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1
Chen, Wei, Sheng Xiong, Jin Li, Xiuying Li, Yuan Liu, Chunbin Zou, and Rama K. Mallampalli. "The Ubiquitin E3 Ligase SCF-FBXO24 Recognizes Deacetylated Nucleoside Diphosphate Kinase A To Enhance Its Degradation." Molecular and Cellular Biology 35, no. 6 (January 12, 2015): 1001–13. http://dx.doi.org/10.1128/mcb.01185-14.
Повний текст джерелаАнотація:
The Skp-Cul-F box (SCF) ubiquitin E3 ligase machinery recognizes predominantly phosphodegrons or, less commonly, an (I/L)Q molecular signature within substrates to facilitate their recruitment in mediating protein ubiquitination and degradation. Here, we examined the molecular signals that determine the turnover of the multifunctional enzyme nucleoside diphosphate kinase A (NDPK-A) that controls cell proliferation. NDPK-A protein exhibits a half-life of ∼6 h in HeLa cells and is targeted for ubiquitylation through actions of the F-box protein FBXO24. SCF-FBXO24 polyubiquitinates NDPK-A at K85, and two NH 2 -terminal residues, L55 and K56, were identified as important molecular sites for FBXO24 interaction. Importantly, K56 acetylation impairs its interaction with FBXO24, and replacing K56 with Q56, an acetylation mimic, reduces NDPK-A FBXO24 binding capacity. The acetyltransferase GCN5 catalyzes K56 acetylation within NDPK-A, thereby stabilizing NDPK-A, whereas GCN5 depletion in cells accelerates NDPK-A degradation. Cellular expression of an NDPK-A acetylation mimic or FBXO24 silencing increases NDPK-A life span which, in turn, impairs cell migration and wound healing. We propose that lysine acetylation when presented in the appropriate context may be recognized by some F-box proteins as a unique inhibitory molecular signal for their recruitment to restrict substrate degradation.
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Yuan, Lamei, Zhi Song, Xiong Deng, Zhijian Yang, Yan Yang, Yi Guo, Hongwei Lu, and Hao Deng. "Genetic Analysis of FBXO2, FBXO6, FBXO12, and FBXO41 Variants in Han Chinese Patients with Sporadic Parkinson’s Disease." Neuroscience Bulletin 33, no. 5 (March 24, 2017): 510–14. http://dx.doi.org/10.1007/s12264-017-0122-5.
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Chen, Wei, Denghui Gao, Long Xie, Anling Wang, Hui Zhao, Chaowan Guo, Yunqi Sun, Yanfeng Nie, An Hong, and Sheng Xiong. "SCF-FBXO24 regulates cell proliferation by mediating ubiquitination and degradation of PRMT6." Biochemical and Biophysical Research Communications 530, no. 1 (September 2020): 75–81. http://dx.doi.org/10.1016/j.bbrc.2020.06.007.
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Zhang, Yuan-Meng, Ling-Bing Meng, Si-Jun Yu, and Dong-Xing Ma. "Identification of potential crucial genes in monocytes for atherosclerosis using bioinformatics analysis." Journal of International Medical Research 48, no. 4 (April 2020): 030006052090927. http://dx.doi.org/10.1177/0300060520909277.
Повний текст джерелаАнотація:
Objective To use bioinformatics tools to screen for gene biomarkers from monocytes, which play an important role in the pathogenesis of atherosclerosis. Methods Two expression profiling datasets (GSE27034 and GSE10195) were obtained from the Gene Expression Omnibus dataset and the differentially expressed genes (DEGs) between atherosclerotic human peripheral blood mononuclear cells (PBMC) samples and control subjects were screened using GEO2R. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted for the DEGs. STRING and MCODE plug-in of Cytoscape were used for constructing a protein–protein interaction network and analysing hub genes. Results The two datasets had 237 DEGs in common between non-atherosclerotic- and atherosclerotic PBMC samples. Functional annotation demonstrated that these DEGs were mainly enriched in protein binding, positive regulation of transcription from RNA polymerase II promoter, nucleus and viral carcinogenesis. Five hub genes, FBXL4, UBOX5, KBTBD6, FZR1 and FBXO2, were identified. Conclusion This present bioinformatics analysis identified that the FBXL4, UBOX5, KBTBD6 and FBXO21 genes might play vital roles in the pathogenesis of atherosclerosis. These four genes might represent new biomarkers for the diagnosis and treatment of atherosclerosis.
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Angeli, Franca, Russell Wyborski, Bill Chen, Rama Mallampalli, and Michael Lark. "P157 FBXO3-FBXL2 AXIS MODULATORS AS A NOVEL CLASS OF ORAL SMALL MOLECULE COMPOUNDS FOR THE TREATMENT OF CROHN’S DISEASE." Inflammatory Bowel Diseases 26, Supplement_1 (January 2020): S6. http://dx.doi.org/10.1093/ibd/zaa010.014.
Повний текст джерелаАнотація:
Abstract Background Ubiquitination is a common post-translational modification, tagging proteins for degradation. The ubiquitin proteasome system is activated in Crohn’s Disease (CD), and modulation of its components might be a novel strategy for therapeutic intervention to control inflammation. The conjugation of ubiquitin to a target protein is orchestrated by a series of enzymatic reactions, the last step being catalyzed by a selective ubiquitin E3 ligase. Among ubiquitin E3 ligases, Fbxl2 serves as a sentinel gatekeeper to limit inflammation by targeting and enhancing the degradation of tumor necrosis factor receptor associated factors (TRAF) proteins, which link cell surface signals (through NFkB signaling) to cytokine secretion. In addition, Fbxl2 controls the ubiquitination of the inflammasome, NOD-like receptor protein 3 (NLRP3), which mediates the release of interleukin (IL)1β and IL18 (Fig.1). Fbxl2 protein itself is ubiquitinated and degraded by another protein, called Fbxo3. Inhibition of Fbxo3 results in increased Fbxl2 levels and decreased TRAF and NLRP3. Individuals with a natural occurring polymorphism within Fbxo3V221Ihave decreased lipopolysaccharide(LPS)-induced cytokine (TNFα, IL1β & IL6) production, Fbxo3 and TRAF levels and increased Fbxl2, providing human genetic target validation for Fbxo3-Fbxl2 axis modulation in inflammatory conditions. BC1261 is a first-in-class, selective, orally available Fbxo3 inhibitor in clinical development for the treatment of CD. Methods and Results In vitro data demonstrated that BC1261 binds to Fbxo3, prevents Fbxo3/Fbxl2 association and produces a concentration-dependent inhibition of LPS-induced release of TNFα and IL1β from human peripheral blood mononuclear cells. In an acute DSS-mouse model, administration of BC-1261 via drinking water ad libitum (30 μg/ml) or by daily intraperitoneal (IP) injection (150 μg) for 5 days resulted in the attenuation of the shortening of colonic length, tissue damage (Fig2.A), and TNFα and IL6 tissue levels (Fig.2B). In a repeated DSS-mouse model, BC-1261 (10 mg/kg bid) given orally for 19 days was comparable to the positive control (anti-p40) in the DAI composite (Fig2.C), stool consistency (Fig2.D), and histopathology score in the proximal colon (Fig2.E). BC-1261 also lead to clinically relevant reductions on endoscopy score (Fig2.F). Conclusion BC1261, a Fbxo3 inhibitor, was efficacious in both acute and chronic preclinical models of colitis. Taken together, these results suggest the potential utility of selective Fbxo3-Fbxl2 modulation in the treatment of CD.
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Masle-Farquhar, Etienne, Amanda Russell, Yangguang Li, Fen Zhu, Lixin Rui, Robert Brink, and Christopher C. Goodnow. "Loss-of-function of Fbxo10, encoding a post-translational regulator of BCL2 in lymphomas, has no discernible effect on BCL2 or B lymphocyte accumulation in mice." PLOS ONE 16, no. 4 (April 29, 2021): e0237830. http://dx.doi.org/10.1371/journal.pone.0237830.
Повний текст джерелаАнотація:
Regulation of the anti-apoptotic BCL2 protein determines cell survival and is frequently abnormal in B cell lymphomas. An evolutionarily conserved post-translational mechanism for over-expression of BCL2 in human B cell lymphomas and the BCL2 paralogue CED-9 in Caenorhabditis elegans results from loss-of-function mutations in human FBXO10 and its C.elegans paralogue DRE-1, a BCL2/CED-9-binding subunit of the SKP-CULLIN-FBOX (SCF) ubiquitin ligase. Here, we tested the role of FBXO10 in BCL2 regulation by producing mice with two different CRISPR/Cas9-engineered Fbxo10 mutations: an Asp54Lys (E54K) missense mutation in the FBOX domain and a Cys55SerfsTer55 frameshift (fs) truncating mutation. Mice homozygous for either mutant allele were born at the expected Mendelian frequency and appeared normal in body weight and appearance as adults. Spleen B cells from homozygous mutant mice did not have increased BCL2 protein, nor were the numbers of mature B cells or germinal centre B cells increased as would be expected if BCL2 was increased. Other lymphocyte subsets that are also regulated by BCL2 levels also displayed no difference in frequency in homozygous Fbxo10 mutant mice. These results support one of two conclusions: either FBXO10 does not regulate BCL2 in mice, or it does so redundantly with other ubiquitin ligase complexes. Possible candidates for the latter include FBXO11 or ARTS-XIAP. The difference between the role of FBXO10 in regulating BCL2 protein levels in C. elegans and in human DLBCL, relative to single-gene deficient mouse leukocytes, should be further investigated.
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Manfiolli, Adriana O., Ana Leticia G. C. Maragno, Munira M. A. Baqui, Sami Yokoo, Felipe R. Teixeira, Eduardo B. Oliveira, and Marcelo D. Gomes. "FBXO25-associated Nuclear Domains: A Novel Subnuclear Structure." Molecular Biology of the Cell 19, no. 5 (May 2008): 1848–61. http://dx.doi.org/10.1091/mbc.e07-08-0815.
Повний текст джерелаАнотація:
Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.
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Guo, Fengjie, Xiaoyu Jiang, Domenico Roberti, Lixin Rui, and Izidore S. Lossos. "FBXO10 Targets HGAL for Degradation." Blood 126, no. 23 (December 3, 2015): 3904. http://dx.doi.org/10.1182/blood.v126.23.3904.3904.
Повний текст джерелаАнотація:
Abstract Expression of the Human Germinal center Associated Lymphoma (HGAL) gene is restricted to germinal center (GC) B-lymphocytes and GC-derived lymphomas. HGAL expression identifies lymphomas characterized by a better prognosis. We previously showed that HGAL is a unique adaptor protein that regulates both cell motility and B-cell receptor (BCR) signaling, processes that are central for successful completion of the GC reaction. In our previous studies we demonstrated that upon BCR activation HGAL binds to and increases Syk kinase activity, resulting in enhanced BCR signaling. Syk induces HGAL phosphorylation, leading to its redistribution from lipid rafts to the bulk membrane and cytoplasm, followed by its rapid degradation. The mechanism of HGAL degradation is currently unknown. To elucidate the mechanism of HGAL degradation, we hypothesized that HGAL is degraded by a SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complex. Consequently, we examined the ability of HGAL to be recruited to the SCF by screening a panel of F-box proteins transfected into HEK293T cells by reciprocal coimmunoprecipitations (Co-IPs). These studies revealed that HGAL specifically bound only FBXO10. Concordantly, HGAL Co-IPed with endogenous SKP1 and CUL1 in the presence of FBXO10. Furthermore, a dose dependent reduction of HGAL levels was observed in Raji cells transfected with increasing quantities of FBXO10 expressing plasmid. In contrast, none of the other FBXO11 tested F-box proteins affected HGAL levels. The reduction in HGAL protein level by FBXO10 was due to enhanced proteolysis, as demonstrated by the decrease in HGAL protein half-life and the rescue of its levels by either addition of proteasome inhibitor MG132 or expression of a dominant-negative CUL1 mutant (CUL1 (1-242)). Furthermore, depletion of FBXO10 by two short hairpin RNA (shRNA) constructs in Raji cells increased HGAL stability and half-life. SCF complexes mediates protein degradation by polyubiquitination. We therefore examined if HGAL protein can be ubiquinated. To this end we cotransfected 293T cells with HGAL-V5, FBXO10-FLAG, HA-ubiquitin with or without CUL1 (1-242) and blotted immunoprecipitated HGAL for ubiquitin. FBXO10 expression markedly increased HGAL ubiquitination and this was significantly decreased in cells coexpressing CUL1 (1-242). Similarly, HGAL ubiquitination was markedly decreased in cells expressing FBXO10 (FBXO10- ΔF-box), in which the F-box motif, necessary for linking the target protein to the SCF ubiquitin ligase moieties, is deleted. These results support the function of FBXO10 in binding HGAL and facilitating its ubiquitination by the SCF complex prior to its degradation. We next analyzed the functional significance of HGAL degradation by FBXO10 on BCR activation, as measured by intracellular and transmembrane Ca2+ mobilization. Overexpression of FBXO10 in Raji cells expressing endogenous HGAL led to decreased levels of HGAL and in Ca2+ mobilization. In contrast, FBXO10 did not affect BCR-induced Ca2+ mobilization in Raji cells in which HGAL was deleted by crispr/Cas9 targeting. These results suggest that FBXO10 affects BCR signaling via regulating HGAL levels. While FBXO10 mediates HGAL degradation post BCR-induced HGAL phosphorylation, FBXO10 also bound HGAL in the absence of BCR activation. The binding of FBXO10 to HGAL was unaffected in Raji cells incubated with anti-IgM antibodies. Treatment of cellular lysates with l-phosphatase did not affect FBXO10 binding to HGAL. These findings indicate that FBXO10-dependent degradation of HGAL is phosphorylation independent. In contrast, HGAL degradation by FBXO10 was dependent on HGAL lipid raft localization, since HGAL myristoylation and palmitoylation mutants (G2A, C43A/C45A, and G2A/C43A/C45A) exhibited decreased Co-IP with the FBXO10 protein. To further map FBXO10 binding motifs of HGAL, we generated HGAL deletion and mutant constructs and examined their ability to Co-IP with FBXO10. These studies revealed that HGAL amino acids 91-95(HRVLC) mediate HGAL binding to FBXO10. In summary, our results demonstrate that SCFFBXO10 is the ubiquitin ligase for HGAL and regulates BCR signaling via controlling HGAL expression. Some diffuse large cell lymphomas expressing HGAL harbor inactivating mutations or express low levels of FBXO10, thus predisposing to enhanced BCR signaling- a phenomenon implicated in lymphomagenesis. Disclosures No relevant conflicts of interest to declare.
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Qie, Shuo. "The E3 Ubiquitin Ligase Fbxo4 Functions as a Tumor Suppressor: Its Biological Importance and Therapeutic Perspectives." Cancers 14, no. 9 (April 25, 2022): 2133. http://dx.doi.org/10.3390/cancers14092133.
Повний текст джерелаАнотація:
Fbxo4, also known as Fbx4, belongs to the F-box protein family with a conserved F-box domain. Fbxo4 can form a complex with S-phase kinase-associated protein 1 and Cullin1 to perform its biological functions. Several proteins are identified as Fbxo4 substrates, including cyclin D1, Trf1/Pin2, p53, Fxr1, Mcl-1, ICAM-1, and PPARγ. Those factors can regulate cell cycle progression, cell proliferation, survival/apoptosis, and migration/invasion, highlighting their oncogenic or oncogene-like activities. Therefore, Fbxo4 is defined as a tumor suppressor. The biological functions of Fbxo4 make it a potential candidate for developing new targeted therapies. This review summarizes the gene and protein structure of Fbxo4, the mechanisms of how its expression and activity are regulated, and its substrates, biological functions, and clinicopathological importance in human cancers.
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Qie, Shuo. "The E3 Ubiquitin Ligase Fbxo4 Functions as a Tumor Suppressor: Its Biological Importance and Therapeutic Perspectives." Cancers 14, no. 9 (April 25, 2022): 2133. http://dx.doi.org/10.3390/cancers14092133.
Повний текст джерелаАнотація:
Fbxo4, also known as Fbx4, belongs to the F-box protein family with a conserved F-box domain. Fbxo4 can form a complex with S-phase kinase-associated protein 1 and Cullin1 to perform its biological functions. Several proteins are identified as Fbxo4 substrates, including cyclin D1, Trf1/Pin2, p53, Fxr1, Mcl-1, ICAM-1, and PPARγ. Those factors can regulate cell cycle progression, cell proliferation, survival/apoptosis, and migration/invasion, highlighting their oncogenic or oncogene-like activities. Therefore, Fbxo4 is defined as a tumor suppressor. The biological functions of Fbxo4 make it a potential candidate for developing new targeted therapies. This review summarizes the gene and protein structure of Fbxo4, the mechanisms of how its expression and activity are regulated, and its substrates, biological functions, and clinicopathological importance in human cancers.
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Wong, Siau Yen, Peiran Lu, Lei Wu, Mckale Montgomery, Winyoo Chowanadisai, Edralin Lucas, Brenda Smith, Stephen Clarke, and Dingbo Lin. "Hepatic F-box Only Protein 2 (FBXO2) Might Not Mediate Glucose Homeostasis in Obese Diabetic Mice." Current Developments in Nutrition 4, Supplement_2 (May 29, 2020): 1281. http://dx.doi.org/10.1093/cdn/nzaa058_039.
Повний текст джерелаАнотація:
Abstract Objectives F-box only protein 2 (FBXO2) is an E3 ubiquitin protein ligase highly expressed in the brain and cochlea, hepatic FBXO2 protein levels are very low in healthy mice. It was reported that hepatic FBXO2 elevation causes hyperglycemia in obese mice. Here, we investigated whether hepatic FBXO2 expression was enhanced in diabetic human samples; and if so, whether deficiency of FBXO2 could improve blood glucose control in diet-induced obese diabetic mice. Methods Human liver specimen samples from healthy and type 2 diabetic human subjects were subject to transcriptomics and proteomics. The results were confirmed by real-time QPCR and immunoblotting, respectively. Male FBXO2 whole-body knockout (KO) and C57BL/6 J mice (WT, genetic background) at 6 weeks of age were fed a high fat (26% kCal)/high sucrose (15% kCal)/high fructose (15% kCal) diet for 8 weeks. Food intake and body weight gain were monitored weekly. Glucose tolerance and insulin tolerance tests were performed at the 7th week of dietary intervention. At the termination of intervention, mice were euthanized, and blood and liver tissues were collected for further assessment. Primary mouse embryonic fibroblasts (MEF) cells were isolated from WT and KO mice. Mitochondrial superoxide was detected by MitoSOX live cell staining. Results Transcriptomics and proteomics results showed vast shifts in pathways involved in energy metabolism and mitochondrial function, and significant elevation of FBXO2 expression at mRNA and protein levels in diabetic human livers. However, deficiency of FBXO2 did not alter mitochondrial superoxide production in MEF cells and did not cause an elevation of the fasting blood glucose level before a diet feeding. Moreover, both strains had significantly elevated fasting blood glucose levels after the high-fat diet intervention but did not differ by strains. The insulin sensitivity had no significant difference between strains after diet intervention. Conclusions The results suggested that FBXO2 depletion did not alter glucose homeostasis in diet-induced obese mouse models, though diabetic humans had a highly elevated hepatic FBXO2 level. Funding Sources N/A.
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Chang, Shu-Chun, Chin-Sheng Hung, Bo-Xiang Zhang, Tsung-Han Hsieh, Wayne Hsu, and Jeak Ling Ding. "A Novel Signature of CCNF-Associated E3 Ligases Collaborate and Counter Each Other in Breast Cancer." Cancers 13, no. 12 (June 8, 2021): 2873. http://dx.doi.org/10.3390/cancers13122873.
Повний текст джерелаАнотація:
Breast cancer (BRCA) malignancy causes major fatalities amongst women worldwide. SCF (Skp1-cullin-F-box proteins) E3 ubiquitin ligases are the most well-known members of the ubiquitination–proteasome system (UPS), which promotes cancer initiation and progression. Recently, we demonstrated that FBXL8, a novel F-box protein (SCFF-boxes) of SCF E3 ligase, accelerates BRCA advancement and metastasis. Since SCFF-boxes is a key component of E3 ligases, we hypothesized that other SCFF-boxes besides FBXL8 probably collaborate in regulating breast carcinogenesis. In this study, we retrospectively profiled the transcriptome of BRCA tissues and found a notable upregulation of four SCFF-box E3 ligases (FBXL8, FBXO43, FBXO15, and CCNF) in the carcinoma tissues. Similar to FBXL8, the knockdown of FBXO43 reduced cancer cell viability and proliferation, suggesting its pro-tumorigenic role. The overexpression of CCNF inhibited cancer cell progression, indicating its anti-tumorigenic role. Unexpectedly, CCNF protein was markedly downregulated in BRCA tissues, although its mRNA level was high. We showed that both E3 ligases, FBXL8 and FZR1, pulled down CCNF. Double knockdown of FBXL8 and FZR1 caused CCNF accumulation. On the other hand, CCNF itself pulled down a tumorigenic factor, RRM2, and CCNF overexpression reduced RRM2. Altogether, we propose a signature network of E3 ligases that collaboratively modulates CCNF anti-cancer activity. There is potential to target BRCA through modulation of the partnership axes of (i) CCNF-FBXL8, (ii) CCNF-FZR1, and (iii) CCNF-RRM2, particularly, via CCNF overexpression and activation and FBXL8/FZR1 suppression.
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Lee, Eric K., Zhaorui Lian, Kurt D'Andrea, Richard Letrero, WeiQi Sheng, Shujing Liu, J. Nathaniel Diehl, et al. "The FBXO4 Tumor Suppressor Functions as a Barrier to BrafV600E-Dependent Metastatic Melanoma." Molecular and Cellular Biology 33, no. 22 (September 9, 2013): 4422–33. http://dx.doi.org/10.1128/mcb.00706-13.
Повний текст джерелаАнотація:
Cyclin D1–cyclin-dependent kinase 4/6 (CDK4/6) dysregulation is a major contributor to melanomagenesis. Clinical evidence has revealed that p16INK4A, an allosteric inhibitor of CDK4/6, is inactivated in over half of human melanomas, and numerous animal models have demonstrated that p16INK4Adeletion promotes melanoma. FBXO4, a specificity factor for the E3 ligase that directs timely cyclin D1 proteolysis, has not been studied in melanoma. We demonstrate that Fbxo4 deficiency induces Braf-driven melanoma and that this phenotype depends on cyclin D1 accumulation in mice, underscoring the importance of this ubiquitin ligase in tumor suppression. Furthermore, we have identified a substrate-binding mutation,FBXO4I377M, that selectively disrupts cyclin D1 degradation while preserving proteolysis of the other known FBXO4 substrate, TRF1. The I377M mutation and Fbxo4 deficiency result in nuclear accumulation of cyclin D1, a key transforming neoplastic event. Collectively, these data provide evidence that FBXO4 dysfunction, as a mechanism for cyclin D1 overexpression, is a contributor to human malignancy.
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Dhar, Ruby, Goura Kishor Rath, and Subhradip Karmakar. "FBXO4 as a novel ubiquitin ligase that targets Cyclin D in the pathogenesis of breast cancer." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e14722-e14722. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e14722.
Повний текст джерелаАнотація:
e14722 Background: Members of ubiquitin ligase family are reported to be associated with cancer. Genome wide RNAi screen using Dharmacon on target Plus RNAi library identified FBXO4 as one of the potential hit . We therefore aimed to study its detailed role on proliferation of MCF7 breast cancer cells with special focus on cell cycle genes. We want to expand it further and study its involvement in context to E2 signaling in breast cancer. We hypothesized that increase in CCND1 expression, could mimic some of the E2 transcriptional regulation and that CCND1 overexpression could lead to E2 independent ER alpha binding to estrogen response element (ERE). Methods: Cell lines: MCF7 grown in MEM alpha supplemented with 10% FBS . For stimulation studies, cells were maintained in phenol free MEM alpha supplemented with 10% FBS and 10 nM estradiol. RNAi knockdown studies: On-target plus siRNA were used to knockdown FBXO4 in MCF7 cells. Western Blot: Western Blot was performed following standard protocols. IHC/Tissue Array: Paraffin embedded malignant breast cancer (ER/PR/EGFR +ve) tissue samples that are scored by pathologist were used for the study. Gene Expression Studies: Microarray and NGS based approach. Results: FBXO4 has been identified as a potential hit in our RNAi screen showing a hyper-proliferative phenotype in MCF7 cellsupon its knockdown. Western blot and ubiquitination assay identified cyclin D (CCND1) as a dedicated FBXO4 target as revealed by confocal microscopy where FBXO4 knockdown showed an enrichment of CCND1 in cytoplasm. Ingenuity pathway analysis and ChIP assay revealed an exploitation of E2 signaling and enhanced recruitment of ER alpha to estrogen response element (ERE) in FBXO4 depleted cells. CCND1 further seems to co-occupy ESR binding motifs thereby representing ER functions. These effects were observed even in absence of any E2 stimulation. Further we observed a significant overlap of differentially expressed genes in E2 stimulated and FBXO4 knockdown cells that were nullified by CCND1 siRNA. Conclusions: Our study elucidated that CCND1 is spared from degradation following loss of FBXO4 which in turn leads to a hyper-proliferative state. Our findings co-related with TCGA and METABRIC databases indicating a poor patient survival and relapse in FBXO4 low and CCND1 high cohorts of ER +ve breast cancer thereby implying a diagnostic value. Along with traditional hormonal therapy, an effective targeted therapy in breast cancer should be aimed to restore FBXO4 levels.
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Lin, T. B., M. C. Hsieh, C. Y. Lai, J. K. Cheng, Y. P. Chau, T. Ruan, G. D. Chen, and H. Y. Peng. "Fbxo3-Dependent Fbxl2 Ubiquitination Mediates Neuropathic Allodynia through the TRAF2/TNIK/GluR1 Cascade." Journal of Neuroscience 35, no. 50 (December 16, 2015): 16545–60. http://dx.doi.org/10.1523/jneurosci.2301-15.2015.
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Akiyama, Mari. "Roles of Two F-Box Proteins: FBXL14 in the Periosteum and FBXW2 at Elastic Fibers." Osteology 3, no. 1 (January 5, 2023): 1–10. http://dx.doi.org/10.3390/osteology3010001.
Повний текст джерелаАнотація:
I previously reported that F-box/leucine-rich repeat protein 14 (FBXL14) expressed in periosteum-derived cells, and F-box and WD-40 domain-containing protein 2 (FBXW2) in the periosteum form a fiber-like structure. Here, two culture medium conditions, that is, media with and without ascorbic acid, were compared during explant culture. In the absence of ascorbic acid, the expression patterns of osteocalcin, FBXW2, and elastin were compared using fluorescent immunostaining during weeks 3–5. By observing the periosteum, cambium layer and bone, I demonstrated FBXL14 expression in micro-vessels and bone lacuna. Fluorescent immunostaining revealed that, without ascorbic acid, the FBXL14 layer was thin. Conversely, in the presence of ascorbic acid, FBXL14 formed a thick membrane-like structure inside the periosteum, and the multilayer of periosteum-derived cells (PDCs) was strong. The expression patterns of osteocalcin and FBXW2 were similar. Elastin retained its fiber structure for up to five weeks. Although osteocalcin and FBXW2 were expressed in regions similar to elastin, they could not retain their fiber structures. In conclusion, FBXL14 appears to play a role in preparing a native scaffold for forming a multilayered sheet of PDCs inside the periosteum. FBXW2 and osteocalcin appear to separate from elastic fibers during calcification.
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Selander, Erik, Jan Heuschele, and Ann I. Larsson. "Hydrodynamic properties and distribution of bait downstream of a zooplankton trap." Journal of Plankton Research 39, no. 6 (May 2, 2017): 1020–27. http://dx.doi.org/10.1093/plankt/fbx024.
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Yoshida, Yukiko, Sayaka Yasuda, Toshiharu Fujita, Maho Hamasaki, Arisa Murakami, Junko Kawawaki, Kazuhiro Iwai, et al. "Ubiquitination of exposed glycoproteins by SCFFBXO27 directs damaged lysosomes for autophagy." Proceedings of the National Academy of Sciences 114, no. 32 (July 25, 2017): 8574–79. http://dx.doi.org/10.1073/pnas.1702615114.
Повний текст джерелаАнотація:
Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy.
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Zhou, Huijun, Chong Zeng, Jie Liu, Haijun Luo, and Wei Huang. "F-Box Protein 43, Stabilized by N6-Methyladenosine Methylation, Enhances Hepatocellular Carcinoma Cell Growth and Invasion via Promoting p53 Degradation in a Ubiquitin Conjugating Enzyme E2 C -Dependent Manner." Cancers 15, no. 3 (February 2, 2023): 957. http://dx.doi.org/10.3390/cancers15030957.
Повний текст джерелаАнотація:
The roles of F-box protein 43 (FBXO43) in carcinogenesis have been rarely revealed. The present study investigates the expression, function, and underlying mechanism of FBXO43 in hepatocellular carcinoma (HCC). Firstly, the expression and clinical significance of FBXO43 in HCC were investigated bioinformatically and experimentally using online omics data and local tissue samples. The role of N6-methyladenosine modification (m6A) of mRNA in regulating FBXO43 expression and the effects of m6A/FBXO43 axis alteration on cell proliferation and invasion were investigated further. Moreover, the underlying mechanism of the oncogenic FBXO43 was also explored. The results demonstrated that FBXO43 was significantly upregulated in HCC and was positively correlated with advanced progression and poor prognosis in patients. METTL3 and IGF2BP2 expressions were positively correlated with FBXO43 expression and served as the writer and reader of FBXO43 m6A, respectively, which stabilized and upregulated FBXO43 mRNA in HCC. FBXO43 silencing significantly reduced cell proliferation and invasion, and ectopic expression of FBXO43 could significantly restore the inhibitory effects caused by METTL3 and IGF2BP2 depletion in HCC cells. Mechanistically, FBXO43 depletion reduced the expression of UBE2C, a p53 ubiquitin-conjugating enzyme, suppressed proteasomal degradation of p53, and thus inhibited cell proliferation and invasion in HCC. In summary, the present study revealed that METTL3/IGF2BP2 mediated m6A contributed to the upregulation of FBXO43 that promoted the malignant progression of HCC by stimulating p53 degradation in a UBE2C-dependent manner, highlighting the promising application of FBXO43 as a target in HCC treatment.
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Deng, Chao, Hongzhi Li, and Qingmin Li. "F-box protein 17 promotes glioma progression by regulating glycolysis pathway." Bioscience, Biotechnology, and Biochemistry 86, no. 4 (January 19, 2022): 455–63. http://dx.doi.org/10.1093/bbb/zbac008.
Повний текст джерелаАнотація:
ABSTRACT F-box protein 17 (FBXO17) is associated with high-grade glioma and acted as a promotor of glioma development. This study investigated the effect and underlying pathway of FBXO17 on glioma. The Cancer Genome Atlas database was applied to analyze FBXO17 expression information in glioma. First, high FBXO17 expressions are associated with glioma and poor prognosis. Then, FBXO17 was upregulated in glioma cells. Meanwhile, knock-down of FBXO17 inhibited cell proliferation, migration, and invasion, but increased the cell apoptosis. Besides, knock-down of FBXO17 inhibited mitochondrial membrane potential and increased reactive oxygen species. Furthermore, knock-down of FBXO17 decreased level of adenosine triphosphate, glucose, lactate, GLUT1, HK2, PFKP, PKM2, and LDHA. In conclusion, FBXO17 was high expression in glioma, and FBXO17 regulates glioma by regulating glycolysis pathway, providing novel theoretical for the treatment of glioma.
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Mo, Angela, Linda Ya-Ting Chang, Gerben Duns, Xuan Wang, Gregg Morin, Tammy Lau, Rod Docking, et al. "Elucidating the Mechanisms of Leukemogenesis Driven By FBXO11 Depletion." Blood 138, Supplement 1 (November 5, 2021): 3328. http://dx.doi.org/10.1182/blood-2021-145384.
Повний текст джерелаАнотація:
Abstract Mutations in SKP1 and CUL1 (Zhang et. al. Oncol Lett 2018), which encode components of the SKP1-CUL1-F-BOX (SCF) ubiquitin E3-ligase complex, have previously been reported or characterized in AML. FBXO11, which encodes the substrate recognizing component, however, has not been studied in AML. We performed whole exome sequencing and RNA-seq on140 clinical AML samples and identified recurrent inactivating mutations in FBXO11. Of the components of the SCF FBXO11 complex, FBXO11 transcript expression is most significantly reduced in AML samples compared to normal. We show that loss of FBXO11 drives leukemogenesis through dysregulation of the novel target, LONP1, by reducing mitochondrial potential and promoting self-renewal. We found that UPS mutations co-occur with AML1-ETO (RUNX1-RUNX1T1) fusions and RAS mutations. Fbxo11 knockdown in mouse hematopoietic stem/progenitor cells (HSPC) cooperated with AML1-ETO to generate serially transplantable AML in mice. FBXO11 depletion in human cord-blood derived CD34+ cells (CD34+ CB), combined with AML1-ETO and a KRAS mutant, promoted stem cell maintenance and myeloid malignancy in a human xenotransplant model. Mass spectrometry analysis of FLAG-FBXO11 co-immunoprecipitating proteins in K562 cells identified mitochondrial protease, LONP1, as a top target. LONP1 protein expression did not vary with FBXO11 loss or overexpression, suggesting that LONP1 is not a degradation target of the SCF FBXO11complex. Knockdown of either FBXO11 or LONP1 resulted in myeloid bias in CD34+ CB in vitro, pointing to an activating role of FBXO11 on LONP1. Both FBXO11 and LONP1 depletion reduced mitochondrial membrane potential (MMP) in CD34+ CB and myeloid cell lines, aligning with the stemness phenotypes observed with FBXO11 depletion, as long-term hematopoietic stem cells (LT-HSCs) are characterized by low MMP (Mansell et. al. Cell Stem Cell 2021), and disruption of MMP promotes self-renewal in HSCs (Vannini et. al. Nat Commun 2016). As FBXO11 neddylates p53 to regulate transcription (Abida et. al. J. Biol. Chem 2007), we examined protein neddylation, and detected increased neddylation in immunoprecipitated LONP1 from FLAG-FBXO11-expressing K562 cells. As, neddylation regulates protein activation (Wu et. al. Nature 2005), our findings suggest that FBXO11 neddylation of LONP1 activates LONP1 to maintain mitochondrial function. Consequently, loss of FBXO11 function primes HSPC for self-renewal by reduction of MMP. To clarify the regulatory relationship between FBXO11 and LONP1, we performed RNA-seq on CD34+ CB cells expressing combinations of shRNAs targeting FBXO11 or LONP1, with overexpression of FLAG -FBXO11 or LONP1. Unsupervised clustering revealed that LONP1-overexpressing samples clustered with controls, suggesting that LONP1 requires modification by FBXO11 for functional effects. Using gene set enrichment analysis, we found that both FBXO11 and LONP1 depletion enriched for HSC and LSC (leukemic stem cell) gene sets. Knockdown of LONP1 reversed the effect of FLAG-FBXO11 overexpression, supporting a model of LONP1 being a downstream mediator of FBXO11 function. Both FBXO11 and LONP1 depletion enriched for a gene set composed of mitochondrial electron transport chain complex (ETC) genes, potentially reflecting a transcriptional response to loss of functional ETC activity, as suggested by accumulation of misfolded ETC proteins with knockdown of LONP1 (Ghosh et. al. Oncogene 2019). In this work, we demonstrate the leukemogenic effects of FBXO11 loss. We draw a novel connection between the UPS and the mitochondrial protease system with the identification of LONP1 as an FBXO11 target that regulates hematopoiesis. Disclosures No relevant conflicts of interest to declare.
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Habel, Nadia, Najla El-Hachem, Frédéric Soysouvanh, Hanene Hadhiri-Bzioueche, Serena Giuliano, Sophie Nguyen, Pavel Horák, et al. "FBXO32 links ubiquitination to epigenetic reprograming of melanoma cells." Cell Death & Differentiation 28, no. 6 (January 18, 2021): 1837–48. http://dx.doi.org/10.1038/s41418-020-00710-x.
Повний текст джерелаАнотація:
AbstractUbiquitination by serving as a major degradation signal of proteins, but also by controlling protein functioning and localization, plays critical roles in most key cellular processes. Here, we show that MITF, the master transcription factor in melanocytes, controls ubiquitination in melanoma cells. We identified FBXO32, a component of the SCF E3 ligase complex as a new MITF target gene. FBXO32 favors melanoma cell migration, proliferation, and tumor development in vivo. Transcriptomic analysis shows that FBXO32 knockdown induces a global change in melanoma gene expression profile. These include the inhibition of CDK6 in agreement with an inhibition of cell proliferation and invasion upon FBXO32 silencing. Furthermore, proteomic analysis identifies SMARC4, a component of the chromatin remodeling complexes BAF/PBAF, as a FBXO32 partner. FBXO32 and SMARCA4 co-localize at loci regulated by FBXO32, such as CDK6 suggesting that FBXO32 controls transcription through the regulation of chromatin remodeling complex activity. FBXO32 and SMARCA4 are the components of a molecular cascade, linking MITF to epigenetics, in melanoma cells.
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Saiga, Toru, Takaichi Fukuda, Masaki Matsumoto, Hirobumi Tada, Hirotaka James Okano, Hideyuki Okano, and Keiichi I. Nakayama. "Fbxo45 Forms a Novel Ubiquitin Ligase Complex and Is Required for Neuronal Development." Molecular and Cellular Biology 29, no. 13 (April 27, 2009): 3529–43. http://dx.doi.org/10.1128/mcb.00364-09.
Повний текст джерелаАнотація:
ABSTRACT Fbxo45 is an F-box protein that is restricted to the nervous system. Unlike other F-box proteins, Fbxo45 was found not to form an SCF complex as a result of an amino acid substitution in the consensus sequence for Cul1 binding. Proteomics analysis revealed that Fbxo45 specifically associates with PAM (protein associated with Myc), a RING finger-type ubiquitin ligase. Mice deficient in Fbxo45 were generated and found to die soon after birth as a result of respiratory distress. Fbxo45 − / − embryos show abnormal innervation of the diaphragm, impaired synapse formation at neuromuscular junctions, and aberrant development of axon fiber tracts in the brain. Similar defects are also observed in mice lacking Phr1 (mouse ortholog of PAM), suggesting that Fbxo45 and Phr1 function in the same pathway. In addition, neuronal migration was impaired in Fbxo45 − / − mice. These results suggest that Fbxo45 forms a novel Fbxo45-PAM ubiquitin ligase complex that plays an important role in neural development.
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Lai, C. Y., Y. C. Ho, M. C. Hsieh, H. H. Wang, J. K. Cheng, Y. P. Chau, and H. Y. Peng. "Spinal Fbxo3-Dependent Fbxl2 Ubiquitination of Active Zone Protein RIM1 Mediates Neuropathic Allodynia through CaV2.2 Activation." Journal of Neuroscience 36, no. 37 (September 14, 2016): 9722–38. http://dx.doi.org/10.1523/jneurosci.1732-16.2016.
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Wang, Ning, Qian Song, Hai Yu та Gang Bao. "Overexpression of FBXO17 Promotes the Proliferation, Migration and Invasion of Glioma Cells Through the Akt/GSK-3β/Snail Pathway". Cell Transplantation 30 (1 січня 2021): 096368972110073. http://dx.doi.org/10.1177/09636897211007395.
Повний текст джерелаАнотація:
FBXO17 is a newly studied F-box protein associated with high-grade glioma. However, its exact role in glioma remains unclear. In the present study, we aimed to investigate the role of FBXO17 in glioma both in vitro and in vivo and explore the underlying mechanism. Our results showed that FBXO17 mRNA and protein levels were upregulated in glioma cells including U87, U251, SHG44, and U-118-MG cells as compared to the HA1800 cells. Downregulation of FBXO17 significantly suppressed the cellular behaviors of glioma cells including cell proliferation, migration, and invasion. In addition, FBXO17 knockdown induced E-cadherin expression and inhibited N-cadherin and vimentin expression at mRNA and protein levels in glioma cells. In contrast, overexpression of FBXO17 promoted cell proliferation, migration, invasion and EMT process. Furthermore, FBXO17 regulated the Akt/GSK-3β/snail signaling pathway in glioma cells with significant changes in the expression levels of p-Akt, p-GSK-3β and snail. Additionally, inhibition of Akt by LY294002 reversed the effects of FBXO17 overexpression on cellular behaviors of glioma cells. Finally, in vivo mouse xenograft assay proved that downregulation of FBXO17 suppresses the tumorigenesis of glioma. In conclusion, these findings demonstrated that FBXO17 acted as a promotor of glioma development via modulating Akt/GSK-3β/snail signaling pathway.
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Choppara, Srinadh, Sunil K. Malonia, Ganga Sankaran, Michael R. Green, and Manas Kumar Santra. "Degradation of FBXO31 by APC/C is regulated by AKT- and ATM-mediated phosphorylation." Proceedings of the National Academy of Sciences 115, no. 5 (January 17, 2018): 998–1003. http://dx.doi.org/10.1073/pnas.1705954115.
Повний текст джерелаАнотація:
The F-box protein FBXO31 is a tumor suppressor that is encoded in 16q24.3, for which there is loss of heterozygosity in various solid tumors. FBXO31 serves as the substrate-recognition component of the SKP/Cullin/F-box protein class of E3 ubiquitin ligases and has been shown to direct degradation of pivotal cell-cycle regulatory proteins including cyclin D1 and the p53 antagonist MDM2. FBXO31 levels are normally low but increase substantially following genotoxic stress through a mechanism that remains to be determined. Here we show that the low levels of FBXO31 are maintained through proteasomal degradation by anaphase-promoting complex/cyclosome (APC/C). We find that the APC/C coactivators CDH1 and CDC20 bind to a destruction-box (D-box) motif present in FBXO31 to promote its polyubiquitination and degradation in a cell-cycle–regulated manner, which requires phosphorylation of FBXO31 on serine-33 by the prosurvival kinase AKT. Following genotoxic stress, phosphorylation of FBXO31 on serine-278 by another kinase, the DNA damage kinase ATM, results in disruption of its interaction with CDH1 and CDC20, thereby preventing FBXO31 degradation. Collectively, our results reveal how alterations in FBXO31 phosphorylation, mediated by AKT and ATM, underlie physiological regulation of FBXO31 levels in unstressed and genotoxically stressed cells.
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Sahasrabuddhe, Anagh Anant, Xiaofei Chen, Kaiyu Ma, Rui Wu, Richa Kapoor, Rishi Raj Chhipa, Xiao Zhang, et al. "A Novel FBXO45-Gef-H1 Axis Controls Oncogenic Signaling in B-Cell Lymphoma." Blood 138, Supplement 1 (November 5, 2021): 711. http://dx.doi.org/10.1182/blood-2021-151245.
Повний текст джерелаАнотація:
Abstract Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.
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Brewer, Kelly, Isabel Nip, Justin Bellizzi, Jessica Costa-Guda, and Andrew Arnold. "Molecular analysis of cyclin D1 modulators PRKN and FBX4 as candidate tumor suppressors in sporadic parathyroid adenomas." Endocrine Connections 10, no. 3 (March 2021): 302–8. http://dx.doi.org/10.1530/ec-21-0055.
Повний текст джерелаАнотація:
Objective Primary hyperparathyroidism is most often caused by a sporadic single-gland parathyroid adenoma (PTA), a tumor type for which cyclin D1 is the only known and experimentally validated oncoprotein. However, the molecular origins of its frequent overexpression have remained mostly elusive. In this study, we explored a potential tumorigenic mechanism that could increase cyclin D1 stability through a defect in molecules responsible for its degradation. Methods We examined two tumor suppressor genes known to modulate cyclin D1 ubiquitination, PRKN and FBXO4 (FBX4), for evidence of classic two-hit tumor suppressor inactivation within a cohort of 82 PTA cases. We examined the cohort for intragenic inactivating and splice site mutations by Sanger sequencing and for locus-associated loss of heterozygosity (LOH) by microsatellite analysis. Results We identified no evidence of bi-allelic tumor suppressor inactivation of PRKN or FBXO4 via inactivating mutation or splice site perturbation, neither in combination with nor independent of LOH. Among the 82 cases, we encountered previously documented benign single nucleotide polymorphisms (SNPs) in 35 tumors at frequencies similar to those reported in the germlines of the general population. Eight cases exhibited intragenic LOH at the PRKN locus, in some cases extending to cover at least an additional 1.7 Mb of chromosome 6q25-26. FBXO4 was not affected by LOH. Conclusion: The absence of evidence for specific bi-allelic inactivation in PRKN and FBXO4 in this sizeable cohort suggests that these genes only rarely, if ever, serve as classic driver tumor suppressors responsible for the growth of PTAs.
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Pighi, Chiara, Mara Compagno, Qi Wang, Taek-Chin Cheong, Teresa Poggio, Fernanda Langellotto, Paola Francia di Celle, Alberto Zamò, and Roberto Chiarle. "FBXO11, a Regulator of BCL6 Stability, Is Recurrently Mutated in Burkitt Lymphoma." Blood 126, no. 23 (December 3, 2015): 3673. http://dx.doi.org/10.1182/blood.v126.23.3673.3673.
Повний текст джерелаАнотація:
Abstract INTRODUCTION: We recently described inactivating mutations of the FBXO11 gene in Diffuse Large B Cell Lymphoma (DLBCL). One major function of FBXO11 is to regulate BCL6 stability as well as other targets such as SNAIL. BCL6 acts as an oncogene in several human B-cell lymphomas and its expression levels can be increased by several mechanisms including chromosomal translocations, point mutations in the promoter region and reduced degradation by inactivation of FBXO11. Thus, FBXO11 acts as an oncosuppressor in DLBCL by promoting the accumulation of BCL6. However, a clear and complete picture of the distribution of FBXO11 mutations in different lymphoma subtypes is missing, and the in vivo functions of FBXO11 in normal tissue and lymphoma development are completely lacking. In the present work, we investigated the frequency and distribution of FBXO11 mutations in an extended panel of human BCL6 positive lymphoma and we functionally validated novel FBXO11 mutations for their ability to induce BCL6 degradation. METHODS: We sequenced the entire FBXO11 coding sequence by classical Sanger sequencing in 100 cases of Follicular Lymphoma (FL), 36 cases of Burkitt Lymphoma (BL), 8 BL cell lines and 8 Anaplastic Large cell lymphoma (ALCL) cell lines, which are typically BCL6-positive lymphomas. Moreover, we sequenced 50 cases of Marginal Zone B cell Lymphoma (MZL), which show variable expression of BCL6. To investigate whether the FBXO11 mutations interfere with FBXO11 activity we generated complementary DNA constructs containing these mutations. Wild-type FBXO11 or FBXO11 mutants were expressed in HEK-293T cells with constructs expressing BCL6 or SNAIL. Twenty-four hours after transfection, HEK-293T cells were treated with cycloheximide and harvested at different time points to check substrates expression and degradation by immunoblotting. RESULTS: BL carried the highest frequency of FBXO11 mutations (12/44 cases analyzed, 27%), whereas FL and MZL were very rarely affected by FBXO11 mutations (1/100 FL and 1/50 MZL). The analysis identified several mutations, either located in the coding sequence mostly in the CASH (functional) domains or located in intronic sequences controlling exon splicing. Sequence changes included missense mutations (n=8), deletions (n=2), a frameshift deletion introducing a premature stop codon (n=1) and nucleotide substitutions at consensus splice donor sites (n=2) and acceptor site (n=1). Remarkably, these last mutations represent the first report of splice site mutations affecting the FBXO11 gene. By cDNA amplification and sequencing, we demonstrated skipping of entire exons: the splice site mutations located at the donor site of exon 15 and 19 lead to the loss of the exon 15 and 19 respectively, while the splice site mutation affecting the acceptor site of exon 16 results in the deletion of the adjacent exon 16. Two of the point mutations (K631N and Y122C), affecting one case of FL and MZL respectively, were also found in the previous panel of DLBCLs (Duan et al, Nature 2012). Both cases consisted of a low grade component adjacent to an area of high grade transformation. By microdissection, we separately studied the two components of each of these cases to demonstrate that the mutation was present only in the high grade component. Finally, to validate all the mutations found in BL, we generated mutant constructs corresponding to each of them. By this approach, we showed that all the mutations identified are loss of function variants because they impaired the FBXO11 ability to promote BCL6 and SNAIL degradation. CONCLUSIONS: We identified several FBXO11 novel mutations in a large panel of BCL6-positive human lymphomas. All the mutations identified were inactivating the FBXO11 ability to induce BCL6 and SNAIL degradation. In contrast, mutations of FBXO11 are rare in low grade FL and MZL and, when present, are associated with high grade transformation. Together with our previous findings, this study showed that FBXO11 is mostly mutated in aggressive lymphomas such as DLBCL and BL, thus suggesting that FBXO11 mutations could contribute to the de-novo onset or transformation into high grade lymphoma. Disclosures No relevant conflicts of interest to declare.
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Li, Yunfeng, Kai Jin, Eric Bunker, Xiaojuan Zhang, Xuemei Luo, Xuedong Liu, and Bing Hao. "Structural basis of the phosphorylation-independent recognition of cyclin D1 by the SCFFBXO31 ubiquitin ligase." Proceedings of the National Academy of Sciences 115, no. 2 (December 26, 2017): 319–24. http://dx.doi.org/10.1073/pnas.1708677115.
Повний текст джерелаАнотація:
Ubiquitin-dependent proteolysis of cyclin D1 is associated with normal and tumor cell proliferation and survival. The SCFFBXO31 (Skp1–Cul1–Rbx1–FBXO31) ubiquitin ligase complex mediates genotoxic stress-induced cyclin D1 degradation. Previous studies have suggested that cyclin D1 levels are maintained at steady state by phosphorylation-dependent nuclear export and subsequent proteolysis in the cytoplasm. Here we present the crystal structures of the Skp1–FBXO31 complex alone and bound to a phosphorylated cyclin D1 C-terminal peptide. FBXO31 possesses a unique substrate-binding domain consisting of two β-barrel motifs, whereas cyclin D1 binds to FBXO31 by tucking its free C-terminal carboxylate tail into an open cavity of the C-terminal FBXO31 β-barrel. Biophysical and functional studies demonstrate that SCFFBXO31 is capable of recruiting and ubiquitinating cyclin D1 in a phosphorylation-independent manner. Our findings provide a conceptual framework for understanding the substrate specificity of the F-box protein FBXO31 and the mechanism of FBXO31-regulated cyclin D1 protein turnover.
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Pighi, Chiara, Taek-Chin Cheong, Mara Compagno, Enrico Patrucco, Maddalena Arigoni, Martina Olivero, Qi Wang, et al. "Frequent mutations of FBXO11 highlight BCL6 as a therapeutic target in Burkitt lymphoma." Blood Advances 5, no. 23 (December 8, 2021): 5239–57. http://dx.doi.org/10.1182/bloodadvances.2021005682.
Повний текст джерелаАнотація:
Abstract The expression of BCL6 in B-cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions, or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase that is involved in the degradation of BCL6, and it is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11 mutations are found in 23% of patients with Burkitt lymphoma (BL). FBXO11 mutations impaired BCL6 degradation, and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of 1 or 2 copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B-cell lymphoma onset, providing experimental evidence that FBXO11 is a haploinsufficient oncosuppressor in B-cell lymphoma. In wild-type and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degraders or inhibitors partially impaired lymphoma growth in vitro and in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11 mutations in BL, we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches, such as BCL6 degraders and direct MYC inhibition, could be exploited as a targeted therapy for BL.
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Matsushima, Norio, Shintaro Takatsuka, Hiroki Miyashita, and Robert H. Kretsinger. "Leucine Rich Repeat Proteins: Sequences, Mutations, Structures and Diseases." Protein & Peptide Letters 26, no. 2 (February 20, 2019): 108–31. http://dx.doi.org/10.2174/0929866526666181208170027.
Повний текст джерелаАнотація:
Mutations in the genes encoding Leucine Rich Repeat (LRR) containing proteins are associated with over sixty human diseases; these include high myopia, mitochondrial encephalomyopathy, and Crohn’s disease. These mutations occur frequently within the LRR domains and within the regions that shield the hydrophobic core of the LRR domain. The amino acid sequences of fifty-five LRR proteins have been published. They include Nod-Like Receptors (NLRs) such as NLRP1, NLRP3, NLRP14, and Nod-2, Small Leucine Rich Repeat Proteoglycans (SLRPs) such as keratocan, lumican, fibromodulin, PRELP, biglycan, and nyctalopin, and F-box/LRR-repeat proteins such as FBXL2, FBXL4, and FBXL12. For example, 363 missense mutations have been identified. Replacement of arginine, proline, or cysteine by another amino acid, or the reverse, is frequently observed. The diverse effects of the mutations are discussed based on the known structures of LRR proteins. These mutations influence protein folding, aggregation, oligomerization, stability, protein-ligand interactions, disulfide bond formation, and glycosylation. Most of the mutations cause loss of function and a few, gain of function.
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Xu, Peng, Daniel C. Scott, Xing Tang, Yu Yao, Yong-Dong Wang, Wenjian Bi, Lance E. Palmer, et al. "FBXO11 Activates Erythroid Gene Transcription By Degrading Heterochromatin-Associated Protein BAHD1." Blood 132, Supplement 1 (November 29, 2018): 529. http://dx.doi.org/10.1182/blood-2018-99-117362.
Повний текст джерелаАнотація:
Abstract Mature red blood cells (RBC) contain approximately 95% cytosolic hemoglobin for the purpose of blood oxygen transport. This specialized state is achieved during erythropoiesis by regulated gene expression and protein degradation. During late-stage erythropoiesis, ubiquitin ligases eliminate unnecessary proteins and maintain quality control by degrading unstable proteins, including unpaired hemoglobin subunits. However, ubiquitin ligases are expressed at all stages of erythropoiesis and the functions of most are unknown. To study ubiquitin ligases involved in RBC formation, we performed a Cas9/single guide (sg) RNA screen for functional ubiquitin-proteasome components in HUDEP-2 cells, an immortalized human cell line that proliferates as immature erythroblasts and can be induced to undergo terminal maturation. We identified the E3 ubiquitin ligase FBXO11 as a top-ranked candidate. FBXO11 is a member of the F-box protein family that assembles into a SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. Depletion of FBXO11 by 2 different sgRNAs in HUDEP-2 cells inhibited erythroid maturation, as evidenced by reduced hemoglobinization, failure to induce the maturation marker Band3 and persistence of immature cell morphology. In primary human CD34+ cells, suppression of FBXO11 expression by Cas9 + two independent sgRNAs inhibited erythroid maturation, as evidenced by reduced Band3 expression (5.3% vs. 15.6% for non-targeting sgRNA, P < 0.001; n=4). RNA-seq analysis of FBXO11-depleted HUDEP-2 cells revealed 951 decreased transcripts (enriched for erythroid genes) and 339 increased transcripts (enriched for genes expressed in activated T-cells) compared to control cells expressing non-targeting gRNA (P < 0.05). Thus, FBXO11 is required for erythroid maturation and facilitates erythroid gene expression. We sought to establish how FBXO11 modulates erythropoiesis and erythroid gene expression by identifying the relevant ubiquitination substrate(s). Combined quantitative proteome analysis with RNA-seq of FBXO11-depleted HUDEP-2 cells identified several proteins that are upregulated with no change in their corresponding mRNA. We tested whether reduction of these candidate substrates could alleviate the erythroid maturation block conferred by FBXO11 depletion. In FBXO11 gene-disrupted HUDEP-2 cells, suppression of the heterochromatin-associated protein BAHD1 partially rescued hemoglobinization and Band3 expression (4.2% for Cas9 + non-targeting sgRNA vs. 21.7% for Cas9 + BAHD1 sgRNAs, P < 0.01; n=3) . Conversely, stable overexpression of V5-epitope-tagged BAHD1 in WT HUDEP-2 cells reduced Band3 expression from 25.0% to 11.4% (P < 0.001; n=3) and inhibited hemoglobinization. Transcriptome analysis demonstrated a significant inverse correlation between genes deregulated by BAHD1-V5 overexpression and FBXO11 deficiency in HUDEP-2 cells, particularly for erythroid genes that are downregulated (P < 0.0001). BAHD1, named after its bromo-adjacent homology domain that interacts with H3K27me3, is part of a transcriptional repressor complex. We showed that BAHD1 and FBXO11 co-immunoprecipitated in cells and that BAHD1 amino (N)-terminal segments of 188 or 240 amino acids were robustly modified with ubiquitin by SCFFBXO11 complex. Chromatin immunoprecipitation-sequencing analysis of BAHD1-V5-expressing WT HUDEP-2 cells showed strong enrichment for BAHD1 occupancy on erythroid gene promoters that were downregulated by FBXO11-deficiency (P < 0.0001). We next investigated whether a specific set of histone marks distinguish FBXO11-regulated genes in normal erythroblasts. We found that most FBXO11-regulated genes in both HUDEP-2 and primary CD34+ derived erythroblasts harbor histone marks H3K4me3 and H3K27me3, indicating a "bivalent" epigenetic state that supports low level transcription in stem or progenitor cells. Together, these data indicate that FBXO11 activates expression of erythroid genes by ubiquitinating and degrading bivalent promoter-bound BAHD1 repressor complexes with likely resolution to a monovalent transcriptionally active state. Overall, our findings identify FBXO11 as a ubiquitin ligase that utilizes a novel mechanism to activate erythroid genes during RBC formation. This newly identified pathway may contribute to known activities of FBXO11 as a tumor suppressor and developmental regulator in non-erythroid tissues. Disclosures No relevant conflicts of interest to declare.
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Hughes, David C., Leslie M. Baehr, Julia R. Driscoll, Sarah A. Lynch, David S. Waddell, and Sue C. Bodine. "Identification and characterization of Fbxl22, a novel skeletal muscle atrophy-promoting E3 ubiquitin ligase." American Journal of Physiology-Cell Physiology 319, no. 4 (October 1, 2020): C700—C719. http://dx.doi.org/10.1152/ajpcell.00253.2020.
Повний текст джерелаАнотація:
Muscle-specific E3 ubiquitin ligases have been identified in muscle atrophy-inducing conditions. The purpose of the current study was to explore the functional role of F-box and leucine-rich protein 22 (Fbxl22), and a newly identified splice variant (Fbxl22–193), in skeletal muscle homeostasis and neurogenic muscle atrophy. In mouse C2C12 muscle cells, promoter fragments of the Fbxl22 gene were cloned and fused with the secreted alkaline phosphatase reporter gene to assess the transcriptional regulation of Fbxl22. The tibialis anterior muscles of male C57/BL6 mice (12–16 wk old) were electroporated with expression plasmids containing the cDNA of two Fbxl22 splice variants and tissues collected after 7, 14, and 28 days. Gastrocnemius muscles of wild-type and muscle-specific RING finger 1 knockout (MuRF1 KO) mice were electroporated with an Fbxl22 RNAi or empty plasmid and denervated 3 days posttransfection, and tissues were collected 7 days postdenervation. The full-length gene and novel splice variant are transcriptionally induced early (after 3 days) during neurogenic muscle atrophy. In vivo overexpression of Fbxl22 isoforms in mouse skeletal muscle leads to evidence of myopathy/atrophy, suggesting that both are involved in the process of neurogenic muscle atrophy. Knockdown of Fbxl22 in the muscles of MuRF1 KO mice resulted in significant additive muscle sparing 7 days after denervation. Targeting two E3 ubiquitin ligases appears to have a strong additive effect on protecting muscle mass loss with denervation, and these findings have important implications in the development of therapeutic strategies to treat muscle atrophy.
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Malonia, Sunil K., Parul Dutta, Manas Kumar Santra, and Michael R. Green. "F-box protein FBXO31 directs degradation of MDM2 to facilitate p53-mediated growth arrest following genotoxic stress." Proceedings of the National Academy of Sciences 112, no. 28 (June 29, 2015): 8632–37. http://dx.doi.org/10.1073/pnas.1510929112.
Повний текст джерелаАнотація:
The tumor suppressor p53 plays a critical role in maintaining genomic stability. In response to genotoxic stress, p53 levels increase and induce cell-cycle arrest, senescence, or apoptosis, thereby preventing replication of damaged DNA. In unstressed cells, p53 is maintained at a low level. The major negative regulator of p53 is MDM2, an E3 ubiquitin ligase that directly interacts with p53 and promotes its polyubiquitination, leading to the subsequent destruction of p53 by the 26S proteasome. Following DNA damage, MDM2 is degraded rapidly, resulting in increased p53 stability. Because of the important role of MDM2 in modulating p53 function, it is critical to understand how MDM2 levels are regulated. Here we show that the F-box protein FBXO31, a candidate tumor suppressor encoded in 16q24.3 for which there is loss of heterozygosity in various solid tumors, is responsible for promoting MDM2 degradation. Following genotoxic stress, FBXO31 is phosphorylated by the DNA damage serine/threonine kinase ATM, resulting in increased levels of FBXO31. FBXO31 then interacts with and directs the degradation of MDM2, which is dependent on phosphorylation of MDM2 by ATM. FBXO31-mediated loss of MDM2 leads to elevated levels of p53, resulting in growth arrest. In cells depleted of FBXO31, MDM2 is not degraded and p53 levels do not increase following genotoxic stress. Thus, FBXO31 is essential for the classic robust increase in p53 levels following DNA damage.
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Lim, Ji Ye, Eunju Kim, Collin M. Douglas, Marvin Wirianto, Chorong Han, Kaori Ono, Sun Young Kim, et al. "The circadian E3 ligase FBXL21 regulates myoblast differentiation and sarcomere architecture via MYOZ1 ubiquitination and NFAT signaling." PLOS Genetics 18, no. 12 (December 27, 2022): e1010574. http://dx.doi.org/10.1371/journal.pgen.1010574.
Повний текст джерелаАнотація:
Numerous molecular and physiological processes in the skeletal muscle undergo circadian time-dependent oscillations in accordance with daily activity/rest cycles. The circadian regulatory mechanisms underlying these cyclic processes, especially at the post-transcriptional level, are not well defined. Previously, we reported that the circadian E3 ligase FBXL21 mediates rhythmic degradation of the sarcomere protein TCAP in conjunction with GSK-3β, and Psttm mice harboring an Fbxl21 hypomorph allele show reduced muscle fiber diameter and impaired muscle function. To further elucidate the regulatory function of FBXL21 in skeletal muscle, we investigated another sarcomere protein, Myozenin1 (MYOZ1), that we identified as an FBXL21-binding protein from yeast 2-hybrid screening. We show that FBXL21 binding to MYOZ1 led to ubiquitination-mediated proteasomal degradation. GSK-3β co-expression and inhibition were found to accelerate and decelerate FBXL21-mediated MYOZ1 degradation, respectively. Previously, MYOZ1 has been shown to inhibit calcineurin/NFAT signaling important for muscle differentiation. In accordance, Fbxl21 KO and MyoZ1 KO in C2C12 cells impaired and enhanced myogenic differentiation respectively compared with control C2C12 cells, concomitant with distinct effects on NFAT nuclear localization and NFAT target gene expression. Importantly, in Psttm mice, both the levels and diurnal rhythm of NFAT2 nuclear localization were significantly diminished relative to wild-type mice, and circadian expression of NFAT target genes associated with muscle differentiation was also markedly dampened. Furthermore, Psttm mice exhibited significant disruption of sarcomere structure with a considerable excess of MYOZ1 accumulation in the Z-line. Taken together, our study illustrates a pivotal role of FBXL21 in sarcomere structure and muscle differentiation by regulating MYOZ1 degradation and NFAT2 signaling.
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Cleveland, Beth M., and Gregory M. Weber. "Effects of insulin-like growth factor-I, insulin, and leucine on protein turnover and ubiquitin ligase expression in rainbow trout primary myocytes." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 298, no. 2 (February 2010): R341—R350. http://dx.doi.org/10.1152/ajpregu.00516.2009.
Повний текст джерелаАнотація:
The effects of insulin-like growth factor-I (IGF-I), insulin, and leucine on protein turnover and pathways that regulate proteolytic gene expression and protein polyubiquitination were investigated in primary cultures of 4-day-old rainbow trout myocytes. Supplementing media with 100 nM IGF-I increased protein synthesis by 13% ( P < 0.05) and decreased protein degradation by 14% ( P < 0.05). Treatment with 1 μM insulin increased protein synthesis by 13% ( P < 0.05) and decreased protein degradation by 17% ( P < 0.05). Supplementing media containing 0.6 mM leucine with an additional 2.5 mM leucine did not increase protein synthesis rates but reduced rates of protein degradation by 8% ( P < 0.05). IGF-I (1 nM–100 nM) and insulin (1 nM-1 μM) independently reduced the abundance of ubiquitin ligase mRNA in a dose-dependent manner, with maximal reductions of ∼70% for muscle atrophy F-box (Fbx) 32, 40% for Fbx25, and 25% for muscle RING finger-1 (MuRF1, P < 0.05). IGF-I and insulin stimulated phosphorylation of FOXO1 and FOXO4 ( P < 0.05), which was inhibited by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, and decreased the abundance of polyubiquitinated proteins by 10–20% ( P < 0.05). Supplementing media with leucine reduced Fbx32 expression by 25% ( P < 0.05) but did not affect Fbx25 nor MuRF1 transcript abundance. Serum deprivation decreased rates of protein synthesis by 60% ( P < 0.05), increased protein degradation by 40% ( P < 0.05), and increased expression of all ubiquitin ligases. These data suggest that, similar to mammals, the inhibitory effects of IGF-I and insulin on proteolysis occur via P I3-kinase/protein kinase B signaling and are partially responsible for the ability of these compounds to promote protein accretion.
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Zhang, Liping, Zihong Chen, Ying Wang, David J. Tweardy, and William E. Mitch. "Stat3 activation induces insulin resistance via a muscle-specific E3 ubiquitin ligase Fbxo40." American Journal of Physiology-Endocrinology and Metabolism 318, no. 5 (May 1, 2020): E625—E635. http://dx.doi.org/10.1152/ajpendo.00480.2019.
Повний текст джерелаАнотація:
Cellular mechanisms causing insulin resistance (IR) in chronic kidney disease (CKD) are poorly understood. One potential mechanism is that CKD-induced inflammation activates the signal transducer and activator of transcription 3 (Stat3) in muscle. We uncovered increased p-Stat3 in muscles of mice with CKD or mice fed high-fat diet (HFD). Activated Stat3 stimulates the expression of Fbxo40, a muscle-specific E3 ubiquitin ligase that stimulates ubiquitin conjugation leading to degradation of insulin receptor substrate 1 (IRS1). Evidence that Stat3 activates Fbxo40 includes 1) potential Stat3 binding sites in Fbxo40 promoters; 2) Stat3 binding to the Fbxo40 promoter; and 3) constitutively active Stat3 stimulating both Fbxo40 expression and its promoter activity. We found that IL-6 activates Stat3 in myotubes, increasing Fbxo40 expression with reduced IRS1 and p-Akt. Knockdown Fbxo40 using siRNA from myotubes results in higher levels of IRS1 and p-Akt despite the presence of IL-6. We treated mice with a small-molecule inhibitor of Stat3 (TTI-101) and found improved glucose tolerance and insulin signaling in skeletal muscles of mice with CKD or fed an HFD. Finally, we uncovered improved glucose tolerance in mice with muscle-specific Stat3 KO versus results in Stat3f/f mice in response to the HFD. Thus Stat3 activation in muscle increases IR in mice. Inhibition of Stat3 by TTI-101 could be developed into clinical strategies to improve muscle insulin signaling in inflammation and other catabolic diseases.
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Akiyama, Mari. "Characterization of the F-box Proteins FBXW2 and FBXL14 in the Initiation of Bone Regeneration in Transplants given to Nude Mice." Open Biomedical Engineering Journal 12, no. 1 (October 18, 2018): 75–89. http://dx.doi.org/10.2174/1874120701812010075.
Повний текст джерелаАнотація:
Background: Cultured bovine-periosteum-derived cells can form three-dimensional structures on tissue culture dishes without artificial scaffolding material, can induce bone regeneration in vivo. The utility of cultured bovine-periosteum-derived cells for bone tissue regeneration after their transplantation into nude mice has been reported, the precise F-box molecular mechanism was unclear. Objective: The aim of this study was to investigate the specific F-box proteins required for bone regeneration by cultured bovine-periosteum-derived cells in vitro. Methods: In the present study, periosteum tissue and cultured periosteum-derived cells were cultured for 5 weeks in vitro and then embedded in collagen gel with a green tissue-marking dye. Electrophoresis and immunohistochemistry were used to identify the specific F-box proteins required for tissue bone regeneration. Results: The bovine-periosteum-derived cells were observed to form bone shortly after the expression of F-box proteins. After the initial phase of bone formation, the expression of the F-box proteins ceased. FBXW2 was shown to be expressed in the periosteum, but not in cultured periosteum-derived cells. Furthermore, FBXL14 disappeared during bone formation. Conclusions: Bone regeneration requires progenitor cells, such as bovine-periosteum-derived cells and the activation of the F-box Proteins FBXW2 and FBXL14, over time the expression of these proteins ceases. Further scientific and clinical trials are needed to investigate how the F-box Proteins can be used therapeutically to treat osteoporosis and osteonecrosis.
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Schieber, Michael, and John D. Crispino. "FBXO11 Is a Candidate Tumor Suppressor in the Transformation of MDS to Secondary AML." Blood 134, Supplement_1 (November 13, 2019): 4217. http://dx.doi.org/10.1182/blood-2019-124007.
Повний текст джерелаАнотація:
Myelodysplastic syndrome (MDS) is a heterogenous myeloid lineage malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of secondary transformation to acute myeloid leukemia (sAML). Genomic sequencing of large MDS cohorts has led to the identification of recurrent genetic abnormalities that carry independent prognostic significance and overlap with mutational changes in sAML. However, no set of mutations is sufficient to predict the transformation of MDS raising the question of how an identical genotype produces MDS in one patient and sAML in another? We hypothesize there are therapeutically targetable cellular processes altered by the initiating genetic changes in MDS that predict transformation to sAML. To uncover novel cellular pathways involved in MDS transformation, we performed an unbiased genome-wide CRISPR/Cas9 in the human MDS-L cell line. MDS-L was established from bone marrow mononuclear cells in a 52-year-old male patient and requires IL3-containing media for growth in vitro (Figure 1A). GFP expressing MDS-L cells were transduced with Cas9 and a sgRNA against GFP to confirm functional Cas9 expression in MDS-L (Figures 1B and C). In Cas9 expressing MDS-L cells, we then transduced the Brunello sgRNA CRISPR library and subjected the cells to IL-3 starvation for 4 weeks. Cells surviving IL-3 starvation were then expanded and harvested for genomic DNA. High throughput sequencing of the barcoded DNA produced raw reads that were analyzed using the PinAPL-Py web-based software. sgRNAs appearing in duplicate with absolute read counts over 1000 or in triplicate over 100 were considered significant. We identified 5 genes that conferred resistance to IL-3 starvation, which included FBXO11 (Figure 1D). The Fbox protein FBXO11 is a component of the SCF ubiquitin ligase complex and regulates its substrates via ubiquitination and proteasomal degradation. FBXO11 is mutated in up to 20% of diffuse large B-cell lymphomas and its loss in breast cancer models leads to increased metastases. Therefore, we hypothesized FBXO11 may also function as a tumor suppressor in the transformation of MDS to AML. We confirmed in the Bloodspot gene expression database that there are decreased levels of FBXO11 in a variety of AML samples, including complex karyotype, compared to normal HSCs. To validate the results of the screen, we synthesized two sgRNAs targeting FBXO11, transduced these into MDS-L cells, and detected reduced FBXO11 expression (Figure 1E). Loss of FBXO11 expression promoted survival in IL-3 free media, confirming the selection readout of the screen (Figure 1F). We then designed a silent mutation in the shorter isoform of FBXO11 (FBXO11v1sm1) that rendered resistance to CRISPR/Cas9 (Figure 1G) and observed that overexpression of FBXO11v1sm1 re-sensitized cells to cytokine starvation (Figure 1H). Whether there are different functions between FBXO11 variant 1 and 4 are currently being explored. We are actively performing RNA sequencing and ubiquitin proteomics in FBXO11 knockout cells to identify its downstream targets and assaying for reduced expression of FBXO11 in primary patient MDS and AML samples. Based on our studies, we predict that SCF ubiquitin ligase component FBXO11 is a tumor suppressor regulating the transformation of MDS to secondary AML. Figure 1 Disclosures Crispino: Sierra Oncology: Consultancy; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Research Funding; Scholar Rock: Research Funding.
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Schneider, Christof, Ning Kon, Letizia Amadori, Qiong Shen, Friederike H. Schwartz, Benjamin Tischler, Marion Bossennec, et al. "FBXO11 inactivation leads to abnormal germinal-center formation and lymphoproliferative disease." Blood 128, no. 5 (August 4, 2016): 660–66. http://dx.doi.org/10.1182/blood-2015-11-684357.
Повний текст джерелаАнотація:
Key Points FBXO11 loss in mice enhances GC B-cell formation and leads to increased BCL6 expression. FBXO11 inactivation, mimicking genetic alterations identified in human lymphomas, represents an alternative mechanism of BCL6 deregulation.
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Cleveland, Beth M., and Jason P. Evenhuis. "Molecular characterization of atrogin-1/F-box protein-32 (FBXO32) and F-box protein-25 (FBXO25) in rainbow trout (Oncorhynchus mykiss): Expression across tissues in response to feed deprivation." Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 157, no. 3 (November 2010): 248–57. http://dx.doi.org/10.1016/j.cbpb.2010.06.010.
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Angeli, Franca, Russell Wyborski, Bill Chen, Rama Mallampalli, and Michael Lark. "P157 FBXO3-FBXL2 AXIS MODULATORS AS A NOVEL CLASS OF ORAL SMALL MOLECULE COMPOUNDS FOR THE TREATMENT OF CROHN’S DISEASE." Gastroenterology 158, no. 3 (February 2020): S9—S10. http://dx.doi.org/10.1053/j.gastro.2019.11.061.
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Fang, Xiaoguang, Wenchao Zhou, Qiulian Wu, Zhi Huang, Yu Shi, Kailin Yang, Cong Chen, et al. "Deubiquitinase USP13 maintains glioblastoma stem cells by antagonizing FBXL14-mediated Myc ubiquitination." Journal of Experimental Medicine 214, no. 1 (December 6, 2016): 245–67. http://dx.doi.org/10.1084/jem.20151673.
Повний текст джерелаАнотація:
Glioblastoma is the most lethal brain tumor and harbors glioma stem cells (GSCs) with potent tumorigenic capacity. The function of GSCs in tumor propagation is maintained by several core transcriptional regulators including c-Myc. c-Myc protein is tightly regulated by posttranslational modification. However, the posttranslational regulatory mechanisms for c-Myc in GSCs have not been defined. In this study, we demonstrate that the deubiquitinase USP13 stabilizes c-Myc by antagonizing FBXL14-mediated ubiquitination to maintain GSC self-renewal and tumorigenic potential. USP13 was preferentially expressed in GSCs, and its depletion potently inhibited GSC proliferation and tumor growth by promoting c-Myc ubiquitination and degradation. In contrast, overexpression of the ubiquitin E3 ligase FBXL14 induced c-Myc degradation, promoted GSC differentiation, and inhibited tumor growth. Ectopic expression of the ubiquitin-insensitive mutant T58A–c-Myc rescued the effects caused by FBXL14 overexpression or USP13 disruption. These data suggest that USP13 and FBXL14 play opposing roles in the regulation of GSCs through reversible ubiquitination of c-Myc.
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Gao, Yu, Xinyu Xiao, Jing Luo, Jianwei Wang, Qiling Peng, Jing Zhao, Ning Jiang, and Yong Zhao. "E3 Ubiquitin Ligase FBXO3 Drives Neuroinflammation to Aggravate Cerebral Ischemia/Reperfusion Injury." International Journal of Molecular Sciences 23, no. 21 (November 7, 2022): 13648. http://dx.doi.org/10.3390/ijms232113648.
Повний текст джерелаАнотація:
Ischemic stroke, one of the most universal causes of human mortality and morbidity, is pathologically characterized by inflammatory cascade, especially during the progression of ischemia/reperfusion (I/R) injury. F-Box Protein 3 (FBXO3), a substrate-recognition subunit of SKP1-cullin 1-F-box protein (SCF) E3 ligase complexes, has recently been proven to be severed as an underlying pro-inflammatory factor in pathological processes of diverse diseases. Given these considerations, the current study aims at investigating whether FBXO3 exerts impacts on inflammation in cerebral I/R injury. In this study, first, it was verified that FBXO3 protein expression increased after a middle cerebral artery occlusion/reperfusion (MCAO/R) model in Sprague–Dawley (SD) rats and was specifically expressed in neurons other than microglia or astrocytes. Meanwhile, in mouse hippocampal neuronal cell line HT22 cells, the elevation of FBXO3 protein was observed after oxygen and glucose deprivation/reoxygenation (OGD/R) treatment. It was also found that interference of FBXO3 with siRNA significantly alleviated neuronal damage via inhibiting the inflammatory response in I/R injury both in vivo and in vitro. The FBXO3 inhibitor BC-1215 was used to confirm the pro-inflammatory effect of FBXO3 in the OGD/R model as well. Furthermore, by administration of FBXO3 siRNA and BC-1215, FBXO3 was verified to reduce the protein level of Homeodomain-Interacting Protein Kinase 2 (HIPK2), likely through the ubiquitin–proteasome system (UPS), to aggravate cerebral I/R injury. Collectively, our results underline the detrimental effect FBXO3 has on cerebral I/R injury by accelerating inflammatory response, possibly through ubiquitylating and degrading HIPK2. Despite the specific interaction between FBXO3 and HIPK2 requiring further study, we believe that our data suggest the therapeutic relevance of FBXO3 to ischemic stroke and provide a new perspective on the mechanism of I/R injury.
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Watanabe, Koki, Kanae Yumimoto, and Keiichi I. Nakayama. "FBXO21 mediates the ubiquitylation and proteasomal degradation of EID1." Genes to Cells 20, no. 8 (June 18, 2015): 667–74. http://dx.doi.org/10.1111/gtc.12260.
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Cai, Lili, Liang Liu, Lihui Li, and Lijun Jia. "SCFFBXO28-mediated self-ubiquitination of FBXO28 promotes its degradation." Cellular Signalling 65 (January 2020): 109440. http://dx.doi.org/10.1016/j.cellsig.2019.109440.
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Jiang, Gui-Yang, Xiu-Peng Zhang, Liang Wang, Xu-Yong Lin, Juan-Han Yu, En-Hua Wang, and Yong Zhang. "FBXO25 promotes cell proliferation, invasion, and migration of NSCLC." Tumor Biology 37, no. 10 (September 5, 2016): 14311–19. http://dx.doi.org/10.1007/s13277-016-5298-1.
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Ren, Jun, Mingming Sun, Hao Zhou, Amir Ajoolabady, Yuan Zhou, Jun Tao, James R. Sowers, and Yingmei Zhang. "FUNDC1 interacts with FBXL2 to govern mitochondrial integrity and cardiac function through an IP3R3-dependent manner in obesity." Science Advances 6, no. 38 (September 2020): eabc8561. http://dx.doi.org/10.1126/sciadv.abc8561.
Повний текст джерелаАнотація:
Defective mitophagy is causally linked to obesity complications. Here, we identified an interaction between mitophagy protein FUNDC1 (FUN14 domain containing 1) and receptor subunit of human SCF (SKP1/cullin/F-box protein) ubiquitin ligase complex FBXL2 as a gatekeeper for mitochondrial Ca2+ homeostasis through degradation of IP3R3 (inositol 1,4,5-trisphosphate receptor type 3). Loss of FUNDC1 in FUNDC1−/− mice accentuated high-fat diet–induced cardiac remodeling, functional and mitochondrial anomalies, cell death, rise in IP3R3, and Ca2+ overload. Mass spectrometry and co-immunoprecipitation analyses revealed an interaction between FUNDC1 and FBXL2. Truncated mutants of Fbox (Delta-F-box) disengaged FBXL2 interaction with FUNDC1. Activation or transfection of FBXL2, inhibition of IP3R3 alleviated, whereas disruption of FBXL2 localization sensitized lipotoxicity-induced cardiac damage. FUNDC1 deficiency accelerated and decelerated palmitic acid–induced degradation of FBXL2 and IP3R3, respectively. Our data suggest an essential role for interaction between FUNDC1 and FBXL2 in preserving mitochondrial Ca2+ homeostasis and cardiac function in obese hearts.
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Liu, Lin, Kun Liu, Yanzhe Yan, Zhuangzhuang Chu, Yi Tang, and Chunbo Tang. "Two Transcripts of FBXO5 Promote Migration and Osteogenic Differentiation of Human Periodontal Ligament Mesenchymal Stem Cells." BioMed Research International 2018 (2018): 1–12. http://dx.doi.org/10.1155/2018/7849294.
Повний текст джерелаАнотація:
Objectives. Enhanced migration and osteogenic differentiation of mesenchymal stem cells (MSCs) are beneficial for MSC-mediated periodontal tissue regeneration, a promising method for periodontitis treatment. FBXO5, a member of the F-box protein family, is involved in the osteogenic differentiation of MSCs. Here, we investigated the effect of FBXO5 on human periodontal ligament stem cells (hPDLSCs). Materials and Methods. hPDLSCs were isolated from periodontal ligament tissue. Lentivirus FBXO5 shRNA was used to silence FBXO5 expression. Two transcripts of FBXO5 were overexpressed and transduced into hPDLSCs via retroviral infection. Migration and osteogenic differentiation of hPDLSCs were evaluated using the scratch migration assay, alkaline phosphatase (ALP) activity, ALP staining, alizarin red staining, western blotting, and real-time polymerase chain reaction. Results. The expression of FBXO5 was upregulated after osteogenic induction in hPDLSCs. FBXO5 knockdown attenuated migration, inhibited ALP activity and mineralization, and decreased RUNX2, OSX, and OCN expression, while the overexpression of two transcript isoforms significantly accelerated migration, enhanced ALP activity and mineralization, and increased RUNX2, OSX, and OCN expression in hPDLSCs. Conclusions. Both isoforms of FBXO5 promoted the migration and osteogenic differentiation potential of hPDLSCs, which identified a potential target for improving periodontal tissue regeneration.