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.

• 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

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.

• 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

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.

• 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

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

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.

• 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

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.

• MeSH
• adhezivita MeSH
• adsorpce MeSH
• Alzheimerova nemoc diagnóza   MeSH
• aminokyselinové motivy 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

Systémová enzymoterapie je definována jako léčebná metoda, která zahrnuje perorální aplikaci kombinovaných enzymových směsí ve formě acidorezistentních tablet. Hlavní účinné látky těchto přípravků tvoří proteolytické enzymy původu živočišného (trypsin a chymotrypsin) a rostlinného (bromelain a papain). Základní podmínkou systémového účinku takto aplikovaných proteáz je jejich podávání nalačno a následné vstřebávání střevní sliznicí, při kterém zůstává zachována jejich enzymatická aktivita. Léčivé přípravky určené pro systémovou enzymoterapii jsou známy svými účinky protizánětlivými, protiotokovými, analgetickými a působí též imunomodulačně. Díky svému fibrinolytickému a antiagregačnímu účinku příznivě ovlivňují reologické vlastnosti krve. Současné podávání přípravků určených pro systémovou enzymoterapii společně s antibiotiky (ale i s dalšími léčivy) zlepšuje jejich tkáňovou dostupnost (efekt vehikula). Léčivé přípravky pro systémovou enzymoterapii Wobenzym® a Phlogenzym® jsou v České republice registrovány jako volně prodejné léky. Oborem, kde má využití systémové enzymoterapie v ČR nejdelší tradici, je patrně sportovní medicína.

Systemic enzyme therapy is defined as a therapeutic method using oral application of combined enzyme mixtures in the form of acid resistant tablets. The key active substances in these tablets are proteolytic enzymes of both animal (trypsin and chymotrypsin) and plant (bromelain and papain) origin. The systemic effect of thus applied proteases fully depends on their administration before meals and on their absorption through the intestinal mucosa, after which their enzymatic activity is preserved. Therapeutic agents for systemic enzyme therapy are known for their anti inflammatory, anti edematous, analgesic, and immunomodulatory properties. Thanks to their fibrinolytic and anti aggregation effects, they also improve the rheologic properties of the blood. Parallel application of systemic enzyme therapy and antibiotics (but also other drugs) improves their tissue availability (effect of the vehiculum). Therapeutic agents for systemic enzyme therapy Wobenzym® and Phlogenzym® are registered in the Czech Republic as over the counter drugs. The longest tradition of systemic enzyme therapy use in the Czech Republic can probably be seen in the sport medicine.

• MeSH
• alfa-makroglobuliny MeSH
• aplikace orální MeSH
• bromelainy farmakokinetika   metabolismus   MeSH
• chymotrypsin farmakokinetika   metabolismus   MeSH
• cytokiny účinky léků   MeSH
• enzymoterapie * MeSH
• imunitní systém enzymologie   MeSH
• klinické zkoušky kontrolované jako téma MeSH
• lidé MeSH
• makrofágy MeSH
• papain farmakokinetika   metabolismus   MeSH
• proteasy * farmakokinetika   metabolismus   MeSH
• střevní sliznice účinky léků   MeSH
• trypsin farmakokinetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

• MeSH
• biologické přípravky * MeSH
• chymotrypsin * izolace a purifikace   MeSH
• dějiny 20. století MeSH
• farmaceutická technologie * metody   normy   organizace a řízení   pracovní síly   trendy   výchova   MeSH
• farmacie * dějiny   metody   normy   organizace a řízení   pracovní síly   trendy   MeSH
• inzulin * izolace a purifikace   MeSH
• klinické laboratorní techniky * metody   normy   využití   MeSH
• lidé MeSH
• mladiství MeSH
• orgány z umělého a biologického materiálu * MeSH
• pankreatin * izolace a purifikace   MeSH
• pepsin A * MeSH
• školy * normy   organizace a řízení   pracovní síly   MeSH
• studenti * dějiny   MeSH
• trypsin * izolace a purifikace   MeSH
• učební obory * metody   organizace a řízení   pracovní síly   trendy   MeSH
• výzkum * MeSH
• Check Tag
• dějiny 20. století MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• Publikační typ
• historické články MeSH

Zatiaľčo u dospelých sa ako hlavný etiologický faktor chronickej pankreatitídy uvádza nadmerná konzumácia alkoholu, u detí sa okrem anatomických anomálií pankreasu a žlčových ciest uplatňujú predovšetkým genetické faktory. Cieľ: Charakterizovať frekvenciu výskytu mutácií jednotlivých génov asociovaných so vznikom rekurentnej akútnej pankreatitídy alebo chronickej pankreatitídy u detí a následne sledovať genotypovo-fenotypové korelácie u týchto pa­cientov. Materiál a metodika: Do štúdie bolo zaradených 21 detí s dia­gnózou rekurentnej akútnej pankreatitídy/chronickej pankreatitídy nejasnej etiológie, ktoré boli v období 2008–2013 vyšetrené na II. detskej klinike LF UK a DFNsP v Bratislave. Molekulovo-genetická analýza génov PRSS1, SPINK1 a CTRC sa uskutočnila v spolupráci s Oddelením lekárskej genetiky Onkologického ústavu sv. Alžbety. Výsledky: Rodin­ná anamnéza bola pozitívna v piatich prípadoch. U dvoch pa­cientov bola dokázaná prítomnosť mutácie p.R122H génu PRSS1; jeden pa­cient je zmiešaný heterozygot pre mutácie p.G208A (PRS­S1) a p.G60G (CTRC). Mutácia p.N34S génu SPINK1 sa potvrdila u šiestich pa­cientov (dvaja homozygotní a štyria heterozygotní), z nich u štyroch bola potvrdená aj mutácia p.G60G (CTRC) v heterozygotnom stave. Jeden pa­cient je homozygot pre mutáciu p.G60G; jeden heterozygot pre p.R254W a jeden heterozygot pre mutáciu p.G214R génu CTRC. Záver: V súbore 21 detských pa­cientov s rekurentnou akútnou pankreatitídou/chronickou pankreatitídou neznámej etiológie sme zaznamenali vysoký výskyt kauzálnych mutácií asociovaných so vznikom ochorenia. Uvedené výsledky potvrdzujú dôležitosť genetického vyšetrenia u detí s idiopatickou rekurentnou akútnou pankreatitídou /chronickou pankreatitídou.

While in adults the major etiological factor of chronic pancreatitis is excessive alcohol consumption, in children, together with anatomical anomalies of the pancreas and biliary tract, genetic factors seem to be crucial. Aim: Our aim was to investigate the frequency of mutations in genes associated with the development of recurrent acute pancreatitis or chronic pancreatitis in children and then monitor the genotype-phenotype correlations in the patients. Material and methods: Twenty-one children with recurrent acute pancreatitis or chronic pancreatitis of unknown etiology diagnosed between 2008 and 2013 at the 2nd Department of Pediatrics, University Children’s Hospital, Bratislava, were enrolled in the study. Molecular genetic analysis of PRSS1, SPINK1 and CTRC genes was performed in cooperation with the Department of Medical Genetics at St. Elisabeth’s Institute of Oncology. Results: Family history was positive in five cases. In 2 out of 21 patients, the p.R122H mutation of PRSS1 gene was found; one patient was a trans-heterozygous carrier of the p.G208A (PRSS1) and p.G60G (CTRC) variants. The p.N34S mutation of SPINK1 gene was seen in six patients (two homozygous and four heterozygous), out of which four were trans-heterozygotes with the p.G60G (CTRC) variant. One patient was homozygous for p.G60G, one was heterozygous for p.R254W and one was heterozygous for p.G214R variant of the CTRC gene. Conclusion: In a group of 21 pediatric patients with recurrent acute pancreatitis/chronic pancreatitis of unknown etiology, a high prevalence of causal mutations associated with the development of the disease was found. These results confirm the importance of genetic testing in children with idiopathic recurrent acute pancreatitis/chronic pancreatitis. Key words: chronic pancreatitis – hereditary pancreatitis – genetic predisposition to disease – genetic analysis – pancreatitis in children The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE „uniform requirements“ for bio­­­­medical papers. Submitted: 5. 11. 2015 Accepted: 29. 11. 2015

• Klíčová slova
• hereditární pankreatitída,
• MeSH
• akutní nemoc MeSH
• chronická pankreatitida * genetika   MeSH
• chymotrypsin genetika   MeSH
• dítě MeSH
• fenotyp MeSH
• genetická predispozice k nemoci * MeSH
• genetické testování MeSH
• genotyp MeSH
• lidé MeSH
• mladiství MeSH
• mutace MeSH
• mutační analýza DNA * MeSH
• pankreatitida genetika   MeSH
• předškolní dítě MeSH
• recidiva MeSH
• rodokmen MeSH
• transportní proteiny genetika   MeSH
• trypsin genetika   MeSH
• trypsinogen genetika   MeSH
• věk při počátku nemoci MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH

Oil bodies, lipid-storage organelles, are stabilized by a number of specific proteins. These proteins are very hydrophobic, which complicates their identification by "classical" proteomic protocols using trypsin digestion. Due to the lack of trypsin cleavage sites, the achievable protein coverage is limited or even insufficient for reliable protein identification. To identify such proteins and to enhance their coverage, we introduced a modified method comprising standard three-step procedure (SDS-PAGE, in-gel digestion, and LC-MS/MS analysis). In this method, chymotrypsin, single or in combination with trypsin, was used, which enabled to obtain proteolytic peptides from the hydrophobic regions and to identify new oil bodies' proteins. Our method can be easily applied to identification of other hydrophobic proteins.

• MeSH
• Arabidopsis metabolismus   MeSH
• chromatografie kapalinová MeSH
• chymotrypsin metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• hmotnostní spektrometrie MeSH
• hydrofobní a hydrofilní interakce * MeSH
• lipidy * MeSH
• organely metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• proteomika metody   MeSH
• trypsin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH