extracellular enzymatic activity
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BACKGROUND: Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood. METHODS: We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested. RESULTS: MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed. CONCLUSIONS: Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins. GENERAL SIGNIFICANCE: Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins.
- MeSH
- cévní endotel cytologie metabolismus MeSH
- dimerizace MeSH
- dusičnany metabolismus MeSH
- extracelulární matrix - proteiny chemie metabolismus MeSH
- fibronektiny metabolismus MeSH
- glykosaminoglykany metabolismus MeSH
- kolagen typu IV metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- oxidační stres MeSH
- peroxidasa metabolismus MeSH
- tyrosin metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Malignant gliomas exhibit abnormal expression of proteolytic enzymes that may participate in the uncontrolled cell proliferation and aberrant interactions with the brain extracellular matrix. The multifunctional membrane bound serine aminopeptidase dipeptidyl peptidase (DPP)-IV has been linked to the development and progression of several malignancies, possibly both through the enzymatic and nonenzymatic mechanisms. In this report we demonstrate the expression of DPP-IV and homologous proteases fibroblast activation protein, DPP8 and DPP9 in primary cell cultures derived from high-grade gliomas, and show that the DPP-IV-like enzymatic activity is negatively associated with their in vitro growth. More importantly, the DPP-IV positive subpopulation isolated from the primary cell cultures using immunomagnetic separation exhibited slower proliferation. Forced expression of the wild as well as the enzymatically inactive mutant DPP-IV in glioma cell lines resulted in their reduced growth, migration and adhesion in vitro, as well as suppressed glioma growth in an orthotopic xenotransplantation mouse model. Microarray analysis of glioma cells with forced DPP-IV expression revealed differential expression of several candidate genes not linked to the tumor suppressive effects of DPP-IV in previous studies. Gene set enrichment analysis of the differentially expressed genes showed overrepresentation of gene ontology terms associated with cell proliferation, cell adhesion and migration. In conclusion, our data show that DPP-IV may interfere with several aspects of the malignant phenotype of glioma cells in great part independent of its enzymatic activity.
- MeSH
- buněčná adheze MeSH
- buněčný cyklus MeSH
- dipeptidasy genetika metabolismus MeSH
- dipeptidylpeptidasa 4 genetika metabolismus MeSH
- dipeptidylpeptidasy a tripeptidylpeptidasy genetika metabolismus MeSH
- gliom enzymologie genetika MeSH
- imunomagnetická separace MeSH
- lidé MeSH
- mutace MeSH
- myši MeSH
- nádorové buňky kultivované MeSH
- pohyb buněk MeSH
- primární buněčná kultura MeSH
- proliferace buněk MeSH
- regulace genové exprese u nádorů MeSH
- signální transdukce genetika MeSH
- stanovení celkové genové exprese MeSH
- transfekce MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Background: The rumen microbiota is one of the most complex consortia of anaerobes, involving archaea, bacteria, protozoa, fungi and phages. They are very effective at utilizing plant polysaccharides, especially cellulose and hemicelluloses. The most important hemicellulose decomposers are clustered with the genus Butyrivibrio. As the related species differ in their range of hydrolytic activities and substrate preferences, Butyrivibrio fibrisolvens was selected as one of the most effective isolates and thus suitable for proteomic studies on substrate comparisons in the extracellular fraction. The B. fibrisolvens genome is the biggest in the butyrivibria cluster and is focused on "environmental information processing" and "carbohydrate metabolism". Methods: The study of the effect of carbon source on B. fibrisolvens 3071 was based on cultures grown on four substrates: xylose, glucose, xylan, xylan with 25% glucose. The enzymatic activities were studied by spectrophotometric and zymogram methods. Proteomic study was based on genomics, 2D electrophoresis and nLC/MS (Bruker Daltonics) analysis. Results: Extracellular β-endoxylanase as well as xylan β-xylosidase activities were induced with xylan. The presence of the xylan polymer induced hemicellulolytic enzymes and increased the protein fraction in the interval from 40 to 80 kDa. 2D electrophoresis with nLC/MS analysis of extracellular B. fibrisolvens 3071 proteins found 14 diverse proteins with significantly different expression on the tested substrates. Conclusion: The comparison of four carbon sources resulted in the main significant changes in B. fibrisolvens proteome occurring outside the fibrolytic cluster of proteins. The affected proteins mainly belonged to the glycolysis and protein synthesis cluster.
- Publikační typ
- časopisecké články MeSH
The release of neutrophil extracellular traps (NETs) is one of the weapons neutrophils have in their armory. NETs consist of extracellular chromatin fibers decorated with a plethora of cytoplasmic and granular proteins, such as the antimicrobial serine protease neutrophil elastase (NE). Because the first description of NETs as beneficial to the host, reports on their double-faced role in health and disease have considerably increased recently. On one hand, NETs reportedly trap and kill bacteria and also participate in the resolution of the acute inflammation associated with infection and with tissue damage. On the other hand, numerous negative aspects of NETs contribute to the etiopathogenesis of autoimmune disorders. Employing soluble and solid fluorescent substrates, we demonstrate the interaction of NE with aggregated NETs (aggNETs), the limitation of its enzymatic activity and the containment of the enzyme from surrounding tissues. These events prevent the spread of inflammation and tissue damage. The detection of DNase 1-dependent elevation of NE activity attests the continuous presence of patrolling neutrophils forming NETs and aggNETs even under conditions physiologic conditions.
- MeSH
- aktivace enzymů MeSH
- deoxyribonukleasa I metabolismus MeSH
- deoxyribonukleasy metabolismus MeSH
- extracelulární pasti imunologie metabolismus MeSH
- leukocytární elastasa metabolismus MeSH
- lidé MeSH
- myši MeSH
- neutrofily imunologie metabolismus MeSH
- tělesné tekutiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Meningiomas are tumors derived from arachnoid cap cells that represent approximately 30% of all intracranial tumors. In this study, we investigated 22 human meningiomas for the expression of dipeptidyl peptidase (DPP)-IV activity and/or structure homologs (DASH), including canonical DPP-IV/CD26, fibroblast activation protein-alpha (FAPalpha), DPP8 and DPP9. DPP-IV-like enzymatic activity, including all enzymatically-active DASH molecules, was found in all 18 benign meningiomas WHO grade I and IV atypical meningiomas WHO grade II by continuous rate fluorimetric assay in tissue homogenates and catalytic enzyme histochemistry in situ. In atypical meningiomas, this activity was significantly higher and was associated with higher cell proliferation as detected by Ki67 antigen immunohistochemistry. The expression of DPP-IV/CD26 and FAPalpha demonstrated by real-time RT-PCR and immunohistochemistry was low. As shown histochemically, it occurred most often on the surface of fibrous bundles and whorls rich in extracellular matrix. Compared to DPP-IV/CD26 and FAPalpha, the expression of DPP8 and DPP9 was higher and, in addition, it was present also in the cells inside these structures. Expression of CXCR4, the receptor of pro-proliferative chemokine stromal cell-derived factor-1alpha (SDF-1alpha), DPP-IV substrate, was found in all tumors, suggesting higher values in atypical grade II samples. This is the first report on the expression status of dipeptidyl peptidase-IV and related molecules in meningiomas. It shows that DPP8 and DPP9 prevail over canonical DPP-IV/CD26 and FAPalpha in all examined patients. In addition, the study suggests an increase of DPP-IV-like enzymatic activity in these tumors of WHO grade II.
- MeSH
- dipeptidasy genetika metabolismus MeSH
- dipeptidylpeptidasa 4 genetika metabolismus MeSH
- dipeptidylpeptidasy a tripeptidylpeptidasy genetika metabolismus MeSH
- dospělí MeSH
- exprese genu MeSH
- imunohistochemie MeSH
- izoenzymy genetika metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- membránové proteiny genetika metabolismus MeSH
- meningeální nádory enzymologie genetika patologie MeSH
- meningeom enzymologie genetika patologie MeSH
- messenger RNA analýza MeSH
- nádorové biomarkery analýza MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- senioři MeSH
- serinové endopeptidasy genetika metabolismus MeSH
- stanovení celkové genové exprese MeSH
- želatinasy genetika metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
Fungal β-N-acetylhexosaminidases are inducible extracellular enzymes with many biotechnological applications. The enzyme from Penicillium oxalicum has unique enzymatic properties despite its close evolutionary relationship with other fungal hexosaminidases. It has high GalNAcase activity, tolerates substrates with the modified N-acyl group better and has some other unusual catalytic properties. In order to understand these features, we performed isolation, biochemical and enzymological characterization, molecular cloning and molecular modelling. The native enzyme is composed of two catalytic units (65 kDa each) and two propeptides (15 kDa each), yielding a molecular weight of 160 kDa. Enzyme deglycosylated by endoglycosidase H had comparable activity, but reduced stability. We have cloned and sequenced the gene coding for the entire hexosaminidase from P. oxalicum. Sufficient sequence identity of this hexosaminidase with the structurally solved enzymes from bacteria and humans with complete conservation of all catalytic residues allowed us to construct a molecular model of the enzyme. Results from molecular dynamics simulations and substrate docking supported the experimental kinetic and substrate specificity data and provided a molecular explanation for why the hexosaminidase from P. oxalicum is unique among the family of fungal hexosaminidases.
- MeSH
- beta-N-acetylhexosaminidasy chemie genetika izolace a purifikace metabolismus MeSH
- fungální proteiny chemie genetika izolace a purifikace metabolismus MeSH
- glykosylace MeSH
- katalytická doména MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- konzervovaná sekvence MeSH
- mannosyl-glykoprotein endo-beta-N-acetylglukosaminidasa metabolismus MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- Penicillium enzymologie genetika MeSH
- prekurzory enzymů chemie genetika izolace a purifikace metabolismus MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční seřazení MeSH
- simulace molekulární dynamiky MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Zymography is an electrophoretic method in which proteins are separated in a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE). This method is used for the detection of enzymatic activity and molecular characterization of proteins. In contrast to the standard SDS-PAGE method, a substrate is incorporated into the gel during zymography, which is subsequently cleaved by target proteases. Many studies have focused on the development and progression of inflammatory diseases affecting the gastrointestinal tract, emphasizing the role of the largest group of proteases, matrix metalloproteinases (MMPs). The most used classification of this group of enzymes (by researchers in MMP biology) is based in part on the historical evaluation of the substrate specificity of MMPs and in part on the cellular localization of MMPs. MMPs are thus classified into the groups of collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs (MT-MMPs), and others. An important group of MMPs are gelatinases which are involved in the breakdown of collagen type IV and gelatin of extracellular matrix and participate in the regulation of various physiological or pathological processes such as morphogenesis, angiogenesis, tissue repair, cirrhosis, arthritis, and metastasis. The present study's objective was to determine the amount of active MMP-9 and MMP-2 forms in tissue samples using zymography. The patient group was according to histology findings divided into the benign tumor (control) group (8 patients), and the malignant tumor group (24 patients). The respondents in the malignant tumor group were further divided according to the standard TNM classification. The results of this study confirmed that MMP-2, unlike MMP-9, can be used as a prognostic biomarker of CRC, because only the expression of active MMP-2 confirmed statistically significant differences between individual stages of CRC. Moreover, MMP-2 seems to play a more important role in higher stages of CRC. Substantial disparities in the determination of active MMPs between the observed groups support the assumption for the integration of zymography into clinical diagnostics of CRC together with molecular and other studies.
Nutritional factors which exhibit antioxidant properties, such as those contained in green plants, may be protective against cancer. Chlorophyll and other tetrapyrrolic compounds which are structurally related to heme and bilirubin (a bile pigment with antioxidant activity) are among those molecules which are purportedly responsible for these effects. Therefore, the aim of our study was to assess both the antiproliferative and antioxidative effects of chlorophylls (chlorophyll a/b, chlorophyllin, and pheophytin a) in experimental pancreatic cancer. Chlorophylls have been shown to produce antiproliferative effects in pancreatic cancer cell lines (PaTu-8902, MiaPaCa-2, and BxPC-3) in a dose-dependent manner (10-125 μmol/L). Chlorophylls also have been observed to inhibit heme oxygenase (HMOX) mRNA expression and HMOX enzymatic activity, substantially affecting the redox environment of pancreatic cancer cells, including the production of mitochondrial/whole-cell reactive oxygen species, and alter the ratio of reduced-to-oxidized glutathione. Importantly, chlorophyll-mediated suppression of pancreatic cancer cell viability has been replicated in in vivo experiments, where the administration of chlorophyll a resulted in the significant reduction of pancreatic tumor size in xenotransplanted nude mice. In conclusion, this data suggests that chlorophyll-mediated changes on the redox status of pancreatic cancer cells might be responsible for their antiproliferative and anticancer effects and thus contribute to the decreased incidence of cancer among individuals who consume green vegetables.
- MeSH
- antioxidancia metabolismus MeSH
- chlorofyl farmakologie MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- feofytiny metabolismus MeSH
- glutathion metabolismus MeSH
- glutathiondisulfid metabolismus MeSH
- hemová oxygenasa (decyklizující) metabolismus MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- mitochondrie účinky léků metabolismus MeSH
- nádorové buněčné linie MeSH
- nádory slinivky břišní metabolismus MeSH
- oxidace-redukce účinky léků MeSH
- peroxid vodíku metabolismus MeSH
- protinádorové látky farmakologie MeSH
- superoxidy metabolismus MeSH
- Synechocystis chemie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Glutamate carboxypeptidase II (GCPII), also known as prostate-specific membrane antigen (PSMA) or folate hydrolase, is a metallopeptidase expressed predominantly in the human brain and prostate. GCPII expression is considerably increased in prostate carcinoma, and the enzyme also participates in glutamate excitotoxicity in the brain. Therefore, GCPII represents an important diagnostic marker of prostate cancer progression and a putative target for the treatment of both prostate cancer and neuronal disorders associated with glutamate excitotoxicity. For the development of novel therapeutics, mouse models are widely used. However, although mouse GCPII activity has been characterized, a detailed comparison of the enzymatic activity and tissue distribution of the mouse and human GCPII orthologs remains lacking. In this study, we prepared extracellular mouse GCPII and compared it with human GCPII. We found that mouse GCPII possesses lower catalytic efficiency but similar substrate specificity compared with the human protein. Using a panel of GCPII inhibitors, we discovered that inhibition constants are generally similar for mouse and human GCPII. Furthermore, we observed highest expression of GCPII protein in the mouse kidney, brain, and salivary glands. Importantly, we did not detect GCPII in the mouse prostate. Our data suggest that the differences in enzymatic activity and inhibition profile are rather small; therefore, mouse GCPII can approximate human GCPII in drug development and testing. On the other hand, significant differences in GCPII tissue expression must be taken into account when developing novel GCPII-based anticancer and therapeutic methods, including targeted anticancer drug delivery systems, and when using mice as a model organism.
- Publikační typ
- časopisecké články MeSH
The effect of cyclosporin A (CsA) on imatinib treated Bcr-Abl positive K562 cells was studied. Similarly to other authors we found that imatinib induced apoptosis and erythroid differentiation in K562 cells. While its low concentrations induced predominantly erythroid differentiation, higher concentrations induced apoptosis. We found that CsA significantly potentiated cytotoxic effects of imatinib. A detailed analysis revealed that CsA shifted the balance between differentiation and apoptosis in favour of apoptosis. Our findings indicated that the observed effect of CsA was mediated neither through inhibition of ERK1/2 (extracellular signal-regulated kinases 1/2), nor through inhibition of p38 MAPK. We further observed that CsA might sensitise cells to apoptosis due to a changed cellular redox status as combined treatment of cells with imatinib and CsA resulted in a dramatic decrease of the ratio between reduced (GSH) and oxidised (GSSG) glutathione GSH/GSSG and in a significant suppression of thioredoxin reductase enzymatic activity. Our results indicated that K562 cells did not express detectable level of P-glycoprotein (P-gp). In addition, CsA did not affect significantly the intracellular level of imatinib. Therefore we excluded the possibility that CsA increased sensitivity of cells to imatinib by the inhibition of P-gp-mediated drug efflux or by another mechanism involving modulation of intracellular drug concentration.
- MeSH
- apoptóza účinky léků MeSH
- bcr-abl fúzní proteiny analýza MeSH
- buňky K562 MeSH
- cyklosporin farmakologie MeSH
- extracelulárním signálem regulované MAP kinasy antagonisté a inhibitory MeSH
- glutathion analýza MeSH
- glutathiondisulfid analýza MeSH
- imidazoly farmakologie MeSH
- lidé MeSH
- P-glykoprotein analýza fyziologie MeSH
- piperaziny farmakologie MeSH
- protinádorové látky farmakologie MeSH
- pyridiny farmakologie MeSH
- pyrimidiny farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH