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/enzimologiaRESUMO
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.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Sequência de Aminoácidos , Arrestinas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HEK293 , Células HeLa , Humanos , Cinética , Substâncias Luminescentes/química , Substâncias Luminescentes/metabolismo , Medições Luminescentes/métodos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , beta-Arrestina 2 , beta-Arrestinas , beta-Lactamases/metabolismoRESUMO
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/metabolismoRESUMO
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.