Functional characterization of p.Ser420Phe CRY2 variant

The circadian clock is an innate mechanism that enables organisms to adapt to the rhythmic changes of the environment. In mammalian cells, this mechanism is achieved by the interaction of four core clock proteins (CRYs, PERs, CLOCK, and BMAL1). Human clock-gene variations are associated with numerous behavioral and physiological diseases, such as sleep, metabolism, addiction, neurological disorders, and endocrine and metabolic. However, little is known about the possible effects of variations at the molecular level. Currently, the functional consequence of these variations and the strength of their association with the disease remains unclear. The functional analyses of variants help us to understand the role of amino acid residues in the structure and function of proteins. In this study, rare CRY2 variants were selected from the 1000 Genomes Project, and the Ensembl database. SNPs were filtered based on the prediction of pathogenicity using in silico tools. Using the 3D structure of the CRY2 protein, the variant (p.Ser420Phe CRY2) located in the vicinity of the coiled-coil-like helix (CC helix) was functionally characterized. We have found that the p.Ser420Phe CRY2 had reduced repression activity on CLOCK/BMAL1-driven transcription and showed rescued circadian rhythm with a shorter period than wild type in Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cell line. Moreover, the variant had reduced affinity to other core clock proteins CLOCK and PER2 and could not properly localize in the nucleus using biochemical methods. Further stability results suggested that the p.Ser420Phe CRY2 variant was degraded by a noncanonical mechanism. Collectively, our results show that the CC helix region plays an important role in the degradation of CRY2, interaction with other core clock proteins, also nuclear shuttling.