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FBXO31, a member of F-box protein family, has been shown to play an important role in preventing tumorigenesis by preserving genomic stability during cell proliferations as well as upon genotoxic stresses. Inactivation of FBXO31 due to loss of heterozygosity is associated with various cancers, including ovarian cancer, one of the deadliest forms of gynecological cancers. However, the role and regulation of FBXO31 in ovarian cancer remained elusive. Here, using biochemical and molecular biology techniques, we show that c-Myc suppresses the mRNA levels of FBXO31 in ovarian cancer cell lines and mouse model. Chromatin immunoprecipitation experiment showed that c-Myc is recruited to the promoter region of FBXO31 and prevents FBXO31 mRNA synthesis. In contrast, FBXO31 maintains the c-Myc expression at an optimum through proteasome pathway. FBXO31 interacts with and facilitates the polyubiquitination of c-Myc through the SCF complex and thereby inhibits ovarian cancer growth both in vitro and in vivo. Moreover, FBXO31-mediated proteasomal degradation of c-Myc is unique. Unlike other negative regulators, FBXO31 recognizes c-Myc in phosphorylation independent manner to direct its degradation. Further, expression levels analysis revealed that c-Myc and FBXO31 share a converse correlation of expression in ovarian cancer cell lines and patient samples. We observed an increase in the expression levels of c-Myc with a concomitant decrease in the levels of FBXO31 in higher grades of ovarian cancer patient samples. In conclusion, our study demonstrated that oncogene c-Myc impairs the tumor-suppressive functions of FBXO31 to promote ovarian cancer progression, and therefore c-Myc-FBXO31 axis can be explored to develop better cancer therapy. This article is protected by copyright. All rights reserved.

Despite the high morbidity and mortality rates associated with colorectal cancer (CRC), the aberrant genes and mechanisms driving CRC pathogenesis remain poorly understood. Chromosome instability (CIN), or ongoing changes in chromosome numbers, is a predominant form of genome instability associated with ~85% of CRCs, suggesting it may be a key mechanism driving CRC oncogenesis. CIN enables the acquisition of copy number alterations conferring selective growth, proliferation and survival advantages that promote cellular transformation. Despite these associations, the aberrant genes underlying CIN remain largely unknown. Candidate CIN gene FBXO7 encodes an F-box protein, a subunit of the SKP1-CUL1-FBOX (SCF) complex that confers substrate specificity to the complex and targets proteins for subsequent degradation by the 26S proteasome. Recently, the genes encoding the three core SCF complex members were identified as CIN genes; however, it is unknown whether F-box proteins exhibit similar integral roles in maintaining chromosome stability. Using short- small interfering RNA (siRNA) and long- (CRISPR/Cas9) term approaches, we show that reduced FBXO7 expression induces CIN in various colonic epithelial cell contexts, whereas FBXO7 knockout clones also exhibit hallmarks associated with cellular transformation, namely increased clonogenic and anchorage-independent growth. Collectively, these data demonstrate that FBXO7 is required to maintain genome stability identifying FBXO7 a novel CIN gene whose reduced expression may contribute to CRC development and progression.

Non-obstructive azoospermia (NOA) represents one of the most serious forms of male infertility caused by spermatogenic failure. Despite multiple genes found to be associated with human NOA, the genetic basis of this idiopathic disease remains largely unknown. FBXO43 is a direct inhibitor of the anaphase-promoting complex/cyclosome (APC/C) E3 ligase and crucially important in mouse spermatogenesis. In this study, for the first time, we identified a homozygous nonsense mutation in FBXO43 c.1747C > T:p.Gln583X in two NOA brothers from a Chinese consanguineous family via whole-exome sequencing. FBXO43 was absent from testicular tissue of the proband, and FBXO43-immunostaining signals were invisible in the affected seminiferous tubules. Furthermore, in humans, FBXO43 defects cause meiotic arrest within early diplotene of prophase I. The results here demonstrate the pathogenicity of this loss-of-function mutation and confirmed that spermatocytes were unable to complete meiotic divisions without FBXO43 in humans. In mouse testicular protein extracts, three subunits of the APC/C, including ANAPC2, ANAPC8 and ANAPC10, were validated to interact directly with FBXO43, whereas no interactions were detected for FBXO43 and SKP1. This study furthers our understanding of the genetic basis of human NOA and provides insights into FBXO43 and male infertility.

Being one of the most prevalent malignancies, hepatocellular carcinoma (HCC) threatens the health of population all over the world. Numerous researches have confirmed that long noncoding RNAs (lncRNAs) play an important role in tumor progression. Nonetheless, the mechanisms of unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1) in HCC remain obscure. Thus, this study aims to investigate the regulatory role and mechanism of UNC5B-AS1 in HCC cells. In our research, UNC5B-AS1 was subjected to gene expression analysis by RT-qPCR. Biological functions of UNC5B-AS1 in HCC cells were measured by MTT, colony formation, EdU and transwell assays. The combination between UNC5B-AS1, lysine demethylase 2A (KDM2A) and miR-4306 was validated by mechanism assays. Result showed UNC5B-AS1 was upregulated in HCC tissues and cells, contributing to the development of cancer staging and survival rate of HCC patients. Moreover, UNC5B-AS1 deficiency inhibited the proliferation, migration and epithelial–mesenchymal transition (EMT) of HCC cells. Furthermore, UNC5B-AS1 could interact with miR-4306 in HCC cells. Similarly, KDM2A was proved as the target gene of miR-4306. Finally, miR-4306 downregulation or KDM2A overexpression reversed the prohibitive role of UNC5B-AS1 knockdown in HCC progression. In short, UNC5B-AS1 accelerates the proliferation, migration and EMT of HCC cells via the regulation of miR-4306/KDM2A axis.

Lung epithelial cell death is a prominent feature of acute lung injury and acute respiratory distress syndrome (ALI/ARDS), which results from severe pulmonary infection leading to respiratory failure. Multiple mechanisms are believed to contribute to the death of epithelia; however, limited data propose a role for epigenetic modifiers. In this study, we report that a chromatin modulator protein arginine N-methyltransferase 4/coactivator-associated arginine methyltransferase 1 (PRMT4/CARM1) is elevated in human lung tissues with pneumonia and in experimental lung injury models. Here PRMT4 is normally targeted for its degradation by an E3 ubiquitin ligase, SCFFBXO9, that interacts with PRMT4 via a phosphodegron to ubiquitinate the chromatin modulator at K228 leading to its proteasomal degradation. Bacterial-derived endotoxin reduced levels of SCFFBXO9 thus increasing PRMT4 cellular concentrations linked to epithelial cell death. Elevated PRMT4 protein caused substantial epithelial cell death via caspase 3-mediated cell death signaling, and depletion of PRMT4 abolished LPS-mediated epithelial cell death both in cellular and murine injury models. These findings implicate a unique molecular interaction between SCFFBXO9 and PRMT4 and its regulation by endotoxin that impacts the life span of lung epithelia, which may play a key role in the pathobiology of tissue injury observed during critical respiratory illness.

The current therapeutic strategy for hyperglycemia is reasonable diet and appropriate exercise with drugs, whose outcome is unsatisfied. Therefore, we aimed to explore the new candidate drug 7-Ethoxyrosmanol (7ERM) on hyperglycemia-induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) were treated with different doses of 7ERM in the presence of 33 mM high glucose to measure cell injury, inflammation, and reactive oxygen species (ROS) production. Then F-box/LRR-repeat protein 7 (FBXL7) knockdown by siRNA or overexpressed by plasmid in HUVECs was assessed its effect in the protective role on hyperglycemia-induced endothelial dysfunction. 7ERM time-dependently increased high glucose-induced cell injury, the secretions of pro-inflammatory cytokines and ROS production in HUVECs. Moreover, high glucose time-dependently increased the FBXL7 expressions, which could be gradually inhibited by 7ERM. FBXL7 knockdown ameliorated high glucose-induced cell injury. On the contrary, FBXL7 over-expression inhibited the protective effect of 7ERM on cell injury. In conclusion, 7ERM effectively attenuates high glucose-induced endothelial dysfunction in HUVECs by regulating FBXL7 expression, indicating its potential as a therapeutic drug to treat hyperglycemia.

Ovarian cancer (OC) is one of the most common cancers among women, characterized by various histological subtypes. Here, we aimed to investigate the biological function of F-box and leucine-rich repeat protein 20 (FBXL20) in the malignant phenotype of OC cells and its related mechanism. The expression of FBXL20 in OC tissue and normal tissue samples was analyzed through the GEPIA database. Quantitative real-time PCR (qRT-PCR), immunohistochemistry (IHC) and Western blot were employed to detect the expression of miR-195-5p and FBXL20 in OC tissues and cell lines. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) experiment and flow cytometry were applied to detect cell proliferation, cell cycle and apoptosis. Bioinformatics analysis and dual-luciferase reporter gene experiments were adopted to predict and validate the targeting relationship between miR-195-5p and FBXL20 mRNA 3'-untranslated region (3'UTR). Correlation between the expressions of miR-195-5p and FBXL20 mRNA was analyzed by Pearson correlation analysis. FBXL20 expression was upregulated in OC, and its high expression level was significantly associated with higher International Federation of Gynecology and Obstetrics (FIGO) stage and poor tumor differentiation. Functionally, overexpression of FBXL20 promoted proliferation, inhibited apoptosis and accelerated the cell cycle in OC cells in comparison to control group, and knockdown of FBXL20 exerted the opposite effects. Mechanistically, miR-195-5p directly targeted FBXL20 and negatively regulated its expression. Pearson correlation analysis indicated that miR-195-5p was negatively correlated with FBXL20 mRNA expression. In addition, overexpression of miR-195-5p reversed the above biological functions of FBXL20 in OC cells. FBXL20, negatively regulated by miR-195-5p, accelerates the proliferation and cell cycle progression of OC cells, and inhibits cell apoptosis, which might act as a prospective prognostic biomarker and a promising therapeutic target for OC.

Parkinson's disease (PD) is one of the most common progressive neurodegenerative diseases. Some microRNAs (miRNAs) play critical roles in the development of many neurological diseases. This study aims to evaluate the clinical significance and biological function of miR-485-3p in the development and progression of PD. The expression of miR-485-3p in serum of PD patients was analyzed by quantitative real-time PCR (qRT-PCR). LPS-treated microglia BV2 cells were used to mimic neuroinflammation in the pathogenesis of PD. The levels of inflammatory cytokines, including IL-1β, IL-6 and TNF-α, were detected by enzyme-linked immunosorbent assay (ELISA). The diagnosis value of miR-485-3p was evaluated by plotting receiver operating characteristic (ROC) curves. A luciferase reporter assay was performed to demonstrate the interaction between miR-485-3p and FBXO45. The results showed that miR-485-3p was significantly up-regulated in serum of PD patients compared with that in both Alzheimer's disease (AD) and healthy cases, and had diagnostic accuracy for PD screening. The activated microglia BV2 cells induced by LPS also had elevated miR-485-3p, and the knockdown of miR-485-3p inhibited the release of pro-inflammatory cytokines. FBXO protein 45 (FBXO45) served as a potential target of miR-485-3p, which was speculated to mediate the function of miR-485-3p. Our results suggest that the up-regulated expression of miR-485-3p in PD may be a novel diagnostic biomarker for PD. Reducing the expression level of miR-485-3p can inhibit the inflammatory responses of BV2 cells, which indicated that miR-485-3p, as a regulator of neuroinflammation, may have the potential as a therapeutic target in PD.

Di Qiao,1 Chi Hu,1 Qiuyan Li,1 Jun Fan2 1Department of Respiratory Medicine, Kunming Tongren Hospital, Kunming City, Yunnan Province, People’s Republic of China; 2Department of Cardiovascular Medicine, Chinese People’s Liberation Army Joint Service Support Unit 920 Hospital, Kunming City, Yunnan Province, People’s Republic of ChinaCorrespondence: Jun FanDepartment of Cardiovascular Medicine, Chinese People’s Liberation Army Joint Service Support Unit 920 Hospital, Block 2, Block 3, Classic Shuangcheng District, Guling Road, Wuhua District, Kunming, 650032, Yunnan Province, People’s Republic of ChinaTel/Fax +86 010-51955890Email asd1331548@163.comBackground: Emerging evidence has reported that circular RNAs (circRNAs) are aberrantly expressed and act as significant regulators in pathological processes of chronic obstructive pulmonary disease (COPD). Here, the purpose of this article was to evaluate and clarify the biological functions and mechanism of circRNA single stranded interacting protein 1 (circ-RBMS1) in cigarette smoke (CS)-induced COPD.Methods: Human bronchial epithelial cells 16HBE treated with or without cigarette smoke extract (CSE) were used in the experimental group in vitro. Levels of circ-RBMS1, microRNA (miR)-197-3p, and F-box only protein 11 (FBXO11) were detected using quantitative real-time polymerase chain reaction and Western blot. The present study used cell counting kit-8 (CCK-8), 5-ethynyl-2ʹ-deoxyuridine (EDU), flow cytometry and Western blot assays to determine the survival of 16HBE cells. The activity of interleukin (IL)-1β, tumor necrosis factor (TNF-α), malondialdehyde (MDA) and superoxide dismutase (SOD) was evaluated using the relative commercial kits. Dual-luciferase activity and RIP assays were used to identify the target relationship between miR-197-3p and circ-RBMS1 or FBXO11.Results: Circ-RBMS1 was highly expressed in COPD patients, and CSE induced an increased expression of circ-RBMS1 in a dose-dependent manner. Functionally, knockdown of circ-RBMS1 attenuated CSE-induced apoptosis, inflammation and oxidative stress in 16HBE cells. Circ-RBMS1 directly targeted miR-197-3p, and miR-197-3p inhibition reversed the effects of circ-RBMS1 knockdown on CSE-induced 16HBE cells. FBXO11 was a target of miR-197-3p. MiR-197-3p overexpression or FBXO11 silencing reduced the apoptosis, inflammation and oxidative stress in CSE-induced 16HBE cells. Moreover, miR-197-3p exerted its effects by targeting FBXO11. Additionally, circ-RBMS1 acted as a sponge for miR-197-3p to positively regulate FBXO11 expression in 16HBE cells.Conclusion: Circ-RBMS1 knockdown alleviated CSE-induced apoptosis, inflammation and oxidative stress in 16HBE cells via miR-197-3p/FBXO11 axis, suggesting a new insight into the pathogenesis of CS-induced COPD.Keywords: circ-RBMS1, miR-197-3p, apoptosis, inflammation, FBXO11, COPD

Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is a type of RNA binding protein that highly expressed in a variety of tumors and plays a vital role in tumor progression. However, its post-translational regulation through ubiquitin-mediated proteolysis and the cellular mechanism responsible for its proteasomal degradation remains unclear. F-box proteins (FBPs) function as the substrate recognition subunits of SCF ubiquitin ligase complexes and directly bind to substrates. The aberrant expression or mutation of FBPs will lead to the accumulation of its substrate proteins that often involved in tumorigenesis. Here we discover FBXO16, an E3 ubiquitin ligase, to be a tumor suppressor in ovarian cancer, and patients with the relatively high expression level of FBXO16 have a better prognosis. Silencing or depleting FBXO16 significantly enhanced ovarian cancer cell proliferation, clonogenic survival, and cell invasion by activating multiple oncogenic pathways. This function requires the F-box domain of FBXO16, through which FBXO16 assembles a canonical SCF ubiquitin ligase complex that constitutively targets hnRNPL for degradation. Depletion of hnRNPL is sufficient to inactive multiple oncogenic signaling regulated by FBXO16 and prevent the malignant behavior of ovarian cancer cells caused by FBXO16 deficiency. FBXO16 interacted with the RRM3 domain of hnRNPL via its C-terminal region to trigger the proteasomal degradation of hnRNPL. Failure to degrade hnRNPL promoted ovarian cancer cell proliferation in vitro and tumor growth vivo, phenocopying the deficiency of FBXO16 in ovarian cancer.

The regulation of proteasome activity is essential to cellular homeostasis and defects have been implicated in various disorders including Parkinson disease. The F-box protein FBXO7 has been implicated in early-onset parkinsonism and has previously been shown to have a regulatory role in proteasome activity and assembly. Here, we report the association of the E3 ubiquitin ligase FBXO7-SCF (SKP1, cullin-1, F-box protein) with the BAG6 complex, consisting of the subunits BAG6, GET4 and UBL4A. We identify the subunit GET4 as a direct interactor of FBXO7 and we show that the subunits GET4 and UBL4A are required for proper proteasome activity. Our findings demonstrate reduced binding of FBXO7 variants to GET4 and that FBXO7 variants bring about reduced proteasome activity. In addition, we find that GET4 is a non-proteolytic substrate of FBXO7, that binding of GET4 to BAG6 is enhanced in the presence of active FBXO7-SCF and that the cytoplasmic localization of the BAG6 complex is dependent on the E3 ubiquitin ligase activity. Taken together, our study shows that the parkinsonism-associated FBXO7 cooperates with the BAG6 complex in proteasome function and determines the subcellular localization of this complex.

Transthyretin (TTR) has been proved to repress neovascularization in diabetic retinopathy environment by regulating the molecules in and downstream of the STAT-4/miR-223–3p/FBXW7 signal pathway; however, the details of its direct targets are still not well understood. The interaction between TTR and a target in nucleus of human retinal microvascular endothelial cells (hRECs), heterogeneous nuclear ribonucleoprotein (hnRNP) A2B1, was screened by immunoprecipitation (IP) and mass spectrum (MS), and it was further confirmed by co-immunoprecipitation (co-IP). Regarding ZDOCK analysis using Discovery Studio, the interface and potential binding sites between TTR and hnRNPA2B1 were simulated; mutants were designed in these regions and five soluble ones were recombinantly expressed and prepared; the interaction between TTR and hnRNPA2B1 were disrupted by several mutated residues. In addition, for several mutated TTRs, the inhibition activities against the proliferation, migration and tube formation of hRECs were absent in vitro. Following the disruption of TTR-hnRNPA2B1, the molecules in and downstream of STAT-4/miR-223–3p/FBXW7 signal pathway, including STAT-4, miR-223–3p, FBXW7 p-Akt and Notch1 could not be regulated by TTR mutants; therefore, a TTR-hnRNPA2B1/STAT-4/miR-223–3p/FBXW7 was proposed. In conclusion, this work suggested that TTR should play a physiological role in diabetic environment by the direct binding with hnRNPA2B1, and it provided a theoretical basis for clinical diagnosis, therapy and further application.

DNAJB9, a member of the heat shock protein 40 family, acts as a multifunctional player involved in the maintenance of their client proteins and cellular homeostasis. However, the mechanistic action of DNAJB9 in human malignancies is yet to be fully understood. In this study, we found that ectopic restoration of DNAJB9 inhibits the migration, invasion, in vivo metastasis, and lung colonization of triple-negative breast cancer (TNBC) cells. Mechanistically, DNAJB9 stabilizes FBXO45 protein by suppressing self-ubiquitination and reduces the abundance of ZEB1 by Lys48-linked polyubiquitination to inhibit the epithelial–mesenchymal transition (EMT) and metastasis. Clinically, the reduction of DNAJB9 expression, concomitant with decreased FBXO45 abundance in breast cancer tissues, correlates with poorer clinical outcomes of patients with breast cancer. Taken together, our results provide a novel insight into the metastasis of TNBC and define a promising therapeutic strategy for cancers with overactive ZEB1 by regulating the DNAJB9–FBXO45 signaling axis.

It has been implied that deregulation of cyclin D1 turnover under stresses can facilitate genomic instability and trigger tumorigenesis. Much focus has been placed on identifying the E3 ligases responsible for mediating cyclin D1 degradation. However, the findings were quite controversial and cell type-dependent. Little is known about how cyclin D1 is regulated in precancerous cells upon DNA damage and which E3 ligases mediate the effects. Here we found cyclin D1 reduction is an early response to DNA damage in immortalized esophageal epithelial cells, with expression dropping to a low level within 1 h after γ-irradiation. Comparison of temporal expression of cyclin D1 upon DNA damage between immortalized NE083-hTERT and NE083-E6E7, the latter being p53/p21-defective, showed that DNA damage-induced rapid cyclin D1 reduction was p53-independent and occurred before p21 accumulation. Overexpression of cyclin D1 in NE083-E6E7 cells could attenuate G0/G1 cell cycle arrest at 1 h after irradiation. Furthermore, rapid reduction of cyclin D1 upon DNA damage was attributed to proteasomal degradation, as evidenced by data showing that proteasomal inhibition by MG132 blocked cyclin D1 reduction while cycloheximide facilitated it. Inhibition of ATM activation and knockdown of E3 ligase adaptor FBX4 reversed cyclin D1 turnover in immortalized NE083-hTERT cells. Further study showed that knockdown of FBX4 facilitated DNA breaks, as indicated by an increase in γ-H2AX foci in esophageal cancer cells. Taken together, the results substantiated a pivotal role of ATM and FBX4 in cyclin D1 proteolysis upon DNA damage in precancerous esophageal epithelial cells, implying that deregulation of the process may contribute to carcinogenesis of esophageal squamous cell carcinoma.

We describe a case of a boy with neurodevelopmental delay and a diffuse large B-cell lymphoma (DLBCL) in whom we discovered a germline de novo 2p16.3 deletion including MSH6 and part of the FBXO11 gene. A causative role for MSH6 in cancer development was excluded based on tumor characteristics. The constitutional FBXO11 deletion explains the neurodevelopmental delay in the patient. The FBXO11 protein is involved in BCL-6 ubiquitination and BCL-6 is required for the germinal center reaction resulting in B cell differentiation. Somatic loss of function alterations of FBXO11 result in BCL-6 overexpression which is a known driver in DLBCL. We therefore consider that a causative relationship between the germline FBXO11 deletion and the development of DLBCL in this boy is conceivable.

The underlying mechanism of orphan nuclear receptor estrogen-related receptor α (ERRα) in breast cancer was investigated by identifying its interaction partners using mass spectrometry. F-box and leucine-rich repeat protein 10 (FBXL10), which modulates various physiological processes, may interact with ERRα in breast cancer. Here, we investigated the interaction between FBXL10 and ERRα, and their protein expression and correlation in breast cancer. Mechanical studies revealed that FBXL10 stabilized ERRα protein levels by reducing its poly-ubiquitylation and promoting its mono-ubiquitylation. The reporter gene assay and examination of ERRα target genes validated the increased transcriptional activity of ERRα due to its increased protein levels by FBXL10. FBXL10 also increased ERRα enrichment at the promoter region of its target genes. Functionally, FBXL10 facilitated the ERRα/peroxisome proliferator-activated receptor gamma coactivator 1 β (PGC1β)-mediated proliferation and tumorigenesis of breast cancer cells in vitro and in vivo. Our results uncovered a molecular mechanism linking the mono-ubiquitylation and protein stability of ERRα to functional interaction with FBXL10. Moreover, a novel regulatory axis of FBXL10 and ERRα regulating the proliferation and tumorigenesis of breast cancer cells was established.

The human orthologue of the tumor suppressor protein FBW7 is encoded by the Drosophila archipelago (ago) gene. Ago is an F-box protein that gives substrate specificity to its SCF ubiquitin ligase complex. It has a central role in multiple biological processes in a tissue-specific manner such as cell proliferation, cellular differentiation, hypoxia-induced gene expression. Here we present a previously unknown tissue-specific role of Ago in spermatid differentiation. We identified a classical mutant of ago which is semi-lethal and male-sterile. During the characterization of ago function in testis, we found that ago plays role in spermatid development, following meiosis. We confirmed spermatogenesis defects by silencing ago by RNAi in testes. The ago mutants show multiple abnormalities in elongating and elongated spermatids, including aberration of the cyst morphology, malformed mitochondrial structures, and individualization defects. Additionally, we determined the subcellular localization of Ago protein with mCherry-Ago transgene in spermatids. Our findings highlight the potential roles of Ago in different cellular processes of spermatogenesis, like spermatid individualization, and regulation of mitochondrial morphology.

The C2H2-type zinc finger transcription factor sensitive to proton rhizotoxicity 1 (STOP1) is crucial for aluminum (Al) resistance in Arabidopsis. The F-box protein Regulation of AtALMT1 Expression 1 (RAE1) was recently reported to regulate the stability of STOP1. There is a unique homolog of RAE1, RAH1 (RAE1 homolog 1), in Arabidopsis, but the biological function of RAH1 is still not known. In this study, we characterize the role of RAH1 and/or RAE1 in the regulation of Al resistance and plant growth. We demonstrate that RAH1 can directly interact with STOP1 and promote its ubiquitination and degradation. RAH1 is preferentially expressed in root caps and various vascular tissues, and its expression is induced by Al and controlled by STOP1. Mutation of RAH1 in rae1 but not the wild-type (WT) background increases the level of STOP1 protein, leading to increased expression of STOP1-regulated genes and enhanced Al resistance. Interestingly, the rah1rae1 double mutant shows reduced plant growth compared with the WT and single mutants under normal conditions, and introduction of stop1 mutation into the double mutant background can rescue its reduced plant growth phenotype. Our results thus reveal that RAH1 plays an unequally redundant role with RAE1 in the modulation of STOP1 stability and plant growth, and dynamic regulation of the STOP1 level is critical for the balance of Al resistance and normal plant growth.

In Rosaceae species, two gametophytic self-incompatibility (GSI) mechanisms are described, the Prunus self-recognition system and the Maleae (Malus/Pyrus/Sorbus) non-self- recognition system. In both systems the pistil component is a S-RNase gene, but from two distinct phylogenetic lineages. The pollen component, always a F-box gene(s), in the case of Prunus is a single gene, and in Maleae there are multiple genes. Previously, the Rosa S-locus was mapped on chromosome 3, and three putative S-RNase genes were identified in the R. chinensis ‘Old Blush’ genome. Here, we show that these genes do not belong to the S-locus region. Using R. chinensis and R. multiflora genomes and a phylogenetic approach, we identified the S-RNase gene, that belongs to the Prunus S-lineage. Expression patterns support this gene as being the S-pistil. This gene is here also identified in R. moschata, R. arvensis, and R. minutifolia low coverage genomes, allowing the identification of positively selected amino acid sites, and thus, further supporting this gene as the S-RNase. Furthermore, genotype–phenotype association experiments also support this gene as the S-RNase. For the S-pollen GSI component we find evidence for multiple F-box genes, that show the expected expression pattern, and evidence for diversifying selection at the F-box genes within an S-haplotype. Thus, Rosa has a non-self-recognition system, like in Maleae species, despite the S-pistil gene belonging to the Prunus S-RNase lineage. These findings are discussed in the context of the Rosaceae GSI evolution. Knowledge on the Rosa S-locus has practical implications since genes controlling floral and other ornamental traits are in linkage disequilibrium with the S-locus.

Pathological cardiac hypertrophy is a crucial cause of cardiac morbidity and mortality worldwide. However, the molecular mechanisms of this disease remain incompletely understood. As a member of E3 ubiquitin ligases, F-box/WD repeat-containing protein 5 (FBXW5) has been implicated in various pathophysiological processes. However, the role of FBXW5 in pathological cardiac hypertrophy remains largely unknown. In this study, decreased expression of FBXW5 was observed in both neonatal rat cardiomyocytes and mouse hearts with hypertrophic remodeling. Gain- and loss-of-function experiments were performed to study the potential function of FBXW5 in pathological cardiac hypertrophy. The in vitro results showed that FBXW5 had a protective effect against cardiac hypertrophy induced by phenylephrine (PE). FBXW5 knockout mice and mice with AAV9-mediated FBXW5 overexpression were generated. Consistent with the in vitro results, FBXW5 deficiency aggravated cardiac hypertrophy induced by pressure overload. FBXW5 overexpression protected mice from hypertrophic stimuli. Remarkably, FBXW5 ameliorated pathological cardiac hypertrophy by directly interacting with the protein transforming growth factor-beta-activated kinase 1 (TAK1) and blocking the mitogen-activated protein kinase (MAPK) signaling pathway. Furthermore, inhibition of TAK1 prevented the effects of FBXW5 on agonist- or pressure overload-induced cardiac hypertrophy. These findings imply that FBXW5 is an essential negative regulator and may be a potential therapeutic target for pathological cardiac hypertrophy.

Background and objectives IgA nephropathy is the most common form of primary GN worldwide. The evidence of geographic and ethnic differences, as well as familial aggregation of the disease, supports a strong genetic contribution to IgA nephropathy. Evidence for genetic factors in IgA nephropathy comes also from genome-wide association patient-control studies. However, few studies have systematically evaluated the contribution of coding variation in IgA nephropathy. Design, setting, participants, & measurements We performed a two-stage exome chip–based association study in 13,242 samples, including 3363 patients with IgA nephropathy and 9879 healthy controls of Han Chinese ancestry. Common variant functional annotation, gene-based low-frequency variants analysis, differential mRNA expression, and gene network integration were also explored. Results We identified three non-HLA gene regions (FBXL21, CCR6, and STAT3) and one HLA gene region (GABBR1) with suggestive significance (Pmeta Conclusions Five novel gene regions with suggestive significance for IgA nephropathy were identified and shed new light for further mechanism investigation.

Germline specification is a fundamental step for human reproduction and this biological phenomenon possesses technical challenges to study in vivo as it occurs immediately after blastocyst implantation. The establishment of in vitro human primordial germ cell-like cells (hPGCLCs) induction system allows sophisticated characterization of human primordial germ cells (hPGCs) development. However, the underlying molecular mechanisms of hPGCLC specification are not fully elucidated. Here, we observed particularly high expression of the histone demethylase KDM2B in male fetal germ cells (FGCs) but not in male somatic cells. Besides, KDM2B shared similar expression pattern with hPGC marker genes in hPGCLCs, suggesting an important role of KDM2B in germ cell development. Although deletion of KDM2B had no significant effects on human embryonic stem cell (hESC)'s pluripotency, loss of KDM2B dramatically impaired hPGCLCs differentiation whereas ectopically expressed KDM2B could efficiently rescue such defect, indicating this defect was due to KDM2B's loss in hPGCLC specification. Mechanistically, as revealed by the transcriptional profiling, KDM2B suppressed the expression of somatic genes thus inhibited somatic differentiation during hPGCLC specification. These data collectively indicate that KDM2B is an indispensable epigenetic regulator for hPGCLC specification, shedding lights on how epigenetic regulations orchestrate transcriptional events in hPGC development for future investigation.

Pink1, Parkin and Fbxo7, three autosomal recessive familial genes of Parkinson's disease (PD), have been implicated in mitophagy pathways for quality control and clearance of damaged mitochondria, but the interplay of these three genes still remains unclear. Here we present that Fbxo7 and Pink1 play a reciprocal role in the regulation of their protein levels. Regardless of the genotypes of Fbxo7, the wild type and the PD familial mutants of Fbxo7 stabilize the processed form of Pink1, supporting the prior study that none of the PD familial mutations in Fbxo7 have an effect on the interaction with Pink1. On the other hand, the interaction of Fbxo7 with Bag2 further facilitates its capability to stabilize Pink1. Intriguingly, the stabilization of Fbxo7 by Pink1 is specifically observed in substantial nigra pars compacta but striatum and cerebral cortex. Taken together, our findings support the notion that Fbxo7 as a scaffold protein has a chaperon activity in the stabilization of proteins.

FBPs are components of the SCF protein E3 ubiquitin ligase and can specifically bind to substrates and thereby regulate multiple tumor behaviors. However, the role of FBPs, FBXO25 in particular, in cutaneous squamous cell carcinoma (cSCC) has not been explored yet. In this study, we found FBXO25 to be highly expressed in cSCC in mice and in vitro, whereas it was significantly less expressed in normal keratinocytes. Stable silencing of FBXO25 in SCC13 cells led to reduced tumor growth, and the knockdown of FBXO25 was accompanied by downregulation of cyclin D1. Correspondingly, stable overexpression of cyclin D1 in FBXO25-deficient SCC13 tumors increased tumor growth, supporting the hypothesis that FBXO25 promotes cSCC growth and metastasis through cyclin D1. Moreover, we found FBXO25 and cyclin D1 interaction to be facilitated through the repressor (Oct-1) of cyclin D1. Our data indicate that Oct-1 interacts directly with FBXO25 and undergoes downregulation, consequently stabilizing cyclin D1 and promoting tumor growth and metastasis.

MicroRNA let‐7b is a potent tumor suppressor and targets crucial oncogenes. Previous studies have shown that let‐7b expression is suppressed in ovarian cancer; however, the regulatory mechanisms of let‐7b in ovarian cancer are still not well defined. The cellular role and targets of let‐7b in ovarian cancer remain elusive. In the present study, we showed that histone demethylase, KDM2B, directly suppressed let‐7b expression by H3K36me2 demethylation. Moreover, let‐7b inhibited EZH2 expression in ovarian cancer cells. Based on these results we know that let‐7b antagonizes the enhancement of EZH2 expression caused by KDM2B overexpression, and its expression is negatively correlated with KDM2B and EZH2 expression. More importantly, proliferation, migration, and wound healing assays showed that let‐7b inhibited ovarian cancer cell proliferation and migration in vitro. Additionally, let‐7b overexpression neutralized KDM2B‐promoted cell proliferation and migration. Furthermore, downregulation of let‐7b increased the xenografted tumor volumes in nude mice that were transplanted with KDM2B‐silenced cells. EZH2 silencing reversed the tumor growth enhancement mediated by inhibition of let‐7b. Last, we show that let‐7b expression is suppressed in ovarian carcinomas and its expression is negatively associated with the clinicopathological features of ovarian cancer, including histological type, histological grade, International Federation of Gynecology and Obstetrics (FIGO) stage, and lymph node metastatic status. In conclusion, in ovarian cancer, let‐7b expression is epigenetically suppressed by high expression of KDM2B. The loss of let‐7b upregulates the expression of EZH2, which promotes ovarian cancer growth in vitro and in vivo., In ovarian cancer, let‐7b expression is epigenetically suppressed by the high expression of KDM2B. The loss of let‐7b upregulates the expression of EZH2, which promotes ovarian cancer growth in vitro and in vivo.

Background Our previous study demonstrated that lysine demethylase 2A (KDM2A) enhances stemness in breast cancer cells. This demethylase is also highly expressed in cancer-associated fibroblasts (CAFs). However, its clinical significance is unclear. Methods The expression of KDM2A in CAFs was studied using immunohistochemical staining and its association with clinicopathological features and patient’s survival was tested. Overexpression and knockdown strategies were used to investigate KDM2A-regulated genes in fibroblasts. Senescent cells were detected by using β-galactosidase staining. The in vivo tumour-promoting activity of stromal KDM2A was confirmed by animal study. Results Increase of stromal KDM2A is associated with advanced tumour stage and poor clinical outcome in breast cancer patients. Cancer-derived cytokines stimulated KDM2A expression in normal fibroblasts and transformed them into CAFs. Upregulation of KDM2A induced p53-dependent senescence in fibroblasts and enhanced the release of cytokines, which reciprocally promoted cancer cell proliferation. Additionally, KDM2A upregulated programmed death-ligand 1 (PD-L1) expression via transcriptional activation in fibroblasts. Knockdown of KDM2A completely abolished the tumour-promoting activity of CAFs on breast tumour growth in vivo and diminished PD-L1 expression in the stroma of tumour tissues. Conclusions Stromal KDM2A plays an oncogenic role in breast cancer and inhibition of KDM2A reduces fibroblast senescence and suppresses tumour growth.

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 C2C12muscle 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.

Myelodysplastic syndrome (MDS) is a heterogeneous myeloid malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of transformation to secondary acute myeloid leukemia (sAML). A number of genetic abnormalities have been identified in MDS and sAML, but sensitive sequencing methods can detect these mutations in nearly all healthy individuals by 60 years of age. To discover novel cellular pathways that accelerate MDS and sAML, we performed a CRISPR/Cas9 screen in the human MDS-L cell line. We report here that loss of the F-Box protein FBXO11, a component of the SCF ubiquitin ligase complex, confers cytokine independent growth to MDS-L cells, suggesting a tumor suppressor role for FBXO11 in myeloid malignancies. Putative FBXO11 substrates are enriched for proteins with functions in RNA metabolism and, of note, spliceosome mutations that are commonly found in MDS/sAML are rare in patients with low FBXO11 expression. We also reveal that loss of FBXO11 leads to significant changes in transcriptional pathways influencing leukocyte proliferation, differentiation, and apoptosis. Last, we find that FBXO11 expression is reduced in patients with secondary AML. We conclude that loss of FBXO11 is a mechanism for disease transformation of MDS into AML, and may represent a future therapeutic target.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that causes adult T-cell leukemia (ATL). The viral protein HTLV-1 basic leucine-zipper factor (HBZ), which is constitutively expressed in all ATL patient cells, contributes toward the development of ATL; however, the underlying mechanism has not been elucidated yet. Here, we identified HS-1-associated protein X-1 (HAX-1) as a novel binding partner of HBZ. Interestingly, HAX-1 specifically associated with HBZ-US, but not HBZ-SI, in the cytoplasm. HBZ suppressed the polyubiquitination levels of HAX-1 protein by inhibiting the association HAX-1 with F-box protein 25 (FBXO25), which is a member of the SCF E3 ubiquitin ligase complex, and promoted the stabilization of HAX-1 levels. In fact, the protein levels of HAX-1 were significantly increased in HTLV-1 infected and the overexpressing HBZ in uninfected T-cell lines. Enhanced HAX-1 correlated well to suppression of caspase 9 processing, suggesting that HBZ may contribute to the enhancement of antiapoptotic function for HAX-1. Our results revealed a role for HBZ on HAX-1 stabilization by abrogating the ubiquitination-mediated degradation pathway, which may play an important role in understanding the potential mechanisms of HTLV-1 related pathogenesis.

Blood-tumour barrier (BTB) has been known to significantly attenuate the efficacy of chemotherapy for glioma. In this report, we identified that insulin-like grown factor 2 mRNA-binding protein 2 (IGF2BP2) was over-expressed in glioma microvessel and glioma endothelial cells (GECs). Knockdown of IGF2BP2 decreased the expression of lncRNA FBXL19-AS1 and tight junction-related proteins, thereby promoting BTB permeability. FBXL19-AS1 was over-expressed and more enriched in the cytoplasm of GECs. In addition, FBXL19-AS1 could bind to 3'-UTR of ZNF765 mRNA and down-regulate ZNF765 mRNA expression through STAU1-mediated mRNA decay (SMD). The low expression of ZNF765 was discovered in GECs and verified to increase BTB permeability by inhibiting the promoter activities of tight junction-related proteins. Meanwhile, ZNF765 also inhibited the transcriptional activity of IGF2BP2, thereby forming a feedback loop in regulating the BTB permeability. Single or combined application of silenced IGF2BP2 and FBXL19-AS1 improved the delivery and antitumor efficiency of doxorubicin (DOX). In general, our study revealed the regulation mechanism of IGF2BP2/FBXL19-AS1/ZNF765 axis on BTB permeability, which may provide valuable insight into treatment strategy for glioma.

Ubiquitin‐dependent protein degradation has been implicated in the control of various cellular processes such as cell cycle control, transcriptional regulation, DNA damage repair, and apoptosis, many of which are involved in the initiation, progression, metastasis, and drug resistance of cancers. E3 ubiquitin ligases are known to be the second most prevalent cancer‐related functional gene family next to protein kinases. Of these, FBXO22, an F‐box receptor subunit of SCF E3 ligase, has recently been proposed to play a critical role in multiple aspects related to cancer development and therapy response. Firstly, FBXO22 is a key regulator of senescence induction through ubiquitylation of p53 for degradation. FBXO22 also acts as a molecular switch for the antagonistic and agonistic actions of selective estrogen receptor modulators (SERM) and determines the sensitivity of breast cancer to SERM by ubiquitylating KDM4B complexed with unliganded or SERMs‐bound estrogen receptor (ER). Furthermore, FBXO22 binds to Bach1, a pro‐metastatic transcription factor, suppressing Bach1‐driven metastasis of lung adenocarcinoma, and loss of FBXO22 facilitates metastasis. These findings, as well as other reports, unveiled strikingly important roles of FBXO22 in cancer development and therapeutic strategy. In this review, we summarize recent findings of how FBXO22 regulates major cancer suppression pathways., Fbxo22 plays strikingly important roles in cancer development and therapeutic strategy through the regulation of p53, KDM4B, and Bach1.

FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1) encodes an F-box protein that regulates photoperiod flowering in Arabidopsis under long-day conditions (LDs). Gibberellin (GA) is also important for regulating flowering under LDs. However, how FKF1 and the GA pathway work in concert in regulating flowering is not fully understood. Here, we showed that the mutation of FKF1 could cause accumulation of DELLA proteins, which are crucial repressors in GA signaling pathway, thereby reducing plant sensitivity to GA in flowering. Both in vitro and in vivo biochemical analyses demonstrated that FKF1 directly interacted with DELLA proteins. Furthermore, we showed that FKF1 promoted ubiquitination and degradation of DELLA proteins. Analysis of genetic data revealed that FKF1 acted partially through DELLAs to regulate flowering under LDs. In addition, DELLAs exerted a negative feedback on FKF1 expression. Collectively, these findings demonstrate that FKF1 promotes flowering partially by negatively regulating DELLA protein stability under LDs, and suggesting a potential mechanism linking the FKF1 to the GA signaling DELLA proteins.

The brain consists of various areas with anatomical features. Neurons communicate with one another via excitatory or inhibitory synaptic transmission. Altered abundance of neurotransmitters, including glutamate and gamma-aminobutyric acid (GABA), in specific brain regions is closely involved in severe neurological diseases, such as schizophrenia and obsessive-compulsive disorder. SCRAPPER, a ubiquitin E3 ligase, regulates synaptic transmission. Scrapper gene deficiency results in defective neurotransmission due to excessive secretion of neurotransmitters. The present study employed matrix-assisted laser desorption/ionization imaging mass spectrometry to analyze the abundance of amino acid neurotransmitters in Scrapper knockout (SCR-KO) mice. SCR-KO mice exhibited significantly increased glutamate levels in the isocortex (CTX), corpus callosum (CC), thalamus (TH), midbrain (MB), cerebellar cortex (CBX), and caudoputamen (CP) and increased GABA levels in the CTX, CC, TH, MB, CBX and hypothalamus (HY) compared with wild-type mice. These findings indicate that Scrapper deficiency leads to upregulated glutamate and GABA levels in multiple regions. Our results show a differential, region-specific effect of Scrapper on the abundance of glutamate and GABA.

Recent studies revealed that the histone demethylase KDM2B regulates the epithelial markers E-Cadherin and ZO-1, the RhoA/B/C-small-GTPases and actin cytoskeleton organization, in DU-145 prostate- and HCT-116 colon-tumor cells. Here we addressed the role of KDM2B in the activation of Focal Adhesion Kinase (FAK)-signaling and its involvement in regulating tumor cell motility. We used RT-PCR for gene transcriptional analysis, Western blotting for the assessment of protein expression and activity and wound-healing assay for the study of cell migration. KDM2B overexpression or silencing controls the activity of FAK in DU-145 prostate- and HCT-116 colon-tumor cells without affecting gene transcription and protein expression of this kinase. Upon KDM2B overexpression in DU-145 cells, significantly enhanced migration was observed, which was abolished in cells pretreated by the specific phosphoinositide-3 kinase (PI3 K) inhibitor LY294002, implying involvement of FAK/PI3 K signaling in the migration process. In line with this, the p85-PI3 K-subunit was downregulated upon knockdown of KDM2B in DU-145 cells, while the opposite effect became evident in KDM2B-overexpressing cells. These results revealed a novel functional role of KDM2B in regulating the activation of the FAK/PI3 K signaling in prostate cancer cells that participates in the control of cell motility.

A generally used chemotherapeutic drug for glioma, a frequently diagnosed brain tumour, is temozolomide (TMZ). Our study investigated the activity of FBXL7 and miR‐152‐5p in glioma. Levels of microRNA‐152‐5p (miR‐152‐5p) and the transcript and protein of FBXL7 were assessed by real‐time PCR and Western blotting, respectively. The migratory and invasive properties of cells were measured by Transwell migration and invasion assay and their viability were examined using CCK‐8 assay. Further, the putative interaction between FBXL7 and miR‐152‐5p were analysed bioinformatically and by luciferase assay. The activities of FBXL7, TMZ and miR‐152‐5p were analysed in vivo singly or in combination, on mouse xenografts, in glioma tumorigenesis. The expression of FBXL7 in glioma tissue is significantly up‐regulated, which is related to the poor prognosis and the grade of glioma. TMZ‐induced cytotoxicity, proliferation, migration and invasion in glioma cells were impeded by the knock‐down of FBXL7 or overexpressed miR‐152‐5p. Furthermore, the expression of miR‐152‐5p reduced remarkably in glioma cells and it exerted its activity through targeted FBXL7. Overexpression of miR‐152‐5p and knock‐down of FBXL7 in glioma xenograft models enhanced TMZ‐mediated anti‐tumour effect and impeded tumour growth. Thus, the miR‐152‐5p suppressed the progression of glioma and associated tumorigenesis, targeted FBXL7 and increased the effect of TMZ‐induced cytotoxicity in glioma cells, further enhancing our knowledge of FBXL7 activity in glioma.

Long noncoding RNA small nucleolus RNA host gene 14 (SNHG14) has been shown to exert oncogenic functions in seceral cancers, but its role in osteosarcoma still unclear. In this present study, we found that SNHG14 was significantly upregulated in osteosarcoma tissues and cell lines. Knockdown SNHG14 expression significantly inhibited osteosarcoma cell proliferation through inducing apoptosis. Further functional assays revealed that SNHG14 knockdown dramatically suppressed cell migration and invasion. Bioinformatics analysis and luciferase assays identified that microRNA-433–3p (miR-433–3p) was a direct target of SNHG14, and directly targeted F-box only protein 22 (FBXO22). Mechanistic analysis demonstrated that SNHG14 acted as a ceRNA in modulating osteosarcoma proliferation, migration and invasion by decoying miR-433–3p to upregulate FBXO22 expression. We also observed that knockdown of FBXO22 and SNHG14 and overexpression of miR-433–3p both dramatically suppressed osteosarcoma cell proliferation, migration and invasion, but induced cell apoptosis. Moreover, the suppresive effect of SNHG14 knockdown on osteosarcoma cell proliferation, invasion and migration was attenuated by miR-433–3p inhibitor. Our findings demonstrated that SNHG14 promoted osteosarcoma progression by acting as a ceRNA for miR-433–3p to regulate FBXO22 expression, suggesting that SNHG14 may serve as a potential therapeutic target in osteosarcoma patients.

Significance The neuron–glial lineage segregation of NC progenitors forming the DRG is essential for peripheral nervous system development. However, hierarchical transcriptional control does not explain how this lineage bifurcation occurs. Here, we provide in vivo evidence that a glial lineage specifier, Sox10, inhibits neurogenesis via a degradation mechanism. A ubiquitin-ligase, Fbxo9, acting downstream of Sox10 destabilizes the proneural Neurog2 protein, leading to the loss of neurogenic capacity in NC progenitors while leaving their glial potential intact. Functional inhibition of Fbxo9 and possibly other members of the F-box family lead to prolonged expression of Neurog2 protein that causes both the expansion of NC progenitors and precocious neuronal differentiation. Our results unravel a previously unrecognized mechanism in neuron–glial cell fate decision., The transcription factor Sox10 is a key regulator in the fate determination of a subpopulation of multipotent trunk neural crest (NC) progenitors toward glial cells instead of sensory neurons in the dorsal root ganglia (DRG). However, the mechanism by which Sox10 regulates glial cell fate commitment during lineage segregation remains poorly understood. In our study, we showed that the neurogenic determinant Neurogenin 2 (Neurog2) exhibited transient overlapping expression with Sox10 in avian trunk NC progenitors, which progressively underwent lineage segregation during migration toward the forming DRG. Gain- and loss-of-function studies revealed that the temporary expression of Neurog2 was due to Sox10 regulation of its protein stability. Transcriptional profiling identified Sox10-regulated F-box only protein (Fbxo9), which is an SCF (Skp1-Cul-F-box)-type ubiquitin ligase for Neurog2. Consistently, overexpression of Fbxo9 in NC progenitors down-regulated Neurog2 protein expression through ubiquitination and promoted the glial lineage at the expense of neuronal differentiation, whereas Fbxo9 knockdown resulted in the opposite phenomenon. Mechanistically, we found that Fbxo9 interacted with Neurog2 to promote its destabilization through the F-box motif. Finally, epistasis analysis further demonstrated that Fbxo9 and probably other F-box members mediated the role of Sox10 in destabilizing Neurog2 protein and directing the lineage of NC progenitors toward glial cells rather than sensory neurons. Altogether, these findings unravel a Sox10–Fbxo9 regulatory axis in promoting the glial fate of NC progenitors through Neurog2 destabilization.

O-GlcNAcylation is a dynamic and reversible post-translational modification of cytonuclear molecules that regulates cellular signaling. Elevated O-GlcNAcylation is a general property of cancer and plays a critical role in cancer progression. We previously showed that the expression of FOXM1, a critical oncogenic transcription factor widely overexpressed in solid tumors, was elevated in MKN45 cells, a human gastric cancer cell line, by the O-GlcNAcase inhibitor Thiamet G (TMG), which induces augmented O-GlcNAcylation. Here, we identified FBXL2 E3 ubiquitin ligase as a new target of O-GlcNAcylation. Consistent with the results in MKN45 cells, FOXM1 expression was increased, accompanied by its decreased ubiquitination and degradation by TMG in the other gastric cancer cell lines, including NUGC-3 cells. We found that FBXL2 ubiquitinated FOXM1, and the interaction with FBXL2 and ubiquitination of FOXM1 were reduced by TMG in NUGC-3 cells. Interestingly, FBXL2 was also ubiquitinated, which was promoted by TMG in the cells. Moreover, FOXM1 expression and cell proliferation were reduced in FBXL2-induced NUGC-3 cells, and the reductions were attenuated by TMG, indicating that FOXM1 was stabilized by O-GlcNAcylation-mediated degradation of FBXL2 to induce cancer progression. These data suggest that elevated O-GlcNAcylation contributes to cancer progression by suppressing FBXL2-mediated degradation of FOXM1.

Summary ERECTA PANICLE 3 (EP3) and ORYZA SATIVA F‐BOX KELCH 1 (OsFBK1) proteins share 57% and 54% sequence identity with the Arabidopsis F‐box protein HAWAIIAN SKIRT (HWS). Previously we showed that EP3 is a functional orthologue of HWS. Here we demonstrate that OsFBK1 is another functional orthologue of HWS and show the complexity of interaction between EP3 and OsFBK1 genes at different developmental stages of the plant. qRT‐PCR expression analyses and studies of EP3‐GFP and OsFBK1‐RFP promoter reporter lines demonstrate that although EP3 and OsFBK1 expression can be detected in the same tissues some cells exclusively express EP3 or OsFBK1 whilst others co‐express both genes. Loss, reduction or gain‐of‐function lines for EP3 and OsFBK1, show that EP3 and OsFBK1 affect plant architecture, organ size, floral organ number and size, floral morphology, pollen viability, grain size and weight. We have identified the putative orthologue genes of the rice microRNA pathway for ORYZA SATIVA DAWDLE (OsDDL) and ORYZA SATIVA SERRATE (OsSE), and demonstrated that EP3 and OsFBK1 affect their transcript levels as well as those of CROWN ROOT DEFECT 1/ORYZA SATIVA Exportin‐5 HASTY (CRD1/OsHST), ORYZA SATIVA DICER‐LIKE 1 (OsDCL) and ORYZA SATIVA WEAVY LEAF1 (OsWAF1). We show that EP3 affects OsPri‐MIR164, OsNAM1 and OsNAC1 transcript levels. OsNAC1 transcripts are modified by OsFBK1, suggesting two independent regulatory pathways, one via EP3 and OsMIR164 and the other via OsFBK1. Our data propose that EP3 and OsFBK1 conjointly play similar roles in rice to how HWS does in Arabidopsis.

F-box proteins (FBPs) are proteins containing an F-box domain and are one of three subunits in SKP1–Cullin1–F-box protein (SCF) E3 ligase complexes. Accumulated evidence has shown that FBP regulates tumorigenesis and the progression of human cancer via ubiquitination and degradation of downstream substrates, which can be oncoproteins or tumor suppressors. Emerging evidence has revealed that noncoding RNAs (ncRNAs) govern the expression of FBP in human cancers. Specifically, microRNAs (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs) have been shown to govern FBP expression in malignancies. Therefore, in this review article, we discuss how miRNAs target FBPs and participate in tumorigenesis and tumor progression. Moreover, we briefly highlight the role of lncRNAs and circRNAs in the regulation of FBPs in cancer. Therefore, targeting ncRNAs could be a novel approach to regulate FBPs for anticancer therapy.

Conditional degron technologies, which allow a protein of interest to be degraded in an inducible manner, are important tools for biological research, and are especially useful for creating conditional loss-of-function mutants of essential genes. The auxin-inducible degron (AID) technology, which utilizes plant auxin signaling components to control protein degradation in non-plant species, is a widely used small-molecular-controlled degradation method in yeasts and animals. However, the currently available AID systems still have room for further optimization. Here, we have improved the AID system for the fission yeast Schizosaccharomyces pombe by optimizing all three components: the AID degron, the small-molecule inducer, and the inducer-responsive F-box protein. We chose a 36-amino-acid sequence of the Arabidopsis IAA17 protein as the degron and employed three tandem copies of it to enhance efficiency. To minimize undesirable side effects of the inducer, we adopted a bulky analog of auxin, 5-adamantyl-IAA, and paired it with the F-box protein OsTIR1 that harbors a mutation (F74A) at the auxin-binding pocket. 5-adamantyl-IAA, when utilized with OsTIR1-F74A, is effective at concentrations thousands of times lower than auxin used in combination with wild-type OsTIR1. We tested our improved AID system on 10 essential genes and achieved inducible lethality for all of them, including ones that could not be effectively inactivated using a previously published AID system. Our improved AID system should facilitate the construction of conditional loss-of-function mutants in fission yeast.

Taxol is commonly used chemotherapy regimen for esophageal squamous cell carcinoma (ESCC). Study of the underlying mechanisms of Taxol chemoresistance provides better understanding of esophageal cancer treatment and may provide a rational molecular target for diagnosis and intervention. Here we showed FBXO31, which was reported to be highly expressed in ESCC and significantly associated with poor prognosis, could regulate ESCC chemosensitivity to Taxol. Silencing of FBXO31 in ESCC cells sensitized cells to Taxol treatment, evidenced by FACS analysis and TUNEL assay, showing as an increased apoptotic population in FBXO31-knockdown cells compared to the control cells. The mass spectrometry data and coimmunoprecipitation results showed FBXO31 could bind with cofilin-1. Cofilin-1 knockdown in FBXO31-overexpression cells reversed FBXO31-induced suppression of cell apoptosis, suggesting FBXO31-mediated Taxol chemoresistance is associated with cofilin-1. Furthermore, in vivo experiments confirmed that knockdown of FBXO31 sensitized ESCC to Taxol treatment. This finding substantiated a pivotal role of FBOX31 in ESCC chemoresistance, indicating that FBXO31 may be a potential indicator or target for drug resistance in ESCC.

High expression of programmed death-ligand 1 (PD-L1) in cancer cells drives immune-independent, cell-intrinsic functions, leading to resistance to DNA-damaging therapies. We find that high expression of the ubiquitin E3 ligase FBXO22 sensitizes nonsmall cell lung cancer (NSCLC) cells to ionizing radiation (IR) and cisplatin, and that activation of FBXO22 by phosphorylation is necessary for this function. Importantly, FBXO22 activates PD-L1 ubiquitination and degradation, which in turn increases the sensitivity of NSCLC cells to DNA damage. Cyclin-dependent kinase 5 (CDK5), aberrantly active in cancer cells, plays a crucial role in increasing the expression of PD-L1 in medulloblastoma [R. D. Dorand et al., Science 353, 399–403 (2016)]. We show in NSCLC cells that inhibiting CDK5 or reducing its expression increases the level of FBXO22, decreases that of PD-L1, and increases the sensitivity of the cells to DNA damage. We conclude that FBXO22 is a substrate of CDK5, and that inhibiting CDK5 reduces PD-L1 indirectly by increasing FBXO22. Pairing inhibitors of CDK5 with immune checkpoint inhibitors may increase the efficacy of immune checkpoint blockade alone or in combination with DNA-damaging therapies.

BackgroundThe function and regulatory mechanism of FBXO43 in breast cancer (BC) are still unclear. Here, we intended to determine the role and mechanism of FBXO43 in BC.MethodsFBXO43 expression in BC was evaluated by analysis of The Cancer Genome Atlas (TCGA). RT-qPCR and western blotting were utilized to detect FBXO43 expression in BC cell lines. Lentivirus was applied to downregulate FBXO43 in human BC cells. Proliferation assays were performed to evaluate the proliferative ability of BC cells. The apoptosis and cell cycle analysis of BC cells were analyzed by flow cytometry. Cell migration and invasion were investigated via Transwell assays. The function of FBXO43 in vivo was evaluated by constructing a xenograft mouse model. The proteins that might interact with FBXO43 in BC were identified by mass spectrometry, bioinformatics analysis, and co-immunoprecipitation (Co-IP) assays. Finally, rescue experiments were conducted to validate the recovery effects of the proteins interacting with FBXO43.ResultsFBXO43 was highly expressed in BC and was significantly downregulated after FBXO43 knockdown. The proliferation, migration, and invasion of BC cells were inhibited, and cell apoptosis was induced by FBXO43 knockdown. In addition, an in vivo experiment indicated that FBXO43 knockdown could inhibit the cell growth of BC. The results of the Co-IP assay showed that FBXO43 interacted with PCNA. Further rescue experiments confirmed that overexpression of PCNA significantly reversed the effects of FBXO43 knockdown on BC cells.ConclusionDownregulation of FBXO43 inhibits the tumor growth of BC by limiting its interaction with PCNA. FBXO43 might be a new potential oncogene and a therapeutic target for BC.

Background Most head and neck cancer (HNC) patients receive radiotherapy (RT) and develop toxicities. This genome-wide association study (GWAS) was designed to identify single nucleotide polymorphisms (SNPs) associated with common acute radiation-induced toxicities (RITs) in an HNC cohort. Methods A two-stage GWAS was performed in 1279 HNC patients treated with RT and prospectively scored for mucositis, xerostomia, sticky saliva, and dysphagia. The area under the curve (AUC) was used to estimate the average load of toxicity during RT. At the discovery study, multivariate linear regression was used in 957 patients, and the top-ranking SNPs were tested in 322 independent replication cohort. Next, the discovery and the replication studies were meta-analyzed. Results A region on 5q21.3 containing 16 SNPs showed genome-wide (GW) significance association at P-value –8 with patient-rated acute xerostomia in the discovery study. The top signal was rs35542 with an adjusted effect size of 0.17*A (95% CI 0.12 to 0.23; P-value –9). The genome wide significant SNPs were located within three genes (EFNA5, FBXL17, and FER). In-silico functional analysis showed these genes may be involved in DNA damage response and co-expressed in minor salivary glands. We found 428 suggestive SNPs (P-value –5) for other toxicities, taken to the replication study. Eleven of them showed a nominal association (P-value Conclusions This GWAS suggested novel SNPs for patient-rated acute xerostomia in HNC patients. If validated, these SNPs and their related functional pathways could lead to a predictive assay to identify sensitive patients to radiation, which may eventually allow a more individualized RT treatment.

Non-small cell lung cancer (NSCLC) is a type of cancer dominated by metastasis-induced death. The transcription factor BTB and CNC homology 1 (Bach1) regulates almost all metastasis steps by activating the transcription of critical metastatic genes. It is urgent to engineer a nanodrug enabling regulation of Bach1 against tumor metastasis. Herein, a minimalist nanodrug integrating chemodynamic therapy (CDT) and Bach1 degradation was reported to prevent metastasis of NSCLC. The nanodrug was achieved by self-assembly of ferrocene (Fc) and Tin protoporphyrin IX (TinPPIX). In our nanodrug, Fc not only triggers the production of highly cytotoxic ∙OH for tumor ablation via Fenton reaction, but also induces heme release from heme-containing proteins to stimulate Bach 1 degradation. Moreover, TinPPIX further augments the free heme level along with amplifies the CDT efficacy by disabling heme oxygenase-1 (HO-1)-mediated heme conversion into antioxidative bilirubin. The results showed that, compared with control group, TinPPIX/Fc nanodrug caused a four-fold increase in heme level, which triggered remarkable Bach1 degradation in Fbxo22-mediated manner and successfully inhibited Bach1-dominated metastasis. Therefore, this nanodrug could powerfully impeded NSCLC progression and metastasis, offering an innovative heme-regulatable chemodynamic therapeutic approach for lung cancer with strong metastasis capability.

The measurement of protein expression level plays a pivotal role in both biological and medical studies. Housekeeping proteins, generally encoded by housekeeping genes are used as loading control proteins to normalize protein expression. Obviously, proper reference standards are essential for adequate analysis of protein expression. However, our study showed that the widely used normalisation proteins, whose expression levels varied greatly among sperm samples, were unsuitable for data standardisation. To uncover the proteins steadily expressed in sperm, we analysed several published transcriptome data of sperm. Seven proteins whose expression levels were relatively stable (co-efficient variation values less than 0.35) were selected and further evaluated by quantitative real-time polymerase chain reaction, Western Blot (WB) and immunocytochemistry. Our results showed that among the classical housekeeping proteins, only β-tubulin remained constant in sperm samples from 85 individuals. Compared with other classical housekeeping proteins such as glyceraldehyde 3-phosphate dehydrogenase, actin and histone H3, Cullin-1 (CUL1) and F-box only protein 7 (FBXO7) seemed to be more suitable to be used as internal controls for WB in sperm protein studies. Combined with the locations of these proteins, CUL1 and FBXO7 were suggested to be used as a housekeeping protein for total proteins.