Updated on 2021/10/15

写真a

 
HIROTA Tsuyoshi
 
Organization
Institute of Transformative Bio-Molecules Designated associate professor
Graduate School
Graduate School of Science
Title
Designated associate professor

Degree 1

  1. 博士(理学) ( 2003.3   東京大学 ) 

Research Areas 1

  1. Life Science / Functional biochemistry

Current Research Project and SDGs 1

  1. 概日時計の分子機構の解析

Research History 9

  1. Japan Science and Technology Agency   PRESTO Researcher (concurrent)

    2014.10 - 2018.3

  2. Nagoya University   Institute of Transformative Bio-Molecules   Designated associate professor

    2014.5

  3. University of Southern California   Researcher

    2013.7 - 2014.4

  4. UC San Diego   Researcher

    2012.7 - 2013.7

  5. UC San Diego   Researcher

    2008.10 - 2012.6

  6. UC San Diego   Researcher

    2007.9 - 2008.9

  7. The Scripps Research Institute   Researcher

    2007.4 - 2007.9

  8. The University of Tokyo   Graduate School of Science   Designated assistant professor

    2003.4 - 2007.3

  9. JSPS   Research Fellow (DC1)

    2000.4 - 2003.3

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Education 1

  1. The University of Tokyo   Grasuate School of Science   Department of Biophysics and Biochemistry

    1998.4 - 2003.3

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    Country: Japan

 

Papers 51

  1. Effects of Cryptochrome-modulating compounds on circadian behavioral rhythms in zebrafish. Reviewed

    Iida M, Nakane Y, Yoshimura T, Hirota T

    Journal of biochemistry     2021.9

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    DOI: 10.1093/jb/mvab096

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  2. Structural differences in the FAD-binding pockets and lid loops of mammalian CRY1 and CRY2 for isoform-selective regulation. Reviewed

    Miller S, Srivastava A, Nagai Y, Aikawa Y, Tama F, Hirota T

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 118 ( 26 )   2021.6

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    The circadian clock is a biological timekeeper that operates through transcription–translation feedback loops in mammals. Cryptochrome 1 (CRY1) and Cryptochrome 2 (CRY2) are highly conserved core clock components having redundant and distinct functions. We recently identified the CRY1- and CRY2-selective compounds KL101 and TH301, respectively, which provide useful tools for the exploration of isoform-selective CRY regulation. However, intrinsic differences in the compound-binding FAD (flavin adenine dinucleotide) pockets between CRY1 and CRY2 are not well understood, partly because of nonoptimal properties of previously reported apo form structures in this particular region constituted by almost identical sequences. Here, we show unliganded CRY1 and CRY2 crystal structures with well-defined electron densities that are largely free of crystal contacts at the FAD pocket and nearby lid loop. We revealed conformational isomerism in key residues. In particular, CRY1 W399 and corresponding CRY2 W417 in the FAD pocket had distinct conformations (“out” for CRY1 and “in” for CRY2) by interacting with the lid loop residues CRY1 Q407 and CRY2 F424, respectively, resulting in different overall lid loop structures. Molecular dynamics simulations supported that these conformations were energetically favorable to each isoform. Isoform-selective compounds KL101 and TH301 preferred intrinsic “out” and “in” conformations of the tryptophan residue in CRY1 and CRY2, respectively, while the nonselective compound KL001 fit to both conformations. Mutations of lid loop residues designed to perturb their isoform-specific interaction with the tryptophan resulted in reversed responses of CRY1 and CRY2 to KL101 and TH301. We propose that these intrinsic structural differences of CRY1 and CRY2 can be targeted for isoform-selective regulation.

    DOI: 10.1073/pnas.2026191118

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  3. Reversible modulation of circadian time with chronophotopharmacology. Reviewed

    Kolarski D, Miró-Vinyals C, Sugiyama A, Srivastava A, Ono D, Nagai Y, Iida M, Itami K, Tama F, Szymanski W, Hirota T, Feringa BL

    Nature communications   Vol. 12 ( 1 ) page: 3164   2021.5

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    The circadian clock controls daily rhythms of physiological processes. The presence of the clock mechanism throughout the body is hampering its local regulation by small molecules. A photoresponsive clock modulator would enable precise and reversible regulation of circadian rhythms using light as a bio-orthogonal external stimulus. Here we show, through judicious molecular design and state-of-the-art photopharmacological tools, the development of a visible light-responsive inhibitor of casein kinase I (CKI) that controls the period and phase of cellular and tissue circadian rhythms in a reversible manner. The dark isomer of photoswitchable inhibitor 9 exhibits almost identical affinity towards the CKIα and CKIδ isoforms, while upon irradiation it becomes more selective towards CKIδ, revealing the higher importance of CKIδ in the period regulation. Our studies enable long-term regulation of CKI activity in cells for multiple days and show the reversible modulation of circadian rhythms with a several hour period and phase change through chronophotopharmacology.

    DOI: 10.1038/s41467-021-23301-x

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  4. Comparing the efficacy and selectivity of Ck2 inhibitors. A phosphoproteomics approach Reviewed

    Christian Borgo, Luca Cesaro, Tsuyoshi Hirota, Keiko Kuwata, Claudio D’Amore, Thomas Ruppert, Renata Blatnik, Mauro Salvi, Lorenzo A. Pinna

    European Journal of Medicinal Chemistry   Vol. 214   page: 113217 - 113217   2021.3

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    DOI: 10.1016/j.ejmech.2021.113217

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  5. Photopharmacological Manipulation of Mammalian CRY1 for Regulation of the Circadian Clock Reviewed

    Dušan Kolarski, Simon Miller, Tsuyoshi Oshima, Yoshiko Nagai, Yugo Aoki, Piermichele Kobauri, Ashutosh Srivastava, Akiko Sugiyama, Kazuma Amaike, Ayato Sato, Florence Tama, Wiktor Szymanski, Ben L. Feringa, Kenichiro Itami, Tsuyoshi Hirota

    Journal of the American Chemical Society   Vol. 143 ( 4 ) page: 2078 - 2087   2021.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society (ACS)  

    DOI: 10.1021/jacs.0c12280

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  6. Reductive stability evaluation of 6-azopurine photoswitches for the regulation of CKIα activity and circadian rhythms Reviewed

    Dušan Kolarski, Akiko Sugiyama, Theo Rodat, Albert Schulte, Christian Peifer, Kenichiro Itami, Tsuyoshi Hirota, Ben L. Feringa, Wiktor Szymanski

    Organic & Biomolecular Chemistry   Vol. 19 ( 10 ) page: 2312 - 2321   2021

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Royal Society of Chemistry ({RSC})  

    DOI: 10.1039/D1OB00014D

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  7. A N-terminally deleted form of the CK2α’ catalytic subunit is sufficient to support cell viability Reviewed

    Christian Borgo, Claudio D’Amore, Luca Cesaro, Kenichiro Itami, Tsuyoshi Hirota, Mauro Salvi, Lorenzo A. Pinna

    Biochemical and Biophysical Research Communications   Vol. 531 ( 3 ) page: 409 - 415   2020.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier {BV}  

    DOI: 10.1016/j.bbrc.2020.07.112

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  8. An Isoform-Selective Modulator of Cryptochrome 1 Regulates Circadian Rhythms in Mammals Reviewed

    Simon Miller, Yoshiki Aikawa, Akiko Sugiyama, Yoshiko Nagai, Aya Hara, Tsuyoshi Oshima, Kazuma Amaike, Steve A. Kay, Kenichiro Itami, Tsuyoshi Hirota

    Cell Chemical Biology   Vol. 27 ( 9 ) page: 1192 - 1198.e5   2020.6

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier {BV}  

    DOI: 10.1016/j.chembiol.2020.05.008

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  9. Small Molecules Modulating Mammalian Biological Clocks: Exciting New Opportunities for Synthetic Chemistry Reviewed

    Amaike, K., Oshima, T., Skoulding, N.S., Toyama, Y., Hirota, T., Itami, K.

    Chem   Vol. 6 ( 9 ) page: 2186 - 2198   2020

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    Language:Japanese   Publishing type:Research paper (scientific journal)  

    DOI: 10.1016/j.chempr.2020.08.011

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  10. Small molecule modulators of the circadian clock function Reviewed

    Hirota Tsuyoshi

    Proceedings for Annual Meeting of The Japanese Pharmacological Society   Vol. 93 ( 0 ) page: 1 - S12-3   2020

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    Publishing type:Research paper (scientific journal)   Publisher:Japanese Pharmacological Society  

    <p>In mammals, circadian rhythms are generated through transcriptional regulatory networks of the clock genes. To search for novel clock modifiers, we applied chemical biology approaches. From hundreds of thousands of small molecules with diverse structure, we identified a number of compounds that potently change the period of the circadian clock in human cells. Among the period lengthening compounds, we previously discovered the first small molecule targeting the core clock protein CRY. The compound KL001 interacts with FAD-binding pocket of CRY and inhibits FBXL3-dependent degradation. By analyzing KL001 derivatives, we found 10 times more potent compound KL044. KL001 and KL044 share carbazole group and act on both CRY1 and CRY2. We further identified novel period lengthening compounds KL101 and TH301 that do not have carbazole group. Surprisingly, we discovered that KL101 is selective against CRY1 while TH301 shows much higher effect on CRY2. To understand molecular basis of the CRY1/CRY2 selectivity, we determined the X-ray crystal structures of CRY1-KL101, CRY1-TH301, CRY2-TH301, and CRY1-KL044 complexes. In this presentation, I will discuss these unique compounds that will enable atomic-level dissection of the functional difference between CRY1 and CRY2 proteins and their selective manipulation.</p>

    DOI: 10.1254/jpssuppl.93.0_1-S12-3

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  11. “Janus” efficacy of CX-5011: CK2 inhibition and methuosis induction by independent mechanisms Reviewed

    D'Amore, C., Moro, E., Borgo, C., Itami, K., Hirota, T., Pinna, L.A., Salvi, M.

    Biochimica et Biophysica Acta - Molecular Cell Research   Vol. 1867 ( 11 ) page: 118807   2020

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    DOI: 10.1016/j.bbamcr.2020.118807

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  12. Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing Reviewed

    Fribourgh, J.L., Srivastava, A., Sandate, C.R., Michael, A.K., Hsu, P.L., Rakers, C., Nguyen, L.T., Torgrimson, M.R., Parico, G.C.G., Tripathi, S., Zheng, N., Lander, G.C., Hirota, T., Tama, F., Partch, C.L.

    eLife   Vol. 9   2020

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    DOI: 10.7554/eLife.55275

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  13. Pharmacological Interventions to Circadian Clocks and Their Molecular Bases Reviewed

    Miller, S., Hirota, T.

    Journal of Molecular Biology   Vol. 432 ( 12 ) page: 3498 - 3514   2020

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    DOI: 10.1016/j.jmb.2020.01.003

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  14. Isoform-selective regulation of mammalian cryptochromes Reviewed

    Miller, S., Son, Y.L., Aikawa, Y., Makino, E., Nagai, Y., Srivastava, A., Oshima, T., Sugiyama, A., Hara, A., Abe, K., Hirata, K., Oishi, S., Hagihara, S., Sato, A., Tama, F., Itami, K., Kay, S.A., Hatori, M., Hirota, T.

    Nature Chemical Biology   Vol. 16 ( 6 ) page: 676 - 685   2020

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    DOI: 10.1038/s41589-020-0505-1

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  15. Identification of pathways that regulate circadian rhythms using a larval zebrafish small molecule screen Reviewed

    Eric A. Mosser, Cindy N. Chiu, T. Katherine Tamai, Tsuyoshi Hirota, Suna Li, May Hui, Amy Wang, Chanpreet Singh, Andrew Giovanni, Steve A. Kay, David A. Prober

    Scientific Reports   Vol. 9 ( 1 ) page: 12405   2019.12

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media {LLC}  

    DOI: 10.1038/s41598-019-48914-7

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    Other Link: http://orcid.org/0000-0003-4876-3608

  16. Casein kinase 1 family regulates PRR5 and TOC1 in the Arabidopsis circadian clock. Reviewed International journal

    Takahiro N Uehara, Yoshiyuki Mizutani, Keiko Kuwata, Tsuyoshi Hirota, Ayato Sato, Junya Mizoi, Saori Takao, Hiromi Matsuo, Takamasa Suzuki, Shogo Ito, Ami N Saito, Taeko Nishiwaki-Ohkawa, Kazuko Yamaguchi-Shinozaki, Takashi Yoshimura, Steve A Kay, Kenichiro Itami, Toshinori Kinoshita, Junichiro Yamaguchi, Norihito Nakamichi

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 116 ( 23 ) page: 11528 - 11536   2019.6

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    The circadian clock provides organisms with the ability to adapt to daily and seasonal cycles. Eukaryotic clocks mostly rely on lineage-specific transcriptional-translational feedback loops (TTFLs). Posttranslational modifications are also crucial for clock functions in fungi and animals, but the posttranslational modifications that affect the plant clock are less understood. Here, using chemical biology strategies, we show that the Arabidopsis CASEIN KINASE 1 LIKE (CKL) family is involved in posttranslational modification in the plant clock. Chemical screening demonstrated that an animal CDC7/CDK9 inhibitor, PHA767491, lengthens the Arabidopsis circadian period. Affinity proteomics using a chemical probe revealed that PHA767491 binds to and inhibits multiple CKL proteins, rather than CDC7/CDK9 homologs. Simultaneous knockdown of Arabidopsis CKL-encoding genes lengthened the circadian period. CKL4 phosphorylated transcriptional repressors PSEUDO-RESPONSE REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1) in the TTFL. PHA767491 treatment resulted in accumulation of PRR5 and TOC1, accompanied by decreasing expression of PRR5- and TOC1-target genes. A prr5 toc1 double mutant was hyposensitive to PHA767491-induced period lengthening. Together, our results reveal posttranslational modification of transcriptional repressors in plant clock TTFL by CK1 family proteins, which also modulate nonplant circadian clocks.

    DOI: 10.1073/pnas.1903357116

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  17. Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth Reviewed

    Oshima, T., Niwa, Y., Kuwata, K., Srivastava, A., Hyoda, T., Tsuchiya, Y., Kumagai, M., Tsuyuguchi, M., Tamaru, T., Sugiyama, A., Ono, N., Zolboot, N., Aikawa, Y., Oishi, S., Nonami, A., Arai, F., Hagihara, S., Yamaguchi, J., Tama, F., Kunisaki, Y., Yagita, K., Ikeda, M., Kinoshita, T., Kay, S.A., Itami, K., Hirota, T.

    Science Advances   Vol. 5 ( 1 ) page: eaau9060   2019

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    DOI: 10.1126/sciadv.aau9060

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  18. Controlling the Circadian Clock with High Temporal Resolution through Photodosing Reviewed

    Kolarski, D., Sugiyama, A., Breton, G., Rakers, C., Ono, D., Schulte, A., Tama, F., Itami, K., Szymanski, W., Hirota, T., Feringa, B.L.

    Journal of the American Chemical Society   Vol. 141 ( 40 ) page: 15784 - 15791   2019

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society ({ACS})  

    DOI: 10.1021/jacs.9b05445

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  19. Chemical Synthesis of Atomically Tailored SUMO E2 Conjugating Enzymes for the Formation of Covalently Linked SUMO-E2-E3 Ligase Ternary Complexes Reviewed International journal

    Zhang, Y., Hirota, T., Kuwata, K., Oishi, S., Gramani, S.G., Bode, J.W.

    Journal of the American Chemical Society   Vol. 141 ( 37 ) page: 14742 - 14751   2019

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    E2 conjugating enzymes are the key catalytic actors in the transfer of ubiquitin, SUMO, and other ubiquitin-like modifiers to their substrate proteins. Their high rates of transfer and promiscuous binding complicate studies of their interactions and binding partners. To access specific, covalently linked conjugates of the SUMO E2 conjugating enzyme Ubc9, we prepared synthetic variants bearing site-specific non-native modifications including the following: (1) replacement of Cys93 to 2,3-diaminopropionic acid to form the amide-linked stable E2-SUMO conjugate, which is known to have high affinity for E3 ligases; (2) a photoreactive group (diazirine) to trap E3 ligases upon UV irradiation; and (3) an N-terminal biotin for purification and detection. To construct these Ubc9 variants in a flexible, convergent manner, we combined the three leading methods: native chemical ligation (NCL), α-ketoacid-hydroxylamine (KAHA) ligation, and serine/threonine ligation (STL). Using the synthetic proteins, we demonstrated the selective formation of Ubc9-SUMO conjugates and the trapping of an E3 ligase (RanBP2) to form the stable, covalently linked SUMO1-Ubc9-RanBP2 ternary complex. The powerful combination of ligation methods-which minimizes challenges of functional group manipulations-will enable chemical probes based on E2 conjugating enzymes to trap E3 ligases and facilitate the synthesis of other protein classes.

    DOI: 10.1021/jacs.9b06820

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  20. Chemical Control of Mammalian Circadian Behavior through Dual Inhibition of Casein Kinase Iα and δ Reviewed

    Lee, J.W., Hirota, T., Ono, D., Honma, S., Honma, K.-I., Park, K., Kay, S.A.

    Journal of Medicinal Chemistry   Vol. 62 ( 4 ) page: 1989 - 1998   2019

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    DOI: 10.1021/acs.jmedchem.8b01541

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  21. Conformational dynamics of human protein kinase CK2α and its effect on function and inhibition Reviewed

    Ashutosh Srivastava, Tsuyoshi Hirota, Stephan Irle, Florence Tama

    Proteins: Structure, Function and Bioinformatics   Vol. 86 ( 3 ) page: 344 - 353   2018.3

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:John Wiley and Sons Inc.  

    Protein kinase, casein kinase II (CK2), is ubiquitously expressed and highly conserved protein kinase that shows constitutive activity. It phosphorylates a diverse set of proteins and plays crucial role in several cellular processes. The catalytic subunit of this enzyme (CK2α) shows remarkable flexibility as evidenced in numerous crystal structures determined till now. Here, using analysis of multiple crystal structures and long timescale molecular dynamics simulations, we explore the conformational flexibility of CK2α. The enzyme shows considerably higher flexibility in the solution as compared to that observed in crystal structure ensemble. Multiple conformations of hinge region, located near the active site, were observed during the dynamics. We further observed that among these multiple conformations, the most populated conformational state was inadequately represented in the crystal structure ensemble. The catalytic spine, was found to be less dismantled in this state as compared to the “open” hinge/αD state crystal structures. The comparison of dynamics in unbound (Apo) state and inhibitor (CX4945) bound state exhibits inhibitor induced suppression in the overall dynamics of the enzyme. This is especially true for functionally important glycine-rich loop above the active site. Together, this work gives novel insights into the dynamics of CK2α in solution and relates it to the function. This work also explains the effect of inhibitor on the dynamics of CK2α and paves way for development of better inhibitors.

    DOI: 10.1002/prot.25444

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  22. Nuclear receptor HNF4A transrepresses CLOCK: BMAL1 and modulates tissue-specific circadian networks Reviewed

    Qu, M., Duffy, T., Hirota, T., Kay, S.A.

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 115 ( 52 ) page: E12305 - E12312   2018

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    DOI: 10.1073/pnas.1816411115

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  23. Global rise of potential health hazards caused by blue light-induced circadian disruption in modern aging societies Reviewed

    Hatori, M., Gronfier, C., Van Gelder, R.N., Bernstein, P.S., Carreras, J., Panda, S., Marks, F., Sliney, D., Hunt, C.E., Hirota, T., Furukawa, T., Tsubota, K.

    npj Aging and Mechanisms of Disease   Vol. 3 ( 1 ) page: 9   2017

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    DOI: 10.1038/s41514-017-0010-2

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  24. Circadian Amplitude Regulation via FBXW7-Targeted REV-ERBα Degradation Reviewed

    Zhao, X., Hirota, T., Han, X., Cho, H., Chong, L.-W., Lamia, K., Liu, S., Atkins, A.R., Banayo, E., Liddle, C., Yu, R.T., Yates, J.R., Kay, S.A., Downes, M., Evans, R.M.

    Cell   Vol. 165 ( 7 ) page: 1644 - 1657   2016

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    Defects in circadian rhythm influence physiology and behavior with implications for the treatment of sleep disorders, metabolic disease, and cancer. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms underpinning amplitude is limited. Here, we show that REV-ERB alpha, a core inhibitory component of clock transcription, is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7. By relieving REV-ERB alpha-dependent repression, FBXW7 provides an unrecognized mechanism for enhancing the amplitude of clock gene transcription. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of REV-ERB alpha is necessary for FBXW7 recognition. Moreover, targeted hepatic disruption of FBXW7 alters circadian expression of core clock genes and perturbs whole-body lipid and glucose levels. This CDK1-FBXW7 pathway controlling REV-ERB alpha repression defines an unexpected molecular mechanism for re-engaging the positive transcriptional arm of the clock, as well as a potential route to manipulate clock amplitude via small molecule CDK1 inhibition.

    DOI: 10.1016/j.cell.2016.05.012

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  25. C-H activation generates period-shortening molecules that target cryptochrome in the mammalian circadian clock Reviewed

    Oshima, T., Yamanaka, I., Kumar, A., Yamaguchi, J., Nishiwaki-Ohkawa, T., Muto, K., Kawamura, R., Hirota, T., Yagita, K., Irle, S., Kay, S.A., Yoshimura, T., Itami, K.

    Angewandte Chemie - International Edition   Vol. 54 ( 24 ) page: 7193 - 7197   2015

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-V C H VERLAG GMBH  

    The synthesis and functional analysis of KL001 derivatives, which are modulators of the mammalian circadian clock, are described. By using cutting-edge C-H activation chemistry, a focused library of KL001 derivatives was rapidly constructed, which enabled the identification of the critical sites on KL001 derivatives that induce a rhythm-changing activity along with the components that trigger opposite modes of action. The first period-shortening molecules that target the cryptochrome (CRY) were thus discovered. Detailed studies on the effects of these compounds on CRY stability implicate the existence of an as yet undiscovered regulatory mechanism.

    DOI: 10.1002/anie.201502942

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  26. Development of Small-Molecule Cryptochrome Stabilizer Derivatives as Modulators of the Circadian Clock Reviewed

    Lee, J.W., Hirota, T., Kumar, A., Kim, N.-J., Irle, S., Kay, S.A.

    ChemMedChem   Vol. 10 ( 9 ) page: 1489 - 1497   2015

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-V C H VERLAG GMBH  

    Small-molecule probes have been playing prominent roles in furthering our understanding of the molecular underpinnings of the circadian clock. We previously discovered a carbazole derivative, KL001 (N-(3-(9H-carbazol-9-yl)-2-hydroxypropyl)-N-(furan-2-ylmethyl)methanesulfonamide), as a stabilizer of the clock protein cryptochrome (CRY). Herein we describe an extensive structure-activity relationship analysis of KL001 derivatives leading to the development of a highly active derivative: 2-(9H-carbazol-9-yl)-N-(2-chloro-6-cyanophenyl)acetamide (KL044). Subsequent 3D-QSAR analysis identified critical features of KL001 derivatives and provided a molecular-level understanding of their interaction with CRY. The electron-rich carbazole, amide/hydroxy linker, sulfonyl group, and electron-withdrawing nitrile moieties contribute to greater biological activity. The hydrogen bonding interactions with Ser394 and His357 as well as stronger CH- interactions with Trp290 make KL044 a better binder than KL001. KL044 lengthened the circadian period, repressed Per2 activity, and stabilized CRY in reporter assays with roughly tenfold higher potency than KL001. Altogether, KL044 is a powerful chemical tool to control the function of the circadian clock through its action on CRY.

    DOI: 10.1002/cmdc.201500260

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  27. Identification of small-molecule modulators of the circadian clock Reviewed

    Hirota, T., Kay, S.A.

    Methods in Enzymology   Vol. 551   page: 267 - 282   2015

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Methods in Enzymology  

    Chemical biology or chemical genetics has emerged as an interdisciplinary research area applying chemistry to understand biological systems. The development of combinatorial chemistry and high-throughput screening technologies has enabled large-scale investigation of the biological activities of diverse small molecules to discover useful chemical probes. This approach is applicable to the analysis of the circadian clock mechanisms through cell-based assays to monitor circadian rhythms using luciferase reporter genes. We and others have established cell-based high-throughput circadian assays and have identified a variety of novel small-molecule modulators of the circadian clock by phenotype-based screening of hundreds of thousands of compounds. The results demonstrated the effectiveness of chemical biology approaches in clock research field. This technique will become more and more common with propagation of high-throughput screening facilities. This chapter describes assay development, screening setups, and their optimization for successful screening campaigns.

    DOI: 10.1016/bs.mie.2014.10.015

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  28. In vivo role of phosphorylation of cryptochrome 2 in the mouse circadian clock Reviewed

    Hirano, A., Kurabayashi, N., Nakagawa, T., Shioi, G., Todo, T., Hirota, T., Fukada, Y.

    Molecular and Cellular Biology   Vol. 34 ( 24 ) page: 4464 - 4473   2014

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC MICROBIOLOGY  

    The circadian clock is finely regulated by posttranslational modifications of clock components. Mouse CRY2, a critical player in the mammalian clock, is phosphorylated at Ser557 for proteasome-mediated degradation, but its in vivo role in circadian organization was not revealed. Here, we generated CRY2(S557A) mutant mice, in which Ser557 phosphorylation is specifically abolished. The mutation lengthened free-running periods of the behavioral rhythms and PER2:: LUC bioluminescence rhythms of cultured liver. In livers from mutant mice, the nuclear CRY2 level was elevated, with enhanced PER2 nuclear occupancy and suppression of E-box-regulated genes. Thus, Ser557 phosphorylation-dependent regulation of CRY2 is essential for proper clock oscillation in vivo.

    DOI: 10.1128/MCB.00711-14

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  29. Spatiotemporal separation of per and CRY posttranslational regulation in the mammalian circadian clock Reviewed

    St. John, P.C., Hirota, T., Kay, S.A., Doyle III, F.J.

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 111 ( 5 ) page: 2040 - 2045   2014

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    Posttranslational regulation of clock proteins is an essential part of mammalian circadian rhythms, conferring sensitivity to metabolic state and offering promising targets for pharmacological control. Two such regulators, casein kinase 1 (CKI) and F-box and leucine-rich repeat protein 3 (FBXL3), modulate the stability of closely linked core clock proteins period (PER) and cryptochrome (CRY), respectively. Inhibition of either CKI or FBXL3 leads to longer periods, and their effects are independent despite targeting proteins with similar roles in clock function. A mechanistic understanding of this independence, however, has remained elusive. Our analysis of cellular circadian clock gene reporters further differentiated between the actions of CKI and FBXL3 by revealing opposite amplitude responses from each manipulation. To understand the functional relationship between the CKI-PER and FBXL3-CRY pathways, we generated robust mechanistic predictions by applying a bootstrap uncertainty analysis to multiple mathematical circadian models. Our results indicate that CKI primarily regulates the accumulating phase of the PER-CRY repressive complex by controlling the nuclear import rate, whereas FBXL3 separately regulates the duration of transcriptional repression in the nucleus. Dynamic simulations confirmed that this spatiotemporal separation is able to reproduce the independence of the two regulators in period regulation, as well as their opposite amplitude effect. As a result, this study provides further insight into the molecular clock machinery responsible for maintaining robust circadian rhythms.

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  30. Biosynthesis and biological actions of pineal neurosteroids in domestic birds Reviewed

    Tsutsui, K., Haraguchi, S., Hatori, M., Hirota, T., Fukada, Y.

    Neuroendocrinology   Vol. 98 ( 2 ) page: 97 - 105   2013

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    The central and peripheral nervous systems have the capacity of synthesizing steroids de novo from cholesterol, the so-called 'neurosteroids'. De novo synthesis of neurosteroids from cholesterol appears to be a conserved property across the subphylum vertebrata. Until recently, it was generally believed that neurosteroids are produced in neurons and glial cells in the central and peripheral nervous systems. However, our recent studies on birds have demonstrated that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol. 7 alpha-Hydroxypregnenolone is a major pineal neurosteroid that stimulates locomotor activity of juvenile birds, connecting light-induced gene expression with locomotion. The other major pineal neurosteroid allopregnanolone is involved in Purkinje cell survival by suppressing the activity of caspase-3, a crucial mediator of apoptosis during cerebellar development. This review is an updated summary of the biosynthesis and biological actions of pineal neurosteroids. Copyright (C) 2013 S. Karger AG, Basel

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  31. New biosynthesis and biological actions of avian neurosteroids. Reviewed International journal

    Kazuyoshi Tsutsui, Shogo Haraguchi, Kazuhiko Inoue, Hitomi Miyabara, Takayoshi Ubuka, Megumi Hatori, Tsuyoshi Hirota, Yoshitaka Fukada

    Journal of experimental neuroscience   Vol. 7 ( 1 ) page: 15 - 29   2013

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    De novo neurosteroidogenesis from cholesterol occurs in the brain of various avian species. However, the biosynthetic pathways leading to the formation of neurosteroids are still not completely elucidated. We have recently found that the avian brain produces 7α-hydroxypregnenolone, a novel bioactive neurosteroid that stimulates locomotor activity. Until recently, it was believed that neurosteroids are produced in neurons and glial cells in the central and peripheral nervous systems. However, our recent studies on birds have demonstrated that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol. 7α-Hydroxypregnenolone is a major pineal neurosteroid that stimulates locomotor activity of juvenile birds, connecting light-induced gene expression with locomotion. The other major pineal neurosteroid allopregnanolone is involved in Purkinje cell survival during development. This paper highlights new aspects of neurosteroid synthesis and actions in birds.

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  32. Real-time in vivo monitoring of circadian E-box enhancer activity: A robust and sensitive zebrafish reporter line for developmental, chemical and neural biology of the circadian clock Reviewed

    Weger, M., Weger, B.D., Diotel, N., Rastegar, S., Hirota, T., Kay, S.A., Strähle, U., Dickmeis, T.

    Developmental Biology   Vol. 380 ( 2 ) page: 259 - 273   2013

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    The circadian clock co-ordinates physiology and behavior with the day/night cycle. It consists of a transcriptional-translational feedback loop that generates self-sustained oscillations in transcriptional activity with a roughly 24 h period via E-box enhancer elements. Numerous in vivo aspects of core clock feedback loop function are still incompletely understood, including its maturation during development, tissue-specific activity and perturbation in disease states. Zebrafish are promising models for biomedical research due to their high regenerative capacity and suitability for in vivo drug screens, and transgenic zebrafish lines are valuable tools to study transcriptional activity in vivo during development.
    To monitor the activity of the core clock feedback loop in vivo, we created a transgenic zebrafish line expressing a luciferase reporter gene under the regulation of a minimal promoter and four E-boxes. This Tg(4xE-box:Luc) line shows robust oscillating reporter gene expression both under light-dark cycles and upon release into constant darkness. Luciferase activity starts to oscillate during the first days of development, indicating that the core clock loop is already functional at an early stage. To test whether the Tg(4xE-box:Luc) line could be used in drug screens aimed at identifying compounds that target the circadian clock in vivo, we examined drug effects on circadian period. We were readily able to detect period changes as low as 0.7 h upon treatment with the period-lengthening drugs lithium chloride and longdaysin in an assay set-up suitable for large-scale screens. Reporter gene mRNA expression is also detected in the adult brain and reveals differential clock activity across the brain, overlapping with endogenous clock gene expression. Notably, core clock activity is strongly correlated with brain regions where neurogenesis takes place and can be detected in several types of neural progenitors.
    Our results demonstrate that the Tg(4xE-box:Luc) line is an excellent tool for studying the regulation of the circadian clock and its maturation in vivo and in real time. Furthermore, it is highly suitable for in vivo screens targeting the core clock mechanism that take into account the complexity of an intact organism. Finally, it allows mapping of clock activity in the brain of a vertebrate model organism with prominent adult neurogenesis and high regeneration capacity. (c) 2013 Elsevier Inc. All rights reserved.

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  33. Identification of small molecule activators of cryptochrome Reviewed

    Hirota, T., Lee, J.W., St. John, P.C., Sawa, M., Iwaisako, K., Noguchi, T., Pongsawakul, P.Y., Sonntag, T., Welsh, D.K., Brenner, D.A., Doyle III, F.J., Schultz, P.G., Kay, S.A.

    Science   Vol. 337 ( 6098 ) page: 1094 - 1097   2012

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    Impairment of the circadian clock has been associated with numerous disorders, including metabolic disease. Although small molecules that modulate clock function might offer therapeutic approaches to such diseases, only a few compounds have been identified that selectively target core clock proteins. From an unbiased cell-based circadian phenotypic screen, we identified KL001, a small molecule that specifically interacts with cryptochrome (CRY). KL001 prevented ubiquitin-dependent degradation of CRY, resulting in lengthening of the circadian period. In combination with mathematical modeling, our studies using KL001 revealed that CRY1 and CRY2 share a similar functional role in the period regulation. Furthermore, KL001-mediated CRY stabilization inhibited glucagon-induced gluconeogenesis in primary hepatocytes. KL001 thus provides a tool to study the regulation of CRY-dependent physiology and aid development of clock-based therapeutics of diabetes.

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  34. A small molecule modulates circadian rhythms through phosphorylation of the period protein Reviewed

    Lee, J.W., Hirota, T., Peters, E.C., Garcia, M., Gonzalez, R., Cho, C.Y., Wu, X., Schultz, P.G., Kay, S.A.

    Angewandte Chemie - International Edition   Vol. 50 ( 45 ) page: 10608 - 10611   2011

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  35. Light-dependent and circadian clock-regulated activation of sterol regulatory element-binding protein, X-box-binding protein 1, and heat shock factor pathways Reviewed

    Hatori, M., Hirota, T., Iitsuka, M., Kurabayashi, N., Haraguchi, S., Kokame, K., Sato, R., Nakai, A., Miyata, T., Tsutsui, K., Fukada, Y.

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 108 ( 12 ) page: 4864 - 4869   2011

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    The circadian clock is phase-delayed or -advanced by light when given at early or late subjective night, respectively. Despite the importance of the time-of-day-dependent phase responses to light, the underlying molecular mechanism is poorly understood. Here, we performed a comprehensive analysis of light-inducible genes in the chicken pineal gland, which consists of light-sensitive clock cells representing a prototype of the clock system. Light stimulated expression of 62 genes and 40 ESTs by &gt;2.5-fold, among which genes responsive to the heat shock and endoplasmic reticulum stress as well as their regulatory transcription factors heat shock factor (HSF) 1, HSF2, and X-box-binding protein 1 (XBP1) were strongly activated when a light pulse was given at late subjective night. In contrast, the light pulse at early subjective night caused prominent induction of E4bp4, a key regulator in the phase-delaying mechanism of the pineal clock, along with activation of a large group of cholesterol biosynthetic genes that are targets of sterol regulatory element-binding protein (SREBP) transcription factor. We found that the light pulse stimulated proteolytic formation of active SREBP-1 that, in turn, transactivated E4bp4 expression, linking SREBP with the light-input pathway of the pineal clock. As an output of light activation of cholesterol biosynthetic genes, we found light-stimulated pineal production of a neurosteroid, 7 alpha-hydroxypregnenolone, demonstrating a unique endocrine function of the pineal gland. Intracerebroventricular injection of 7 alpha-hydroxypregnenolone activated locomotor activities of chicks. Our study on the genome-wide gene expression analysis revealed time-of-day-dependent light activation of signaling pathways and provided molecular connection between gene expression and behavior through neurosteroid release from the pineal gland.

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  36. Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis Reviewed

    Zhang, E.E., Liu, Y., Dentin, R., Pongsawakul, P.Y., Liu, A.C., Hirota, T., Nusinow, D.A., Sun, X., Landais, S., Kodama, Y., Brenner, D.A., Montminy, M., Kay, S.A.

    Nature Medicine   Vol. 16 ( 10 ) page: 1152 - U133   2010

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    During fasting, mammals maintain normal glucose homeostasis by stimulating hepatic gluconeogenesis1. Elevations in circulating glucagon and epinephrine, two hormones that activate hepatic gluconeogenesis, trigger the cAMP-mediated phosphorylation of cAMP response element-binding protein (Creb) and dephosphorylation of the Creb-regulated transcription coactivator-2 (Crtc2)-two key transcriptional regulators of this process(2). Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment(3-6). Circadian control of gene expression is achieved by two transcriptional activators, Clock and Bmal1, which stimulate cryptochrome (Cry1 and Cry2) and Period (Per1, Per2 and Per3) repressors that feed back on Clock-Bmal1 activity. Here we show that Creb activity during fasting is modulated by Cry1 and Cry2, which are rhythmically expressed in the liver. Cry1 expression was elevated during the night-day transition, when it reduced fasting gluconeogenic gene expression by blocking glucagon-mediated increases in intracellular cAMP concentrations and in the protein kinase A-mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry1 inhibited accumulation of cAMP in response to G protein-coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry proteins seemed to modulate GPCR activity directly through interaction with G(s)alpha. As hepatic overexpression of Cry1 lowered blood glucose concentrations and improved insulin sensitivity in insulin-resistant db/db mice, our results suggest that compounds that enhance cryptochrome activity may provide therapeutic benefit to individuals with type 2 diabetes.

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  37. Transcriptional repressor TIEG1 regulates Bmal1 gene through GC box and controls circadian clockwork Reviewed

    Hirota, T., Kon, N., Itagaki, T., Hoshina, N., Okano, T., Fukada, Y.

    Genes to Cells   Vol. 15 ( 2 ) page: 111 - 121   2010

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    The circadian clock controls daily rhythms in many physiologic processes, and the clock oscillation is regulated by external time cues such as light, temperature, and feeding. In mammals, the transcriptional regulation of clock genes underlies the clock oscillatory mechanism, which is operative even in cultured fibroblasts. We previously demonstrated that glucose treatment of rat-1 fibroblasts evokes circadian expression of clock genes with a rapid induction of Tieg1 transcript encoding a transcriptional repressor. Here, we found diurnal variation of both Tieg1 mRNA and nuclear TIEG1 protein levels in the mouse liver with their peaks at day/night transition and midnight, respectively. In vitro experiments showed that TIEG1 bound to Bmal1 gene promoter and repressed its transcriptional activity through two juxtaposed GC boxes near the transcription initiation site. The GC box/TIEG1-mediated repression of Bmal1 promoter was additive to RORE-dependent repression by REV-ERB alpha, a well-known repressor of Bmal1 gene. In cell-based real-time assay, siRNA-mediated knock-down of TIEG1 caused period shortening of cellular bioluminescence rhythms driven by Bmal1-luciferase and Per2-luciferase reporters. These findings highlight an active role of TIEG1 in the normal clock oscillation and GC box-mediated regulation of Bmal1 transcription.

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  38. High-throughput chemical screen identifies a novel potent modulator of cellular circadian rhythms and reveals CKIα as a clock regulatory kinase Reviewed

    Hirota, T., Lee, J.W., Lewis, W.G., Zhang, E.E., Breton, G., Liu, X., Garcia, M., Peters, E.C., Etchegaray, J.-P., Traver, D., Schultz, P.G., Kay, S.A.

    PLoS Biology   Vol. 8 ( 12 ) page: e1000559   2010

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    The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of similar to 120,000 uncharacterized compounds on human cells containing a circadian reporter. The analysis identified a small molecule that potently lengthens the circadian period in a dose-dependent manner. Subsequent analysis showed that the compound also lengthened the period in a variety of cells from different tissues including the mouse suprachiasmatic nucleus, the central clock controlling behavioral rhythms. Based on the prominent period lengthening effect, we named the compound longdaysin. Longdaysin was amenable for chemical modification to perform affinity chromatography coupled with mass spectrometry analysis to identify target proteins. Combined with siRNA-mediated gene knockdown, we identified the protein kinases CKI delta, CKI alpha, and ERK2 as targets of longdaysin responsible for the observed effect on circadian period. Although individual knockdown of CKI delta, CKI alpha, and ERK2 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to longdaysin treatment. We characterized the role of CKI alpha in the clock mechanism and found that CKI alpha-mediated phosphorylation stimulated degradation of a clock protein PER1, similar to the function of CKI delta. Longdaysin treatment inhibited PER1 degradation, providing insight into the mechanism of longdaysin-dependent period lengthening. Using larval zebrafish, we further demonstrated that longdaysin drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed CKI alpha as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. Longdaysin provides novel possibilities in manipulating clock function due to its ability to simultaneously inhibit several key components of this conserved network across species.

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  39. DYRK1A and glycogen synthase kinase 3β, a dual-kinase mechanism directing proteasomal degradation of CRY2 for circadian timekeeping Reviewed

    Kurabayashi, N., Hirota, T., Sakai, M., Sanada, K., Fukada, Y.

    Molecular and Cellular Biology   Vol. 30 ( 7 ) page: 1757 - 1768   2010

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    Circadian molecular oscillation is generated by a transcription/translation-based feedback loop in which CRY proteins play critical roles as potent inhibitors for E-box-dependent clock gene expression. Although CRY2 undergoes rhythmic phosphorylation in its C-terminal tail, structurally distinct from the CRY1 tail, little is understood about how protein kinase(s) controls the CRY2-specific phosphorylation and contributes to the molecular clockwork. Here we found that Ser557 in the C-terminal tail of CRY2 is phosphorylated by DYRK1A as a priming kinase for subsequent GSK-3 beta (glycogen synthase kinase 3 beta)-mediated phosphorylation of Ser553, which leads to proteasomal degradation of CRY2. In the mouse liver, DYRK1A kinase activity toward Ser557 of CRY2 showed circadian variation, with its peak in the accumulating phase of CRY2 protein. Knockdown of Dyrk1a caused abnormal accumulation of cytosolic CRY2, advancing the timing of a nuclear increase of CRY2, and shortened the period length of the cellular circadian rhythm. Expression of an S557A/S553A mutant of CRY2 phenocopied the effect of Dyrk1a knockdown in terms of the circadian period length of the cellular clock. DYRK1A is a novel clock component cooperating with GSK-3 beta and governs the Ser557 phosphorylation-triggered degradation of CRY2.

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  40. A Genome-wide RNAi Screen for Modifiers of the Circadian Clock in Human Cells Reviewed

    Zhang, E.E., Liu, A.C., Hirota, T., Miraglia, L.J., Welch, G., Pongsawakul, P.Y., Liu, X., Atwood, A., Huss III, J.W., Janes, J., Su, A.I., Hogenesch, J.B., Kay, S.A.

    Cell   Vol. 139 ( 1 ) page: 199 - 210   2009

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    Two decades of research identified more than a dozen clock genes and defined a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modifiers, we conducted a genome-wide small interfering RNA screen in a human cellular clock model. Knockdown of nearly 1000 genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and confirmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock regulated, we conclude the clock is interconnected with many aspects of cellular function.

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  41. High-Throughput Screening and Chemical Biology: New Approaches for Understanding Circadian Clock Mechanisms Reviewed

    Hirota, T., Kay, S.A.

    Chemistry and Biology   Vol. 16 ( 9 ) page: 921 - 927   2009

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    Most organisms exhibit daily changes in physiology and metabolism under the control of a cell-autonomous circadian clock. In the core clock mechanism, clock genes form a transcription factor network to generate circadian rhythms of gene expression. Clock protein phosphorylation and histone modifications are also important for the clock regulation. Pharmacological approaches have been making significant contributions to the clock research, for example, in characterizing the roles of protein kinases CKI delta, CKI epsilon, CK2, and GSK-3 beta. Recently, high-throughput circadian functional assays have been established. Chemical biology approaches utilizing high-throughput compound screening together with RNAi-based genomic screening will open a new way for the circadian clock field. Finding a set of compounds that potently affect the clock function will lead to the identification of novel clock components and form the basis for therapeutic strategies directed toward circadian disorders.

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  42. A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3β Reviewed

    Hirota, T., Lewis, W.G., Liu, A.C., Jae, W.L., Schultz, P.G., Kay, S.A.

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 105 ( 52 ) page: 20746 - 20751   2008

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    The circadian clock controls daily oscillations of gene expression at the cellular level. We report the development of a high-throughput circadian functional assay system that consists of luminescent reporter cells, screening automation, and a data analysis pipeline. We applied this system to further dissect the molecular mechanisms underlying the mammalian circadian clock using a chemical biology approach. We analyzed the effect of 1,280 pharmacologically active compounds with diverse structures on the circadian period length that is indicative of the core clock mechanism. Our screening paradigm identified many compounds previously known to change the circadian period or phase, demonstrating the validity of the assay system. Furthermore, we found that small molecule inhibitors of glycogen synthase kinase 3 (GSK-3) consistently caused a strong short period phenotype in contrast to the well-known period lengthening by lithium, another presumed GSK-3 inhibitor. siRNA-mediated knockdown of GSK-3 beta also caused a short period, confirming the phenotype obtained with the small molecule inhibitors. These results clarify the role of GSK-3 beta in the period regulation of the mammalian clockworks and highlight the effectiveness of chemical biology in exploring unidentified mechanisms of the circadian clock.

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  43. Activation of TGF-β/activin signalling resets the circadian clock through rapid induction of Dec1 transcripts Reviewed

    Kon, N., Hirota, T., Kawamoto, T., Kato, Y., Tsubota, T., Fukada, Y.

    Nature Cell Biology   Vol. 10 ( 12 ) page: 1463 - U197   2008

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    The circadian clock is reset by external time cues for synchronization to environmental changes(1). In mammals, the light-input signalling pathway mediated by Per gene induction has been extensively studied(2,3). On the other hand, little is known about resetting mechanisms that are independent of Per induction(4-6). Here we show that activation of activin receptor-like kinase (ALK), triggered by TGF-beta, activin or alkali signals, evoked resetting of the cellular clock independently of Per induction. The resetting was mediated by an immediate-early induction of Dec1, a gene whose physiological role in the function of the circadian clock has been unclear. Acute Dec1 induction was a prerequisite for ALK-mediated resetting and upregulation was dependent on SMAD3, which was phosphorylated for activation in response to the resetting stimuli. Intraperitoneal injection of TGF-beta into wild-type or Dec1-deficient mice demonstrated that Dec1 has an essential role in phase-shift of clock gene expression in the kidney and adrenal gland. These results indicate that ALK-SMAD3-Dec1 signalling provides an input pathway in the mammalian molecular clock.

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  44. Circadian proteomics of the mouse retina Reviewed

    Tsuji, T., Hirota, T., Takemori, N., Komori, N., Yoshitane, H., Fukuda, M., Matsumoto, H., Fukada, Y.

    Proteomics   Vol. 7 ( 19 ) page: 3500 - 3508   2007

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    The circadian clock in the retina regulates a variety of physiological phenomena such as disc shedding and melatonin release. Although these events are critical for retinal functions, it is almost unknown how the circadian clock controls the physiological rhythmicity. To gain insight into the processes, we performed a proteomic analysis using 2-DE to find proteins whose levels show circadian changes. Among 415 retinal protein spots, 11 protein spots showed circadian rhythmicity in their intensities. We performed MALDI-TOF MS and NanoLC-MS/MS analyses and identified proteins contained in the 11 spots. The proteins were related to vesicular transport, calcium-binding, protein degradation, metabolism, RNA-binding, and protein foldings, suggesting the clock-regulation of neurotransmitter release, transportation of the membrane proteins, calcium-binding capability, and so on. We also found a rhythmic phosphorylation of N-ethylmaleimide-sensitive fusion protein and identified one of the amino acid residues modified by phosphorylation. These findings provide a new perspective on the relationship between the physiological functions of the retina and the circadian clock system.

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  45. Phosphorylation of mCRY2 at SER557 in the hypothalamic suprachiasmatic nucleus of the mouse Reviewed

    Kurabayashi, N., Hirota, T., Harada, Y., Sakai, M., Fukada, Y.

    Chronobiology International   Vol. 23 ( 1-2 ) page: 129 - 134   2006

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    Cryptochrome1 and 2 play a critical role in the molecular oscillations of the circadian clocks of central and peripheral tissues in mammals. Mouse Cryptochrome2 (mCRY2) is phosphorylated at Ser557 in the liver, in which the Ser557-phosphorylated form accumulates during the night in parallel with mCRY2 protein. Phosphorylation of mCRY2 at Ser557 allows subsequent phosphorylation at Ser553 by glycogen synthase kinase-3 beta (GSK-3 beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. In the present study, we found that mCRY2 is phosphorylated at Ser557 also in the region of the mouse brain containing the suprachiasmatic nucleus (SCN), the central circadian clock tissue. Daily fluctuation of the Ser557-phosphorylation level in the SCN region suggests an important role of sequential phosphorylation of Ser557 and Ser553 in the rhythmic degradation of mCRY2 in both central and peripheral clocks of mice.

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  46. Ser-557-phosphorylated mCRY2 is degraded upon synergistic phosphorylation by glycogen synthase kinase-3β Reviewed

    Harada, Y., Sakai, M., Kurabayashi, N., Hirota, T., Fukada, Y.

    Journal of Biological Chemistry   Vol. 280 ( 36 ) page: 31714 - 31721   2005

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    Cryptochrome 1 and 2 act as essential components of the central and peripheral circadian clocks for generation of circadian rhythms in mammals. Here we show that mouse cryptochrome 2 (mCRY2) is phosphorylated at Ser-557 in the liver, a well characterized peripheral clock tissue. The Ser-557- phosphorylated form accumulates in the liver during the night in parallel with mCRY2 protein, and the phosphorylated form reaches its maximal level at late night, preceding the peak-time of the protein abundance by similar to 4 h in both light-dark cycle and constant dark conditions. The Ser-557- phosphorylated form of mCRY2 is localized in the nucleus, whereas mCRY2 protein is located in both the cytoplasm and nucleus. Importantly, phosphorylation of mCRY2 at Ser-557 allows subsequent phosphorylation at Ser-553 by glycogen synthase kinase-3 beta (GSK-3 beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. As assessed by phosphorylation of GSK-3 beta at Ser-9, which negatively regulates the kinase activity, GSK-3 beta exhibits a circadian rhythm in its activity with a peak from late night to early morning when Ser-557 of mCRY2 is highly phosphorylated. Altogether, the present study demonstrates an important role of sequential phosphorylation at Ser-557/Ser-553 for destabilization of mCRY2 and illustrates a model that the circadian regulation of mCRY2 phosphorylation contributes to rhythmic degradation of mCRY2 protein.

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  47. Resetting mechanism of central and peripheral circadian clocks in mammals Reviewed

    Hirota, T., Fukada, Y.

    Zoological Science   Vol. 21 ( 4 ) page: 359 - 368   2004

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    Almost all organisms on earth exhibit diurnal rhythms in physiology and behavior under the control of autonomous time-measuring system called circadian clock. The circadian clock is generally reset by environmental time cues, such as light, in order to synchronize with the external 24-h cycles. In mammals, the core oscillator of the circadian clock is composed of transcription/translation-based negative feedback loops regulating the cyclic expression of a limited number of clock genes (such as Per, Cry, Bmal1, etc.) and hundreds of output genes in a well-concerted manner. The central clock controlling the behavioral rhythm is localized in the hypothalamic suprachiasmatic nucleus (SCN), and peripheral clocks are present in other various tissues. The phase of the central clock is amenable to ambient light signal captured by the visual rod-cone photoreceptors and non-visual melanopsin in the retina. These light signals are transmitted to the SCN through the retinohypothalamic tract, and transduced therein by mitogen-activated protein kinase and other signaling molecules to induce Per gene expression, which eventually elicits phase-dependent phase shifts of the clock. The central clock controls peripheral clocks directly and indirectly by virtue of neural, humoral, and other signals in a coordinated manner. The change in feeding time resets the peripheral clocks in a SCN-independent manner, possibly by food metabolites and body temperature rhythms. In this article, we will provide an overview of recent molecular and genetic studies on the resetting mechanism of the central and peripheral circadian clocks in mammals.

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  48. p38 Mitogen-activated Protein Kinase Regulates Oscillation of Chick Pineal Circadian Clock Reviewed

    Hayashi, Y., Sanada, K., Hirota, T., Shimizu, F., Fukada, Y.

    Journal of Biological Chemistry   Vol. 278 ( 27 ) page: 25166 - 25171   2003

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    Extracellular signal-regulated kinase (ERK) and p38 are members of the mitogen-activated protein kinase (MAPK) family, and in some cases these kinases serve for closely related cellular functions within a cell. In a wide range of animal clock structures, ERK plays an important role in the circadian time-keeping mechanism. Here we found that immunoreactivity to p38 protein was uniformly distributed among cells in the chick pineal gland. On the other hand, a constant level of activated p38 was detected over the day, predominantly in the follicular and parafollicular pinealocytes that are potential circadian clock-containing cells. Chronic application of SB203580, a selective and reversible inhibitor of p38, to the cultured chick pineal cells markedly lengthened the period of the circadian rhythm of the melatonin release (up to 28.7 h). Noticeably, despite no significant temporal change of activated p38 level, a 4-h pulse treatment with SB203580 delayed the phase of the rhythm only when delivered during the subjective day. These results indicate a time-of-day-specific role of continuously activated p38 in the period length regulation of the chick pineal clock and suggest temporally separated regulation of the clock by two MAPKs, nighttime-activated ERK and daytime-working p38.

    DOI: 10.1074/jbc.M212726200

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  49. Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expression in cultured rat-1 fibroblasts Reviewed

    Hirota, T., Okano, T., Kokame, K., Shirotani-Ikejima, H., Miyata, T., Fukada, Y.

    Journal of Biological Chemistry   Vol. 277 ( 46 ) page: 44244 - 44251   2002

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    In mammals, peripheral circadian clocks are present in most tissues, but little is known about how these clocks are synchronized with the ambient 24-h cycles. By using rat-1 fibroblasts, a model cell system of the peripheral clock, we found that an exchange of the culture medium triggered circadian gene expression that was preceded by slow down-regulation of Per1 and Per2 mRNA levels. This profile contrasts to the immediate up-regulation of these genes often observed for clock resetting. The screening of factor(s) responsible for the down-regulation revealed glucose as a key component triggering the circadian rhythm. The requirement of both glucose metabolism and RNA/protein synthesis for the down-regulation suggests the involvement of gene(s) immediately up-regulated by glucose metabolism. An analysis with high density oligonucleotide microarrays identified &gt;100 glucose-regulated genes. We found among others immediately up-regulated genes encoding transcriptional regulators TIEG1, VDUP1, and HES1, in addition to cooperatively regulated genes that are associated with cholesterol biosynthesis and cell cycle. The immediate up-regulation of Tieg1 and Vdup1 expression was dependent on glucose metabolism but not on protein synthesis, suggesting that the transcriptional regulators mediate the glucose-induced down-regulation of Per1 and Per2 expression. These results illustrate a novel mode of peripheral clock resetting by external glucose, a major food metabolite.

    DOI: 10.1074/jbc.M206233200

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  50. Chicken pineal clock genes: Implication of BMAL2 as a bidirectional regulator in circadian clock oscillation Reviewed

    Okano, T., Yamamoto, K., Okano, K., Hirota, T., Kasahara, T., Sasaki, M., Takanaka, Y., Fukada, Y.

    Genes to Cells   Vol. 6 ( 9 ) page: 825 - 836   2001

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:BLACKWELL SCIENCE LTD  

    Background: In a transcription/translation-based autoregulatory feedback loop of vertebrate circadian clock systems, a BMAL1-CLOCK heterodimer is a positive regulator for the transcription of the negative element gene Per. The chicken pineal gland represents a photosensitive clock tissue, but the pineal clock genes constituting the oscillator loop have been less well characterized.
    Results: We identified expression of the Per2, Bmal1, Bmal2 and Clock genes in the chicken pineal gland. Messenger RNA levels of these genes exhibited overt circadian rhythms in the pineal cells, both in vivo and in culture. In vitro functional analyses revealed the formation of cBMAL1-cCLOCK and cBMAL2-cCLOCK heteromers. Both of the cBMAL-cCLOCK heteromers activated E-box element-dependent transcription, which was negatively regulated by cPER2 in luciferase assays. Co-expression of cCLOCK, cBMAL1 and cBMAL2 co-operatively activated E-box element-dependent transcription, and a greater level of expression of cBMAL2 inhibited the activation. In the cultured pineal cells, an over-expression of either cBMAL1 or cBMAL2 disrupted the circadian rhythm of melatonin production.
    Conclusion: The functional characterization of the chicken pineal clock molecules supports the key roles of BMAL1, BMAL2 and CLOCK which contribute to the E-box-dependent transcriptional regulation in the circadian clock system.

    DOI: 10.1046/j.1365-2443.2001.00462.x

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  51. Effect of brefeldin A on melatonin secretion of chick pineal cells Reviewed

    Hirota, T., Kagiwada, S., Kasahara, T., Okano, T., Murata, M., Fukada, Y.

    Journal of Biochemistry   Vol. 129 ( 1 ) page: 51 - 59   2001

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:JAPANESE BIOCHEMICAL SOC  

    Melatonin is secreted from the pineal gland in a circadian manner. It is well established that the synthesis of melatonin shows a diurnal rhythm reflecting a daily change in serotonin N-acetyltransferase (NAT) activity, and the overall secretion of melatonin requires a cellular release process, which is poorly understood. To investigate the possible involvement of Golgi-derived vesicles in the release, we examined the effect of brefeldin A (BFA), a reversible inhibitor of Golgi-mediated secretion, on melatonin secretion of cultured chick pineal cells. We show here that treatment with BFA completely disassembles the Golgi apparatus and reduces melatonin secretion. In more detailed time course experiments, however, the inhibition of melatonin secretion is only observed after the removal of BFA in parallel with the reassembly of the Golgi apparatus. This inhibition of melatonin secretion is not accompanied by accumulation of melatonin in the cells. These observations indicate that chick pineal melatonin is released independently of the Golgi-derived vesicles, and suggest inhibition of melatonin synthesis after the removal of BFA. By measuring the activities and mRNA levels of melatonin-synthesizing enzymes, we found that the removal of BFA specifically inhibits NAT activity at the protein level. On the other hand, BFA causes no detectable phase-shift of the chick pineal oscillator regulating the circadian rhythm of melatonin secretion. The results presented here suggest that the Golgi-mediated vesicular transport is involved in neither the melatonin release nor the time-keeping mechanism of the circadian oscillator, but rather contributes to the regulation of NAT activity.

    DOI: 10.1093/oxfordjournals.jbchem.a002836

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Books 1

  1. 分子・細胞・個体レベルにおける動物の光環境応答とサーカディアンリズム

    深田 吉孝, 岡野 俊行, 仲村 厚志, 和田 恭高, 広田 毅, 小島 大輔( Role: Sole author)

    深田吉孝  2007 

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MISC 44

  1. Chemical biology of the circadian clock and its application to drug discovery

    Hirota Tsuyoshi, Matsuda Tomohiro

    MEDCHEM NEWS   Vol. 31 ( 2 ) page: 62 - 67   2021

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    Language:Japanese   Publisher:The Pharmaceutical Society of Japan  

    The circadian clock is a biological system ubiquitous to almost all organisms on the earth, and controls daily rhythms in our body. Many physiological functions are rhythmically regulated by the cycles of expression, post-translational modification, and degradation of the clock proteins constituting the circadian clock. Disruption of circadian rhythms due to shift work and social jet lag is getting worse in modern society, and has been related with not only sleep disorders, but also various diseases, such as cancer and metabolic disorders. Small-molecule compounds targeting the clock-related proteins will provide a useful tool for uncovering the molecular mechanisms of circadian-related diseases and their treatments. In this article, we introduce the unique circadian clock modulating compounds and their potential for drug discovery.

    DOI: 10.14894/medchem.31.2_62

    CiNii Article

  2. How CRY Regulates the Clock: Structural Studies of a Dynamic Mammalian Circadian Complex

    Sandate C., Fribourgh J., Michael A., Srivastava A., Hura G., Schneidman-Duhovny D., Tripathi S., Takahashi J., Lander G., Hirota T., Tama F., Partch C.

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 76   page: A122 - A122   2020.8

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    DOI: 10.1107/S0108767320098785

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  3. An Isoform-Selective Modulator of Cryptochrome 1 Regulates Circadian Rhythms in Mammals. Reviewed

    Miller S, Aikawa Y, Sugiyama A, Nagai Y, Hara A, Oshima T, Amaike K, Kay SA, Itami K, Hirota T

    Cell chemical biology   Vol. 27 ( 9 ) page: 1192-1198.e5   2020.9

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    DOI: 10.1016/j.chembiol.2020.05.008

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  4. Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing. Reviewed

    Fribourgh JL, Srivastava A, Sandate CR, Michael AK, Hsu PL, Rakers C, Nguyen LT, Torgrimson MR, Parico GCG, Tripathi S, Zheng N, Lander GC, Hirota T, Tama F, Partch CL

    eLife   Vol. 9   2020.2

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    DOI: 10.7554/eLife.55275

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  5. Identification of pathways that regulate circadian rhythms using a larval zebrafish small molecule screen. Reviewed

    Mosser EA, Chiu CN, Tamai TK, Hirota T, Li S, Hui M, Wang A, Singh C, Giovanni A, Kay SA, Prober DA

    Scientific reports   Vol. 9 ( 1 ) page: 12405   2019.8

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    DOI: 10.1038/s41598-019-48914-7

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  6. Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth. Reviewed

    Oshima T, Niwa Y, Kuwata K, Srivastava A, Hyoda T, Tsuchiya Y, Kumagai M, Tsuyuguchi M, Tamaru T, Sugiyama A, Ono N, Zolboot N, Aikawa Y, Oishi S, Nonami A, Arai F, Hagihara S, Yamaguchi J, Tama F, Kunisaki Y, Yagita K, Ikeda M, Kinoshita T, Kay SA, Itami K, Hirota T

    Science advances   Vol. 5 ( 1 ) page: eaau9060   2019.1

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    DOI: 10.1126/sciadv.aau9060

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  7. Nuclear receptor HNF4A transrepresses CLOCK:BMAL1 and modulates tissue-specific circadian networks. Reviewed

    Qu M, Duffy T, Hirota T, Kay SA

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 115 ( 52 ) page: E12305-E12312   2018.12

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    DOI: 10.1073/pnas.1816411115

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  8. Conformational dynamics of human protein kinase CK2α and its effect on function and inhibition. Reviewed

      Vol. 86 ( 3 ) page: 344-353   2018.3

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    DOI: 10.1002/prot.25444

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  9. Global rise of potential health hazards caused by blue light-induced circadian disruption in modern aging societies. Reviewed

    Hatori M, Gronfier C, Van Gelder RN, Bernstein PS, Carreras J, Panda S, Marks F, Sliney D, Hunt CE, Hirota T, Furukawa T, Tsubota K

    NPJ aging and mechanisms of disease   Vol. 3   page: 9   2017

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    DOI: 10.1038/s41514-017-0010-2

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  10. Circadian Amplitude Regulation via FBXW7-Targeted REV-ERBα Degradation. Reviewed

      Vol. 165 ( 7 ) page: 1644-1657   2016.6

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    DOI: 10.1016/j.cell.2016.05.012

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  11. Development of Small-Molecule Cryptochrome Stabilizer Derivatives as Modulators of the Circadian Clock. Reviewed

    Lee JW, Hirota T, Kumar A, Kim NJ, Irle S, Kay SA

    ChemMedChem   Vol. 10 ( 9 ) page: 1489-97   2015.9

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    DOI: 10.1002/cmdc.201500260

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  12. C-H activation generates period-shortening molecules that target cryptochrome in the mammalian circadian clock. Reviewed

    Oshima T, Yamanaka I, Kumar A, Yamaguchi J, Nishiwaki-Ohkawa T, Muto K, Kawamura R, Hirota T, Yagita K, Irle S, Kay SA, Yoshimura T, Itami K

    Angewandte Chemie (International ed. in English)   Vol. 54 ( 24 ) page: 7193-7   2015.6

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    DOI: 10.1002/anie.201502942

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  13. Identification of small-molecule modulators of the circadian clock. Reviewed

    Hirota T, Kay SA

    Methods in enzymology   Vol. 551   page: 267-82   2015

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    DOI: 10.1016/bs.mie.2014.10.015

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  14. In vivo role of phosphorylation of cryptochrome 2 in the mouse circadian clock. Reviewed

    Hirano A, Kurabayashi N, Nakagawa T, Shioi G, Todo T, Hirota T, Fukada Y

    Molecular and cellular biology   Vol. 34 ( 24 ) page: 4464-73   2014.12

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    DOI: 10.1128/MCB.00711-14

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  15. Spatiotemporal separation of PER and CRY posttranslational regulation in the mammalian circadian clock. Reviewed

    St John PC, Hirota T, Kay SA, Doyle FJ 3rd

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 111 ( 5 ) page: 2040-5   2014.2

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    DOI: 10.1073/pnas.1323618111

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  16. Real-time in vivo monitoring of circadian E-box enhancer activity: a robust and sensitive zebrafish reporter line for developmental, chemical and neural biology of the circadian clock. Reviewed

    Developmental biology   Vol. 380 ( 2 ) page: 259-73   2013.8

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    DOI: 10.1016/j.ydbio.2013.04.035

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  17. Biosynthesis and biological actions of pineal neurosteroids in domestic birds. Reviewed

    Tsutsui K, Haraguchi S, Hatori M, Hirota T, Fukada Y

    Neuroendocrinology   Vol. 98 ( 2 ) page: 97-105   2013

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    DOI: 10.1159/000353782

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  18. New biosynthesis and biological actions of avian neurosteroids. Reviewed

    Tsutsui K, Haraguchi S, Inoue K, Miyabara H, Ubuka T, Hatori M, Hirota T, Fukada Y

    Journal of experimental neuroscience   Vol. 7   page: 15-29   2013

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    DOI: 10.4137/JEN.S11148

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  19. Identification of small molecule activators of cryptochrome. Reviewed

    Hirota T, Lee JW, St John PC, Sawa M, Iwaisako K, Noguchi T, Pongsawakul PY, Sonntag T, Welsh DK, Brenner DA, Doyle FJ 3rd, Schultz PG, Kay SA

    Science (New York, N.Y.)   Vol. 337 ( 6098 ) page: 1094-7   2012.8

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    DOI: 10.1126/science.1223710

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  20. A small molecule modulates circadian rhythms through phosphorylation of the period protein. Reviewed

    Lee JW, Hirota T, Peters EC, Garcia M, Gonzalez R, Cho CY, Wu X, Schultz PG, Kay SA

    Angewandte Chemie (International ed. in English)   Vol. 50 ( 45 ) page: 10608-11   2011.11

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    DOI: 10.1002/anie.201103915

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  21. Light-dependent and circadian clock-regulated activation of sterol regulatory element-binding protein, X-box-binding protein 1, and heat shock factor pathways. Reviewed

    Hatori M, Hirota T, Iitsuka M, Kurabayashi N, Haraguchi S, Kokame K, Sato R, Nakai A, Miyata T, Tsutsui K, Fukada Y

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 108 ( 12 ) page: 4864-9   2011.3

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    DOI: 10.1073/pnas.1015959108

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  22. High-throughput chemical screen identifies a novel potent modulator of cellular circadian rhythms and reveals CKIα as a clock regulatory kinase. Reviewed

      Vol. 8 ( 12 ) page: e1000559   2010.12

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    DOI: 10.1371/journal.pbio.1000559

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  23. Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis. Reviewed

    Zhang EE, Liu Y, Dentin R, Pongsawakul PY, Liu AC, Hirota T, Nusinow DA, Sun X, Landais S, Kodama Y, Brenner DA, Montminy M, Kay SA

    Nature medicine   Vol. 16 ( 10 ) page: 1152-6   2010.10

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    DOI: 10.1038/nm.2214

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  24. DYRK1A and glycogen synthase kinase 3beta, a dual-kinase mechanism directing proteasomal degradation of CRY2 for circadian timekeeping. Reviewed

    Kurabayashi N, Hirota T, Sakai M, Sanada K, Fukada Y

    Molecular and cellular biology   Vol. 30 ( 7 ) page: 1757-68   2010.4

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    DOI: 10.1128/MCB.01047-09

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  25. Transcriptional repressor TIEG1 regulates Bmal1 gene through GC box and controls circadian clockwork. Reviewed

    Hirota T, Kon N, Itagaki T, Hoshina N, Okano T, Fukada Y

    Genes to cells : devoted to molecular & cellular mechanisms   Vol. 15 ( 2 ) page: 111-21   2010.2

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    DOI: 10.1111/j.1365-2443.2009.01371.x

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  26. A genome-wide RNAi screen for modifiers of the circadian clock in human cells. Reviewed

    Zhang EE, Liu AC, Hirota T, Miraglia LJ, Welch G, Pongsawakul PY, Liu X, Atwood A, Huss JW 3rd, Janes J, Su AI, Hogenesch JB, Kay SA

    Cell   Vol. 139 ( 1 ) page: 199-210   2009.10

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    DOI: 10.1016/j.cell.2009.08.031

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  27. High-throughput screening and chemical biology: new approaches for understanding circadian clock mechanisms. Reviewed

    Hirota T, Kay SA

    Chemistry & biology   Vol. 16 ( 9 ) page: 921-7   2009.9

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    DOI: 10.1016/j.chembiol.2009.09.002

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  28. A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3beta. Reviewed

    Hirota T, Lewis WG, Liu AC, Lee JW, Schultz PG, Kay SA

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 105 ( 52 ) page: 20746-51   2008.12

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    DOI: 10.1073/pnas.0811410106

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  29. Activation of TGF-beta/activin signalling resets the circadian clock through rapid induction of Dec1 transcripts. Reviewed

    Kon N, Hirota T, Kawamoto T, Kato Y, Tsubota T, Fukada Y

    Nature cell biology   Vol. 10 ( 12 ) page: 1463-9   2008.12

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    DOI: 10.1038/ncb1806

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  30. 細胞外pHのアルカリ性化はDec1遺伝子の誘導を介して概日時計の位相をリセットする

    金 尚宏, 広田 毅, 河本 健, 加藤 幸夫, 坪田 匡史, 深田 吉孝

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   Vol. 81回・31回   page: 4T16 - 4   2008.11

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  31. ニワトリ松果体におけるコレステロール合成系酵素群の遺伝子発現解析

    飯塚倫子, 倉林伸博, 羽鳥恵, 広田毅, 原口省吾, 筒井和義, 深田吉孝

    日本比較生理生化学会大会予稿集   Vol. 30th   page: 23   2008

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    J-GLOBAL

  32. Circadian proteomics of the mouse retina. Reviewed

    Tsuji T, Hirota T, Takemori N, Komori N, Yoshitane H, Fukuda M, Matsumoto H, Fukada Y

    Proteomics   Vol. 7 ( 19 ) page: 3500-8   2007.10

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    DOI: 10.1002/pmic.200700272

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  33. Sequential phosphorylation of mouse cryptochrome2

    Nobuhiro Kurabayashi, Tsuyoshi Hirota, Yoko Harada, Mihiko Sakai, Yoshitaka Fukada

    ZOOLOGICAL SCIENCE   Vol. 23 ( 12 ) page: 1194 - 1194   2006.12

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    Web of Science

  34. 【ここまで分かった生物時計の分子ネットワーク リン酸化による時計タンパク質の制御機構から睡眠・肥満への関与まで】時計タンパク質の多段階リン酸化

    広田 毅, 深田 吉孝

    実験医学   Vol. 24 ( 4 ) page: 452 - 459   2006.3

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    概日時計の自律的な発振メカニズムにおいては,時計遺伝子の転写・翻訳とともに時計タンパク質のリン酸化が重要な役割を果たす.いくつかの時計タンパク質は,複数のキナーゼによって段階的にリン酸化され,その分解速度や細胞内局在,転写調節などの機能が緻密に調節されている.本稿では,時計タンパク質をリン酸化するキナーゼの同定,およびリン酸化サイトの決定に関する最近の知見を紹介するとともに,多段階リン酸化による時計タンパク質の制御機構を概説する(著者抄録)

  35. Phosphorylation of mCRY2 at Ser557 in the hypothalamic suprachiasmatic nucleus of the mouse. Reviewed

    Kurabayashi N, Hirota T, Harada Y, Sakai M, Fukada Y

    Chronobiology international   Vol. 23 ( 1-2 ) page: 129-34   2006

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    DOI: 10.1080/07420520500464478

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  36. Ser-557-phosphorylated mCRY2 is degraded upon synergistic phosphorylation by glycogen synthase kinase-3 beta. Reviewed

    Harada Y, Sakai M, Kurabayashi N, Hirota T, Fukada Y

    The Journal of biological chemistry   Vol. 280 ( 36 ) page: 31714-21   2005.9

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    DOI: 10.1074/jbc.M506225200

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  37. Resetting mechanism of central and peripheral circadian clocks in mammals. Reviewed

    Hirota T, Fukada Y

    Zoological science   Vol. 21 ( 4 ) page: 359-68   2004.4

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    DOI: 10.2108/zsj.21.359

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  38. p38 mitogen-activated protein kinase regulates oscillation of chick pineal circadian clock. Reviewed

    Hayashi Y, Sanada K, Hirota T, Shimizu F, Fukada Y

    The Journal of biological chemistry   Vol. 278 ( 27 ) page: 25166-71   2003.7

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    DOI: 10.1074/jbc.M212726200

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  39. Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expression in cultured Rat-1 fibroblasts. Reviewed

    Hirota T, Okano T, Kokame K, Shirotani-Ikejima H, Miyata T, Fukada Y

    The Journal of biological chemistry   Vol. 277 ( 46 ) page: 44244-51   2002.11

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    DOI: 10.1074/jbc.M206233200

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  40. 分子時計の生化学 グルコース投与によるrat-1細胞の時計遺伝子発現における概日リズムの誘導

    広田 毅, 岡野 俊行, 小亀 浩市, 池島 裕子, 谷, 宮田 敏行, 深田 吉孝

    生化学   Vol. 74 ( 8 ) page: 643 - 643   2002.8

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  41. Effect of brefeldin A on melatonin secretion of chick pineal cells Reviewed

    Hirota, T., Kagiwada, S., Kasahara, T., Okano, T., Murata, M., Fukada, Y.

    Journal of Biochemistry   Vol. 129 ( 1 ) page: 51-59   2001

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    Melatonin is secreted from the pineal gland in a circadian manner. It is well established that the synthesis of melatonin shows a diurnal rhythm reflecting a daily change in serotonin N-acetyltransferase (NAT) activity, and the overall secretion of melatonin requires a cellular release process, which is poorly understood. To investigate the possible involvement of Golgi-derived vesicles in the release, we examined the effect of brefeldin A (BFA), a reversible inhibitor of Golgi-mediated secretion, on melatonin secretion of cultured chick pineal cells. We show here that treatment with BFA completely disassembles the Golgi apparatus and reduces melatonin secretion. In more detailed time course experiments, however, the inhibition of melatonin secretion is only observed after the removal of BFA in parallel with the reassembly of the Golgi apparatus. This inhibition of melatonin secretion is not accompanied by accumulation of melatonin in the cells. These observations indicate that chick pineal melatonin is released independently of the Golgi-derived vesicles, and suggest inhibition of melatonin synthesis after the removal of BFA. By measuring the activities and mRNA levels of melatonin-synthesizing enzymes, we found that the removal of BFA specifically inhibits NAT activity at the protein level. On the other hand, BFA causes no detectable phase-shift of the chick pineal oscillator regulating the circadian rhythm of melatonin secretion. The results presented here suggest that the Golgi-mediated vesicular transport is involved in neither the melatonin release nor the time-keeping mechanism of the circadian oscillator, but rather contributes to the regulation of NAT activity.

    DOI: 10.1093/oxfordjournals.jbchem.a002836

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  42. Chicken pineal clock genes: Implication of BMAL2 as a bidirectional regulator in circadian clock oscillation Reviewed

    Okano, T., Yamamoto, K., Okano, K., Hirota, T., Kasahara, T., Sasaki, M., Takanaka, Y., Fukada, Y.

    Genes to Cells   Vol. 6 ( 9 ) page: 825-836   2001

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    Background: In a transcription/translation-based autoregulatory feedback loop of vertebrate circadian clock systems, a BMAL1-CLOCK heterodimer is a positive regulator for the transcription of the negative element gene Per. The chicken pineal gland represents a photosensitive clock tissue, but the pineal clock genes constituting the oscillator loop have been less well characterized.
    Results: We identified expression of the Per2, Bmal1, Bmal2 and Clock genes in the chicken pineal gland. Messenger RNA levels of these genes exhibited overt circadian rhythms in the pineal cells, both in vivo and in culture. In vitro functional analyses revealed the formation of cBMAL1-cCLOCK and cBMAL2-cCLOCK heteromers. Both of the cBMAL-cCLOCK heteromers activated E-box element-dependent transcription, which was negatively regulated by cPER2 in luciferase assays. Co-expression of cCLOCK, cBMAL1 and cBMAL2 co-operatively activated E-box element-dependent transcription, and a greater level of expression of cBMAL2 inhibited the activation. In the cultured pineal cells, an over-expression of either cBMAL1 or cBMAL2 disrupted the circadian rhythm of melatonin production.
    Conclusion: The functional characterization of the chicken pineal clock molecules supports the key roles of BMAL1, BMAL2 and CLOCK which contribute to the E-box-dependent transcriptional regulation in the circadian clock system.

    DOI: 10.1046/j.1365-2443.2001.00462.x

    Web of Science

    Scopus

  43. ブレフェルジンAを用いたニワトリ松果体細胞のメラトニン分泌機構の解析

    広田 毅, 鍵和田 聡, 笠原 和起, 岡野 俊行, 村田 昌之, 深田 吉孝

    生化学   Vol. 72 ( 8 ) page: 1016 - 1016   2000.8

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    Language:Japanese   Publisher:(公社)日本生化学会  

  44. 松果体とサーカディアンリズム

    深田吉孝, 広田毅

    Clinical Neuroscience特集「サーカディアンリズムのしくみと働き」   Vol. 18 ( 10 ) page: 1147 - 1149   2000

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KAKENHI (Grants-in-Aid for Scientific Research) 9

  1. 革新的な機能調節化合物の創製による概日時計システムの統合的な理解と制御

    Grant number:21H04766  2021.4 - 2024.3

    廣田 毅

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    Authorship:Principal investigator 

    Grant amount:\42510000 ( Direct Cost: \32700000 、 Indirect Cost:\9810000 )

    睡眠・覚醒など様々な生理機能の日内リズムを支配する概日時計について、私たちが世界に先駆けて発見した独自の時計調節化合物を用い、新たな時計タンパク質の発見や概日リズムの自在な制御を可能にする新技術を開発する。これらの革新的な技術を用いて、分子メカニズム解析から組織・個体における機能制御を統合的に行い、従来の分子遺伝学研究を超えて概日時計システムの作動原理を徹底解剖する。

  2. Mechanistic study on circadian clock system regulated by heme/CO crosstalk

    Grant number:20H02871  2020.4 - 2023.3

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    Authorship:Coinvestigator(s) 

  3. ケージド時計調節化合物を用いた概日リズムの光制御と細胞間相互作用の解析

    Grant number:20K21269  2020.7 - 2022.3

    挑戦的研究(萌芽)

    廣田 毅

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    Authorship:Principal investigator 

    Grant amount:\6500000 ( Direct Cost: \5000000 、 Indirect Cost:\1500000 )

    申請者は概日リズムの周期を変化させる新規化合物を発見して鍵となる制御機構を明らかにしてきた。多細胞から成る概日時計システムの理解に向けて解明すべき重要課題が、細胞間の相互作用である。本研究では時計調節化合物のケージド誘導体を用い、狙ったタイミングに狙った細胞で概日時計を定量的に操作する新技術を生み出す。これを概日リズムの1細胞イメージングに応用し、細胞間相互作用の時空間的な解析に挑戦する。

  4. Development and characterization of small molecules for analysis of circadian rhythm amplitude

    Grant number:18K19171  2018.6 - 2020.3

    Grant-in-Aid for Challenging Research (Exploratory)

    Hirota Tsuyoshi

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    Authorship:Principal investigator 

    Grant amount:\6240000 ( Direct Cost: \4800000 、 Indirect Cost:\1440000 )

    Amplitude of the circadian rhythm is reduced by metabolic disease and aging. While circadian amplitude plays an important role in the regulation of physiological outputs, its molecular mechanism is poorly understood. In this study, we tried to approach the molecular mechanism of circadian amplitude regulation by applying chemical biology methods. From chemical screening, we identified new compounds that increase rhythm amplitude and analyzed its mechanism of action. Further study will facilitate the understanding of amplitude regulation.

  5. 新規化合物を用いて迫る概日時計タンパク質CRY1とCRY2の特異性の分子基盤

    Grant number:18H02402  2018.4 - 2021.3

    廣田 毅

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    Authorship:Principal investigator 

    Grant amount:\17550000 ( Direct Cost: \13500000 、 Indirect Cost:\4050000 )

    睡眠・覚醒や代謝など、多様な生理現象は体内に存在する概日時計に支配されて一日周期のリズムを示す。申請者はケミカルバイオロジーを応用し、概日時計の機能を調節する新規化合物を発見して鍵となる制御機構を明らかにしてきた。そのひとつである化合物KL001は、概日時計の発振に中心的な役割を果たす時計タンパク質CRYを標的とする。さらに、KL001誘導体の解析から非常に高い活性を持つKL044を見出した。KL001とKL044は共にCRY1とCRY2の両者に作用する。これに対し、全く異なる化学構造を持つ新たな周期延長化合物AとBを表現型スクリーニングから見出し、CRY1選択的に作用することを発見した。本研究ではこれらのユニークな化合物を用い、CRY1とCRY2の違いを生み出すメカニズムに迫る。本年度はまず、CRY2により強く作用する化合物として同定したCの特異性を、Cry1およびCry2ノックアウト細胞を用いてPer2レポーターの抑制活性ならびに周期延長活性を指標に解明した。さらにCRY2の発現・精製系を構築し、CRY2と化合物Cとの複合体の構造をX線結晶構造解析によって明らかにした。化合物Cの誘導体が概日リズムの周期に与える作用を細胞レベルで評価して構造活性相関を解析し、活性に必要な化合物の特徴を抽出して立体構造との対応を明らかにすると共に、結晶構造におけるタンパク質と化合物の相互作用様式が溶液中でも成り立つことを示した。さらに、CRY1とCRY2の間で違いのあるアミノ酸残基を入れ替えた変異体を作製し、化合物AとCに対する感受性の変化を解析することによって、選択性に必要な領域を見出した。
    CRY2に選択性をもつ化合物として見出したCとCRY2との複合体の構造を決定し、順調に進展している。さらに、CRY1とCRY2の選択性に必要な領域を見出すことに成功した。
    化合物Bの解析を進め、化合物AならびにCと比較することにより、CRY1とCRY2の違いを生み出す分子基盤を解析する。

  6. Development and characterization of small molecules that regulate clock protein function for analysis of the circadian clock mechanism

    Grant number:15H05590  2015.4 - 2018.3

    Hirota Tsuyoshi

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    Authorship:Principal investigator 

    Grant amount:\23530000 ( Direct Cost: \18100000 、 Indirect Cost:\5430000 )

    The circadian clock regulates daily rhythms of physiological processes through temporal and spatial organization from molecular to organismal levels. In this study, we focused on stability and complex formation of the clock proteins PER and CRY that play central role in the regulation of circadian period. We searched for regulatory compounds targeting these processes and discovered new compound that selectively control CRY1 function. In parallel, we analyzed post-translational modification of clock proteins by SUMO and identified CRY SUMOylation. By using original tools, we are going to reveal and control the molecular mechanism of the circadian clock.

  7. 概日時計タンパク質CRYの機能を調節する低分子化合物

    Grant number:26891011  2014.8 - 2015.3

    廣田 毅

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    Authorship:Principal investigator 

    Grant amount:\1430000 ( Direct Cost: \1100000 、 Indirect Cost:\330000 )

    本研究では、時計タンパク質CRYの安定性を変化させる新規の化合物を同定し、それをプローブとしてCRYの機能調節機構を解明することを目指す。そのために本年度は化合物スクリーニング系のセットアップを行った。CRY1とルシフェラーゼの融合タンパク質であるCRY1-LUCを安定発現するHEK293細胞を用い、申請者が発見したCRYを安定化する化合物KL001の効果を指標に細胞数、培地量、化合物添加のタイミング、処理時間などを検討し、384ウェルプレートを用いたアッセイ条件を最適化した。化合物処理によるCRY1-LUCの蓄積量の変化を用いたエンドポイントアッセイとすることで、半減期を測定するよりも手順を簡略化し、ハイスループット化への目途をつけた。コントロールとしてLUCを安定発現する細胞を用い、化合物がルシフェラーゼに与える影響を評価することで、CRYに対する作用を明らかにする。さらに名古屋大学トランスフォーマティブ生命分子研究所の化合物ライブラリーセンターと共同で、384ウェルプレートに対応した細胞分注機およびプレートリーダーを導入した。現在、化合物投与に用いる微量分注機をセットアップしているところである。機器の準備が整い次第、化合物ライブラリーセンターが所有する化合物のうち1万種類をスクリーニングし、CRYの発現量を増加あるいは減少させる化合物がどの程度の頻度で出現するかを見積もる予定である。
    26年度が最終年度であるため、記入しない。
    26年度が最終年度であるため、記入しない。

  8. 哺乳類の末梢組織における概日時計のグルコース応答

    Grant number:16770091  2004 - 2005

    廣田 毅

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    Authorship:Principal investigator 

    Grant amount:\3200000 ( Direct Cost: \3200000 )

    交付申請書に記載した研究実施計画に沿って研究を行い、下記の成果を得た。
    TIEG1蛋白質の部分配列を有する抗原蛋白質を作製し、ウサギに免疫した。得られた抗血清をアフィニティー精製し、抗TIEG1抗体とした。この抗体を用いてウエスタンブロット解析した結果、TIEG1は主に核内に存在する約65kDaの蛋白質で、rat-1細胞における発現量はグルコース刺激によって急速に上昇することが判明した。TIEG1蛋白質量はグルコース投与の2時間後にピークに達し、これはTieg1 mRNAのピークから約1時間遅れていたものの、mRNAと蛋白質の発現プロファイルが互いに似ていたことから、TIEG1蛋白質の合成・分解は比較的速いと考えられた。さらに、生体内においてもTIEG1が時計入力因子として働く可能性を検証するため、マウスを2日間絶食させた後に餌を2時間与え、摂食が肝臓におけるTIEG1蛋白質量に与える効果を解析した。その結果、餌を与えた場合は絶食を続けた場合と比べ、TIEG1蛋白質量が有意に上昇することがわかった。一方、Per1およびBmal1遺伝子の発現量は摂食によって低下したことから、TIEG1は生体内においてもPer1・Bmal1遺伝子の抑制因子として時計入力に関与すると考えられた。
    以上の解析と並行して、時計蛋白質E4BP4およびCRY2のリン酸化動態を解析した。ニワトリE4BP4のSer182およびマウスCRY2のSer557のリン酸化は、各蛋白質のプロテアソームを介した分解を導く。本研究では、Ser182リン酸化型のE4BP4に対する抗体を作製し、ニワトリ松果体においてSer182がリン酸化されていることを見出すとともに、Ser557リン酸化型のCRY2に対する抗体を用い、マウスの肝臓だけでなく視交叉上核においてもSer557のリン酸化量が顕著な概日リズムを示すことを明らかにした。

  9. Molecular, cellular and in vivo analyses of photoresponses and circadian rhythms in animals

    Grant number:14104003  2002 - 2006

    FUKADA Yoshitaka

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    Authorship:Coinvestigator(s) 

    (1)Activation of Gil induced a phase-shift of the chick pineal circadian clock in a manner similar to that induced by light. In the promoter region of pinopsin gene, we identified a light-responsive element responsible for its light-dependent transcriptional activation.
    (2)Farnesylation of retinal rod G-protein transducin plays an important role in not only the cellular light-signaling but also the light-adaptation. We found that GRK7-1 expressed in the zebrafish retinal cones shows opsin-phosphorylating activity dozens-fold higher than that of rod-specific GRK1A.
    (3)Chick pineal and frog retinal MAPK was circadian-activated showing the peak activity at night. Within the mouse SCN, the profiles of the temporal and light activation of MAPK are both different between the dorsomedial and central regions of the SCN. We identified amino acid residues in BMAL1, CRY1 and CRY2 to be phosphorylated by MAPK and their phosphorylation-dependent functional changes. We also found that SCOP expressed abundantly in the SCN is a negative regulator of K-Ras and downstream MAPK. It was shown that p38 MAPK regulates the molecular clock in the daytime to have the phase-advancing effect.
    (4)When the chicks entrained to 12L : 12D conditions were exposed to prolonged light extending into the early night, pineal E4BP4 protein was kept at a high level, which delayed the morning induction of Per2 and delayed the phase of the pineal clock. E4BP4 was phosphorylated by CKIε, leading to its proteasomal degradation.
    (5)We found that glucose-stimulation of rat-1 cells resets the clock by a mechanism to which Tieg 1 and Vdup 1 contribute. TIEG 1 protein was in fact up-regulated upon glucose administration and it directly acts on the promoter of Bmall to inhibit the transcription.
    (6)Within the promoter region of pineal opsin exorhodopsin gene, we identified a cis-acting element, PIPE, that drives pineal-specific gene expression in the zebrafish.

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