SCF Ubiquitin Ligase Targets Cryptochromes at Their Cofactor Pocket

1] Department of Pharmacology, Box 357280, University of Washington, Seattle, Washington 98195, USA [2] Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA.
Nature (Impact Factor: 41.46). 03/2013; 496(7443). DOI: 10.1038/nature11964
Source: PubMed


The cryptochrome (CRY) flavoproteins act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCF(FBXL3) ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitination and degradation. However, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound and FBXL3-SKP1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Notably, the F-box protein FBXL3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved carboxy-terminal tail and burying its PER-binding interface. This novel F-box-protein-substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitination.

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Available from: Nabiha Huq Saifee, Jun 22, 2014
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    • "Interestingly, FBXL3 cannot undergo SCF complex formation in the absence of its CRY substrates (Yumimoto et al., 2013). X-ray crystallography revealed that FBXL3 binds to the FAD-binding pocket of mammalian CRY, which may also be bound by FAD or PER proteins (Czarna et al., 2013; Xing et al., 2013), which suggests a mechanism for the protection of CRYs from degradation in the presence of PER (Yagita et al., 2002). "
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    ABSTRACT: Circadian rhythms, endogenous cycles of about 24 h in physiology, are generated by a master clock located in the suprachiasmatic nucleus of the hypothalamus and other clocks located in the brain and peripheral tissues. Circadian disruption is known to increase the incidence of various illnesses, such as mental disorders, metabolic syndrome, and cancer. At the molecular level, periodicity is established by a set of clock genes via autoregulatory translation-transcription feedback loops. This clock mechanism is regulated by post-translational modifications such as phosphorylation and ubiquitination, which set the pace of the clock. Ubiquitination in particular has been found to regulate the stability of core clock components but also other clock protein functions. Mutation of genes encoding ubiquitin ligases can cause either elongation or shortening of the endogenous circadian period. Recent research has also started to uncover roles for deubiquitination in the molecular clockwork. Here, we review the role of the ubiquitin pathway in regulating the circadian clock and we propose that ubiquitination is a key element in a clock protein modification code that orchestrates clock mechanisms and circadian behavior over the daily cycle.
    Frontiers in Molecular Neuroscience 08/2014; 7:69. DOI:10.3389/fnmol.2014.00069 · 4.08 Impact Factor
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    • "Our structure also shows that binding of FBXL3 or PER2 to CRY is mutually exclusive and suggests that PER2 stabilizes CRY1 by shielding it from FBXL3 . Strikingly , neither mPER2 ( this study ) nor FBXL3 ( Xing et al . , 2013 ) binds in close proximity of the AMPK phosphorylation site Ser71 , suggesting that AMPK regulates PER2 and FBXL3 binding in a more indirect manner . Notably , Cys412 appears highly reactive in switching functions because it not only forms the intramolecular disulfide bond to Cys363 in apo - mCRY1 but also an intermolecular disul - fide"
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    ABSTRACT: Period (PER) proteins are essential components of the mammalian circadian clock. They form complexes with cryptochromes (CRY), which negatively regulate CLOCK/BMAL1-dependent transactivation of clock and clock-controlled genes. To define the roles of mammalian CRY/PER complexes in the circadian clock, we have determined the crystal structure of a complex comprising the photolyase homology region of mouse CRY1 (mCRY1) and a C-terminal mouse PER2 (mPER2) fragment. mPER2 winds around the helical mCRY1 domain covering the binding sites of FBXL3 and CLOCK/BMAL1, but not the FAD binding pocket. Our structure revealed an unexpected zinc ion in one interface, which stabilizes mCRY1-mPER2 interactions in vivo. We provide evidence that mCRY1/mPER2 complex formation is modulated by an interplay of zinc binding and mCRY1 disulfide bond formation, which may be influenced by the redox state of the cell. Our studies may allow for the development of circadian and metabolic modulators.
    Cell 05/2014; 157(5):1203-15. DOI:10.1016/j.cell.2014.03.057 · 32.24 Impact Factor
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    • "Cys414 is part of a well conserved CP dipeptide motif across CRY-proteins and phyla, indicating its importance for CRY1 protein functionality. Based on the solved crystal-structure of murine CRY2, the CP motif appears situated within a co-factor binding pocket regulating CRY1 stability through the interaction with the ubiquitin ligase complex SCFFBXL3, flavin adenine dinucleotide (FAD) and PERs [34]. Although both the catalytic function with PER proteins and repression of the CLOCK/BMAL1 complex appears unaffected by the Cys414-Ala point mutation [12], [32], circadian rhythms and cellular function (in e.g. "
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    ABSTRACT: Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3-20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1(AP)). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders.
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