Publications by authors named "Takeaki Ozawa"

119 Publications

A Split-Luciferase-Based Cell Fusion Assay for Evaluating the Myogenesis-Promoting Effects of Bioactive Molecules.

Methods Mol Biol 2021 ;2274:79-87

Department of Chemistry, School of Science, The University of Tokyo, Tokyo, Japan.

A split-luciferase-based cell fusion assay enables high-throughput screening of myogenesis-promoting chemicals in chemical libraries. The assay consists of two C2C12 myoblast-derived cell lines (N- and C-cells), each of which stably expresses either an N- or C-terminal split-firefly luciferase (FLuc) fragment fused to a naturally split DnaE intein (N- and C-probes, respectively). The fusion of N- and C-cells during myogenesis induces bioluminescence (BL) in the cytosol due to a stable reconstitution of the split-FLuc. Thus, the myogenesis-promoting effects of a chemical compound can be determined through the enhanced BL intensity. Here, we describe the preparation of N- and C-cells and determination of the myogenesis-promoting effects of imatinib using a 96-well microplate-based assay.
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http://dx.doi.org/10.1007/978-1-0716-1258-3_8DOI Listing
June 2021

Quantitative Determination and Imaging of Gα Signaling in Live Cells via Split-Luciferase Complementation.

Methods Mol Biol 2021 ;2274:69-78

Institute of Pharmacy, University of Regensburg, Regensburg, Germany.

G Protein-coupled receptors (GPCRs) transduce signals elicited by bioactive chemical agents (ligands), such as hormones, neurotransmitters, or cytokines, across the cellular membrane. Upon ligand binding, the receptor undergoes structural rearrangements, which cause the activation of G proteins. This triggers the activation of signaling cascades involving amplification, which takes place after every stage of the cascade. Consequently, signals from early stages can be masked when the activation of the signaling cascade is probed remote (distal) from the receptor. This led to the development of several techniques, which probe the activation of such signaling cascades as proximal to the receptor as possible. However, these methods often require specialized equipment or are limited in throughput. By applying split-luciferase complementation to the interaction between the Gα protein and its effector the phospholipase C-β3 (PLC-β3), we introduce a protocol with a conventional plate reader at high throughput. The method is applicable to live cells and additionally allows imaging of the probe by bioluminescence microscopy.
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http://dx.doi.org/10.1007/978-1-0716-1258-3_7DOI Listing
June 2021

Quantitative Analysis of Membrane Receptor Trafficking Manipulated by Optogenetic Tools.

Methods Mol Biol 2021 ;2274:15-23

Department of Chemistry, School of Science, The University of Tokyo, Tokyo, Japan.

Membrane receptors play a crucial role in transmitting external signals inside cells. Signal molecule-bound receptors activate multiple downstream pathways, the dynamics of which are modulated by intracellular trafficking. A significant contribution of β-arrestin to intracellular trafficking has been suggested, but the underlying mechanism is poorly understood. Here, we describe a protocol for manipulating β-arrestin-regulated membrane receptor trafficking using photo-induced dimerization of cryptochrome-2 from Arabidopsis thaliana and its binding partner CIBN. Additionally, the protocol guides analytical methods to quantify the changes in localization and modification of membrane receptors during trafficking.
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http://dx.doi.org/10.1007/978-1-0716-1258-3_2DOI Listing
June 2021

Functional Modulation of Receptor Proteins on Cellular Interface with Optogenetic System.

Adv Exp Med Biol 2021 ;1293:247-263

Department of Chemistry, School of Science, The University of Tokyo, Tokyo, Japan.

In multicellular organisms, living cells cooperate with each other to exert coordinated complex functions by responding to extracellular chemical or physical stimuli via proteins on the plasma membrane. Conventionally, chemical signal transduction or mechano-transduction has been investigated by chemical, genetic, or physical perturbation; however, these methods cannot manipulate biomolecular reactions at high spatiotemporal resolution. In contrast, recent advances in optogenetic perturbation approaches have succeeded in controlling signal transduction with external light. The methods have enabled spatiotemporal perturbation of the signaling, providing functional roles of the specific proteins. In this chapter, we summarize recent advances in the optogenetic tools that modulate the function of a receptor protein. While most optogenetic systems have been devised for controlling ion channel conductivities, the present review focuses on the other membrane proteins involved in chemical transduction or mechano-transduction. We describe the properties of natural or artificial photoreceptor proteins used in optogenetic systems. Then, we discuss the strategies for controlling the receptor protein functions by external light. Future prospects of optogenetic tool development are discussed.
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http://dx.doi.org/10.1007/978-981-15-8763-4_15DOI Listing
February 2021

Long-term single cell bioluminescence imaging with C-3 position protected coelenterazine analogues.

Org Biomol Chem 2021 01 3;19(3):579-586. Epub 2020 Nov 3.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.

Bioluminescence is a powerful imaging modality for monitoring biological phenomena both in vitro and in vivo. Bioluminescence imagin (BLI) is becoming a seamless imaging technology covering the range from cells to organs of small animals. Long-term imaging at the single cell level would lead to a true understanding of the dynamics of life phenomena. This work presents a long-term single cell bioluminescence imaging technology accomplished with C-3 position protected furimazines (FMZs), a CTZ analogues, which generate intense blue emission when paired with a highly stable engineered luciferase, Nanoluc. Four types of FMZs protected at the C-3 position have been synthesized. The type and steric bulkiness of the protection group strongly contributed to storage stability and the kinetics of the bioluminescence reactions of the analogues in human living cells. In particular, two developed FMZ analogues resulted in significantly longer bioluminescence emission with higher S/N ratio than FMZ at single cell level. Long-term bioluminescence single cell imaging technology with the developed FMZ analogues will lead to seamless imaging in the range from cells to organs of small animals.
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http://dx.doi.org/10.1039/d0ob02020fDOI Listing
January 2021

Advanced Bioluminescence System for In Vivo Imaging with Brighter and Red-Shifted Light Emission.

Int J Mol Sci 2020 Sep 7;21(18). Epub 2020 Sep 7.

Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

In vivo bioluminescence imaging (BLI), which is based on luminescence emitted by the luciferase-luciferin reaction, has enabled continuous monitoring of various biochemical processes in living animals. Bright luminescence with a high signal-to-background ratio, ideally red or near-infrared light as the emission maximum, is necessary for in vivo animal experiments. Various attempts have been undertaken to achieve this goal, including genetic engineering of luciferase, chemical modulation of luciferin, and utilization of bioluminescence resonance energy transfer (BRET). In this review, we overview a recent advance in the development of a bioluminescence system for in vivo BLI. We also specifically examine the improvement in bioluminescence intensity by mutagenic or chemical modulation on several beetle and marine luciferase bioluminescence systems. We further describe that intramolecular BRET enhances luminescence emission, with recent attempts for the development of red-shifted bioluminescence system, showing great potency in in vivo BLI. Perspectives for future improvement of bioluminescence systems are discussed.
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http://dx.doi.org/10.3390/ijms21186538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555964PMC
September 2020

Fluorescent H Receptor Squaramide-Type Antagonists: Synthesis, Characterization, and Applications.

ACS Med Chem Lett 2020 Aug 20;11(8):1521-1528. Epub 2020 Jul 20.

Institute of Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.

Fluorescence labeled ligands have been gaining importance as molecular tools, enabling receptor-ligand-binding studies by various fluorescence-based techniques. Aiming at red-emitting fluorescent ligands for the hHR, a series of squaramides labeled with pyridinium or cyanine fluorophores (-) was synthesized and characterized. The highest hHR affinities in radioligand competition binding assays were obtained in the case of pyridinium labeled antagonists - (p: 7.71-7.76) and cyanine labeled antagonists and (p: 7.67, 7.11). These fluorescent ligands proved to be useful tools for binding studies (saturation and competition binding as well as kinetic experiments), using confocal microscopy, flow cytometry, and high content imaging. Saturation binding experiments revealed p values comparable to the p values. The fluorescent probes , , and could be used to localize H receptors in HEK cells and to determine the binding affinities of unlabeled compounds.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429974PMC
August 2020

Synergetic Roles of Formyl Peptide Receptor 1 Oligomerization in Ligand-Induced Signal Transduction.

ACS Chem Biol 2020 09 6;15(9):2577-2587. Epub 2020 Sep 6.

School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

G protein-coupled receptors (GPCRs) transduce extracellular signals into cells by interacting with G proteins and arrestins. Emerging evidence suggests that GPCRs on the plasma membrane are in a dynamic equilibrium among monomers, dimers, and larger oligomers. Nevertheless, the role of the oligomer formation in the GPCR signal transduction remains unclear. Using multicolor single-molecule live-cell imaging, we show a dynamic interconversion between small and large oligomer states of a chemoattractant GPCR, Formyl Peptide Receptor 1 (FPR1), and its binding affinity with G protein. Full agonist stimulation increased a fraction of large FPR1 oligomers, which allowed for prolonged FPR1-G protein interaction. The G protein interaction with FPR1 was most stabilized at the full agonist-bound large FPR1 oligomers. Based on these results, we propose that G protein-mediated signal transduction may be regulated synergistically by the ligand-binding and FPR1 oligomerization. Cooperative signal control induced by receptor oligomerization is anticipated as a target for drug discovery.
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http://dx.doi.org/10.1021/acschembio.0c00631DOI Listing
September 2020

Light-mediated control of Gene expression in mammalian cells.

Neurosci Res 2020 Mar 7;152:66-77. Epub 2020 Jan 7.

Research Center for Dynamic Living Systems, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8501, Japan; Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan; Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, 606-8501, Japan. Electronic address:

Taking advantage of the recent development of genetically-defined photo-activatable actuator molecules, cellular functions, including gene expression, can be controlled by exposure to light. Such optogenetic strategies enable precise temporal and spatial manipulation of targeted single cells or groups of cells at a level hitherto impossible. In this review, we introduce light-controllable gene expression systems exploiting blue or red/far-red wavelengths and discuss their inherent properties potentially affecting induced downstream gene expression patterns. We also discuss recent advances in optical devices that will extend the application of optical gene expression control technologies into many different areas of biology and medicine.
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http://dx.doi.org/10.1016/j.neures.2019.12.018DOI Listing
March 2020

Enhanced bioluminescent sensor for longitudinal detection of CREB activation in living cells.

Photochem Photobiol Sci 2019 Nov 1;18(11):2740-2747. Epub 2019 Oct 1.

Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is associated with memory formation and controls cell survival and proliferation via regulation of downstream gene expression in tumorigenesis. As a transcription factor, CREB binds to cAMP response elements. Phosphorylation of CREB triggers transcriptional activation of CREB downstream genes following the interaction of the kinase-inducible domain (KID) of CREB with the KID interaction domain (KIX) of CREB-binding protein. Nevertheless, because of the lack of single-cell analytical techniques, little is known about spatiotemporal regulation of CREB phosphorylation. To analyze CREB activation in single living cells, we developed genetically encoded bioluminescent sensors using luciferase-fragment complementation: the sensors are designed based on KID-KIX interaction with a single-molecule format. The luminescence intensity of the sensor, designated as CREX (a sensor of CREB activation based on KID(CREB)-KIX interaction), increased by phosphorylation of CREB. Moreover, the luminescence intensity of CREX was sufficient to detect CREB activation in live-cell bioluminescence imaging for single-cell analysis because of the higher sensitivity. CREX sensor is expected to contribute to elucidation of the spatiotemporal regulation of CREB phosphorylation by applying single-cell analysis.
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http://dx.doi.org/10.1039/c9pp00249aDOI Listing
November 2019

Photocleavable Cadherin Inhibits Cell-to-Cell Mechanotransduction by Light.

ACS Chem Biol 2019 10 20;14(10):2206-2214. Epub 2019 Sep 20.

Department of Chemistry, Graduate School of Science , The University of Tokyo , 7-3-1 Hongo , Bunkyo-ku, Tokyo 113-0033 , Japan.

Precise integration of individual cell behaviors is indispensable for collective tissue morphogenesis and maintenance of tissue integrity. Organized multicellular behavior is achieved mechanical coupling of individual cellular contractility, mediated by cell adhesion molecules at the cell-cell interface. Conventionally, gene depletion or laser microsurgery has been used for functional analysis of intercellular mechanotransduction. Nevertheless, these methods are insufficient to investigate either the spatiotemporal dynamics or the biomolecular contribution in cell-cell mechanical coupling within collective multicellular behaviors. Herein, we present our effort in adaption of PhoCl for attenuation of cell-to-cell tension transmission mediated by E-cadherin. To release intercellular contractile tension applied on E-cadherin molecules with external light, a genetically encoded photocleavable module called PhoCl was inserted into the intracellular domain of E-cadherin, thereby creating photocleavable cadherin (PC-cadherin). In response to light illumination, the PC-cadherin cleaved into two fragments inside cells, resulting in attenuating mechanotransduction at intercellular junctions in living epithelial cells. Light-induced perturbation of the intercellular tension balance with surrounding cells changed the cell shape in an epithelial cell sheet. The method is expected to enable optical manipulation of force-mediated cell-to-cell communications in various multicellular behaviors, which contributes to a deeper understanding of embryogenesis and oncogenesis.
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http://dx.doi.org/10.1021/acschembio.9b00460DOI Listing
October 2019

[H]UR-DEBa176: A 2,4-Diaminopyrimidine-Type Radioligand Enabling Binding Studies at the Human, Mouse, and Rat Histamine H Receptors.

J Med Chem 2019 09 30;62(17):8338-8356. Epub 2019 Aug 30.

Institute of Pharmacy , University of Regensburg , D-93053 Regensburg , Germany.

Differences in sequence homology between human (h), mouse (m), and rat (r) histamine H receptors (HR) cause discrepancies regarding affinities, potencies, and/or efficacies of ligands and therefore compromise translational animal models and the applicability of radioligands. Aiming at a radioligand enabling robust and comparative binding studies at the h/m/rHRs, 2,4-diaminopyrimidines were synthesized and pharmacologically investigated. The most notable compounds identified were two (partial) agonists with comparable potencies at the h/m/rHRs: UR-DEBa148 (-neopentyl-4-(1,4,6,7-tetrahydro-5-imidazo[4,5-]pyridin-5-yl)pyrimidin-2-amine bis(2,2,2-trifluoroacetate), ), the most potent [pEC (reporter gene assay) = 9.9/9.6/10.3] compound in the series being slightly G-protein biased and UR-DEBa176 [()-4-[3-(dimethylamino)pyrrolidin-1-yl]--neopentylpyrimidin-2-amine bis(2,2,2-trifluoroacetate), , pEC (reporter gene assay) = 8.7/9.0/9.2], a potential "cold" form of a tritiated HR ligand. After radiolabeling, binding studies with [H]UR-DEBa176 ([H]) at the h/m/rHRs revealed comparable values (41/17/22 nM), low nonspecific binding (11-17%, ∼), and fast associations/dissociations (25-30 min) and disclosed [H]UR-DEBa176 as useful molecular tool to determine h/m/rHR binding affinities for HR ligands.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01342DOI Listing
September 2019

Parallelized shifted-excitation Raman difference spectroscopy for fluorescence rejection in a temporary varying system.

J Biophotonics 2019 12 28;12(12):e201960028. Epub 2019 Aug 28.

Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan.

A fluorescence background is one of the common interference factors of the Raman spectroscopic analysis in the biology field. Shifted-excitation Raman difference spectroscopy (SERDS), in which a slow (typically 1 Hz) modulation to excitation wavelength is coupled with a sequential acquisition of alternating shifted-excitation spectra, has been used to separate Raman scattering from excitation-shift insensitive background. This sequential method is susceptible to spectral change and thus is limited only to stable samples. We incorporated a fast laser modulation (200 Hz) and a mechanical streak camera into SERDS to effectively parallelize the SERDS measurement in a single exposure. The developed system expands the scope of SERDS to include temporary varying system. The proof of concept is demonstrated using highly fluorescent samples, including living algae. Quantitative performance in fluorescence rejection and the robustness of the method to the dynamic spectral change during the measurement are manifested.
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http://dx.doi.org/10.1002/jbio.201960028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065630PMC
December 2019

A Detection Method for GLUT4 Exocytosis Based on Spontaneous Split Luciferase Complementation.

Anal Sci 2019 Aug 5;35(8):835-838. Epub 2019 Jul 5.

Department of Chemistry, School of Science, The University of Tokyo.

Glucose transporter 4 (GLUT4) is an insulin-regulated glucose transporter, which is vital for blood glucose homeostasis. To clarify the physiological roles of GLUT4, quantitative measurement of GLUT4 exocytosis is indispensable. Herein, we show a rapid detection system for GLUT4 on the cell surface using spontaneous split-luciferase reconstitution. Upon insulin-induced GLUT4 exocytosis, GLUT4 was exposed outside, where luciferase is reconstituted and emitted luminescence. Pretreatment with inhibitors reduced the insulin-induced signal elevation. The results indicate that the developed method is applicable to high-throughput analysis on GLUT4 trafficking, which will greatly accelerate comprehensive research on the physiological roles of GLUT4.
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http://dx.doi.org/10.2116/analsci.19C003DOI Listing
August 2019

Phenotype Profiling for Forensic Purposes: Determining Donor Sex Based on Fourier Transform Infrared Spectroscopy of Urine Traces.

Anal Chem 2019 05 23;91(9):6288-6295. Epub 2019 Apr 23.

Department of Chemistry , University at Albany, SUNY , 1400 Washington Avenue , Albany , New York 12222 , United States.

Forensic science is an important field of analytical chemistry where vibrational spectroscopy, in particular Fourier transform infrared spectroscopy and Raman spectroscopy, present advantages as they have a nondestructive nature, high selectivity, and no need for sample preparation. Herein, we demonstrate a method for determination of donor sex, based on attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy of dry urine traces. Trace body fluid evidence is of special importance to the modern criminal investigation as a source of individualizing DNA evidence. However, individual identification of a urine donor is generally difficult because of the small amount of DNA. Therefore, the development of an innovative method to provide phenotype information about the urine donor-including sex-is highly desirable. In this study, we developed a multivariate discriminant model for the ATR FT-IR spectra of dry urine to identify the donor sex. Rigorous selection of significant wavenumbers on the spectrum using a genetic algorithm enabled superb discrimination performance for the model and conclusively indicated a chemical origin for donor sex differences, which was supported by physiological knowledge. Although further investigations need to be conducted, this proof-of-concept study demonstrates the great potential of the developed methodology for phenotype profiling based on the analysis of urine traces.
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http://dx.doi.org/10.1021/acs.analchem.9b01058DOI Listing
May 2019

Using redox-sensitive mitochondrial cytochrome Raman bands for label-free detection of mitochondrial dysfunction.

Analyst 2019 Apr;144(8):2531-2540

Department of Applied Visual Science, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

Mitochondrial activity is a widely used criterion to judge the metabolic condition of a living specimen. Numerous methods have been developed for related analyses, including the detection of O2 consumption, trans-membrane potential, and ATP production. In this study, we demonstrate that the redox state of cytochromes can serve as a sensitive mitochondrial activity indicator in glutamate-stressed neuronal cells. Mitochondrial dysfunction was detected by Raman imaging as early as 30 min after glutamate-stress induction. By comparing this result with other commonly used mitochondrial function assays, we found Raman imaging has a similar sensitivity to ATP production and trans-membrane potential assays. Other viability tests, such as MTT assay and ROS production tests, showed a slower response than our method. A thorough understanding of cytochrome dynamics with our new method will help establish Raman spectroscopy as a competitive clinical diagnosis tool for neurodegenerative diseases involving mitochondrial dysfunction.
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http://dx.doi.org/10.1039/c8an02213eDOI Listing
April 2019

Cooperative interaction among BMAL1, HSF1, and p53 protects mammalian cells from UV stress.

Commun Biol 2018 22;1:204. Epub 2018 Nov 22.

Department of Physiology & Advanced Research Center for Medical Science, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo, 143-8540, Japan.

The circadian clock allows physiological systems to adapt to their changing environment by synchronizing their timings in response to external stimuli. Previously, we reported clock-controlled adaptive responses to heat-shock and oxidative stress and showed how the circadian clock interacts with BMAL1 and HSF1. Here, we present a similar clock-controlled adaptation to UV damage. In response to UV irradiation, HSF1 and tumor suppressor p53 regulate the expression of the clock gene in a time-dependent manner. UV irradiation first activates the HSF1 pathway, which subsequently activates the p53 pathway. Importantly, BMAL1 regulates both HSF1 and p53 through the BMAL1-HSF1 interaction to synchronize the cellular clock. Based on these findings and transcriptome analysis, we propose that the circadian clock protects cells against the UV stress through sequential and hierarchical interactions between the circadian clock, the heat shock response, and a tumor suppressive mechanism.
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http://dx.doi.org/10.1038/s42003-018-0209-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250677PMC
November 2018

A split luciferase-based probe for quantitative proximal determination of Gα signalling in live cells.

Sci Rep 2018 11 21;8(1):17179. Epub 2018 Nov 21.

Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, D-93053, Regensburg, Germany.

The earlier an activation of a G protein-dependent signalling cascade at a G protein-coupled receptor (GPCR) is probed, the less amplificatory effects contribute to the measured signal. This is especially useful in case of a precise quantification of agonist efficacies, and is of paramount importance, when determining agonist bias in relation to the β-arrestin pathway. As most canonical assays with medium to high throughput rely on the quantification of second messengers, and assays affording more proximal readouts are often limited in throughput, we developed a technique with a proximal readout and sufficiently high throughput that can be used in live cells. Split luciferase complementation (SLC) was applied to assess the interaction of Gα with its effector phospholipase C-β3. The resulting probe yielded an excellent Z' value of 0.7 and offers a broad and easy applicability to various Gα-coupling GPCRs (hHR, hMR, hNTSR), expressed in HEK293T cells, allowing the functional characterisation of agonists and antagonists. Furthermore, the developed sensor enabled imaging of live cells by luminescence microscopy, as demonstrated for the hMR. The versatile SLC-based probe is broadly applicable e.g. to the screening and the pharmacological characterisation of GPCR ligands as well as to molecular imaging.
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http://dx.doi.org/10.1038/s41598-018-35615-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249299PMC
November 2018

Preferential Photoreaction in a Porous Crystal, Metal-Macrocycle Framework: Pd-Mediated Olefin Migration over [2+2] Cycloaddition.

J Am Chem Soc 2018 12 26;140(48):16610-16614. Epub 2018 Nov 26.

Department of Chemistry, Graduate School of Science , The University of Tokyo , 7-3-1 Hongo , Bunkyo-ku, Tokyo 113-0033 , Japan.

A nanosized confined space with well-defined functional surfaces has great potential to control the efficiency and selectivity of catalytic reactions. Herein we report that a 1,6-diene, which normally forms an intramolecular [2+2] cycloadduct under photoirradiation, preferentially undergoes a photoinduced olefin migration in a porous crystal, metal-macrocycle framework (MMF), and alternatively [2+2] cycloaddition is completely inhibited in the confined space. A plausible reaction mechanism for olefin migration triggered by the photoinduced dissociation of the Pd-Cl bond is suggested based on UV-vis diffuse reflectance spectroscopy, single-crystal XRD, and MS-CASPT2 calculation. The substrate scope of the photoinduced olefin migration in MMF was also examined using substituted allylbenzene derivatives.
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http://dx.doi.org/10.1021/jacs.8b08534DOI Listing
December 2018

Activation of caspase-3 during Chlamydia trachomatis-induced apoptosis at a late stage.

Can J Microbiol 2019 Feb 18;65(2):135-143. Epub 2018 Oct 18.

a Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, North-12, West-5, Kita-ku, Sapporo 060-0812, Japan.

The obligate intracellular bacterium Chlamydia trachomatis activates the host cell apoptosis pathway at a late stage of its developmental cycle. However, whether caspase-3, which is a key enzyme of apoptosis, is activated in Chlamydia-infected cells remains unknown. Here, we established HEp-2 cells stably expressing cFluc-DEVD, which is a caspase-3 substrate sequence inserted into cyclic firefly luciferase, and then monitored the dynamics of caspase-3 activity in cells infected with Chlamydia. Transfected cells without infection showed a significant increase in luciferase activity due to stimulation with staurosporine, an inducer of apoptosis. Activation was significantly blocked by addition of caspase inhibitor z-VAD-fmk. Furthermore, as expected, Chlamydia infection caused a significant increase in luciferase activation at 36-48 h postinfection with a contrastive decrease at 24 h postinfection, which is already well known. Such activation caused by the infection was much stronger when the amount of bacteria was increased. Thus, caspase-3 activation was accurately monitored by the luciferase activity in HEp-2 cells constitutively expressing the cFluc-DEVD probe. Furthermore, our data showed that C. trachomatis activates caspase-3 in host cells at a late stage of infection.
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http://dx.doi.org/10.1139/cjm-2018-0408DOI Listing
February 2019

In Search of NPY YR Antagonists: Incorporation of Carbamoylated Arginine, Aza-Amino Acids, or d-Amino Acids into Oligopeptides Derived from the C-Termini of the Endogenous Agonists.

ACS Omega 2017 Jul 14;2(7):3616-3631. Epub 2017 Jul 14.

Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, Regensburg D-93053, Germany.

The cross-linked pentapeptides (2,7)-diaminooctanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide) ((2,7)-BVD-74D, (2,7)-) and octanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide) () as well as the pentapeptide Ac-Tyr-Arg-Leu-Arg-Tyr-amide () were previously described as neuropeptide Y Y receptor (YR) partial agonists. Here, we report on a series of analogues of (2,7)- and in which Arg, Leu, or Arg were replaced by the respective aza-amino acids. The replacement of Arg in with a carbamoylated arginine building block and the extension of the N-terminus by an additional arginine led to the high-affinity hexapeptide Ac-Arg-Tyr--[(4-aminobutyl)aminocarbonyl]Arg-Leu-Arg-Tyr-amide (), which was used as a precursor for a d-amino acid scan. The target compounds were investigated for YR functional activity in assays with complementary readouts: aequorin Ca and β-arrestin 1 or β-arrestin 2 assays. In contrast to the parent compounds, which are YR agonists, several ligands were able to suppress the effect elicited by the endogenous ligand pancreatic polypeptide and therefore represent a novel class of peptide YR antagonists.
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http://dx.doi.org/10.1021/acsomega.7b00451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044894PMC
July 2017

A robust split-luciferase-based cell fusion screening for discovering myogenesis-promoting molecules.

Analyst 2018 Jul;143(14):3472-3480

Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Myogenesis-promoting chemicals are an important source of new pharmaceuticals for the treatment of skeletal muscle atrophy that impairs quality of life. This report presents a robust and quantitative bioluminescence-based assay for screening myogenesis-promoting compounds in chemical libraries. The assay system consists of two stable C2C12 myoblast cell lines, each of which expresses either an N-terminal or a C-terminal split luciferase fragment fused to a naturally split DnaE intein as an indicator for cell fusion. Cell fusion during myogenesis induces bioluminescence in the cytosol because of the reconstitution of luciferases. The luminescence intensity quantitatively represents the progress in the cell fusion and therefore indicates the extent of myogenesis. We applied this assay system to a high-throughput screening of myogenesis-promoting compouns in 1191 pharmacologically proven bioactive small molecules, which revealed two chemical compounds as myogenesis-promoting compounds: Imatinib and Doxazosin mesylate. The assay system enabled a robust and quantitative evaluation of the extent of myogenesis through simple luminescence measurements, and is expected to be widely applicable for high-throughput screening of cell fusion-promoting and inhibiting molecules.
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http://dx.doi.org/10.1039/c8an00285aDOI Listing
July 2018

Light-controllable Transcription System by Nucleocytoplasmic Shuttling of a Truncated Phytochrome B.

Photochem Photobiol 2018 09 12;94(5):1071-1076. Epub 2018 Jul 12.

Department of Chemistry, School of Science, The University of Tokyo, Tokyo, Japan.

Transcriptional regulation is a useful strategy for gene therapy and for biomedical research. Unlike chemically regulated transcriptional approaches, spatiotemporal control of transcription using optogenetic tools is a powerful technology for the analysis of single cells. For light to penetrate into tissues, it is desired to use photoreceptors absorbing red/far-red light with a low-molecular mass applicable for the use of virus vectors, and a photoswitch using the photoreceptor needs to be constructed as a single expression vector. Herein, we describe an optogenetic tool based on Arabidopsis thaliana phytochrome (Phy) B and its binding partner, phytochrome-interacting factor (PIF) 6. We generated a truncated PhyB, which allowed for reversible association with PIF6 by red/far-red light illumination. The red light illumination only for 5 min induced PhyB translocation from the cytoplasm into the nucleus by the association with PIF6, resulting in transcriptional activation based on Gal4 DNA-binding domain and the upstream activating sequence of Gal system. The nucleocytoplasmic shuttling vector using PhyB and PIF6 might be applicable for transcriptional regulation in tissue experiments.
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http://dx.doi.org/10.1111/php.12955DOI Listing
September 2018

Soft and Robust Identification of Body Fluid Using Fourier Transform Infrared Spectroscopy and Chemometric Strategies for Forensic Analysis.

Sci Rep 2018 05 31;8(1):8459. Epub 2018 May 31.

Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Bunkyo, Tokyo, 113-0033, Japan.

Body fluid (BF) identification is a critical part of a criminal investigation because of its ability to suggest how the crime was committed and to provide reliable origins of DNA. In contrast to current methods using serological and biochemical techniques, vibrational spectroscopic approaches provide alternative advantages for forensic BF identification, such as non-destructivity and versatility for various BF types and analytical interests. However, unexplored issues remain for its practical application to forensics; for example, a specific BF needs to be discriminated from all other suspicious materials as well as other BFs, and the method should be applicable even to aged BF samples. Herein, we describe an innovative modeling method for discriminating the ATR FT-IR spectra of various BFs, including peripheral blood, saliva, semen, urine and sweat, to meet the practical demands described above. Spectra from unexpected non-BF samples were efficiently excluded as outliers by adopting the Q-statistics technique. The robustness of the models against aged BFs was significantly improved by using the discrimination scheme of a dichotomous classification tree with hierarchical clustering. The present study advances the use of vibrational spectroscopy and a chemometric strategy for forensic BF identification.
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http://dx.doi.org/10.1038/s41598-018-26873-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5981217PMC
May 2018

Establishing a Split Luciferase Assay for Proteinkinase G (PKG) Interaction Studies.

Int J Mol Sci 2018 Apr 12;19(4). Epub 2018 Apr 12.

Department of Pharmacology and Toxicology, University of Regensburg, 93053 Regensburg, Germany.

Nitric oxide (NO/cyclic guanosine monophosphate (cGMP)-regulated cellular mechanisms are involved in a variety of (patho-) physiological processes. One of the main effector molecules in this system, proteinkinase G (PKG), serves as a molecular switch by phosphorylating different target proteins and thereby turning them on or off. To date, only a few interaction partners of PKG have been described although the identification of protein-protein interactions (PPI) is indispensable for the understanding of cellular processes and diseases. Conventionally used methods to detect PPIs exhibit several disadvantages, e.g., co-immunoprecipitations, which depend on suitable high-affinity antibodies. Therefore, we established a cell-based protein-fragment complementation assay (PCA) for the identification of PKG target proteins. Here, a reporter protein ( luciferase) is split into two fragments and fused to two different possible interaction partners. If interaction occurs, the reporter protein is functionally complemented and the catalyzed reaction can then be quantitatively measured. By using this technique, we confirmed the regulator of G-Protein signaling 2 (RGS2) as an interaction partner of PKGIα (a PKG-isoform) following stimulation with 8-Br-cGMP and 8-pCPT-cGMP. Hence, our results support the conclusion that the established approach could serve as a novel tool for the rapid, easy and cost-efficient detection of novel PKG target proteins.
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http://dx.doi.org/10.3390/ijms19041180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979328PMC
April 2018

Nano-Materials for Bioimaging.

Authors:
Takeaki Ozawa

Anal Sci 2018;34(2):125-126

Department of Chemistry, School of Science, The University of Tokyo.

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http://dx.doi.org/10.2116/analsci.34.125DOI Listing
May 2018

Unique Roles of β-Arrestin in GPCR Trafficking Revealed by Photoinducible Dimerizers.

Sci Rep 2018 01 12;8(1):677. Epub 2018 Jan 12.

Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

Intracellular trafficking of G protein-coupled receptors (GPCRs) controls their localization and degradation, which affects a cell's ability to adapt to extracellular stimuli. Although the perturbation of trafficking induces important diseases, these trafficking mechanisms are poorly understood. Herein, we demonstrate an optogenetic method using an optical dimerizer, cryptochrome (CRY) and its partner protein (CIB), to analyze the trafficking mechanisms of GPCRs and their regulatory proteins. Temporally controlling the interaction between β-arrestin and β2-adrenergic receptor (ADRB2) reveals that the duration of the β-arrestin-ADRB2 interaction determines the trafficking pathway of ADRB2. Remarkably, the phosphorylation of ADRB2 by G protein-coupled receptor kinases is unnecessary to trigger clathrin-mediated endocytosis, and β-arrestin interacting with unphosphorylated ADRB2 fails to activate mitogen-activated protein kinase (MAPK) signaling, in contrast to the ADRB2 agonist isoproterenol. Temporal control of β-arrestin-GPCR interactions will enable the investigation of the unique roles of β-arrestin and the mechanism by which it regulates β-arrestin-specific trafficking pathways of different GPCRs.
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http://dx.doi.org/10.1038/s41598-017-19130-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766490PMC
January 2018

A genetic screen to discover SUMOylated proteins in living mammalian cells.

Sci Rep 2017 12 12;7(1):17443. Epub 2017 Dec 12.

Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

Post-translational modification by the Small Ubiquitin-related Modifier (SUMO) is indispensable for diverse biological mechanisms. Although various attempts have been made to discover novel SUMO substrate proteins to unveil the roles of SUMOylation, the reversibility of SUMOylation, and the differences in the SUMOylation level still makes it difficult to explore infrequently-SUMOylated proteins in mammalian cells. Here, we developed a method to screen for mammalian SUMOylated proteins using the reconstitution of split fluorescent protein fragments in living mammalian cells. Briefly, the cells harboring cDNAs of SUMOylated proteins were identified by the reconstituted fluorescence emission and separated by cell sorting. The method successfully identified 36 unreported SUMO2-substrate candidates with distinct intracellular localizations and functions. Of the candidates, we found Atac2, a histone acetyltransferase, was SUMOylated at a lysine 408, and further modified by multiple SUMOs without isoform specificity. Because the present method is applicable to other SUMO isoforms and mammalian cell-types, it could contribute to a deeper understanding of the role of SUMOylation in various biological contexts.
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http://dx.doi.org/10.1038/s41598-017-17450-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727073PMC
December 2017

Real-Time Fluorescence Imaging of Single-Molecule Endogenous Noncoding RNA in Living Cells.

Methods Mol Biol 2018 ;1649:337-347

Department of Chemistry, School of Science, The University of Tokyo, Tokyo, 113-0033, Japan.

Visualizing RNA in living cells is increasingly important to facilitate accumulation of knowledge about the relation between specific RNA dynamics and physiological events. Single-molecule fluorescence imaging of target RNAs is an excellent approach to analyzing intracellular RNA motion, but it requires special techniques for probe design and microscope setup. Herein, we present a principle and protocol of an RNA visualization probe based on an RNA binding protein of the Pumilio homology domain (PUM-HD). We also describe the setup and operation of a microscope, and introduce an application to visualize telomeric repeats-containing RNA with telomeres and a telomere-related protein: hnRNPA1. This imaging technique is applicable to visualization of different RNAs, especially including repetitive sequences, in living cells.
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http://dx.doi.org/10.1007/978-1-4939-7213-5_22DOI Listing
June 2018