FBXL3 is a gene in humans and mice that encodes the F-box/LRR-repeat protein 3 (FBXL3).[1][2]
FBXL3 is a member of the F-box protein family, which constitutes one of the four subunits in the SCFubiquitin ligase complex.[3]
The FBXL3 protein participates in the negative feedback loop responsible for generating molecular circadian rhythms in mammals by binding to the CRY1 and CRY2 proteins to facilitate their polyubiquitination by the SCF complex and their subsequent degradation by the proteasome.[4] FBXL3 also plays a role in the related loop that regulates the transcription of the BMAL1 gene.[5]
The Fbxl3 gene function was independently identified in 2007 by three groups, lead by Joseph S. Takahashi, Dr. Patrick Nolan and Michael Hastings, and Lucio Busino respectively. Takahashi used forward geneticsN-ethyl-N-nitrosourea (ENU) mutagenesis to screen for mice with varied circadian activity which led to the discovery of the Overtime (Ovtm) mutant of the Fbxl3 gene. Nolan discovered the Fbxl3 mutation After hours (Afh) by a forward screen assessing wheel activity behavior of mutagenized mice.[4] Busino discovered that the FBXL3 protein is necessary for the reactivation of the CLOCK and BMAL1 protein heterodimer by inducing the degradation of CRY proteins.[6]
Overtime
Mice with the homozygous mutation of Ovtm, free run with an intrinsic period of 26 hours. Overtime is a loss of function mutation caused by a substitution of isoleucine to threonine in the region of FBXL3 that binds to CRY. In mice with this mutation, levels of the proteins PER1 and PER2 are decreased, while levels of CRY proteins do not differ from those of wild type mice. The stabilization of CRY protein levels leads to continued repression of Per1 and Per2 transcription and translation. [4][7]
After-hours
The After-hours mutation is a substitution of cysteine to serine at position 358. Similar to Overtime, the mutation occurs in the region where FBXL3 binds to CRY. Mice homozygous for the Afh mutation have a free running period of about 27 hours. The Afh mutation delays the rate of CRY protein degradation, therefore affecting the transcription of PER2 protein. [4][8]
Fbxl21
The closest homologue to Fbxl3 is Fbxl21 as it also binds to the CRY1 and CRY2 proteins. Predominantly localized to the cytosol, Fbxl21 antagonizes the action of Fbxl3 through ubiquitination and stabilization of CRY proteins instead of leading it to degradation.[9]
Characteristics
The human FBXL3 gene is located on the long arm of chromosome 13 at position 22.3.[10] The protein is composed of 428 amino acids and has a mass of 48,707 Daltons.[11] The FBXL3 protein contains an F-box domain, characterized by a 40 amino acid motif that mediates protein-protein interactions, and several tandem leucine-rich repeats used for substrate recognition. It has eight post-translational modification sites involving ubiquitination and four sites involving phosphorylation. The FBXL3 protein is predominantly localized to the nucleus. It is one of four subunits of a ubiquitin ligase complex called SKP1-CUL1-F-box-protein, which includes the proteins CUL1, SKP1, and RBX1. [7][3]
Function
The FBXL3 protein plays a role in the negative feedback loop of the mammalian molecular circadian rhythm. The PER and CRY proteins inhibit the transcription factors CLOCK and BMAL1. The degradation of PER and CRY prevent the inhibition of the CLOCK and BMAL1 protein heterodimer. In the nucleus, the FBXL3 protein targets CRY1 and CRY2 for polyubiquitination, which triggers the degradation of the proteins by the proteasome.[4] FBXL3 binds to CRY2 by occupying its flavin adenine dinucleotide (FAD) cofactor pocket with a C-terminal tail and buries the PER-binding interface on the CRY2 protein.[12]
The FBXL3 protein is also involved in a related feedback loop that regulates the transcription of the Bmal1 gene. Bmal1 expression is regulated by the binding of REV-ERBα and RORα proteins to retinoic acid-related orphan receptor response elements (ROREs) in the Bmal1promoter region. The binding of the REV-ERBα protein to the promoter represses expression, while RORα binding activates expression.[5] FBXL3 decreases the repression of Bmal1 transcription by inactivating the REV-ERBα and HDAC3 repressor complex.[13]
The FBXL3 protein has also been found to cooperatively degrade c-MYC when bound to CRY2. The c-MYC protein is a transcription factor important in regulating cell proliferation. The CRY2 protein can function as a co-factor for the FBXL3 ligase complex and interacts with phosphorylated c-MYC. This interaction promotes the ubiquitination and degradation of the c-MYC protein. [14]
↑Cenciarelli C, Chiaur DS, Guardavaccaro D, Parks W, Vidal M, Pagano M (October 1999). "Identification of a family of human F-box proteins". Current Biology. 9 (20): 1177–9. doi:10.1016/S0960-9822(00)80020-2. PMID10531035.
↑Chiaur DS, Murthy S, Cenciarelli C, Parks W, Loda M, Inghirami G, Demetrick D, Pagano M (Jun 2000). "Five human genes encoding F-box proteins: chromosome mapping and analysis in human tumors". Cytogenetics and Cell Genetics. 88 (3–4): 255–8. doi:10.1159/000015532. PMID10828603.
↑ 5.05.1Ko CH, Takahashi JS (October 2006). "Molecular components of the mammalian circadian clock". Human Molecular Genetics. 15 Spec No 2 (Review Issue 2): R271–7. doi:10.1093/hmg/ddl207. PMID16987893.
↑Busino L, Bassermann F, Maiolica A, Lee C, Nolan PM, Godinho SI, Draetta GF, Pagano M (May 2007). "SCFFbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins". Science. 316 (5826): 900–4. doi:10.1126/science.1141194. PMID17463251.
↑ 7.07.1Siepka SM, Yoo SH, Park J, Lee C, Takahashi JS (2007). "Genetics and neurobiology of circadian clocks in mammals". Cold Spring Harbor Symposia on Quantitative Biology. 72 (1): 251–9. doi:10.1101/sqb.2007.72.052. PMID18419282.
↑Godinho SI, Maywood ES, Shaw L, Tucci V, Barnard AR, Busino L, Pagano M, Kendall R, Quwailid MM, Romero MR, O'neill J, Chesham JE, Brooker D, Lalanne Z, Hastings MH, Nolan PM (May 2007). "The after-hours mutant reveals a role for Fbxl3 in determining mammalian circadian period". Science. 316 (5826): 897–900. doi:10.1126/science.1141138. PMID17463252.
↑Hirano A, Yumimoto K, Tsunematsu R, Matsumoto M, Oyama M, Kozuka-Hata H, Nakagawa T, Lanjakornsiripan D, Nakayama KI, Fukada Y (February 2013). "FBXL21 regulates oscillation of the circadian clock through ubiquitination and stabilization of cryptochromes". Cell. 152 (5): 1106–18. doi:10.1016/j.cell.2013.01.054. PMID23452856.
↑Sato K, Yoshida K (November 2010). "Augmentation of the ubiquitin-mediated proteolytic system by F-box and additional motif-containing proteins (Review)". International Journal of Oncology. 37 (5): 1071–6. doi:10.3892/ijo_00000758. PMID20878054.
↑ 12.012.1Xing W, Busino L, Hinds TR, Marionni ST, Saifee NH, Bush MF, Pagano M, Zheng N (April 2013). "SCF(FBXL3) ubiquitin ligase targets cryptochromes at their cofactor pocket". Nature. 496 (7443): 64–8. doi:10.1038/nature11964. PMID23503662.
↑ 13.013.113.2Shi G, Xing L, Liu Z, Qu Z, Wu X, Dong Z, Wang X, Gao X, Huang M, Yan J, Yang L, Liu Y, Ptácek LJ, Xu Y (March 2013). "Dual roles of FBXL3 in the mammalian circadian feedback loops are important for period determination and robustness of the clock". Proceedings of the National Academy of Sciences of the United States of America. 110 (12): 4750–5. doi:10.1073/pnas.1302560110. PMID23471982.
↑ 14.014.114.2Huber AL, Papp SJ, Chan AB, Henriksson E, Jordan SD, Kriebs A, Nguyen M, Wallace M, Li Z, Metallo CM, Lamia KA (November 2016). "CRY2 and FBXL3 Cooperatively Degrade c-MYC". Molecular Cell. 64 (4): 774–789. doi:10.1016/j.molcel.2016.10.012. PMID27840026.
↑Cenciarelli C, Chiaur DS, Guardavaccaro D, Parks W, Vidal M, Pagano M (October 1999). "Identification of a family of human F-box proteins". Current Biology. 9 (20): 1177–9. doi:10.1016/S0960-9822(00)80020-2. PMID10531035.