Circadian proteolysis by the E3 ubiquitin ligase gp78 controls CYP3A4 activity in human hepatocytes

The pharmacokinetics and pharmacodynamics of many drugs vary with the time of administration, reflecting circadian rhythms in drug-target sensitivity and in drug concentrations at target sites. Cytochrome P450 3A4 (CYP3A4), which metabolizes approximately half of all clinically used drugs, exhibits circadian oscillations in expression and activity driven in part by transcriptional regulation via the clock output factors DBP and E4BP4. However, the amplitude of its enzymatic activity substantially exceeds that of its mRNA, implying additional post-transcriptional control, but the underlying mechanism remains largely unknown. Herein, we report that the endoplasmic reticulum-associated E3 ubiquitin ligase gp78 acts as a key regulator of circadian CYP3A4 activity in human hepatocytes. In clock-synchronized HepaRG cells, CYP3A4 protein and enzymatic activity oscillated in phase despite the absence of mRNA rhythms. Using HepaRG cells stably expressing CYP3A4 from a constitutive CMV promoter, CYP3A4 protein stability varied with the time of day, further supporting post-transcriptional control of its circadian rhythm. The protein levels of gp78 oscillated antiphasic to those of CYP3A4, and overexpression of wild-type gp78, but not its catalytically inactive RING-finger mutant, reduced CYP3A4 levels and abolished the rhythmicity, indicating that the ubiquitin ligase activity of gp78 is required for rhythmic CYP3A4 degradation. Our findings reveal gp78-mediated proteolysis as a key contributor to circadian CYP3A4 activity and provide a mechanistic basis for the dosing-time dependence of CYP3A4 substrate pharmacokinetics.