USP2-45 is a circadian clock output effector regulating calcium absorption at the post- Translational level

The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs) oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2), whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output effector. We report here that Usp2 is a clock output effector related to bodily Ca²⁺ homeostasis, a feature that is conserved across evolution. Drosophila with a whole-body knockdown of the orthologue of Usp2, CG14619 (dUsp2-kd), predominantly die during pupation but are rescued by dietary Ca²⁺ supplementation. Usp2-KO mice show hyperabsorption of dietary Ca²⁺ in small intestine, likely due to strong overexpression of the membrane scaffold protein NHERF4, a regulator of the Ca²⁺ channel TRPV6 mediating dietary Ca²⁺uptake. In this tissue, USP2-45 is found in membrane fractions and negatively regulates NHERF4 protein abundance in a rhythmic manner at the protein level. In clock mutant animals (Cry1/Cry2-dKO), rhythmic USP2-45 expression is lost, as well as the one of NHERF4, confirming the inverse relationship between USP2-45 and NHERF4 protein levels. Finally, USP2-45 interacts in vitro with NHERF4 and endogenous Clathrin Heavy Chain. Taken together these data prompt us to define USP2- 45 as the first clock output effector acting at the post-translational level at cell membranes and possibly regulating membrane permeability of Ca²⁺.