Structural Maintenance of Chromosomes (SMC) complexes are important for many aspects of the chromosomal organization. Unlike cohesin and condensin, the SMC5/6 complex contains a variant RING domain carried by its Nse1 subunit. RING domains are characteristic for ubiquitin ligases, and human NSE1 has been shown to possess ubiquitin-ligase activity in vitro. However, other studies were unable to show such activity. Here, we confirm Nse1 ubiquitin-ligase activity using purified Schizosaccharomyces pombe proteins. We demonstrate that the Nse1 ligase activity is stimulated by Nse3 and Nse4. We show that Nse1 specifically utilizes Ubc13/Mms2 E2 enzyme and interacts directly with ubiquitin. We identify the Nse1 mutation (R188E) that specifically disrupts its E3 activity and demonstrate that the Nse1-dependent ubiquitination is particularly important under replication stress. Moreover, we determine Nse4 (lysine K181) as the first known SMC5/6-associated Nse1 substrate. Interestingly, abolition of Nse4 modification at K181 leads to suppression of DNA-damage sensitivity of other SMC5/6 mutants. Altogether, this study brings new evidence for Nse1 ubiquitin ligase activity, significantly advancing our understanding of this enigmatic SMC5/6 function.
- MeSH
- chromozomální proteiny, nehistonové metabolismus MeSH
- lidé MeSH
- ligasy metabolismus MeSH
- proteiny buněčného cyklu metabolismus MeSH
- Schizosaccharomyces pombe - proteiny metabolismus MeSH
- transportní proteiny metabolismus MeSH
- ubikvitin metabolismus MeSH
- ubikvitinace imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We adopted an approach based on peptide immobilized pH gradient-isoelectric focusing (IPG-IEF) separation, coupled with LC-MS/MS, in order to maximize coverage of the beer proteome. A lager beer brewed using traditional Czech technology was degassed, desalted and digested. Tryptic peptides were separated by isoelectric focusing on an immobilized pH gradient strip and, after separation, the gel strip was divided into seven equally sized parts. Peptides extracted from gel fractions were analyzed by LC-MS/MS. This approach resulted in a three-fold increase in the number of proteins identified (over 1700) when compared to analysis of unfractionated beer processed by a filter-aided sample preparation (FASP). Over 1900 protein groups (PGs) in total were identified by both approaches. SIGNIFICANCE: The study significantly extends knowledge about the beer proteome and demonstrates its complexity. Detailed knowledge of the protein content, especially gluten proteins, will enhance the evaluation of potential health risks related to beer consumption (coeliac disease) and will contribute to improving beer quality.
- MeSH
- chromatografie kapalinová MeSH
- gluteny analýza MeSH
- isoelektrická fokusace MeSH
- pivo analýza MeSH
- proteom analýza MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Československo MeSH
The incorporation of histone H3 with an acetylated lysine 56 (H3K56ac) into the nucleosome is important for chromatin remodeling and serves as a marker of new nucleosomes during DNA replication and repair in yeast. However, in human cells, the level of H3K56ac is greatly reduced, and its role during the cell cycle is controversial. Our aim was to determine the potential of H3K56ac to regulate cell cycle progression in different human cell lines. A significant increase in the number of H3K56ac foci, but not in H3K56ac protein levels, was observed during the S and G2 phases in cancer cell lines, but was not observed in embryonic stem cell lines. Despite this increase, the H3K56ac signal was not present in late replication chromatin, and H3K56ac protein levels did not decrease after the inhibition of DNA replication. H3K56ac was not tightly associated with the chromatin and was primarily localized to active chromatin regions. Our results support the role of H3K56ac in transcriptionally active chromatin areas but do not confirm H3K56ac as a marker of newly synthetized nucleosomes in DNA replication.
- MeSH
- buněčný cyklus genetika fyziologie MeSH
- chromatin metabolismus MeSH
- G2 fáze genetika MeSH
- histony metabolismus MeSH
- HL-60 buňky MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- nukleozomy metabolismus MeSH
- replikace DNA genetika fyziologie MeSH
- S fáze genetika MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The MAGE (Melanoma-associated antigen) protein family members are structurally related to each other by a MAGE-homology domain comprised of 2 winged helix motifs WH/A and WH/B. This family specifically evolved in placental mammals although single homologs designated NSE3 (non-SMC element) exist in most eukaryotes. NSE3, together with its partner proteins NSE1 and NSE4 form a tight subcomplex of the structural maintenance of chromosomes SMC5-6 complex. Previously, we showed that interactions of the WH/B motif of the MAGE proteins with their NSE4/EID partners are evolutionarily conserved (including the MAGEA1-NSE4 interaction). In contrast, the interaction of the WH/A motif of NSE3 with NSE1 diverged in the MAGE paralogs. We hypothesized that the MAGE paralogs acquired new RING-finger-containing partners through their evolution and form MAGE complexes reminiscent of NSE1-NSE3-NSE4 trimers. In this work, we employed the yeast 2-hybrid system to screen a human RING-finger protein library against several MAGE baits. We identified a number of potential MAGE-RING interactions and confirmed several of them (MDM4, PCGF6, RNF166, TRAF6, TRIM8, TRIM31, TRIM41) in co-immunoprecipitation experiments. Among these MAGE-RING pairs, we chose to examine MAGEA1-TRIM31 in detail and showed that both WH/A and WH/B motifs of MAGEA1 bind to the coiled-coil domain of TRIM31 and that MAGEA1 interaction stimulates TRIM31 ubiquitin-ligase activity. In addition, TRIM31 directly binds to NSE4, suggesting the existence of a TRIM31-MAGEA1-NSE4 complex reminiscent of the NSE1-NSE3-NSE4 trimer. These results suggest that MAGEA1 functions as a co-factor of TRIM31 ubiquitin-ligase and that the TRIM31-MAGEA1-NSE4 complex may have evolved from an ancestral NSE1-NSE3-NSE4 complex.
- MeSH
- biologické modely MeSH
- chromatografie kapalinová MeSH
- domény RING-prstů MeSH
- HEK293 buňky MeSH
- imunoprecipitace MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- multimerizace proteinu MeSH
- multiproteinové komplexy metabolismus MeSH
- nádorové proteiny chemie metabolismus MeSH
- peptidové fragmenty chemie metabolismus MeSH
- peptidy chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- tandemová hmotnostní spektrometrie MeSH
- techniky dvojhybridového systému MeSH
- transportní proteiny metabolismus MeSH
- ubikvitinligasy chemie metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The characterization of the immune response of chickens to Salmonella infection is usually limited to the quantification of expression of genes coding for cytokines, chemokines or antimicrobial peptides. However, processes occurring in the cecum of infected chickens are likely to be much more diverse. In this study we have therefore characterized the transcriptome and proteome in the chicken cecum after infection with Salmonella Enteritidis. Using a combination of 454 pyrosequencing, protein mass spectrometry and quantitative real-time PCR, we identified 48 down- and 56 up-regulated chicken genes after Salmonella Enteritidis infection. The most inducible gene was that coding for MMP7, exhibiting a 5952 fold induction 9 days post-infection. An induction of greater than 100 fold was observed for IgG, IRG1, SAA, ExFABP, IL-22, TRAP6, MRP126, IFNγ, iNOS, ES1, IL-1β, LYG2, IFIT5, IL-17, AVD, AH221 and SERPIN B. Since prostaglandin D2 synthase was upregulated and degrading hydroxyprostaglandin dehydrogenase was downregulated after the infection, prostaglandin must accumulate in the cecum of chickens infected with Salmonella Enteritidis. Finally, above mentioned signaling was dependent on the presence of a SPI1-encoded type III secretion system in Salmonella Enteritidis. The inflammation lasted for 2 weeks after which time the expression of the "inflammatory" genes returned back to basal levels and, instead, the expression of IgA and IgG increased. This points to an important role for immunoglobulins in the restoration of homeostasis in the cecum after infection.
- MeSH
- cékum imunologie metabolismus MeSH
- hmotnostní spektrometrie veterinární MeSH
- kur domácí * MeSH
- nemoci drůbeže genetika imunologie mikrobiologie MeSH
- nemoci úst genetika imunologie mikrobiologie veterinární MeSH
- northern blotting veterinární MeSH
- polymerázová řetězová reakce veterinární MeSH
- přirozená imunita * MeSH
- proteom imunologie MeSH
- ptačí proteiny genetika imunologie MeSH
- regulace genové exprese * MeSH
- Salmonella enteritidis imunologie MeSH
- salmonelová infekce u zvířat genetika imunologie mikrobiologie MeSH
- sekvenční analýza DNA veterinární MeSH
- transkriptom MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein or peptide sample losses could accompany all steps of the proteomic analysis workflow. We focused on suppression of sample adsorptive losses during sample storage in autosampler vials. We examined suppression capabilities of six different sample injection solutions and seven types of autosampler vial surfaces using a model sample (tryptic digest of six proteins, 1 fmol per protein). While the vial material did not play an essential role, the choice of appropriate composition of sample injection solution reduced adsorptive losses substantially. The combination of a polypropylene vial and solution of poly(ethylene glycol) (PEG) (0.001%) or a mixture of high concentrated urea and thiourea (6 M and 1 M) as injection solutions (both acidified with formic acid (FA) (0.1%)) provided the best results in terms of number of significantly identified peptides (p < 0.05). These conclusions were confirmed by analyses of a real sample with intermediate complexity (in-gel digest from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)). Addition of PEG into the real sample solution proved to prevent higher losses, concerning mainly hydrophobic peptides, during up to 48 h storage in the autosampler in comparison with a formic acid solution and even with a solution of highly concentrated urea and thiourea. Using PEG for several months was not accompanied by any adverse effect to the liquid chromatography system.
- MeSH
- adsorpce MeSH
- chromatografie kapalinová normy MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- formiáty MeSH
- hydrofobní a hydrofilní interakce MeSH
- močovina MeSH
- odběr biologického vzorku normy MeSH
- peptidové fragmenty izolace a purifikace normy MeSH
- polyethylenglykoly MeSH
- proteiny chemie MeSH
- průtoková injekční analýza normy MeSH
- thiomočovina MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mycobacterium avium subsp. avium (MAA) and Mycobacterium avium subsp. hominissuis (MAH) are the most common mycobacterial species isolated from granulomatous lesions in swine in countries with controlled bovine tuberculosis. This study is focused on the immunological aspect of MAA and MAH infection in pigs. We detected induction of humoral and cell-mediated immunity in experimentally infected pigs. Specific antibodies were analyzed in serum by ELISA and the IFN-γ release assay was used for evaluation of cell-mediated immunity. While MAA induced a significant increase of both types of immune responses, MAH-infected pigs had an unvarying level of specific antibodies and showed low cell-mediated immunity with high individual variability. The subsequent in vitro experiment confirmed the lower immunogenicity of the MAH strain in comparison to MAA. MAH-infected porcine monocyte-derived macrophages showed a weaker induction of pro-inflammatory mediators in comparison to MAA, which included mRNA for IL-1β, TNF-α, IL-23p19, IL-18 and chemokines CCL-3, CCL-5, CXCL-8 and CXCL-10. Additionally, qualitative proteomic analysis revealed 28 proteins exclusively in MAA and 7 proteins unique to MAH. In conclusion, closely related M. avium subspecies MAA and MAH showed different capacities to stimulate the porcine immune system. From a diagnostic point of view, the IFN-γ release assay showed higher sensitivity than the detection of specific antibodies by ELISA and seems to be an effective tool for discrimination of MAA-infected pigs. In the case of MAH infection, the IFN-γ release assay could fail because of the low immunogenic capacity of the MAH strain.
- MeSH
- buněčná imunita MeSH
- interleukin-18 genetika imunologie metabolismus MeSH
- interleukin-23 - podjednotka p19 genetika imunologie MeSH
- makrofágy imunologie MeSH
- mediátory zánětu imunologie MeSH
- Mycobacterium avium klasifikace izolace a purifikace fyziologie MeSH
- nemoci prasat genetika imunologie mikrobiologie MeSH
- prasata MeSH
- TNF-alfa genetika imunologie MeSH
- tuberkulóza imunologie veterinární MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cryptogein is a proteinaceous elicitor secreted by Phytophthora cryptogea that can induce resistance to P. parasitica in tobacco plants. On the basis of previous computer modelling experiments, by site-directed mutagenesis a series of cryptogein variants was prepared with altered abilities to bind sterols, phospholipids or both. The sterol binding and phospholipid transfer activities corresponded well with the previously reported structural data. Induction of the synthesis of reactive oxygen species (ROS) in tobacco cells in suspension and proteomic analysis of intercellular fluid changes in tobacco leaves triggered by these mutant proteins were not proportional to their ability to bind or transfer sterols and phospholipids. However, changes in the intercellular proteome corresponded to transcription levels of defence genes and resistance to P. parasitica and structure-prediction of mutants did not reveal any significant changes in protein structure. These results suggest, contrary to previous proposals, that the sterol-binding ability of cryptogein and its mutants, and the associated conformational change in the ω-loop, might not be principal factors in either ROS production or resistance induction. Nevertheless, the results support the importance of the ω-loop for the interaction of the protein with the high affinity binding site on the plasma membrane.
- MeSH
- fosfolipidy metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- imunita rostlin imunologie MeSH
- kyselina chlorogenová analýza MeSH
- listy rostlin genetika imunologie parazitologie fyziologie MeSH
- mutace MeSH
- nemoci rostlin imunologie parazitologie MeSH
- Phytophthora genetika metabolismus patogenita MeSH
- proteiny genetika metabolismus MeSH
- proteomika metody MeSH
- reaktivní formy kyslíku metabolismus MeSH
- rekombinantní proteiny MeSH
- seskviterpeny analýza MeSH
- steroly metabolismus MeSH
- tabák genetika imunologie parazitologie fyziologie MeSH
- vazba proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Východiska: Termín biomarker se v posledních letech stal, zvláště pak ve spojení s termínem klinická proteomika, jedním z nejfrekventovanějších pojmů v oblasti biomedicínského výzkumu. Předmětem této práce byl výběr vhodné metody frakcionace krevní plazmy umožňující oddělení majoritních proteinů od nízkoabundantní proteinové frakce před následnou proteomickou analýzou pomocí dvourozměrné gelové elektroforézy (2-DE) s cílem zlepšit rozlišení 2-DE mapy a identifikaci proteinů hmotnostní spektrometrií. Materiál a metody: Nejprve bylo provedeno paralelní srovnání dvou metod frakcionace krevní plazmy (depleční kolona MARS vs ProteoMiner) na souboru 10 vzorků. Na základě výsledků srovnávacích experimentů byla k analýze vzorků 18 pacientů nemocných mnohočetným myelomem a léčených režimy s bortezomibem použita frakcionační metoda ProteoMiner v kombinaci s 2-DE. Soubor pacientů byl rozdělen na dvě skupiny: skupina pacientů rezistentních na chemoterapii (9 pacientů – progrese onemocnění, stabilizace onemocnění) a skupina pacientů s pozitivní léčebnou odpovědí (9 pacientů – kompletní a parciální remise). Výsledky a závěr: Metoda ProteoMiner umožnila na 2-DE mapách signifikantně zvýšit počet proteinových spotů ve srovnání s imunodepleční metodou MARS (Multiple Affinity Removal System). Mezi skupinami pacientů chemorezistentních a pacientů citlivých na léčbu bortezomibem bylo pomocí analýzy obrazu 2-DE gelů zjištěno 15 rozdílových proteinových spotů, které byly analyzovány hmotnostní spektrometrií. V těchto spotech bylo identifikováno 10 proteinů. Sedm proteinů vykazovalo signifikantně nižší hladinu proteinu ve skupině chemosenzitivních pacientů (sérový amyloid P, fibrinogen – gama řetězec, retinol-vazebný protein 4, komplement faktor C4-A, apolipoprotein E, karboxypeptidáza N, komplement faktoru H – příbuzný protein 1) a 3 proteiny vykazovaly signifikantně vyšší hladinu proteinu nebo byly detekovány pouze ve skupině chemosenzitivních pacientů (sérová paraoxonáza 1, alfa-1-antitrypsin a komplement faktor B).
Backgrounds: Recently, the term biomarker has become, especially in connection with the term clinical proteomics, one of the most frequent terms in the field of biomedical research. The aim of this work was to select an appropriate pre-fractionation method of blood plasma prior to a subsequent proteomic analysis of low-abundant fraction of proteins by two dimensional gel electrophoresis (2-DE) and mass spectrometry to improve the resolution of 2-DE maps and protein identification. Materials and Methods: First, we compared two prefractionation methods (MARS versus ProteoMiner) preceding 2-DE analysis using 10 blood plasma samples. Based on the results of the comparative experiments, low-abundant plasma protein fractions from 18 multiple myeloma patients treated with bortezomib were analyzed. Patients were divided into two groups: a group resistant to chemotherapy (9 patients – disease progression, stable disease) and a group with positive clinical response (9 patients – complete and partial remission). Results and Conclusion: Samples prefractioned by ProteoMiner method yielded 2-DE maps with a significantly increased number of detected protein spots, as compared to immunodepletion method MARS (Multiple Affinity Removal System). Between groups of chemoresistant and sensitive patients treated with bortezomib, 15 differently intense spots were revealed by image analysis. These spots were found to correspond to 10 proteins, as confirmed by mass spectrometry. Seven proteins had significantly lower protein level in the group of chemosensitive patients (serum amyloid P, fibrinogen – gamma chain, retinol-binding protein 4, complement factor C4-A, apolipoprotein E, carboxypeptidase N and complement factor H-related protein 1) and 3 proteins showed significantly higher levels of protein (or were only detected) in the group of chemosensitive patients (serum paraoxonase 1, alpha-1-antitrypsin and complement factor B).
- Klíčová slova
- elektroforéza gelová dvourozměrná, protein,
- MeSH
- 2D gelová elektroforéza metody využití MeSH
- biomedicínský výzkum MeSH
- bortezomib MeSH
- farmakoterapie MeSH
- financování organizované MeSH
- krevní plazma účinky léků MeSH
- krevní proteiny izolace a purifikace účinky léků MeSH
- kyseliny boronové aplikace a dávkování škodlivé účinky terapeutické užití MeSH
- lidé MeSH
- mnohočetný myelom farmakoterapie krev MeSH
- nádorové biomarkery krev MeSH
- proteomika metody MeSH
- pyraziny aplikace a dávkování škodlivé účinky terapeutické užití MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody využití MeSH
- statistika jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
