FBXO32-mediated degradation of PTEN promotes lung adenocarcinoma progression.

FBXO32, a member of the F-box protein family, is known to play both oncogenic and tumor-suppressive roles in different cancers. However, the functions and the molecular mechanisms regulated by FBXO32 in lung adenocarcinoma (LUAD) remain unclear. Here, we report that FBXO32 is overexpressed in LUAD compared with normal lung tissues, and high expression of FBXO32 correlates with poor prognosis in LUAD patients. Firstly, we observed with a series of functional experiments that FBXO32 alters the cell cycle and promotes the invasion and metastasis of LUAD cells. We further corroborate our findings using in vivo mouse models of metastasis and confirmed that FBXO32 positively regulates LUAD tumor metastasis. Using a proteomic-based approach combined with computational analyses, we found a positive correlation between FBXO32 and the PI3K/AKT/mTOR pathway, and identified PTEN as a FBXO32 interactor. More important, FBXO32 binds PTEN via its C-terminal substrate binding domain and we also validated PTEN as a bona fide FBXO32 substrate. Finally, we demonstrated that FBXO32 promotes EMT and regulates the cell cycle by targeting PTEN for proteasomal-dependent degradation. In summary, our study highlights the role of FBXO32 in promoting the PI3K/AKT/mTOR pathway via PTEN degradation, thereby fostering lung adenocarcinoma progression.

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.

FBXO28 promotes cell proliferation, migration and invasion via upregulation of the TGF-beta1/SMAD2/3 signaling pathway in ovarian cancer.

BACKGROUND: Ovarian cancer is one of the most common gynecological malignancies due to the lack of early symptoms, early diagnosis and limited screening. Therefore, it is necessary to understand the molecular mechanism underlying the occurrence and progression of ovarian cancer and to identify a basic biomarker for the early diagnosis and clinical treatment of ovarian cancer. METHODS: The association between FBXO28 and ovarian cancer prognosis was analyzed using Kaplan‒Meier survival analysis. The difference in FBXO28 mRNA expression between normal ovarian tissues and ovarian tumor tissues was obtained from The Cancer Genome Atlas (TCGA), and Genotype-Tissue Expression (GTEx) cohorts. The expression levels of the FBXO28 protein in ovarian cancer tissues and normal ovarian tissues were measured via immunohistochemical staining. Western blotting was used to determine the level of FBXO28 expression in ovarian cancer cells. The CCK-8, the colony formation, Transwell migration and invasion assays were performed to evaluate cell proliferation and motility. RESULTS: We found that a higher expression level of FBXO28 was associated with poor prognosis in ovarian cancer patients. Analysis of the TCGA and GTEx cohorts showed that the FBXO28 mRNA level was lower in normal ovarian tissue samples than in ovarian cancer tissue samples. Compared with that in normal ovarian tissues or cell lines, the expression of FBXO28 was greater in ovarian tumor tissues or tumor cells. The upregulation of FBXO28 promoted the viability, proliferation, migration and invasion of ovarian cancer cells. Finally, we demonstrated that FBXO28 activated the TGF-beta1/Smad2/3 signaling pathway in ovarian cancer. CONCLUSIONS: In conclusion, FBXO28 enhanced oncogenic function via upregulation of the TGF-beta1/Smad2/3 signaling pathway in ovarian cancer.

COP9 signalosome-mediated deneddylation of CULLIN1 is necessary for SCFEBF1 assembly in Arabidopsis thaliana.

Functions of the SKP1-CUL1-F box (SCF) ubiquitin E3 ligases are essential in plants. The F box proteins (FBPs) are substrate receptors that recruit substrates and assemble an active SCF complex, but the regulatory mechanism underlying the FBPs binding to CUL1 to activate the SCF cycle is not fully understood. We show that Arabidopsis csn1-10 is defective in SCFEBF1-mediated PIF3 degradation during de-etiolation, due to impaired association of EBF1 with CUL1 in csn1-10. EBF1 preferentially associates with un-neddylated CUL1 that is deficient in csn1-10 and the EBF1-CUL1 binding is rescued by the neddylation inhibitor MLN4924. Furthermore, we identify a subset of FBPs with impaired binding to CUL1 in csn1-10, indicating their assembly to form SCF complexes may depend on COP9 signalosome (CSN)-mediated deneddylation of CUL1. This study reports that a key role of CSN-mediated CULLIN deneddylation is to gate the binding of the FBP-substrate module to CUL1, thus initiating the SCF cycle of substrate ubiquitination.

MiR-431 promotes cardiomyocyte proliferation by targeting FBXO32 expression.

BACKGROUND: The induction of cardiomyocyte (CM) proliferation is a promising approach for cardiac regeneration following myocardial injury. MicroRNAs (miRNAs) have been reported to regulate CM proliferation. In particular, miR-431 expression decreases during cardiac development, according to Gene Expression Omnibus (GEO) microarray data. However, whether miR-431 regulates CM proliferation has not been thoroughly investigated. METHODS: We used integrated bioinformatics analysis of GEO datasets to identify the most significantly differentially expressed miRNAs. Real-time quantitative PCR and fluorescence in situ hybridization were performed to determine the miRNA expression patterns in hearts. Gain- and loss-of-function assays were conducted to detect the role of miRNA in CM proliferation. Additionally, we detected whether miR-431 affected CM proliferation in a myocardial infarction model. The TargetScan, miRDB and miRWalk online databases were used to predict the potential target genes of miRNAs. Luciferase reporter assays were used to study miRNA interactions with the targeting mRNA. RESULTS: First, we found a significant reduction in miR-431 levels during cardiac development. Then, by overexpression and inhibition of miR-431, we demonstrated that miR-431 promotes CM proliferation in vitro and in vivo, as determined by immunofluorescence assays of 5-ethynyl-2'-deoxyuridine (EdU), pH3, Aurora B and CM count, whereas miR-431 inhibition suppresses CM proliferation. Then, we found that miR-431 improved cardiac function post-myocardial infarction. In addition, we identified FBXO32 as a direct target gene of miR-431, with FBXO32 mRNA and protein expression being suppressed by miR-431. FBXO32 inhibited CM proliferation. Overexpression of FBXO32 blocks the enhanced effect of miR-431 on CM proliferation, suggesting that FBXO32 is a functional target of miR-431 during CM proliferation. CONCLUSION: In summary, miR-431 promotes CM proliferation by targeting FBXO32, providing a potential molecular target for preventing myocardial injury.

Protective effect of Luffa cylindrica Roemer against dexamethasone-induced muscle atrophy in primary rat skeletal muscle cells.

Glucocorticoids (GCs) are commonly used in the treatment of chronic inflammatory conditions. However, the administration of high doses and long-term use of GCs can induce muscle atrophy (MA) in patients, leading to a decline in quality of life and increased mortality. MA leads to protein degradation in skeletal muscle, resulting in a reduction of muscle mass. This process is triggered by GCs like dexamethasone (DEX), which induce the expression of E3 ubiquitin ligases, namely Atrogin-1 and muscle RING-finger protein-1 (MuRF1). In this study, we examined the anti-MA potential of Luffa cylindrica Roemer (LCR) on DEX-treated primary skeletal myotubes. Primary skeletal myotubes stimulated with LCR alone resulted in a significant upregulation of myotube development, characterized by an increase in both the number and diameter of myotubes. Contrastingly, combined treatment with LCR and DEX reduced the expression of Atrogin-1, while treatment with DEX alone induced the expression of MuRF1. Furthermore, LCR treatment successfully restored the number and diameter of myotubes that had been diminished by DEX treatment. These findings suggest that LCR holds potential for treating MA, as an accelerating effect on muscle development and anti-MA effects on primary skeletal muscle cells were observed.

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.

3' UTR Deletion of FBXO28 in a Patient with Brain Abnormalities and Developmental Delay.

Constitutional deletions of chromosome 1q42 region are rare. The phenotype spectrum associated with this copy number change is variable, including developmental delay, intellectual disability, seizures, and dysmorphology. This study describes a patient with developmental delays and brain abnormalities. G-banded karyotype, FISH, SNP oligonucleotide microarray analysis (SOMA), and whole exome sequencing analysis were performed. Postnatal reanalysis of prenatal SOMA and follow-up parental testing revealed a paternally inherited 63 kb deletion at 1q42.11 in the patient. We characterized the clinical features of this patient, providing insight into the clinical phenotype associated with deletions of the 1q42.11 sub-band. Our study provides new evidence supporting the potential functional importance of the FBXO28 3' UTR region and the hypothesis that FBXO28 is a critical gene in the pathogenesis of chromosome 1q41q42 microdeletion syndrome. It also highlights the different goals and reporting criteria between prenatal and postnatal microarray tests.

C-terminal sequence stability profiling in Saccharomyces cerevisiae reveals protective protein quality control pathways.

Protein quality control (PQC) mechanisms are essential for degradation of misfolded or dysfunctional proteins. An essential part of protein homeostasis is recognition of defective proteins by PQC components and their elimination by the ubiquitin-proteasome system, often concentrating on protein termini as indicators of protein integrity. Changes in amino acid composition of C-terminal ends arise through protein disintegration, alternative splicing, or during the translation step of protein synthesis from premature termination or translational stop-codon read-through. We characterized reporter protein stability using light-controlled exposure of the random C-terminal peptide collection (CtPC) in budding yeast revealing stabilizing and destabilizing features of amino acids at positions -5 to -1 of the C terminus. The (de)stabilization properties of CtPC-degrons depend on amino acid identity, position, as well as composition of the C-terminal sequence and are transferable. Evolutionary pressure toward stable proteins in yeast is evidenced by amino acid residues under-represented in cytosolic and nuclear proteins at corresponding C-terminal positions, but over-represented in unstable CtPC-degrons, and vice versa. Furthermore, analysis of translational stop-codon read-through peptides suggested that such extended proteins have destabilizing C termini. PQC pathways targeting CtPC-degrons involved the ubiquitin-protein ligase Doa10 and the cullin-RING E3 ligase SCFDas1 (Skp1-Cullin-F-box protein). Overall, our data suggest a proteome protection mechanism that targets proteins with unnatural C termini by recognizing a surprisingly large number of C-terminal sequence variants.

Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway.

Background: Advanced glycation end products (AGEs) impair vascular physiology in Diabetes mellitus (DM). However, the underlying mechanisms remain unclear. Vascular large conductance calcium-activated potassium (BK) channels play important roles in coronary arterial function.Purpose: Our study aimed to investigate the regulatory role of AGEs in BK channels.Research Design: Using gavage of vehicle (V, normal saline) or aminoguanidine (A) for 8 weeks, normal and diabetic rats were divided into four groups: C+V group, DM+V group, C+A group, and DM+A group.Study Sample: Coronary arteries from different groups of rats and human coronary smooth muscle cells were used in this study.Data Collection and Analysis: Data were presented as mean ± SEM (standard error of mean). Student's t-test was used to compare data between two groups. One-way ANOVA with post-hoc LSD analysis was used to compare data between multiple groups.Results: Compared to the C+V group, vascular contraction induced by iberiotoxin (IBTX), a BK channel inhibitor, was impaired, and BK channel densities decreased in the DM+V group. However, aminoguanidine administration reduced the impairment. Protein expression of BK-ß1, phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), and protein kinase B (PKB or Akt) were down-regulated, while F-box protein 32 (FBXO32) expression increased in the DM+V group and in high glucose (HG) cultured human coronary smooth muscle cells. Treatment with aminoguanidine in vitro and in vivo could reverse the above protein expression. The effect of aminoguanidine on the improvement of BK channel function by inhibiting the generation of AGEs was reversed by adding MK2206 (Akt inhibitor) or Compound C (AMPK inhibitor) in HG conditions in vitro.Conclusions: AGEs aggravate BK channel dysfunction via the AMPK/Akt/FBXO32 signaling pathway.

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.

Isolation and characterization of ß-transducin repeat-containing protein ligands screened using a high-throughput screening system.

ß-transducin repeat-containing protein (ß-TrCP) is an F-box protein subunit of the E3 Skp1-Cullin-F box (SCF) type ubiquitin-ligase complex, and provides the substrate specificity for the ligase. To find potent ligands of ß-TrCP useful for the proteolysis targeting chimera (PROTAC) system using ß-TrCP in the future, we developed a high-throughput screening system for small molecule ß-TrCP ligands. We screened the chemical library utilizing the system and obtained several hit compounds. The effects of the hit compounds on in vitro ubiquitination activity of SCFß-TrCP1 and on downstream signaling pathways were examined. Hit compounds NPD5943, NPL62020-01, and NPL42040-01 inhibited the TNFα-induced degradation of IκBα and its phosphorylated form. Hence, they inhibited the activation of the transcription activity of NF-κB, indicating the effective inhibition of ß-TrCP by the hit compounds in cells. Next, we performed an in silico analysis of the hit compounds to determine the important moieties of the hit compounds. Carboxyl groups of NPL62020-01 and NPL42040-01 and hydroxyl groups of NPD5943 created hydrogen bonds with ß-TrCP similar to those created by intrinsic target phosphopeptides of ß-TrCP. Our findings enhance our knowledge of useful small molecule ligands of ß-TrCP and the importance of residues that can be ligands of ß-TrCP.

Ethanol Extract of Citrus grandis 'Tomentosa' Exerts Anticancer Effects by Targeting Skp2/p27 Pathway in Non-Small Cell Lung Cancer.

SCOPE: This study aims to investigate the anticancer properties of Citrus grandis 'Tomentosa' (CGT) in non-small cell lung cancer (NSCLC). METHODS AND RESULTS: The ethanol extract of CGT (CGTE) is prepared by using anhydrous ethanol and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), revealing that the main chemical components in CGTE are flavonoids and coumarins, such as naringin, rhoifolin, apigenin, bergaptol, and osthole. CGTE at concentrations without inducing cell death significantly inhibits cell proliferation via inducing cell cycle G1 phase arrest by MTT, colony formation, and flow cytometry assays, implying that CGT has anticancer potential. CGTE markedly inhibits the activity of Skp2-SCF E3 ubiquitin ligase, decreases the protein level of Skp2, and promotes the accumulation of p27 by co-immunoprecipitation (co-IP) and in vivo ubiquitination assay; whereas Skp2 overexpression rescues the effects of CGTE in NSCLC cells. In subcutaneous LLC allograft and A549 xenograft mouse models, CGTE, without causing obvious side effects in mice, significantly inhibits lung tumor growth by targeting the Skp2/p27 signaling pathway. CONCLUSION: These findings demonstrate that CGTE efficiently inhibits NSCLC proliferation both in vitro and in vivo by targeting the Skp2/p27 signaling pathway, suggesting that CGTE may serve as a therapeutic candidate for NSCLC treatment.

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.

Promotive Effect of FBXO32 on the Odontoblastic Differentiation of Human Dental Pulp Stem Cells.

Odontoblastic differentiation of human dental pulp stem cells (hDPSCs) is crucial for the intricate formation and repair processes in dental pulp. Until now, the literature is not able to demonstrate the role of ubiquitination in the odontoblastic differentiation of hDPSCs. This study investigated the role of F-box-only protein 32 (FBXO32), an E3 ligase, in the odontoblastic differentiation of hDPSCs. The mRNA expression profile was obtained from ribonucleic acid sequencing (RNA-Seq) data and analyzed. Immunofluorescence and immunohistochemical staining identify the FBXO32 expression in human dental pulp and hDPSCs. Small-hairpin RNA lentivirus was used for FBXO32 knockdown and overexpression. Odontoblastic differentiation of hDPSCs was determined via alkaline phosphatase activity, Alizarin Red S staining, and mRNA and protein expression levels were detected using real-time quantitative polymerase chain reaction and Western blotting. Furthermore, subcutaneous transplantation in nude mice was performed to evaluate the role of FBXO32 in mineralization in vivo using histological analysis. FBXO32 expression was upregulated in the odontoblast differentiated hDPSCs as evidenced by RNA-Seq data analysis. FBXO32 was detected in hDPSCs and the odontoblast layer of the dental pulp. Increased FBXO32 expression in hDPSCs during odontoblastic differentiation was confirmed. Through lentivirus infection method, FBXO32 downregulation in hDPSCs attenuated odontoblastic differentiation in vitro and in vivo, whereas FBXO32 upregulation promoted the hDPSCs odontoblastic differentiation, without affecting proliferation and migration. This study demonstrated, for the first time, the promotive role of FBXO32 in regulating the odontoblastic differentiation of hDPSCs, thereby providing novel insights into the regulatory mechanisms during odontoblastic differentiation in hDPSCs.