F-box Proteins Function Research Articles

Exploring the role of FBXL fbxl gene family in Soybean: Implications for plant height and seed size regulation.

F-box proteins constitute a significant family in eukaryotes and, as a component of the Skp1p-cullin-F-box complex, are considered critical for cellular protein degradation and other biological processes in plants. Despite their importance, the functions of F-box proteins, particularly those with C-terminal leucine-rich repeat (LRR) domains, remain largely unknown in plants. Therefore, the present study conducted genome-wide identification and in silico characterization of F-BOX proteins with C-terminal LRR domains in soybean (Glycine max L.) (GmFBXLs). A total of 45 GmFBXLs were identified. The phylogenetic analysis showed that GmFBXLs could be subdivided into ten subgroups and exhibited a close relationship with those from Arabidopsis thaliana, Cicer aretineum, and Medicago trunculata. It was observed that most cis-regulatory elements in the promoter regions of GmFBXLs are involved in hormone signalling, stress responses, and developmental stages. In silico transcriptome data illustrated diverse expression patterns of the identified GmFBXLs across various tissues, such as shoot apical meristem, flower, green pods, leaves, nodules, and roots. Overexpressing (OE) GmFBXL12 in Tianlong No.1 cultivar resulted in a significant difference in seed size, number of pods, and number of seeds per plant, indicated a potential increase in yield compared to wild type. This study offers valuable perspectives into the role of FBXLs in soybean, serving as a foundation for future research. Additionally, the identified OE lines represent valuable genetic resources for enhancing seed-related traits in soybean.

Multiple F-Box Proteins Collectively Regulate Cell Development and Pathogenesis in the Human Pathogen Cryptococcus neoformans.

The ubiquitin-proteasome system (UPS) mediates intracellular proteins degradation that influences various cellular functions in eukaryotic cells. The UPS is also involved in the development and virulence of pathogenic fungi. F-box proteins, which are part of the SCF (Skp1-Cullin-F-box protein) ligase, are a key component of UPS and are essential for the recognition of specific substrates. In this study, we identified 20 F-box proteins in C. neoformans and obtained deletion mutants for 19 of them. A comprehensive phenotypic analysis of these mutants revealed the diverse function of F-box proteins in stress response, cell size regulation, sexual reproduction, antifungal drug resistance, and fungal virulence in C. neoformans. The importance of three F-box proteins: Fbp4, Fbp8, and Fbp11, in these cellular functions were characterized in detail. This study provides an overall view of the F-box gene family in C. neoformans, which will lead to a better understanding of the function of fungal SCF E3 ligase-mediated UPS in fungal development and pathogenesis.

Ultrasound-targeted microbubble destruction-mediated silencing of FBXO11 suppresses development of pancreatic cancer

Ultrasound-targeted microbubble destruction (UTMD) has been a promising noninvasive tool for organ- or tissue-specific gene or drug delivery. This study aimed to explore the function of F-box protein 11 (FBXO11), an E3 ubiquitin ligase, in the development of pancreatic cancer (PCa). Differentially expressed genes in PCa were identified using the GSE62452 and GSE28735 datasets, and FBXO11 was significantly highly expressed in PCa. UTMD-mediated FBXO11 silencing significantly suppressed growth activity, epithelial-mesenchymal transition, migration, and invasion while reduced apoptosis of PCa cells in vitro and reduced the growth and metastasis of xenograft tumors in vivo. Importantly, UTMD-mediated sh-FBXO11 showed more pronounced tumor-suppressive effects than direct administration of sh-FBXO11 alone. The potential substrates of FBXO11 as an E3 ubiquitin ligase were predicted using the Ubibrowser. TP53 was predicted and validated as a downstream substrate of FBXO11. FBXO11 induced ubiquitination and degradation of the tumor suppressor protein TP53 to induce PCa progression. In conclusion, this study suggests that silencing of FBXO11, especially that mediated by UTMD, might suppress the malignant biological behaviors of PCa cells and serve as a potential therapeutic strategy for PCa management.

FBXO32 targets PHPT1 for ubiquitination to regulate the growth of EGFR mutant lung cancer.

Phosphohistidine phosphatase 1 (PHPT1) is an oncogene that has been reported to participate in multiple tumorigenic processes. As yet, however, the role of PHPT1 in lung cancer development remains uncharacterized. RNA sequencing assay and 18 pairs of tumor and normal tissues from patients were analyzed to reveal the upregulation of PHPT1 in lung cancer, followed by confirming the biological function in vitro and in vivo. Next, Gene Set Enrichment Analysis, lung cancer samples, apoptosis assay, mass spectrometry experiments and western blotting were used to investigate the molecular mechanism underlying PHPT1 driven progression in epidermal growth factor receptor (EGFR)-mutant lung cancer. Finally, we performed cellular and animal experiments to explore the tumor suppressive function of F-box protein 32 (FBXO32). We found that PHPT1 is overexpressed in lung cancer patients and correlates with a poor overall survival. In addition, we found that the expression of PHPT1 is elevated in EGFR-mutant lung cancer cells and primary patient samples. Inhibition of PHPT1 expression in EGFR mutant lung cancer cells significantly decreased their proliferation and clonogenicity, and suppressed their in vitro tumor growth. Mechanistic studies revealed that activation of the ERK/MAPK pathway is driven by PHPT1. PHPT1 is required for maintaining drug resistance to erlotinib in EGFR mutant lung cancer cells. We found that FBXO32 acts as an E3 ubiquitin ligase for PHPT1, and that knockdown of FBXO32 leads to PHPT1 accumulation, activation of the ERK/MAPK pathway and promotion of the proliferation, clonogenicity and growth of lung cancer cells. Our findings indicate that PHPT1 may serve as a biomarker and therapeutic target for acquired erlotinib resistance in lung cancer patients carrying EGFR mutations.

Diverse Evolution in 111 Plant Genomes Reveals Purifying and Dosage Balancing Selection Models for F-Box Genes.

The F-box proteins function as substrate receptors to determine the specificity of Skp1-Cul1-F-box ubiquitin ligases. Genomic studies revealed large and diverse sizes of the F-box gene superfamily across plant species. Our previous studies suggested that the plant F-box gene superfamily is under genomic drift evolution promoted by epigenomic programming. However, how the size of the superfamily drifts across plant genomes is currently unknown. Through a large-scale genomic and phylogenetic comparison of the F-box gene superfamily covering 110 green plants and one red algal species, I discovered four distinct groups of plant F-box genes with diverse evolutionary processes. While the members in Clusters 1 and 2 are species/lineage-specific, those in Clusters 3 and 4 are present in over 46 plant genomes. Statistical modeling suggests that F-box genes from the former two groups are skewed toward fewer species and more paralogs compared to those of the latter two groups whose presence frequency and sizes in plant genomes follow a random statistical model. The enrichment of known Arabidopsis F-box genes in Clusters 3 and 4, along with comprehensive biochemical evidence showing that Arabidopsis members in Cluster 4 interact with the Arabidopsis Skp1-like 1 (ASK1), demonstrates over-representation of active F-box genes in these two groups. Collectively, I propose purifying and dosage balancing selection models to explain the lineage/species-specific duplications and expansions of F-box genes in plant genomes. The purifying selection model suggests that most, if not all, lineage/species-specific F-box genes are detrimental and are thus kept at low frequencies in plant genomes.

F-Box Proteins and Cancer.

Controlled protein degradation is essential for the operation of a variety of cellular processes including cell division, growth, and differentiation. Identification of the relations between ubiquitin ligases and their substrates is key to understanding the molecular basis of cancer development and to the discovery of novel targets for cancer therapeutics. F-box proteins function as the substrate recognition subunits of S-phase kinase-associated protein 1 (SKP1)−Cullin1 (CUL1)−F-box protein (SCF) ubiquitin ligase complexes. Here, we summarize the roles of specific F-box proteins that have been shown to function as tumor promoters or suppressors. We also highlight proto-oncoproteins that are targeted for ubiquitylation by multiple F-box proteins, and discuss how these F-box proteins are deployed to regulate their cognate substrates in various situations.

The soybean F-box protein GmFBX176 regulates ABA-mediated responses to drought and salt stress

As key components of the E3 ubiquitin ligase in the Skp1/Cullin or CDC53/F-box (SCF) complex, F-box proteins are involved in plant abiotic stress responses. However, the functions of F-box proteins in soybean in response to abiotic stress remain unclear. In this study, the soybean F-box protein-encoding gene GmFBX176 was cloned and functionally analyzed. GmFBX176 localized to the nucleus, was widely expressed in tissues (with the highest level in the roots), and responded to polyethylene glycol (PEG) treatment, abscisic acid (ABA) treatment, and salt stress at the transcriptional level. GmFBX176-overexpressing Arabidopsis had lower survival rates; faster water loss rates; lower proline, sucrose and chlorophyll contents; greater malondialdehyde (MDA) contents; greater relative electrical conductivity (REC); and greater drought stress sensitivity than did wild-type (WT) Arabidopsis. Moreover, GmFBX176-overexpressing Arabidopsis had lower survival rates, lower chlorophyll content, lower superoxide dismutase (SOD) activity, greater sensitivity to salt stress, longer roots, greater stomatal opening rates, and lower sensitivity to ABA than did WT Arabidopsis. Transcriptome sequencing and quantitative real-time PCR (qPCR) analyses revealed that GmFBX176 affects the transcript levels of stress response genes. These results indicate that GmFBX176 regulates ABA-mediated responses to drought and salt stress.

MicroRNA regulation of F-box proteins and its role in cancer.

MicroRNAs (miRNAs) are small endogenous non-coding RNAs, which play critical roles in cancer development by suppressing gene expression at the post-transcriptional level. In general, oncogenic miRNAs are upregulated in cancer, while miRNAs that act as tumor suppressors are downregulated, leading to decreased expression of tumor suppressors and upregulated oncogene expression, respectively. F-box proteins function as the substrate-recognition components of the SKP1-CUL1-F-box (SCF)-ubiquitin ligase complex for the degradation of their protein targets by the ubiquitin-proteasome system. Therefore F-box proteins and miRNAs both negatively regulate target gene expression post-transcriptionally. Since each miRNA is capable of fine-tuning the expression of multiple target genes, multiple F-box proteins may be suppressed by the same miRNA. Meanwhile, one F-box proteins could be regulated by several miRNAs in different cancer types. In this review, we will focus on miRNA-mediated downregulation of various F-box proteins, the resulting stabilization of F-box protein substrates and the impact of these processes on human malignancies. We provide insight into how the miRNA: F-box protein axis may regulate cancer progression and metastasis. We also consider the broader role of F-box proteins in the regulation of pathways that are independent of the ubiquitin ligase complex and how that impacts on oncogenesis. The area of miRNAs and the F-box proteins that they regulate in cancer is an emerging field and will inform new strategies in cancer treatment.

Small molecule therapeutics targeting F-box proteins in cancer.

The ubiquitin proteasome system (UPS) plays vital roles in maintaining protein equilibrium mainly through proteolytic degradation of targeted substrates. The archetypical SCF ubiquitin E3 ligase complex contains a substrate recognition subunit F-box protein that recruits substrates to the catalytic ligase core for its polyubiquitylation and subsequent proteasomal degradation. Several well-characterized F-box proteins have been demonstrated that are tightly linked to neoplasia. There is mounting information characterizing F-box protein-substrate interactions with the rationale to develop unique therapeutics for cancer treatment. Here we review that how F-box proteins function in cancer and summarize potential small molecule inhibitors for cancer therapy.

The Involvement of Wheat F-Box Protein Gene TaFBA1 in the Oxidative Stress Tolerance of Plants

As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants’ tolerance to multiple stress conditions.

Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome.

The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40-50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of the Skp1-cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes (F-box genes for short) were identified and further subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain. The F-box genes were distributed in all 17 apple chromosomes with various densities and tended to form gene clusters. Spatial expression profile analysis revealed that F-box genes have organ-specific expression and are widely expressed in all organs. Proteins that contained the galactose oxidase domain were highly expressed in leaves, flowers and seeds. From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. The results of this study helped to elucidate the functions of F-box proteins, especially in Rosaceae plants.

The Functions of F-box Proteins in Regulating the Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) has been unraveled to regulate the tumor invasion and metastasis processes. In this review, in order to better understand the regulatory mechanisms of EMT, we describe that F-box proteins could be critically involved in regulating the EMT process in human cancers. Specifically, we discuss how these F-box proteins directly control the stability of EMT regulators such as E-cadherin, β-catenin, Twist, Slug, Snail and ZEB. Moreover, we summary mechanistically how the F-box proteins govern EMT progression through regulation of EMT inducers including Notch, NF-κB, Akt, Hedgehog, mTOR, and HIF-1α. Therefore, targeting these F-box proteins could be useful for treating human cancers.

Proteomic identification of potential target proteins regulated by the SCFFbp1‐mediated proteolysis pathway in Fusarium oxysporum

F-box proteins function in the recruitment of proteins for SCF ubiquitination and proteasome degradation. Here, we studied the role of Fbp1, a nonessential F-box protein of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. The Δfbp1 mutant showed a significant delay in the production of wilt symptoms on tomato plants and was impaired in invasive growth on cellophane membranes and on living plant tissue. To search for target proteins recruited by Fbp1, a combination of sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) was used to compare proteins in mycelia of the wild-type and Δfbp1 mutant. The proteomic approach identified 41 proteins differing significantly in abundance between the two strains, 17 of which were more abundant in the Δfbp1 mutant, suggesting a possible regulation by proteasome degradation. Interestingly, several of the identified proteins were related to vesicle trafficking. Microscopic analysis revealed an impairment of the Δfbp1 strain in directional growth and in the structure of the Spitzenkörper, suggesting a role of Fbp1 in hyphal orientation. Our results indicate that Fbp1 regulates protein turnover and pathogenicity in F. oxysporum.

Characterization of the Cullin7 E3 ubiquitin ligase — Heterodimerization of cullin substrate receptors as a novel mechanism to regulate cullin E3 ligase activity

Cul1 and Cul7 are cullin E3 ubiquitin ligase scaffold proteins. Cul1 is known to form a complex with the RING domain protein Rbx1 and one of approximately 70 different F-box proteins. F-box proteins function as substrate receptor subunits and recruit numerous substrates for poly-ubiquitination. Similarly to Cul1, Cul7 interacts with Rbx1, however, only one F-box protein, Fbxw8, has been shown to bind to Cul7. To date only few Cul7 E3 ubiquitin ligase substrates, including cyclin D1, IRS-1 and GRASP65, have been reported, and using Fbxw8 affinity purification, we were unable to identify additional substrate proteins. Here we provide evidence for a model in which Cul7-Rbx1 can promote the ubiquitination of Cul1 substrates by forming high order complexes with Cul1-Rbx1. Binding of Cul1-Rbx1 to Cul7-Rbx1 is mediated via heterodimerization of Fbxw8 with other F-box proteins which function to recruit substrates into the E3 ligase complex. The formation of this high order complex is likely to increase polyubiquitination efficiency.

The utility F-box for protein destruction.

A signature feature of all living organisms is their utilization of proteins to construct molecular machineries that undertake the complex network of cellular activities. The abundance of a protein element is temporally and spatially regulated in two opposing aspects: de novo synthesis to manufacture the required amount of the protein, and destruction of the protein when it is in excess or no longer needed. One major route of protein destruction is coordinated by a set of conserved molecules, the F-box proteins, which promote ubiquitination in the ubiquitin-proteasome pathway. Here we discuss the functions of F-box proteins in several cellular scenarios including cell cycle progression, synapse formation, plant hormone responses, and the circadian clock. We particularly emphasize the mechanisms whereby F-box proteins recruit specific substrates and regulate their abundance in the context of SCF E3 ligases. For some exceptions, we also review how F-box proteins function through non-SCF mechanisms.

MAX2 participates in an SCF complex which acts locally at the node to suppress shoot branching

The Arabidopsis gene ORE9/MAX2 encodes an F-box leucine-rich repeat protein. F-box proteins function as the substrate-recruiting subunit of SCF-type ubiquitin E3 ligases in protein ubiquitination. One of several phenotypes of max2 mutants, the highly branched shoot, is identical to mutants at three other MAX loci. Reciprocal grafting, double mutant analysis and gene cloning suggest that all MAX genes act in a common pathway, where branching suppression depends on MAX2 activity in the shoot, in response to an acropetally mobile signal that requires MAX3, MAX4 and MAX1 for its production. Here, we further investigate the site and mode of action of MAX2 in branching. Transcript analysis and a translational MAX2-GUS fusion indicate that MAX2 is expressed throughout the plant, most highly in developing vasculature, and is nuclear-localized in many cell types. Analysis of cell autonomy shows that MAX2 acts locally, either in the axillary bud, or in adjacent stem or petiole tissue. Expression of MAX2 from the CaMV 35S promoter complements the max2 mutant, does not affect branching in a wild-type background and partially rescues increased branching in the max1, max3 and max4 backgrounds. Expression of mutant MAX2, lacking the F-box domain, under the CaMV 35S promoter does not complement max2, and dominant-negatively affects branching in the wild-type background. Myc-epitope-tagged MAX2 interacts with the core SCF subunits ASK1 and AtCUL1 in planta. We conclude that axillary shoot growth is controlled locally, at the node, by an SCF(MAX2), the action of which is enhanced by the mobile MAX signal.

F-Box Proteins in Rice. Genome-Wide Analysis, Classification, Temporal and Spatial Gene Expression during Panicle and Seed Development, and Regulation by Light and Abiotic Stress

F-box proteins constitute a large family in eukaryotes and are characterized by a conserved F-box motif (approximately 40 amino acids). As components of the Skp1p-cullin-F-box complex, F-box proteins are critical for the controlled degradation of cellular proteins. We have identified 687 potential F-box proteins in rice (Oryza sativa), the model monocotyledonous plant, by a reiterative database search. Computational analysis revealed the presence of several other functional domains, including leucine-rich repeats, kelch repeats, F-box associated domain, domain of unknown function, and tubby domain in F-box proteins. Based upon their domain composition, they have been classified into 10 subfamilies. Several putative novel conserved motifs have been identified in F-box proteins, which do not contain any other known functional domain. An analysis of a complete set of F-box proteins in rice is presented, including classification, chromosomal location, conserved motifs, and phylogenetic relationship. It appears that the expansion of F-box family in rice, in large part, might have occurred due to localized gene duplications. Furthermore, comprehensive digital expression analysis of F-box protein-encoding genes has been complemented with microarray analysis. The results reveal specific and/or overlapping expression of rice F-box protein-encoding genes during floral transition as well as panicle and seed development. At least 43 F-box protein-encoding genes have been found to be differentially expressed in rice seedlings subjected to different abiotic stress conditions. The expression of several F-box protein-encoding genes is also influenced by light. The structure and function of F-box proteins in plants is discussed in light of these results and the published information. These data will be useful for prioritization of F-box proteins for functional validation in rice.

F-box proteins: the key to protein degradation

The eukaryotic protein degradation pathway involves the ubiquitin (Ub) modification of substrates targeted for degradation by the 26S proteasome. The addition of Ub, a process called ubiquitination, is mediated by enzymes including the E3 Ub ligases which transfer the Ub to targeted substrates. A major type of E3 Ub ligases, the SCF (Skp-Cullin-F-box) complex, is composed of four major components: Skp1, Cul1/Cdc53, Roc1/Rbx1/Hrt1, and an F-box protein. The F-box component of the SCF machineries is responsible for recognizing different substrates for ubiquitination. Interaction with components of the SCF complex is mediated through the F-box motif of the F-box protein while it associates with phosphorylated substrates through its second protein-protein interaction motif such as Trp-Asp (WD) repeats or leucine-rich repeats (LRRs). By targeting diverse substrates, F-box proteins exert controls over stability of proteins and regulate the mechanisms for a wide-range of cellular processes. Here we discuss the importance of F-box proteins by providing a general overview and examples of how F-box proteins function in various cellular settings such as tissue development, cell proliferation, and cell death, in the modeling organism Drosophila.

The Function of F-box Protein in Plant Growth and Development

Ubiquitin-mediated proteolysis is involved in many biological processes in eukaryotes. SCF complex is a very important ubiquitin E3 ligase which has been exploited very well in plants. F-box protein characterized by an F-box motif is a subunit of SCF complex, which works as determinant in substrate recognition. Currently, many F-box proteins have been identified in plants which are involving in hormone (e.g., ethylene, auxin, gibberellin and jasmonate ) signal transduction and biological processes, such as self-incompatibility and floral development. F-box proteins may also participate stress response in plants. Recent study suggested that the Arabidopsis F-box protein TIR1 is an auxin receptor. Therefore, F-box protein mediated proteolysis may be an important gene expression mechanism in plants.

A Molecular Basis for Stabilization of the von Hippel-Lindau (VHL) Tumor Suppressor Protein by Components of the VHL Ubiquitin Ligase

The multiprotein von Hippel-Lindau (VHL) tumor suppressor (CBC(VHL), Cul2-Elongin BC-VHL) and SCF (Skp1-Cul1/Cdc53-F-box protein) complexes are members of structurally related families of E3 ubiquitin ligases that use a heterodimeric module composed of a member of the Cullin protein family and the RING finger protein Rbx1 (ROC1/Hrt1) to activate ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. VHL and F-box proteins function as the substrate recruitment subunits of CBC(VHL) and SCF complexes, respectively. In cells, many F-box proteins are short lived and are proposed to be ubiquitylated by an autocatalytic mechanism and destroyed by the proteasome following assembly into SCF complexes. In contrast, the VHL protein is stabilized by interaction with the Elongin B and C subunits of CBC(VHL) in cells. In this report, we have presented direct biochemical evidence that unlike the F-box protein Cdc4, which is ubiquitylated in vitro by Cdc34 in the context of the SCF, the VHL protein is protected from Ubc5-catalyzed ubiquitylation following assembly into the CBC(VHL) complex. CBC(VHL) is continuously required for negative regulation of hypoxia-inducible transcription factors in normoxic cells and of SCF complexes, many of which function only transiently during the cell cycle or in response to cellular signals. Our findings provide a molecular basis for the different modes of cellular regulation of VHL and F-box proteins and are consistent with the known roles of CBC(VHL).