Luciferase reporter assays represent a simple and sensitive experimental system in cell and molecular biology to study multiple biological processes. However, the application of these assays is often limited by the costs of conventional luminometer instruments and the versatility of their use in different experimental conditions. Therefore, we aimed to develop a small, affordable luminometer allowing continuous measurement of luciferase activity, designed for inclusion into various kinds of tissue culture incubators. Here, we introduce LuminoCell-an open-source platform for the construction of an affordable, sensitive, and portable luminometer capable of real-time monitoring in-cell luciferase activity. The LuminoCell costs $40, requires less than 1 h to assemble, and it is capable of performing real-time sensitive detection of both magnitude and duration of the activity of major signalling pathways in cell cultures, including receptor tyrosine kinases (EGF and FGF), WNT/β-catenin, and NF-κB. In addition, we show that the LuminoCell is suitable to be used in cytotoxicity assays as well as for monitoring periodic circadian gene expression.
Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.
- MeSH
- buňky NIH 3T3 MeSH
- katalýza MeSH
- kinetika MeSH
- konformace proteinů MeSH
- luciferasy renil chemie genetika metabolismus MeSH
- luciferasy chemie genetika metabolismus MeSH
- mutace MeSH
- mutageneze MeSH
- myši MeSH
- proteinové inženýrství * MeSH
- savci MeSH
- simulace molekulární dynamiky * MeSH
- stabilita enzymů MeSH
- teplota MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- aktivace enzymů účinky léků MeSH
- antivirové látky * analýza farmakologie MeSH
- Cercopithecus aethiops MeSH
- Gammaherpesvirinae * enzymologie genetika MeSH
- inhibiční koncentrace 50 MeSH
- luciferasy metabolismus MeSH
- myši MeSH
- preklinické hodnocení léčiv * metody MeSH
- replikace viru účinky léků MeSH
- Vero buňky MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Bisphenol S (BPS) is heat-stable structural analog of bisphenol A (BPA), a known endocrine disruptor. Due to the effort to replace BPA with BPS, it is essential to know if BPS is suitable non-toxic replacement without reported deleterious effects of BPA. Since most of the BPA effects are ascribed to its ability to activate nuclear receptors, we screened some prominent members of this family in order to confirm or refute some recent findings. We found that BPS insignificantly activated aryl hydrocarbon receptor (AhR) in reporter gene assay and no induction of AhR target gene CYP1A1 was observed in human hepatocytes (HH). BPS was able to act like an antagonist of pregnane X receptor (PXR) in reporter gene assay, but the expression of PXR target gene CYP3A4, was only moderately affected in HH. While BPS antagonized dexamethasone-inducible glucocorticoid receptor (GR)-dependent luciferase activity in reporter gene assay (IC50=52μM), it was not able to antagonize dexamethasone effects on GR-target genes, including GILZ, NFKBIA and IL-6. Synergistic effect of BPS (range 0.001-100μM) and DHT (100nM) was observed at androgen receptor (AR) activity level only. In conclusion, we show that BPS had only limited effect on tested nuclear receptors. Moreover, submicromolar concentrations of BPS affected activated AR. Thus, due to the low levels of exposure for humans, BPS is probably of no regulatory concern. However, further investigation should delineate possible impact on male/female development or sexual functions.
- MeSH
- cytochrom P-450 CYP1A1 genetika metabolismus MeSH
- cytochrom P-450 CYP3A genetika metabolismus MeSH
- dexamethason toxicita MeSH
- dihydrotestosteron toxicita MeSH
- endokrinní disruptory toxicita MeSH
- fenoly toxicita MeSH
- genetická transkripce MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- hepatocyty účinky léků metabolismus MeSH
- interleukin-6 genetika metabolismus MeSH
- lidé MeSH
- luciferasy genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- NFKB inhibitor alfa genetika metabolismus MeSH
- proliferace buněk účinky léků MeSH
- receptory aromatických uhlovodíků genetika metabolismus MeSH
- receptory cytoplazmatické a nukleární genetika metabolismus MeSH
- receptory glukokortikoidů genetika metabolismus MeSH
- reportérové geny MeSH
- steroidní receptory antagonisté a inhibitory genetika metabolismus MeSH
- sulfony toxicita MeSH
- transkripční faktory genetika metabolismus MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Pregnane X receptor (PXR) is a major transcriptional regulator of xenobiotic metabolism and transport pathways in the liver and intestines, which are critical for protecting organisms against potentially harmful xenobiotic and endobiotic compounds. Inadvertent activation of drug metabolism pathways through PXR is known to contribute to drug resistance, adverse drug-drug interactions, and drug toxicity in humans. In both humans and rodents, PXR has been implicated in non-alcoholic fatty liver disease, diabetes, obesity, inflammatory bowel disease, and cancer. Because of PXR's important functions, it has been a therapeutic target of interest for a long time. More recent mechanistic studies have shown that PXR is modulated by multiple PTMs. Herein we provide the first investigation of the role of acetylation in modulating PXR activity. Through LC-MS/MS analysis, we identified lysine 109 (K109) in the hinge as PXR's major acetylation site. Using various biochemical and cell-based assays, we show that PXR's acetylation status and transcriptional activity are modulated by E1A binding protein (p300) and sirtuin 1 (SIRT1). Based on analysis of acetylation site mutants, we found that acetylation at K109 represses PXR transcriptional activity. The mechanism involves loss of RXRα dimerization and reduced binding to cognate DNA response elements. This mechanism may represent a promising therapeutic target using modulators of PXR acetylation levels. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.
- MeSH
- acetylace MeSH
- aktivace transkripce * MeSH
- buňky Hep G2 MeSH
- DNA chemie metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- klonování DNA MeSH
- lidé MeSH
- luciferasy genetika metabolismus MeSH
- lysin chemie metabolismus MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- mutageneze cílená MeSH
- posttranslační úpravy proteinů * MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- reportérové geny MeSH
- responzivní elementy MeSH
- sekundární struktura proteinů MeSH
- sirtuin 1 genetika metabolismus MeSH
- steroidní receptory chemie genetika metabolismus MeSH
- strukturní homologie proteinů MeSH
- transkripční faktory p300-CBP genetika metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Mechanisms controlling the metabotropic γ-aminobutyric acid receptor (GABAB) cell surface stability are still poorly understood. In contrast with many other G protein-coupled receptors (GPCR), it is not subject to agonist-promoted internalization, but is constitutively internalized and rapidly down-regulated. In search of novel interacting proteins regulating receptor fate, we report that the ubiquitin-specific protease 14 (USP14) interacts with the GABAB(1b)subunit's second intracellular loop. Probing the receptor for ubiquitination using bioluminescence resonance energy transfer (BRET), we detected a constitutive and phorbol 12-myristate 13-acetate (PMA)-induced ubiquitination of the receptor at the cell surface. PMA also increased internalization and accelerated receptor degradation. Overexpression of USP14 decreased ubiquitination while treatment with a small molecule inhibitor of the deubiquitinase (IU1) increased receptor ubiquitination. Treatment with the internalization inhibitor Dynasore blunted both USP14 and IU1 effects on the receptor ubiquitination state, suggesting a post-endocytic site of action. Overexpression of USP14 also led to an accelerated degradation of GABABin a catalytically independent fashion. We thus propose a model whereby cell surface ubiquitination precedes endocytosis, after which USP14 acts as an ubiquitin-binding protein that targets the ubiquitinated receptor to lysosomal degradation and promotes its deubiquitination.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- buněčná membrána účinky léků metabolismus MeSH
- endocytóza účinky léků MeSH
- HEK293 buňky MeSH
- hydrazony farmakologie MeSH
- lidé MeSH
- luciferasy genetika metabolismus MeSH
- luminescentní proteiny genetika metabolismus MeSH
- lyzozomy metabolismus MeSH
- molekulární sekvence - údaje MeSH
- posttranslační úpravy proteinů * MeSH
- proteinkinasa C genetika metabolismus MeSH
- proteolýza MeSH
- receptory GABA-B genetika metabolismus MeSH
- reportérové geny MeSH
- sekvence aminokyselin MeSH
- signální transdukce MeSH
- tetradekanoylforbolacetát farmakologie MeSH
- thiolesterasa ubikvitinu genetika metabolismus MeSH
- ubikvitin genetika metabolismus MeSH
- ubikvitinace MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Williams-Beuren syndrome-associated transcription factor TFII-I plays a critical regulatory role in bone and neural tissue development and in immunity, in part by regulating cell proliferation in response to mitogens. Mdm2, a cellular oncogene responsible for the loss of p53 tumor suppressor activity in a significant proportion of human cancers, was identified in this study as a new binding partner for TFII-I and a negative regulator of TFII-I-mediated transcription. These findings suggest a new p53-independent mechanism by which increased Mdm2 levels found in human tumors could influence cancer cells. In addition to that, we present data indicating that TFII-I is an important cellular regulator of transcription from the immediate-early promoter of human cytomegalovirus, a promoter sequence frequently used in mammalian expression vectors, including vectors for gene therapy. Our observation that Mdm2 over-expression can decrease the ability of TFII-I to activate the CMV promoter might have implications for the efficiency of experimental gene therapy based on CMV promoter-derived vectors in cancers with Mdm2 gene amplification.
- MeSH
- beta-galaktosidasa genetika metabolismus MeSH
- Cytomegalovirus genetika metabolismus MeSH
- genetická transkripce MeSH
- HEK293 buňky MeSH
- lidé MeSH
- luciferasy genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- proliferace buněk MeSH
- promotorové oblasti (genetika) MeSH
- protoonkogenní proteiny c-mdm2 genetika metabolismus MeSH
- regulace genové exprese u nádorů * MeSH
- reportérové geny MeSH
- signální transdukce MeSH
- transkripční faktory TFII genetika metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plant-derived smoke and certain smoke compounds improve seed germination and enhance seedling growth of many species. Thus, smoke-infused water and the active smoke-derived compounds have the potential to be used in different agricultural and horticultural applications. However, despite these interesting and potentially practical properties, it should also be ascertained whether such compounds may pose a health risk, particularly if they are to be used in the production of food or fodder crops. Amongst some of the aspects that would be important to understand are any possible genotoxic properties that the compounds may possess due to potential carry-over effects. Here, we report on a genotoxicity study of 3,4,5-trimethylfuran-2(5H)-one, a compound from plant-derived smoke previously shown to have germination inhibitory activity. Using two in vitro tests, namely the bacterial VITOTOX® test (with/without S9 metabolic activation) and the cytome assay on human C3A cells, no genotoxicity or toxicity was found. Furthermore, these results support a previous study where a related smoke-derived compound with germination promoting properties was investigated.
- MeSH
- 4-nitrochinolin-1-oxid toxicita MeSH
- benzopyreny toxicita MeSH
- chinolony toxicita MeSH
- furany farmakologie MeSH
- hepatocyty cytologie účinky léků metabolismus MeSH
- klíčení účinky léků MeSH
- lidé MeSH
- luciferasy genetika metabolismus MeSH
- mutageny farmakologie MeSH
- nádorové buněčné linie MeSH
- reportérové geny MeSH
- rostliny účinky léků MeSH
- Salmonella typhimurium účinky léků genetika růst a vývoj MeSH
- semena rostlinná účinky léků růst a vývoj MeSH
- SOS odpověď (genetika) účinky léků MeSH
- testy genotoxicity MeSH
- transkripční faktory genetika metabolismus MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this paper we investigated the effects of several drugs used in transplant medicine, i.e. cyclosporine A, tacrolimus, rapamycin, everolimus, mycophenolate mofetil, fluvastatin and rosuvastatin, on the expression of major drug-metabolizing enzymes in human hepatocytes. Moreover, we tested the ability of these drugs to affect transcriptional activity of glucocorticoid (GR) and aryl hydrocarbon receptor (AhR). We found that most of tested compounds did not induce expression of CYP1A1/1A2/3A4/2A6/2B6/2C9 mRNAs in human hepatocytes. Slight induction was observed for CYP2A6/2C9 mRNAs and CYP2A6 protein in the rapamycin-treated hepatocytes. Decrease of CYP2A6 and CYP2B6 proteins was observed in rosuvastatin-treated cells. Mycophenolate mofetil antagonized the effects of dexamethasone on GR but it potentiated the action of dioxin on AhR. Induction of CYP1A1 mRNA in HepG2 cells by dioxin was modestly antagonized by mycophenolate mofetil, while the induction by benzo[a]pyren or S-omeprazole was significantly potentiated by this drug. In general, tested compounds can be considered safe in the terms of possible drug-drug interaction caused by induction of drug-metabolizing cytochromes P450. Nevertheless, mycophenolate mofetil is of possible concern and its combination with drugs, environmental pollutants or food constituents, which activate AhR, may represent a significant toxicological risk.
- MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- dexamethason farmakologie MeSH
- dospělí MeSH
- hepatocyty účinky léků metabolismus MeSH
- imunologické faktory farmakologie MeSH
- kultivované buňky MeSH
- lékové interakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- luciferasy genetika metabolismus MeSH
- messenger RNA metabolismus MeSH
- polychlorované dibenzodioxiny farmakologie MeSH
- receptory aromatických uhlovodíků genetika MeSH
- receptory glukokortikoidů genetika MeSH
- systém (enzymů) cytochromů P-450 genetika MeSH
- xenobiotika metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Androgen receptor plays multiple physiological and pathological roles in human organism. In the current paper, we describe construction and characterization of a novel stably transfected human reporter cell line AIZ-AR for assessment of transcriptional activity of human androgen receptor. Cell line AIZ-AR is derived from human prostate carcinoma epithelial cell line 22Rv1 that was transfected with reporter plasmid containing 3 copies of androgen response regions (ARRs) followed by a single copy of androgen response element (ARE) from the promoter region of human prostate specific antigen (PSA) gene. AIZ-AR cells remained fully functional for more than 60 days and over 25 passages in the culture and even after cryopreservation. Time-course analyses showed that AIZ-AR cells allow detection of AR ligands as soon as after 8 hours of the treatment. We performed dose-response analyses with 23 steroids in 96-well plate format. We observed activation of AR by androgens, but not by estrogens and mineralocorticoids. Some glucocorticoids and progesterone also induced luciferase, but their potencies were 2-3 orders of magnitude weaker as compared to androgens. Taken together, we have developed a rapid, sensitive, selective, high-throughput and reproducible tool for detection of human AR ligands, with potential use in pharmacological and environmental applications.
- MeSH
- androgenní receptory genetika metabolismus MeSH
- antagonisté androgenních receptorů farmakologie MeSH
- buněčné klony MeSH
- časové faktory MeSH
- cinnamáty farmakologie MeSH
- dihydrotestosteron farmakologie MeSH
- genetická transkripce * účinky léků MeSH
- hygromycin B analogy a deriváty farmakologie MeSH
- kryoprezervace MeSH
- lidé MeSH
- luciferasy metabolismus MeSH
- nádorové buněčné linie MeSH
- reportérové geny * MeSH
- steroidy farmakologie MeSH
- transfekce * MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
