ABSTRACT
A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types: n-hexane-acetonitrile-water (10:2:8, v/v), ethyl acetate-acetonitrile-water (10:2:8, v/v), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v). The lower phase of the n-hexane-acetonitrile-water (10:2:8, v/v) was used as the stationary phase, while its upper phase, as well as ethyl acetate-acetonitrile-water (10:2:8), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds from Centipeda minima, using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products.
Subject(s)
Asteraceae/chemistry , Chromatography, Liquid/methods , Countercurrent Distribution/methods , Plant Extracts/analysis , Solvents/chemistry , 1-Butanol/chemistry , Acetates/chemistry , Acetonitriles/chemistry , Antioxidant Response Elements/drug effects , Biological Assay , Cell Survival/drug effects , Chromatography, Liquid/instrumentation , Countercurrent Distribution/instrumentation , Crotonates/isolation & purification , Genes, Reporter/drug effects , Hep G2 Cells , Hexanes/chemistry , Humans , Luciferases/chemistry , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Quercetin/analogs & derivatives , Quercetin/isolation & purification , Sesquiterpenes/isolation & purification , Water/chemistryABSTRACT
The limited therapeutic options and increasing drug-resistance call for next-generation influenza antivirals. Due to the essential function in viral replication and high sequence conservation among influenza viruses, influenza polymerase PA-PB1 protein-protein interaction becomes an attractive drug target. Here, we developed an in vitro split luciferase complementation-based assay to speed up screening of PA-PB1 interaction inhibitors. By screening 10,000 compounds, we identified two PA-PB1 interaction inhibitors, R160792 and R151785, with potent and broad-spectrum antiviral activity against a panel of influenza A and B viruses, including amantadine-, oseltamivir-, or dual resistant strains. Further mechanistic study reveals that R151785 inhibits PA nuclear localization, reduces the levels of viral RNAs and proteins, and inhibits viral replication at the intermediate stage, all of which are in line with its antiviral mechanism of action. Overall, we developed a robust high throughput-screening assay for screening broad-spectrum influenza antivirals targeting PA-PB1 interaction and identified R151785 as a promising antiviral drug candidate.
Subject(s)
Antiviral Agents/chemistry , DNA-Directed RNA Polymerases/chemistry , Luciferases/chemistry , Nucleic Acid Synthesis Inhibitors/chemistry , Orthomyxoviridae/enzymology , Viral Proteins/chemistry , Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , High-Throughput Screening Assays , Humans , In Vitro Techniques , Molecular Docking Simulation , Molecular Structure , Nucleic Acid Synthesis Inhibitors/pharmacology , Protein Binding , Structure-Activity RelationshipABSTRACT
Firefly luciferase is an enzyme that has found ubiquitous use in biological assays in high-throughput screening (HTS) campaigns. The inhibition of luciferase in such assays could lead to a false positive result. This issue has been known for a long time, and there have been significant efforts to identify luciferase inhibitors in order to enhance recognition of false positives in screening assays. However, although a large amount of publicly accessible luciferase counterscreen data is available, to date little effort has been devoted to building a chemoinformatic model that can identify such molecules in a given data set. In this study we developed models to identify these molecules using various methods, such as molecular docking, SMARTS screening, pharmacophores, and machine learning methods. Among the structure-based methods, the pharmacophore-based method showed promising results, with a balanced accuracy of 74.2%. However, machine-learning approaches using associative neural networks outperformed all of the other methods explored, producing a final model with a balanced accuracy of 89.7%. The high predictive accuracy of this model is expected to be useful for advising which compounds are potential luciferase inhibitors present in luciferase HTS assays. The models developed in this work are freely available at the OCHEM platform at http://ochem.eu .
Subject(s)
Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , High-Throughput Screening Assays/methods , Luciferases/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , False Positive Reactions , Luciferases/chemistry , Luciferases/metabolism , Molecular Docking Simulation , Protein ConformationABSTRACT
Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that induces highly selective immunogenic cell death. It is based on an antibody-photoabsorber conjugate (APC) that is activated by NIR light. The purpose of this study was to investigate the effects of NIR-PIT as measured by luciferase-luciferin photon-counting and fluorescence imaging. Six days after subcutaneous injection of A431-luc-GFP cells tumors formed in a xenograft mouse model. The EGFR-targeting antibody, panitumumab, was conjugated to the photoabsorber, IRDye-700DX (pan-IR700), and was intravenously administered to tumor-bearing mice. Serial luciferase-luciferin photon-counting images and both green fluorescent protein (GFP) and IR700 fluorescence images were obtained from the same mice before and after NIR-PIT treatment (0, 10, 20, 30 min (early phase), and 24, 48 h (late phase) after NIR light exposure). Optical signal intensities were compared for each modality. IR700 fluorescence and luciferase-luciferin photon-counting images showed decreased intensities in both the early and late phases after NIR-PIT (p < 0.01). On the other hand, GFP fluorescence images showed decreased intensities only in the late phase (p < 0.01). In the early phase, GFP fluorescence images showed smaller intensity reductions compared to IR700 fluorescence and luciferase-luciferin photon-counting (p < 0.01), while in the late phase, IR700 fluorescence showed smaller intensity reductions than luciferase-luciferin photon-counting and GFP fluorescence (p < 0.05), due to redistribution of pan-IR700 within the tumor bed. In conclusion, luciferase-luciferin photon-counting imaging is suitable to evaluate early phase NIR-PIT effects, while both luciferase-luciferin photon-counting and GFP reflected later phase effects.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Carcinoma, Squamous Cell/diagnostic imaging , ErbB Receptors/antagonists & inhibitors , Immunotherapy/methods , Phototherapy/methods , Animals , Antibodies, Monoclonal/chemistry , Benzothiazoles/chemistry , Biomarkers, Tumor , Carcinoma, Squamous Cell/therapy , Cell Line, Tumor , Female , Humans , Immunoconjugates/chemistry , Immunoconjugates/therapeutic use , Indoles , Infrared Rays/therapeutic use , Luciferases/chemistry , Mice , Mice, Nude , Optical Imaging/methods , Organosilicon Compounds , Panitumumab , Photosensitizing Agents/chemistry , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Ebolaviruses cause severe hemorrhagic fever with high case fatality rates. Despite recent progress, there is a continued need for the development of antivirals against these viruses. Reporter-expressing ebolaviruses, which can be generated using reverse genetics systems, are powerful tools for antiviral screening. While viruses expressing fluorescent reporters are amenable for this purpose and can be used for high-content imaging-type screens, as an alternative, luciferase-expressing reporter viruses have recently been developed and have the advantages of being extremely easy to use and having short assay times. Here we provide a detailed protocol for the use of such a luciferase-expressing reporter virus for antiviral screening in a 96-well format, with parallel assessment of cytotoxicity of the screened compounds.
Subject(s)
Antiviral Agents/isolation & purification , Drug Evaluation, Preclinical/methods , Ebolavirus/drug effects , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Ebolavirus/chemistry , Ebolavirus/pathogenicity , Humans , Luciferases/chemistry , Luciferases/genetics , Virus Replication/drug effectsABSTRACT
α-synuclein (α-SYN) is a major pathologic contributor to Parkinson's disease (PD). Multiplication of α-SYN encoding gene (SNCA) is correlated with early onset of the disease underlining the significance of its transcriptional regulation. Thus, monitoring endogenous transcription of SNCA is of utmost importance to understand PD pathology. We developed a stable cell line expressing α-SYN endogenously tagged with NanoLuc luciferase reporter using CRISPR/Cas9-mediated genome editing. This allows efficient measurement of transcriptional activity of α-SYN in its native epigenetic landscape which is not achievable using exogenous transfection-based luciferase reporter assays. The NanoLuc activity faithfully monitored the transcriptional regulation of SNCA following treatment with different drugs known to regulate α-SYN expression; while exogenous promoter-reporter assays failed to reproduce the similar outcomes. To our knowledge, this is the first report showing endogenous monitoring of α-SYN transcription, thus making it an efficient drug screening tool that can be used for therapeutic intervention in PD.
Subject(s)
Gene Editing/methods , Parkinson Disease/drug therapy , Transcription, Genetic , alpha-Synuclein/genetics , CRISPR-Cas Systems/genetics , Cell Line , Drug Evaluation, Preclinical , Gene Expression Regulation , Humans , Luciferases/chemistry , Luciferases/genetics , Parkinson Disease/genetics , Parkinson Disease/pathology , Promoter Regions, Genetic , TransfectionABSTRACT
Metabolic pathways and bioenergetics were described in great detail over half a century ago, and during the past decade there has been a resurgence in integrating these cellular processes with other biological properties of the cell, including growth control, protein kinase cascade signaling, cell cycle division, and autophagy. Since many disease conditions are associated with altered metabolism and production of energy, it is important to develop new approaches to measure these cellular parameters. This chapter summarizes a new and exciting approach based on the Seahorse XF24 Extracelluar Flux analyzer, which takes real time measurements of oxidative phosphorylation and glycolysis in living cells. These bioenergetic profiles are then compared with steady-state levels of cellular ATP as measured by a luciferase assay.
Subject(s)
Adenosine Triphosphate/metabolism , Glycolysis , Metabolic Flux Analysis/methods , Oxidative Phosphorylation , Adenosine Triphosphate/chemistry , Animals , Calibration , Cell Culture Techniques , Cell Line, Tumor , Enzyme Assays/standards , Humans , Hydrolysis , Luciferases/chemistry , Metabolic Flux Analysis/instrumentation , Reference StandardsABSTRACT
Inositol 1,4,5-trisphosphate (IP(3)) is a crucial second messenger that regulates complicated signaling processes in various physiological events. Alteration in its content has been observed in many diseases. Hence, development of a high-throughput screening system to monitor temporal changes of IP(3) is essential for screening of new potential therapeutic compounds. Toward a simple, sensitive and rapid method for measuring IP(3), we describe the development and application of a novel biosensor based on luciferase fragment assisted complementation strategy, which converts the ligand-induced conformational changes to light. Designed sensor comprising the IP(3)-binding core domain of IP(3)-receptor fused between complementary non-functional fragments of firefly luciferase allows direct detection of IP(3) in presence of luciferin substrate both in cell lysate and in living cells. According to the result presented in this manuscript, the screening time was very fast and maximum response was obtained up to 11-fold higher than untreated cells. Moreover, the designed biosensor was able to monitor release of IP(3) upon induction by different inducers like Bradykinin and ATP. The current biosensor not only provides a specific IP(3) detector in vitro but also facilitates monitoring of the response of IP(3) in living organisms.
Subject(s)
Biosensing Techniques/instrumentation , Inositol 1,4,5-Trisphosphate Receptors/chemistry , Luciferases/chemistry , Luminescent Measurements/instrumentation , Equipment Design , Equipment Failure Analysis , Inositol 1,4,5-Trisphosphate , Luciferases/analysis , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Bioluminescence is the biologically active luminescence light producing event encountered in nature. In recent years several new screening methods utilizing bioluminescent cell-based biosensors have been designed demonstrating their utility towards dynamic monitoring of a variety of cellular functions. Because luciferase is unnatural to mammalian physiology, assays utilizing specific substrates to yield a luminescent signal are attractive and serve the purpose with high sensitivity and specificity. Often genetic or chemical modifications in different luciferase-substrate system in use have afforded new functionalities making these assays even more robust. Finally, in the evolving paradigm of molecular imaging, in vivo bioluminescence imaging (BLI) has evolved as a very attractive tool for interrogating human cellular biology in rodent models. In this short review we explore various bioluminescence screening strategies developed and analyze their scope in future drug screening processes.
Subject(s)
Biosensing Techniques/methods , Drug Evaluation, Preclinical/methods , Luciferases/chemistry , Luminescent Measurements/methods , Molecular Imaging/methods , Animals , HumansABSTRACT
Chinese herbal medicines are often applied as an alternative therapy for viral diseases. However, the development of anti-HIV herbal drugs has proceeded slowly, partly because of the lack of a high-throughput system for screening these drugs. The present study evaluated 16 herbal medicines for anti-HIV activities in vitro and in vivo. Herbal medicines were first screened for the ability to regulate C-X-C receptor 4 (CXCR4) and C-C receptor 5 (CCR5) promoter activities. A single-round pseudotyped HIV-luciferase reporter virus system (HIV-Luc) was used to identify potential anti-HIV mechanisms. CD4+ T cells from healthy volunteers were examined for changes in CXCR4 and CCR5 levels. HIV-1 replication was evaluated by ELISA. Spica Prunellae and Herba Andrographitis were found to down-regulate the activities of both the CXCR4 and CCR5 promoters. Also, Spica Prunellae and Herba Andrographitis (>1000 µM) inhibited HIV-1 in a dose-dependent manner. CXCR4 and CCR5 levels were reduced in CD4+ T cells from healthy volunteers (p<0.05). Spica Prunellae and Herba Andrographitis (EC50: 3.18 and 5.49 µg/mL, respectively) could suppress cell fusion and decrease p24 antigen. In conclusion, the data demonstrated that Spica Prunellae and Herba Andrographitis possessed anti-HIV-1 capabilities, perhaps through the inhibition of the CXCR4 and CCR5 promoters and HIV-1 replication.
Subject(s)
Anti-HIV Agents/pharmacology , Drug Evaluation, Preclinical/methods , Drugs, Chinese Herbal/pharmacology , HIV-1/drug effects , Adult , Andrographis/chemistry , Animals , CCR5 Receptor Antagonists , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Cell Survival , Dose-Response Relationship, Drug , Drug Evaluation , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/standards , Enzyme Activation , Enzyme Assays/methods , Enzyme-Linked Immunosorbent Assay , Female , Genes, Reporter , HEK293 Cells , HIV Core Protein p24/metabolism , HIV Infections/virology , HIV-1/pathogenicity , HIV-1/physiology , Humans , Leukocytes, Mononuclear/drug effects , Luciferases/chemistry , Luciferases/genetics , Male , Promoter Regions, Genetic , Prunella/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Receptors, CCR5/metabolism , Receptors, CXCR4/genetics , Receptors, CXCR4/metabolism , Transfection , Virus Replication , Young AdultABSTRACT
Inhibitors of both heat shock proteins Hsp90 and Hsp70 have been identified in assays measuring luciferase refolding containing rabbit reticulocyte lysate or purified chaperone components. Here, we report the discovery of a series of phenoxy-N-arylacetamides that disrupt Hsp70-mediated luciferase refolding by binding to DnaJ, the bacterial homolog of human Hsp40. Inhibitor characterization experiments demonstrated negative cooperativity with respect to DnaJ and luciferase concentration, but varying the concentration of ATP had no effect on potency. Thermal shift analysis suggested a direct interaction with DnaJ, but not with Hsp70. These compounds may be useful tools for studying DnaJ/Hsp40 in various cellular processes.
Subject(s)
Acetamides/chemistry , Acetamides/pharmacology , Escherichia coli Proteins/metabolism , HSP40 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/antagonists & inhibitors , Luciferases/chemistry , Luciferases/metabolism , Adenosine Triphosphate/metabolism , Animals , Drug Evaluation, Preclinical , HSP70 Heat-Shock Proteins/metabolism , Humans , Inhibitory Concentration 50 , Protein Folding , RabbitsABSTRACT
Protein-protein interactions are critical molecular determinants of ion channel function and emerging targets for pharmacological interventions. Yet, current methodologies for the rapid detection of ion channel macromolecular complexes are still lacking. In this study we have adapted a split-luciferase complementation assay (LCA) for detecting the assembly of the voltage-gated Na+ (Nav) channel C-tail and the intracellular fibroblast growth factor 14 (FGF14), a functionally relevant component of the Nav channelosome that controls gating and targeting of Nav channels through direct interaction with the channel C-tail. In the LCA, two complementary N-terminus and C-terminus fragments of the firefly luciferase were fused, respectively, to a chimera of the CD4 transmembrane segment and the C-tail of Nav1.6 channel (CD4-Nav1.6-NLuc) or FGF14 (CLuc-FGF14). Co-expression of CLuc-FGF14 and CD4-Nav1.6-NLuc in live cells led to a robust assembly of the FGF14:Nav1.6 C-tail complex, which was attenuated by introducing single-point mutations at the predicted FGF14:Nav channel interface. To evaluate the dynamic regulation of the FGF14:Nav1.6 C-tail complex by signaling pathways, we investigated the effect of kinase inhibitors on the complex formation. Through a platform of counter screenings, we show that the p38/MAPK inhibitor, PD169316, and the IκB kinase inhibitor, BAY 11-7082, reduce the FGF14:Nav1.6 C-tail complementation, highlighting a potential role of the p38MAPK and the IκB/NFκB pathways in controlling neuronal excitability through protein-protein interactions. We envision the methodology presented here as a new valuable tool to allow functional evaluations of protein-channel complexes toward probe development and drug discovery targeting ion channels implicated in human disorders.
Subject(s)
Ion Channel Gating/physiology , Luminescence , Luminescent Proteins/analysis , Sodium Channels/physiology , Algorithms , Amino Acids/analysis , Animals , Blotting, Western , Cell Survival , DNA/genetics , Fibroblast Growth Factors/genetics , Fireflies/chemistry , Genetic Complementation Test , Genetic Vectors , HEK293 Cells , Humans , Indicators and Reagents , Luciferases/chemistry , Models, Molecular , NAV1.6 Voltage-Gated Sodium Channel , Nerve Tissue Proteins/chemistry , Polymerase Chain Reaction/methods , Sodium Channels/chemistryABSTRACT
Here we engineered transgenic Leishmania infantum that express luciferase, the objectives being to more easily monitor in real time their establishment either in BALB/c mice--the liver and spleen being mainly studied-or in vitro. Whatever stationary phase L. infantum promastigotes population--wild type or engineered to express luciferase-the parasite burden was similar in the liver and the spleen at day 30 post the intravenous inoculation of BALB/c mice. Imaging of L. infantum hosting BALB/C mice provided sensitivity in the range of 20,000 to 40,000 amastigotes/mg tissue, two tissues-liver and spleen-being monitored. Once sampled and processed ex vivo for their luciferin-dependent bioluminescence the threshold sensitivity was shown to range from 1,000 to 6,000 amastigotes/mg tissue. This model further proved to be valuable for in vivo measurement of the efficiency of drugs such as miltefosine and may, therefore, additionally be used to evaluate vaccine-induced protection.
Subject(s)
Leishmania infantum/enzymology , Luciferases/analysis , Parasite Load/methods , Animals , Antiprotozoal Agents/pharmacology , Drug Evaluation, Preclinical/methods , Female , Leishmania infantum/genetics , Leishmania infantum/isolation & purification , Leishmaniasis, Visceral/drug therapy , Leishmaniasis, Visceral/parasitology , Life Cycle Stages , Liver/parasitology , Luciferases/biosynthesis , Luciferases/chemistry , Luciferases/genetics , Luminescent Measurements , Mice , Mice, Inbred BALB C , Organisms, Genetically Modified , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/pharmacology , Protozoan Proteins , Reproducibility of Results , Sensitivity and Specificity , Spectrum Analysis/methods , Spleen/parasitology , Statistics, Nonparametric , Whole Body ImagingABSTRACT
The ability to conditionally turn on a signal or induce a function in the presence of a user-defined RNA target has potential applications in medicine and synthetic biology. Although sequence-specific pumilio repeat proteins can target a limited set of ssRNA sequences, there are no general methods for targeting ssRNA with designed proteins. As a first step toward RNA recognition, we utilized the RNA binding domain of argonaute, implicated in RNA interference, for specifically targeting generic 2-nucleotide, 3' overhangs of any dsRNA. We tested the reassembly of a split-luciferase enzyme guided by argonaute-mediated recognition of newly generated nucleotide overhangs when ssRNA is targeted by a designed complementary guide sequence. This approach was successful when argonaute was utilized in conjunction with a pumilio repeat and expanded the scope of potential ssRNA targets. However, targeting any desired ssRNA remained elusive as two argonaute domains provided minimal reassembled split-luciferase. We next designed and tested a second hierarchical assembly, wherein ssDNA guides are appended to DNA hairpins that serve as a scaffold for high affinity zinc fingers attached to split-luciferase. In the presence of a ssRNA target containing adjacent sequences complementary to the guides, the hairpins are brought into proximity, allowing for zinc finger binding and concomitant reassembly of the fragmented luciferase. The scope of this new approach was validated by specifically targeting RNA encoding VEGF, hDM2, and HER2. These approaches provide potentially general design paradigms for the conditional reassembly of fragmented proteins in the presence of any desired ssRNA target.
Subject(s)
Luciferases/chemistry , RNA/chemistry , DNA/chemistry , DNA/genetics , Humans , Luciferases/genetics , Luciferases/metabolism , Models, Molecular , Protein Conformation , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/genetics , RNA/genetics , Receptor, ErbB-2/chemistry , Receptor, ErbB-2/genetics , Vascular Endothelial Growth Factors/chemistry , Vascular Endothelial Growth Factors/geneticsABSTRACT
The identification of a novel pyrazolidine-3,5-dione based scaffold hit compound as Farnesoid X receptor (FXR) partial or full agonist has been accomplished by means of virtual screening techniques. A series of pyrazolidine-3,5-dione derivatives (1a-u and 7) was designed, synthesized, and evaluated by a cell-based luciferase transactivation assay for their agonistic activities against FXR. Most of them showed agonistic potencies and 10 of them (1a, 1b, 1d-f, 1j, 1n, 1t, 5b, and 7) exhibited lower EC(50) values than the reference drug CDCA. Molecular modeling studies for the representative compounds 1a, 1d, 1f, 1j, 1n, 1u, 5b, and 7 were also presented. The novel structural scaffold has provided a new direction for finding potent and selective FXR partial and full agonists (referred to as 'selective bile acid receptor modulators', SBARMs).
Subject(s)
DNA-Binding Proteins/agonists , Drug Evaluation, Preclinical/methods , Pyrazoles/chemical synthesis , Receptors, Cytoplasmic and Nuclear/agonists , Transcription Factors/agonists , Bile Acids and Salts/chemistry , Chemistry, Pharmaceutical/methods , DNA-Binding Proteins/chemistry , Dose-Response Relationship, Drug , Drug Design , Humans , Hydrogen Bonding , Ligands , Luciferases/chemistry , Models, Chemical , Models, Molecular , Molecular Conformation , Pyrazoles/pharmacology , Receptors, Cytoplasmic and Nuclear/chemistry , Transcription Factors/chemistry , Transcriptional ActivationABSTRACT
Hyperthermia is used to treat various malignancies, including esophageal, stomach and rectal cancer. Since hyperthermia alone has produced limited results, much attention has been focused on combining hyperthermia with chemotherapy and on searching for substances able to sensitize tumor cells to hyperthermia-induced damage. Here, we show that vitamins K1 and K2 (VK1, VK2) inhibited the expression of heat-shock protein 72 (Hsp72) but did not affect the constitutive expression of Hsc70 or calnexin in vitro and in vivo. VK1 and VK2 sensitized A549 cells to heat-shock induced cell death, while the compounds alone had no effect on cell viability. The suppression of Hsp72 was apparently at the protein level because the mRNA expression of Hsp72 was unchanged. Moreover, the chaperone activity of Hsp72 was compromised after heat-shock when cells were pre-treated with VK2. The effect of VK2 on Hsp72 suppression, however, was also observed in normal mouse tissue after the mice were subjected to whole-body hyperthermia. To eliminate this side effect, local hyperthermia was performed on tumors in mice. The pre-treatment with VK2 potentiated the effect of local hyperthermia on tumor growth suppression. The findings here that VK1 and VK2 inhibit heat-shock-induced Hsp72 suggest their possible use as an adjuvant for hyperthermia in cancer therapy.
Subject(s)
HSP72 Heat-Shock Proteins/genetics , Hot Temperature , Hyperthermia, Induced/methods , Vitamins/pharmacology , Xenograft Model Antitumor Assays/methods , Animals , Apoptosis/drug effects , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , Combined Modality Therapy , Dose-Response Relationship, Drug , Down-Regulation , Female , Gene Expression/drug effects , HSP72 Heat-Shock Proteins/metabolism , HeLa Cells , Humans , Luciferases/chemistry , Luciferases/genetics , Luciferases/metabolism , Mice , Mice, Inbred ICR , Mice, Nude , Protein Folding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transfection , Vitamin K 1/pharmacology , Vitamin K 2/pharmacologyABSTRACT
The emerging technique of bioluminescence resonance energy transfer (BRET) allows us to detect protein interactions in live cells and in real time, thus providing a new window into cellular signal transduction processes. We present experimental protocols for expressing fusion proteins between luciferase and fluorescent proteins that are the basis for BRET measurement, as well as for measuring and imaging BRET in a variety of cell types. Despite our focus on plant cells, the techniques described here are easily adaptable to other cell systems that have yet to benefit from the BRET technique.
Subject(s)
Luminescent Proteins/chemistry , Plant Proteins/analysis , Protein Interaction Mapping/methods , Signal Transduction , Arabidopsis/chemistry , Arabidopsis/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Energy Transfer , Luciferases/chemistry , Luminescent Measurements , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Onions/chemistry , Onions/genetics , Plant Proteins/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Nicotiana/chemistry , Nicotiana/geneticsABSTRACT
A new method employing the classical techniques of chemical modification of proteins and the new technology of mass spectrometry, known as pulsed-alkylation mass spectrometry (PA/MS), has been developed to probe the dynamic structure of folding intermediates and folded complexes of proteins under a variety of conditions. This method is fast and simple, and the results are easily interpreted. PA/MS may provide an alternative to H/D exchange monitored either by NMR or by electrospray ionization mass spectrometry for some experiments; for others, it may provide access to questions not readily answered by available methods. The objective of PA/MS is to determine simultaneously the location and the extent of labeling of functional groups in a protein by measuring the reactivity of cysteines with N-ethylmaleimide, within the context of the conformation of the protein under specific conditions. The method can also be applied to chemical modification of other amino acid residues employing any of a vast array of reagents, depending upon the specifics of the protein under investigation. The enormous range of reactivity of the thiol groups of the cysteinyl residues in proteins and the change in reactivity upon denaturation or conformational rearrangement afford a large signal change that can be correlated with changes in accessibility of the thiol group. The information obtained from the correlation of observed thiol reactivity with the local environment of each cysteinyl residue in the structure of the folded protein can be supplemented by results obtained from fluorescence, circular dichroism, or other methods, to develop an understanding of the structure and dynamics of altered conformational states. With bacterial luciferase as a model system, we have applied PA/MS to investigate the structural differences between the native heterodimeric enzyme and a folding intermediate that is well-populated in 2 M urea. The thiol residues at positions 307, 324, and 325 of the alpha subunit were much more reactive with N-ethylmaleimide in the presence of 2 M urea than in the native enzyme, suggesting that the C-terminal region of the alpha subunit was less tightly packed in the folding intermediate. The apparent unfolding of the C-terminal region of the alpha subunit of the alphabeta structure in 2 M urea appears to mimic the unfolding of the C-terminal domain of the free alpha subunit, also in 2 M urea, described by Noland, B. W., Dangott, L. J., and Baldwin, T. O. (1999) Biochemistry 38, 16136-16145. The approach described here should be applicable to a wide array of problems that have in common the need to determine the locations of conformational changes in proteins. Application of PA/MS to the investigation of the relative thermodynamic stability of the coordination complexes of zinc within each of the six zinc-finger domains of MRE-binding transcription factor-1 (Zn(6) MTF-zf) in its free and DNA-bound forms is presented in the companion paper in this issue [Apuy, J. L., Chen, X., Russell, D. H., Baldwin, T. O., and Giedroc, D. P. (2001) Biochemistry 40, 15164-15175].
Subject(s)
Luciferases/chemistry , Mass Spectrometry/methods , Protein Folding , Proteins/chemistry , Alkylation , Amino Acid Sequence , Chymotrypsin , Cysteine/chemistry , Enzyme Stability , Luciferases/genetics , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Subunits , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Thermodynamics , Vibrio/enzymology , Vibrio/geneticsABSTRACT
Bioluminescence of the medusa Periphylla is based on the oxidation of coelenterazine catalyzed by luciferase. Periphylla has two types of luciferase: the soluble form luciferase L, which causes the exumbrellar bioluminescence display of the medusa, and the insoluble aggregated form, which is stored as particulate material in the ovary, in an amount over 100 times that of luciferase L. The eggs are especially rich in the insoluble luciferase, which drastically decreases upon fertilization. The insoluble form could be solubilized by 2-mercaptoethanol, yielding a mixture of luciferase oligomers with molecular masses in multiples of approximately 20 kDa. Those having the molecular masses of 20 kDa, 40 kDa, and 80 kDa were isolated and designated, respectively, as luciferase A, luciferase B, and luciferase C. The luminescence activities of Periphylla luciferases A, B, and C were 1.2 approximately 4.1 x 10(16) photon/mg. s, significantly higher than any coelenterazine luciferase known, and the quantum yields of coelenterazine catalyzed by these luciferases (about 0.30 at 24 degrees C) are comparable to that catalyzed by Oplophorus luciferase (0.34 at 22 degrees C), which has been considered the most efficient coelenterazine luciferase until now. Luciferase L (32 kDa) could also be split by 2-mercaptoethanol into luciferase A and an accessory protein (approx. 12 kDa), as yet uncharacterized. Luciferases A, B, and C are highly resistant to inactivation: their luminescence activities are only slightly diminished at pH 1 and pH 11 and are enhanced in the presence of 1 approximately 2 M guanidine hydrochloride; but they are less stable to heating than luciferase L, which is practically unaffected by boiling.
Subject(s)
Imidazoles , Luciferases/isolation & purification , Ovary/enzymology , Scyphozoa/enzymology , Animals , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Female , Guanidine/chemistry , Hot Temperature , Hydrogen-Ion Concentration , Luciferases/chemistry , Luciferases/metabolism , Luminescent Measurements , Mercaptoethanol/chemistry , Molecular Weight , Pyrazines/metabolism , Scyphozoa/metabolismABSTRACT
The activity regenerating luciferin from the luminescent product oxyluciferin was found in the protein fraction of a lantern extract from Photinus pyralis. The protein, luciferin-regenerating enzyme (LRE), was purified to homogeneity by ammonium sulfate precipitation followed by successive column chromatography on Ultrogel AcA34, S-Sepharose FF, Q-Sepharose FF, TSKgel super Q 5pw and TSKgel G3000 SW(XL). This enzyme was a single polypeptide with a molecular mass of 38 kDa. LRE converted oxyluciferin to 2-cyano-6-hydroxybenzothiazole and thioglycolic acid. In the presence of d-cysteine, 2-cyano-6-hydroxybenzothiazole was turned over into luciferin. The same activities were detected in the extracts from two Japanese fireflies, Luciola cruciata and Luciola lateralis. We have cloned a cDNA encoding LRE from poly(A)+ RNA of the lantern of P. pyralis using reverse transcription-polymerase chain reaction, 5'-RACE (rapid amplification of cDNA ends) and 3'-RACE. The primary structure of LRE from P. pyralis deduced from the nucleotide sequence was shown to consist of 308 amino acids with a molecular weight of 33,619. The cDNA was successfully expressed under the control of the tac promoter in Escherichia coli.