title: USP7 and TDP-43: Pleiotropic Regulation of Cryptochrome Protein Stability Paces the Oscillation of the Mammalian Circadian Clock.
abstract: Mammalian Cryptochromes, CRY1 and CRY2, function as principal regulators of a transcription-translation-based negative feedback loop underlying the mammalian circadian clockwork. An F-box protein, FBXL3, promotes ubiquitination and degradation of CRYs, while FBXL21, the closest paralog of FBXL3, ubiquitinates CRYs but leads to stabilization of CRYs. Fbxl3 knockout extremely lengthened the circadian period, and deletion of Fbxl21 gene in Fbxl3-deficient mice partially rescued the period-lengthening phenotype, suggesting a key role of CRY protein stability for maintenance of the circadian periodicity. Here, we employed a proteomics strategy to explore regulators for the protein stability of CRYs. We found that ubiquitin-specific protease 7 (USP7 also known as HAUSP) associates with CRY1 and CRY2 and stabilizes CRYs through deubiquitination. Treatment with USP7-specific inhibitor or Usp7 knockdown shortened the circadian period of the cellular rhythm. We identified another CRYs-interacting protein, TAR DNA binding protein 43 (TDP-43), an RNA-binding protein. TDP-43 stabilized CRY1 and CRY2, and its knockdown also shortened the circadian period in cultured cells. The present study identified USP7 and TDP-43 as the regulators of CRY1 and CRY2, underscoring the significance of the stability control process of CRY proteins for period determination in the mammalian circadian clockwork.
authors.affiliation: Department of Biological Sciences, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Arisa
authors.initials: A
authors.lastname: Hirano
authors.affiliation: Department of Biological Sciences, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Nakagawa
authors.affiliation: Department of Biological Sciences, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Hikari
authors.initials: H
authors.lastname: Yoshitane
authors.affiliation: Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Oyama
authors.affiliation: Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kozuka-Hata
authors.affiliation: Department of Biological Sciences, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Darin
authors.initials: D
authors.lastname: Lanjakornsiripan
authors.affiliation: Department of Biological Sciences, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Fukada
mesh_terms: D000818:Animals; D057906:Circadian Clocks / Q000235:genetics*; D056931:Cryptochromes / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism; D025461:Feedback, Physiological; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D055550:Protein Stability; D059748:Proteolysis; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D043222:Ubiquitin Thiolesterase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000076084:Ubiquitin-Specific Peptidase 7; D054875:Ubiquitination
chemical_list: D056931:Cryptochromes; D004268:DNA-Binding Proteins; D044783:F-Box Proteins; D008156:Luciferases; D046988:Proteasome Endopeptidase Complex; D034741:RNA, Small Interfering; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7; C538963:CRY1 protein, human; C538966:CRY2 protein, human; C519795:FBXL3 protein, human; C578077:Tardbp protein, mouse; C464569:USP7 protein, human