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1.
Nat Methods ; 21(3): 406-410, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38253843

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 , Mamíferos
2.
J Biol Chem ; 295(15): 5124-5135, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32107310

RESUMO

G protein-coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the ß-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Membrana Celular/metabolismo , Luciferases/metabolismo , Luminescência , Fragmentos de Peptídeos/análise , Receptores Adrenérgicos beta 2/metabolismo , Regulação Alostérica , Ligação Competitiva , Transferência de Energia , Células HEK293 , Humanos , Cinética , Ligantes , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Transporte Proteico
3.
Anal Chem ; 93(12): 5177-5184, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33730483

RESUMO

Enzyme-linked immunosorbent assays (ELISAs) are used extensively for the detection and quantification of biomolecules in clinical diagnostics as well as in basic research. Although broadly used, the inherent complexities of ELISAs preclude their utility for straightforward point-of-need testing, where speed and simplicity are essential. With this in mind, we developed a bioluminescence-based immunoassay format that provides a sensitive and simple method for detecting biomolecules in clinical samples. We utilized a ternary, split-NanoLuc luciferase complementation reporter consisting of two small peptides (11mer, 13mer) and a 17 kDa polypeptide combined with a luminogenic substrate to create a complete, shelf-stable add-and-read assay detection reagent. Directed evolution was used to optimize reporter constituent sequences to impart chemical and thermal stability, as well as solubility, while formulation optimization was applied to stabilize an all-in-one reagent that can be reconstituted in aqueous buffers or sample matrices. The result of these efforts is a robust, first-generation bioluminescence-based homogenous immunoassay reporter platform where all assay components can be configured into a stable lyophilized cake, supporting homogeneous, rapid, and sensitive one-step biomolecule quantification in complex human samples. This technology represents a promising alternative immunoassay format with significant potential to bring critical diagnostic molecular detection testing closer to the point-of-need.


Assuntos
Testes Imunológicos , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoensaio , Indicadores e Reagentes , Luciferases/genética
4.
Nat Methods ; 12(7): 661-663, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26030448

RESUMO

Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein-coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Receptores Acoplados a Proteínas G/metabolismo , Fluorescência , Células HEK293 , Humanos , Ligantes , Receptores Adrenérgicos beta 2/metabolismo
5.
Anal Biochem ; 555: 67-72, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29733811

RESUMO

Neddylation is a posttranslational modification that regulates protein stability, activity, and subcellular localization. Here, we describe a new tool for exploring the neddylation cycle of cullin1 (Cul1) directly in a cellular context. This assay utilizes the NanoLuc® Binary Technology (NanoBiT) to monitor the covalent neddylation status of Cul1. A stable clonal cell line derived from HEK293 was developed that expressed a C-terminus LgBiT tagged-Cul1 and N-terminus SmBiT tagged-Nedd8. Using this cell line, we screened inhibitors that are known to disrupt Nedd8 biology and demonstrated that both inhibitors of Nedd8-activating enzyme (NAE) and Constitutive photomorphogenesis 9 signalosome (CSN) complex produce concentration and time dependent signal decreases and increases, respectively. The kinetics of both responses could be monitored in real time and demonstrated that modulation of the Nedd8 pathway occurs rapidly. Further characterization of the cellular components of this cell line was performed in order to quantify the various levels of Cul1, Nedd8 and NAE and determined to be near endogenous levels. There was no difference between control and stably transfected cell lines in viability studies of NAE and CSN inhibitors. Taken together, these results suggest that the NanoBiT assay can be used to monitor Cul1 neddylation specifically and in real time.


Assuntos
Bioensaio/métodos , Proteínas Culina/metabolismo , Proteína NEDD8/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Culina/genética , Células HCT116 , Células HEK293 , Humanos , Proteína NEDD8/genética
6.
Org Biomol Chem ; 15(40): 8559-8567, 2017 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-28972606

RESUMO

We report the synthesis and characterization of novel coelenterazine analogues that demonstrate a red-shift in their bioluminescent emission with NanoLuc luciferase. These coelenterazines can be tuned to shift the bioluminescent emission from blue light in the native system. In particular, direct attachment of an aryl moiety to the imidazopyrazinone core of furimazine at the C8 position provides a significant red-shift while maintaining reasonable light output. In addition, modification of the C6 aryl moiety provided additive red-shifts, and by combining the most promising modifications we report a coelenterazine with a maximum emission near 600 nm with NanoLuc. Finally, we show that this new bioluminescent system is capable of efficient BRET to far-red fluorophores. We anticipate these new principles of NanoLuc substrate design will impact applications that depend on shifting the colour of emission to the red, most notably in vivo bioluminescent imaging.


Assuntos
Imidazóis/química , Luciferases/química , Substâncias Luminescentes/química , Pirazinas/química , Imidazóis/metabolismo , Luciferases/metabolismo , Substâncias Luminescentes/metabolismo , Medições Luminescentes , Estrutura Molecular , Pirazinas/metabolismo
7.
Chemistry ; 22(30): 10369-75, 2016 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-27305599

RESUMO

The growing popularity of bioluminescent assays has highlighted the need for coelenterazine analogues possessing properties tuned for specific applications. However, the structural diversity of known coelenterazine analogues has been limited by current syntheses. Known routes for the preparation of coelenterazine analogues employ harsh reaction conditions that limit access to many substituents and functional groups. Novel synthetic routes reported here establish simple and robust methods for synthesis and investigation of structurally diverse marine luciferase substrates. Specifically, these new routes allow synthesis of coelenterazine analogues containing various heterocyclic motifs and substituted aromatic groups with diverse electronic substituents at the R(2) position. Interesting analogues described herein were characterized by their physicochemical properties, bioluminescent half-life, light output, polarity and cytotoxicity. Some of the analogues represent leads that can be utilized in the development of improved bioluminescent systems.

8.
Anal Biochem ; 489: 1-8, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26278171

RESUMO

Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs.


Assuntos
Proteínas de Artrópodes/metabolismo , Endocitose , Ensaios de Triagem em Larga Escala/métodos , Luciferases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Descoberta de Drogas/métodos , Endocitose/efeitos dos fármacos , Corantes Fluorescentes/química , Genes Reporter/efeitos dos fármacos , Células HEK293 , Humanos , Interleucina-6/química , Interleucina-6/genética , Interleucina-6/metabolismo , Cinética , Ligantes , Luciferases/química , Luciferases/genética , Microscopia Confocal , Microscopia de Fluorescência , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Sinais Direcionadores de Proteínas/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
9.
J Proteome Res ; 11(2): 564-75, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22149079

RESUMO

Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization.


Assuntos
RNA Polimerases Dirigidas por DNA/química , Sondas Moleculares/química , Subunidades Proteicas/análise , Proteômica/métodos , Linhagem Celular , Núcleo Celular , Biologia Computacional , Citoplasma , RNA Polimerases Dirigidas por DNA/metabolismo , Bases de Dados de Proteínas , Células HEK293 , Humanos , Espectrometria de Massas , Microscopia de Fluorescência , Sondas Moleculares/metabolismo , Complexos Multiproteicos
10.
Protein Expr Purif ; 76(2): 154-64, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21129486

RESUMO

Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins.


Assuntos
Cromatografia de Afinidade/métodos , Clonagem Molecular/métodos , Proteínas Quinases/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Western Blotting , Técnicas de Cultura de Células , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Proteínas Imobilizadas/metabolismo , Proteínas Quinases/análise , Proteínas Quinases/metabolismo , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo
11.
ACS Chem Biol ; 16(2): 404-413, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33543920

RESUMO

Identification of physiologically relevant targets for lead compounds emerging from drug discovery screens is often the rate-limiting step toward understanding their mechanism of action and potential for undesired off-target effects. To this end, we developed a streamlined chemical proteomic approach utilizing a single, photoreactive cleavable chloroalkane capture tag, which upon attachment to bioactive compounds facilitates selective isolation of their respective cellular targets for subsequent identification by mass spectrometry. When properly positioned, the tag does not significantly affect compound potency and membrane permeability, allowing for binding interactions with the tethered compound (probe) to be established within intact cells under physiological conditions. Subsequent UV-induced covalent photo-cross-linking "freezes" the interactions between the probe and its cellular targets and prevents their dissociation upon cell lysis. Targets cross-linked to the capture tag are then efficiently enriched through covalent capture onto HaloTag coated beads and subsequent selective chemical release from the solid support. The tag's built-in capability for selective enrichment eliminates the need for ligation of a capture tag, thereby simplifying the workflow and reducing variability introduced through additional operational steps. At the same time, the capacity for adequate cross-linking without structural optimization permits modular assembly of photoreactive chloroalkane probes, which reduces the burden of customized chemistry. Using three model compounds, we demonstrate the capability of this approach to identify known and novel cellular targets, including those with low affinity and/or low abundance as well as membrane targets with several transmembrane domains.


Assuntos
Marcadores de Afinidade/química , Azidas/química , Reagentes de Ligações Cruzadas/química , Diazometano/análogos & derivados , Hidrocarbonetos Clorados/química , Proteômica/métodos , Marcadores de Afinidade/efeitos da radiação , Azidas/efeitos da radiação , Cromatografia Líquida , Reagentes de Ligações Cruzadas/efeitos da radiação , Dasatinibe/análogos & derivados , Dasatinibe/farmacologia , Dasatinibe/efeitos da radiação , Diazometano/efeitos da radiação , Histona Desacetilases/análise , Histona Desacetilases/química , Humanos , Hidrocarbonetos Clorados/efeitos da radiação , Hidrolases/química , Células K562 , Espectrometria de Massas , Propranolol/análogos & derivados , Propranolol/farmacologia , Propranolol/efeitos da radiação , Proteínas Quinases/análise , Proteínas Quinases/química , Receptores Adrenérgicos alfa 2/análise , Receptores Adrenérgicos alfa 2/química , Raios Ultravioleta , Vorinostat/análogos & derivados , Vorinostat/farmacologia , Vorinostat/efeitos da radiação
12.
Sci Rep ; 10(1): 8953, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488146

RESUMO

The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically, it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose, a large set of proteins representing diverse structures and functions, some of which are known or potential drug targets, were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86% of the targets, as determined by luminescence-based plate assays, blotting, and imaging. In order to determine whether endogenously tagged proteins yield more representative models, cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases, only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches.


Assuntos
Medições Luminescentes/métodos , Proteínas Luminescentes/análise , Proteínas/análise , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Plasmídeos
13.
PLoS One ; 15(12): e0243747, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33315907

RESUMO

Firefly luciferase-based ATP detection assays are frequently used as a sensitive, cost-efficient method for monitoring hygiene in many industrial settings. Solutions of detection reagent, containing a mixture of a substrate and luciferase enzyme that produces photons in the presence of ATP, are relatively unstable and maintain only a limited shelf life even under refrigerated conditions. It is therefore common for the individual performing a hygiene test to manually prepare fresh reagent at the time of monitoring. To simplify sample processing, a liquid detection reagent with improved thermal stability is needed. The engineered firefly luciferase, Ultra-Glo™, fulfills one aspect of this need and has been valuable for hygiene monitoring because of its high resistance to chemical and thermal inactivation. However, solutions containing both Ultra-Glo™ luciferase and its substrate luciferin gradually lose the ability to effectively detect ATP over time. We demonstrate here that dehydroluciferin, a prevalent oxidative breakdown product of luciferin, is a potent inhibitor of Ultra-Glo™ luciferase and that its formation in the detection reagent is responsible for the decreased ability to detect ATP. We subsequently found that dialkylation at the 5-position of luciferin (e.g., 5,5-dimethylluciferin) prevents degradation to dehydroluciferin and improves substrate thermostability in solution. However, since 5,5-dialkylluciferins are poorly utilized by Ultra-Glo™ luciferase as substrates, we used structural optimization of the luciferin dialkyl modification and protein engineering of Ultra-Glo™ to develop a luciferase/luciferin pair that shows improved total reagent stability in solution at ambient temperature. The results of our studies outline a novel luciferase/luciferin system that could serve as foundations for the next generation of bioluminescence ATP detection assays with desirable reagent stability.


Assuntos
Luciferina de Vaga-Lumes/química , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Trifosfato de Adenosina/química , Alquilação , Indicadores e Reagentes , Luciferases de Vaga-Lume/química , Especificidade por Substrato , Temperatura
14.
Wellcome Open Res ; 5: 20, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32587898

RESUMO

Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLuc â ® Binary Technology (NanoBiT â ®) was originally developed to detect protein-protein interactions in live mammalian cells. Here we report the successful translation of the NanoBit cellular assay into a biochemical, cell-free format using mammalian cell lysates. We show that the assay is suitable for the detection of both strong and weak protein interactions such as those involving the binding of RAS oncoproteins to either RAF or phosphoinositide 3-kinase (PI3K) effectors respectively, and that it is also effective for the study of poorly soluble protein domains such as the RAS binding domain of PI3K. Furthermore, the RAS interaction assay is sensitive and responds to both strong and weak RAS inhibitors. Our data show that the assay is robust, reproducible, cost-effective, and can be adapted for small and large-scale screening approaches. The NanoBit Biochemical Assay offers an attractive tool for drug screening against challenging protein-protein interaction targets, including the interaction of RAS with PI3K.

15.
Protein Expr Purif ; 68(1): 110-20, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19464373

RESUMO

Over-expression and purification of soluble and functional proteins remain critical challenges for many aspects of biomolecular research. To address this, we have developed a novel protein tag, HaloTag7, engineered to enhance expression and solubility of recombinant proteins and to provide efficient protein purification coupled with tag removal. HaloTag7 was designed to bind rapidly and covalently with a unique synthetic linker to achieve an essentially irreversible attachment. The synthetic linker may be attached to a variety of entities such as fluorescent dyes and solid supports, permitting labeling of fusion proteins in cell lysates for expression screening, and efficient capture of fusion proteins onto a purification resin. The combination of covalent capture with rapid binding kinetics overcomes the equilibrium-based limitations associated with traditional affinity tags and enables efficient capture even at low expression levels. Following immobilization on the resin, the protein of interest is released by cleavage at an optimized TEV protease recognition site, leaving HaloTag7 bound to the resin and pure protein in solution. Evaluation of HaloTag7 for expression of 23 human proteins in Escherichia coli relative to MBP, GST and His(6)Tag revealed that 74% of the proteins were produced in soluble form when fused to HaloTag7 compared to 52%, 39% and 22%, respectively, for the other tags. Using a subset of the test panel, more proteins fused to HaloTag7 were successfully purified than with the other tags, and these proteins were of higher yield and purity.


Assuntos
Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Humanos , Projetos Piloto , Proteínas Recombinantes de Fusão/genética , Solubilidade
16.
Methods Mol Biol ; 1888: 45-71, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30519940

RESUMO

Intracellular target affinity and residence time are fundamental aspects of pharmacological mechanism (Lu and Tonge, Curr Opin Chem Biol 14:467-474, 2010). Although various robust biochemical approaches exist to measure these binding characteristics, analysis of compound binding with isolated targets may not accurately reflect engagement in the milieu of living cells. To realize the influence of cellular context, methods are needed that are capable of quantifying affinity and residence time in the presence of the intracellular factors that may impact target engagement. Bioluminescence resonance energy transfer (BRET) offers a solution for intracellular target engagement when quantitative metrics or kinetic analyses are required.


Assuntos
Descoberta de Drogas/métodos , Transferência Ressonante de Energia de Fluorescência , Medições Luminescentes , Técnicas de Cultura de Células , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala , Humanos , Medições Luminescentes/métodos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Permeabilidade , Reprodutibilidade dos Testes
17.
Sci Rep ; 9(1): 7046, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31065015

RESUMO

Ligand binding assays routinely employ fluorescently-labeled protein ligands to quantify the extent of binding. These ligands are commonly generated through chemical modification of accessible lysine residues, which often results in heterogeneous populations exhibiting variable binding properties. This could be remedied by quantitative, site-specific labeling. Recently, we reported on a single-step method integrating recombinant protein purification with 2-cyanobenzothiazole (CBT) condensation for labeling a proteolytically exposed N-terminal cysteine. Here, using three growth factors, we show that unlike random lysine labeling, this site-specific approach yielded homogeneous populations of growth factors that were quantitatively labeled at their N-termini and retained their binding characteristics. We demonstrate the utility of this labeling method through the development of a novel assay that quantifies the capacity of antibodies to block receptor-ligand interactions (i.e. antibody blockade). The assay uses bioluminescence resonance energy transfer (BRET) to detect binding of CBT-labeled growth factors to their cognate receptors genetically fused to NanoLuc luciferase. The ability of antibodies to block these interactions is quantified through decrease in BRET. Using several antibodies, we show that the assay provides reliable quantification of antibody blockade in a cellular context. As demonstrated here, this simple method for generating uniformly-labeled proteins has potential to promote more accurate and robust ligand binding assays.


Assuntos
Anticorpos Bloqueadores/análise , Corantes Fluorescentes/química , Proteômica/métodos , Anticorpos Bloqueadores/metabolismo , Becaplermina/genética , Becaplermina/metabolismo , Benzopiranos/química , Benzotiazóis/química , Cetuximab/farmacologia , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Células HEK293 , Humanos , Indóis/química , Ligantes , Medições Luminescentes/métodos , Nitrilas/química , Panitumumabe/farmacologia , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
18.
Cell Chem Biol ; 26(6): 830-841.e9, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30956148

RESUMO

Vascular endothelial growth factor (VEGF) is an important mediator of endothelial cell proliferation and angiogenesis via its receptor VEGFR2. A common tumor associated with elevated VEGFR2 signaling is infantile hemangioma that is caused by a rapid proliferation of vascular endothelial cells. The current first-line treatment for infantile hemangioma is the ß-adrenoceptor antagonist, propranolol, although its mechanism of action is not understood. Here we have used bioluminescence resonance energy transfer and VEGFR2 genetically tagged with NanoLuc luciferase to demonstrate that oligomeric complexes involving VEGFR2 and the ß2-adrenoceptor can be generated in both cell membranes and intracellular endosomes. These complexes are induced by agonist treatment and retain their ability to couple to intracellular signaling proteins. Furthermore, coupling of ß2-adrenoceptor to ß-arrestin2 is prolonged by VEGFR2 activation. These data suggest that protein-protein interactions between VEGFR2, the ß2-adrenoceptor, and ß-arrestin2 may provide insight into their roles in health and disease.


Assuntos
Receptores Adrenérgicos beta 2/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Células Cultivadas , Corantes Fluorescentes/química , Células HEK293 , Humanos , Ligantes , Luciferases/química , Luciferases/metabolismo , Ligação Proteica , Receptores Adrenérgicos beta 2/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
19.
Cell Chem Biol ; 25(10): 1208-1218.e5, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30057299

RESUMO

Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF165a, VEGF165b, and VEGF121a. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A isoforms displayed high affinity for VEGFR2. Only VEGF165a-TMR bound to NanoLuc-NRP1 with a similar high affinity (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF165a > VEGF189a > VEGF145a. VEGF165b, VEGF-Ax, VEGF121a, and VEGF111a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2. These data emphasize the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signaling.


Assuntos
Neuropilina-1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transferência de Energia , Corantes Fluorescentes/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligantes , Medições Luminescentes , Ligação Proteica , Isoformas de Proteínas/metabolismo , Rodaminas/metabolismo
20.
ACS Chem Biol ; 13(2): 467-474, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-28892606

RESUMO

Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Proteínas Luminescentes/genética , Oligopeptídeos/genética , Proteínas Adaptadoras de Transdução de Sinal , Anticorpos/química , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Proteína 9 Associada à CRISPR/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Fatores de Transcrição de Resposta de Crescimento Precoce/metabolismo , Genes Reporter/genética , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Leupeptinas/farmacologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Luciferases/metabolismo , Luminescência , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Streptococcus pyogenes/enzimologia
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