F-box only protein 2 exacerbates non-alcoholic fatty liver disease by targeting the hydroxyl CoA dehydrogenase alpha subunit.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a major health burden with an increasing global incidence. Unfortunately, the unavailability of knowledge underlying NAFLD pathogenesis inhibits effective preventive and therapeutic measures. AIM: To explore the molecular mechanism of NAFLD. METHODS: Whole genome sequencing (WGS) analysis was performed on liver tissues from patients with NAFLD (n = 6) and patients with normal metabolic conditions (n = 6) to identify the target genes. A NAFLD C57BL6/J mouse model induced by 16 wk of high-fat diet feeding and a hepatocyte-specific F-box only protein 2 (FBXO2) overexpression mouse model were used for in vivo studies. Plasmid transfection, co-immunoprecipitation-based mass spectrometry assays, and ubiquitination in HepG2 cells and HEK293T cells were used for in vitro studies. RESULTS: A total of 30982 genes were detected in WGS analysis, with 649 up-regulated and 178 down-regulated. Expression of FBXO2, an E3 ligase, was upregulated in the liver tissues of patients with NAFLD. Hepatocyte-specific FBXO2 overexpression facilitated NAFLD-associated phenotypes in mice. Overexpression of FBXO2 aggravated odium oleate (OA)-induced lipid accumulation in HepG2 cells, resulting in an abnormal expression of genes related to lipid metabolism, such as fatty acid synthase, peroxisome proliferator-activated receptor alpha, and so on. In contrast, knocking down FBXO2 in HepG2 cells significantly alleviated the OA-induced lipid accumulation and aberrant expression of lipid metabolism genes. The hydroxyl CoA dehydrogenase alpha subunit (HADHA), a protein involved in oxidative stress, was a target of FBXO2-mediated ubiquitination. FBXO2 directly bound to HADHA and facilitated its proteasomal degradation in HepG2 and HEK293T cells. Supplementation with HADHA alleviated lipid accumulation caused by FBXO2 overexpression in HepG2 cells. CONCLUSION: FBXO2 exacerbates lipid accumulation by targeting HADHA and is a potential therapeutic target for NAFLD.

ELK4 Promotes Cell Cycle Progression and Stem Cell-like Characteristics in HPV-associated Cervical Cancer by Regulating the FBXO22/PTEN Axis

Background: Cervical cancer (CC) is a prevalent gynecological carcinoma, and patients infected with human papillomavirus (HPV) have a higher morbidity rate. Aims: To explore the effects of ETS-like transcription factor 4 (ELK4) in patients with HPV+ CC. Study design: In vitro cell lines and human-sample study. Methods: The ELK4 levels in human tissue (65 HPV+ CC tissue and 25 HPV− normal cervical tissue) and cell lines (human cervical epithelial immortalized cell line H8 and CC cell lines HeLa [HPV18], CaSki [HPV16], and SiHa [HPV−]) were quantified using qRT-PCR and western blot assay. ELK4 knockdown transfection was effective and confirmed by western blotting. The MTT and EDU assays were used to evaluate cell viability and proliferation, respectively. Flow cytometry was used to detect the CC cell cycle stage. Stem cell markers, such as cluster of differentiation 133 (CD133), CD44, and aldehyde dehydrogenase 1, and the cervicospheres formed were measured. ChIP-qPCR and luciferase activity experiments were used to assess the bond between ELK4 and F-box protein 22 (FBXO22). Results: ELK4 was highly expressed in the HPV+ CC tissue. CC cells with ELK4 knockdown had lower viability and proliferation than the control cells. ELK4 knockdown blocked the progression of the cell cycle from G1 to S phase. ELK4 knockdown suppressed the stem cell-like characteristics of the HPV+ CC cells. ELK4 bonded with the FBXO22 promoter, inhibiting the levels of phosphatase and tensin homolog (PTEN). Conclusion: ELK4 facilitated cell cycle progression and stem cell-like characteristics by regulating the FBXO22/PTEN axis. Thus, ELK4 could be a potential therapeutic target to arrest the progress of HPV-associated CC.

FBXO10 prevents chronic unpredictable stress-induced behavioral despair and cognitive impairment through promoting RAGE degradation.

AIMS: Depression is one of the leading causes of disability worldwide. The receptor for advanced glycosylation end products (RAGE) is closely related to chronic stress and is a target of F-box protein O10 (FBXO10) which promotes the degradation of RAGE by ubiquitination. Here, we explored the role of FBXO10 and RAGE in chronic unpredictable stress (CUS)-induced behavioral despair, cognitive impairment, neuroinflammation, and the polarization microglia. METHODS: Male C57BL/6 mice with or without infusion of viral in the medial prefrontal cortex (PFC) were subjected to CUS. Then the mice were exposed to forced swim test, sucrose consumption test, novelty-suppressed feeding test, and temporal object recognition task to assess the behavioral despair and cognitive impairment. Inflammatory cytokines and the neurotrophic factor brain-derived neurotrophic factor (BDNF) levels in PFC were assessed by enzyme-linked immunosorbent assay. Immunofluorescence and immunohistochemistry staining were performed to observe the activation and phenotypic transformation of microglia in PFC. LPS-induced cell model was constructed to explore the effect of FBXO10/RAGE axis in the polarization of microglia in vitro. RESULTS: FBXO10 promoted RAGE degradation by ubiquitination in BV2 cells. FBXO10 protein levels were reduced whereas RAGE protein levels were enhanced in CUS mice. FBXO10 overexpression or RAGE knockdown inhibited proinflammatory cytokine release, promoted BDNF expression, mitigated the depressive-like and cognitive impairment behaviors, and affected the polarization of microglia induced by CUS exposure. FBXO10/RAGE axis promoted the polarization of microglia from the M1 to the M2 phenotype in vitro. Moreover, p38 MAPK and NF-κΒ were identified to be the downstream effect factors for FBXO10/RAGE axis. CONCLUSIONS: FBXO10 administration prevents CUS-induced behavioral despair, cognitive impairment, neuroinflammation, and the polarization of microglia through decreasing the accumulation of RAGE, p38 MAPK, and NF-κΒ, suggesting potential therapeutic strategies for the prevention and treatment of depression.

Electron microscopy structure of human APC/C(CDH1)-EMI1 reveals multimodal mechanism of E3 ligase shutdown.

The anaphase-promoting complex/cyclosome (APC/C) is a ~1.5-MDa multiprotein E3 ligase enzyme that regulates cell division by promoting timely ubiquitin-mediated proteolysis of key cell-cycle regulatory proteins. Inhibition of human APC/C(CDH1) during interphase by early mitotic inhibitor 1 (EMI1) is essential for accurate coordination of DNA synthesis and mitosis. Here, we report a hybrid structural approach involving NMR, electron microscopy and enzymology, which reveal that EMI1's 143-residue C-terminal domain inhibits multiple APC/C(CDH1) functions. The intrinsically disordered D-box, linker and tail elements, together with a structured zinc-binding domain, bind distinct regions of APC/C(CDH1) to synergistically both block the substrate-binding site and inhibit ubiquitin-chain elongation. The functional importance of intrinsic structural disorder is explained by enabling a small inhibitory domain to bind multiple sites to shut down various functions of a 'molecular machine' nearly 100 times its size.

FBXW7 influences murine intestinal homeostasis and cancer, targeting Notch, Jun, and DEK for degradation.

The Fbxw7 (F-box/WD repeat-containing protein 7; also called CDC4, Sel10, Ago, and Fbw7) component of the SCF (Skp1/Cullin/F-box protein) E3 ubiquitin ligase complex acts as a tumor suppressor in several tissues and targets multiple transcriptional activators and protooncogenes for ubiquitin-mediated degradation. To understand Fbxw7 function in the murine intestine, in this study, we specifically deleted Fbxw7 in the murine gut using Villin-Cre (Fbxw7(ΔG)). In wild-type mice, loss of Fbxw7 in the gut altered homeostasis of the intestinal epithelium, resulted in elevated Notch and c-Jun expression, and induced development of adenomas at 9-10 mo of age. In the context of APC (adenomatous polyposis coli) deficiency (Apc(Min/+) mice), loss of Fbxw7 accelerated intestinal tumorigenesis and death and promoted accumulation of ß-catenin in adenomas at late but not early time points. At early time points, Fbxw7 mutant tumors showed accumulation of the DEK protooncogene. DEK expression promoted cell division and altered splicing of tropomyosin (TPM) RNA, which may also influence cell proliferation. DEK accumulation and altered TPM RNA splicing were also detected in FBXW7 mutant human colorectal tumor tissues. Given their reduced lifespan and increased incidence of intestinal tumors, Apc(Min/+)Fbxw7(ΔG) mice may be used for testing carcinogenicity and drug screening.

Ex vivo maintenance of hematopoietic stem cells by quiescence induction through Fbxw7α overexpression.

Cell-cycle quiescence in hematopoietic stem cells (HSCs) is essential for maintaining stemness by protecting cells from differentiation or senescence. F-box and WD-40 domain protein 7 (Fbxw7) maintains HSCs and suppresses leukemogenesis by mediating ubiquitin-dependent degradation of cell-cycle activators and oncoproteins. Fbxw7α was shown to be the preferentially expressed Fbxw7 isoform in primitive HSCs. Forced Fbxw7α expression in lineage marker Sca-1(+)c-Kit(+) cells led to cell-cycle dormancy by reducing the protein levels of the Fbxw7 substrates c-Myc, Notch1, and phosphorylated S6 (a key downstream element of mTOR). Hypoxia, an essential factor for HSC quiescence, suppressed c-Myc in an Fbxw7α-dependent manner. Fbxw7α-overexpressing lineage marker Sca-1(+)c-Kit(+) cells sustained high reconstitution capacities during in vitro culture. These data suggest that Fbxw7α sustains HSC dormancy through c-Myc, Notch1, and the mTOR pathways. The modulation of Fbxw7α expression or activity represents a promising new tool for ex vivo HSC maintenance.

Complex regulation of cell-cycle inhibitors by Fbxw7 in mouse embryonic fibroblasts.

The F-box protein Fbxw7 (also known as Fbw7, SEL-10, hCdc4 or hAgo) mediates the ubiquitylation and thereby contributes to the degradation of proteins that positively regulate cell cycle. Conditional ablation of Fbxw7 in mouse embryonic fibroblasts (MEFs) induces cell-cycle arrest accompanied by abnormal accumulation of the intracellular domain of Notch1 (NICD1) and c-Myc. However, the molecular mechanisms by which the accumulation of NICD1 and c-Myc induces cell-cycle arrest have remained unclear. We have now examined the expression of cell-cycle inhibitors in Fbxw7-deficient MEFs and found that the abundance of p27(Kip1) and p57(Kip2) is paradoxically decreased. This phenomenon appears to be attributable to the accumulation of NICD1, given that it was recapitulated by overexpression of NICD1 and blocked by ablation of RBP-J. Conversely, the expression of p16(Ink4a) and p19(ARF) was increased in an NICD1-independent manner in Fbxw7-null MEFs. The increased expression of p19(ARF) was recapitulated by overexpression of c-Myc and abolished by ablation of c-Myc, suggesting that the accumulation of c-Myc is primarily responsible for that of p19(ARF). In contrast, the upregulation of p16(Ink4a) appeared to be independent of c-Myc. These results indicate that cell-cycle inhibitors undergo complex regulation by the Fbxw7-mediated proteolytic system.

SCFCdc4 acts antagonistically to the PGC-1alpha transcriptional coactivator by targeting it for ubiquitin-mediated proteolysis.

Peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1alpha (PGC-1alpha) is a highly regulated transcriptional coactivator that coordinates energy metabolism in mammals. Misregulation of PGC-1alpha has been implicated in the pathogenesis of several human diseases, including diabetes, obesity, and neurological disorders. We identified SCF(Cdc4) as an E3 ubiquitin ligase that regulates PGC-1alpha through ubiquitin-mediated proteolysis. PGC-1alpha contains two Cdc4 phosphodegrons that bind Cdc4 when phosphorylated by Glycogen Synthase Kinase 3beta (GSK3beta) and p38 MAPK, leading to SCF(Cdc4)-dependent ubiquitylation and proteasomal degradation of PGC-1alpha. Furthermore, SCF(Cdc4) negatively regulates PGC-1alpha-dependent transcription. We demonstrate that RNAi-mediated reduction of Cdc4 in primary neurons results in an increase of endogenous PGC-1alpha protein, while ectopic expression of Cdc4 leads to a reduction of endogenous PGC-1alpha protein. Finally, under conditions of oxidative stress in neurons, Cdc4 levels are decreased, leading to an increase in PGC-1alpha protein and PGC-1alpha-dependent transcription. These results suggest that attenuation of SCF(Cdc4)-dependent proteasomal degradation of PGC-1alpha has a role in mediating the PGC-1alpha-dependent transcriptional response to oxidative stress.