BACKGROUND: An impairment of the 5-fluorouracil (5-FU) catabolic pathway, represented by alterations in the dihydropyrimidine dehydrogenase (DPYD) gene, is considered a crucial factor contributing to the development of 5-FU-related toxicity. The β-ureidopropionase (BUP1) enzyme catalyzes the final step in the 5-FU catabolic pathway; however, alterations in the UPB1 gene coding for the BUP1 enzyme have not yet been analyzed in fluoropyrimidine (FP)-treated patients suffering from 5-FU-related toxicity. METHODS: We have performed a mutation analysis of the entire coding sequence of UPB1 based on denaturing high-performance liquid chromatography in 113 cancer patients treated by FP-containing regimes. These patients included 67 individuals suffering from severe 5-FU-related toxicity and 46 individuals with excellent tolerance of chemotherapy. RESULTS: Nine UPB1 variants were detected in the subpopulation of patients with severe toxicity, including a novel mutation affecting the coding sequence (c.872_873+11del13). An analysis of UPB1 variants on 5-FU-related toxicity in the population of all analyzed patients revealed an association between the c.-80C>G (rs2070474) variant and gastrointestinal toxicity. A strong positive correlation was found between the carriers of the c.-80 GG genotype and the development of severe (grade 3-4) mucositis (OR = 7.5; 95% CI = 2.60 - 21.60; p = 0.0002). CONCLUSION: Our results suggest that UPB1 variants may contribute to the development of 5-FU-related toxicity in some FP-treated patients; however, the role of UPB1 alterations is probably less significant than that of DPYD alterations.

• MeSH
• DNA vazebné proteiny genetika   MeSH
• dospělí MeSH
• fluoruracil škodlivé účinky   MeSH
• genotyp MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace * MeSH
• protinádorové antimetabolity škodlivé účinky   MeSH
• senioři MeSH
• transkripční faktory genetika   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

Human cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.

• MeSH
• cytomegalovirové infekce genetika   imunologie   virologie   MeSH
• Cytomegalovirus genetika   imunologie   fyziologie   MeSH
• DNA vazebné proteiny chemie   genetika   imunologie   MeSH
• imunitní únik MeSH
• lidé MeSH
• proteiny chemie   genetika   imunologie   MeSH
• proteomika MeSH
• stabilita proteinů MeSH
• transkripční faktory chemie   genetika   imunologie   MeSH
• virové proteiny chemie   genetika   imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The flavoprotein WrbA from Escherichia coli is considered to be the prototype of a new family of multimeric flavodoxin-like proteins that are implicated in cell protection against oxidative stress. The present study is aimed at structural characterization of the E. coli protein with respect to its recently revealed oxidoreductase activity. Crystals of WrbA holoprotein in complex with the oxidized flavin cofactor (FMN) were obtained using standard vapour-diffusion techniques. Deep yellow tetragonal crystals obtained from differing crystallization conditions display different space groups and unit-cell parameters. X-ray crystal structures of the WrbA holoprotein have been determined to resolutions of 2.0 and 2.6 A.

• MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• Escherichia coli metabolismus   MeSH
• financování organizované MeSH
• flavinmononukleotid chemie   metabolismus   MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• proteiny z Escherichia coli chemie   metabolismus   MeSH
• represorové proteiny chemie   metabolismus   MeSH

The TRPS1 protein is a potent regulator of proliferation, differentiation, and apoptosis. The TRPS1 gene aberrations are strongly associated with rare trichorhinophalangeal syndrome (TRPS) development. We have conducted MLPA analysis to capture deletion within the crucial 8q24.1 chromosomal region in combination with mutation analysis of TRPS1 gene including core promoter, 5'UTR, and 3'UTR sequences in nine TRPS patients. Low complexity or extent of untranslated regulatory sequences avoided them from analysis in previous studies. Amplicon based next generation sequencing used in our study bridge over these technical limitations. Finally, we have made extended in silico analysis of TRPS1 gene regulatory sequences organization. Single contiguous deletion and an intragenic deletion intervening several exons were detected. Mutation analysis revealed five TRPS1 gene aberrations (two structural rearrangements, two nonsense mutations, and one missense substitution) reaching the overall detection rate of 78%. Several polymorphic variants were detected within the analysed regulatory sequences but without proposed pathogenic effect. In silico analysis suggested alternative promoter usage and diverse expression effectivity for different TRPS1 transcripts. Haploinsufficiency of TRPS1 gene was responsible for most of the TRPS phenotype. Structure of TRPS1 gene regulatory sequences is indicative of generally low single allele expression and its tight control.

• MeSH
• 3' nepřekládaná oblast genetika   MeSH
• 5' nepřekládaná oblast genetika   MeSH
• dítě MeSH
• DNA vazebné proteiny chemie   genetika   MeSH
• dospělí MeSH
• haploinsuficience MeSH
• Langerův-Giedionův syndrom genetika   MeSH
• lidé MeSH
• mladý dospělý MeSH
• mutační analýza DNA * MeSH
• předškolní dítě MeSH
• promotorové oblasti (genetika) genetika   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• transkripční faktory chemie   genetika   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

DNMT1 is the maintenance DNA methyltransferase shown to be essential for embryonic development and cellular growth and differentiation in many somatic tissues in mammals. Increasing evidence has also suggested a role for DNMT1 in repressing gene expression through interactions with specific transcription factors. Previously, we identified DNMT1 as an interacting partner of the TR2/TR4 nuclear receptor heterodimer in erythroid cells, implicated in the developmental silencing of fetal β-type globin genes in the adult stage of human erythropoiesis. Here, we extended this work by using a biotinylation tagging approach to characterize DNMT1 protein complexes in mouse erythroleukemic cells. We identified novel DNMT1 interactions with several hematopoietic transcription factors with essential roles in erythroid differentiation, including GATA1, GFI-1b and FOG-1. We provide evidence for DNMT1 forming distinct protein subcomplexes with specific transcription factors and propose the existence of a "core" DNMT1 complex with the transcription factors ZBP-89 and ZNF143, which is also present in non-hematopoietic cells. Furthermore, we identified the short (17a.a.) PCNA Binding Domain (PBD) located near the N-terminus of DNMT1 as being necessary for mediating interactions with the transcription factors described herein. Lastly, we provide evidence for DNMT1 serving as a co-repressor of ZBP-89 and GATA1 acting through upstream regulatory elements of the PU.1 and GATA1 gene loci.

• MeSH
• buněčná diferenciace genetika   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• DNA-(cytosin-5-)methyltransferasa genetika   metabolismus   MeSH
• DNA-(cytosin-5)-methyltransferasa 1 MeSH
• erytroidní buňky chemie   metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• metylace DNA genetika   MeSH
• multiproteinové komplexy genetika   metabolismus   MeSH
• myši MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• regulace genové exprese genetika   MeSH
• represorové proteiny genetika   metabolismus   MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• transkripční faktor GATA1 genetika   metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation. It was followed by a necrosis-like cell death [K. Kuzelová, D. Grebenová, M. Pluskalová, I. Marinov, Z. Hrkal, J. Photochem. Photobiol. B 73 (2004) 67-78]. ALA-PDT led to the perturbation of the Hsp90/p23 multichaperone complex of which the Bcr-Abl is the client protein. Bcr-Abl protein was suppressed whereas the bcr-abl mRNA level was not affected. Further on, we observed several changes in the cytoskeleton organization. We detected ALA-PDT-mediated disruption of filamental actin structure using FITC-Phalloidin staining. In connection with this we uncovered certain cytoskeleton organizing proteins involved in the cell response to the treatment. Among these proteins, Septin2, which plays a role in maintaining actin bundles, was suppressed. Another one, PDZ-LIM domain protein 1 (CLP36) was altered. This protein acts as an adaptor molecule for LIM-kinase which phosphorylates and thus inactivates cofilin. Cofilin was indeed dephosphorylated and could thus be activated and operate as an actin-depolymerizing factor. We propose the scheme of molecular response of K562 cells to ALA-PDT.

• MeSH
• buněčná smrt účinky léků   účinky záření   MeSH
• buňky K562 patologie   účinky léků   účinky záření   MeSH
• časové faktory MeSH
• cytoskelet patologie   účinky léků   účinky záření   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• faloidin chemie   MeSH
• financování organizované MeSH
• fluorescein-5-isothiokyanát MeSH
• fotosenzibilizující látky farmakologie   MeSH
• kyselina aminolevulová farmakologie   MeSH
• lidé MeSH
• Lim-kinasy MeSH
• messenger RNA metabolismus   MeSH
• mikrofilamentové proteiny metabolismus   MeSH
• molekulární chaperony metabolismus   MeSH
• onkogenní proteiny metabolismus   MeSH
• proteinkinasy fyziologie   MeSH
• proteiny tepelného šoku HSP90 metabolismus   MeSH
• regulace genové exprese MeSH
• světlo MeSH
• transkripční faktory MeSH
• transportní proteiny metabolismus   MeSH
• tyrosinkinasy metabolismus   účinky léků   účinky záření   MeSH
• Check Tag
• lidé MeSH

OBJECTIVES: The acute respiratory syndrome, known as COVID-19, is characterised by high morbidity and increased mortality. Genetic factors may partially explain the differences in susceptibility to and severity of COVID-19. METHODS: We have analysed common functional polymorphisms within the OAS1 (rs4767027), TMPRSS6 (rs855791), DPP4 (rs3788979), and ZNF335 (rs3848719) genes in SARS-CoV-2 positive subjects (n = 521, different disease severity) and in population controls (n = 2,559 subjects, COVID-19 status unknown). RESULTS: Neither DPP4 nor ZNF335 were associated with disease susceptibility or severity in the Czech population in any of the models used for calculation. T allele carriers of the OAS1 polymorphism seem to be protective against symptomatic COVID-19 (p = 0.002 calculated for trend; asymptomatic, symptomatic, hospitalised). Similarly, within the TMPRSS6, minor TT homozygotes associated with lower plasma Fe concentrations were underrepresented in the overall patient group (p = 0.044; OR = 0.77, 95% CI: 0.59-0.99), and the difference was mainly driven by the severe COVID-19 subjects. In general, risky homozygotes of these two polymorphisms were less frequent than expected in the group of hospitalised COVID-19 survivors. CONCLUSIONS: Common variants within OAS1 (rs4767027) and TMPRSS6 (rs855791) play some role in COVID-19 pathology in the Czech Caucasian population. Whether the depletion of minor allele carriers of these two variants is associated with increased COVID-19 mortality, needs to be analysed in an external confirmatory study.

• MeSH
• 2',5'-oligoadenylátsynthetasa MeSH
• COVID-19 * genetika   MeSH
• dipeptidylpeptidasa 4 MeSH
• DNA vazebné proteiny MeSH
• jednonukleotidový polymorfismus MeSH
• lidé MeSH
• membránové proteiny MeSH
• SARS-CoV-2 MeSH
• serinové endopeptidasy genetika   MeSH
• transkripční faktory MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH

Reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream uORFs to retain post-termination 40S subunits on mRNA. Its efficiency depends on surrounding cis-acting sequences, uORF elongation rates, various initiation factors, and the intercistronic distance. To unravel effects of cis-acting sequences, we investigated previously unconsidered structural properties of one such a cis-enhancer in the mRNA leader of GCN4 using yeast genetics and biochemistry. This leader contains four uORFs but only uORF1, flanked by two transferrable 5' and 3' cis-acting sequences, and allows efficient reinitiation. Recently we showed that the 5' cis-acting sequences stimulate reinitiation by interacting with the N-terminal domain (NTD) of the eIF3a/TIF32 subunit of the initiation factor eIF3 to stabilize post-termination 40S subunits on uORF1 to resume scanning downstream. Here we identify four discernible reinitiation-promoting elements (RPEs) within the 5' sequences making up the 5' enhancer. Genetic epistasis experiments revealed that two of these RPEs operate in the eIF3a/TIF32-dependent manner. Likewise, two separate regions in the eIF3a/TIF32-NTD were identified that stimulate reinitiation in concert with the 5' enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 5' enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 5' enhancer's stimulatory activity is strictly dependent on and thus follows the 3' enhancer's activity. These findings allow us to propose for the first time a model of events required for efficient post-termination resumption of scanning. Strikingly, structurally similar RPE was predicted and identified also in the 5' leader of reinitiation-permissive uORF of yeast YAP1. The fact that it likewise operates in the eIF3a/TIF32-dependent manner strongly suggests that at least in yeasts the underlying mechanism of reinitiation on short uORFs is conserved.

• MeSH
• 5' nepřekládaná oblast MeSH
• 5' přiléhající oblast DNA MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• malé podjednotky ribozomu eukaryotické genetika   metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• otevřené čtecí rámce genetika   MeSH
• regulační oblasti nukleových kyselin MeSH
• ribozomální proteiny genetika   metabolismus   MeSH
• ribozomy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• transkripční faktory bZIP genetika   metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zesilovače transkripce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Reduced nitric oxide (NO)/cGMP signalling is observed in age-related vascular disease. We hypothesize that this disturbed signalling involves effects of genomic instability, a primary causal factor in aging, on vascular smooth muscle cells (VSMCs) and that the underlying mechanism plays a role in human age-related vascular disease. To test our hypothesis, we combined experiments in mice with genomic instability resulting from the defective nucleotide excision repair gene ERCC1 (Ercc1(d/-) mice), human VSMC cultures and population genome-wide association studies (GWAS). Aortic rings of Ercc1(d/-) mice showed 43% reduced responses to the soluble guanylate cyclase (sGC) stimulator sodium nitroprusside (SNP). Inhibition of phosphodiesterase (PDE) 1 and 5 normalized SNP-relaxing effects in Ercc1(d/-) to wild-type (WT) levels. PDE1C levels were increased in lung and aorta. cGMP hydrolysis by PDE in lungs was higher in Ercc1(d/-) mice. No differences in activity or levels of cGMP-dependent protein kinase 1 or sGC were observed in Ercc1(d/-) mice compared with WT. Senescent human VSMC showed elevated PDE1A and PDE1C and PDE5 mRNA levels (11.6-, 9- and 2.3-fold respectively), which associated with markers of cellular senescence. Conversely, PDE1 inhibition lowered expression of these markers. Human genetic studies revealed significant associations of PDE1A single nucleotide polymorphisms with diastolic blood pressure (DBP; β=0.28, P=2.47×10(-5)) and carotid intima-media thickness (cIMT; β=-0.0061, P=2.89×10(-5)). In summary, these results show that genomic instability and cellular senescence in VSMCs increase PDE1 expression. This might play a role in aging-related loss of vasodilator function, VSMC senescence, increased blood pressure and vascular hypertrophy.

• MeSH
• arteriae carotides enzymologie   patologie   MeSH
• celogenomová asociační studie MeSH
• cyklické nukleotidfosfodiesterasy, typ 1 antagonisté a inhibitory   genetika   metabolismus   MeSH
• cyklické nukleotidfosfodiesterasy, typ 5 genetika   metabolismus   MeSH
• DNA vazebné proteiny nedostatek   genetika   MeSH
• endonukleasy nedostatek   genetika   MeSH
• fenotyp MeSH
• genetická predispozice k nemoci MeSH
• guanosinmonofosfát cyklický metabolismus   MeSH
• hydrolýza MeSH
• hyperplazie MeSH
• hypertenze enzymologie   genetika   patofyziologie   MeSH
• inhibitory fosfodiesterasy 5 farmakologie   MeSH
• intimomediální šíře tepenné stěny MeSH
• jednonukleotidový polymorfismus MeSH
• krevní tlak MeSH
• kultivované buňky MeSH
• lidé MeSH
• myocyty hladké svaloviny účinky léků   enzymologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• nemoci arterie carotis enzymologie   genetika   patologie   MeSH
• regulace genové exprese u nádorů MeSH
• stárnutí buněk MeSH
• stárnutí genetika   metabolismus   MeSH
• svaly hladké cévní účinky léků   enzymologie   MeSH
• systémy druhého messengeru MeSH
• techniky in vitro MeSH
• vazodilatace * účinky léků   MeSH
• vazodilatancia farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mutations in the 5'-regulatory region of BCL6 were suggested to play a role in non-Hodgkin's lymphoma (NHL) progression and in the transformation of follicular lymphoma to more aggressive diffuse large B-cell type. The aim of this study was to explore association between polymorphism G397C in the 5'-region of BCL6 and both incidence and progression of NHL in 154 NHL cases and 362 controls. Neither frequencies of the rare BCL6 allele 397C nor particular genotypes differed significantly between NHL cases and controls. There was no significant association of histological type of NHL and clinical characteristics with this polymorphism.

• MeSH
• 5' přiléhající oblast DNA genetika   MeSH
• DNA vazebné proteiny genetika   MeSH
• financování organizované MeSH
• jednonukleotidový polymorfismus MeSH
• lidé MeSH
• nehodgkinský lymfom diagnóza   genetika   MeSH
• prognóza MeSH
• riziko MeSH
• rizikové faktory MeSH
• Check Tag
• lidé MeSH