Atrogin-1 promotes muscle homeostasis by regulating levels of endoplasmic reticulum chaperone BiP.

Skeletal muscle wasting results from numerous pathological conditions affecting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role of E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss-of-function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 resulted in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fiber integrity. We further implicated a disruption in atrogin-1-mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We revealed that BiP was not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorated pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicate atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy and highlight atrogin-1's essential role in maintaining muscle homeostasis.

ROS in diabetic atria regulate SK2 degradation by Atrogin-1 through the NF-κB signaling pathway.

One of the independent risk factors for atrial fibrillation is diabetes mellitus (DM); however, the underlying mechanisms causing atrial fibrillation in DM are unknown. The underlying mechanism of Atrogin-1-mediated SK2 degradation and associated signaling pathways are unclear. The aim of this study was to elucidate the relationship among reactive oxygen species (ROS), the NF-κB signaling pathway, and Atrogin-1 protein expression in the atrial myocardia of DM mice. We found that SK2 expression was downregulated comitant with increased ROS generation and enhanced NF-κB signaling activation in the atrial cardiomyocytes of DM mice. These observations were mimicked by exogenously applicating H2O2 and by high glucose culture conditions in HL-1 cells. Inhibition of ROS production by diphenyleneiodonium chloride or silencing of NF-κB by siRNA decreased the protein expression of NF-κB and Atrogin-1 and increased that of SK2 in HL-1 cells with high glucose culture. Moreover, chromatin immunoprecipitation assay demonstrated that NF-κB/p65 directly binds to the promoter of the FBXO32 gene (encoding Atrogin-1), regulating the FBXO32 transcription. Finally, we evaluated the therapeutic effects of curcumin, known as a NF-κB inhibitor, on Atrogin-1 and SK2 expression in DM mice and confirmed that oral administration of curcumin for 4 weeks significantly suppressed Atrogin-1 expression and protected SK2 expression against hyperglycemia. In summary, the results from this study indicated that the ROS/NF-κB signaling pathway participates in Atrogin-1-mediated SK2 regulation in the atria of streptozotocin-induced DM mice.

DNMT1-mediated regulating on FBXO32 promotes the progression of glioma cells through the regulation of SKP1 activity.

Glioma, a prevalent and serious form of brain cancer, is associated with dysregulation of DNA methylation, where DNA methyltransferase-1 (DNMT1) plays a significant role in glioma progression. However, the involvement of F-box protein 32 (FBXO32) in glioma and its regulation by DNMT1-mediated methylation remain poorly understood. In this study, we investigated FBXO32 expression in glioma cells with high DNMT1 expression using the online dataset and correlated it with patient survival. Then impact of elevated FBXO32 expression on cell proliferation, migration, and invasion was evaluated, along with the examination of EMT-related proteins. Furthermore, a xenograft model established by injecting glioma cells stably transfected with FBXO32 was used to evaluate tumor growth, volume, and weight. The ChIP assay was employed to study the interaction between DNMT1 and the FBXO32 promoter, revealing that DNMT1 negatively correlated with FBXO32 expression in glioma cells and promoted FBXO32 promoter methylation. Moreover, we investigated the interaction between FBXO32 and SKP1 using Co-IP and GST pulldown assays, discovering that FBXO32 acts as an E3 ubiquitin ligase and promotes SKP1 ubiquitination, leading to its degradation. Interestingly, our findings demonstrated that high FBXO32 expression was associated with improved overall survival in glioma patients. Knockdown of DNMT1 in glioma cells increased FBXO32 expression and suppressed malignant phenotypes, suggesting that FBXO32 functions as a tumor suppressor in glioma. In conclusion, this study reveals a novel regulatory mechanism involving DNMT1-mediated FBXO32 expression in glioma cells, where FBXO32 acts as an E3 ubiquitin ligase to degrade SKP1 via ubiquitination. This FBXO32-mediated regulation of SKP1 activity contributes to the progression of glioma cells. These findings provide important insights into the molecular mechanisms underlying glioma progression and may hold promise for the development of targeted therapies for glioma patients.

Orphan quality control by an SCF ubiquitin ligase directed to pervasive C-degrons.

Selective protein degradation typically involves substrate recognition via short linear motifs known as degrons. Various degrons can be found at protein termini from bacteria to mammals. While N-degrons have been extensively studied, our understanding of C-degrons is still limited. Towards a comprehensive understanding of eukaryotic C-degron pathways, here we perform an unbiased survey of C-degrons in budding yeast. We identify over 5000 potential C-degrons by stability profiling of random peptide libraries and of the yeast C­terminome. Combining machine learning, high-throughput mutagenesis and genetic screens reveals that the SCF ubiquitin ligase targets ~40% of degrons using a single F-box substrate receptor Das1. Although sequence-specific, Das1 is highly promiscuous, recognizing a variety of C-degron motifs. By screening for full-length substrates, we implicate SCFDas1 in degradation of orphan protein complex subunits. Altogether, this work highlights the variety of C-degron pathways in eukaryotes and uncovers how an SCF/C-degron pathway of broad specificity contributes to proteostasis.

Cullin-associated and neddylation-dissociated 1 regulate reprogramming of lipid metabolism through SKP1-Cullin-1-F-boxFBXO11 -mediated heterogeneous nuclear ribonucleoprotein A2/B1 ubiquitination and promote hepatocellular carcinoma.

BACKGROUND: Enhanced de novo lipogenesis is essential for hepatocellular carcinoma (HCC). Abnormally high cullin-associated and neddylation-dissociated 1 (CAND1) expression is associated with poor clinical prognosis in HCC. The SKP1-Cullin-1-F-box (SCF) complex consists of the SKP1, Cullin-1 and F-box proteins (FBPs) and performs multiple functions including adipogenesis. SCF complex was modulated by CAND1, but Whether and how the CAND1 promotes HCC by regulating SCF complex and lipogenesis are unknown. METHODS: HCC samples were used to analyze the correlations between CAND1 expression and clinicopathological characteristics such as survival and prognosis. The in vitro functions of CAND1, FBXO11 and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) were measured by cell proliferation, colony formation and migration assays. The in vivo functions were tested in multiple mouse liver cancer models including patient-derived xenograft (PDX), cell line-derived xenograft and AKT/NRASV12-induced primary liver cancer models. Injections of adeno-associated virus targeting CAND1 (AAV-shCAND1) were performed to evaluate the therapeutic efficacy of targeting CAND1. RNA-Seq and lipidomic assays followed by serial biochemical experiments including mass spectrometry, immunoprecipitation and GST pull-down were performed to dissect the underlying mechanisms. RESULTS: CAND1 promoted the expression of lipid synthesis genes by disrupting SCF complex assembly and lipid accumulation. Furthermore, we identified hnRNPA2B1 as a novel F-box protein 11 (FBXO11)-binding partner. FBXO11 directly bound to hnRNPA2B1 and promoted hnRNPA2B1 ubiquitination and subsequent degradation. Our evaluations of the therapeutic efficacy of AAV-shCAND1 injections confirmed that targeting the CAND1-SCFFBXO11 -hnRNPA2B1A signalling axis was therapeutically effective. CAND1 downregulation significantly reduced the tumour burden in a primary mouse liver cancer model and a PDX model. CONCLUSIONS: Our results highlight that CAND1 is associated with poor prognosis in HCC and regulates lipid metabolic reprogramming by dissociating the SCF complex. Targeting the CAND1-SCFFBXO11 -hnRNPA2B1 axis may be a novel strategy for HCC treatment.

FBXO28 suppresses liver cancer invasion and metastasis by promoting PKA-dependent SNAI2 degradation.

FBXO28 is a member of F-box proteins that are the substrate receptors of SCF (SKP1, CULLIN1, F-box protein) ubiquitin ligase complexes. Despite the implications of its role in cancer, the function of FBXO28 in epithelial-mesenchymal transition (EMT) process and metastasis for cancer remains largely unknown. Here, we report that FBXO28 is a critical negative regulator of migration, invasion and metastasis in human hepatocellular carcinoma (HCC) in vitro and in vivo. FBXO28 expression is upregulated in human epithelial cancer cell lines relative to mesenchymal counterparts. Mechanistically, by directly binding to SNAI2, FBXO28 functions as an E3 ubiquitin ligase that targets the substrate for degradation via ubiquitin proteasome system. Importantly, we establish a cooperative function for PKA in FBXO28-mediated SNAI2 degradation. In clinical HCC specimens, FBXO28 protein levels positively whereas negatively correlate with PKAα and SNAI2 levels, respectively. Low FBXO28 or PRKACA expression is associated with poor prognosis of HCC patients. Together, these findings elucidate the novel function of FBXO28 as a critical inhibitor of EMT and metastasis in cancer and provide a mechanistic rationale for its candidacy as a new prognostic marker and/or therapeutic target in human aggressive HCC.

Neurochemistry evaluated by magnetic resonance spectroscopy in a patient with FBXO28-related developmental and epileptic encephalopathy.

BACKGROUND: Mutations in the FBXO28 gene, which encodes FBXO28, one of the F-box protein family, may cause developmental and epileptic encephalopathy (DEE). FBXO28-related DEE is radiologically characterized by cerebral atrophy, delayed/abnormal myelination, and brain malformation; however, no neurochemical analyses have been reported. CASE REPORT: A female Japanese infant presented with severe psychomotor delay, epileptic spasms, and visual impairment. Whole-exome sequencing revealed a de novo variant of the FBXO28 gene, leading to the diagnosis of FBXO28-related DEE. Magnetic resonance (MR) spectroscopy at 6, 12, and 32 months revealed decreased N-acetylaspartate and choline-containing compounds and increased levels of myoinositol. CONCLUSION: MR spectroscopy revealed neurochemical derangement in FBXO28-related DEE, that is, disturbed myelination secondary to neuronal damage with astrogliosis.

Structural and mechanistic insights into the CAND1-mediated SCF substrate receptor exchange.

Modular SCF (SKP1-CUL1-Fbox) ubiquitin E3 ligases orchestrate multiple cellular pathways in eukaryotes. Their variable SKP1-Fbox substrate receptor (SR) modules enable regulated substrate recruitment and subsequent proteasomal degradation. CAND proteins are essential for the efficient and timely exchange of SRs. To gain structural understanding of the underlying molecular mechanism, we reconstituted a human CAND1-driven exchange reaction of substrate-bound SCF alongside its co-E3 ligase DCNL1 and visualized it by cryo-EM. We describe high-resolution structural intermediates, including a ternary CAND1-SCF complex, as well as conformational and compositional intermediates representing SR- or CAND1-dissociation. We describe in molecular detail how CAND1-induced conformational changes in CUL1/RBX1 provide an optimized DCNL1-binding site and reveal an unexpected dual role for DCNL1 in CAND1-SCF dynamics. Moreover, a partially dissociated CAND1-SCF conformation accommodates cullin neddylation, leading to CAND1 displacement. Our structural findings, together with functional biochemical assays, help formulate a detailed model for CAND-SCF regulation.

FBXO28 promotes proliferation, invasion, and metastasis of pancreatic cancer cells through regulation of SMARCC2 ubiquitination.

The E3 ligase F-box only protein 28 (FBXO28) belongs to the F-box family of proteins that play a critical role in tumor development. However, the potential function of FBXO28 in pancreatic cancer (PC) and its molecular mechanism remain unclear. In this study, we examined FBXO28 expression in PC and its biological role and explored the mechanism of FBXO28-mediated proliferation, invasion, and metastasis of PC cells. Compared with paracancerous tissues and human normal pancreatic ductal epithelial cells, FBXO28 was highly expressed in PC tissues and cell lines. High expression of FBXO28 was negatively correlated with the survival prognosis of patients with PC. Functional assays indicated that FBXO28 promoted PC cell proliferation, invasion, and metastasis in vitro and in vivo. Furthermore, immunoprecipitation-mass spectrometry was used to identify SMARCC2 as the target of FBXO28; upregulation of SMARCC2 can reverse the effect of overexpression of FBXO28 on promoting the proliferation, invasion, and metastasis of PC cells. Mechanistically, FBXO28 inhibited SMARCC2 expression in post-translation by increasing SMARCC2 ubiquitination and protein degradation. In conclusion, FBXO28 has a potential role in PC, possibly promoting PC progression through SMARCC2 ubiquitination. Thus, FBXO28 might be a potential treatment target in PC.

The atypical ubiquitin ligase RNF31 stabilizes c-Myc via epigenetic inactivation of FBXO32 and promotes cancer development.

RNF31, an atypical E3 ubiquitin ligase of the RING-between-RING protein family, is one of the important components of the linear ubiquitin chain complex LUBAC. It plays a carcinogenic role in a variety of cancers by promoting cell proliferation, invasion and inhibiting apoptosis. However, the specific molecular mechanism by which RNF31 exerts its cancer-promoting effects is still unclear. By analyzing the expression profile of RNF31-depleted cancer cells, we found that loss of RNF31 significantly resulted in the inactivation of the c-Myc pathway. We further showed that RNF31 played an important role in the maintenance of c-Myc protein levels in cancer cells by extending the half-life of c-Myc protein and reducing its ubiquitination. c-Myc protein levels are tightly regulated by the ubiquitin proteasome, in which the E3 ligase FBXO32 is required to mediate its ubiquitin-dependent degradation. We found that RNF31 inhibited the transcription of FBXO32 through EZH2-mediated trimethylation of histone H3K27 in the FBXO32 promoter region, leading to the stabilization and activation of c-Myc protein. Under this circumstance, the expression of FBXO32 was significantly increased in RNF31-deficient cells, promoting the degradation of c-Myc protein, inhibiting cell proliferation and invasion, increasing cell apoptosis, and ultimately blocking the progression of tumors. Consistent with these results, the reduced malignancy phenotype caused by RNF31 deficiency could be partially reversed by overexpression of c-Myc or further knockdown of FBXO32. Together, our results reveal a key association between RNF31 and epigenetic inactivation of FBXO32 in cancer cells, and suggest that RNF31 may be a promising target for cancer therapy.

Urotensin II can Induce Skeletal Muscle Atrophy Associated with Upregulating Ubiquitin-Proteasome System and Inhibiting the Differentiation of Satellite Cells in CRF Mice.

Skeletal muscle wasting and atrophy is highly prevalent in chronic renal failure (CRF) and increases the risk of mortality. According to our previous study, we speculate that urotensin II (UII) can induce skeletal muscle atrophy by upregulating ubiquitin-proteasome system(UPS) in CRF. C2C12 mouse myoblast cells were differentiated into myotubes, and myotubes were exposed to different concentrations of UII. Myotube diameters, myosin heavy chain(MHC), p-Fxo03A, skeletal muscle-specific E3 ubiquitin ligases such as muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx/atrogin1) were detected. Three animal models (the sham operation mice as normal control (NC) group, wild-type C57BL/6 mice with 5/6 nephrectomy (WT CRF) group, UII receptor gene knock out (UT KO) mice with 5/6 nephrectomy (UT KO CRF) group) were designed. Cross-sectional area (CSA) of skeletal muscle tissues in three animal models were measured, and western blot detected protein of UII, p-Fxo03A, MAFbx and MuRF1, and immunofluorescence assays explored the satellite cell marker of Myod1 and Pax7, and PCR arrays detected the muscle protein degradation genes, protein synthesis genes and the genes which were involved in muscle components. UII could decrease mouse myotube diameters, and upregulate dephosphorylated Fxo03A protein. MAFbx and MuRF1 were higher in WT CRF group than that in NC group, but after UII receptor gene was knocked out (UT KO CRF), their expressions were downregulated. UII could inhibit the expression of Myod1 but not Pax7 in animal study. We first demonstrate that skeletal muscle atrophy induced by UII associated with upregulating ubiquitin-proteasome system and inhibiting the differentiation of satellite cells in CRF mice.

Downregulation of BASP1 Promotes Temozolomide Resistance in Gliomas via Epigenetic Activation of the FBXO32/NF-κB/MGMT Axis.

The chemoresistance of temozolomide-based therapy is a serious limitation for lasting effective treatment of gliomas, while the underlying mechanisms remain unclear. In this study, we showed that downregulation of BASP1 correlated negatively with the response to temozolomide therapy and disease-free survival (DFS) of patients with gliomas. Silencing BASP1 significantly enhanced the temozolomide resistance of glioma cells both in vitro and in vivo through repair of temozolomide-induced DNA damage via activation of the FBXO32/NF-κB/MGMT axis in both MGMT-methylated and -unmethylated gliomas. We demonstrated that loss of BASP1 resulted in removal of TRIM37/EZH2 complex-induced repressive histone modifications, including H2A-ub and H3K27me3, but addition of WDR5/MLL complex-mediated active histone modifications, including H3K4me3 and H3K9ac, on the FBXO32 promoter, which elicited in FBXO32 upregulation and further activated NF-κB/MGMT signaling via ubiquitin-dependent degradation of IκBα. Importantly, treatment with OICR-9429, an antagonist of the WDR5-MLL interaction, impaired the FBXO32/NF-κB/MGMT axis-mediated repair of temozolomide-induced DNA damage, leading to significant apoptosis of BASP1-downregulated glioma cells. These findings shed light on the molecular mechanism underlying BASP1-mediated epigenetic transcriptional repression and may represent a potential strategy in the fight against temozolomide-resistant gliomas. IMPLICATIONS: BASP1 downregulation promotes temozolomide resistance in gliomas through WDR5/MLL complex-mediated epigenetic activation of the FBXO32/NF-κB/MGMT axis, providing new target for improving outcomes in patients with temozolomide-resistant gliomas.

The wheat TaF-box3, SCF ubiquitin ligase component, participates in the regulation of flowering time in transgenic Arabidopsis.

Histone methylation is actively involved in plant flowering time and is regulated by a myriad of genetic pathways that integrate endogenous and exogenous signals. We identified an F-box gene from wheat (Triticum aestivum L.) and named it TaF-box3. Transcript expression analysis showed that TaF-box3 expression was gradually induced during the floret development and anthesis stages (WS2.5-10). Furthermore, ubiquitination assays have shown that TaF-box3 is a key component of the SCF ubiquitin ligase complex. TaF-box3 overexpression in Arabidopsis resulted in an early flowering phenotype and different cell sizes in leaves compared to the WT. Furthermore, the transcript level of a flowering time-related gene was significantly reduced in TaF-box3 overexpressing plants, which was linked with lower histone H3 Lys4 trimethylation (H3K4me3) and H3 Lys36 trimethylation (H3K36me3). Overexpression of TaF-box3 in Arabidopsis was shown to be involved in the regulation of flowering time by demethylating FLC chromatin, according to ChIP experiments. Protein analysis confirmed that TaMETS interacts with TaF-box3 and is ubiquitinated and degraded in a TaF-box3-dependnent manner. Based on these findings, we propose that TaF-box3 has a positive role in flowering time, which leads to a better understanding of TaF-box3 physiological mechanism in Arabidopsis.

AtTLP2, a Tubby-like protein, plays intricate roles in abiotic stress signalling.

KEY MESSAGE: The Arabidopsis Tubby-like protein (TLP) encoding gene, AtTLP2, plays intricate roles during ABA-dependent abiotic stress signalling, particularly salt and dehydration stress responses. TLPs (Tubby-like proteins) are a small group of eukaryotic proteins characterized by the presence of a Tubby domain. The plant TLPs have been widely shown to play important roles during abiotic stress signaling. In this study, we investigated the role of an Arabidopsis TLP, AtTLP2, in mediating abiotic stress responses. Both attlp2 null mutant and overexpression (OE) lines, in Arabidopsis, were studied which indicated the role of the gene also in development. The attlp2 mutant showed an overall dwarfism, while its overexpression caused enhanced growth. AtTLP2 localized to the plasma membrane (PM) and showed nuclear translocation in response to dehydration stress. The protein interacted with ASK1 and ASK2, but failed to show transactivation activity in yeast. AtTLP2 was transcriptionally induced by stress, caused by salt, dehydration and ABA. The attlp2 mutant was insensitive to ABA, but hypersensitive to oxidative stress at all stages of growth. ABA insensitivity conferred tolerance to salt and osmotic stresses at the germination and early seedling growth stages, but caused hypersensitivity to salt and drought stresses at advanced stages of growth. The OE lines were more sensitive to ABA, causing increased sensitivity to most stresses at the seed germination stage, but conferring tolerance to salt and osmotic stresses at more advanced stages of development. The stomata of the attlp2 mutant were less responsive to ABA and H2O2, while that of the OE lines exhibited greater sensitivity. Several ABA-regulated stress responsive marker genes were found to be downregulated in the mutant, but upregulated in the OE lines. The study establishes that AtTLP2 plays intricate roles in abiotic stress signaling, and the response may be largely ABA dependent.

F-box receptor mediated control of substrate stability and subcellular location organizes cellular development of Aspergillus nidulans.

Fungal growth and development are coordinated with specific secondary metabolism. This coordination requires 8 of 74 F-box proteins of the filamentous fungus Aspergillus nidulans. F-box proteins recognize primed substrates for ubiquitination by Skp1-Cul1-Fbx (SCF) E3 ubiquitin RING ligases and degradation by the 26S proteasome. 24 F-box proteins are found in the nuclear fraction as part of SCFs during vegetative growth. 43 F-box proteins interact with SCF proteins during growth, development or stress. 45 F-box proteins are associated with more than 700 proteins that have mainly regulatory roles. This corroborates that accurate surveillance of protein stability is prerequisite for organizing multicellular fungal development. Fbx23 combines subcellular location and protein stability control, illustrating the complexity of F-box mediated regulation during fungal development. Fbx23 interacts with epigenetic methyltransferase VipC which interacts with fungal NF-κB-like velvet domain regulator VeA that coordinates fungal development with secondary metabolism. Fbx23 prevents nuclear accumulation of methyltransferase VipC during early development. These results suggest that in addition to their role in protein degradation, F-box proteins also control subcellular accumulations of key regulatory proteins for fungal development.

A novel likely pathogenic variant in the FBXO32 gene associated with dilated cardiomyopathy according to whole­exome sequencing.

BACKGROUND: Familial dilated cardiomyopathy (DCM) is a genetic heart disorder characterized by progressive heart failure and sudden cardiac death. Over 250 genes have been reported in association with DCM; nonetheless, the genetic cause of most DCM patients has been unknown. The goal of the present study was to determine the genetic etiology of familial DCM in an Iranian family. METHODS: Whole-exome sequencing was performed to identify the underlying variants in an Iranian consanguineous family with DCM. The presence of the candidate variant was confirmed and screened in available relatives by PCR and Sanger sequencing. The pathogenic effect of the candidate variant was assessed by bioinformatics analysis, homology modeling, and docking. RESULTS: One novel likely pathogenic deletion, c.884_886del: p.Lys295del, in F-box only protein 32 (muscle-specific ubiquitin-E3 ligase protein; FBXO32) was identified. Based on bioinformatics and modeling analysis, c.884_886del was the most probable cause of DCM in the studied family. CONCLUSIONS: Our findings indicate that variants in FBXO32 play a role in recessive DCM. Variants in FBXO32 may disturb the degradation of target proteins in the ubiquitin-proteasome system and lead to severe DCM. We suggest considering this gene variants in patients with recessively inherited DCM.

The SCF-FBXW7 E3 ubiquitin ligase triggers degradation of histone 3 lysine 4 methyltransferase complex component WDR5 to prevent mitotic slippage.

During prolonged mitotic arrest induced by antimicrotubule drugs, cell fate decision is determined by two alternative pathways, one leading to cell death and the other inducing premature escape from mitosis by mitotic slippage. FBWX7, a member of the F-box family of proteins and substrate-targeting subunit of the SKP1-CUL1-F-Box E3 ubiquitin ligase complex, promotes mitotic cell death and prevents mitotic slippage, but molecular details underlying these roles for FBWX7 are unclear. In this study, we report that WDR5 (WD-repeat containing protein 5), a component of the mixed lineage leukemia complex of histone 3 lysine 4 methyltransferases, is a substrate of FBXW7. We determined by coimmunoprecipitation experiments and in vitro binding assays that WDR5 interacts with FBXW7 in vivo and in vitro. SKP1-CUL1-F-Box-FBXW7 mediates ubiquitination of WDR5 and targets it for proteasomal degradation. Furthermore, we find that WDR5 depletion counteracts FBXW7 loss of function by reducing mitotic slippage and polyploidization. In conclusion, our data elucidate a new mechanism in mitotic cell fate regulation, which might contribute to prevent chemotherapy resistance in patients after antimicrotubule drug treatment.

Identification of an E3 ligase that targets the catalytic subunit of RNA Polymerase I upon transcription stress.

RNA Polymerase I (Pol I) synthesizes rRNA, which is the first and rate-limiting step in ribosome biogenesis. Factors governing the stability of the polymerase complex are not known. Previous studies characterizing Pol I inhibitor BMH-21 revealed a transcriptional stress-dependent pathway for degradation of the largest subunit of Pol I, RPA194. To identify the E3 ligase(s) involved, we conducted a cell-based RNAi screen for ubiquitin pathway genes. We establish Skp-Cullin-F-box protein complex F-box protein FBXL14 as an E3 ligase for RPA194. We show that FBXL14 binds to RPA194 and mediates RPA194 ubiquitination and degradation in cancer cells treated with BMH-21. Mutation analysis in yeast identified lysines 1150, 1153, and 1156 on Rpa190 relevant for the protein degradation. These results reveal the regulated turnover of Pol I, showing that the stability of the catalytic subunit is controlled by the F-box protein FBXL14 in response to transcription stress.

An integrative pan-cancer analysis revealing the difference in small ring finger family of SCF E3 ubiquitin ligases.

Background: The SCF (Skp1-cullin-F-box proteins) complex is the largest family of E3 ubiquitin ligases that mediate multiple specific substrate proteins degradation. Two ring-finger family members RBX1/ROC1 and RBX2/RNF7/SAG are small molecular proteins necessary for ubiquitin ligation activity of the multimeric SCF complex. Accumulating evidence indicated the involvement of RBX proteins in the pathogenesis and development of cancers, but no research using pan-cancer analysis for evaluating their difference has been directed previously. Methods: We investigated RBX1/2 expression patterns and the association with clinicopathological features, and survivals of cancer patients obtained from the TCGA pan-cancer data. The binding energies of RBX1/2-CUL1 complexes were preliminarily calculated by using molecular dynamics simulations. Meanwhile, we assessed their immune infiltration level across numerous databases, including TISIDB and Timer database. Results: High expression levels of RBX1/2 were observed in most cancer types and correlated with poor prognosis of patients analyzed. Nonetheless, exceptions were observed: RBX2 expression in KICH was higher than normal renal tissues and played a detrimental role in KICH. The expression of RBX1 was not associated with the prognostic risk of KICH. Moreover, the combination of RBX1 and CUL1 expression is more stable than that of RBX2 and CUL1. RBX1/2 expression showed their own specific characteristics in tumor pathological stages and grades, copy number variation and immune components. Conclusions: These findings not only indicated that the difference of RBX1/2 might result in varying degrees of tumor progression, but also suggested that they might serve as biomarkers for immune infiltration in cancers, shedding new light on therapeutics of cancers.