RESUMO
A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.
Assuntos
Ligação Proteica/efeitos dos fármacos , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bioensaio/métodos , Técnicas Biossensoriais , Linhagem Celular Tumoral , Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Transporte Proteico/efeitos dos fármacos , Células U937RESUMO
We combined fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G protein-coupled receptors, receptor tyrosine kinases, and ion channels, which were previously not suitable for high-throughput screening by flow cytometry. The system has been validated using the ß2-adrenergic receptor (ß2AR) system and extended to other GPCRs. When a chemical library containing â¼ 1200 off-patent drugs was screened against cells expressing FAP-tagged ß2AR, all known ß2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a nontraditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de-orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification.
Assuntos
Técnicas Biossensoriais , Sistemas Computacionais , Citometria de Fluxo/métodos , Corantes Fluorescentes/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Endocitose , Humanos , Transporte Proteico , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Células U937RESUMO
Monitoring the trafficking of multiple proteins simultaneously in live cells is of great interest because many receptor proteins are found to function together with others in the same cell. However, existing fluorescent labeling techniques have restricted the mechanistic study of functional receptor pairs. We have expanded a hybrid system combining fluorogen-activating protein (FAP) technology and high-throughput flow cytometry to a new type of biosensor that is robust, sensitive, and versatile. This provides the opportunity to study multiple trafficking proteins in the same cell. Human beta2 adrenergic receptor (ß2AR) fused with FAP AM2.2 and murine C-C chemokines receptor type 5 fused with FAP MG13 was chosen for our model system. The function of the receptor and the binding between MG13 and fluorogen MG-2p have been characterized by flow cytometry and confocal microscopy assays. The binding of fluorogen and the FAP pair is highly specific, while both FAP-tagged fusion proteins function similarly to their wild-type counterparts. The system has successfully served as a counter screen assay to eliminate false positive compounds identified in a screen against NIH Molecular Libraries Small Molecule Repository targeting regulators of the human ß2AR.
Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Proteínas Recombinantes de Fusão/química , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Quimiocina CCL5/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo/métodos , Humanos , Concentração Inibidora 50 , Isoproterenol/farmacologia , Ligação Proteica , Receptores Adrenérgicos beta 2/biossíntese , Receptores CCR5/agonistas , Receptores CCR5/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Corantes de Rosanilina/química , Células U937RESUMO
We developed a platform combining fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the ß2-adrenergic receptor (ß2AR) system. When a chemical library containing â¼1200 off-patent drugs was screened against cells expressing FAP-tagged ß2ARs, all 33 known ß2AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner. Results indicated that the platform identified ligands of target proteins regardless of the associated signaling pathway; therefore, this approach presents opportunities to search for biased receptor modulators and is suitable for screening of multiplexed targets for improved efficiency. The results revealed that ligands may be biased with respect to the rate or duration of receptor internalization and that receptor internalization may be independent of activation of the mitogen-activated protein kinase pathway.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Ligação Competitiva , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/genética , Humanos , Ligantes , Transporte Proteico , Receptores Adrenérgicos beta 2/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transfecção , Células U937RESUMO
As yeast cultures enter stationary phase in rich, glucose-based medium, differentiation of two major subpopulations of cells, termed quiescent and nonquiescent, is observed. Differences in mRNA abundance between exponentially growing and stationary-phase cultures and quiescent and nonquiescent cells are known, but little was known about protein abundance in these cells. To measure protein abundance in exponential and stationary-phase cultures, the yeast GFP-fusion library (4159 strains) was examined during exponential and stationary phases, using high-throughput flow cytometry (HyperCyt). Approximately 5% of proteins in the library showed twofold or greater changes in median fluorescence intensity (abundance) between the two conditions. We examined 38 strains exhibiting two distinct fluorescence-intensity peaks in stationary phase and determined that the two fluorescence peaks distinguished quiescent and nonquiescent cells, the two major subpopulations of cells in stationary-phase cultures. GFP-fusion proteins in this group were more abundant in quiescent cells, and half were involved in mitochondrial function, consistent with the sixfold increase in respiration observed in quiescent cells and the relative absence of Cit1p:GFP in nonquiescent cells. Finally, examination of quiescent cell-specific GFP-fusion proteins revealed symmetry in protein accumulation in dividing quiescent and nonquiescent cells after glucose exhaustion, leading to a new model for the differentiation of these cells.
Assuntos
Proteômica , Saccharomyces cerevisiae/fisiologia , Ciclo Celular/fisiologia , Citometria de Fluxo , Regulação Fúngica da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Consumo de Oxigênio , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cells in glucose-limited Saccharomyces cerevisiae cultures differentiate into quiescent (Q) and nonquiescent (NQ) fractions before entering stationary phase. To understand this differentiation, Q and NQ cells from 101 deletion-mutant strains were tested for viability and reproductive capacity. Eleven mutants that affected one or both phenotypes in Q or NQ fractions were identified. NQ fractions exhibit a high level of petite colonies, and nine mutants affecting this phenotype were identified. Microarray analysis revealed >1300 mRNAs distinguished Q from NQ fractions. Q cell-specific mRNAs encode proteins involved in membrane maintenance, oxidative stress response, and signal transduction. NQ-cell mRNAs, consistent with apoptosis in these cells, encode proteins involved in Ty-element transposition and DNA recombination. More than 2000 protease-released mRNAs were identified only in Q cells, consistent with these cells being physiologically poised to respond to environmental changes. Our results indicate that Q and NQ cells differentiate significantly, with Q cells providing genomic stability and NQ cells providing nutrients to Q cells and a regular source of genetic diversity through mutation and transposition. These studies are relevant to chronological aging, cell cycle, and genome evolution, and they provide insight into complex responses that even simple organisms have to starvation.