Deubiquitinating enzyme USP9X regulates cellular clock function by modulating the ubiquitination and degradation of a core circadian protein BMAL1

Abstract

Living organisms on the earth maintain a roughly 24 h circadian rhythm, which is regulated by circadian clock genes and their protein products. Post-translational modifications of core clock proteins could affect the circadian behavior. Although ubiquitination of core clock proteins was studied extensively, the reverse process, deubiquitination, has only begun to unfold and the role of this regulation on circadian function is not completely understood. Here, we use affinity purification and mass spectrometry analysis to identify probable ubiquitin carboxyl-terminal hydrolase FAF-X (USP9X) as an interacting protein of the core clock protein aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL or BMAL1). Through biochemical experiments, we discover that USP9X reduces BMAL1 ubiquitination, enhances its stability, and increases its protein level, leading to the elevated transcriptional activity. Bioluminescence measurement reveals that USP9X knockdown decreases the amplitude of the cellular circadian rhythm but the period and phase are not affected. Our experiments find a new regulator for circadian clock at the post-translational level and demonstrate a different regulatory function for the circadian clock through the deubiquitination and the up-regulation of the core clock protein BMAL1 in the positive limb of the transcription-translation feedback loop.

E3 ubiquitin ligase HRD1 modulates the circadian clock through regulation of BMAL1 stability

“…BMAL1 preferentially interacts with the zinc finger domain, but not the conventional substrate recognition domain in TRAF2 (39). Additionally, ubiquitin carboxyl-terminal hydrolase FAF-X, a deubiquitinating enzyme, was found to modulate the ubiquitination and degradation of BMAL1 (40). Although several ubiquitinating and deubiquitinating enzymes have previously been identified to mediate BMAL1 degradation, it is still of interest to investigate novel E3s that influence BMAL1 protein degradation.…”

Cereblon suppresses the lipopolysaccharide-induced inflammatory response by promoting the ubiquitination and degradation of c-Jun

“…c-Jun and CRBN antibodies were used for immunoprecipitation of endogenous proteins according to a procedure described previously (59). Briefly, cell lysates were precleared with protein A/G-agarose beads and incubated with 1.6 g of control IgG, c-Jun, or CRBN antibodies at 4°C for 16 h. Prewashed protein A/G-agarose beads (40 l) were mixed with the above cell lysate and incubated at 4°C for 4 h. The protein A/G-agarose beads were washed three times with TBST and once with modified RIPA buffer, and proteins were eluted by heating the beads with 80 l of 2ϫ SDS sample loading buffer.…”

The E3 ubiquitin ligase STUB1 attenuates cell senescence by promoting the ubiquitination and degradation of the core circadian regulator BMAL1

“…Genetic knockout of Bmal1 in mice completely disrupts the circadian rhythm in constant darkness (28). Modulating BMAL1 by E2, E3, and deubiquitinase may affect the mouse circadian rhythm or cellular clock behaviors (30)(31)(32)(33). Since our experiments were carried out in HEK293T cells, which are deficient of CLOCK, we did not test the effect of STUB1 on cellular circadian behavior.…”

“…Dysregulation of circadian clock is associated with premature aging in Bmal1 knockout mice (29). Many enzymes in the UPS interact with BMAL1 and regulate its ubiquitination and degradation (30)(31)(32)(33). However, the E3 ubiquitin ligases in the upstream signaling pathways of BMAL1 and their roles in the regulation of cell senescence have not been explored.…”