Abstract
Dysregulation of the circadian rhythm is associated with many diseases, including diabetes, obesity, and cancer. Aryl hydrocarbon receptor nuclear translocator-like protein 1 (Arntl or Bmal1) is the only clock gene whose loss disrupts circadian locomotor behavior in constant darkness. BMAL1 levels are affected by proteasomal inhibition and by several enzymes in the ubiquitin-proteasome system, but the exact molecular mechanism remains unclear. Here, using immunoprecipitation and MS analyses, we discovered an interaction between BMAL1 and ubiquitin-conjugating enzyme E2 O (UBE2O), an E3-independent E2 ubiquitin-conjugating enzyme (i.e. hybrid E2/E3 enzyme). Biochemical experiments with cell lines and animal tissues validated this specific interaction and uncovered that UBE2O expression reduces BMAL1 levels by promoting its ubiquitination and degradation. Moreover, UBE2O expression/knockdown diminished/increased, respectively, BMAL1-mediated transcriptional activity but did not affect BMAL1 gene expression. Bioluminescence experiments disclosed that UBE2O knockdown elevates the amplitude of the circadian clock in human osteosarcoma U2OS cells. Furthermore, mapping of the BMAL1-interacting domain in UBE2O and analyses of BMAL1 stability and ubiquitination revealed that the conserved region 2 (CR2) in UBE2O significantly enhances BMAL1 ubiquitination and decreases BMAL1 protein levels. A Cys-to-Ser substitution experiment identified the critical Cys residue in the CR2 domain responsible for BMAL1 ubiquitination. This work identifies UBE2O as a critical regulator in the ubiquitin-proteasome system, which modulates BMAL1 transcriptional activity and circadian function by promoting BMAL1 ubiquitination and degradation under normal physiological conditions.
Highlights
Dysregulation of the circadian rhythm is associated with many diseases, including diabetes, obesity, and cancer
From the comparison of peptide spectral matches (PSMs) between the mock and BMAL1 immunoprecipitates, we identified 24 BMAL1-interacting proteins that were potentially involved in the ubiquitin– proteasome system (UPS) (Fig. 1B)
The transcriptional activity of BMAL1 could be modulated by post-translational modifications (PTMs), such as acetylation, phosphorylation, and SUMOylation
Summary
It has been shown that BMAL1 has diverse roles in the regulation of protein synthesis, cellular senescence, reproductive ability, and cancer progression. Only a couple of enzymes in the UPS have been found to regulate BMAL1 ubiquitination, which cannot fully explain the fact that BMAL1 participates in diverse cellular processes in cells from different tissues. From the comparison of peptide spectral matches (PSMs) between the mock and BMAL1 immunoprecipitates, we identified 24 BMAL1-interacting proteins that were potentially involved in the UPS (Fig. 1B). An MS/MS spectrum of one tryptic peptide provided annotated b-ions and y-ions for its confident identification (Fig. 1C). We further validated the interaction between UBE2O and BMAL1 using four biochemical experiments. FLAG-BMAL1 was expressed without or with MycUBE2O in HEK293T cells, and FLAG-BMAL1 was immunoprecipitated for immunoblotting (Fig. 1D). 26S protease regulatory subunit 6A 26S proteasome non-ATPase regulatory subunit 12 26S proteasome non-ATPase regulatory subunit 2 26S proteasome non-ATPase regulatory subunit 3 COP9 signalosome complex subunit 6 Cullin-1 F-box-like/WD repeat-containing protein TBL1XR1 HECT, UBA and WWE domain containing 1 Isoform 2 of 26S proteasome non-ATPase regulatory subunit 1 Isoform 2 of Cullin-associated NEDD8-dissociated protein 1 Isoform 2 of Zinc finger protein 174 Isoform 3 of Zinc finger protein 207 Proteasome 26S ATPase subunit 5 variant (Fragment) proteasome 26S ATPase subunit 6 Proteasome 26S non-ATPase subunit 11 variant (Fragment) proteasome activator subunit 2 Proteasome beta 1 subunit variant (Fragment) similar to Zinc finger protein 679 Ubiquitin carboxyl-terminal hydrolase 38 Ubiquitin carboxyl-terminal hydrolase L5 Ubiquitin-conjugating enzyme E2O Ubiquitin-like modifier-activating enzyme 1 WD repeat-containing protein SAZD Zinc finger protein 44 (KOX 7), isoform CRA_a
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