Novel assays to monitor gene expression and protein-protein interactions in rice using the bioluminescent protein, NanoLuc.
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ABSTRACT:
SUBMITTER: Taoka KI
PROVIDER: S-EPMC8215459 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundProtein data over circadian time scale is scarce for clock transcription factors. Further work in this direction is required for refining quantitative clock models. However, gathering highly resolved dynamics of low-abundance transcription factors has been a major challenge in the field. In this work we provide a new tool that could help this major issue. Bioluminescence is an important tool for gathering data on circadian gene expression. It allows data collection over extended time periods for low signal levels, thanks to a large signal-to-noise ratio. However, the main reporter so far used, firefly luciferase (FLUC), presents some disadvantages for reporting total protein levels. For example, the rapid, post-translational inactivation of this luciferase will result in underestimation of protein numbers. A more stable reporter protein could in principle tackle this issue. We noticed that NanoLUC might fill this gap, given its reported brightness and the stability of both enzyme and substrate. However, no data in plant systems on the circadian time scale had been reported.ResultsWe tested NanoLUC activity under different scenarios that will be important for generating highly quantitative data. These include enzyme purification for calibration curves, expression in transient plant systems, stable transgenic plants and in planta time series over circadian time scales. Furthermore, we show that the difference in substrate use between firefly luciferase and NanoLUC allows tracking of two different reporters from the same samples. We show this by exploring the impact of a BOAp:BOA-NanoLUC construct transformed into a Col-0 CCA1p:FLUC background.ConclusionsWe concluded that NanoLUC reporters are compatible with established instrumentation and protocols for firefly luciferase. Overall, our results provide guidelines for researchers gathering dynamic protein data over different time scales and experimental setups.
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Project description:Assays for ATPases have been enabled for high-throughput screening (HTS) by employing firefly luciferase to detect the remaining ATP in the assay. However, for any enzyme assay, measurement of product formation is a more sensitive assay design. Recently, technologies that allow detection of the ADP product from ATPase reactions have been described using fluorescent methods of detection. We describe here the characterization of a bioluminescent assay that employs firefly luciferase in a coupled-enzyme assay format to enable detection of ADP levels from ATPase assays (ADP-Glo, Promega Corp.). We determined the performance of the ADP-Glo assay in 1,536-well microtiter plates using the protein kinase Clk4 and a 1,352 member kinase focused combinatorial library. The ADP-Glo assay was compared to the Clk4 assay performed using a bioluminescence ATP-depletion format (Kinase-Glo, Promega Corp). We performed this analysis using quantitative HTS (qHTS) where we determined potency values for all library members and identified approximately 300 compounds with potencies ranging from as low as 50 nM to >10 microM, yielding a robust dataset for the comparison. Both assay formats showed high performance (Z'-factors approximately 0.9) and showed a similar potency distribution for the actives. We conclude that the bioluminescence ADP detection assay system is a viable generic alternative to the widely used ATP-depletion assay for ATPases and discuss the advantages and disadvantages of both approaches.
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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:FRET is a powerful approach to study the interactions of fluorescent molecules, and numerous methods have been developed to measure FRET in cells. Here, we present a method based on a donor molecule's photoswitching properties, which are slower in the presence vs. the absence of an acceptor. The technique, photoswitching FRET (psFRET), is similar to an established but underutilized method called photobleaching FRET (pbFRET), with the major difference being that the molecules are switched "off" rather than photobleached. The psFRET technique has some of the FRET imaging advantages normally attributed to fluorescence lifetime imaging microscopy (FLIM), such as monitoring only donor fluorescence. However, it can be performed on a conventional widefield microscope, requires less illumination light to photoswitch off than photobleaching, and can be photoswitched "on" again to repeat the experiment. We present data testing the validity of the psFRET approach to quantify FRET in cells and demonstrate its use in imaging protein-protein interactions and fluorescent protein-based biosensors.
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Project description:Protein-protein interactions play a crucial role in biological processes such as cell-cell adhesion, immune system-pathogen interactions, and sensory perception. Understanding the structural determinants of protein-protein complex formation and obtaining quantitative estimates of their dissociation constant (KD) are essential for the study of these interactions and for the discovery of new therapeutics. At the same time, it is equally important to characterize protein-protein interactions in a high-throughput fashion. Here, we use a modified thermal scanning assay to test interactions of wild type (WT) and mutant variants of N-terminal fragments (EC1+2) of cadherin-23 and protocadherin-15, two proteins essential for inner-ear mechanotransduction. An environmentally sensitive fluorescent dye (SYPRO orange) is used to monitor melting temperature (Tm) shifts of protocadherin-15 EC1+2 (pcdh15) in the presence of increasing concentrations of cadherin-23 EC1+2 (cdh23). These Tm shifts are absent when we use proteins containing deafness-related missense mutations known to disrupt cdh23 binding to pcdh15, and are increased for some rationally designed mutants expected to enhance binding. In addition, surface plasmon resonance binding experiments were used to test if the Tm shifts correlated with changes in binding affinity. We used this approach to find a double mutation (cdh23(T15E)- pcdh15(G16D)) that enhances binding affinity of the cadherin complex by 1.98 kJ/mol, roughly two-fold that of the WT complex. We suggest that the thermal scanning methodology can be used in high-throughput format to quickly compare binding affinities (KD from nM up to 100 μM) for some heterodimeric protein complexes and to screen small molecule libraries to find protein-protein interaction inhibitors and enhancers.
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Project description:We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand-receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand-receptor interaction studies.
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