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FAD Regulates CRYPTOCHROME Protein Stability and Circadian Clock in Mice
Arisa Hirano1, Daniel Braas2, Ying-Hui Fu3
1Department of Neurology, University of California, San Francisco, CA 94143, USA.
Abstract:
The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD) binding pocket of CRYPTOCHROME2 (CRY2), a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin kinase (Rfk), an FAD biosynthetic enzyme, enhanced CRY degradation. RFK protein levels and FAD concentrations oscillate in the nucleus, suggesting that they are subject to circadian control. Knockdown of Rfk combined with a riboflavin-deficient diet altered the CRY levels in mouse liver and the expression profiles of clock and clock-controlled genes (especially those related to metabolism including glucose homeostasis). We conclude that light-independent mechanisms of FAD regulate CRY and contribute to proper circadian oscillation of metabolic genes in mammals.
Insights
Flavin adenine dinucleotide (FAD) stabilizes CRY proteins, regulating the circadian clock and metabolism. Disrupting FAD synthesis impacts CRY levels and metabolic gene expression, crucial for mammalian circadian rhythms.
Area of Science:
- Chronobiology
- Molecular Biology
- Metabolic Regulation
Background:
- The circadian clock governs metabolic and physiological rhythms, including sleep-wake cycles.
- A mutation in CRYPTOCHROME2 (CRY2) linked to advanced sleep phase highlighted the potential role of flavin adenine dinucleotide (FAD).
Purpose of the Study:
- To investigate the role of FAD in mediating circadian clock functions and metabolism.
- To understand how FAD levels influence CRY protein stability and circadian gene expression.
Main Methods:
- Examined the effect of FAD on CRY protein stability.
- Investigated the impact of Riboflavin kinase (Rfk) knockdown on CRY degradation.
- Analyzed nuclear oscillations of RFK and FAD.
- Assessed changes in CRY levels and gene expression in mouse liver under Rfk knockdown and riboflavin deficiency.
Main Results:
- FAD enhances CRY protein stability, increasing CRY levels.
- Knockdown of Rfk, an FAD biosynthetic enzyme, accelerates CRY degradation.
- RFK and FAD exhibit nuclear oscillations, indicating circadian control.
- Rfk knockdown and riboflavin deficiency alter hepatic CRY levels and disrupt circadian expression of metabolic genes, including those involved in glucose homeostasis.
Conclusions:
- FAD acts as a light-independent regulator of CRY proteins.
- FAD contributes to the proper circadian oscillation of metabolic genes in mammals.
- These findings reveal a novel link between FAD metabolism and circadian regulation of physiology.