USP7 and TDP-43: Pleiotropic Regulation of Cryptochrome Protein Stability Paces the Oscillation of the Mammalian Circadian Clock

Arisa Hirano; Tomoki Nakagawa; Hikari Yoshitane; Masaaki Oyama; Hiroko Kozuka-Hata; Darin Lanjakornsiripan; Yoshitaka Fukada

doi:10.1371/journal.pone.0154263

USP7 and TDP-43: Pleiotropic Regulation of Cryptochrome Protein Stability Paces the Oscillation of the Mammalian Circadian Clock

PLoS One. 2016 Apr 28;11(4):e0154263. doi: 10.1371/journal.pone.0154263. eCollection 2016.

Authors

Arisa Hirano¹, Tomoki Nakagawa¹, Hikari Yoshitane¹, Masaaki Oyama², Hiroko Kozuka-Hata², Darin Lanjakornsiripan¹, Yoshitaka Fukada¹

Affiliations

¹ Department of Biological Sciences, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
² Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Circadian Clocks / genetics*
Cryptochromes / genetics*
Cryptochromes / metabolism
DNA-Binding Proteins / antagonists & inhibitors
DNA-Binding Proteins / genetics*
DNA-Binding Proteins / metabolism
F-Box Proteins / genetics
F-Box Proteins / metabolism
Feedback, Physiological
Gene Expression Regulation
Genes, Reporter
HEK293 Cells
Humans
Luciferases / genetics
Luciferases / metabolism
Mice
NIH 3T3 Cells
Proteasome Endopeptidase Complex / metabolism
Protein Stability
Proteolysis
RNA, Small Interfering / genetics
RNA, Small Interfering / metabolism
Signal Transduction
Ubiquitin Thiolesterase / antagonists & inhibitors
Ubiquitin Thiolesterase / genetics*
Ubiquitin Thiolesterase / metabolism
Ubiquitin-Specific Peptidase 7
Ubiquitination

Substances

CRY1 protein, human
CRY2 protein, human
Cryptochromes
DNA-Binding Proteins
F-Box Proteins
FBXL3 protein, human
RNA, Small Interfering
TDP-43 protein, mouse
Luciferases
USP7 protein, human
Ubiquitin Thiolesterase
Ubiquitin-Specific Peptidase 7
Proteasome Endopeptidase Complex

Grants and funding

This work was supported by Ministry of Education, Culture, Sports, Science and Technology, MEXT (Japan), Japan Society of the Promotion of Science, Grants-in-Aid for Scientific Research, https://www.jsps.go.jp/english/e-grants/, grant numbers 24227001 and 15H01173, Recipient Y.F.; Ministry of Education, Culture, Sports, Science and Technology, MEXT (Japan), Japan Society of the Promotion of Science, Research Fellowships for Young Researchers, http://www.jsps.go.jp/english/e-pd/index.html, Recipient A.H.