2021/07/12 更新

写真a

サトウ ケイタ
佐藤 恵太
SATO Keita
所属
医歯薬学域 助教
職名
助教
プロフィール
生物が持つ光受容タンパク質の分子特性と生理機能に興味があります。

学位

  • 博士(理学) ( 京都大学 )

研究キーワード

  • 光生物学

  • 視覚

  • ロドプシン

  • 非視覚性光受容

  • 光遺伝学

研究分野

  • ライフサイエンス / 動物生理化学、生理学、行動学

学歴

  • 京都大学大学院理学研究科    

    2007年4月 - 2012年3月

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  • 京都大学理学部    

    2003年4月 - 2007年3月

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経歴

  • 岡山大学医歯薬学総合研究科   細胞組織学分野   助教

    2016年5月 - 現在

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  • 京都大学大学院理学研究科   Graduate School of Science   研究員

    2012年4月 - 2016年4月

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所属学協会

 

論文

  • The Opsin 3/Teleost multiple tissue opsin system: mRNA localization in the retina and brain of medaka (Oryzias latipes). 国際誌

    Keita Sato, Khine Nwe Nwe, Hideyo Ohuchi

    The Journal of comparative neurology   2021年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Wiley  

    The photoreceptor protein, opsin, is one of the major components for vision and photoreceptive function in animals. Although many opsins have been discovered from animal genomes, only a few non-image-forming functions mediated by opsins have been identified. Understanding the mRNA distribution of photoreceptor proteins is one crucial step in uncovering their photoreceptive function in animals. Here we focus on the medaka fish (Oryzias latipes) Opsin 3 (Opn3)/Teleost multiple opsin (Tmt) system, which constitutes a separate phylogenetic group, having putative blue light photoreceptors for non-image-forming functions. In medaka, there is one opn3 and five tmt-opsin orthologs. The expression pattern of the opn3/tmt-opsins in the retina and brain was investigated by in situ hybridization. mRNAs for opn3/tmt-opsins were distributed in the retinal ganglion cells as well as interneurons and specific brain nuclei. Specifically, hybridization signals were observed in the glutamate decarboxylase 1 (gad1)-expressing amacrine cells for opn3, tmt1a, tmt1b, and tmt2, in the caudal lobe of the cerebellum for tmt1b and tmt2, in the cranial nerve nuclei for opn3, tmt1a, tmt1b, tmt2, and in the rostral pars distalis (adenohypophysis) for opn3. These expression patterns suggest that blue light sensing in the fish retina and brain may be involved in the integration of visual inputs, vestibular function, somatosensation, motor outputs, and pituitary endocrine regulation. This article is protected by copyright. All rights reserved.

    DOI: 10.1002/cne.25106

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  • KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells. 国際誌

    Yuki Kanzaki, Hirofumi Fujita, Keita Sato, Mio Hosokawa, Hiroshi Matsumae, Fumio Shiraga, Yuki Morizane, Hideyo Ohuchi

    Investigative ophthalmology & visual science   61 ( 5 )   38 - 38   2020年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Purpose: The purpose of this study was to establish and analyze a cell model of Leber congenital amaurosis type 16 (LCA16), which is caused by mutations in the KCNJ13 gene encoding Kir7.1, an inward-rectifying potassium ion channel. Methods: The two guide RNAs specific to the target sites in the KCNJ13 gene were designed and KCNJ13 knock-out (KO) human-induced pluripotent stem cells (hiPSCs) were generated using the CRISPR/Cas9 system. The KCNJ13-KO hiPSCs were differentiated into retinal pigment epithelial cells (hiPSC-RPEs). The KCNJ13-KO in hiPSC-RPEs was confirmed by immunostaining. Phagocytic activity of hiPSC-RPEs was assessed using the uptake of fluorescently labeled porcine photoreceptor outer segments (POSs). Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain reaction. Results: Most of the translated region of the KCNJ13 gene was deleted in the KCNJ13-KO hiPSCs by the CRISPR/Cas9 system, and this confirmed that the Kir7.1 protein was not present in RPE cells induced from the hiPSCs. Expression of RPE marker genes such as BEST1 and CRALBP was retained in the wild-type (WT) and in the KCNJ13-KO hiPSC-RPE cells. However, phagocytic activity and expression of phagocytosis-related genes in the KCNJ13-null hiPSC-RPE cells were significantly reduced compared to those of WT. Conclusions: We succeeded in generating an RPE model of LCA16 using hiPSCs. We suggest that Kir7.1 is required for phagocytosis of POSs by RPE cells and that impaired phagocytosis in the absence of Kir7.1 would be involved in the retinal degeneration found in LCA16.

    DOI: 10.1167/iovs.61.5.38

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  • Dickkopf3 (Dkk3) is required for maintaining the integrity of secretory vesicles in the mouse adrenal medulla. 国際誌

    Munenori Habuta, Hirofumi Fujita, Keita Sato, Tetsuya Bando, Junji Inoue, Yoichi Kondo, Satoru Miyaishi, Hiromi Kumon, Hideyo Ohuchi

    Cell and tissue research   379 ( 1 )   157 - 167   2020年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    REIC (reduced expression in immortalized cells) has been identified as a gene whose expression was reduced in immortalized cultured cells. The REIC gene is identical to Dickkopf-3 (Dkk3), which encodes a secreted glycoprotein belonging to the Dkk family. Previously, we showed that Dkk3 protein is present in the mouse adrenal medulla. However, its role in this tissue has not been elucidated. To explore it, we performed electron microscopic (EM) studies and RNA-sequencing (RNA-seq) analysis on Dkk3-null adrenal glands. EM studies showed that the number of dense core secretory vesicles were significantly reduced and empty vesicles were increased in the medulla endocrine cells. Quantitative PCR (qPCR) analysis showed relative expression levels of chromogranin A (Chga) and neuropeptide Y (Npy) were slightly but significantly reduced in the Dkk3-null adrenal glands. From the result of RNA-seq analysis as a parallel study, we selected three of the downregulated genes, uncoupled protein-1 (Ucp1), growth arrest and DNA-damage-inducible 45 gamma (Gadd45g), and Junb with regard to the estimated expression levels. In situ hybridization confirmed that these genes were regionally expressed in the adrenal gland. However, expression levels of these three genes were not consistent as revealed by qPCR. Thus, Dkk3 maintains the integrity of secreting vesicles in mouse adrenal medulla by regulating the expression of Chga and Npy.

    DOI: 10.1007/s00441-019-03113-8

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  • Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. 国際誌

    Munenori Habuta, Akihiro Yasue, Ken-Ichi T Suzuki, Hirofumi Fujita, Keita Sato, Hitomi Kono, Ayuko Takayama, Tetsuya Bando, Satoru Miyaishi, Seiichi Oyadomari, Eiji Tanaka, Hideyo Ohuchi

    PloS one   15 ( 10 )   e0240333   2020年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known that Fibroblast growth factor 10 (Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in the Fgf10-CRISPR F0 mice. However, how the lung phenotype in the Fgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in the Fgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functional Fgf10 genotypes. Firstly, according to a previous report, Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of the Fgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 ± 6.2% in type I, 25.3 ± 2.7% in type II, and 54.3 ± 9.5% in type III (mean ± standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in the Fgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functional Fgf10 genotypes. These data suggest the Fgf10 gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, the Fgf10-CRISPR F0 mouse would present an ideal experimental system to explore it.

    DOI: 10.1371/journal.pone.0240333

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  • Localization of the ultraviolet-sensor Opn5m and its effect on myopia-related gene expression in the late-embryonic chick eye. 国際誌

    Mutsuko Kato, Keita Sato, Munenori Habuta, Hirofumi Fujita, Tetsuya Bando, Yuki Morizane, Fumio Shiraga, Satoru Miyaishi, Hideyo Ohuchi

    Biochemistry and biophysics reports   19   100665 - 100665   2019年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Recent studies show that exposure to ultraviolet (UV) light suppresses ocular elongation, which causes myopia development. However, the specific mechanisms of this process have not been elucidated. A UV-sensor, Opsin 5 (Opn5) mRNA was shown to be present in extraretinal tissues. To test the possibility that UV-signals mediated by Opn5 would have a direct effect on the outer connective tissues of the eye, we first examined the expression patterns of a mammalian type Opn5 (Opn5m) in the late-embryonic chicken eye. Quantitative PCR showed Opn5m mRNA expression in the cornea and sclera. The anti-Opn5m antibody stained a small subset of cells in the corneal stroma and fibrous sclera. We next assessed the effect of UV-A (375 nm) irradiation on the chicken fibroblast cell line DF-1 overexpressing chicken Opn5m. UV-A irradiation for 30 min significantly increased the expression of Early growth response 1 (Egr1), known as an immediate early responsive gene, and of Matrix metalloproteinase 2 (Mmp2) in the presence of retinal chromophore 11-cis-retinal. In contrast, expression of Transforming growth factor beta 2 and Tissue inhibitor of metalloproteinase 2 was not significantly altered. These results indicate that UV-A absorption by Opn5m can upregulate the expression levels of Egr1 and Mmp2 in non-neuronal, fibroblasts. Taken together with the presence of Opn5m in the cornea and sclera, it is suggested that UV-A signaling mediated by Opn5 in the extraretinal ocular tissues could influence directly the outer connective tissues of the chicken late-embryonic eye.

    DOI: 10.1016/j.bbrep.2019.100665

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  • Evolutionary history of teleost intron-containing and intron-less rhodopsin genes. 国際誌

    Chihiro Fujiyabu, Keita Sato, Ni Made Laksmi Utari, Hideyo Ohuchi, Yoshinori Shichida, Takahiro Yamashita

    Scientific reports   9 ( 1 )   10653 - 10653   2019年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Springer Science and Business Media {LLC}  

    Recent progress in whole genome sequencing has revealed that animals have various kinds of opsin genes for photoreception. Among them, most opsin genes have introns in their coding regions. However, it has been known for a long time that teleost retinas express intron-less rhodopsin genes, which are presumed to have been formed by retroduplication from an ancestral intron-containing rhodopsin gene. In addition, teleosts have an intron-containing rhodopsin gene (exo-rhodopsin) exclusively for pineal photoreception. In this study, to unravel the evolutionary origin of the two teleost rhodopsin genes, we analyzed the rhodopsin genes of non-teleost fishes in the Actinopterygii. The phylogenetic analysis of full-length sequences of bichir, sturgeon and gar rhodopsins revealed that retroduplication of the rhodopsin gene occurred after branching of the bichir lineage. In addition, analysis of the tissue distribution and the molecular properties of bichir, sturgeon and gar rhodopsins showed that the abundant and exclusive expression of intron-containing rhodopsin in the pineal gland and the short lifetime of its meta II intermediate, which leads to optimization for pineal photoreception, were achieved after branching of the gar lineage. Based on these results, we propose a stepwise evolutionary model of teleost intron-containing and intron-less rhodopsin genes.

    DOI: 10.1038/s41598-019-47028-4

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  • Opn5L1 is a retinal receptor that behaves as a reverse and self-regenerating photoreceptor. 査読 国際誌

    Keita Sato, Takahiro Yamashita, Hideyo Ohuchi, Atsuko Takeuchi, Hitoshi Gotoh, Katsuhiko Ono, Misao Mizuno, Yasuhisa Mizutani, Sayuri Tomonari, Kazumi Sakai, Yasushi Imamoto, Akimori Wada, Yoshinori Shichida

    Nature communications   9 ( 1 )   1255 - 1255   2018年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Springer Nature  

    Most opsins are G protein-coupled receptors that utilize retinal both as a ligand and as a chromophore. Opsins' main established mechanism is light-triggered activation through retinal 11-cis-to-all-trans photoisomerization. Here we report a vertebrate non-visual opsin that functions as a Gi-coupled retinal receptor that is deactivated by light and can thermally self-regenerate. This opsin, Opn5L1, binds exclusively to all-trans-retinal. More interestingly, the light-induced deactivation through retinal trans-to-cis isomerization is followed by formation of a covalent adduct between retinal and a nearby cysteine, which breaks the retinal-conjugated double bond system, probably at the C11 position, resulting in thermal re-isomerization to all-trans-retinal. Thus, Opn5L1 acts as a reverse photoreceptor. We conclude that, like vertebrate rhodopsin, Opn5L1 is a unidirectional optical switch optimized from an ancestral bidirectional optical switch, such as invertebrate rhodopsin, to increase the S/N ratio of the signal transduction, although the direction of optimization is opposite to that of vertebrate rhodopsin.

    DOI: 10.1038/s41467-018-03603-3

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  • Pinopsin evolved as the ancestral dim-light visual opsin in vertebrates. 査読 国際誌

    Keita Sato, Takahiro Yamashita, Keiichi Kojima, Kazumi Sakai, Yuki Matsutani, Masataka Yanagawa, Yumiko Yamano, Akimori Wada, Naoyuki Iwabe, Hideyo Ohuchi, Yoshinori Shichida

    Communications biology   1 ( 1 )   156 - 156   2018年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Springer Nature  

    Pinopsin is the opsin most closely related to vertebrate visual pigments on the phylogenetic tree. This opsin has been discovered among many vertebrates, except mammals and teleosts, and was thought to exclusively function in their brain for extraocular photoreception. Here, we show the possibility that pinopsin also contributes to scotopic vision in some vertebrate species. Pinopsin is distributed in the retina of non-teleost fishes and frogs, especially in their rod photoreceptor cells, in addition to their brain. Moreover, the retinal chromophore of pinopsin exhibits a thermal isomerization rate considerably lower than those of cone visual pigments, but comparable to that of rhodopsin. Therefore, pinopsin can function as a rhodopsin-like visual pigment in the retinas of these lower vertebrates. Since pinopsin diversified before the branching of rhodopsin on the phylogenetic tree, two-step adaptation to scotopic vision would have occurred through the independent acquisition of pinopsin and rhodopsin by the vertebrate lineage.

    DOI: 10.1038/s42003-018-0164-x

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  • Relationship between somatic mosaicism of Pax6 mutation and variable developmental eye abnormalities-an analysis of CRISPR genome-edited mouse embryos. 査読 国際誌

    Akihiro Yasue, Hitomi Kono, Munenori Habuta, Tetsuya Bando, Keita Sato, Junji Inoue, Seiichi Oyadomari, Sumihare Noji, Eiji Tanaka, Hideyo Ohuchi

    Scientific reports   7 ( 1 )   53 - 53   2017年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Springer Nature  

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a rapid gene-targeting technology that does not require embryonic stem cells. To demonstrate dosage effects of the Pax6 gene on eye formation, we generated Pax6-deficient mice with the CRISPR/Cas system. Eyes of founder embryos at embryonic day (E) 16.5 were examined and categorized according to macroscopic phenotype as class 1 (small eye with distinct pigmentation), class 2 (pigmentation without eye globes), or class 3 (no pigmentation and no eyes). Histologically, class 1 eyes were abnormally small in size with lens still attached to the cornea at E16.5. Class 2 eyes had no lens and distorted convoluted retinas. Class 3 eyes had only rudimentary optic vesicle-like tissues or histological anophthalmia. Genotyping of neck tissue cells from the founder embryos revealed somatic mosaicism and allelic complexity for Pax6. Relationships between eye phenotype and genotype were developed. The present results demonstrated that development of the lens from the surface ectoderm requires a higher gene dose of Pax6 than development of the retina from the optic vesicle. We further anticipate that mice with somatic mosaicism in a targeted gene generated by CRISPR/Cas-mediated genome editing will give some insights for understanding the complexity in human congenital diseases that occur in mosaic form.

    DOI: 10.1038/s41598-017-00088-w

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  • Two UV-Sensitive Photoreceptor Proteins, Opn5m and Opn5m2 in Ray-Finned Fish with Distinct Molecular Properties and Broad Distribution in the Retina and Brain. 査読 国際誌

    Keita Sato, Takahiro Yamashita, Yoshihiro Haruki, Hideyo Ohuchi, Masato Kinoshita, Yoshinori Shichida

    PloS one   11 ( 5 )   e0155339   2016年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Public Library of Science ({PLoS})  

    Opn5 is a group within the opsin family of proteins that is responsible for visual and non-visual photoreception in animals. It consists of several subgroups, including Opn5m, the only subgroup containing members found in most vertebrates, including mammals. In addition, recent genomic information has revealed that some ray-finned fishes carry paralogous genes of Opn5m while other fishes have no such genes. Here, we report the molecular properties of the opsin now called Opn5m2 and its distributions in both the retina and brain. Like Opn5m, Opn5m2 exhibits UV light-sensitivity when binding to 11-cis-retinal and forms a stable active state that couples with Gi subtype of G protein. However, Opn5m2 does not bind all-trans-retinal and exhibits exclusive binding to 11-cis-retinal, whereas many bistable opsins, including fish Opn5m, can bind directly to all-trans-retinal as well as 11-cis-retinal. Because medaka fish has lost the Opn5m2 gene from its genome, we compared the tissue distribution patterns of Opn5m in medaka fish, zebrafish, and spotted gar, in addition to the distribution patterns of Opn5m2 in zebrafish and spotted gar. Opn5m expression levels showed a gradient along the dorsal-ventral axis of the retina, and preferential expression was observed in the ventral retina in the three fishes. The levels of Opn5m2 showed a similar gradient with preferential expression observed in the dorsal retina. Opn5m expression was relatively abundant in the inner region of the inner nuclear layer, while Opn5m2 was expressed in the outer edge of the inner nuclear layer. Additionally, we could detect Opn5m expression in several brain regions, including the hypothalamus, of these fish species. Opn5m2 expression could not be detected in zebrafish brain, but was clearly observed in limited brain regions of spotted gar. These results suggest that ray-finned fishes can generally utilize UV light information for non-image-forming photoreception in a wide range of cells in the retina and brain.

    DOI: 10.1371/journal.pone.0155339

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  • Two Opsin 3-Related Proteins in the Chicken Retina and Brain: A TMT-Type Opsin 3 Is a Blue-Light Sensor in Retinal Horizontal Cells, Hypothalamus, and Cerebellum. 査読 国際誌

    Mutsuko Kato, Takashi Sugiyama, Kazumi Sakai, Takahiro Yamashita, Hirofumi Fujita, Keita Sato, Sayuri Tomonari, Yoshinori Shichida, Hideyo Ohuchi

    PloS one   11 ( 11 )   e0163925   2016年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    Opsin family genes encode G protein-coupled seven-transmembrane proteins that bind a retinaldehyde chromophore in photoreception. Here, we sought potential as yet undescribed avian retinal photoreceptors, focusing on Opsin 3 homologs in the chicken. We found two Opsin 3-related genes in the chicken genome: one corresponding to encephalopsin/panopsin (Opn3) in mammals, and the other belonging to the teleost multiple tissue opsin (TMT) 2 group. Bioluminescence imaging and G protein activation assays demonstrated that the chicken TMT opsin (cTMT) functions as a blue light sensor when forced-expressed in mammalian cultured cells. We did not detect evidence of light sensitivity for the chicken Opn3 (cOpn3). In situ hybridization demonstrated expression of cTMT in subsets of differentiating cells in the inner retina and, as development progressed, predominant localization to retinal horizontal cells (HCs). Immunohistochemistry (IHC) revealed cTMT in HCs as well as in small numbers of cells in the ganglion and inner nuclear layers of the post-hatch chicken retina. In contrast, cOpn3-IR cells were found in distinct subsets of cells in the inner nuclear layer. cTMT-IR cells were also found in subsets of cells in the hypothalamus. Finally, we found differential distribution of cOpn3 and cTMT proteins in specific cells of the cerebellum. The present results suggest that a novel TMT-type opsin 3 may function as a photoreceptor in the chicken retina and brain.

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  • Contribution of Glutamic Acid in the Conserved E/DRY Triad to the Functional Properties of Rhodopsin 査読

    Keita Sato, Takahiro Yamashita, Yoshinori Shichida

    Biochemistry   53 ( 27 )   4420 - 4425   2014年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:American Chemical Society ({ACS})  

    Rhodopsin is a G protein-coupled receptor specialized for photoreception and contains a light-absorbing chromophore retinal that binds to the lysine residue of opsin through a protonated Schiff base linkage. Light converts rhodopsin to an equilibrium mixture of the active state metarhodopsin II (Mu) and its precursor, metarhodopsin I (MI), which have deprotonated and protonated Schiff base chromophores, respectively. This equilibrium was thought to depend on the pK(a) of not the Schiff base chrornophore but glutamic acid E134 in the highly conserved E/DRY triad in helix Ill. We performed mutational analyses of E134 and nearby residues to examine whether the equilibrium is really dependent on the pK(a) of E134 and to obtain dues about the contribution of E134 to the G protein activation characteristics of rhodopsin. All the single mutants at position 134 except for E134D lost the characteristic pH-dependent equilibrium, indicating that the carboxyl group of E134 is responsible for the equilibrium. Interestingly, mutation at position 134 caused little change in the MI or MI spectra or G protein activation efficiency of MII, while it caused a shift of the MI-MII equilibrium. The mutants containing hydrophobic or amide-containing residues at position 134 formed an equilibrium in favor of MII, resulting in an increase in light-induced G protein activation efficiency. On the other hand, the wild type exhibited an opsin activity lower than those of the mutants, which exhibited reasonable light-dependent activities. These results strongly suggest that the evolutionary significance of E134 is not an increase in G protein activity but rather suppression of the opsin activity.

    DOI: 10.1021/bi5003772

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  • Comparative Studies on the Late Bleaching Processes of Four Kinds of Cone Visual Pigments and Rod Visual Pigment 査読

    Keita Sato, Takahiro Yamashita, Yasushi Imamoto, Yoshinori Shichida

    Biochemistry   51 ( 21 )   4300 - 4308   2012年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:American Chemical Society ({ACS})  

    Visual pigments in rod and cone photoreceptor cells of vertebrate retinas are highly diversified photoreceptive proteins that consist of a protein moiety opsin and a light-absorbing chromophore 11-cis-retinal. There are four types of cone visual pigments and a single type of rod visual pigment. The reaction process of the rod visual pigment, rhodopsin, has been extensively investigated, whereas there have been few studies of cone visual pigments. Here we comprehensively investigated the reaction processes of cone visual pigments on a time scale of milliseconds to minutes, using flash photolysis equipment optimized for cone visual pigment photochemistry. We used chicken violet (L-group), chicken blue (M1-group), chicken green (M2-group), and monkey green (L-group) visual pigments as representatives of the respective groups of the phylogenetic tree of cone pigments. The S, M1, and M2 pigments showed the formation of a pH-dependent mixture of meta intermediates, similar to that formed from rhodopsin. Although monkey green (L-group) also formed a mixture of meta intermediates, pH dependency of meta intermediates was not observed. However, meta intermediates of monkey green became pH dependent when the chloride ion bound to the monkey green was replaced with a nitrate ion. These results strongly suggest that rhodopsin and S, M1, and M2 cone visual pigments share a molecular mechanism for activation, whereas the L-group pigment may have a special reaction mechanism involving the chloride-binding site.

    DOI: 10.1021/bi3000885

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  • Spectroscopic Analysis of the Effect of Chloride on the Active Intermediates of the Primate L Group Cone Visual Pigment 査読

    Takefumi Morizumi, Keita Sato, Yoshinori Shichida

    Biochemistry   51 ( 50 )   10017 - 10023   2012年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:American Chemical Society ({ACS})  

    Cone visual pigments responsible for color vision are classified into four groups; among these, the L(LWS) group contains the visual pigments having the most red-shifted lambda(max) and a chloride-binding site in their protein moiety. Binding of chloride results in the socalled "chloride effect", e.g., the red shift of lambda(max) and the faster decay of meta-I. These properties disappear upon replacement of chloride with nitrate. Because the amino acid residue primary responsible for the chloride effect is H197, we have replaced this residue with 19 other amino acids to gain insights into the mechanism creating these properties. Of the 19 single-site mutants, 13 were successfully expressed and bound 11-cis-retinal to form pigments. Eleven of the 13 mutants exhibited a red shift of lambda(max) upon chloride binding, and histidine produced the most red-shifted lambda(max). We classified H197 mutants into three groups according to their properties. The first group of mutants exhibited a chloride effect similar to that of the wild type, while the second group of mutants showed no chloride effect. The third group of mutants exhibited a small shift in lambda(max) and enhanced decay rates of meta-I upon chloride binding. Furthermore, some of the mutants in this group showed meta-I decay faster than that of the wild type and extraordinarily fast decays of meta-I even in the absence of chloride. Interestingly, amino acid residues in the third group of mutants are characterized by their propensity to form beta-sheets. These results suggest that the acquisition of H197 would be due to the most red-shifted absorption maximum, resulting in fast formation of the active state.

    DOI: 10.1021/bi300995s

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  • Vertebrate ancient-long opsin has molecular properties intermediate between those of vertebrate and invertebrate visual pigments. 査読 国際誌

    Keita Sato, Takahiro Yamashita, Hideyo Ohuchi, Yoshinori Shichida

    Biochemistry   50 ( 48 )   10484 - 90   2011年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:American Chemical Society ({ACS})  

    VA/VAL opsin is one of the four kinds of nonvisual opsins that are closely related to vertebrate visual pigments in the phylogenetic tree of opsins. Previous studies indicated that among these opsins, parapinopsin and pinopsin exhibit molecular properties similar to those of invertebrate bistable visual pigments and vertebrate visual pigments, respectively. Here we show that VA/VAL opsin exhibits molecular properties intermediate between those of parapinopsin and pinopsin. VAL opsin from Xenopus tropicalis was expressed in cultured cells, and the pigment with an absorption maximum at 501 nm was reconstituted by incubation with 11-cis-retinal. Light irradiation of this pigment caused cis-to-trans isomerization of the chromophore to form a state having an absorption maximum in the visible region. This state has the ability to activate Gi and Gt types of G proteins. Therefore, the active state of VAL opsin is a visible light-absorbing intermediate, which probably has a protonated retinylidene Schiff base as its chromophore, like the active state of parapinopsin. However, this state was apparently photoinsensitive and did not show reverse reaction to the original pigment, unlike the active state of parapinopsin, and instead similar to that of pinopsin. Furthermore, the Gi activation efficiency of VAL opsin was between those of pinopsin and parapinopsin. Thus, the molecular properties of VA/VAL opsin give insights into the mechanism of conversion of the molecular properties from invertebrate to vertebrate visual pigments.

    DOI: 10.1021/bi201212z

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  • Direct Observation of the pH-Dependent Equilibrium between Metarhodopsins I and II and the pH-Independent Interaction of Metarhodopsin II with Transducin C-Terminal Peptide 査読

    Keita Sato, Takefumi Morizumi, Takahiro Yamashita, Yoshinori Shichida

    Biochemistry   49 ( 4 )   736 - 741   2010年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:American Chemical Society ({ACS})  

    Bovine rhodopsin contains 11-cis-retinal as a light-absorbing chromophore that binds to a lysine residue of the apoprotein opsin via a protonated Schiff base linkage. Light isomerizes 11-cis-retinal into the all-trans form, which eventually leads to the formation of an enzymatically active state, metarhodopsin II (MII). It is widely believed that MII forms a pH-dependent equilibrium with metarhodopsin I (MI), but direct evidence for this equilibrium has not been reported. Here, we confirmed this equilibrium by direct observation of the mutual conversions of MI and MII upon changing the pH of the MI/MII mixture. We also observed a reversible binding of the synthetic peptide constituting the C-terminal 11 amino acids of the transducin alpha-subunit to MII, which resulted in change of the amounts of MI and MII in the equilibrium. Interestingly, addition of the peptide did not induce a simple pK(a) shift but rather induced an increase of the MII fraction at high pH. These results indicate that in addition to the MII that is formed from MI in a pH-dependent manner there also exists another MII, which is in equilibrium with MI in a pH-independent manner and can bind to the peptide. Therefore, there is no need for proton uptake by the protein moiety of opsin for the binding to the peptide.

    DOI: 10.1021/bi9018412

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書籍等出版物

  • Vertebrate Photoreceptors: Functional Molecular Bases

    佐藤恵太, 七田芳則( 担当: 分担執筆 ,  範囲: Chapter 1: Evolution and Diversity of Visual Pigments in Connection with Their Functional Differences)

    Springer  2014年3月 

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MISC

  • Congenital eye anomalies: More mosaic than thought? 国際誌

    Hideyo Ohuchi, Keita Sato, Munenori Habuta, Hirofumi Fujita, Tetsuya Bando

    Congenital anomalies   59 ( 3 )   56 - 73   2019年5月

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    記述言語:英語  

    The eye is a sensory organ that primarily captures light and provides the sense of sight, as well as delivering non-visual light information involving biological rhythms and neurophysiological activities to the brain. Since the early 1990s, rapid advances in molecular biology have enabled the identification of developmental genes, genes responsible for human congenital diseases, and relevant genes of mutant animals with various anomalies. In this review, we first look at the development of the eye, and we highlight seminal reports regarding archetypal gene defects underlying three developmental ocular disorders in humans: (1) holoprosencephaly (HPE), with cyclopia being exhibited in the most severe cases; (2) microphthalmia, anophthalmia, and coloboma (MAC) phenotypes; and (3) anterior segment dysgenesis (ASDG), known as Peters anomaly and its related disorders. The recently developed methods, such as next-generation sequencing and genome editing techniques, have aided the discovery of gene mutations in congenital eye diseases and gene functions in normal eye development. Finally, we discuss Pax6-genome edited mosaic eyes and propose that somatic mosaicism in developmental gene mutations should be considered a causal factor for variable phenotypes, sporadic cases, and de novo mutations in human developmental disorders.

    DOI: 10.1111/cga.12304

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  • 「光受容体」Opn5が示す多様な分子機能

    佐藤 恵太, 山下 高廣, 大内 淑代, 七田 芳則

    生物物理   59 ( 3 )   132 - 136   2019年

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    記述言語:日本語   出版者・発行元:一般社団法人 日本生物物理学会  

    <p>Opsins are animal photoreceptor proteins that use retinal as their chromophore (ligand) and are classified into several groups based on sequence similarity. Opn5 forms one phylogenetically separate opsin group and includes three vertebrate-specific subgroups, Opn5m, Opn5L1 and Opn5L2. In this article, we introduce highly diversified functionalities of vertebrate Opn5. Three Opn5 subgroups show characteristic spectral sensitivity, preference for retinal isomers and photoreaction process, and work as bistable photoreceptor, retinal chemoreceptor or unidirectional photoreceptor. Such broad spectrum of functionalities may be attributed to optimization for respective physiological functions.</p>

    DOI: 10.2142/biophys.59.132

    CiNii Article

    CiNii Books

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    その他リンク: http://search.jamas.or.jp/link/ui/2019266724

講演・口頭発表等

  • KCNJ13遺伝子欠損がヒト多能性幹細胞由来網膜色素上皮細胞の貪食能に及ぼす影響

    神崎勇希, 神崎勇希, 藤田洋史, 佐藤恵太, 細川海音, 松前洋, 白神史雄, 森實祐基, 大内淑代

    日本眼科学会雑誌  2020年 

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    開催年月日: 2020年

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  • 分子組織化学的解析による真骨魚類が持つ複数のロドプシンの進化過程の解明

    佐藤恵太, 藤藪千尋, UTARI Ni Made Laksmi, 大内淑代, 七田芳則, 山下高廣

    日本解剖学会総会・全国学術集会講演プログラム・抄録集  2020年 

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    開催年月日: 2020年

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  • Fgf10モザイク変異体における肺表現型および下肢帯表現型の形態学的解析

    土生田宗憲, 泰江章博, 鈴木賢一, 高山鮎子, 藤田洋史, 板東哲哉, 佐藤恵太, 親泊政一, 田中栄二, 大内淑代

    日本先天異常学会学術集会プログラム・抄録集  2020年 

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    開催年月日: 2020年

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  • ゲノム編集Fgf10モザイク変異体の遺伝子型と表現型解析

    土生田宗憲, 泰江章博, 鈴木賢一, 藤田洋史, 高山鮎子, 佐藤恵太, 板東哲哉, 親泊政一, 田中栄二, 大内淑代

    日本解剖学会総会・全国学術集会講演プログラム・抄録集  2020年 

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    開催年月日: 2020年

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  • REIC/Dickkopf3(Dkk3)の副腎における機能解析

    土生田宗憲, 藤田洋史, 佐藤恵太, 板東哲哉, 公文裕巳, 大内淑代

    日本解剖学会総会・全国学術集会講演プログラム・抄録集  2019年 

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    開催年月日: 2019年

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  • ニワトリ視床下部における光受容タンパク質Opn5L1の分子組織化学的解析

    佐藤恵太, 春木慶洸, 山下高廣, 七田芳則, 大内淑代

    日本下垂体研究会学術集会プログラム・講演要旨集  2019年 

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    開催年月日: 2019年

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  • 非視覚性光受容タンパク質Opn5L1の分子組織化学的解析

    佐藤恵太, 春木慶洸, 山下高廣, 七田芳則, 七田芳則, 大内淑代

    日本解剖学会総会・全国学術集会講演プログラム・抄録集  2019年 

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    開催年月日: 2019年

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  • 神経網膜形成における転写因子Lhx1の下流遺伝子の機能解析

    衣畑俊希, 佐藤恵太, 藤田洋史, 板東哲哉, 濃野勉, 大内淑代

    日本分子生物学会年会プログラム・要旨集(Web)  2019年 

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    開催年月日: 2019年

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  • Lhx1過剰発現による二重網膜誘導モデルを用いた網膜初期発生の研究

    衣畑俊希, 佐藤恵太, 藤田洋史, 板東哲哉, 大内淑代

    日本分子生物学会年会プログラム・要旨集(Web)  2018年 

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    開催年月日: 2018年

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  • ゲノム編集Fgf10モザイク変異体の組織学的解析

    土生田宗憲, 泰江章博, 藤田洋史, 板東哲哉, 佐藤恵太, 親泊政一, 田中栄二, 大内淑代

    日本解剖学会総会・全国学術集会講演プログラム・抄録集  2018年 

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    開催年月日: 2018年

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  • 小型魚類における光受容タンパク質Opn3及びそのホモログtmtオプシンの組織局在

    佐藤恵太, NWE Khine Nwe, NWE Khine Nwe, 大内淑代

    日本解剖学会総会・全国学術集会講演プログラム・抄録集  2018年 

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    開催年月日: 2018年

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  • 紫外線受容視物質オプシン5のニワトリ胚線維芽細胞における作用

    加藤睦子, 加藤睦子, 佐藤恵太, 藤田洋史, 板東哲哉, 森實祐基, 中山正, 白神史雄, 大内淑代

    日本眼科学会雑誌  2018年 

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    開催年月日: 2018年

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  • 脊椎動物がもつ新規光センサーOpn5L1の不活性化状態の発色団構造

    水野操, 水谷泰久, 佐藤恵太, 大内淑代, 山下高廣, 酒井佳寿美, 今元泰, 七田芳則, 七田芳則, 山野由美子, 和田昭盛

    分子科学討論会講演プログラム&要旨(Web)  2018年 

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    開催年月日: 2018年

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  • ピノプシンは薄明視を担う視物質として進化してきた光受容タンパク質である

    佐藤恵太, 山下高廣, 小島慧一, 松谷優樹, 酒井佳寿美, 柳川正隆, 山野由美子, 和田昭盛, 岩部直之, 大内淑代, 七田芳則, 七田芳則

    日本分子生物学会年会プログラム・要旨集(Web)  2018年 

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    開催年月日: 2018年

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  • メダカ下垂体に発現する光受容タンパク質の分子組織化学的解析

    佐藤恵太, 菱池政展, 大内淑代

    日本下垂体研究会学術集会プログラム・講演要旨集  2018年 

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    開催年月日: 2018年

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  • Fgf10モザイク変異体における肺表現型の形態学的解析

    土生田宗憲, 泰江章博, 藤田洋史, 板東哲哉, 佐藤恵太, 親泊政一, 田中栄二, 大内淑代

    日本先天異常学会学術集会プログラム・抄録集  2018年 

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    開催年月日: 2018年

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  • Analysis of CRISPR genome-edited founder mice shows relationship between somatic mosaicism of Pax6 mutation and variable developmental eye abnormalities

    Akihiro Yasue, Hitomi Kono, Munenori Habuta, Tetsuya Bando, Keita Sato, Junji Inoue, Seiichi Oyadomari, Sumihare Noji, Eiji Tanaka, Hideyo Ohuchi

    Mechanisms of Development  2017年7月  Elsevier {BV}

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    開催年月日: 2017年7月

    記述言語:英語  

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  • CRISPR/Cas9システムにより作製したモザイク変異マウスの組織学的解析

    土生田宗憲, 泰江章博, 藤田洋史, 板東哲哉, 佐藤恵太, 親泊政一, 田中栄二, 大内淑代

    日本生化学会大会(Web)  2017年 

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    開催年月日: 2017年

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  • 新規オプシン5様視物質(cOpn5n)のニワトリ網膜における発現様式

    大内淑代, 山下高廣, 湯本茜, 佐藤恵太, 井上順治, 友成さゆり, 野地澄晴, 七田芳則

    日本組織細胞化学会総会・学術集会講演プログラム・予稿集  2013年 

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    開催年月日: 2013年

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  • Opn5の分子機構多様性とその応用 招待

    佐藤恵太

    ISSP ワークショップ「レチナールタンパク質の光機能発現の物理と化学」  2019年9月 

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    会議種別:シンポジウム・ワークショップ パネル(指名)  

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  • Opn5類縁タンパク質の光遺伝学への応用可能性 招待

    佐藤恵太

    第90回日本動物学会大会  2019年9月 

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    会議種別:口頭発表(招待・特別)  

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  • 脊椎動物の光受容体Opn5L1は逆行性・自己再生能をもつ新しいタイプのオプシンである

    第56回日本生物物理学会年会  2018年9月 

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    会議種別:口頭発表(招待・特別)  

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  • Opn5L1, a reverse photoreceptor with light-induced covalent chromophore modification 招待

    佐藤 恵太

    International Symposium on Biophysics of Rhodopsins  2017年5月 

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    記述言語:英語   会議種別:口頭発表(招待・特別)  

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  • Opn5L1 is a photocyclic GPCR 招待

    佐藤 恵太

    第53回日本生物物理学会年会  2015年9月 

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    記述言語:英語   会議種別:口頭発表(招待・特別)  

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▼全件表示

受賞

  • 若手奨励賞

    2018年9月   日本生物物理学会   脊椎動物の光受容体Opn5L1は逆行性・自己再生能をもつ新しいタイプのオプシンである

    佐藤 恵太

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    受賞区分:国内学会・会議・シンポジウム等の賞 

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  • Poster Award

    2016年   Gordon Research Conference: Photosensory Receptors & Signal Transduction   Opn5L1 is a reverse and self-regenerating photoreceptor with light-induced covalent chromophore modification

    佐藤 恵太

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  • Best Poster Award

    2015年   FASEB Science Research Conference “The Biology and Chemistry of Vision”   Non-mammalian photoreceptor protein Opn5L1 is a photocyclic G protein-coupled receptor

    佐藤 恵太

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共同研究・競争的資金等の研究

  • 眼球伸長におけるFGF10と紫光Opsin5システムの機能解明

    研究課題/領域番号:20K21655  2020年07月 - 2023年03月

    日本学術振興会  科学研究費助成事業 挑戦的研究(萌芽)  挑戦的研究(萌芽)

    大内 淑代, 藤田 洋史, 佐藤 恵太

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    配分額:6370000円 ( 直接経費:4900000円 、 間接経費:1470000円 )

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  • 魚類下垂体がもつ光受容能の分子機構の解明、その応用による内分泌系光操作技術の開発

    研究課題/領域番号:20K08885  2020年04月 - 2023年03月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    佐藤 恵太, 神田 真司

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    配分額:4420000円 ( 直接経費:3400000円 、 間接経費:1020000円 )

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  • 逆方向光受容体Opn5L1の生理機能の同定

    研究課題/領域番号:17K15159  2017年04月 - 2020年03月

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    佐藤 恵太

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    担当区分:研究代表者  資金種別:競争的資金

    配分額:4420000円 ( 直接経費:3400000円 、 間接経費:1020000円 )

    本研究ではメダカをモデル動物とし、光受容分子オプシン5-like1(Opn5L1)の生理機能を明らかにするため、Opn5L1発現細胞においてレポータータンパク質の遺伝子を発現する遺伝子組み換えメダカを作製し、解析することを試みている。前年度までの課題として、遺伝子導入に用いた遺伝子コンストラクトの導入効率が悪いこと、スクリーニングにかかる手間と時間が過大であること、Opn5L1プロモーターの転写活性が低いことなどから、解析に用いる事が可能な遺伝子組み換えメダカを得ることができていなかった。
    最終年度は、より効率的にトランスジェニックメダカを作製するためのベクターの構築を行った。まずスクリーニングの手間をなるべく省くため、ゼブラフィッシュ心筋特異的遺伝子(cmlc2)プロモーターに蛍光タンパク質(EGFPまたはmRFP)とSV40ポリAを連結した配列を搭載した。これにより、遺伝子導入の可否を孵化前の段階で心臓の蛍光の有無によって判別できる。Opn5L1プロモーターの転写活性が低いために、レポーター遺伝子の発現量が十分でない問題を解決するため、CRE/loxpシステムを導入した。Opn5L1プロモーターにCREリコンビナーゼを、メダカβアクチンプロモーターにloxp-STOP-loxpとレポーター遺伝子を連結したベクターをそれぞれ構築した。これらの遺伝子を導入したメダカにおいては、βアクチンプロモーターからの転写は通常STOP配列で停止しているが、Opn5L1発現細胞ではSTOP配列が除去され、レポーター遺伝子が発現すると期待される。またOpn5L1プロモーター-CREやβアクチンプロモーター-loxP-STOP-loxP-レポーターの配列は、cmlc2プロモーターや、ゲノム上で組み込まれた領域の近傍の遺伝子の影響を避けるため、インシュレータ配列で挟み込むように設計した。
    現在これらのプラスミドをメダカ卵へのマイクロインジェクションにより導入中である。

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  • 光受容タンパク質オプシンにおけるレチナールとシステインの共役付加反応をもたらすメカニズムの解明とその応用

    2016年04月 - 2017年03月

    公益財団法人 日本科学協会  笹川科学研究助成 

    佐藤 恵太

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    担当区分:研究代表者  資金種別:競争的資金

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  • 光受容蛋白質ロドプシンに見るG蛋白質共役型受容体の活性化状態形成機構の最適化

    2009年04月 - 2012年03月

    日本学術振興会  特別研究員奨励費 

    佐藤 恵太

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    担当区分:研究代表者  資金種別:競争的資金

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  • 光受容蛋白質ロドプシンに見るG蛋白質共役型受容体の活性化状態形成機構の最適化

    研究課題/領域番号:09J01046  2009年 - 2011年

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    佐藤 恵太

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    配分額:2800000円 ( 直接経費:2800000円 )

    VA/VALオプシンは分子系統樹上において、脊椎動物型視物質が多様化する前に分岐したと考えられる蛋白質であり、その性質を決定することで脊椎動物型視物質に共有される性質が、祖先型蛋白質が進化する過程のどの段階で獲得されたかについて情報を得られると考えた。
    これまでの知見として、脊椎動物型視物質と、無脊椎動物型ロドプシンやパラピノプシンの性質を比較すると、脊椎動物型視物質の活性状態は紫外部に、無脊椎動物型ロドプシンやパラピノプシンなどの活性状態は可視部に吸収極大があることが知られている。さらに無脊椎動物型ロドプシンやパラピノプシンの活性状態に特有の性質として、直接全トランス型レチナールと結合しても形成されること、光があたると不活性な状態に戻ることが知られている。また、G蛋白質活性化効率を比較すると無脊椎動物型ロドプシンやパラピノプシンの活性化効率は脊椎動物型ロドプシンの1/50から1/20程度であると言われている。
    アフリカツメガエルのVALオプシンの性質を調べた結果、全トランス型レチナールの直接結合能は極めて低く、活性状態は光に対して非感受性である一方でその吸収極大は可視波長にあり、且つそのG蛋白質活性化効率はウシロドプシンと比較しておよそ1/5程度であった。これらの結果はVALオプシンの活性状態はある部分において脊椎動物型視物質に近く、ある部分においてパラピノプシンや無脊椎動物型ロドプシンに近いことを示す。この結果から、無脊椎動物型ロドプシン様の活性化メカニズムを持つオプシンから脊椎動物型視物質様の活性化メカニズムを持つオプシンへの変遷は段階的に起こったものであり、その途上にある祖先型蛋白質からVALオプシンが分岐して現れた可能性が考えられる。さらに、活性状態の性質の変化に対応する重要なアミノ酸の置換が複数存在することが考えられる。

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