Luciferase reporter assays to monitor interferon signaling modulation by SARS-CoV-2 proteins.
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ABSTRACT:
SUBMITTER: Hirschenberger M
PROVIDER: S-EPMC8361205 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Nuclear receptors play important roles in many cellular functions through control of gene transcription. It is also a large target class for drug discovery. Luciferase reporter assays are frequently used to study nuclear receptor function because of their wide dynamic range, low endogenous activity, and ease of use. Recent improvements of luciferase genes and vectors have further enhanced their utilities. Here we applied these improvements to two reporter formats for studying nuclear receptors. The first assay contains a Murine Mammary Tumor Virus promoter upstream of a destabilized luciferase. The presence of response elements for nuclear hormone receptor in this promoter allows the studies of endogenous and/or exogenous full length receptors. The second assay contains a ligand binding domain (LBD) of a nuclear receptor fused to the GAL4 DNA binding domain (DBD) on one vector and multiple Gal4 Upstream Activator Sequences (UAS) upstream of luciferase reporter on another vector. We showed that codon optimization of luciferase reporter genes increased expression levels in conjunction with the incorporation of protein destabilizing sequences into luciferase led to a larger assay dynamic range in both formats. The optimum number of UAS to generate the best response was determined. The expression vector for nuclear receptor LBD/GAL4 DBD fusion also constitutively expresses a Renilla luciferase-neo(R) fusion protein, which provides selection capability (G418 resistance, neo(R)) as well as an internal control (Renilla luciferase). This dual-luciferase format allowed detecting compound cytotoxicity or off-target change in expression during drug screening, therefore improved data quality. These luciferase reporter assays provided better research and drug discovery tools for studying the functions of full length nuclear receptors and ligand binding domains.
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Project description:Sirtuin 3 (SIRT3) is a major regulator of oncometabolism. Indeed, the activity of SIRT3 significantly affects the response to oxidative stress, glycolytic proficiency, and tumorigenic potential of malignant cells. Thus, a system to accurately measure the transcriptional activity of the SIRT3 promoter could facilitate the identification of novel antineoplastic agents or have diagnostic applications. Here, we describe all the steps involved in the development of a luciferase-based reporter system to measure the activation of the human SIRT3 promoter, encompassing the design of appropriate primers, the cloning of the promoter fragment, and its site-directed mutagenesis. We validated this system in human embryonic kidney 293T cells, taking advantage of the renowned ability of the transcription factor estrogen-related receptor ? to transactivate SIRT3. Moreover, here we demonstrate that SIRT3 expression is responsive to rapamycin, a small inhibitor of mammalian target of rapamycin that has been extensively employed as a caloric restriction mimetic. Finally, we provide an overview of the complementary molecular biology techniques that might be employed to further verify the reliability of this system.
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Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-β) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.
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Project description:BackgroundPer- and polyfluoroalkyl substances (PFAS), organophosphate esters (OPEs), and polybrominated diphenyl ethers (PBDEs) are hormone-disrupting chemicals that migrate from building materials into air and dust.ObjectivesWe aimed to quantify the hormonal activities of 46 dust samples and identify chemicals driving the observed activities.MethodsWe evaluated associations between hormonal activities of extracted dust in five cell-based luciferase reporter assays and dust concentrations of 42 measured PFAS, OPEs, and PBDEs, transformed as either raw or potency-weighted concentrations based on Tox21 high-throughput screening data.ResultsAll dust samples were hormonally active, showing antagonistic activity toward peroxisome proliferator-activated receptor (PPARγ2) (100%; 46 of 46 samples), thyroid hormone receptor (TRβ) (89%; 41 samples), and androgen receptor (AR) (87%; 40 samples); agonist activity on estrogen receptor (ERα) (96%; 44 samples); and binding competition with thyroxine (T4) on serum transporter transthyretin (TTR) (98%; 45 samples). Effects were observed with as little as 4μg of extracted dust. In regression models for each chemical class, interquartile range increases in potency-weighted or unknown-potency chemical concentrations were associated with higher hormonal activities of dust extracts (potency-weighted: ΣPFAS-TRβ, ↑28%, p<0.05; ΣOPEs-TRβ, ↑27%, p=0.08; ΣPBDEs-TRβ, ↑20%, p<0.05; ΣPBDEs-ERα, ↑7.7%, p=0.08; unknown-potency: ΣOPEs-TTR, ↑34%, p<0.05; ΣOPEs-AR, ↑13%, p=0.06), adjusted for chemicals with active, inactive, and unknown Tox21 designations.DiscussionAll indoor dust samples exhibited hormonal activities, which were associated with PFAS, PBDE, and OPE levels. Reporter gene cell-based assays are relatively inexpensive, health-relevant evaluations of toxic loads of chemical mixtures that building occupants are exposed to. https://doi.org/10.1289/EHP8054.
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Project description:Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure-activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.
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Project description:Mutagens and oxidative agents damage biomolecules, such as DNA; therefore, detecting genotoxic and oxidative chemicals is crucial for maintaining human health. To address this, we have developed several types of yeast-based reporter assays designed to detect DNA damage and oxidative stress. This study aimed to develop a novel yeast-based assay using a codon-optimized stable or unstable NanoLuc luciferase (yNluc and yNluCP) gene linked to a DNA damage- or oxidative stress-responsive promoter, enabling convenient sensing genotoxicity or oxidative stress, respectively. End-point luciferase assays using yeasts with a chromosomally integrated RNR3 promoter (PRNR3)-driven yNluc gene exhibited high levels of chemiluminescence via NanoLuc luciferase and higher fold induction by hydroxyurea than a multi-copy plasmid-based assay. Additionally, the integrated reporter system detected genotoxicity caused by four different types of chemicals. Oxidants (hydrogen peroxide, tert-butyl hydroperoxide, and menadione) were successfully detected through transient expressions of luciferase activity in real-time luciferase assay using yeasts with a chromosomally integrated TRX2 promoter (PTRX2)-linked yNlucCP gene. However, the luciferase activity was gradually induced in yeasts with a multi-copy reporter plasmid, and their expression profiles were notably distinct from those observed in chromosomally integrated yeasts. The responses of yNlucCP gene against three oxidative chemicals, but not diamide and zinc oxide suspension, were observed using chromosomally integrated reporter yeasts. Given that yeast cells with chromosomally integrated PRNR3-linked yNluc and PTRX2-linked yNlucCP genes express strong chemiluminescence signals and are easily maintained and handled without restrictive nutrient medium, these yeast strains with NanoLuc reporters may prove useful for screening potential genotoxic and oxidative chemicals.
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Project description:SARS-CoV-2 main protease (Mpro) is one of the most extensively exploited drug targets for COVID-19. Structurally disparate compounds have been reported as Mpro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed Mpro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based FlipGFP assay. Collectively, our results have shown that majority of the Mpro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to Mpro, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit Mpro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery.
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Project description:Effective methods for monitoring eukaryotic gene expression and regulation based on bioluminescence - the emission of light by living organisms - are well established. Typically, the expression of a gene of interest is reported on with high sensitivity and over a wide dynamic range by the emission of light from a variety of engineered luciferase genes from beetles and marine organisms. The luciferase reporter genes are expressed downstream of the target gene or promoter and detected after exogenous addition of luciferin substrates. We describe a novel bioluminescence reporter method for the simultaneous monitoring of two genes expressing engineered firefly luciferase variants that emit readily distinguishable green and red light signals. The key feature is the selectivity of the enzymes for two luciferin substrates that determine each emission color. To validate our method, we performed a complex promoter transactivation experiment side-by-side with the Dual-Luciferase Reporter protocol and obtained essentially identical results. Additional comparative experiments demonstrated that our assay system provided improvements in background, cell normalization, and detectability compared to representative available methods. With access to a luminometer equipped with two optical filters, this method is an excellent choice for genetic reporter assays that can be performed with a single reagent solution.
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Project description:Hypoxia has been identified as a contributing factor in the pathophysiology of several diseases and oxygen regulation is important during stem cell development, particularly in early embryogenesis. One aspect that has emerged is the role of hypoxia-inducible factors, or HIFs in regulating the effect of hypoxia. Studies in our laboratory sought to examine the hypoxic regulation of HIF activity in placental trophoblast cells, through the use of dual-reporter luciferase assays. Our study demonstrates that hypoxic conditions cause a significant increase in the level of constitutive luciferase reporter activity. We also show that this induction is not a cell type or species-specific phenomenon and provides an alternative method for normalizing transfection efficiency in luciferase assays under hypoxic conditions. Our results suggest that in studies dealing with hypoxic conditions, caution should be used when interpreting measurements of transcriptional activity by traditional dual-reporter assays.
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