Isoform-selective regulation of mammalian cryptochromes

Abstract

CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY–compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.

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Fig. 1: Phenylpyrazole derivatives lengthen circadian period.
Fig. 2: KL101 and TH301 are selective against CRY1 and CRY2.
Fig. 3: KL101 and TH301 interact with the FAD-binding pocket of CRY1.
Fig. 4: C-terminal region of CRY is required for the effects of KL101 and TH301.
Fig. 5: CRY exon 10 affects selectivity of KL101 and TH301.
Fig. 6: KL101 and TH301 promote brown adipocyte differentiation.

Data availability

The final coordinates of CRY1, CRY1–KL044, CRY1–KL101, CRY1–TH301 and CRY2–TH301 were deposited into the Protein Data Bank with the accession numbers 6KX4, 6KX5, 6KX6, 6KX7 and 6KX8, respectively. Further data are available from the corresponding authors upon request.

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Acknowledgements

We thank N. Ono, Y. Niwa, A. Shiba, N. Kadofusa and Y. Aoki for technical assistance; T. Senda for technical advice; J.S. Takahashi (UT Southwestern) for Per2::Luc knock-in mice; T. Todo (Osaka University) for Cry1/Cry2 double knockout mice; and H.R. Ueda (University of Tokyo) for Cry1/Cry2 double knockout cells and pMU2-P(Cry1)-FLAG-I/RRE-Cry1 plasmid. This work was supported in part by JST PRESTO Grant No. JPMJPR14LA (T.H.); JSPS Grants No. 15H05590 and No. 18H02402 (T.H.); the Takeda Science Foundation (T.H.); the Suzuken Memorial Foundation (T.H.); AMED Grant No. JP19gm6110026 (M.H.); a JST PRESTO Grant (M.H.); JSPS Grants No. 16H06174, No. 19H03266 and No. 19K22693 (M.H.); the Uehara Memorial Foundation (M.H.); the Sumitomo Foundation (M.H.); the Astellas Foundation for Research on Metabolic Disorders (M.H.); the Cell Science Research Foundation (M.H.); JSPS Grant No. 18K06316 (Y.L.S.); and a MEXT PDIS Grant (S.O.). X-ray diffraction data collection and preliminary experiments were carried out at beamlines BL44XU of SPring-8 (proposals No. 2017A6743 and No. 2017B6743), BL41XU of SPring-8 (proposal No. 2018B1011) and BL-17A of the Photon Factory (proposals No. 2016R-63 and No. 2017G563). Recombinant CRY expression and beamline experiments were supported in part by BINDS from AMED (support No. JP19am0101074-0055 and No. JP19am0101071-0529).

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T.H. and M.H. conceptualized and administrated the project. S.M., Y.A., A. Srivastava and K.H. performed structural biology experiments. T.O. synthesized GO series compounds. Y.L.S. and M.H. conducted BAT experiments. E.M., Y.N., A. Sugiyama, A.H. and T.H. performed all other experiments. K.A., S.O. and A. Sato provided reagents and facilities. S.M., Y.L.S., Y.N., A. Srivastava, S.H., M.H. and T.H. validated data. T.H., M.H., S.M. and Y.L.S. visualized data and wrote the manuscript. T.H., M.H., S.M., Y.L.S., A. Srivastava, S.O. and S.H. edited the manuscript. All authors read and approved the manuscript. T.H., M.H., S.A.K., K.I. and F.T. supervised the project. T.H., M.H., Y.L.S. and S.O. acquired funding.

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Correspondence to Megumi Hatori or Tsuyoshi Hirota.

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Miller, S., Son, Y.L., Aikawa, Y. et al. Isoform-selective regulation of mammalian cryptochromes. Nat Chem Biol 16, 676–685 (2020). https://doi.org/10.1038/s41589-020-0505-1

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