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1.
Nat Commun ; 15(1): 4790, 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38839752

ABSTRACT

Cancer cells are often addicted to serine synthesis to support growth. How serine synthesis is regulated in cancer is not well understood. We recently demonstrated protein arginine methyltransferase 1 (PRMT1) is upregulated in hepatocellular carcinoma (HCC) to methylate and activate phosphoglycerate dehydrogenase (PHGDH), thereby promoting serine synthesis. However, the mechanisms underlying PRMT1 upregulation and regulation of PRMT1-PHGDH axis remain unclear. Here, we show the E3 ubiquitin ligase F-box-only protein 7 (FBXO7) inhibits serine synthesis in HCC by binding PRMT1, inducing lysine 37 ubiquitination, and promoting proteosomal degradation of PRMT1. FBXO7-mediated PRMT1 downregulation cripples PHGDH arginine methylation and activation, resulting in impaired serine synthesis, accumulation of reactive oxygen species (ROS), and inhibition of HCC cell growth. Notably, FBXO7 is significantly downregulated in human HCC tissues, and inversely associated with PRMT1 protein and PHGDH methylation level. Overall, our study provides mechanistic insights into the regulation of cancer serine synthesis by FBXO7-PRMT1-PHGDH axis, and will facilitate the development of serine-targeting strategies for cancer therapy.


Subject(s)
Carcinoma, Hepatocellular , F-Box Proteins , Liver Neoplasms , Phosphoglycerate Dehydrogenase , Protein-Arginine N-Methyltransferases , Serine , Ubiquitination , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Humans , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/genetics , F-Box Proteins/metabolism , F-Box Proteins/genetics , Serine/metabolism , Serine/biosynthesis , Phosphoglycerate Dehydrogenase/metabolism , Phosphoglycerate Dehydrogenase/genetics , Cell Line, Tumor , Animals , Repressor Proteins/metabolism , Repressor Proteins/genetics , Mice , Cell Proliferation , Methylation , Gene Expression Regulation, Neoplastic , Mice, Nude , Male , HEK293 Cells , Female , Hep G2 Cells
2.
Cell Biochem Funct ; 42(4): e4020, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38702967

ABSTRACT

The regulatory potential of long noncoding RNA (lncRNA) FBXL19-AS1 has been highlighted in various cancers, but its effect on triple-negative breast cancer (TNBC) remains unclear. Here, we aimed to elucidate the role of FBXL19-AS1 in TNBC and its underlying mechanism. RT-qPCR was employed to detect the expressions of FBXL19-AS1 and miR-378a-3p in tissues and cells. Immunohistochemical staining and western blot were utilized to detect the expression levels of proteins. Cell activities were detected using flow cytometry, CCK-8, and transwell assay. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were deployed to investigate interactions of different molecules. Protein-protein interaction (PPI) network, gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathways were used to analyze the downstream pathway. In vivo xenograft model was conducted to detect the effect of FBXL19-AS1 on tumor growth. FBXL19-AS1 was overexpressed in TNBC tissues and cell lines compared with counterparts. FBXL19-AS1 knockdown suppressed TNBC cell activities, whereas its overexpression exhibited the opposite effect. Mechanistically, FBXL19-AS1 was found to interact with miR-378a-3p. Further analysis revealed that miR-378a-3p exerted tumor-suppressive effects in TNBC cells. Additionally, miR-378a-3p targeted and downregulated the expression of ubiquitin aldehyde binding 2 (OTUB2), a deubiquitinase associated with TNBC progression. In vivo experiments substantiated the inhibitory effects of FBXL19-AS1 knockdown on TNBC tumorigenesis, and a miR-378a-3p inhibitor partially rescued these effects. The downstream pathway of the miR-378a-3p/OTUB2 axis was explored, revealing connections with proteins involved in modifying other proteins, removing ubiquitin molecules, and influencing signaling pathways, including the Hippo signaling pathway. Western blot analysis confirmed changes in YAP and TAZ expression levels, indicating a potential regulatory network. In summary, FBXL19-AS1 promotes exacerbation in TNBC by suppressing miR-378a-3p, leading to increased OTUB2 expression. The downstream mechanism may be related to the Hippo signaling pathway. These findings propose potential therapeutic targets for TNBC treatment.


Subject(s)
MicroRNAs , RNA, Long Noncoding , Triple Negative Breast Neoplasms , Animals , Female , Humans , Mice , Cell Line, Tumor , Cell Proliferation , Deubiquitinating Enzymes/metabolism , F-Box Proteins/metabolism , F-Box Proteins/genetics , Gene Expression Regulation, Neoplastic , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/metabolism , MicroRNAs/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Triple Negative Breast Neoplasms/genetics
3.
BMC Cancer ; 24(1): 619, 2024 May 21.
Article in English | MEDLINE | ID: mdl-38773471

ABSTRACT

BACKGROUND: Breast cancer is one of the common malignancies in women. Evidence has demonstrated that FBXO45 plays a pivotal role in oncogenesis and progression. However, the role of FBXO45 in breast tumorigenesis remains elusive. Exploration of the regulatory mechanisms of FBXO45 in breast cancer development is pivotal for potential therapeutic interventions in patients with breast cancer. METHODS: Hence, we used numerous approaches to explore the functions of FBXO45 and its underlaying mechanisms in breast cancer pathogenesis, including CCK-8 assay, EdU assay, colony formation analysis, apoptosis assay, RT-PCR, Western blotting, immunoprecipitation, ubiquitination assay, and cycloheximide chase assay. RESULTS: We found that downregulation of FBXO45 inhibited cell proliferation, while upregulation of FBXO45 elevated cell proliferation in breast cancer. Silencing of FBXO45 induced cell apoptosis, whereas overexpression of FBXO45 inhibited cell apoptosis in breast cancer. Moreover, FBXO45 interacted with BIM and regulated its ubiquitination and degradation. Furthermore, knockdown of FBXO45 inhibited cell proliferation via regulation of BIM pathway. Notably, overexpression of FBXO45 facilitated tumor growth in mice. Strikingly, FBXO45 expression was associated with poor survival of breast cancer patients. CONCLUSION: Our study could provide the rational for targeting FBXO45 to obtain benefit for breast cancer patients. Altogether, modulating FBXO45/Bim axis could be a promising strategy for breast cancer therapy.


Subject(s)
Apoptosis , Bcl-2-Like Protein 11 , Breast Neoplasms , Cell Proliferation , Disease Progression , F-Box Proteins , Ubiquitination , Humans , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/genetics , Female , Animals , F-Box Proteins/metabolism , F-Box Proteins/genetics , Mice , Bcl-2-Like Protein 11/metabolism , Bcl-2-Like Protein 11/genetics , Cell Line, Tumor , Proteolysis , Gene Expression Regulation, Neoplastic , Mice, Nude
4.
Biomolecules ; 14(5)2024 May 03.
Article in English | MEDLINE | ID: mdl-38785960

ABSTRACT

Histone demethylases, enzymes responsible for removing methyl groups from histone proteins, have emerged as critical players in regulating gene expression and chromatin dynamics, thereby influencing various cellular processes. LSD2 and LSD1 have attracted considerable interest among these demethylases because of their associations with cancer. However, while LSD1 has received significant attention, LSD2 has not been recognized to the same extent. In this study, we conduct a comprehensive comparison between LSD2 and LSD1, with a focus on exploring LSD2's implications. While both share structural similarities, LSD2 possesses unique features as well. Functionally, LSD2 shows diverse roles, particularly in cancer, with tissue-dependent roles. Additionally, LSD2 extends beyond histone demethylation, impacting DNA methylation, cancer cell reprogramming, E3 ubiquitin ligase activity and DNA damage repair pathways. This study underscores the distinct roles of LSD2, providing insights into their contributions to cancer and other cellular processes.


Subject(s)
DNA Methylation , Epigenesis, Genetic , Histone Demethylases , Neoplasms , Histone Demethylases/metabolism , Histone Demethylases/genetics , Humans , Neoplasms/genetics , Neoplasms/metabolism , DNA Methylation/genetics , Histones/metabolism , Histones/genetics , DNA Repair , Gene Expression Regulation, Neoplastic , F-Box Proteins , Jumonji Domain-Containing Histone Demethylases
5.
Mol Plant Pathol ; 25(6): e13459, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38808386

ABSTRACT

F-box protein is a subunit of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which plays a critical role in regulating different pathways in plant immunity. In this study, we identified the rice (Oryza sativa) F-box protein OsFBX156, which targets the heat shock protein 70 (OsHSP71.1) to regulate resistance to the rice blast fungus Magnaporthe oryzae. Overexpression of OsFBX156 or knockout of OsHSP71.1 in rice resulted in the elevation of pathogenesis-related (PR) genes and an induction burst of reactive oxygen species (ROS) after flg22 and chitin treatments, thereby enhancing resistance to M. oryzae. Furthermore, OsFBX156 can promote the degradation of OsHSP71.1 through the 26S proteasome pathway. This study sheds lights on a novel mechanism wherein the F-box protein OsFBX156 targets OsHSP71.1 for degradation to promote ROS production and PR gene expression, thereby positively regulating rice innate immunity.


Subject(s)
Disease Resistance , F-Box Proteins , Oryza , Plant Diseases , Plant Proteins , Ubiquitination , Oryza/microbiology , Oryza/metabolism , Oryza/genetics , Plant Diseases/microbiology , Plant Diseases/immunology , Plant Proteins/metabolism , Plant Proteins/genetics , Disease Resistance/genetics , F-Box Proteins/metabolism , F-Box Proteins/genetics , Reactive Oxygen Species/metabolism , Gene Expression Regulation, Plant , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Plant Immunity/genetics , Ascomycota/pathogenicity
6.
Front Biosci (Landmark Ed) ; 29(5): 202, 2024 May 24.
Article in English | MEDLINE | ID: mdl-38812312

ABSTRACT

OBJECTIVE: The F-box protein (FBXO) family plays a key role in the malignant progression of tumors. However, the biological functions and clinical value of the FBXO family in liver cancer remain unclear. Our study comprehensively assessed the clinical value of the FBXO family in hepatocellular carcinoma (HCC) and constructed a novel signature based on the FBXO family to predict prognosis and guide precision immunotherapy. METHODS: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases were utilized to investigate the expression characteristics and prognostic value of the FBXO family in HCC. A predictive model based on the FBXO family using TCGA database; and its predictive ability was validated using the ICGC database. Further analyses revealed that this predictive model can independently predict the overall survival (OS) rate of patients with HCC. We further analyzed the association of this predictive model with signaling pathways, clinical pathological features, somatic mutations, and immune therapy responses. Finally, we validated the biological functions of cyclin F (CCNF) through in vitro experiments. RESULTS: A predictive model involving three genes (CCNF, FBXO43, and FBXO45) was constructed, effectively identifying high and low-risk patients with differences in OS, clinicopathological characteristics, somatic mutations, and immune cell infiltration status. Additionally, knock-down of CCNF in HCC cell lines reduced cell proliferation in vitro, suggesting that CCNF may be a potential therapeutic target for HCC. CONCLUSIONS: The predictive model based on the FBXO family can effectively predict OS and the immune therapy response in HCC. Additionally, CCNF is a potential therapeutic target for HCC.


Subject(s)
Carcinoma, Hepatocellular , F-Box Proteins , Liver Neoplasms , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , F-Box Proteins/genetics , F-Box Proteins/metabolism , Prognosis , Male , Female , Cell Line, Tumor , Middle Aged , Gene Expression Regulation, Neoplastic , Cyclins/genetics , Cyclins/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Proliferation/genetics , Databases, Genetic
7.
Nat Commun ; 15(1): 3894, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38719837

ABSTRACT

The F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCFMet30 ubiquitin ligase during cadmium stress. Several highly conserved cysteine residues within the Met30 F-box contribute to binding of cadmium with a KD of 8 µM. Binding induces a conformational change that allows for Met30 autoubiquitylation, which in turn leads to recruitment of the segregase Cdc48/p97/VCP followed by active SCFMet30 disassembly. The resulting inactivation of SCFMet30 protects cells from cadmium stress. Our results show that F-box domains participate in regulation of SCF ligases beyond formation of the Skp1 binding interface.


Subject(s)
Cadmium , Protein Binding , SKP Cullin F-Box Protein Ligases , Cadmium/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , SKP Cullin F-Box Protein Ligases/genetics , Valosin Containing Protein/metabolism , Valosin Containing Protein/genetics , Saccharomyces cerevisiae/metabolism , Stress, Physiological , F-Box Proteins/metabolism , F-Box Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Ubiquitination , Protein Domains , Humans , S-Phase Kinase-Associated Proteins/metabolism , S-Phase Kinase-Associated Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics
8.
COPD ; 21(1): 2342797, 2024 12.
Article in English | MEDLINE | ID: mdl-38712759

ABSTRACT

Objective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11's role in CS-induced airway epithelial cell cytotoxicity.Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs-human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits.Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner.Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death.HIGHLIGHTSLung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression.STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity.STK11 depletion augments CS-induced airway epithelial cell cytotoxicity.CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.


Subject(s)
AMP-Activated Protein Kinases , Epithelial Cells , F-Box Proteins , Protein Serine-Threonine Kinases , Smoke , Animals , Humans , Male , Mice , AMP-Activated Protein Kinase Kinases , Cell Line , Cigarette Smoking/adverse effects , Cycloheximide/pharmacology , Epithelial Cells/metabolism , Epithelial Cells/drug effects , F-Box Proteins/metabolism , F-Box Proteins/genetics , Leupeptins/pharmacology , Mice, Inbred C57BL , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Proteolysis/drug effects , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/genetics , Respiratory Mucosa/metabolism , Respiratory Mucosa/drug effects , RNA, Small Interfering , Smoke/adverse effects
9.
Proc Natl Acad Sci U S A ; 121(17): e2314353121, 2024 Apr 23.
Article in English | MEDLINE | ID: mdl-38635634

ABSTRACT

Auxin regulates plant growth and development through downstream signaling pathways, including the best-known SCFTIR1/AFB-Aux/IAA-ARF pathway and several other less characterized "noncanonical" pathways. Recently, one SCFTIR1/AFB-independent noncanonical pathway, mediated by Transmembrane Kinase 1 (TMK1), was discovered through the analyses of its functions in Arabidopsis apical hook development. Asymmetric accumulation of auxin on the concave side of the apical hook triggers DAR1-catalyzed release of the C-terminal of TMK1, which migrates into the nucleus, where it phosphorylates and stabilizes IAA32/34 to inhibit cell elongation, which is essential for full apical hook formation. However, the molecular factors mediating IAA32/34 degradation have not been identified. Here, we show that proteins in the CYTOKININ INDUCED ROOT WAVING 1 (CKRW1)/WAVY GROWTH 3 (WAV3) subfamily act as E3 ubiquitin ligases to target IAA32/34 for ubiquitination and degradation, which is inhibited by TMK1c-mediated phosphorylation. This antagonistic interaction between TMK1c and CKRW1/WAV3 subfamily E3 ubiquitin ligases regulates IAA32/34 levels to control differential cell elongation along opposite sides of the apical hook.


Subject(s)
Arabidopsis Proteins , Arabidopsis , F-Box Proteins , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Indoleacetic Acids/metabolism , Signal Transduction , Ubiquitins/metabolism , Gene Expression Regulation, Plant , F-Box Proteins/genetics , F-Box Proteins/metabolism
10.
Cell Cycle ; 23(3): 262-278, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38597826

ABSTRACT

Esophageal cancer (EC) is a common and serious form of cancer, and while DNA methyltransferase-1 (DNMT1) promotes DNA methylation and carcinogenesis, the role of F-box protein 32 (FBXO32) in EC and its regulation by DNMT1-mediated methylation is still unclear. FBXO32 expression was examined in EC cells with high DNMT1 expression using GSE163735 dataset. RT-qPCR assessed FBXO32 expression in normal and EC cells, and impact of higher FBXO32 expression on cell proliferation, migration, and invasion was evaluated, along with EMT-related proteins. The xenograft model established by injecting EC cells transfected with FBXO32 was used to evaluate tumor growth, apoptosis, and tumor cells proliferation and metastasis. Chromatin immunoprecipitation (ChIP) assay was employed to study the interaction between DNMT1 and FBXO32. HitPredict, co-immunoprecipitation (Co-IP), and Glutathione-S-transferase (GST) pulldown assay analyzed the interaction between FBXO32 and cyclin dependent kinase 9 (CDK9). Finally, the ubiquitination assay identified CDK9 ubiquitination, and its half-life was measured using cycloheximide and confirmed through western blotting. DNMT1 negatively correlated with FBXO32 expression in esophageal cells. High FBXO32 expression was associated with better overall survival in patients. Knockdown of DNMT1 in EC cells increased FBXO32 expression and suppressed malignant phenotypes. FBXO32 repressed EC tumor growth and metastasis in mice. Enrichment of DNMT1 in FBXO32 promoter region led to increased DNA methylation and reduced transcription. Mechanistically, FBXO32 degraded CDK9 through promoting its ubiquitination.


Subject(s)
Cell Proliferation , DNA (Cytosine-5-)-Methyltransferase 1 , Epigenesis, Genetic , Esophageal Neoplasms , F-Box Proteins , Gene Expression Regulation, Neoplastic , Mice, Nude , Esophageal Neoplasms/pathology , Esophageal Neoplasms/genetics , Esophageal Neoplasms/mortality , Esophageal Neoplasms/metabolism , Humans , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/genetics , Animals , Cell Proliferation/genetics , F-Box Proteins/metabolism , F-Box Proteins/genetics , Cell Line, Tumor , Epigenesis, Genetic/genetics , Mice , DNA Methylation/genetics , Ubiquitination , Cell Movement/genetics , Apoptosis/genetics , Mice, Inbred BALB C , Cell Survival/genetics , Female , Male
11.
Arch Microbiol ; 206(5): 209, 2024 Apr 08.
Article in English | MEDLINE | ID: mdl-38587657

ABSTRACT

The F-box proteins in fungi perform diverse functions including regulation of cell cycle, circadian clock, development, signal transduction and nutrient sensing. Genome-wide analysis revealed 10 F-box genes in Puccinia triticina, the causal organism for the leaf rust disease in wheat and were characterized using in silico approaches for revealing phylogenetic relationships, gene structures, gene ontology, protein properties, sequence analysis and gene expression studies. Domain analysis predicted functional domains like WD40 and LRR at C-terminus along with the obvious presence of F-box motif in N-terminus. MSA showed amino acid replacements, which might be due to nucleotide substitution during replication. Phylogenetic analysis revealed the F-box proteins with similar domains to be clustered together while some sequences were spread out in different clades, which might be due to functional diversity. The clustering of Puccinia triticina GG705409 with Triticum aestivum TaAFB4/TaAFB5 in a single clade suggested the possibilities of horizontal gene transfer during the coevolution of P. triticina and wheat. Gene ontological annotation categorized them into three classes and were functionally involved in protein degradation through the protein ubiquitination pathway. Protein-protein interaction network revealed F-box proteins to interact with other components of the SCF complex involved in protein ubiquitination. Relative expression analysis of five F-box genes in a time course experiment denoted their involvement in leaf rust susceptible wheat plants. This study provides information on structure elucidation of F-box proteins of a basidiomycetes plant pathogenic fungi and their role during pathogenesis.


Subject(s)
Basidiomycota , F-Box Proteins , Phylogeny , Puccinia , Basidiomycota/genetics , F-Box Proteins/genetics
12.
Cancer Lett ; 591: 216848, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38604312

ABSTRACT

FBXO43 is a member of the FBXO subfamily of F-box proteins, known to be a regulatory hub during meiosis. A body of data showed that FBXO43 is overexpressed in a number of human cancers. However, whether and how FBXO43 affects cell cycle progression and growth of cancer cells remain elusive. In this study, we provide first piece of evidence, showing a pivotal role of FBXO43 in cell cycle progression and growth of cancer cells. Specifically, FBXO43 acts as a positive cell cycle regulator with an oncogenic activity in variety types of human cancer, including non-small cell lung cancer, hepatocellular carcinoma and sarcoma. Mechanistically, FBXO43 interacts with phosphorylated SKP2 induced by AKT1, leading to reduced SKP2 auto-ubiquitylation and subsequent proteasome degradation. Taken together, our study demonstrates that FBXO43 promotes cell cycle progression by stabilizing SKP2, and FBXO43 could serve as a potential anti-cancer target.


Subject(s)
Cell Cycle , F-Box Proteins , Proto-Oncogene Proteins c-akt , S-Phase Kinase-Associated Proteins , Ubiquitination , Humans , S-Phase Kinase-Associated Proteins/metabolism , S-Phase Kinase-Associated Proteins/genetics , F-Box Proteins/metabolism , F-Box Proteins/genetics , Proto-Oncogene Proteins c-akt/metabolism , Cell Line, Tumor , Cell Proliferation , Phosphorylation , Animals , Mice , Proteolysis , Gene Expression Regulation, Neoplastic , Neoplasms/pathology , Neoplasms/metabolism , Neoplasms/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Proteasome Endopeptidase Complex/metabolism , HEK293 Cells , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/genetics
13.
Clinics (Sao Paulo) ; 79: 100365, 2024.
Article in English | MEDLINE | ID: mdl-38677194

ABSTRACT

OBJECTIVE: This study explored the pharmacological mechanism of Tanshinone IIA (TAN IIA) in the treatment of Osteoarthritis (OA), which provided a certain reference for further research and clinical application of Tan IIA in OA. METHODS: CHON-001 cells were stimulated with 10 µg/mL IL-1ß for 48 h and treated with 10 µM TAN IIA for 48 h. Cellular viability and apoptosis were evaluated by CCK-8 assay and flow cytometry, and Cleaved caspase-3 was measured by Immunoblot assay and RT-qPCR. TNF-α, IL-6, and iNOS in CHON-001 cells were determined by RT-qPCR and ELISA. To further verify the effect of TAN IIA on OA, a rat model of OA in vivo was established by right anterior cruciate ligament transection. TAN IIA was administered at 50 mg/kg or 150 mg/kg for 7 weeks. The degree of cartilage destruction in OA rats was observed by TUNEL and HE staining. Cleaved caspase-3 and FBXO11 were measured by immunohistochemical staining, RT-qPCR, and Immunoblot. TNF-α, IL-6, and iNOS in chondrocytes of OA rats were detected by ELISA. RESULTS: IL-1ß stimulated CHON-001 cell apoptosis and inflammation, and TAN IIA had anti-apoptosis and anti-inflammatory effects on IL-1ß-regulated CHON-001 cells. TAN IIA down-regulated FBXO11 and inhibited PI3K/AKT and NF-κB pathways, thereby alleviating apoptotic and inflammatory reactions in CHON-001 cells under IL-1ß treatment. Moreover, TAN IIA treatment improved chondrocyte apoptosis and inflammations in OA rats. CONCLUSION: TAN IIA inhibits PI3K/Akt and NF-κB pathways by down-regulating FBXO11 expression, alleviates chondrocyte apoptosis and inflammation, and delays the progression of OA.


Subject(s)
Abietanes , Apoptosis , Chondrocytes , Interleukin-1beta , Osteoarthritis , Chondrocytes/drug effects , Chondrocytes/metabolism , Animals , Abietanes/pharmacology , Apoptosis/drug effects , Interleukin-1beta/metabolism , Interleukin-1beta/pharmacology , Osteoarthritis/drug therapy , Osteoarthritis/pathology , Osteoarthritis/metabolism , Male , F-Box Proteins/metabolism , Rats, Sprague-Dawley , Inflammation/drug therapy , Inflammation/metabolism , NF-kappa B/metabolism , Cell Survival/drug effects , Rats , Signal Transduction/drug effects , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Caspase 3/metabolism
14.
Int J Mol Sci ; 25(8)2024 Apr 09.
Article in English | MEDLINE | ID: mdl-38673728

ABSTRACT

BTB and CNC homology 1 (BACH1) represses the expression of genes involved in the metabolism of iron, heme and reactive oxygen species. While BACH1 is rapidly degraded when it is bound to heme, it remains unclear how BACH1 degradation is regulated under other conditions. We found that FBXO22, a ubiquitin ligase previously reported to promote BACH1 degradation, polyubiquitinated BACH1 only in the presence of heme in a highly purified reconstitution assay. In parallel to this regulatory mechanism, TANK binding kinase 1 (TBK1), a protein kinase that activates innate immune response and regulates iron metabolism via ferritinophagy, was found to promote BACH1 degradation when overexpressed in 293T cells. While TBK1 phosphorylated BACH1 at multiple serine and threonine residues, BACH1 degradation was observed with not only the wild-type TBK1 but also catalytically impaired TBK1. The BACH1 degradation in response to catalytically impaired TBK1 was not dependent on FBXO22 but involved both autophagy-lysosome and ubiquitin-proteasome pathways judging from its suppression by using inhibitors of lysosome and proteasome. Chemical inhibition of TBK1 in hepatoma Hepa1 cells showed that TBK1 was not required for the heme-induced BACH1 degradation. Its inhibition in Namalwa B lymphoma cells increased endogenous BACH1 protein. These results suggest that TBK1 promotes BACH1 degradation in parallel to the FBXO22- and heme-dependent pathway, placing BACH1 as a downstream effector of TBK1 in iron metabolism or innate immune response.


Subject(s)
Basic-Leucine Zipper Transcription Factors , F-Box Proteins , Heme , Protein Serine-Threonine Kinases , Proteolysis , Receptors, Cytoplasmic and Nuclear , Humans , Heme/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , F-Box Proteins/metabolism , F-Box Proteins/genetics , HEK293 Cells , Ubiquitination , Cell Line, Tumor , Lysosomes/metabolism , Autophagy , Proteasome Endopeptidase Complex/metabolism
15.
Mol Plant ; 17(5): 772-787, 2024 May 06.
Article in English | MEDLINE | ID: mdl-38581129

ABSTRACT

The phytohormone auxin plays a pivotal role in governing plant growth and development. Although the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) receptors function in both the nucleus and cytoplasm, the mechanism governing the distribution of TIR1/AFBs between these cellular compartments remains unknown. In this study, we demonstrate that auxin-mediated oxidation of TIR1/AFB2 is essential for their targeting to the nucleus. We showed that small active molecules, reactive oxygen species (ROS) and nitric oxide (NO), are indispensable for the nucleo-cytoplasmic distribution of TIR1/AFB2 in trichoblasts and root hairs. Further studies revealed that this process is regulated by the FERONIA receptor kinase-NADPH oxidase signaling pathway. Interestingly, ROS and NO initiate oxidative modifications in TIR1C140/516 and AFB2C135/511, facilitating their subsequent nuclear import. The oxidized forms of TIR1C140/516 and AFB2C135/511 play a crucial role in enhancing the function of TIR1 and AFB2 in transcriptional auxin responses. Collectively, our study reveals a novel mechanism by which auxin stimulates the transport of TIR1/AFB2 from the cytoplasm to the nucleus, orchestrated by the FERONIA-ROS signaling pathway.


Subject(s)
Arabidopsis Proteins , Arabidopsis , F-Box Proteins , Indoleacetic Acids , Oxidation-Reduction , Protein Serine-Threonine Kinases , Signal Transduction , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Cell Nucleus/metabolism , F-Box Proteins/metabolism , Gene Expression Regulation, Plant , Indoleacetic Acids/metabolism , Nitric Oxide/metabolism , Phosphotransferases/metabolism , Reactive Oxygen Species/metabolism , Receptors, Cell Surface/metabolism
16.
Plant Physiol Biochem ; 210: 108592, 2024 May.
Article in English | MEDLINE | ID: mdl-38569422

ABSTRACT

The present study investigates the phytotoxic potential of azelaic acid (AZA) on Arabidopsis thaliana roots. Effects on root morphology, anatomy, auxin content and transport, gravitropic response and molecular docking were analysed. AZA inhibited root growth, stimulated lateral and adventitious roots, and altered the root apical meristem by reducing meristem cell number, length and width. The treatment also slowed down the roots' gravitropic response, likely due to a reduction in statoliths, starch-rich organelles involved in gravity perception. In addition, auxin content, transport and distribution, together with PIN proteins' expression and localisation were altered after AZA treatment, inducing a reduction in auxin transport and its distribution into the meristematic zone. Computational simulations showed that AZA has a high affinity for the auxin receptor TIR1, competing with auxin for the binding site. The AZA binding with TIR1 could interfere with the normal functioning of the TIR1/AFB complex, disrupting the ubiquitin E3 ligase complex and leading to alterations in the response of the plant, which could perceive AZA as an exogenous auxin. Our results suggest that AZA mode of action could involve the modulation of auxin-related processes in Arabidopsis roots. Understanding such mechanisms could lead to find environmentally friendly alternatives to synthetic herbicides.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Dicarboxylic Acids , F-Box Proteins , Gravitropism , Indoleacetic Acids , Plant Roots , Receptors, Cell Surface , Arabidopsis/metabolism , Arabidopsis/drug effects , Arabidopsis/growth & development , Indoleacetic Acids/metabolism , Arabidopsis Proteins/metabolism , Plant Roots/metabolism , Plant Roots/drug effects , Plant Roots/growth & development , Gravitropism/drug effects , Dicarboxylic Acids/metabolism , F-Box Proteins/metabolism , Receptors, Cell Surface/metabolism , Binding Sites , Biological Transport/drug effects , Molecular Docking Simulation
17.
Proc Natl Acad Sci U S A ; 121(12): e2309902121, 2024 Mar 19.
Article in English | MEDLINE | ID: mdl-38483988

ABSTRACT

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.


Subject(s)
F-Box Proteins , Neoplasms , Humans , F-Box-WD Repeat-Containing Protein 7/genetics , F-Box-WD Repeat-Containing Protein 7/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Epidermal Growth Factor/genetics , Epidermal Growth Factor/pharmacology , Epidermal Growth Factor/metabolism , Proteomics , ErbB Receptors/genetics , ErbB Receptors/metabolism , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , F-Box Proteins/genetics
18.
J Cell Biol ; 223(5)2024 May 06.
Article in English | MEDLINE | ID: mdl-38451221

ABSTRACT

Polycomb repressive complexes regulate developmental gene programs, promote DNA damage repair, and mediate pericentromeric satellite repeat repression. Expression of pericentromeric satellite repeats has been implicated in several cancers and diseases, including facioscapulohumeral dystrophy (FSHD). Here, we show that DUX4-mediated transcription of HSATII regions causes nuclear foci formation of KDM2A/B-PRC1 complexes, resulting in a global loss of PRC1-mediated monoubiquitination of histone H2A. Loss of PRC1-ubiquitin signaling severely impacts DNA damage response. Our data implicate DUX4-activation of HSATII and sequestration of KDM2A/B-PRC1 complexes as a mechanism of regulating epigenetic and DNA repair pathways.


Subject(s)
DNA Repair , Homeodomain Proteins , Multiprotein Complexes , Cell Nucleus/genetics , Epigenomics , Histones/genetics , Humans , F-Box Proteins/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Cell Cycle Proteins/metabolism , Homeodomain Proteins/metabolism , Multiprotein Complexes/metabolism
19.
J Cell Sci ; 137(7)2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38477340

ABSTRACT

Axonal transport in neurons is essential for cargo movement between the cell body and synapses. Caenorhabditis elegans UNC-104 and its homolog KIF1A are kinesin-3 motors that anterogradely transport precursors of synaptic vesicles (pre-SVs) and are degraded at synapses. However, in C. elegans, touch neuron-specific knockdown of the E1 ubiquitin-activating enzyme, uba-1, leads to UNC-104 accumulation at neuronal ends and synapses. Here, we performed an RNAi screen and identified that depletion of fbxb-65, which encodes an F-box protein, leads to UNC-104 accumulation at neuronal distal ends, and alters UNC-104 net anterograde movement and levels of UNC-104 on cargo without changing synaptic UNC-104 levels. Split fluorescence reconstitution showed that UNC-104 and FBXB-65 interact throughout the neuron. Our theoretical model suggests that UNC-104 might exhibit cooperative cargo binding that is regulated by FBXB-65. FBXB-65 regulates an unidentified post-translational modification (PTM) of UNC-104 in a region beside the cargo-binding PH domain. Both fbxb-65 and UNC-104, independently of FBXB-65, regulate axonal pre-SV distribution, transport of pre-SVs at branch points and organismal lifespan. FBXB-65 regulates a PTM of UNC-104 and the number of motors on the cargo surface, which can fine-tune cargo transport to the synapse.


Subject(s)
Axonal Transport , Caenorhabditis elegans Proteins , F-Box Proteins , Kinesins , Animals , Axonal Transport/physiology , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , F-Box Proteins/metabolism , Kinesins/metabolism , Nerve Tissue Proteins/metabolism , Pleckstrin Homology Domains , Protein Processing, Post-Translational
20.
Cancer Res ; 84(11): 1764-1780, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38471099

ABSTRACT

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) plays a key role in tumor progression and response to therapy. The dense PDAC stroma causes hypovascularity, which leads to hypoxia. Here, we showed that hypoxia drives long-lasting epithelial-mesenchymal transition (EMT) in PDAC primarily through a positive-feedback histone methylation-MAPK signaling axis. Transformed cells preferentially underwent EMT in hypoxic tumor regions in multiple model systems. Hypoxia drove a cell autonomous EMT in PDAC cells, which, unlike EMT in response to growth factors, could last for weeks. Furthermore, hypoxia reduced histone demethylase KDM2A activity, suppressed PP2 family phosphatase expression, and activated MAPKs to post-translationally stabilize histone methyltransferase NSD2, leading to an H3K36me2-dependent EMT in which hypoxia-inducible factors played only a supporting role. Hypoxia-driven EMT could be antagonized in vivo by combinations of MAPK inhibitors. Collectively, these results suggest that hypoxia promotes durable EMT in PDAC by inducing a histone methylation-MAPK axis that can be effectively targeted with multidrug therapies, providing a potential strategy for overcoming chemoresistance. SIGNIFICANCE: Integrated regulation of histone methylation and MAPK signaling by the low-oxygen environment of pancreatic cancer drives long-lasting EMT that promotes chemoresistance and shortens patient survival and that can be pharmacologically inhibited. See related commentary by Wirth and Schneider, p. 1739.


Subject(s)
Carcinoma, Pancreatic Ductal , Epithelial-Mesenchymal Transition , Histones , Pancreatic Neoplasms , Humans , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/drug therapy , Mice , Histones/metabolism , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/drug therapy , Animals , Methylation , MAP Kinase Signaling System , Cell Line, Tumor , Tumor Microenvironment , Mice, Nude , Xenograft Model Antitumor Assays , Cell Hypoxia , Tumor Hypoxia , Hypoxia/metabolism , F-Box Proteins , Jumonji Domain-Containing Histone Demethylases
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