Mutant Luciferase Research Articles

PunctaFinder: An algorithm for automated spot detection in fluorescence microscopy images.

Fluorescence microscopy has revolutionized biological research by enabling the visualization of subcellular structures at high resolution. With the increasing complexity and volume of microscopy data, there is a growing need for automated image analysis to ensure efficient and consistent interpretation. In this study, we introduce PunctaFinder, a novel Python-based algorithm designed to detect puncta, small bright spots, in raw fluorescence microscopy images without image denoising or signal enhancement steps. Furthermore, unlike other available spot detectors, PunctaFinder not only detects puncta, but also defines the cytoplasmic region, making it a valuable tool to quantify target molecule localization in cellular contexts. PunctaFinder is a widely applicable punctum detector and size estimator, as evidenced by its successful detection of Atg9-positive vesicles, lipid droplets, aggregates of a destabilized luciferase mutant, and the nuclear pore complex. Notably, PunctaFinder excels in detecting puncta in images with a relatively low resolution and signal-to-noise ratio, demonstrating its capability to identify dim puncta and puncta of dynamic target molecules. PunctaFinder reliably detects puncta in fluorescence microscopy images where automated analysis was not possible before, providing researchers with an efficient and robust method for punctum quantification in fluorescence microscopy images.

High-content analysis of proteostasis capacity in cellular models of amyotrophic lateral sclerosis (ALS)

Disrupted proteome homeostasis (proteostasis) in amyotrophic lateral sclerosis (ALS) has been a major focus of research in the past two decades. However, the proteostasis processes that become disturbed in ALS are not fully understood. Obtaining more detailed knowledge of proteostasis disruption in association with different ALS-causing mutations will improve our understanding of ALS pathophysiology and may identify novel therapeutic targets and strategies for ALS patients. Here we describe the development and use of a novel high-content analysis (HCA) assay to investigate proteostasis disturbances caused by the expression of several ALS-causing gene variants. This assay involves the use of conformationally-destabilised mutants of firefly luciferase (Fluc) to examine protein folding/re-folding capacity in NSC-34 cells expressing ALS-associated mutations in the genes encoding superoxide dismutase-1 (SOD1A4V) and cyclin F (CCNFS621G). We demonstrate that these Fluc isoforms can be used in high-throughput format to report on reductions in the activity of the chaperone network that result from the expression of SOD1A4V, providing multiplexed information at single-cell resolution. In addition to SOD1A4V and CCNFS621G, NSC-34 models of ALS-associated TDP-43, FUS, UBQLN2, OPTN, VCP and VAPB mutants were generated that could be screened using this assay in future work. For ALS-associated mutant proteins that do cause reductions in protein quality control capacity, such as SOD1A4V, this assay has potential to be applied in drug screening studies to identify candidate compounds that can ameliorate this deficiency.

A dual-luciferase bioluminescence system for the assessment of cellular therapies

Bioluminescence imaging is a well-established platform for evaluating engineered cell therapies in preclinical studies. However, despite the discovery of new luciferases and substrates, optimal combinations to simultaneously monitor two cell populations remain limited. This makes the functional assessment of cellular therapies cumbersome and expensive, especially in preclinical invivo models. In this study, we explored the potential of using a green bioluminescence-emitting click beetle luciferase, CBG99, and a red bioluminescence-emitting firefly luciferase mutant, Akaluc, together to simultaneously monitor two cell populations. Using various chimeric antigen receptor Tcells and tumor pairings, we demonstrate that these luciferases are suitable for real-time tracking of two cell types using 2D and 3D cultures invitro and experimental models invivo. Our data show the broad compatibility of this dual-luciferase (duo-luc) system with multiple bioluminescence detection equipment ranging from benchtop spectrophotometers to live animal imaging systems. Although this study focused on investigating complex CAR Tcells and tumor cell interactions, this duo-luc system has potential utility for the simultaneous monitoring of any two cellular components-for example, to unravel the impact of a specific genetic variant on clonal dominance in a mixed population of tumor cells.

Multiple Origins of Bioluminescence in Beetles and Evolution of Luciferase Function.

Bioluminescence in beetles has long fascinated biologists, with diverse applications in biotechnology. To date, however, our understanding of its evolutionary origin and functional variation mechanisms remains poor. To address these questions, we obtained high-quality reference genomes of luminous and nonluminous beetles in 6 Elateroidea families. We then reconstructed a robust phylogenetic relationship for all luminous families and related nonluminous families. Comparative genomic analyses and biochemical functional experiments suggested that gene evolution within Elateroidea played a crucial role in the origin of bioluminescence, with multiple parallel origins observed in the luminous beetle families. While most luciferase-like proteins exhibited a conserved nonluminous amino acid pattern (TLA346 to 348) in the luciferin-binding sites, luciferases in the different luminous beetle families showed divergent luminous patterns at these sites (TSA/CCA/CSA/LVA). Comparisons of the structural and enzymatic properties of ancestral, extant, and site-directed mutant luciferases further reinforced the important role of these sites in the trade-off between acyl-CoA synthetase and luciferase activities. Furthermore, the evolution of bioluminescent color demonstrated a tendency toward hypsochromic shifts and variations among the luminous families. Taken together, our results revealed multiple parallel origins of bioluminescence and functional divergence within the beetle bioluminescent system.

Molecular evolution of Cypridina noctiluca secretory luciferase for production of spectrum-shifted luminescence-emitting mutants and their application in nuclear receptor-reporter assays.

Luciferase is a popular enzyme used for biological analyses, such as reporter assays. In addition to a conventional reporter assay using a pair of firefly and Renilla luciferases, a simple multicolor reporter assay using multiple firefly or beetle luciferases emitting different color luminescence with a single substrate has been reported. Secretory luciferases have also been used for convenient sample preparation in reporter assays; however, reporter assay using secretory luciferase mutants that emit spectrum-shifted luminescence have not yet been reported. In this study, we generated blue- and red-shifted (-16 and 12 nm) luminescence-emitting Cypridina secretory luciferase (CLuc) mutants using multiple cycles of random and site-directed mutagenesis. Even for red-shifted CLuc mutant, which exhibited relatively low activity and stability, its enzymatic activity was sufficiently high for a luciferase assay (3.26 × 106 relative light unit/s), light emission was sufficiently prolonged (half-life is 3 min), and stability at 37°C was high. We independently determined the luminescence of these CLuc mutants using a luminometer with an optical filter. Finally, we replaced the commonly used reporters, firefly and Renilla luciferases used in a conventional nuclear receptor-reporter assay with these CLuc mutants and established a secretory luciferase-based single-substrate dual-color nuclear receptor-reporter assay.

Super-Stable Metal-Organic Framework (MOF)/Luciferase Paper-Sensing Platform for Rapid ATP Detection.

Adenosine triphosphate (ATP) determination has been used for many decades to assess microbial contamination for hygiene monitoring in different locations and workplace environments. Highly sophisticated methods have been reported, yet commercially available kits rely on a luciferase-luciferin system and require storage and shipping at controlled temperatures (+4 or -20 °C). The applicability of these systems is limited by the need for a secure cold chain, which is not always applicable, especially in remote areas or low-resource settings. In this scenario, easy-to-handle and portable sensors would be highly valuable. Prompted by this need, we developed a bioluminescence paper biosensor for ATP monitoring in which a new luciferase mutant was combined with a metal-organic framework (MOF); i.e., zeolitic imidazolate framework-8 (ZIF-8). A paper biosensor was developed, ZIF-8@Luc paper sensor, and interfaced with different portable light detectors, including a silicon photomultiplier (SiPM) and smartphones. The use of ZIF-8 not only provided a five-fold increase in the bioluminescence signal, but also significantly improved the stability of the sensor, both at +4 and +28 °C. The ATP content in complex biological matrices was analyzed with the ZIF-8@Luc paper sensor, enabling detection down to 7 × 10-12 moles of ATP and 8 × 10-13 moles in bacterial lysates and urine samples, respectively. The ZIF-8@Luc sensor could, therefore, be applied in many fields in which ATP monitoring is required such as the control of microbial contamination.

Effects of synonymous mutations on kinetic properties and structure of firefly luciferase: Molecular dynamics simulation, molecular docking, RNA folding, and experimental study

Although synonymous mutations have long been thought to lack striking results, a growing body of research shows these mutations have highly variable effects. In this study, the impact of synonymous mutations in the development of thermostable luciferase was investigated using a combination of experimental and theoretical approaches. Using bioinformatics analysis, the codon usage features in the Lampyridae family's luciferases were studied and four synonymous mutations of Arg in luciferase were created. An exciting result was that the analysis of kinetic parameters showed a slight increase in the thermal stability of the mutant luciferase. AutoDock Vina, %MinMax algorithm, and UNAFold Server were used to perform molecular docking, folding rate, and RNA folding, respectively. Here, it was assumed that in the region (Arg337) with a moderate propensity for coil, synonymous mutation altered the rate of translation, which in turn may lead to a slight change in the structure of the enzyme. According to the molecular dynamics simulation data, local minor global flexibility is observed in the context of the protein conformation. A plausible explanation is that this flexibility may strengthen hydrophobic interactions due to its sensitivity to a molecular collision. Accordingly, thermostability originated mainly from hydrophobic interaction.

Biochemical Analysis Leads to Improved Orthogonal Bioluminescent Tools.

Engineered luciferase-luciferin pairs have expanded the number of cellular targets that can be visualized in tandem. While light production relies on selective processing of synthetic luciferins by mutant luciferases, little is known about the origin of selectivity. The development of new and improved pairs requires a better understanding of the structure-function relationship of bioluminescent probes. In this work, we report a biochemical approach to assessing and optimizing two popular bioluminescent pairs: Cashew/d-luc and Pecan/4'-BrLuc. Single mutants derived from Cashew and Pecan revealed key residues for selectivity and thermal stability. Stability was further improved through a rational addition of beneficial residues. In addition to providing increased stability, the known stabilizing mutations surprisingly also improved selectivity. The resultant improved pair of luciferases are >100-fold selective for their respective substrates and highly thermally stable. Collectively, this work highlights the importance of mechanistic insight for improving bioluminescent pairs and provides significantly improved Cashew and Pecan enzymes which should be immediately suitable for multicomponent imaging applications.

Red-Shifted Coumarin Luciferins for Improved Bioluminescence Imaging.

Multicomponent bioluminescence imaging in vivo requires an expanded collection of tissue-penetrant probes. Toward this end, we generated a new class of near-infrared (NIR) emitting coumarin luciferin analogues (CouLuc-3s). The scaffolds were easily accessed from commercially available dyes. Complementary mutant luciferases for the CouLuc-3 analogues were also identified. The brightest probes enabled sensitive imaging in vivo. The CouLuc-3 scaffolds are also orthogonal to popular bioluminescent reporters and can be used for multicomponent imaging applications. Collectively, this work showcases a new set of bioluminescent tools that can be readily implemented for multiplexed imaging in a variety of biological settings.

Novel Nanozeolitic Imidazolate Framework (ZIF-8)-Luciferase Biocomposite for Nanosensing Applications.

The identification of new strategies to improve the stability of proteins is of utmost importance for a number of applications, from biosensing to biocatalysis. Metal-organic frameworks (MOFs) have been shown as a versatile host platform for the immobilization of proteins, with the potential to protect proteins in harsh conditions. In this work, a new thermostable luciferase mutant has been selected as a bioluminescent protein model to investigate the suitability of MOFs to improve its stability and prompt its applications in real-world applications, for example, ATP detection in portable systems. The luciferase has been immobilized onto zeolitic imidazolate framework-8 (ZIF-8) to obtain a bioluminescent biocomposite with enhanced performance. The biocomposite ZIF-8@luc has been characterized in harsh conditions (e.g., high temperature, non-native pH, etc.). Bioluminescence properties confirmed that MOF enhanced the luciferase stability at acidic pH, in the presence of organic solvents, and at -20 °C. To assess the feasibility of this approach, the recyclability, storage stability, precision, and Michaelis-Menten constants (Km) for ATP and d-luciferin have been also evaluated. As a proof of principle, the suitability for ATP detection was investigated and the biocomposite outperformed the free enzyme in the same experimental conditions, achieving a limit of detection for ATP down to 0.2 fmol.

A Luciferase Mutant with Improved Brightness and Stability for Whole-Cell Bioluminescent Biosensors and In Vitro Biosensing.

The availability of new bioluminescent proteins with tuned properties, both in terms of emission wavelength, kinetics and protein stability, is highly valuable in the bioanalytical field, with the potential to improve the sensitivity and analytical performance of the currently used methods for ATP detection, whole-cell biosensors, and viability assays among others. We present a new luciferase mutant, called BgLuc, suitable for developing whole-cell biosensors and in vitro biosensors characterized by a bioluminescence maximum of 548 nm, narrow emission bandwidth, favorable kinetic properties, and excellent pH- and thermo-stabilities at 37 and 45 °C and pH from 5.0 to 8.0. We assessed the suitability of this new luciferase for whole-cell biosensing with a cell-based bioreporter assay for Nuclear Factor-kappa B (NF-kB) signal transduction pathway using 2D and 3D human embryonic kidney (HEK293T) cells, and for ATP detection with the purified enzyme. In both cases the luciferase showed suitable for sensitive detection of the target analytes, with better or similar performance than the commercial counterparts.

The dual role of p62 in ferroptosis of glioblastoma according to p53 status

BackgroundFerroptosis plays a key role in human cancer, but its function and mechanism in glioma is not clear. P62/SQSTM1 was reported to inhibit ferroptosis via the activation of NRF2 signaling pathway. In this study we reveal a dual role of p62 in ferroptosis of glioblastoma (GBM) according to p53 status.MethodLipid peroxidation analysis, transmission electron microscopy (TEM), GSH assay were performed to determine the level of ferroptosis. Western blot and qPCR were obtained to detect the expression of ferroptosis markers. Construction of mutant plasmids, immunoprecipitation, luciferase assay and rescue-experiments were performed to explore the regulatory mechanism.ResultsP62 overexpression facilitates ferroptosis and inhibits SLC7A11 expression in p53 mutant GBM, while attenuates ferroptosis and promotes SLC7A11 expression in p53 wild-type GBM. P62 associates with p53 and inhibits its ubiquitination. The p53-NRF2 association and p53-mediated suppression of NRF2 antioxidant activity are diversely regulated by p62 according to p53 status. P53 mutation status is required for the dual regulation of p62 on ferroptosis. In wild-type p53 GBM, the classical p62-mediated NRF2 activation pathway plays a major regulatory role of ferroptosis, leading to increased SLC7A11 expression, resulting in a anti-ferroptosis role. In mutant p53 GBM, stronger interaction of mutant-p53/NRF2 by p62 enhance the inhibitory effect of mutant p53 on NRF2 signaling, which reversing the classical p62-mediated NRF2 activation pathway, together with increased p53’s transcriptional suppression on SLC7A11 by p62, leading to a decrease of SLC7A11, resulting in a pro-ferroptosis role.ConclusionTogether, this study shows novel molecular mechanisms of ferroptosis regulated by p62; the mutation status of p53 is an important factor that determines the therapeutic response to p62-mediated ferroptosis-targeted therapies in GBM.

Improved bioluminescence-based endotoxin measurement method using a salt-resistant luciferase mutant

Endotoxin is a component of the cell wall of gram-negative bacteria and causes fever and shock symptoms upon entering the bloodstream. We previously demonstrated that the bioluminescence-based Limulus amebocyte lysate test is highly sensitive and rapid for measuring endotoxin. However, as the firefly luciferase reaction is inhibited in the presence of sodium chloride, the endotoxin detection method did not meet the validation guidelines under medical dialysis conditions (range of 75–125% of the measured values tested in water). Here, we used a salt-resistant luciferase mutant, which met the criteria for validation of endotoxin measurement.

Saccharomyces cerevisiae Fpr1 functions as a chaperone to inhibit protein aggregation

Peptidyl prolyl isomerases (PPIases) accelerate the rate limiting step of protein folding by catalyzing cis/trans isomerization of peptidyl prolyl bonds. The larger PPIases have been shown to be multi-domain proteins, with functions other than isomerization of the proline-containing peptide bond. Recently, a few smaller PPIases have also been described for their ability to stabilize folding intermediates. The yeast Fpr1 (FK506-sensitive proline rotamase) is a homologue of the mammalian prolyl isomerase FKBP12 (FK506-binding protein of 12 kDa). Its ability to stabilize stressed cellular proteins has not been reported yet. We had earlier reported upregulation of Fpr1 in yeast cells exposed to proteotoxic stress conditions. In this work, we show that yeast Fpr1 exhibits characteristics typical of a general chaperone of the proteostasis network. Aggregation of mutant huntingtin fragment was higher in Fpr1-deleted as compared to parental yeast cells. Overexpression of Fpr1 led to reduced protein aggregation by decreasing the amount of oligomers and diverting the aggregation pathway towards the formation of detergent-soluble species. This correlated well with higher survival of these cells. Purified and enzymatically active yeast Fpr1 was able to inhibit aggregation of mutant huntingtin fragment and luciferase in vitro in a concentration-dependent manner; suggesting a direct action for aggregation inhibitory action of Fpr1. Overexpression of yeast Fpr1 was able to protect E. coli cells against thermal shock. This work establishes the role of Fpr1 in the protein folding network and will be used for the identification of novel pharmacological leads in disease conditions.

Multicomponent Bioluminescence Imaging with Naphthylamino Luciferins.

Bioluminescent tools have been used for decades to image processes in complex tissues and preclinical models. However, few distinct probes are available to probe multicellular interactions. We and others are addressing this limitation by engineering new luciferases that can selectively process synthetic luciferin analogues. In this work, we explored naphthylamino luciferins as orthogonal bioluminescent probes. Three analogues were prepared using an optimized synthetic route. The luciferins were found to be robust emitters with native luciferase in vitro and in cellulo. We further screened the analogues against libraries of luciferase mutants to identify unique enzyme-substrate pairs. The new probes can be used in conjunction with existing bioluminescent tools for multi-component imaging.

Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride.

Firefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study was aimed at obtaining mutant luciferase enzymes resistant to the sodium chloride inhibition. We first obtained two mutant luciferase enzymes whose inhibition were alleviated and determined the mutations to be Val288Ile and Glu488Val. Under medical dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride. The mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The luciferase CR may be extensively useful in any bioassay which includes firefly luciferase and is employed in the presence of sodium chloride.

Orthogonal Bioluminescent Probes from Disubstituted Luciferins.

Bioluminescence imaging with luciferase-luciferin pairs is routinely used to monitor cellular functions. Multiple targets can be visualized in tandem using luciferases that process unique substrates, but only a handful of such orthogonal probes are known. Multiplexed studies require additional robust, light-emitting molecules. In this work, we report new luciferins for orthogonal imaging that comprise disubstituted cores. These probes were found to be bright emitters with various engineered luciferases. The unique patterns of light output also provided insight into enzyme-substrate interactions necessary for productive emission. Screening studies identified mutant luciferases that could preferentially process the disubstituted analogues, enabling orthogonal imaging with existing bioluminescent reporters. Further mutational analyses revealed the origins of substrate selectivity. Collectively, this work provides insights into luciferase-luciferin features relevant to bioluminescence and expands the number of probes for multicomponent tracking.

How to Select Firefly Luciferin Analogues for In Vivo Imaging.

Bioluminescence reactions are widely applied in optical in vivo imaging in the life science and medical fields. Such reactions produce light upon the oxidation of a luciferin (substrate) catalyzed by a luciferase (enzyme), and this bioluminescence enables the quantification of tumor cells and gene expression in animal models. Many researchers have developed single-color or multicolor bioluminescence systems based on artificial luciferin analogues and/or luciferase mutants, for application in vivo bioluminescence imaging (BLI). In the current review, we focus on the characteristics of firefly BLI technology and discuss the development of luciferin analogues for high-resolution in vivo BLI. In addition, we discuss the novel luciferin analogues TokeOni and seMpai, which show potential as high-sensitivity in vivo BLI reagents.

V-ATPase Inhibition Decreases Mutant Androgen Receptor Activity in Castrate-resistant Prostate Cancer.

Prostate cancer is critically dependent on androgen receptor (AR) signaling. Despite initial responsiveness to androgen deprivation, most patients with advanced prostate cancer subsequently progress to a clinically aggressive castrate-resistant prostate cancer (CRPC) phenotype, typically associated with expression of splice-variant or mutant AR forms. Although current evidence suggests that the vacuolar-ATPase (V-ATPase), a multiprotein complex that catalyzes proton transport across intracellular and plasma membranes, influences wild-type AR function, the effect of V-ATPase inhibition on variant AR function is unknown.Inhibition of V-ATPase reduced AR function in wild-type and mutant AR luciferase reporter models. In hormone-sensitive prostate cancer cell lines (LNCaP, DuCaP) and mutant AR CRPC cell lines (22Rv1, LNCaP-F877L/T878A), V-ATPase inhibition using bafilomycin-A1 and concanamycin-A reduced AR expression, and expression of AR target genes, at mRNA and protein levels. Furthermore, combining chemical V-ATPase inhibition with the AR antagonist enzalutamide resulted in a greater reduction in AR downstream target expression than enzalutamide alone in LNCaP cells. To investigate the role of individual subunit isoforms, siRNA and CRISPR-Cas9 were used to target the V1C1 subunit in 22Rv1 cells. Whereas transfection with ATP6V1C1-targeted siRNA significantly reduced AR protein levels and function, CRISPR-Cas9-mediated V1C1 knockout showed no substantial change in AR expression, but a compensatory increase in protein levels of the alternate V1C2 isoform.Overall, these results indicate that V-ATPase dysregulation is directly linked to both hormone-responsive prostate cancer and CRPC via impact on AR function. In particular, V-ATPase inhibition can reduce AR signaling regardless of mutant AR expression.

Application of Taxol Nanomicelles with Lyp-1 Target in Targeted Therapy of Colon Cancer

As a new type of non-viral gene drug carrier, paclitaxel with Lyp-1 target has unique transmembrane ability due to its unique structure. In this paper, amino acids and surfactants are used to disperse SWCNTs in water, and non-covalent interactions are used to adsorb paclitaxel to the surface of SWCNTs. DSPE-PEG-Maleimide is then connected to NGR to achieve active targeting. To investigate the effect of NGR-SWCNTs-Paclitaxel on isolated cells, and to observe the antitumor effect of NGR-SWCNTs-Paclitaxel on S180 colon cancer mice in vivo, we provide theoretical and experimental basis for targeted cancer treatment. The luciferase activity test results showed that mi R-218 mimics had no significant effect on the intensity of the blank reporter plasmid group and p MIR-REPORT/UTR mutant luciferase activity, but in mi R-218 mimics and p MIR-REPORT/UTR Luciferase activity decreased after co-transfection of wild-type plasmids into cells. The validation results of the luciferase activity analysis system showed that mi R-218 was able to bind to Sp13’UTR. Overexpression of mi R-218 can significantly reduce the expression level of Sp1 protein but has no significant effect on Sp1 m RNA level, indicating that mi R-218 can target the regulation of Sp1 expression at the translation level.