RESUMO
Highly sensitive self-cleavable trimethyl lock quinone-luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H-dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.
Assuntos
Luciferina de Vaga-Lumes/análogos & derivados , Substâncias Luminescentes/metabolismo , NADP/metabolismo , Quinonas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Luciferina de Vaga-Lumes/análise , Luciferina de Vaga-Lumes/metabolismo , Humanos , Substâncias Luminescentes/análise , Medições Luminescentes , NADP/análise , Quinonas/análiseRESUMO
Identification of proteins resolved by SDS-PAGE depends on robust in-gel protein digestion and efficient peptide extraction, requirements that are often difficult to achieve. A lengthy and laborious procedure is an additional challenge of protein identification in gel. We show here that with the use of the mass spectrometry compatible surfactant sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, the challenges of in-gel protein digestion are effectively addressed. Peptide quantitation based on stable isotope labeling showed that the surfactant induced 1.5-2 fold increase in peptide recovery. Consequently, protein sequence coverage was increased by 20-30%, on average, and the number of identified proteins saw a substantial boost. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from gel eliminating the need for postdigestion extraction. This study shows that the surfactant provides an efficient means of improving protein identification in gel and streamlining the in-gel digestion procedure requiring no extra handling steps or special equipment.
Assuntos
Proteínas/metabolismo , Tensoativos/química , Géis/química , Géis/metabolismo , Estrutura Molecular , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tensoativos/metabolismoRESUMO
Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.
RESUMO
Five novel firefly luciferin analogues in which the benzothiazole ring system of the natural substrate was replaced with benzimidazole, benzofuran, benzothiophene, benzoxazole, and indole were synthesized. The fluorescence, bioluminescence, and kinetic properties of the compounds were evaluated with recombinant Photinus pyralis wild type luciferase. With the exception of indole, all of the substrates containing heterocycle substitutions produced readily measurable flashes of light with luciferase. Compared to that of luciferin, the intensities ranged from 0.3 to 4.4% in reactions with varying pH optima and times to reach maximal intensity. The heteroatom changes influenced both the fluorescence and bioluminescence emission spectra, which displayed maxima of 479-528 and 518-574 nm, respectively. While there were some interesting trends in the spectroscopic and bioluminescence properties of this group of structurally similar substrate analogues, the most significant findings were associated with the benzothiophene-containing compound. This synthetic substrate produced slow decay glow kinetics that increased the total light-based specific activity of luciferase more than 4-fold versus the luciferin value. Moreover, over the pH range of 6.2-9.4, the emission maximum is 523 nm, an unusual 37 nm blue shift compared to that of the natural substrate. The extraordinary bioluminescence properties of the benzothiophene luciferin should translate into greater sensitivity for analyte detection in a wide variety of luciferase-based applications.
Assuntos
Luciferina de Vaga-Lumes/química , Compostos Heterocíclicos/química , Luminescência , Espectrofotometria UltravioletaRESUMO
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.
Assuntos
Crustáceos/enzimologia , Genes Reporter , Luciferases/análise , Luciferases/genética , Engenharia de Proteínas , Pirazinas/metabolismo , Animais , Linhagem Celular , Crustáceos/química , Crustáceos/genética , Crustáceos/metabolismo , Estabilidade Enzimática , Vaga-Lumes/enzimologia , Expressão Gênica , Humanos , Luciferases/metabolismo , Substâncias Luminescentes/análise , Substâncias Luminescentes/metabolismo , Modelos Moleculares , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Renilla/enzimologia , TemperaturaRESUMO
INTRODUCTION: The cytochrome P450s (CYPs) are central to ADME studies because of their central role in drug metabolism. Proper CYP assay design and a correct understanding of CYP assay selectivity are critical for generating and interpreting biologically relevant data during drug development. Bioluminescent CYP assays use luminogenic probe substrates that have the unique property of producing photons in a second reaction with luciferase. AREAS COVERED: This article presents the general design principles for in vitro CYP assays. Specifically, the article focuses on the bioluminescent approach that couples CYP activity with photon production. EXPERT OPINION: Highly selective luminogenic substrates for CYP1A1, CYP1A2, CYP2C9, CYP3A4, CYP3A7, CYP4A and CYP4F have been developed with utility for interrogating the roles of these enzymes in biochemical and cell-based formats. These selective substrates are part of a larger collection of probes that deliver CYP inhibition and induction data that predict in vivo drug interactions. Furthermore, they support highly sensitive, rapid and scalable assays for cell-based and cell-free biochemical applications, which offer an alternative and often enabling option over conventional assay strategies.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Substâncias Luminescentes/metabolismo , Medições Luminescentes/métodos , Inibidores das Enzimas do Citocromo P-450 , Interações Medicamentosas , Humanos , Luciferases/metabolismo , Microssomos Hepáticos/enzimologia , Sondas Moleculares/metabolismo , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
Detailed knowledge of drug metabolism is relevant information provided by preclinical drug development research. Oxidative enzymes such as those belonging to P450 family of cytochromes (CYP) play a prominent role in drug metabolism. Here, we propose an innovative method based on bioluminescence in vivo imaging which has the potential to simplify the in vivo measurement of CYP activity also providing a dynamic measure of the effects of a drug on a specific P450 enzyme complex in a living mouse. The method is based on a pro-luciferin which can be converted into the active luciferase substrate by a specific P450 activity. The pro-luciferin is administered to a luciferase reporter mouse which produces luminescent signals in relation to the cytochrome activity present in each tissue. The photon emission generated can be easily localized and quantified by optical imaging. To demonstrate the validity of the system, we pharmacologically induced hepatic Cyp3a in the reporter mouse and proved that pro-luciferin administration generates a Cyp3a selective signal in the chest area that can be efficiently detected by optical imaging. The kind of tool generated has the potential to be exploited for the study of additional CYPs.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Acetais/metabolismo , Animais , Dexametasona/farmacologia , Luciferina de Vaga-Lumes/análogos & derivados , Luciferina de Vaga-Lumes/metabolismo , Genes Reporter , Fígado/efeitos dos fármacos , Fígado/metabolismo , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Substâncias Luminescentes/metabolismo , Medições Luminescentes , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Distribuição TecidualRESUMO
Among the many molecular imaging techniques, reporter gene imaging has been a dynamic area of research. The HaloTag protein is a modified haloalkane dehalogenase which was designed to covalently bind to synthetic ligands (i.e. the HaloTag ligands [HTL]). Covalent bond formation between the HaloTag protein and the chloroal-kane within the HTL occurs rapidly under physiological conditions, which is highly specific and essentially irreversible. Over the years, HaloTag technology has been investigated for various applications such as in vitro/in vivo imaging, protein purification/trafficking, high-throughput assays, among others. The goal of this study is to explore the use of the HaloTag protein as a novel reporter gene for positron emission tomography (PET) imaging. By attaching a HaloTag -reactive chloroalkane to 1, 4, 7-triazacyclononane-N, N', N"-triacetic acid (NOTA) through hydrophilic linkers, the resulting NOTA-conjugated HTLs were labeled with (64)Cu and tested for PET imaging in living mice bearing 4T1-HaloTag-ECS tumors, which stably express the HaloTag protein on the cell surface. Significantly higher uptake of (64)Cu-NOTA-HTL-S (which contains a short hydrophilic linker) in the 4T1-HaloTag-ECS than the non-HaloTag-expressing 4T1 tumors was observed, which demonstrated the HaloTag specificity of (64)Cu-NOTA-HTL-S and warranted future investigation of the HaloTag protein as a PET reporter gene.
RESUMO
Cytochrome P450 (P450) assays use probe substrates to interrogate the influence of new chemical entities toward P450 enzymes. We report the synthesis and study of a family of bioluminogenic luciferin acetal substrates that are oxidized by P450 enzymes to form luciferase substrates. The luciferin acetals were screened against a panel of purified P450 enzymes. In particular, one proluciferin acetal has demonstrated sensitive and selective CYP3A4-catalyzed oxidation to a luciferin ester-K(m) and k(cat) are 2.88 µM and 5.87 pmol metabolite · min(-1) · pmol enzyme(-1), respectively. The proluciferin acetal was used as a probe substrate to measure IC(50) values of known inhibitors against recombinant CYP3A4 or human liver microsomes. IC(50) values for the known inhibitors correlate strongly with IC(50) values calculated from the traditional high-performance liquid chromatography-based probe substrate testosterone. Luciferin acetals are rapidly oxidized to unstable hemi-orthoesters by CYP3A resulting in luciferin esters and, therefore, are conducive to simple rapid CYP3A bioluminescent assays.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Cromatografia Líquida de Alta Pressão , Inibidores do Citocromo P-450 CYP3A , Humanos , Concentração Inibidora 50 , Microssomos Hepáticos/enzimologia , Sondas Moleculares , Especificidade por SubstratoRESUMO
A bioluminescent general protease assay was developed using a combination of five luminogenic peptide substrates. The peptide-conjugated luciferin substrates were combined with luciferase to form a homogeneous, coupled-enzyme assay. This single-reagent format minimized backgrounds, gave stable signals, and reached peak sensitivity within 30 min. The bioluminescent assay was used to detect multiple proteases representing serine, cysteine, and metalloproteinase classes. The range of proteases detected was broader and the sensitivity greater, when compared with a standard fluorescent assay based on cleavage of the whole protein substrate casein. Fifteen of twenty proteases tested had signal-to-background ratios >10 with the bioluminescent method, compared with only seven proteases with the fluorescent approach. The bioluminescent assay also achieved lower detection limits (≤100 pg) than fluorescent methods. During protein purification processes, especially for therapeutic proteins, even trace levels of contamination can impact the protein's stability and activity. This sensitive, bioluminescent, protease assay should be useful for applications in which contaminating proteases are detrimental and protein purity is essential.
Assuntos
Luciferina de Vaga-Lumes/análogos & derivados , Corantes Fluorescentes/metabolismo , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes/métodos , Peptídeo Hidrolases/análise , Caseínas/química , Caseínas/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Substâncias Luminescentes/química , Modelos Químicos , Oligopeptídeos/metabolismo , Peptídeo Hidrolases/classificação , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/normas , Sensibilidade e EspecificidadeRESUMO
Chromophore-assisted light inactivation (CALI) is a potentially powerful tool for the acute disruption of a target protein inside living cells with high spatiotemporal resolution. This technology, however, has not been widely utilized, mainly because of the lack of an efficient chromophore as the photosensitizing agent for singlet oxygen ((1)O(2)) generation and the difficulty of covalently labeling the target protein with the chromophore. Here we choose eosin as the photosensitizing chromophore showing 11-fold more production of ((1)O(2)) than fluorescein and about 5-fold efficiency in CALI of ß-galactosidase by using an eosin-labeled anti-ß-galactosidase antibody compared with the fluorescein-labeled one. To covalently label target protein with eosin, we synthesize a membrane-permeable eosin ligand for HaloTag technology, demonstrating easy labeling and efficient inactivation of HaloTag-fused PKC-γ and aurora B in living cells. These antibody- and HaloTag-based CALI techniques using eosin promise effective biomolecule inactivation that is applicable to many cell biological assays in living cells.
Assuntos
Amarelo de Eosina-(YS)/farmacologia , Fármacos Fotossensibilizantes/farmacologia , beta-Galactosidase/antagonistas & inibidores , Aurora Quinase B , Aurora Quinases , Corantes Fluorescentes/farmacologia , Células HeLa , Humanos , Lasers , Ligantes , Luz , Proteína Quinase C/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/efeitos da radiação , Oxigênio Singlete , beta-Galactosidase/imunologia , beta-Galactosidase/efeitos da radiaçãoRESUMO
As a means of reliably measuring intracellular pH, we have precisely targeted 5(and 6-) carboxyseminaphthorhodafluor to cellular subcompartments. This was accomplished by combining the well-established pH-sensitive dye with a protein-based reporter system. When expressed in cells, the reporter protein is designed to covalently bind ligands composed of a functional group and a reactive linker. In order to make a pH-sensitive ligand, we chemically coupled the pH sensor to a reactive linker. Several ligands of differing linker lengths were made and tested for their pH responsiveness in vitro. The most responsive of these ligands was then evaluated for its efficacy in live cell labeling and its use as an intracellular pH sensor for ratiometric confocal microscopy. Here we show that we could target this pH sensor within mammalian cells exclusively to either the nucleus or cytoplasm. Exhibiting the versatility of this reporter technology, we were also able to specifically limit pH sensor labeling to within the trafficking pathway of integrins and directly measure pH of this environment. Results correspond well with previously published reports. Both the simplicity and flexibility of the technology used in this study make possible the development of diverse targeted microenvironmental sensors or other moieties of interest.
Assuntos
Benzopiranos/análise , Técnicas Biossensoriais/métodos , Naftóis/análise , Rodaminas/análise , Frações Subcelulares/química , Benzopiranos/química , Transporte Biológico , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Estrutura Molecular , Naftóis/química , Rodaminas/químicaRESUMO
HaloTag is a protein fusion tag which was genetically engineered to covalently bind a series of specific synthetic ligands. All ligands carry two groups, the reactive group and the functional/reporter group. The reactive group, the choloroalkane, is the same in all the ligands and is involved in binding to the HaloTag. The functional reporter group is variable and can carry many different moieties including fluorescent dyes, affinity handles like biotin or solid surfaces such as agarose beads. Thus, HaloTag can serve either as a labeling tag or as a protein immobilization tag depending on which ligand is bound to it. Here, we describe a procedure for immobilization of HaloTag fusion proteins and how immobilized proteins can be used to study protein-protein and protein-DNA interactions in vivo and in vitro.
Assuntos
DNA/química , Proteínas/química , Clonagem Molecular , Ligantes , Ligação ProteicaRESUMO
A set of 6'-alkylated aminoluciferins are shown to be bioluminescent substrates for Ultra-Glo and QuantiLum luciferases. These studies demonstrate that both the engineered and wild-type firefly luciferases tolerate much greater steric bulk at the 6' position of luciferin than has been previously reported. The nature of the alkyl substituent strongly affects the strength of the bioluminescent signal, which varies widely based on size, shape, and charge. Several compounds were observed to generate more light than the corresponding unsubstituted 6'-aminoluciferin. Determination of Michaelis-Menten constants for the substrates with Ultra-Glo indicated that the variation arises primarily from differences in V max, ranging from 1.33 x 10 (4) to 332 x 10 (4) relative light units, but in some cases K m (0.73-10.8 microM) also plays a role. Molecular modeling results suggest that interactions of the side chain with a hydrogen-bonding network at the base of the luciferin binding pocket may influence substrate-enzyme binding.
Assuntos
Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/metabolismo , Luciferases/metabolismo , Alquilação , Animais , Domínio Catalítico , Cinética , Luz , Luciferases/química , Luciferases de Vaga-Lume/genética , Luminescência , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.
Assuntos
Técnicas Biossensoriais/métodos , Células/citologia , Corantes Fluorescentes/química , Medições Luminescentes/métodos , Proteínas Luminescentes/química , Coloração e Rotulagem , Animais , Sítios de Ligação , Células/metabolismo , DNA/análise , DNA/química , DNA/metabolismo , Enzimas Imobilizadas , Humanos , Hidrocarbonetos Clorados/química , NF-kappa B/análise , NF-kappa B/metabolismo , Proteínas/análise , Proteínas/química , Proteínas/metabolismo , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The ability to specifically label proteins within living cells can provide information about their dynamics and function. To study a membrane protein, we fused a multi-functional reporter protein, HaloTag, to the extracellular domain of a truncated integrin. RESULTS: Using the HaloTag technology, we could study the localization, trafficking and processing of an integrin-HaloTag fusion, which we showed had cellular dynamics consistent with native integrins. By labeling live cells with different fluorescent impermeable and permeable ligands, we showed spatial separation of plasma membrane and internal pools of the integrin-HaloTag fusion, and followed these protein pools over time to study bi-directional trafficking. In addition to combining the HaloTag reporter protein with different fluorophores, we also employed an affinity tag to achieve cell capture. CONCLUSION: The HaloTag technology was used successfully to study expression, trafficking, spatial separation and real-time translocation of an integrin-HaloTag fusion, thereby demonstrating that this technology can be a powerful tool to investigate membrane protein biology in live cells.
Assuntos
Bioensaio/métodos , Corantes Fluorescentes/metabolismo , Genes Reporter/genética , Proteômica/métodos , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem/métodos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Citoplasma/metabolismo , Humanos , Integrinas/metabolismo , Proteínas Luminescentes/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genéticaRESUMO
This article describes a novel two-step homogeneous bioluminescent assay for monoamine oxidase (MAO) that is simple, sensitive, and amenable to high-throughput screening. In the first step, MAO reacts with an aminopropylether analog of methyl ester luciferin. In the second step, a luciferin detection reagent inactivates MAO and converts the product of the first step into a luminescent signal. The amount of light produced is proportional to the amount of MAO and the time of incubation in the first step, but the luminescent signal is stable in the second step with a half-life greater than 5h. The assay has high precision, is more sensitive than current fluorescent methods, and can accurately measure the binding constants of known substrates and inhibitors. An automated screen of the Sigma-RBI Library of Pharmacologically Active Compounds (LOPAC(1280)) revealed a surprisingly high percentage of MAO inhibitors (16%) with a low false hit rate (0.9%). This implies that a significant number of compounds interact with the MAO enzymes and suggests that it is important to include MAO assays in drug metabolism studies. Other advantages of this bioluminescent assay over comparable fluorescent assays are discussed.
Assuntos
Medições Luminescentes/métodos , Inibidores da Monoaminoxidase/metabolismo , Monoaminoxidase/análise , Monoaminoxidase/metabolismo , Animais , Cinética , Luciferases/metabolismo , Luciferases/farmacocinética , Redes e Vias Metabólicas , Éteres Metílicos/química , Éteres Metílicos/farmacocinética , Camundongos , Mitocôndrias Hepáticas/metabolismo , Sensibilidade e EspecificidadeRESUMO
New highly sensitive latent bioluminescent luciferin substrates were designed and synthesized for monitoring mammalian glutathione S-transferase (GST) and Schistosoma japonicum enzyme activities.
Assuntos
Luciferina de Vaga-Lumes/química , Glutationa Transferase/química , Medições Luminescentes , Animais , Luciferina de Vaga-Lumes/síntese química , Glutationa/química , Indicadores e Reagentes , Estrutura Molecular , Schistosoma/enzimologia , Sensibilidade e EspecificidadeRESUMO
Novel bioluminogenic substrates were designed for probing monoamine oxidase (MAO) activity based on a simple and effective beta-elimination strategy. By modifying the amino group and the central core of luciferin derivatives, we have developed a series of substrates useful for assays of MAO A or B, or both. One of these substrates, exhibiting low Km values and high signal-to-background ratios with both isozymes, was shown to accurately measure the Ki values of known MAO inhibitors. This substrate is a key component in the development of a highly sensitive homogeneous MAO assay for high-throughput screening (HTS) of compounds in drug discovery and for monitoring MAO activity in complex biological systems. This design strategy should be applicable to fluorogenic MAO substrates and could broaden the structural requirements of substrates for other enzyme assays.