RESUMO
Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present "translation-activating RNAs" (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs. We minimize the taRNA scaffold to 94 nucleotides, identify two translation initiation factor proteins responsible for taRNA activity, and validate the technology by amplifying SYNGAP1 expression, a haploinsufficiency disease target, in patient-derived cells. Finally, taRNAs are suitable for delivery as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a general and compact nucleic acid-based technology to upregulate protein production from endogenous mRNAs, and may open up possibilities for therapeutic RNA research.
Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas , Animais , Camundongos , Humanos , Regulação para Cima , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sítios Internos de Entrada Ribossomal , Mamíferos/genéticaRESUMO
The spread of SARS-CoV-2 and ongoing COVID-19 pandemic underscores the need for new treatments. Here we report that cannabidiol (CBD) inhibits infection of SARS-CoV-2 in cells and mice. CBD and its metabolite 7-OH-CBD, but not THC or other congeneric cannabinoids tested, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after viral entry, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD inhibits SARS-CoV-2 replication in part by up-regulating the host IRE1α RNase endoplasmic reticulum (ER) stress response and interferon signaling pathways. In matched groups of human patients from the National COVID Cohort Collaborative, CBD (100 mg/ml oral solution per medical records) had a significant negative association with positive SARS-CoV-2 tests. This study highlights CBD as a potential preventative agent for early-stage SARS-CoV-2 infection and merits future clinical trials. We caution against use of non-medical formulations including edibles, inhalants or topicals as a preventative or treatment therapy at the present time.
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Antivirais/farmacologia , Canabidiol/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , COVID-19/virologia , Canabidiol/química , Canabidiol/metabolismo , Chlorocebus aethiops , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Células Epiteliais/virologia , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Interferons/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , SARS-CoV-2/fisiologia , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
Ligand-dependent biosensors are valuable tools for coupling the intracellular concentrations of small molecules to easily detectable readouts such as absorbance, fluorescence, or cell growth. While ligand-dependent biosensors are widely used for monitoring the production of small molecules in engineered cells and for controlling or optimizing biosynthetic pathways, their application to directed evolution for biocatalysts remains underexplored. As a consequence, emerging continuous evolution technologies are rarely applied to biocatalyst evolution. Here, we develop a panel of ligand-dependent biosensors that can detect a range of small molecules. We demonstrate that these biosensors can link enzymatic activity to the production of an essential phage protein to enable biocatalyst-dependent phage-assisted continuous evolution (PACE) and phage-assisted continuous selection (PACS). By combining these phage-based evolution and library selection technologies, we demonstrate that we can evolve enzyme variants with improved and expanded catalytic properties. Finally, we show that the genetic diversity resulting from a highly mutated PACS library is enriched for active enzyme variants with altered substrate scope. These results lay the foundation for using phage-based continuous evolution and selection technologies to engineer biocatalysts with novel substrate scope and reactivity.
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The continued toll of COVID-19 has halted the smooth functioning of civilization on a global scale. With a limited understanding of all the essential components of viral machinery and the lack of structural information of this new virus, initial drug discovery efforts had limited success. The availability of high-resolution crystal structures of functionally essential SARS-CoV-2 proteins, including 3CLpro, supports the development of target-specific therapeutics. 3CLpro, the main protease responsible for the processing of viral polypeptide, plays a vital role in SARS-CoV-2 viral replication and translation and is an important target in other coronaviruses. Additionally, 3CLpro is the target of repurposed drugs, such as lopinavir and ritonavir. In this study, target proteins were retrieved from the protein data bank (PDB IDs: 6 M03, 6LU7, 2GZ7, 6 W63, 6SQS, 6YB7, and 6YVF) representing different open states of the main protease to accommodate macromolecular substrate. A hydroxyethylamine (HEA) library was constructed from harvested chemical structures from all the series being used in our laboratories for screening against malaria and Leishmania parasites. The database consisted of â¼1000 structure entries, of which 70% were new to ChemSpider at the time of screening. This in-house library was subjected to high throughput virtual screening (HTVS), followed by standard precision (SP) and then extra precision (XP) docking (Schrodinger LLC 2021). The ligand strain and complex energy of top hits were calculated by Molecular Mechanics Generalized Born Surface Area (MM/GBSA) method. Promising hit compounds (n = 40) specifically binding to 3CLpro with high energy and average MM/GBSA scores were then subjected to (100-ns) MD simulations. Using this sequential selection followed by an in-silico validation approach, we found a promising HEA-based compound (N,N'-((3S,3'S)-piperazine-1,4-diylbis(3-hydroxy-1-phenylbutane-4,2-diyl))bis(2-(5-methyl-1,3-dioxoisoindolin-2-yl)-3-phenylpropanamide)), which showed high in vitro antiviral activity against SARS-CoV-2. Further to reduce the size of the otherwise larger ligand, a pharmacophore-based predicted library of â¼42 derivatives was constructed, which were added to the previous compound library and rescreened virtually. Out of several hits from the predicted library, two compounds were synthesized, tested against SARS-CoV-2 culture, and found to have markedly improved antiviral activity.
Assuntos
Antivirais/química , Proteases 3C de Coronavírus/antagonistas & inibidores , Etilaminas/química , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Proteases 3C de Coronavírus/metabolismo , Etilaminas/metabolismo , Etilaminas/farmacologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , SARS-CoV-2/isolamento & purificação , Termodinâmica , Células VeroRESUMO
There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Coronavirus Humano OC43/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , SARS-CoV-2/efeitos dos fármacos , Tiazóis/farmacologia , Células A549 , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Benzamidas , COVID-19/virologia , Domínio Catalítico , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Coronavirus Humano OC43/fisiologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Células HEK293 , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Transgênicos , Testes de Sensibilidade Microbiana , Piperidinas , Piridinas , SARS-CoV-2/enzimologia , SARS-CoV-2/fisiologia , Tiazóis/química , Tiazóis/metabolismo , Tiazóis/uso terapêutico , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV-2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARS-CoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.
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Studies of biological function demand probes that can report on processes in real time and in physiological environments. Bioluminescent tools are uniquely suited for this purpose, as they enable sensitive imaging in cells and tissues. Bioluminescent reporters can also be monitored continuously over time without detriment, as excitation light is not required. Rather, light emission derives from luciferase-luciferin reactions. Several engineered luciferases and luciferins have expanded the scope of bioluminescence imaging in recent years. Multicomponent tracking remains challenging, though, due to a lack of streamlined methods to visualize combinations of bioluminescent reporters. Conventional approaches image one luciferase at a time. Consequently, short-term changes in cell growth or gene expression cannot be easily captured. Here, we report a strategy for rapid, multiplexed imaging with a wide range of luciferases and luciferins. Sequential addition of orthogonal luciferins, followed by substrate unmixing, enabled facile detection of multiple luciferases in vitro and in vivo. Multicomponent imaging in mice was also achieved on the minutes-to-hours time scale.
Assuntos
Medições Luminescentes , Animais , Células HEK293 , Humanos , Sondas Moleculares , Especificidade por SubstratoRESUMO
The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to expand. Papain-like protease (PLpro) is one of two SARS-CoV-2 proteases potentially targetable with antivirals. PLpro is an attractive target because it plays an essential role in cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex, and disruption of host responses. We report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the SARS-CoV-2 enzyme. We determined the high resolution structure of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors. This collection of structures details inhibitors recognition and interactions providing fundamental molecular and mechanistic insight into PLpro. All compounds inhibit the peptidase activity of PLpro in vitro, some block SARS-CoV-2 replication in cell culture assays. These findings will accelerate structure-based drug design efforts targeting PLpro to identify high-affinity inhibitors of clinical value.
Assuntos
Papaína/metabolismo , Peptídeo Hidrolases/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Antivirais/farmacologia , Humanos , Mutação , Poliproteínas/metabolismo , Especificidade por Substrato , Replicação Viral/efeitos dos fármacosRESUMO
SARS-CoV-2, the virus that causes COVID-19 consists of several enzymes with essential functions within its proteome. Here, we focused on repurposing approved and investigational drugs/compounds. We targeted seven proteins with enzymatic activities known to be essential at different stages of the viral cycle including PLpro, 3CLpro, RdRP, Helicase, ExoN, NendoU, and 2'-O-MT. For virtual screening, energy minimization of a crystal structure of the modeled protein was carried out using the Protein Preparation Wizard (Schrodinger LLC 2020-1). Following active site selection based on data mining and COACH predictions, we performed a high-throughput virtual screen of drugs and investigational molecules (nâ¯=â¯5903). The screening was performed against viral targets using three sequential docking modes (i.e., HTVS, SP, and XP). Virtual screening identified â¼290 potential inhibitors based on the criteria of energy, docking parameters, ligand, and binding site strain and score. Drugs specific to each target protein were further analyzed for binding free energy perturbation by molecular mechanics (prime MM-GBSA) and pruning the hits to the top 32 candidates. The top lead from each target pool was further subjected to molecular dynamics simulation using the Desmond module. The resulting top eight hits were tested for their SARS-CoV-2 anti-viral activity in-vitro. Among these, a known inhibitor of protein kinase C isoforms, Bisindolylmaleimide IX (BIM IX), was found to be a potent inhibitor of SARS-CoV-2. Further, target validation through enzymatic assays confirmed 3CLpro to be the target. This is the first study that has showcased BIM IX as a COVID-19 inhibitor thereby validating our pipeline.
Assuntos
Antivirais/administração & dosagem , Proteases 3C de Coronavírus/antagonistas & inibidores , Sistemas de Liberação de Medicamentos/normas , Indóis/administração & dosagem , Maleimidas/administração & dosagem , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Antivirais/metabolismo , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos/métodos , Reposicionamento de Medicamentos/normas , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos , Indóis/química , Indóis/metabolismo , Maleimidas/química , Maleimidas/metabolismo , Simulação de Acoplamento Molecular/métodos , Simulação de Acoplamento Molecular/normas , Estrutura Secundária de Proteína , Reprodutibilidade dos Testes , SARS-CoV-2/químicaRESUMO
All aspects of mRNA lifetime and function, including its stability, translation into protein, and trafficking through the cell, are tightly regulated through coordinated post-transcriptional modifications and interactions with a multitude of RNA effector proteins. Despite the increasing recognition of RNA regulation as a critical layer of mammalian gene expression control and its increasing excitement as a therapeutic target, tools to study and control RNA regulatory mechanisms with temporal precision in their endogenous environment are lacking. Here, we present small molecule-inducible RNA-targeting effectors based on our previously developed CRISPR/Cas-inspired RNA targeting system (CIRTS). The CIRTS biosensor platform is based on guide RNA (gRNA)-dependent RNA binding domains that interact with a target transcript using Watson-Crick-Franklin base pair interactions. Addition of a small molecule recruits an RNA effector to the target transcript, thereby eliciting a local effect on the transcript. In this work, we showcase that these CIRTS biosensors can trigger inducible RNA editing, degradation, or translation on target transcripts in a small molecule-dependent manner. We further go on to show that the CIRTS RNA base editor biosensor can induce RNA base editing in a small molecule-controllable manner in vivo. Collectively this work provides a new set of tools to probe the dynamics of RNA regulatory systems and control gene expression at the RNA level.
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There is an urgent need for anti-viral agents that treat SARS-CoV-2 infection. The shortest path to clinical use is repurposing of drugs that have an established safety profile in humans. Here, we first screened a library of 1,900 clinically safe drugs for inhibiting replication of OC43, a human beta-coronavirus that causes the common-cold and is a relative of SARS-CoV-2, and identified 108 effective drugs. We further evaluated the top 26 hits and determined their ability to inhibit SARS-CoV-2, as well as other pathogenic RNA viruses. 20 of the 26 drugs significantly inhibited SARS-CoV-2 replication in human lung cells (A549 epithelial cell line), with EC50 values ranging from 0.1 to 8 micromolar. We investigated the mechanism of action for these and found that masitinib, a drug originally developed as a tyrosine-kinase inhibitor for cancer treatment, strongly inhibited the activity of the SARS-CoV-2 main protease 3CLpro. X-ray crystallography revealed that masitinib directly binds to the active site of 3CLpro, thereby blocking its enzymatic activity. Mastinib also inhibited the related viral protease of picornaviruses and blocked picornaviruses replication. Thus, our results show that masitinib has broad anti-viral activity against two distinct beta-coronaviruses and multiple picornaviruses that cause human disease and is a strong candidate for clinical trials to treat SARS-CoV-2 infection.
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Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small-molecule-based imaging agents are widely used in biological studies, and small-molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small-molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show that the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure protein-protein interactions and protein-protein interaction inhibitor engagement. We demonstrate that the esterase-based reporter system is compatible with other commonly used split reporter imaging systems for the simultaneous detection of multiple protein-protein interactions. Finally, we develop a system for selective small-molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show that the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications.
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Enzymes are powerful catalysts for site-selective C-H bond functionalization. Identifying suitable enzymes for this task and for biocatalysis in general remains challenging, however, due to the fundamental difficulty of predicting catalytic activity from sequence information. In this study, family-wide activity profiling was used to obtain sequence-function information on flavin-dependent halogenases (FDHs). This broad survey provided a number of insights into FDH activity, including halide specificity and substrate preference, that were not apparent from the more focused studies reported to date. Regions of FDH sequence space that are most likely to contain enzymes suitable for halogenating small-molecule substrates were also identified. FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compounds were characterized and used for preparative-scale late-stage C-H functionalization. In many cases, these enzymes provide activities that required several rounds of directed evolution to accomplish in previous efforts, highlighting that this approach can achieve significant time savings for biocatalyst identification and provide advanced starting points for further evolution.
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New applications for bioluminescence imaging require an expanded set of luciferase enzymes and luciferin substrates. Here, we report two novel luciferins for use in vitro and in cells. These molecules comprise regioisomeric pyridone cores that can be accessed from a common synthetic route. The analogues exhibited unique emission spectra with firefly luciferase, although photon intensities remained weak. Enhanced light outputs were achieved by using mutant luciferase enzymes. One of the luciferin-luciferase pairs produced light on par with native probes in live cells. The pyridone analogues and complementary luciferases add to a growing set of designer probes for bioluminescence imaging.
Assuntos
Luciferina de Vaga-Lumes/análogos & derivados , Luciferases de Vaga-Lume/genética , Substâncias Luminescentes/química , Mutação , Imagem Óptica/métodos , Piridonas/química , Animais , Vaga-Lumes/química , Vaga-Lumes/enzimologia , Células HEK293 , Humanos , Isomerismo , Luciferases de Vaga-Lume/química , Luminescência , Medições Luminescentes/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate "hits" were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multicomponent imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.
Assuntos
Luciferina de Vaga-Lumes/química , Luciferases/química , Medições Luminescentes , Animais , Células Cultivadas , Vaga-Lumes , Luciferina de Vaga-Lumes/síntese química , Luciferina de Vaga-Lumes/metabolismo , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Estrutura Molecular , Engenharia de ProteínasRESUMO
We report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed by using a common synthetic route. All analogues produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed by computation and photophysical measurements. The brightest brominated luciferin was further evaluated in cell and animal models. At low doses, the analogue outperformed the native substrate in cells. The remaining luciferins, although weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.
Assuntos
Luciferina de Vaga-Lumes/metabolismo , Medições Luminescentes , Animais , Linhagem Celular Tumoral , Vaga-Lumes/enzimologia , Luciferina de Vaga-Lumes/análogos & derivados , Luciferina de Vaga-Lumes/síntese química , Células HEK293 , Halogenação , Humanos , Cinética , Luz , Luciferases de Vaga-Lume/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Bioluminescence imaging with luciferase enzymes and luciferin small molecules is a well-established technique for tracking cells and other biological features in rodent models. Despite its popularity, bioluminescence has long been hindered by a lack of distinguishable probes. Here we present a method to rapidly identify new substrate-selective luciferases for multicomponent imaging. Our strategy relies on parallel screening of luciferin analogues with panels of mutant enzymes. The compiled data set is then analyzed in silico to uncover mutually orthogonal sets. Using this approach, we screened 159 mutant enzymes with 12 luciferins. Thousands of orthogonal pairs were revealed with sufficient selectivity for use in biological environments. Over 100 pairs were validated in vitro, and three were applied in cell and animal models. The parallel screening method is both generalizable and scalable and will streamline the search for larger collections of orthogonal probes.
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Toxoplasma gondii is an obligate intracellular parasite that infects all nucleated cell types in diverse warm-blooded organisms. Many of the surface antigens and effector molecules secreted by the parasite during invasion and intracellular growth are modified by glycans. Glycosylated proteins in the nucleus and cytoplasm have also been reported. Despite their prevalence, the complete inventory and biological significance of glycosylated proteins in Toxoplasma remain unknown. In this study, we aimed to globally profile parasite glycoproteins using a bioorthogonal chemical reporter strategy. This strategy involves the metabolic incorporation of unnatural functional groups (i.e., "chemical reporters") into Toxoplasma glycans, followed by covalent labeling with visual probes or affinity tags. The two-step approach enables the visualization and identification of newly biosynthesized glycoconjugates in the parasite. Using a buffer that mimics intracellular conditions, extracellular Toxoplasma tachyzoites were found to metabolize and incorporate unnatural sugars (equipped with bioorthogonal functional groups) into diverse proteins. Covalent chemistries were used to visualize and retrieve these labeled structures. Subsequent mass spectrometry analysis revealed 89 unique proteins. This survey identified novel proteins as well as previously characterized proteins from lectin affinity analyses.
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Metabolismo dos Carboidratos , Glicoproteínas/análise , Proteínas de Protozoários/análise , Toxoplasma/metabolismo , Coloração e RotulagemRESUMO
Cell-cell interactions underlie fundamental biological processes but remain difficult to visualize over long times and large distances in tissues and live organisms. Bioluminescence imaging with luciferase-luciferin pairs is sufficiently sensitive to image cells in vivo but lacks the spatial resolution to identify cellular locations and interactions. To repurpose this technology for visualizing cellular networks, we developed a "caged" luciferin that produces light only when cells are in close contact. This molecule comprises a nitroaromatic core that can be selectively reduced ("uncaged") by one cell type, liberating a luciferin that can be selectively consumed by neighboring, luciferase-expressing cells. When the two cell types are in contact, robust light emission is observed. This imaging strategy will enable the noninvasive visualization of cell-cell interactions relevant to organismal biology.
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Benzotiazóis/metabolismo , Comunicação Celular , Substâncias Luminescentes/metabolismo , Bactérias/enzimologia , Benzotiazóis/análise , Células HEK293 , Humanos , Cinética , Luciferases de Vaga-Lume/metabolismo , Substâncias Luminescentes/análise , Medições Luminescentes , Nitrorredutases/metabolismo , Imagem ÓpticaRESUMO
Cell-cell interactions underlie diverse physiological processes ranging from immune function to cell migration. Dysregulated cellular crosstalk also potentiates numerous pathologies, including infections and metastases. Despite their ubiquity in organismal biology, cell-cell interactions are difficult to examine in tissues and whole animals without invasive procedures. Here, we report a strategy to noninvasively image cell proximity using engineered bioluminescent probes. These tools comprise "split" fragments of Gaussia luciferase (Gluc) fused to the leucine zipper domains of Fos and Jun. When cells secreting the fragments draw near one another, Fos and Jun drive the assembly of functional, light-emitting Gluc. Photon production thus provides a readout on the distance between two cell types. We used the split fragments to visualize cell-cell interactions over time in vitro and in macroscopic models of cell migration. Further application of these tools in live organisms will refine our understanding of cell contacts relevant to basic biology and disease.