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.

Fig 1. USP7 interacts with CRY proteins.

A. Silver staining image of proteins co-purified with FLAG-His-Myc-CRY1 (FHM-CRY1) or FHM-CRY2. NIH3T3 cells expressing FHM-CRY1 or FHM-CRY2 were treated with 10 μM MG132 for 6 hours and lysed with IP Buffer. Cell lysates were subjected to immunoprecipitation using anti-FLAG-M2 agarose beads. FH-LacZ expressed in NIH3T3 cells was used as a control. B. The numbers of proteins co-purified with FHM-CRY1 or FHM-CRY2. Proteins co-purified with FH-LacZ were eliminated from the list of CRY1 and CRY2 interacting proteins. Proteins detected in both CRY1 and CRY2 samples with high MS scores were listed in S1 Table. C. Interaction of USP7 with CRY2 protein. NIH3T3 cells expressing FLAG-CRY2 and/or Myc-USP7 were cultured in the presence of 10 μM MG132 for 6 hours and lysed with IP Buffer. The cell lysates were subjected to immunoprecipitation using anti-FLAG, anti-Myc antibody or normal mouse IgG (negative control) as precipitating antibodies.

Fig 2. USP7 deubiquitinates CRY proteins.

A. In vitro ubiquitination assay. HEK293T/17 cells were transfected with the expression vector of FLAG-CRY2. Forty-two hours after the transfection, the cells were cultured in the presence of 10 μM MG132 for 6 hours and then harvested. FLAG-CRY2 purified from the cell lysate with anti-FLAG M2 agarose beads was incubated with or without a recombinant protein, full-length USP7 or a catalytic domain of USP2 (USP2 CD), for 30 min at 37°C. Recombinant USP2 catalytic domain was used as a positive control [26]. B. In vivo deubiquitination assay in HEK293T/17 cells. The cells were transfected with indicated expression vectors. Forty-two hours after the transfection, the cells were cultured in the presence of 10 μM MG132 for 6 hours and then lysed with IP Buffer. FLAG-CRY2 was purified with anti-FLAG M2 agarose beads, followed by western blotting analysis with anti-CRY2 antibody. An inactive mutant of USP7 (USP7-C223A) was used for a negative control. C. Effect of USP7-specific inhibitor on CRY up-shifted bands. NIH3T3 cells were transfected with the expression vector for Myc-CRY1 or Myc-CRY2. Forty-two hours after the transfection, the cells were cultured in the presence of 20 μM HBX 41108 for 6 hours. The smear bands of Myc-CRY1 or Myc-CRY2 were quantified (means + SEM, n = 3, **: p < 0.01 by Student’s t-test).

Fig 3. USP7 increases the protein levels and stabilities of CRY1 and CRY2.

A. Effect of USP7 expression on CRY protein levels. HEK293T/17 cells were transfected with indicated expression vectors. Forty-eight hours after the transfection, the cells were lysed with SDS-PAGE sample buffer, and the cell lysate was analyzed by western blotting. GFP was used as a control. Quantified data are shown by means + SEM (n = 3, **: p < 0.01 by Student’s t-test). B. Effect of USP7 overexpression on CRY-LUC protein stability. HEK293T/17 cells were transfected with expression vectors for CRY-LUC and Myc-USP7 (or empty control), and cultured for 48 hours. The culture medium was changed to the recording medium containing 0.1 mg/ml cycloheximide. Bioluminescence signals were recorded continuously at 10-min intervals, and the signal was normalized to the value at time 0. Half-lives of CRY1-LUC and CRY2-LUC were calculated by fitting an exponential decay curve to bioluminescence signals, and are shown as means + SEM (n = 3, **: p < 0.01 by Student’s t-test). C. USP7 inhibitor treatment of NIH3T3 cells decreased Myc-CRY1 expression. NIH3T3 cells were transfected with Myc-CRY1 and GFP expression vectors. Forty-two hours after the transfection, the cell were cultured with 10 or 20 μM HBX 41108 for 6 hours. Quantified data are shown as means + SEM (n = 3). D. Usp7 knockdown decreased the starting levels of CRYs-LUC. The decay of the bioluminescence signals was recorded as described in B. The starting levels of CRYs-LUC bioluminescence signals are shown as means + SEM (n = 4, **: p < 0.01 by Student’s t-test). E. Usp7 knockdown decreased CRYs-LUC stability. The bioluminescence signals normalized to the value at time 0 and the half-lives of CRYs-LUC are shown as means + SEM (n = 4, **: p < 0.01 by Tukey’s test or Student’s t-test). F. Effect of USP7 overexpression on CRY2-LUC protein stability in Fbxl3 knockdown cells. HEK293T/17 cells were transfected with indicated plasmid vectors and cultured for 72 hours. The decay of the bioluminescence signals was recorded as described in B. Quantified data are shown as means + SEM (n = 3, *: p < 0.05 by Student’s t-test).

Fig 4. Usp7 knockdown shortens the circadian period of the cellular rhythms.

A. NIH3T3 cells were transfected with a luciferase reporter vector, Bmal1 us0.3-luc, and USP7 expression vector. Twenty-four hours after the transfection, the cellular rhythms were synchronized by 30-min treatment with 0.1 μM dexamethasone (Dex). The culture medium was changed to the recording medium, and bioluminescence signals were recorded continuously. The calculated circadian periods are shown as means + SEM (n = 4, *: p < 0.05 by Student’s t-test). B. Cellular rhythms in Usp7 knockdown cells. Bioluminescence rhythms of Bmal1-luc reporter were recorded as described in A. The calculated circadian periods are shown as means + SEM (n = 4, **: p < 0.01 by Tukey’s test).

Fig 5. TDP-43 interacts with CRY proteins and stabilizes CRY proteins.

A. Effect of TDP-43 expression on CRY protein levels. HEK293T/17 cells were transfected with expression vectors for Myc-CRY1, Myc-CRY2 and FLAG-TDP-43, and cultured for 48 hours. The cells were lysed with SDS-PAGE sample buffer, and the cell lysate was analyzed by western blotting. Quantified data are shown by means + SEM (n = 3, **: p < 0.01 by Student’s t-test). GFP was used as a control. B, C. Interaction of TDP-43 with CRY2 protein. HEK293 cells expressing FLAG-CRY2 were lysed with IP buffer, followed by immunoprecipitation using anti-FLAG antibody. Binding of endogenous TDP-43 with FLAG-CRY2 was detected by western blotting analysis (B). HEK293 cells expressing Myc-CRY2 and/or FLAG-TDP-43 were lysed with IP Buffer. The cell lysates were subjected to immunoprecipitation using anti-FLAG antibody (C). D. Effect of TDP-43 overexpression on CRY-LUC stability. HEK293T/17 cells were transfected with CRYs-LUC and TDP-43 expression vectors and cultured for 48 hours. The culture medium was changed to the recording medium containing 0.1 mg/ml cycloheximide and the bioluminescence signals of CRY-LUC were recorded. Bioluminescence signal at the start time point was set to 1. Half-lives of CRY1-LUC and CRY2-LUC were calculated by fitting an exponential decay curve to bioluminescence signals and shown as means + SEM (n = 4, **: p < 0.01, ***: p < 0.001 by Student’s t-test). E. Tdp-43 knockdown decreased CRY2-LUC stability. The decay of the bioluminescence signals of CRY2-LUC was recorded, and the half-life of CRY2-LUC was calculated as described in D. Quantified data are shown as means + SEM (n = 4, **: p < 0.01 by Student’s t-test). F. Effect of TDP-43 overexpression on CRY2-LUC protein stability in Fbxl3-knockdown cells. HEK293T/17 cells were transfected with indicated expression vectors and cultured for 72 hours. The decay of the bioluminescence signals was recorded as described in D. Quantified data are shown as means + SEM (n = 4, **: p < 0.01 by Student’s t-test).

Fig 6. Tdp-43 knockdown shortens the circadian period.

A. Cellular rhythms in Tdp-43 knockdown cells. Bioluminescence rhythms of Bmal1-luc reporter were recorded as described in Fig 4A. Calculated periods of the cellular rhythms are shown as means + SEM (n = 4, *: p < 0.05, **: p < 0.01 by Student’s t-test). B. A model for period-determination by the control of CRY proteins stabilities. CRY protein destabilizer, FBXL3, accelerates the oscillation of the circadian clock, while CRY stabilizers slow down the oscillation speed.