It has been 30 years since the first member of the protease-activated receptor (PAR) family was discovered. This was followed by the discovery of three other receptors, including PAR2. PAR2 is a G protein-coupled receptor activated by trypsin site-specific proteolysis. The process starts with serine proteases acting between arginine and serine, creating an N-terminus that functions as a tethered ligand that binds, after a conformational change, to the second extracellular loop of the receptor, leading to activation of G-proteins. The physiological and pathological functions of this ubiquitous receptor are still elusive. This review focuses on PAR2 activation and its distribution under physiological and pathological conditions, with a particular focus on the pancreas, a significant producer of trypsin, which is the prototype activator of the receptor. The role in acute or chronic pancreatitis, pancreatic cancer, and diabetes mellitus will be highlighted.
- MeSH
- lidé MeSH
- nemoci slinivky břišní * diagnóza MeSH
- pankreas metabolismus MeSH
- receptor PAR-2 * metabolismus MeSH
- receptory spřažené s G-proteiny MeSH
- trypsin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Specific nuclear sub-compartments that are regions of fundamental processes such as gene expression or DNA repair, contain phosphoinositides (PIPs). PIPs thus potentially represent signals for the localization of specific proteins into different nuclear functional domains. We performed limited proteolysis followed by label-free quantitative mass spectrometry and identified nuclear protein effectors of the most abundant PIP-phosphatidylinositol 4,5-bisphosphate (PIP2). We identified 515 proteins with PIP2-binding capacity of which 191 'exposed' proteins represent a direct PIP2 interactors and 324 'hidden' proteins, where PIP2 binding was increased upon trypsin treatment. Gene ontology analysis revealed that 'exposed' proteins are involved in the gene expression as regulators of Pol II, mRNA splicing, and cell cycle. They localize mainly to non-membrane bound organelles-nuclear speckles and nucleolus and are connected to the actin nucleoskeleton. 'Hidden' proteins are linked to the gene expression, RNA splicing and transport, cell cycle regulation, and response to heat or viral infection. These proteins localize to the nuclear envelope, nuclear pore complex, or chromatin. Bioinformatic analysis of peptides bound in both groups revealed that PIP2-binding motifs are in general hydrophilic. Our data provide an insight into the molecular mechanism of nuclear PIP2 protein interaction and advance the methodology applicable for further studies of PIPs or other protein ligands.
- MeSH
- buněčné jádro metabolismus MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- genová ontologie MeSH
- HeLa buňky MeSH
- hmotnostní spektrometrie * MeSH
- hydrofobní a hydrofilní interakce MeSH
- lidé MeSH
- peptidy metabolismus MeSH
- proteolýza * MeSH
- proteom chemie metabolismus MeSH
- regulace genové exprese MeSH
- sekvence aminokyselin MeSH
- trypsin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The commonly used histological assessment of pathological states of alveolar bone tissues in oral surgery needs laborious and time-consuming processing by an experienced histologist. Therefore, a simpler and faster methodology is required in this field. Following this demand, this paper reports a straightforward approach using the tryptic cleavage of proteins directly in bone without its demineralization, followed by the capillary electrophoresis-ultraviolet detection profiling of the yielded protein digest. Cleavage-derived peptides were separated by capillary electrophoresis in acidic background electrolytes, pH 2.01-2.54. The best resolution of peptide fragments with the highest peak capacity was achieved in the background electrolyte composed of 55 mM H3 PO4 , 14 mM tris(hydroxymethyl)aminomethan, pH 2.01. The differences in the obtained capillary electrophoresis-ultraviolet detection profiles with characteristic patterns for particular bone samples were subsequently discriminated by linear discriminant analysis over principal components. This approach was first verified on porcine bone tissues as model samples; jawbone and calf bone tissues could be discriminated with an accuracy of 100%. Subsequently, the method was capable of differentiating unequivocally between human healthy and inflammatory alveolar bone tissues obtained from oral surgery. This procedure seems to be promising as complement or even an alternative to the traditional histological discrimination between healthy and inflammatory bone tissues in oral surgery.
The dental pulp represents an easily accessible source of adult dental pulp stem cells (DPSCs). The preferred approach to DPSC isolation is enzymatic digestion. However, the duration of the enzymatic activity is crucial. The purpose of this study was to isolate the DPSC populations using this method, characterize their biological properties and proliferation capacity, and to determine their ability to differentiate into mature cells. Before enzymatic digestion using 0.05% trypsin, we used the homogenization method in order to obtain a fine homogenate from the solid pulp tissue. The stem cells were cultivated in modified cultivation medium for mesenchymal adult progenitor cells containing 2% foetal bovine serum, growth factors and insulin-transferrin-selenium supplement. We were successfully able to isolate 10 populations of DPSCs. The vitality of DPSCs did not drop below 90 %. However, the DPSCs showed a significant decrease in the relative telomere length number with increasing passaging (P < 0.05). Isolated DPSCs highly expressed the CD markers: CD29, CD44, CD90, CD13, CD73 and CD166. In contrast, CD markers CD31, CD106, CD34 and CD45 were negative or low positive. We confirmed the high osteogenic and chondrogenic potential of the isolated stem cells. Isolated DPSCs did not show signs of cell degeneration or spontaneous differentiation during the entire cultivation. In addition, we were able to shorten the enzyme activity duration, and we were the first to demonstrate trypsin as the enzyme used for the enzymatic digestion method with the viability over 90 % of isolated DPSCs using this method.
- MeSH
- buněčná diferenciace MeSH
- buněčný rodokmen MeSH
- CD antigeny metabolismus MeSH
- chondrogeneze MeSH
- dospělé kmenové buňky cytologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- osteogeneze MeSH
- proliferace buněk MeSH
- separace buněk metody MeSH
- telomery metabolismus MeSH
- trypsin metabolismus MeSH
- zubní dřeň cytologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The work is focused on the development of microspheres based on the combination of two polysaccharides; chitosan and alginic acid with the aim to allocate, hold, release and protect environmentally sensible molecules. The microspheres were prepared using a solvent-free, low cost and scalable approach and two enzymes; trypsin and protease from Aspergillus Oryzae have been used as a model to evaluate the microspheres peculiarities. The proteins were encapsulated during the microspheres preparation. The relationship between the polysaccharides weight ratio and the morphology, stability and ability of the carrier to allocate the enzymes has been evaluated. The enzymatic activity and the release kinetics were assessed in different conditions to assess the impact of the external environment. Obtained results demonstrate the efficacy of the prepared microspheres to preserve the activity of relevant bioactive compounds which are highly relevant in food, cosmetic and pharmaceutic, but the application is limited due to their high sensibility.
- MeSH
- Aspergillus oryzae enzymologie MeSH
- buňky NIH 3T3 MeSH
- chitosan chemie toxicita MeSH
- enzymy imobilizované chemie metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- kyselina alginová chemie toxicita MeSH
- lidé MeSH
- mikrosféry * MeSH
- myši MeSH
- testování materiálů MeSH
- tobolky MeSH
- trypsin chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The main objective of this study was to develop an effective in-gel trypsin digestion protocol using aqueous-acetonitrile solvent system to facilitate MS analysis and maximize the number of identified proteins from biological samples. The procedure, where 80% acetonitrile was present in the trypsin reaction mixture, increased the number of matched peptides, and allowed the identification of more proteins with higher coverage than the common digestion protocol. Vimentin, annexins, tubulin, actin, peptidyl-prolyl cis-trans isomerase or alpha-enolase are examples of important proteins that change during the progress cancer. These were isolated from human breast cancer cells and were used for this study.
- MeSH
- acetonitrily chemie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny analýza izolace a purifikace metabolismus MeSH
- nádory metabolismus patologie MeSH
- peptidy analýza MeSH
- proteomika metody MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- trypsin metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Trypsin is the protease of choice for protein sample digestion in proteomics. The most typical active forms are the single-chain β-trypsin and the two-chain α-trypsin, which is produced by a limited autolysis of β-trypsin. An additional intra-chain split leads to pseudotrypsin (ψ-trypsin) with three chains interconnected by disulfide bonds, which can be isolated from the autolyzate by ion-exchange chromatography. Based on experimental data with artificial substrates, peptides, and protein standards, ψ-trypsin shows altered kinetic properties, thermodynamic stability and cleavage site preference (and partly also cleavage specificity) compared to the above-mentioned proteoforms. In our laboratory, we have analyzed the performance of bovine ψ-trypsin in the digestion of protein samples with a different complexity. It cleaves predominantly at the characteristic trypsin cleavage sites. However, in a comparison with common tryptic digestion, non-specific cleavages occur more frequently (mostly after the aromatic residues of Tyr and Phe) and more missed cleavages are generated. Because of the preferential cleavages after the basic residues and more developed side specificity, which is not expected to occur for the major trypsin forms (but may appear anyway because of their autolysis), ψ-trypsin produces valuable information, which is complementary in part to data based on a strictly specific trypsin digestion and thus can be unnoticed following common proteomics protocols.
- MeSH
- autolýza MeSH
- kinetika MeSH
- protein - isoformy chemie metabolismus MeSH
- proteolýza MeSH
- skot MeSH
- stabilita enzymů MeSH
- trypsin chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The human amniotic membrane (HAM) is widely used for its wound healing effect in clinical practice, as a feeder for the cell cultivation, or a source of cells to be used in cell therapy. The aim of this study was to find effective and safe enzymatic HAM de-epithelialization method leading to harvesting of both denuded undamaged HAM and viable human amniotic epithelial cells (hAECs). The efficiency of de-epithelialization using TrypLE Express, trypsin/ ethylenediaminetetraacetic (EDTA), and thermolysin was monitored by hematoxylin and eosin staining and by the measurement of DNA concentration. The cell viability was determined by trypan blue staining. Scanning electron microscopy and immunodetection of collagen type IV and laminin α5 chain were used to check the basement membrane integrity. De-epithelialized hAECs were cultured and their stemness properties and proliferation potential was assessed after each passage. The HAM was successfully de-epithelialized using all three types of reagents, but morphological changes in basement membrane and stroma were observed after the thermolysin application. About 60% of cells remained viable using trypsin/EDTA, approximately 6% using TrypLE Express, and all cells were lethally damaged after thermolysin application. The hAECs isolated using trypsin/EDTA were successfully cultured up to the 5th passage with increasing proliferation potential and decreased stem cell markers expression (NANOG, SOX2) in prolonged cell culture. Trypsin/EDTA technique was the most efficient for obtaining both undamaged denuded HAM and viable hAECs for consequent culture.
- MeSH
- amnion cytologie metabolismus patologie MeSH
- DNA analýza izolace a purifikace MeSH
- EDTA chemie MeSH
- epitelové buňky cytologie metabolismus patologie MeSH
- kolagen typu IV metabolismus MeSH
- kultivované buňky MeSH
- laminin metabolismus MeSH
- lidé MeSH
- mikroskopie elektronová rastrovací MeSH
- nanog metabolismus MeSH
- proliferace buněk MeSH
- reepitalizace MeSH
- transkripční faktory SOXB1 metabolismus MeSH
- trypsin metabolismus MeSH
- viabilita buněk MeSH
- Check Tag
- lidé 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
Analysis of glycosylation is challenging due to micro- and macro-heterogeneity of the protein attachment. A combination of LC with MS/MS is one of the most powerful tools for glycopeptide analysis. In this work, we show the effect of various monosaccharide units on the retention time of glycopeptides. Retention behavior of several glycoforms of six peptides obtained from tryptic digest of haptoglobin, hemopexin, and sex hormone-binding globulin was studied on a reversed phase chromatographic column. We observed reduction of the retention time with increasing number of monosaccharide units of glycans attached to the same peptide backbone. Fucosylation of larger glycans provides less significant retention time shift than for smaller ones. Retention times of glycopeptides were expressed as relative retention times. These relative retention times were used for calculation of upper and lower limits of glycopeptide retention time windows under the reversed phase conditions. We then demonstrated on the case of a glycopeptide of haptoglobin that the predicted retention time window boosts confidence of identification and minimizes false-positive identification. Relative retention time, as a qualitative parameter, is expected to improve LC-MS/MS characterization of glycopeptides.
- MeSH
- chromatografie s reverzní fází metody MeSH
- glykopeptidy krev chemie metabolismus MeSH
- glykosylace MeSH
- lidé MeSH
- nanotechnologie metody MeSH
- peptidové fragmenty analýza chemie metabolismus MeSH
- proteomika MeSH
- senzitivita a specificita MeSH
- trypsin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
