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Takeaki Ozawa

Publications and source records attributed to Takeaki Ozawa.

At least 19 recordsLinked to original sources
PubMed2026

Nuclear class 3 PI3K co-activates fasting-specific chromatin remodelling.

Transcriptional remodelling during fasting ensures metabolic adaptation and provides health benefits across species. Although several regulators of fasting-induced transcription and chromatin are known, how nutrient levels directly influence RNA polymerase II (RNAPII) and epigenetic writers remains unclear. Here we show that lipid kinase class 3 phosphatidylinositol 3-kinase (PI3K-3), a master regulator of autophagy, also functions on chromatin as a co-activator of epigenetic writers to promote RNAPII transcription. PI3K-3 overlaps with transcriptionally engaged RNAPII phosphorylated at Ser5 and with Setd1a/COMPASS, the complex that deposits the activating H3K4me3 mark. Nuclear PI3K-3 interacts with RNAPII and Setd1a/COMPASS and promotes their chromatin binding. PI3K-3 loss reduces RNAPII-S5p and H3K4me3 at selected genes, whereas PI3K-3 overexpression co-activates p300/CBP and chromatin-targeted PI3K-3 increases H3K4me3. During starvation, PI3K-3 induces autophagy genes and drives fasted liver towards ketogenesis and lipid degradation. These findings link nutrient stress to chromatin-mediated transcriptional activation.

Chromatin Assembly and Disassembly↗
PubMed2025

Live-Cell Monitoring and Omics Analysis of Liquid-Solid Transitions of Biomolecular Condensates.

Biomolecular condensates, or so-called membraneless organelles, transition from liquid into more solid-like states over time, contributing to the development of pathological conditions. The present study proposes a simple method using photoactive yellow protein (PYP) and its specific fluorescent covalent ligands to distinguish between the liquid and solid states of protein condensates in live cells. The method, compatible with fluorescence-activated cell sorting (FACS), correlates the stiffness of specific protein condensates with their accessibility to PYP ligands, enabling quantitative multicolor monitoring of condensate solidification. We applied this technique to 12 phase-separating proteins and their mutants, finding that TDP-43, particularly its A315T mutant linked to familial amyotrophic lateral sclerosis, most readily forms solid aggregates. Furthermore, this FACS-compatible strategy enabled the isolation of distinct cell populations based on condensate states, allowing for subsequent proteomic and transcriptomic analyses. Our findings demonstrate that condensate solidification is accompanied by the upregulated expression of extracellular matrix proteins, suggesting a previously unrecognized link between solid aggregate formation and extracellular matrix hardening.

Humans↗
PubMed2007

Methods for imaging and analyses of intracellular organelles using fluorescent and luminescent proteins.

One of the important features of eukaryotic cells is the presence of intracellular compartments, organelles. Each organelle plays an essential role in the organelle-specific function; each cooperates with others for maintaining the vital activity of living cells. To analyze the organelle-specific functions, researchers have used many fluorescent and luminescent reporter proteins extensively. The reporter proteins have provided unprecedented insights into the movement of proteins and their interactions in the organelles. In this review, we summarize recently validated methods for visualizing protein localization, dynamics and protein-protein interactions, and for identifying a series of proteins localized in organelles using newly developed reporter proteins.

Animals↗
PubMed2006

Genetically encoded optical probe for detecting release of proteins from mitochondria toward cytosol in living cells and mammals.

We developed a genetically encoded bioluminescence indicator for monitoring the release of proteins from the mitochondria in living cells. The principle of this method is based on reconstitution of split Renilla reniformis luciferase (Rluc) fragments by protein splicing with an Ssp DnaE intein. A target mitochondrial protein connected with an N-terminal fragment of Rluc and an N-terminal fragment of DnaE is expressed in mammalian cells. If the target protein is released from the mitochondria toward the cytosol upon stimulation with a specific chemical, the N-terminal Rluc meets the C-terminal Rluc connected with C-terminal DnaE in the cytosol, and thereby, the full-length Rluc is reconstituted by protein splicing. The extent of release of the target fusion protein is evaluated by measuring activities of the reconstituted Rluc. To test the feasibility of this method, here we monitored the release of Smac/DIABLO protein from mitochondria during apoptosis in living cells and mice. The present method allowed high-throughput screening of an apoptosis-inducing reagent, staurosporine, and imaging of the Smac/DIABLO release in cells and in living mice. This rapid analysis can be used for screening and assaying chemicals that would increase or inhibit the release of mitochondrial proteins in living cells and animals.

Animals↗
PubMed2006

Intein-mediated reporter gene assay for detecting protein-protein interactions in living mammalian cells.

For nondestructive analysis of chemical processes in living mammalian cells, here we developed a new reporter gene assay for detecting cytosolic protein-protein interactions based on protein splicing of transcription factors with DnaE inteins. The protein splicing induces connection of a DNA-binding protein (modified LexA; mLexA) with a transcription activation domain of a herpes simplex virus protein (VP16AD). We thereby circumvented the limitation of earlier methods for monitoring protein-protein interactions, including the two-hybrid systems, protein complementation systems (PCS), and protein reconstitution systems, and rather combined their advantages. To test the applicability of this method, we monitored epidermal growth factor (EGF)-induced interactions on cell membranes of a known partner, an oncogenic product Ras and its target Raf-1. Ras was connected with N-terminal DnaE and mLexA, while Raf-1 was connected with C-terminal DnaE and VP16AD. Upon stimulation with EGF, the interaction between Ras and Raf-1 triggered folding of the DnaEs, thereby inducing protein splicing to form mLexA-VP16AD fusion protein, and transcription of a reporter gene, firefly luciferase. The extent of Ras-Raf-1 interaction was quantified by measuring the luciferase activity. The interaction was not able to be monitored by two-hybrid systems nor by PCS of split firefly luciferases; however, by using the protein splicing elements and the reporter gene, we obtained the bioluminescence signals sufficient for evaluation of the interactions close to cell membranes.

Animals↗
PubMed2005

Quantitative determination of protein nuclear transport induced by phosphorylation or by proteolysis.

Nucleocytoplasmic transport of proteins in eukaryotic cells is a fundamental process for gene expression. The transport is regulated by posttranslational modifications of the proteins, such as ligand-binding, phosphorylation, and proteolysis. For monitoring the nuclear transport of proteins induced by a ligand binding, we have recently developed a genetically encoded bioluminescent indicator based on reconstitution of split fragments of Renilla reniformis (RLuc) by protein splicing with DnaE inteins. We herein describe that the technique is used for detecting phosphorylation- or proteolysis-induced nuclear transports of a target protein. Two model proteins, signal transducer and activator of transcription 3 (STAT3) and sterol-regulatory element binding protein-2 (SREBP-2), were exemplified as phosphorylation- and proteolysis-induced nuclear transport, respectively. Each STAT3 or SREBP-2 is connected with C-terminal halves of RLuc and DnaE. If the protein translocates into the nucleus, the C-terminal fragment of RLuc meets the N-terminal fragment of RLuc, and full-length RLuc is reconstituted by protein splicing in the nucleus. The indicator with SREBP-2 enabled us to quantify the intracellular concentrations of cholesterol. The indicator with STAT3 quantified the extent of the nuclear transport induced by representative cytokines. This simple assay based on protein nuclear transports allows the selection of suitable drugs among candidates and has significant potential for risk assessments, such as carcinogenic chemical screening in vitro and in vivo.

Active Transport, Cell Nucleus↗
PubMed2005

Genetically encoded stress indicator for noninvasively imaging endogenous corticosterone in living mice.

Physical and emotional stress is one of the major controllers of physiological reactions and homeostasis in living animals. A stress hormone, corticosterone, is secreted from the adrenal cortex into the blood vessels when animals sense the stress. The quantitative evaluation of corticosterone in living animals has been limited because of the unavailability of suitable methods in vivo. For a noninvasive molecular imaging of the stress, we developed a method for detecting physiological increases in the endogenous corticosterone caused by exo- and endogenous stress in living animals. We constructed a pair of genetically encoded indicators composed of cDNAs of glucocorticoid receptor (GR), split Renilla luciferase (RLuc), and a Synechocystis sp. DnaE intein. The GR fused with C-terminal halves of RLuc and DnaE is localized in the cytosol, whereas a fusion protein of N-terminal halves of RLuc and DnaE is localized in the nucleus. If corticosterone induces GR translocation into the nucleus, the C-terminal RLuc meets the N-terminal one in the nucleus, and full-length RLuc is reconstituted by protein splicing with DnaE. Cell-based methods provided a quantitative bioluminescence assay of the extent of GR translocation into the nucleus. We further demonstrated that the indicator enabled noninvasive imaging against two different types of imposed stress: a forced swimming and metabolic perturbation caused by 2-deoxy-D-glucose. This stress indicator should be valuable for screening pharmacological compounds and for tools to study the mechanism of physiological stress.

Animals↗
PubMed2005

A genetically encoded indicator for assaying bioactive chemicals that induce nuclear transport of glucocorticoid receptor.

Glucocorticoids, the adrenal steroid hormones secreted during stress, are essential to homeostasis and metabolism in the human body. An impaired glucocorticoid signaling due to dysfunction of the glucocorticoid receptor (GR) by synthetic chemicals can cause diseases and disruptions of the homeostasis and metabolism. Here we demonstrate the development of a method for screening endocrine-disrupting chemicals and potent risk factors of human diseases based on the nuclear trafficking of the GR. We constructed a new assay using a pair of genetic indicators with the full length of the GR, split Renilla luciferase (RLuc), and split DnaE (a protein splicing element). The GR-containing fusion protein with C-terminal halves of DnaE and RLuc is localized in cytosol due to the cytosolic character of the GR, whereas the fusion protein with N-terminal halves of DnaE and RLuc stays in the nucleus due to the cofused nucleus localization signal. On being stimulated with a ligand, the GR is translocated into the cellular nucleus. Thus, a protein splicing occurs in the nucleus by an interaction between the splicing junctions of each DnaE fragment. The enzymatic activities from the reconstituted RLuc allow the ligand-dependent luminescence intensities. The feasibility of the method was evaluated by quantifying the hormonal activities of 20 different kinds of steroids and synthetic chemicals using the NIH 3T3 cells carrying the pair of indicators. The hormonal activities of tested ligands are discussed based on the chemical structure-activity relationship. We found that androgens, testosterone, and 19-nortestosterone weakly induce the nuclear transport of the GR. The current assay allows high-throughput screening of risk chemicals and drug candidates influential to a signal transduction pathway of the GR.

Active Transport, Cell Nucleus↗
PubMed2005

A high-throughput screening of genes that encode proteins transported into the endoplasmic reticulum in mammalian cells.

The compartments of eukaryotic cells maintain a distinct protein composition to perform a variety of specialized functions. We developed a new method for identifying the proteins that are transported to the endoplasmic reticulum (ER) in living mammalian cells. The principle is based on the reconstitution of two split fragments of enhanced green fluorescent protein (EGFP) by protein splicing with DnaE from Synechocystis PCC6803. Complementary DNA (cDNA) libraries fused to the N-terminal halves of DnaE and EGFP are introduced in mammalian cells with retroviruses. If an expressed protein is transported into the ER, the N-terminal half of EGFP meets its C-terminal half in the ER, and full-length EGFP is reconstituted by protein splicing. The fluorescent cells are isolated using fluorescence-activated cell sorting and the cDNAs are sequenced. The developed method was able to accurately identify cDNAs that encode proteins transported to the ER. We identified 27 novel proteins as the ER-targeting proteins. The present method overcomes the limitation of the previous GFP- or epitope-tagged methods, using which it was difficult to identify the ER-targeting proteins in a high-throughput manner.

Animals↗
PubMed2005

Methods of analysis for chemicals that disrupt cellular signaling pathways: risk assessment for potential endocrine disruptors.

Here we present a basic concept and several examples of methods of analysis for chemicals that disrupt cellular signaling pathways, in view of risk assessment for potential endocrine disrupting chemicals (EDCs). The key cellular signaling pathways include 1) ER/coactivator interaction, 2) AR translocation into the nucleus, 3) ER/NO/sGC/cGMP, 4) ER/Akt, 5) ER/Src, 6)ER/Src/Grb2, and 7) ER/Ca2+/CaM/CaMK pathways. These were visualized in relevant live cells using newly developed fluorescent and bioluminescent probes. Changes in cellular signals were thereby observed in nongenomic pathways of steroid hormones upon treatment of the target cells with steroid hormones and related chemicals. This method of analysis appears to be a rational approach to high-throughput prescreening (HTPS) of biohazardous chemicals, EDCs, in particular. Also described was the screening of gene expression by serial analysis of gene expression and gene chips upon applying EDCs to breast cancer cells, mouse livers, and human neuroblastoma NB-1 cells.

Animals↗
PubMed2004

High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase.

Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo. The principle is based on reconstitution of split fragments of Renilla reniformis (Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-based in vitro sensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasive in vivo imaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analyses in vitro and in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.

Active Transport, Cell Nucleus↗
PubMed2003

Locating a protein-protein interaction in living cells via split Renilla luciferase complementation.

For spatial and quantitative kinetic analysis of protein-protein interactions (PPIs) in living mammalian cells, a method was developed in which PPI-induced complementation of split Renilla luciferase triggers spontaneous emission of luminescence using a cell membrane permeable substrate, coelenterazine. This split Renilla luciferase complementation readout was shown to work for locating a PPI between the tyrosine-phosphorylated peptide (Y941) of IRS-1 and the SH2 domain of PI3K among insulin signaling pathways in living Chinese hamster ovary cells overexpressing human insulin receptors (CHO-HIR). It was thereby found that the insulin-stimulated interaction occurred near the plasma membrane in the cytosol.

Amino Acid Sequence↗
PubMed2003

A genetic approach to identifying mitochondrial proteins.

The control of intricate networks within eukaryotic cells relies on differential compartmentalization of proteins. We have developed a method that allows rapid identification of novel proteins compartmentalized in mitochondria by screening large-scale cDNA libraries. The principle is based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing of DnaE derived from Synechocystis sp. PCC6803. The cDNA libraries are expressed in mammalian cells following infection with retrovirus. If a test protein contains a functional mitochondrial targeting signal (MTS), it translocates into the mitochondrial matrix, where EGFP is then formed by protein splicing. The cells harboring this reconstituted EGFP are screened rapidly by fluorescence-activated cell sorting, and the cDNAs are isolated and identified from the cells. The analysis of 258 cDNAs revealed various MTSs, among which we identified new transcripts corresponding to mitochondrial proteins. This method should provide a means to map proteins distributed within intracellular organelles in a broad range of different tissues and disease states.

Amino Acid Sequence↗
PubMed2003

A screening method for estrogens using an array-type DNA glass slide.

A new screening assay was described for the determination of endocrine disrupting chemicals (EDCs), such as synthetic estrogens, with an array-type DNA glass slide having characteristics of 1) a high sample throughput, 2) a compact size allowing a small sample volume, and 3) a sensitive determination based on the estrogen-dependent binding of the human estrogen receptor a (hERalpha) with its estrogen responsive element (ERE; Vit. A2 gene promoter). We devised a glass slide on which a thin agarose gel was mounted. Avidin was then covalently immobilized on each well of the glass slide after the gel was activated by a NaIO4 solution. Also, the biotinylated ERE as a DNA probe was immobilized on the gel layer through avidin-biotin binding. After the estrogen-dependent binding of a yellow fluorescent protein-fused hERalpha (YFP-hERalpha) to ERE on the gel layer, the fluorescence intensity of YFP-hERalpha quantitatively extracted into the gel was directly determined with a fluorescence microplate reader. Pre-incubation of YFP-hERalpha with estrogen at 37 degrees C for 30 min enhanced the estrogen-dependent hERalpha-ERE binding. The determined hormonal activities of estrogens on the interaction of YFP-hERalpha with ERE were as follows in their decreasing order: diethylstilbestrol (DES) > 17beta-estradiol (E2) <==> ethynylestradiol (EE2) > 4-hydroxy tamoxifen (OHT) > clomiphene (Clo). The present method provides a sensitive estrogen-dependent dose-response curve down to approximately 10(-13) M in the case of DES. This method will become a competitive alternative to the conventional in vitro assays, such as a DNA-binding assay using radioisotopes.

Avidin↗
PubMed2002

Fluorescent indicators for imaging protein phosphorylation in single living cells.

To visualize signal transduction based on protein phosphorylation in living cells, we have developed genetically encoded fluorescent indicators, named phocuses. Two different color mutants of green fluorescent protein (GFP) were joined by a tandem fusion domain composed of a substrate domain for the protein kinase of interest, a flexible linker sequence, and a phosphorylation recognition domain that binds with the phosphorylated substrate domain. Intramolecular interaction of the substrate domain and the adjacent phosphorylation recognition domain within a phocus was dependent upon phosphorylation of the substrate domain by protein kinase, which influenced the efficiency of fluorescence resonance energy transfer (FRET) between the GFPs within a phocus. In the present study, we employed phocuses composed of insulin signaling proteins to visualize protein phosphorylation by the insulin receptor. This method may provide a general approach for studying the dynamics of protein phosphorylation-based signal transduction in living cells.

Animals↗