin and chymotrypsin, and antimicrobial peptides (AMPs) attacin and defensins was assessed by quantitative PCR (qPCR). The gene expression of a mucin-like protein in L. longipalpis was increased by L. infantum LPG and E. coli LPS. The gene expression of a galectin was increased in L. longipalpis by L. major LPG, and in P. papatasi by E. coli LPS. Nevertheless, the gene expression of trypsins and chymotrypsins did not significantly change. On the other hand, both L. infantum and L. major LPG significantly enhanced expression of the AMP attacin in both sand fly species and defensin in L. longipalpis. In addition, E. coli LPS increased the expression of attacin and defensin in L. longipalpis. Our study showed that Leishmania LPG and E. coli LPS differentially modulate the expression of sand fly genes involved in gut maintenance and defence. This suggests that the glycoconjugates from microbiota or Leishmania may increase the vector's immune response and the gene expression of a gut coating protein in a permissive vector.

• Klíčová slova
• Bacteria LPS, Digestion, Gut protein, Innate immunity, Leishmania LPG, Lutzomyia, PAMPs, Phlebotomus,
• MeSH
• antimikrobiální peptidy * metabolismus   genetika   MeSH
• chymotrypsin metabolismus   genetika   MeSH
• Escherichia coli genetika   MeSH
• exprese genu MeSH
• gastrointestinální trakt mikrobiologie   parazitologie   metabolismus   MeSH
• glykosfingolipidy metabolismus   MeSH
• hmyz - vektory parazitologie   mikrobiologie   genetika   MeSH
• hmyzí proteiny * genetika   metabolismus   MeSH
• Leishmania infantum * genetika   metabolismus   MeSH
• Leishmania major genetika   metabolismus   MeSH
• lipopolysacharidy * MeSH
• membránové proteiny genetika   metabolismus   MeSH
• muciny metabolismus   genetika   MeSH
• PAMP struktury metabolismus   MeSH
• Phlebotomus genetika   parazitologie   metabolismus   MeSH
• Psychodidae * parazitologie   MeSH
• regulace genové exprese MeSH
• trypsin metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antimikrobiální peptidy * MeSH
• attacin antibacterial protein, insect MeSH Prohlížeč
• chymotrypsin MeSH
• glykosfingolipidy MeSH
• hmyzí proteiny * MeSH
• lipophosphonoglycan MeSH Prohlížeč
• lipopolysacharidy * MeSH
• membránové proteiny MeSH
• muciny MeSH
• PAMP struktury MeSH
• trypsin MeSH

A typical bottom-up proteomic workflow comprises sample digestion with trypsin, separation of the hydrolysate using reversed-phase HPLC, and detection of peptides via electrospray ionization (ESI) tandem mass spectrometry. Despite the advantages and wide usage of protein identification and quantification, the procedure has limitations. Some domains or parts of the proteins may remain inadequately described due to inefficient detection of certain peptides. This study presents an alternative approach based on sample acetylation and mass spectrometry with atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). These ionizations allowed for improved detection of acetylated peptides obtained via chymotrypsin or glutamyl peptidase I (Glu-C) digestion. APCI and APPI spectra of acetylated peptides often provided sequence information already at the full scan level, while fragmentation spectra of protonated molecules and sodium adducts were easy to interpret. As demonstrated for bovine serum albumin, acetylation improved proteomic analysis. Compared to ESI, gas-phase ionizations APCI and APPI made it possible to detect more peptides and provide better sequence coverages in most cases. Importantly, APCI and APPI detected many peptides which passed unnoticed in the ESI source. Therefore, analytical methods based on chymotrypsin or Glu-C digestion, acetylation, and APPI or APCI provide data complementary to classical bottom-up proteomics.

• Klíčová slova
• acetylation, chemical ionization, photoionization, proteomics,
• MeSH
• acetylace MeSH
• atmosférický tlak MeSH
• chymotrypsin * MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací metody   MeSH
• peptidy MeSH
• proteomika * MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chymotrypsin * MeSH
• peptidy MeSH

OBJECTIVE: Alcohol-related pancreatitis is associated with a disproportionately large number of hospitalisations among GI disorders. Despite its clinical importance, genetic susceptibility to alcoholic chronic pancreatitis (CP) is poorly characterised. To identify risk genes for alcoholic CP and to evaluate their relevance in non-alcoholic CP, we performed a genome-wide association study and functional characterisation of a new pancreatitis locus. DESIGN: 1959 European alcoholic CP patients and population-based controls from the KORA, LIFE and INCIPE studies (n=4708) as well as chronic alcoholics from the GESGA consortium (n=1332) were screened with Illumina technology. For replication, three European cohorts comprising 1650 patients with non-alcoholic CP and 6695 controls originating from the same countries were used. RESULTS: We replicated previously reported risk loci CLDN2-MORC4, CTRC, PRSS1-PRSS2 and SPINK1 in alcoholic CP patients. We identified CTRB1-CTRB2 (chymotrypsin B1 and B2) as a new risk locus with lead single-nucleotide polymorphism (SNP) rs8055167 (OR 1.35, 95% CI 1.23 to 1.6). We found that a 16.6 kb inversion in the CTRB1-CTRB2 locus was in linkage disequilibrium with the CP-associated SNPs and was best tagged by rs8048956. The association was replicated in three independent European non-alcoholic CP cohorts of 1650 patients and 6695 controls (OR 1.62, 95% CI 1.42 to 1.86). The inversion changes the expression ratio of the CTRB1 and CTRB2 isoforms and thereby affects protective trypsinogen degradation and ultimately pancreatitis risk. CONCLUSION: An inversion in the CTRB1-CTRB2 locus modifies risk for alcoholic and non-alcoholic CP indicating that common pathomechanisms are involved in these inflammatory disorders.

• Klíčová slova
• Genome wide association study, chronic pancreatitis, genetic rearrangement,
• MeSH
• alkoholická pankreatitida * epidemiologie   genetika   MeSH
• chymotrypsin genetika   MeSH
• dospělí MeSH
• genetická predispozice k nemoci MeSH
• jednonukleotidový polymorfismus MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři 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
• Geografické názvy
• Evropa epidemiologie   MeSH
• Názvy látek
• chymotrypsin B MeSH Prohlížeč
• chymotrypsin MeSH

A synthetic three-fluorophore system with two enzymatically cleavable linkers has been developed for the simultaneous detection of two proteases in a mixture. The probe was designed to afford single excitation/triple emission ratiometric detection through a fluorescence change during the cleavage of a peptide linker. The developed assays were verified for trypsin and chymotrypsin as the model enzymes.

• MeSH
• aminokumariny chemická syntéza   chemie   účinky záření   MeSH
• chymotrypsin analýza   MeSH
• enzymatické testy MeSH
• fenylalanin analogy a deriváty   chemie   MeSH
• fluoresceiny chemická syntéza   chemie   účinky záření   MeSH
• fluorescence MeSH
• fluorescenční barviva chemická syntéza   chemie   účinky záření   MeSH
• hydrolýza MeSH
• lysin analogy a deriváty   chemie   MeSH
• rezonanční přenos fluorescenční energie MeSH
• rhodaminy chemická syntéza   chemie   účinky záření   MeSH
• stabilita léku MeSH
• trypsin analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokumariny MeSH
• chymotrypsin MeSH
• fenylalanin MeSH
• fluoresceiny MeSH
• fluorescenční barviva MeSH
• lysin MeSH
• rhodaminy MeSH
• trypsin MeSH

Rhomboid proteases form one of the most widespread intramembrane protease families. They have been implicated in variety of human diseases. The currently reported rhomboid inhibitors display some selectivity, but their construction involves multistep synthesis protocols. Here, we report benzoxazin-4-ones as novel inhibitors of rhomboid proteases with a covalent, but slow reversible inhibition mechanism. Benzoxazin-4-ones can be synthesized from anthranilic acid derivatives in a one-step synthesis, making them easily accessible. We demonstrate that an alkoxy substituent at the 2-position is crucial for potency and results in low micromolar inhibitors of rhomboid proteases. Hence, we expect that these compounds will allow rapid synthesis and optimization of inhibitors of rhomboids from different organisms.

• Klíčová slova
• Activity-based protein profiling, Benzoxazinones, Inhibitors, Intramembrane proteases, Rhomboid proteases,
• MeSH
• Bacillus subtilis enzymologie   MeSH
• benzoxaziny chemická syntéza   chemie   farmakologie   MeSH
• chymotrypsin antagonisté a inhibitory   MeSH
• DNA vazebné proteiny antagonisté a inhibitory   MeSH
• endopeptidasy MeSH
• enzymatické testy MeSH
• Escherichia coli enzymologie   MeSH
• inhibitory serinových proteinas chemická syntéza   chemie   farmakologie   MeSH
• inhibitory trypsinu chemická syntéza   chemie   farmakologie   MeSH
• membránové proteiny antagonisté a inhibitory   MeSH
• molekulární struktura MeSH
• ortoaminobenzoáty chemie   MeSH
• proteiny z Escherichia coli antagonisté a inhibitory   MeSH
• skot MeSH
• trypsin chemie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzoxaziny MeSH
• chymotrypsin MeSH
• DNA vazebné proteiny MeSH
• endopeptidasy MeSH
• GlpG protein, E coli MeSH Prohlížeč
• inhibitory serinových proteinas MeSH
• inhibitory trypsinu MeSH
• membránové proteiny MeSH
• ortoaminobenzoáty MeSH
• proteiny z Escherichia coli MeSH
• trypsin MeSH

Rhomboids are intramembrane serine proteases with diverse physiological functions in organisms ranging from archaea to humans. Crystal structure analysis has provided a detailed understanding of the catalytic mechanism, and rhomboids have been implicated in various disease contexts. Unfortunately, the design of specific rhomboid inhibitors has lagged behind, and previously described small molecule inhibitors displayed insufficient potency and/or selectivity. Using a computer-aided approach, we focused on the discovery of novel scaffolds with reduced liabilities and the possibility for broad structural variations. Docking studies with the E. coli rhomboid GlpG indicated that 2-styryl substituted benzoxazinones might comprise novel rhomboid inhibitors. Protease in vitro assays confirmed activity of 2-styryl substituted benzoxazinones against GlpG but not against the soluble serine protease α-chymotrypsin. Furthermore, mass spectrometry analysis demonstrated covalent modification of the catalytic residue Ser201, corroborating the predicted mechanism of inhibition and the formation of an acyl enzyme intermediate. In conclusion, 2-styryl substituted benzoxazinones are a novel rhomboid inhibitor scaffold with ample opportunity for optimization.

• Klíčová slova
• Benzoxazinones, Inhibition, Intramembrane proteases, Molecular docking, Rhomboid proteases,
• MeSH
• benzoxaziny chemická syntéza   chemie   MeSH
• chymotrypsin chemie   MeSH
• DNA vazebné proteiny antagonisté a inhibitory   chemie   genetika   MeSH
• Drosophila chemie   MeSH
• endopeptidasy chemie   genetika   MeSH
• enzymatické testy MeSH
• Escherichia coli enzymologie   MeSH
• inhibitory serinových proteinas chemická syntéza   chemie   MeSH
• katalytická doména MeSH
• lidé MeSH
• membránové proteiny antagonisté a inhibitory   chemie   genetika   MeSH
• mutace MeSH
• objevování léků MeSH
• proteiny Drosophily metabolismus   MeSH
• proteiny z Escherichia coli antagonisté a inhibitory   chemie   genetika   MeSH
• serin chemie   MeSH
• simulace molekulového dockingu MeSH
• skot MeSH
• styreny chemická syntéza   chemie   MeSH
• transformující růstový faktor alfa metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alpha-chymotrypsin MeSH Prohlížeč
• benzoxaziny MeSH
• chymotrypsin MeSH
• DNA vazebné proteiny MeSH
• endopeptidasy MeSH
• GlpG protein, E coli MeSH Prohlížeč
• grk protein, Drosophila MeSH Prohlížeč
• inhibitory serinových proteinas MeSH
• membránové proteiny MeSH
• proteiny Drosophily MeSH
• proteiny z Escherichia coli MeSH
• serin MeSH
• styreny MeSH
• transformující růstový faktor alfa MeSH

Chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc), is a major structural component in chitin-containing organism including crustaceans, insects and fungi. Mammals express two chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Here, we report that pig AMCase is stable in the presence of other digestive proteases and functions as chitinolytic enzyme under the gastrointestinal conditions. Quantification of chitinases expression in pig tissues using quantitative real-time PCR showed that Chit1 mRNA was highly expressed in eyes, whereas the AMCase mRNA was predominantly expressed in stomach at even higher levels than the housekeeping genes. AMCase purified from pig stomach has highest activity at pH of around 2-4 and remains active at up to pH 7.0. It was resistant to robust proteolytic activities of pepsin at pH 2.0 and trypsin and chymotrypsin at pH 7.6. AMCase degraded polymeric chitin substrates including mealworm shells to GlcNAc dimers. Furthermore, we visualized chitin digestion of fly wings by endogenous AMCase and pepsin in stomach extract. Thus, pig AMCase can function as a protease resistant chitin digestive enzyme at broad pH range present in stomach as well as in the intestine. These results indicate that chitin-containing organisms may be a sustainable feed ingredient in pig diet.

• MeSH
• chitin metabolismus   MeSH
• chitinasy genetika   izolace a purifikace   metabolismus   MeSH
• chymotrypsin metabolismus   MeSH
• dieta * MeSH
• Drosophila chemie   MeSH
• endopeptidasy metabolismus   MeSH
• gastrointestinální trakt metabolismus   MeSH
• křídla zvířecí chemie   MeSH
• messenger RNA genetika   metabolismus   MeSH
• orgánová specificita MeSH
• pepsinogen A metabolismus   MeSH
• prasata genetika   MeSH
• rozpustnost MeSH
• substrátová specifita MeSH
• Tenebrio MeSH
• tkáňové extrakty MeSH
• trypsin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chitin MeSH
• chitinasy MeSH
• chymotrypsin MeSH
• endopeptidasy MeSH
• messenger RNA MeSH
• pepsinogen A MeSH
• tkáňové extrakty MeSH
• trypsin MeSH

Mass spectrometry coupled with bioaffinity separation techniques is considered a powerful tool for studying protein interactions. This work is focused on epitope analysis of tau protein, which contains two VQIXXK aggregation motifs regarded as crucial elements in the formation of paired helical filaments, the main pathological characteristics of Alzheimer's disease. To identify major immunogenic structures, the epitope extraction technique utilizing protein fragmentation and magnetic microparticles functionalized with specific antibodies was applied. However, the natural adhesiveness of some newly generated peptide fragments devalued the experimental results. Beside presumed peptide fragment specific to applied monoclonal anti-tau antibodies, the epitope extraction repeatedly revealed inter alia tryptic fragment 299-HVPGGGSVQIVYKPVDLSK-317 containing the fibril-forming motif 306-VQIVYK-311. The tryptic fragment pro-aggregation and hydrophobic properties that might contribute to adsorption phenomenon were examined by Thioflavin S and reversed-phase chromatography. Several conventional approaches to reduce the non-specific fragment sorption onto the magnetic particle surface were performed, however with no effect. To avoid methodological complications, we introduced an innovative approach based on altered proteolytic digestion. Simultaneous fragmentation of tau protein by two immobilized proteases differing in the cleavage specificity (TPCK-trypsin and α-chymotrypsin) led to the disruption of motif responsible for undesirable adhesiveness and enabled us to obtain undistorted structural data.

• Klíčová slova
• Epitope extraction, Mass spectrometry, Nonspecific sorption, Tau protein, Thioflavin S assay,
• MeSH
• adhezivita MeSH
• adsorpce MeSH
• Alzheimerova nemoc diagnóza   MeSH
• aminokyselinové motivy MeSH
• benzothiazoly MeSH
• biologické markery chemie   MeSH
• chymotrypsin chemie   MeSH
• epitopy chemie   MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• magnetismus MeSH
• monoklonální protilátky chemie   MeSH
• proteiny tau chemie   MeSH
• proteolýza MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• thiazoly chemie   MeSH
• trypsin chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• alpha-chymotrypsin MeSH Prohlížeč
• benzothiazoly MeSH
• biologické markery MeSH
• chymotrypsin MeSH
• epitopy MeSH
• MAPT protein, human MeSH Prohlížeč
• monoklonální protilátky MeSH
• proteiny tau MeSH
• thiazoly MeSH
• thioflavin T MeSH Prohlížeč
• trypsin MeSH

Hydrophobins are small proteins that play a role in a number of processes during the filamentous fungi growth and development. These proteins are characterized by the self-assembly of their molecules into an amphipathic membrane at hydrophilic-hydrophobic interfaces. Isolation and purification of hydrophobins generally present a challenge in their analysis. Hydrophobin SC3 from Schizophyllum commune was selected as a representative of class I hydrophobins in this work. A novel procedure for selective and effective isolation of hydrophobin SC3 based on solid-phase extraction with polytetrafluoroethylene microparticles loaded in a small self-made microcolumn is reported. The tailored binding of hydrophobins to polytetrafluoroethylene followed by harsh elution conditions resulted in a highly specific isolation of hydrophobin SC3 from the model mixture of ten proteins. The presented isolation protocol can have a positive impact on the analysis and utilization of these proteins including all class I hydrophobins. Hydrophobin SC3 was further subjected to reduction of its highly stable disulfide bonds and to chymotryptic digestion followed by mass spectrometric analysis. The isolation and digestion protocols presented in this work make the analysis of these highly hydrophobic and compact proteins possible.

• Klíčová slova
• Hydrophobin SC3, Isolation, Mass spectrometry, Polytetrafluoroethylene microparticles, Protein analysis,
• MeSH
• albuminy chemie   MeSH
• ananasovník chemie   MeSH
• bromelainy chemie   MeSH
• Canavalia chemie   MeSH
• chymotrypsin chemie   MeSH
• cytochromy c chemie   MeSH
• disulfidy chemie   MeSH
• erytrocyty enzymologie   MeSH
• extrakce na pevné fázi metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• karboanhydrasy chemie   MeSH
• kaseiny chemie   MeSH
• koně MeSH
• konkanavalin A chemie   MeSH
• kur domácí MeSH
• lidé MeSH
• mikrosféry * MeSH
• mléko enzymologie   MeSH
• myokard metabolismus   MeSH
• polytetrafluoroethylen chemie   MeSH
• proteomika metody   MeSH
• Schizophyllum chemie   MeSH
• skot MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• tandemová hmotnostní spektrometrie MeSH
• thermolysin chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• albuminy MeSH
• bromelainy MeSH
• chymotrypsin MeSH
• cytochromy c MeSH
• disulfidy MeSH
• karboanhydrasy MeSH
• kaseiny MeSH
• konkanavalin A MeSH
• polytetrafluoroethylen MeSH
• thermolysin MeSH

The effect of long-term exposure to tributyltin (TBT) on the intestine-related biochemical biomarkers in common carp was investigated in this study. Fish were exposed at sub-lethal concentrations of TBT (75 ng/L, 0.75 and 7.5 μg/L) for 60 days. Multiple biomarkers were measured, including digestive enzymes (trypsin, lipase and amylase), antioxidant responses (malondialdehyde (MDA) and total antioxidative capacity (T-AOC)), RNA/DNA ratio and the expression of digestive-related genes (try, lipc and amy). TBT exposure at 0.75 and 7.5 μg/L led to significantly inhibited activities of all digestive enzymes. At higher concentration of TBT, oxidative stress was apparent as reflected by the significant higher MDA content in the fish intestine, associated with an inhibition of T-AOC activities. After 60 days, the RNA/DNA ratio in fish intestine was significantly lower in groups exposed to TBT at higher concentrations (0.75 and 7.5 μg/L). In addition, the expression levels of try, lipc and amy in intestine of all treated fish were inhibited, even at the environmental concentration (75 ng/L). Our results suggest that long-term exposure to TBT could result in different responses of intestine-related biochemical biomarkers in fish, which could be used as new potential indicators for monitoring residual TBT present in aquatic environment.

• Klíčová slova
• Digestive enzyme, Fish intestine, Gene expression, Organotin compounds, Oxidative stress, RNA/DNA ratio,
• MeSH
• antioxidancia metabolismus   MeSH
• biologické markery metabolismus   MeSH
• chymotrypsin metabolismus   MeSH
• DNA metabolismus   MeSH
• kapři metabolismus   MeSH
• malondialdehyd metabolismus   MeSH
• oxidační stres účinky léků   MeSH
• pankreatická elastasa metabolismus   MeSH
• pesticidy chemie   toxicita   MeSH
• RNA metabolismus   MeSH
• střeva účinky léků   MeSH
• střevní sliznice metabolismus   MeSH
• trialkylcínové sloučeniny chemie   toxicita   MeSH
• trypsin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antioxidancia MeSH
• biologické markery MeSH
• chymotrypsin MeSH
• DNA MeSH
• malondialdehyd MeSH
• pankreatická elastasa MeSH
• pesticidy MeSH
• RNA MeSH
• trialkylcínové sloučeniny MeSH
• tributyltin MeSH Prohlížeč
• trypsin MeSH