RESUMEN
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.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Membrana Celular/metabolismo , Luciferasas/metabolismo , Luminiscencia , Fragmentos de Péptidos/análisis , Receptores Adrenérgicos beta 2/metabolismo , Regulación Alostérica , Unión Competitiva , Transferencia de Energía , Células HEK293 , Humanos , Cinética , Ligandos , Fragmentos de Péptidos/metabolismo , Unión Proteica , Transporte de ProteínasRESUMEN
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.
Asunto(s)
Proteínas de Artrópodos/metabolismo , Endocitosis , Ensayos Analíticos de Alto Rendimiento/métodos , Luciferasas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/metabolismo , Descubrimiento de Drogas/métodos , Endocitosis/efectos de los fármacos , Colorantes Fluorescentes/química , Genes Reporteros/efectos de los fármacos , Células HEK293 , Humanos , Interleucina-6/química , Interleucina-6/genética , Interleucina-6/metabolismo , Cinética , Ligandos , Luciferasas/química , Luciferasas/genética , Microscopía Confocal , Microscopía Fluorescente , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Señales de Clasificación de Proteína/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Dual-specific A-kinase-anchoring protein 2 (D-AKAP2/AKAP10), which interacts at its carboxyl terminus with protein kinase A and PDZ domain proteins, contains two tandem regulator of G-protein signaling (RGS) domains for which the binding partners have remained unknown. We show here that these RGS domains interact with Rab11 and GTP-bound Rab4, the first demonstration of RGS domains binding small GTPases. Rab4 and Rab11 help regulate membrane trafficking through the endocytic recycling pathways by recruiting effector proteins to specific membrane domains. Although D-AKAP2 is primarily cytosolic in HeLa cells, a fraction of the protein localizes to endosomes and can be recruited there to a greater extent by overexpression of Rab4 or Rab11. D-AKAP2 also regulates the morphology of the Rab11-containing compartment, with co-expression causing accumulation of both proteins on enlarged endosomes. Knockdown of D-AKAP2 by RNA interference caused a redistribution of both Rab11 and the constitutively recycling transferrin receptor to the periphery of cells. Knockdown also caused an increase in the rate of transferrin recycling, suggesting that D-AKAP2 promotes accumulation of recycling proteins in the Rab4/Rab11-positive endocytic recycling compartment.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Receptores de Transferrina/química , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab4/química , Línea Celular , Citosol/metabolismo , Endocitosis , Endosomas/metabolismo , Citometría de Flujo , Células HeLa , Humanos , Estructura Terciaria de Proteína , Interferencia de ARN , Transferrina/químicaRESUMEN
Engineering of protein-protein interactions is used to enhance the affinity or specificity of proteins, such as antibodies or protease inhibitors, for their targets. However, fully diversifying all residues in a protein-protein interface is often unfeasible. Therefore, we limited our phage library for the serine protease inhibitor ecotin by restricting it to only tetranomial diversity and then targeted all 20 amino acid residues involved in protein recognition. This resulted in a high-affinity and highly specific plasma kallikrein inhibitor, ecotin-Pkal. To validate this approach we dissected the energetic contributions of each wild type (wt) or mutated surface loop to the binding of either plasma kallikrein (PKal) or membrane-type serine protease 1. The analysis demonstrated that a mutation in one loop has opposing effects depending on the sequence of surrounding loops. This finding stresses the cooperative nature of loop-loop interactions and justifies targeting multiple loops with a limited diversity. In contrast to ecotin wt, the specific loop combination of ecotin-Pkal discriminates the subtle structural differences between the active enzymes, PKal and Factor XIIa, and their respective zymogen forms. We used ecotin-Pkal to specifically inhibit contact activation of human plasma at the level mediated by plasma kallikrein.
Asunto(s)
Calicreína Plasmática/antagonistas & inhibidores , Calicreína Plasmática/metabolismo , Inhibidores de Proteasas/farmacología , Ingeniería de Proteínas/métodos , Activación Enzimática/efectos de los fármacos , Precursores Enzimáticos/antagonistas & inhibidores , Precursores Enzimáticos/metabolismo , Humanos , Mutación , Tiempo de Tromboplastina Parcial , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Proteínas Periplasmáticas/farmacología , Inhibidores de Proteasas/metabolismo , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Proteínas Luminiscentes/genética , Oligopéptidos/genética , Proteínas Adaptadoras Transductoras de Señales , Anticuerpos/química , Transferencia de Energía por Resonancia de Bioluminiscencia , Proteína 9 Asociada a CRISPR/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Genes Reporteros/genética , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Leupeptinas/farmacología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Luciferasas/metabolismo , Luminiscencia , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Streptococcus pyogenes/enzimologíaRESUMEN
Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 µM) and association constants (kon = 500 M(-1) s(-1), koff = 0.2 s(-1)) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction between SME-1 ß-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and ß-arrestin-2, were rapid, reversible, and robust to temperature (21-37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Secuencia de Aminoácidos , Arrestinas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Células HeLa , Humanos , Cinética , Sustancias Luminiscentes/química , Sustancias Luminiscentes/metabolismo , Mediciones Luminiscentes/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Arrestina beta 2 , beta-Arrestinas , beta-Lactamasas/metabolismoRESUMEN
Ecotin is a dimeric periplasmic protein from Escherichia coli that has been shown to inhibit potently many trypsin-fold serine proteases of widely varying substrate specificity. To help elucidate the physiological function of ecotin, we examined the family of ecotin orthologues, which are present in a subset of Gram-negative bacteria. Phylogenetic analysis suggested that ecotin has an exogenous target, possibly neutrophil elastase. Recombinant protein was expressed and purified from E. coli, Yersinia pestis and Pseudomonas aeruginosa, all species that encounter the mammalian immune system, and also from the plant pathogen Pantoea citrea. Notably, the Pa. citrea variant inhibits neutrophil elastase 1000-fold less potently than the other orthologues. All four orthologues are dimeric proteins that potently inhibit (<10 pM) the pancreatic digestive proteases trypsin and chymotrypsin, while showing more variable inhibition (5 pM to 24 microM) of the blood proteases Factor Xa, thrombin and urokinase-type plasminogen activator. To test whether ecotin does, in fact, protect bacteria from neutrophil elastase, an ecotin-deficient strain was generated in E. coli. This strain is significantly more sensitive in cell-killing assays to human neutrophil elastase, which causes increased permeability of the outer membrane that persists even during renewed bacterial growth. Ecotin affects primarily the ability of E. coli to recover and grow following treatment with neutrophil elastase, rather than the actual rate of killing. This suggests that an important part of the antimicrobial mechanism of neutrophil elastase may be a periplasmic bacteriostatic effect of protease that has translocated across the damaged outer membrane.
Asunto(s)
Proteínas de Escherichia coli/fisiología , Bacterias Gramnegativas/metabolismo , Elastasa de Leucocito/antagonistas & inhibidores , Proteínas Periplasmáticas/fisiología , Inhibidores de Serina Proteinasa/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/ultraestructura , Humanos , Cinética , Elastasa de Leucocito/farmacología , Datos de Secuencia Molecular , Pantoea/genética , Pantoea/metabolismo , Proteínas Periplasmáticas/genética , Filogenia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/genética , Especificidad de la Especie , Yersinia pestis/genética , Yersinia pestis/metabolismoRESUMEN
Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.
Asunto(s)
Crustáceos/enzimología , Genes Reporteros , Luciferasas/análisis , Luciferasas/genética , Ingeniería de Proteínas , Pirazinas/metabolismo , Animales , Línea Celular , Crustáceos/química , Crustáceos/genética , Crustáceos/metabolismo , Estabilidad de Enzimas , Luciérnagas/enzimología , Expresión Génica , Humanos , Luciferasas/metabolismo , Sustancias Luminiscentes/análisis , Sustancias Luminiscentes/metabolismo , Modelos Moleculares , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Renilla/enzimología , TemperaturaRESUMEN
The second messenger cAMP is a key mediator of signal transduction following activation of G-protein coupled receptors. Investigations on Gs-coupled receptors would benefit from a second messenger assay that allows continuous monitoring of kinetic changes in cAMP concentration over a broad dynamic range. To accomplish this, we have evolved a luminescent biosensor for cAMP to better encompass the physiological concentration ranges present in living cells. When compared to an immunoassay, the evolved biosensor construct was able to accurately track both the magnitude and kinetics of cAMP change using a far less labor intensive format. We demonstrate the utility of this construct to detect a broad range of receptor activity, together with showing suitability for use in high-throughput screening.