RESUMO
The fungal bioluminescence pathway can be reconstituted in other organisms allowing luminescence imaging without exogenously supplied substrate. The pathway starts from hispidin biosynthesis-a step catalyzed by a large fungal polyketide synthase that requires a posttranslational modification for activity. Here, we report identification of alternative compact hispidin synthases encoded by a phylogenetically diverse group of plants. A hybrid bioluminescence pathway that combines plant and fungal genes is more compact, not dependent on availability of machinery for posttranslational modifications, and confers autonomous bioluminescence in yeast, mammalian, and plant hosts. The compact size of plant hispidin synthases enables additional modes of delivery of autoluminescence, such as delivery with viral vectors.
Assuntos
Luminescência , Plantas , Animais , MamíferosRESUMO
Bioluminescence resonance energy transfer (BRET) is one of the most promising approaches used for noninvasive imaging of protein-protein interactions in vivo. Recently, our team has discovered a genetically encodable bioluminescent system from the fungus Neonothopanus nambi and identified a novel luciferase that represents an imaging tool orthogonal to other luciferin-luciferase systems. We demonstrated the possibility of using the fungal luciferase as a new BRET donor by creating fused pairs with acceptor red fluorescent proteins, of which tdTomato provided the highest BRET efficiency. Using this new BRET system, we also designed a mTOR pathway specific rapamycin biosensor by integrating the FRB and FKBP12 protein dimerization system. We demonstrated the specificity and efficacy of the new fungal luciferase-based BRET combination for application in mammalian cell culture that will provide the unique opportunity to perform multiplexed BRET assessment in the future.
RESUMO
The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.
Assuntos
Eucariotos , Luminescência , Animais , MamíferosRESUMO
This study demonstrates the development of a humanized luciferase imaging reporter based on a recently discovered mushroom luciferase (Luz) from Neonothopanus nambi. In vitro and in vivo assessments showed that human-codon-optimized Luz (hLuz) has significantly higher activity than native Luz in various cancer cell types. The potential of hLuz in non-invasive bioluminescence imaging was demonstrated by human tumor xenografts subcutaneously and by the orthotopic lungs xenograft in immunocompromised mice. Luz enzyme or its unique 3OH-hispidin substrate was found to be non-cross-reacting with commonly used luciferase reporters such as Firefly (FLuc2), Renilla (RLuc), or nano-luciferase (NLuc). Based on this feature, a non-overlapping, multiplex luciferase assay using hLuz was envisioned to surpass the limitation of dual reporter assay. Multiplex reporter functionality was demonstrated by designing a new sensor construct to measure the NF-κB transcriptional activity using hLuz and utilized in conjunction with two available constructs, p53-NLuc and PIK3CA promoter-FLuc2. By expressing these constructs in the A2780 cell line, we unveiled a complex macromolecular regulation of high relevance in ovarian cancer. The assays performed elucidated the direct regulatory action of p53 or NF-κB on the PIK3CA promoter. However, only the multiplexed assessment revealed further complexities as stabilized p53 expression attenuates NF-κB transcriptional activity and thereby indirectly influences its regulation on the PIK3CA gene. Thus, this study suggests the importance of live cell multiplexed measurement of gene regulatory function using more than two luciferases to address more realistic situations in disease biology.
Assuntos
Agaricales , Neoplasias Ovarianas , Humanos , Animais , Feminino , Camundongos , NF-kappa B , Linhagem Celular Tumoral , Proteína Supressora de Tumor p53/genética , Luciferases/genética , Agaricales/genética , Classe I de Fosfatidilinositol 3-QuinasesRESUMO
Hispidin is a polyketide found in plants and fungi. In bioluminescent fungi, hispidin serves as a precursor of luciferin and is produced by hispidin synthases. Previous studies revealed that hispidin synthases differ in orthologous polyketide synthases from non-bioluminescent fungi by the absence of two domains with predicted ketoreductase and dehydratase activities. Here, we investigated the hypothesis that the loss of these domains in evolution led to the production of hispidin and the emergence of bioluminescence. We cloned three orthologous polyketide synthases from non-bioluminescent fungi, as well as their truncated variants, and assessed their ability to produce hispidin in a bioluminescence assay in yeast. Interestingly, expression of the full-length enzyme hsPKS resulted in dim luminescence, indicating that small amounts of hispidin are likely being produced as side products of the main reaction. Deletion of the ketoreductase and dehydratase domains resulted in no luminescence. Thus, domain truncation by itself does not appear to be a sufficient step for the emergence of efficient hispidin synthases from orthologous polyketide synthases. At the same time, the production of small amounts of hispidin or related compounds by full-length enzymes suggests that ancestral fungal species were well-positioned for the evolution of bioluminescence.
Assuntos
Policetídeo Sintases , Pironas , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Óxido Nítrico Sintase/metabolismo , Fungos/genética , Fungos/metabolismo , Hidroliases/metabolismoRESUMO
We report a systematic comparison of 19 plant promoters and 20 promoter-terminator combinations in two expression systems: agroinfiltration in Nicotiana benthamiana leaves, and Nicotiana tabacum BY-2 plant cell packs. The set of promoters tested comprised those not present in previously published work, including several computationally predicted synthetic promoters validated here for the first time. The expression of EGFP driven by different promoters varied by more than two orders of magnitude and was largely consistent between two tested Nicotiana systems. We confirmed previous reports of significant modulation of expression by terminators, as well as synergistic effects of promoters and terminators. Additionally, we observed non-linear effects of gene dosage on expression level. The dataset presented here can inform the design of genetic constructs for plant engineering and transient expression assays.
Assuntos
Nicotiana , Plantas , Nicotiana/genética , Regiões Promotoras Genéticas , Plantas/genética , Folhas de Planta/genética , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genéticaRESUMO
Studies of protein fitness landscapes reveal biophysical constraints guiding protein evolution and empower prediction of functional proteins. However, generalisation of these findings is limited due to scarceness of systematic data on fitness landscapes of proteins with a defined evolutionary relationship. We characterized the fitness peaks of four orthologous fluorescent proteins with a broad range of sequence divergence. While two of the four studied fitness peaks were sharp, the other two were considerably flatter, being almost entirely free of epistatic interactions. Mutationally robust proteins, characterized by a flat fitness peak, were not optimal templates for machine-learning-driven protein design - instead, predictions were more accurate for fragile proteins with epistatic landscapes. Our work paves insights for practical application of fitness landscape heterogeneity in protein engineering.
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Aptidão Genética , Modelos Genéticos , Mutação , Proteínas/genéticaRESUMO
The phase of the cell cycle determines numerous aspects of cancer cell behaviour including invasiveness, ability to migrate and responsiveness to cytotoxic drugs. To non-invasively monitor progression of cell cycle in vivo, a family of genetically encoded fluorescent indicators, FUCCI (fluorescent ubiquitination-based cell cycle indicator), has been developed. Existing versions of FUCCI are based on fluorescent proteins of two or more different colors fused to cell-cycle-dependent degradation motifs. Thus, FUCCI-expressing cells emit light of different colors in different phases providing a robust way to monitor cell cycle progression by fluorescence microscopy and flow cytometry but limiting the possibility to simultaneously visualize other markers. To overcome this limitation, we developed a single-color variant of FUCCI, called FUCCI-Red, which utilizes two red fluorescent proteins with distinct fluorescence lifetimes, mCherry and mKate2. Similarly to FUCCI, these proteins carry cell cycle-dependent degradation motifs to resolve G1 and S/G2/M phases. We showed utility of FUCCI-Red by visualizing cell cycle progression of cancer cells in 2D and 3D cultures and monitoring development of tumors in vivo by confocal and fluorescence lifetime imaging microscopy (FLIM). Single-channel registration and red-shifted spectra make FUCCI-Red sensor a promising instrument for multiparameter in vivo imaging applications, which was demonstrated by simultaneous detection of cellular metabolic state using endogenous fluorescence in the blue range.
Assuntos
Ciclo Celular , Neoplasias do Colo/patologia , Corantes Fluorescentes/química , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Imagem Individual de Molécula/métodos , Animais , Proliferação de Células , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína Vermelha FluorescenteRESUMO
Bioluminescence-based technologies are among the most commonly used methods to quantify and visualise physiology at the cellular and organismal levels. However, the potential of bioluminescence beyond reporter technologies remains largely unexplored. Here, we provide an overview of the emerging approaches employing bioluminescence as a biological light source that triggers physiological events and controls cell behaviour and discuss its possible future application in synthetic biology.
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Genetically encoded photosensitizers are increasingly used as optogenetic tools to control cell fate or trigger intracellular processes. A monomeric red fluorescent protein called SuperNova has been recently developed, however, it demonstrates suboptimal characteristics in most phototoxicity-based applications. Here, we applied directed evolution to this protein and identified SuperNova2, a protein with S10R substitution that results in enhanced brightness, chromophore maturation and phototoxicity in bacterial and mammalian cell cultures.
Assuntos
Proteínas Luminescentes , Fármacos Fotossensibilizantes/farmacologia , Escherichia coli/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/farmacologia , Mutação , Optogenética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteína Vermelha FluorescenteRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Autoluminescent plants engineered to express a bacterial bioluminescence gene cluster in plastids have not been widely adopted because of low light output. We engineered tobacco plants with a fungal bioluminescence system that converts caffeic acid (present in all plants) into luciferin and report self-sustained luminescence that is visible to the naked eye. Our findings could underpin development of a suite of imaging tools for plants.
Assuntos
Luciferina de Vaga-Lumes/metabolismo , Nicotiana/genética , Plantas Geneticamente Modificadas/metabolismo , Ácidos Cafeicos/metabolismo , Fungos/genética , Fungos/metabolismoRESUMO
For most animals, feeding includes two behaviors: foraging to find a food patch and food intake once a patch is found. The nematode Caenorhabditis elegans is a useful model for studying the genetics of both behaviors. However, most methods of measuring feeding in worms quantify either foraging behavior or food intake, but not both. Imaging the depletion of fluorescently labeled bacteria provides information on both the distribution and amount of consumption, but even after patch exhaustion a prominent background signal remains, which complicates quantification. Here, we used a bioluminescent Escherichia coli strain to quantify C. elegans feeding. With light emission tightly coupled to active metabolism, only living bacteria are capable of bioluminescence, so the signal is lost upon ingestion. We quantified the loss of bioluminescence using N2 reference worms and eat-2 mutants, and found a nearly 100-fold increase in signal-to-background ratio and lower background compared to loss of fluorescence. We also quantified feeding using aggregating npr-1 mutant worms. We found that groups of npr-1 mutants first clear bacteria from within the cluster before foraging collectively for more food; similarly, during large population swarming, only worms at the migrating front are in contact with bacteria. These results demonstrate the usefulness of bioluminescent bacteria for quantifying feeding and generating insights into the spatial pattern of food consumption.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Ingestão de Alimentos/genética , Receptores de Neuropeptídeo Y/genética , Receptores Nicotínicos/genética , Animais , Caenorhabditis elegans/microbiologia , Ingestão de Alimentos/fisiologia , Escherichia coli/química , Comportamento Alimentar/fisiologia , Proteínas Mutantes/genética , Mutação/genéticaRESUMO
Characterizing the fitness landscape, a representation of fitness for a large set of genotypes, is key to understanding how genetic information is interpreted to create functional organisms. Here we determined the evolutionarily-relevant segment of the fitness landscape of His3, a gene coding for an enzyme in the histidine synthesis pathway, focusing on combinations of amino acid states found at orthologous sites of extant species. Just 15% of amino acids found in yeast His3 orthologues were always neutral while the impact on fitness of the remaining 85% depended on the genetic background. Furthermore, at 67% of sites, amino acid replacements were under sign epistasis, having both strongly positive and negative effect in different genetic backgrounds. 46% of sites were under reciprocal sign epistasis. The fitness impact of amino acid replacements was influenced by only a few genetic backgrounds but involved interaction of multiple sites, shaping a rugged fitness landscape in which many of the shortest paths between highly fit genotypes are inaccessible.
Assuntos
Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Aptidão Genética , Leveduras/genética , Leveduras/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Epistasia Genética , Proteínas Fúngicas/química , Genes Fúngicos , Genótipo , Hidroliases/química , Hidroliases/genética , Hidroliases/metabolismo , Modelos Genéticos , Modelos Moleculares , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Despite being widely used in reporter technologies, bioluminescent systems are largely understudied. Of at least forty different bioluminescent systems thought to exist in nature, molecular components of only seven light-emitting reactions are known, and the full biochemical pathway leading to light emission is only understood for two of them. Here, we provide a succinct overview of currently known bioluminescent systems highlighting available tools for research and discussing future applications.
Assuntos
Fenômenos Bioquímicos/genética , Luciferases/genética , Proteínas Luminescentes/genética , Biologia de Sistemas , Luciferases/química , Proteínas Luminescentes/química , Imagem Molecular/tendênciasRESUMO
Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.
Assuntos
Fungos/genética , Proteínas Luminescentes/genética , Sequência de Aminoácidos , Animais , Vias Biossintéticas/genética , Ácidos Cafeicos , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Duplicação Gênica/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Alinhamento de Sequência , Xenopus laevisRESUMO
We present protein-PAINT - the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes.
RESUMO
Rapid development of new microscopy techniques exposed the need for genetically encoded fluorescent tags with special properties. Recent works demonstrated the potential of fluorescent proteins with tryptophan-based chromophores. We applied rational design and random mutagenesis to the monomeric red fluorescent protein FusionRed and found two groups of mutants carrying a tryptophan-based chromophore: with yellow (535 nm) or orange (565 nm) emission. On the basis of the properties of proteins, a model synthetic chromophore, and a computational modeling, we concluded that the presence of a ketone-containing chromophore in different isomeric forms can explain the observed yellow and orange phenotypes.
Assuntos
Corantes Fluorescentes/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Coloração e Rotulagem/métodos , Triptofano/química , Cor , Simulação por Computador , Corantes Fluorescentes/síntese química , Concentração de Íons de Hidrogênio , Cetonas/química , Estrutura Molecular , Isoformas de Proteínas/química , Proteína Vermelha FluorescenteRESUMO
Fitness landscapes depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence or in different sequences. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.
Assuntos
Aptidão Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Animais , Epistasia Genética , Evolução Molecular , Fluorescência , Estudos de Associação Genética , Genótipo , Hidrozoários/química , Hidrozoários/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , FenótipoRESUMO
Genetically encoded photosensitizers, proteins that produce reactive oxygen species when illuminated with visible light, are increasingly used as optogenetic tools. Their applications range from ablation of specific cell populations to precise optical inactivation of cellular proteins. Here, we report an orange mutant of red fluorescent protein KillerRed that becomes toxic when illuminated with blue or green light. This new protein, KillerOrange, carries a tryptophan-based chromophore that is novel for photosensitizers. We show that KillerOrange can be used simultaneously and independently from KillerRed in both bacterial and mammalian cells offering chromatic orthogonality for light-activated toxicity.