SCFFBXO28-mediated self-ubiquitination of FBXO28 promotes its degradation

The F-box protein is the substrate recognition subunit of SCF (SKP1/CUL1/F-box) E3 ubiquitin ligase complex, a multicomponent RING-type E3 ligase involved in the regulation of numerous cellular processes by targeting critical regulatory proteins for ubiquitination. However, whether and how F-box proteins are regulated is largely unknown. Here we report that FBXO28, a poorly characterized F-box protein, is a novel substrate of SCF E3 ligase. Pharmaceutical or genetic inhibition of neddylation pathway that is required for the activation of SCF stabilizes FBXO28 and prolongs its half-life. Meanwhile, FBXO28 is subjected to ubiquitination and cullin1-based SCF complex promotes FBXO28 degradation. Moreover, deletion of F-box domain stabilizes FBXO28 and knockdown of endogenous FBXO28 strongly upregulates exogenous FBXO28 expression. Taken together, these data reveal that SCF^FBXO28 is the E3 ligase responsible for the self-ubiquitination and proteasomal degradation of FBXO28, providing a new clue for the upstream signaling regulation for F-box proteins.     

FBXO6-mediated RNASET2 ubiquitination and degradation governs the development of ovarian cancer

RNASET2 (Ribonuclease T2) functions as a tumor suppressor in preventing ovarian tumorigenesis. However, the mechanisms underlying the regulation of RNASET2 protein are completely unknown. Here we identified the F-box protein FBXO6, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the ubiquitin E3 ligase for RNASET2. We found that the interaction between FBXO6 and RNASET2 induced RNASET2 instability through the ubiquitin-mediated proteasome degradation pathway. FBXO6 promoted K48-dependent ubiquitination of RNASET2 via its FBA domain. Through analysis of the TCGA dataset, we found that FBXO6 was significantly increased in ovarian cancer tissues and the high expression of FBXO6 was related to the poor overall survival (OS) of ovarian cancer patients at advanced stages. An inverse correlation between the protein levels of FBXO6 and RNASET2 was observed in clinic ovarian cancer samples. Depletion of FBXO6 promoted ovarian cancer cells proliferation, migration, and invasion, which could be partially reversed by RNASET2 silencing. Thus, our data revealed a novel FBXO6-RNASET2 axis, which might contribute to the development of ovarian cancer. We propose that inhibition of FBXO6 might represent an effective therapeutic strategy for ovarian cancer treatment.Subject terms: Oncogenes, Ubiquitin ligases     

The p38-interacting Protein (p38IP) Regulates G2/M Progression by Promoting α-Tubulin Acetylation via Inhibiting Ubiquitination-induced Degradation of the Acetyltransferase GCN5

p38-interacting protein (p38IP) is a component of the GCN5 histone acetyltransferase-containing coactivator complex (GCN5-SAGA complex). It remains unclear whether p38IP or GCN5-SAGA is involved in cell cycle regulation. Using RNA interference to knock down p38IP, we observed that cells were arrested at the G₂/M phase, exhibiting accumulation of cyclins, shrunken spindles, and hypoacetylation of α-tubulin. Further analysis revealed that knockdown of p38IP led to proteasome-dependent degradation of GCN5. GCN5 associated with and acetylated α-tubulin, and recovering GCN5 protein levels in p38IP knockdown cells by ectopic expression of GCN5 efficiently reversed α-tubulin hypoacetylation and G₂/M arrest. During the G₂/M transition, the association of α-tubulin with GCN5 increased, and the acetylation of α-tubulin reached a peak. Biochemical analyses demonstrated that the interaction between p38IP and GCN5 depended on the p38IP N terminus (1–381 amino acids) and GCN5 histone acetyltransferase domain and bromodomain. The p38IP N terminus could effectively reverse p38IP depletion-induced GCN5 degradation, thus recovering α-tubulin acetylation and G₂/M progression. p38IP-mediated suppression of GCN5 ubiquitination most likely occurs via nuclear sequestration of GCN5. Our data indicate that the GCN5-SAGA complex is required for G₂/M progression, mainly because p38IP promotes the acetylation of α-tubulin by preventing the degradation of GCN5, in turn facilitating the formation of the mitotic spindle.     

FBXO16-mediated hnRNPL ubiquitination and degradation plays a tumor suppressor role in ovarian cancer

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.Subject terms: Ovarian cancer, Oncogenes     

FBXO44-Mediated Degradation of RGS2 Protein Uniquely Depends on a Cullin 4B/DDB1 Complex

The ubiquitin-proteasome system for protein degradation plays a major role in regulating cell function and many signaling proteins are tightly controlled by this mechanism. Among these, Regulator of G Protein Signaling 2 (RGS2) is a target for rapid proteasomal degradation, however, the specific enzymes involved are not known. Using a genomic siRNA screening approach, we identified a novel E3 ligase complex containing cullin 4B (CUL4B), DNA damage binding protein 1 (DDB1) and F-box protein 44 (FBXO44) that mediates RGS2 protein degradation. While the more typical F-box partners CUL1 and Skp1 can bind FBXO44, that E3 ligase complex does not bind RGS2 and is not involved in RGS2 degradation. These observations define an unexpected DDB1/CUL4B-containing FBXO44 E3 ligase complex. Pharmacological targeting of this mechanism provides a novel therapeutic approach to hypertension, anxiety, and other diseases associated with RGS2 dysregulation.     

The F-box Protein FBXO25 Promotes the Proteasome-dependent Degradation of ELK-1 Protein

FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens.     

The F-box Protein FBXO44 Mediates BRCA1 Ubiquitination and Degradation

BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCF^FBXO44) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCF^FBXO44 reduces BRCA1 protein level. Taken together, our work strongly suggests that SCF^FBXO44 is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCF^FBXO44-mediated BRCA1 degradation might contribute to sporadic breast tumor development.     

The Centrosomal E3 Ubiquitin Ligase FBXO31-SCF Regulates Neuronal Morphogenesis and Migration

Neuronal development requires proper migration, polarization and establishment of axons and dendrites. Growing evidence identifies the ubiquitin proteasome system (UPS) with its numerous components as an important regulator of various aspects of neuronal development. F-box proteins are interchangeable subunits of the Cullin-1 based E3 ubiquitin ligase, but only a few family members have been studied. Here, we report that the centrosomal E3 ligase FBXO31-SCF (Skp1/Cullin-1/F-box protein) regulates neuronal morphogenesis and axonal identity. In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth. Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex. Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.     

cDNA cloning and expression analysis of a novel human F-box domain containing gene†

F-box proteins are a large family of eukaryotic proteins that are characterized by an approximately 40 amino acid motif. Some F-box proteins are critical for the controlled degradation of cellular regulatory proteins. Here we report that a novel member of F-box proteins, FBXO35 gene, was cloned and identified during the large-scale sequencing analysis from a human fetal brain cDNA library. FBXO35 gene shares amino acid similarity with several putative mouse genes not only in F-box domain but also in the rest of the sequence, which indicates that FBXO35 might also contain some other unknown conserved domain. RT-PCR analysis indicated that FBXO35 gene had a ubiquitously low expression pattern in most human adult tissues. According to bioinformatics analysis, the FBXO35 gene was found located in chromosome 3p21.     

Identification of a molecular component of the mitochondrial acetyltransferase programme: a novel role for GCN5L1

SIRT3 (sirtuin 3) modulates respiration via the deacetylation of lysine residues in electron transport chain proteins. Whether mitochondrial protein acetylation is controlled by a counter-regulatory program has remained elusive. In the present study we identify an essential component of this previously undefined mitochondrial acetyltransferase system. We show that GCN5L1 [GCN5 (general control of amino acid synthesis 5)-like 1; also known as Bloc1s1] counters the acetylation and respiratory effects of SIRT3. GCN5L1 is mitochondrial-enriched and displays significant homology with a prokaryotic acetyltransferase. Genetic knockdown of GCN5L1 blunts mitochondrial protein acetylation, and its reconstitution in intact mitochondria restores protein acetylation. GCN5L1 interacts with and promotes acetylation of SIRT3 respiratory chain targets and reverses global SIRT3 effects on mitochondrial protein acetylation, respiration and bioenergetics. The results of the present study identify GCN5L1 as a critical prokaryote-derived component of the mitochondrial acetyltransferase programme.     

Proteomic identification of common SCF ubiquitin ligase FBXO6-interacting glycoproteins in three kinds of cells

FBOX6 ubiquitin ligase complex is involved in the endoplasmic reticulum-associated degradation pathway by mediating the ubiquitination of glycoproteins. FBXO6 interacts with the chitobiose in unfolded N-glycoprotein, pointing glycoproteins toward E2 for ubiquitination. Although the glycoprotein-recognizing mechanism of FBXO6 is well documented, its bona fide interacting glycoproteins are largely unknown. Here we utilized a protein purification approach combined with LC-MS to systematically identify the FBXO6-interacting glycoproteins. Following identification of 39 proteins that specifically interact with FBXO6 in all three different cell lines, 293T, HeLa and Jurkat cells, we compared the protein complex organization between wild-type FBXO6 and its mutant, which fails to recognize glycoproteins. Combining these databases, 29 highly confident glycoproteins that interact with FBXO6 in an N-glycan dependent manner are identified. Our data provide valuable information for the discovery of the interacting glycoproteins of FBXO6 and also demonstrate the potential of these approaches as general platforms for the global discovery of interacting glycoproteins of other FBAs (F-box associated regions) containing F-box proteins.     

F-Box Only Protein 31 (FBXO31) Negatively Regulates p38 Mitogen-activated Protein Kinase (MAPK) Signaling by Mediating Lysine 48-linked Ubiquitination and Degradation of Mitogen-activated Protein Kinase Kinase 6 (MKK6)

The p38 MAPK signal transduction pathway plays an important role in inflammatory and stress responses. MAPKK6 (MKK6), a dual specificity protein kinase, is a p38 activator. Activation of the MKK6-p38 pathway is kept in check by multiple layers of regulations, including autoinhibition, dimerization, scaffold proteins, and Lys-63-linked polyubiquitination. However, the mechanisms underlying deactivation of MKK6-p38, which is crucial for maintaining the magnitude and duration of signal transduction, are not well understood. Lys-48-linked ubiquitination, which marks substrates for proteasomal degradation, is an important negative posttranslational regulatory machinery for signal pathway transduction. Here we report that the accumulation of F-box only protein 31 (FBXO31), a component of Skp1·Cul1·F-box protein E3 ligase, negatively regulated p38 activation in cancer cells upon genotoxic stresses. Our results show that FBXO31 binds to MKK6 and mediates its Lys-48-linked polyubiquitination and degradation, thereby functioning as a negative regulator of MKK6-p38 signaling and protecting cells from stress-induced cell apoptosis. Taken together, our findings uncover a new mechanism of deactivation of MKK6-p38 and substantiate a novel regulatory role of FBXO31 in stress response.     

FBXO31 modulates activation of hepatic stellate cells and liver fibrogenesis by promoting ubiquitination of Smad7

Liver fibrosis is a critical pathological process in the early stage of many liver diseases, including hepatic cirrhosis and liver cancer. However, the molecular mechanism is not fully revealed. In this study, we investigated the role of F-box protein 31 (FBXO31) in liver fibrosis. We found FBXO31 upregulated in carbon tetrachloride (CCl₄) induced liver fibrosis and in activated hepatic stellate cells, induced by transforming growth factor-β (TGF-β). The enforced expression of FBXO31 caused enhanced proliferation and increased expression of α-smooth muscle actin (α-SMA) and Col-1 in HSC-T6 cells. Conversely, suppression of FBXO31 resulted in inhibition of proliferation and decreased accumulation of α-SMA and Col-1 in HSC-T6 cells. In addition, upregulation of FBXO31 in HSC-T6 cells decreased accumulation of Smad7, the negative regulator of the TGF-β/smad signaling pathway, and suppression of the FBXO31 increased accumulation of Smad7. Immunofluorescence staining showed FBXO31 colocalized with Smad7 in HSC-T6 cells and in liver tissues of BALB/c mice treated with CCl₄. Immunoprecipitation demonstrated FBXO31 interacted with Smad7. Moreover, FBXO31 enhanced ubiquitination of Smad7. In conclusion, FBXO31 modulates activation of HSCs and liver fibrogenesis by promoting ubiquitination of Smad7.     

Inter- and intra-molecular interactions of Arabidopsis thaliana DELLA protein RGL1

The phytohormone gibberellin and the DELLA proteins act together to control key aspects of plant development. Gibberellin induces degradation of DELLA proteins by recruitment of an F-box protein using a molecular switch: a gibberellin-bound nuclear receptor interacts with the N-terminal domain of DELLA proteins, and this event primes the DELLA C-terminal domain for interaction with the F-box protein. However, the mechanism of signalling between the N- and C-terminal domains of DELLA proteins is unresolved. In the present study, we used in vivo and in vitro approaches to characterize di- and tri-partite interactions of the DELLA protein RGL1 (REPRESSOR OF GA1-3-LIKE 1) of Arabidopsis thaliana with the gibberellin receptor GID1A (GIBBERELLIC ACID-INSENSITIVE DWARF-1A) and the F-box protein SLY1 (SLEEPY1). Deuterium-exchange MS unequivocally showed that the entire N-terminal domain of RGL1 is disordered prior to interaction with the GID1A; furthermore, association/dissociation kinetics, determined by surface plasmon resonance, predicts a two-state conformational change of the RGL1 N-terminal domain upon interaction with GID1A. Additionally, competition assays with monoclonal antibodies revealed that contacts mediated by the short helix Asp-Glu-Leu-Leu of the hallmark DELLA motif are not essential for the GID1A-RGL1 N-terminal domain interaction. Finally, yeast two- and three-hybrid experiments determined that unabated communication between N- and C-terminal domains of RGL1 is required for recruitment of the F-box protein SLY1.     

FBXO6介导的ERO1L泛素化降解对膀胱癌细胞增殖、迁移和侵袭的作用研究

目的探究F盒蛋白6 （FBXO6）对膀胱癌细胞的作用及其作用机制。 方法体外培养人正常膀胱上皮细胞株（SV-HUC-1）和人膀胱癌细胞株（T24）。用过表达载体阴性对照（oe-NC）、过表达FBXO6 （oe-FBXO6）、过表达内质网氧化还原蛋白-1样蛋白（oe-ERO1L）及oe-FBXO6和oe-ERO1L慢病毒液（MOI = 20）感染T24细胞。RT-qPCR检测细胞FBXO6和ERO1L mRNA表达;放线菌酮（CHX）蛋白合成抑制实验检测T24细胞ERO1L蛋白稳定性;免疫共沉淀（Co-IP）实验检测FBXO6对ERO1L泛素化调控;Western blot检测细胞FBXO6和ERO1L蛋白表达;CCK-8检测细胞活力;克隆形成实验检测细胞增殖;Transwell实验检测细胞迁移和侵袭。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。 结果与SV-HUC-1相比,T24细胞中FBXO6 mRNA （1.00±0.05比0.33±0.02）和蛋白表达（1.00±0.11比0.31±0.03）均降低（P均< 0.05）,而ERO1L mRNA （1.00±0.05比2.70±0.12）和蛋白表达（1.00±0.16比3.27±0.09）均升高（P均< 0.05）。FBXO6可降低ERO1L蛋白稳定性并促进ERO1L泛素化。与空白对照和oe-NC相比,oe-FBXO6细胞中FBXO6 mRNA （1.00±0.06比3.74±0.18）和蛋白表达（1.00±0.10比2.25±0.06）均升高,ERO1L蛋白表达（0.99±0.08比0.21±0.03）,细胞活力、克隆形成数[（78.00±3.00）比（41.67±2.52）个]、迁移[（150.67±5.03）比（91.67±5.51）个]和侵袭细胞数[（122.00±7.00）比（74.67±5.51）个]均降低（P均< 0.05）;与oe-NC相比,oe-ERO1L细胞中ERO1L蛋白表达（1.01±0.06比2.58±0.02）、细胞活力、克隆形成数[ （78.00±3.00）比（121.67±7.64）个]、迁移[（150.67±5.03）比（230.33±12.01）个]和侵袭细胞数[（122.00±7.00）比（203.00± 11.53）个]均升高（P均< 0.05）;与oe-FBXO6相比,oe-FBXO6+oe-ERO1L细胞中ERO1L蛋白表达（0.54±0.02比1.02±0.06）,细胞活力、克隆形成数[（41.67±2.52）比（62.00±3.61）个]、迁移[（91.67±5.51）比（131.67±6.03）个]和侵袭细胞数[（74.67±5.51）比（102.67±7.51）个]均升高（P均< 0.05）。 结论FBXO6通过介导ERO1L泛素化降解抑制膀胱癌细胞增殖、迁移和侵袭。