RESUMO
Cell adhesion to the extracellular matrix and its natural outcome of cell spreading, along with the maintenance of barrier activity, are essential behaviors of epithelial cells, including retinal pigment epithelium (RPE). Disruptions in these characteristics can result in severe vision-threatening diseases such as diabetic macular edema and age-related macular degeneration. However, the precise mechanisms underlying how RPE cells regulate their barrier integrity and cell spreading are not fully understood. This study aims to elucidate the relative importance of upper glycolytic components in governing these cellular behaviors of RPE cells. Electric Cell-Substrate Impedance Sensing (ECIS) technology was utilized to assess in real-time the effects of targeting various upper glycolytic enzymes on RPE barrier function and cell spreading by measuring cell resistance and capacitance, respectively. Specific inhibitors used included WZB117 for Glut1 inhibition, Lonidamine for Hexokinase inhibition, PFK158 for PFKFB3/PFK axis inhibition, and TDZD-8 for Aldolase inhibition. Additionally, the viability of RPE cells was evaluated using a lactate dehydrogenase (LDH) cytotoxicity assay. The most significant decrease in electrical resistance and increase in capacitance of RPE cells were observed due to dose-dependent inhibition of Glut1 using WZB117, as well as Aldolase inhibition with TDZD-8. LDH level analysis at 24-72 h post-treatment with WZB117 (1 and 10 µM) or TDZD-8 (1 µM) showed no significant difference compared to the control, indicating that the disruption of RPE functionality was not attributed to cell death. Lastly, inhibition of other upper glycolytic components, including PFKFB3/PFK with PFK158 or Hexokinase with Lonidamine, did not significantly affect RPE cell behavior. This study provides insights into the varied roles of upper glycolytic components in regulating the functionality of RPE cells. Specifically, it highlights the critical roles of Glut1 and Aldolase in preserving barrier integrity and promoting RPE cell adhesion and spreading. Such understanding will guide the development of safe interventions to treat RPE cell dysfunction in various retinal disorders.
Assuntos
Glicólise , Epitélio Pigmentado da Retina , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/citologia , Glicólise/efeitos dos fármacos , Humanos , Transportador de Glucose Tipo 1/metabolismo , Hexoquinase/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Impedância Elétrica , Fosfofrutoquinase-2/metabolismo , Fosfofrutoquinase-2/antagonistas & inibidoresRESUMO
BACKGROUND: Human hexokinase 2 (HK2) plays an important role in regulating Warburg effect, which metabolizes glucose to lactate acid even in the presence of ample oxygen and provides intermediate metabolites to support cancer cell proliferation and tumor growth. HK2 overexpression has been observed in various types of cancers and targeting HK2-driven Warburg effect has been suggested as a potential cancer therapeutic strategy. Given that epigenetic enzymes utilize metabolic intermediates as substrates or co-factors to carry out post-translational modification of histones and nucleic acids modifications in cells, we hypothesized that altering HK2 expression could impact the epigenome and, consequently, chromatin stability in yeast. To test this hypothesis, we established genetic models with different yeast hexokinase 2 (HXK2) expression in Saccharomyces cerevisiae yeast cells and investigated the effect of HXK2-dependent metabolism on parental nucleosome transfer, a key DNA replication-coupled epigenetic inheritance process, and chromatin stability. RESULTS: By comparing the growth of mutant yeast cells carrying single deletion of hxk1Δ, hxk2Δ, or double-loss of hxk1Δ hxk2Δ to wild-type cells, we firstly confirmed that HXK2 is the dominant HXK in yeast cell growth. Surprisingly, manipulating HXK2 expression in yeast, whether through overexpression or deletion, had only a marginal impact on parental nucleosome assembly, but a noticeable trend with decrease chromatin instability. However, targeting yeast cells with 2-deoxy-D-glucose (2-DG), a clinical glycolysis inhibitor that has been proposed as an anti-cancer treatment, significantly increased chromatin instability. CONCLUSION: Our findings suggest that in yeast cells lacking HXK2, alternative HXKs such as HXK1 or glucokinase 1 (GLK1) play a role in supporting glycolysis at a level that adequately maintains epigenomic stability. While our study demonstrated an increase in epigenetic instability with 2-DG treatment, the observed effect seemed to occur dependent on non-glycolytic function of Hxk2. Thus, additional research is needed to identify the molecular mechanism through which 2-DG influences chromatin stability.
Assuntos
Cromatina , Epigênese Genética , Hexoquinase , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Hexoquinase/metabolismo , Hexoquinase/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Cromatina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Nucleossomos/metabolismo , Regulação Fúngica da Expressão GênicaRESUMO
Glycolysis is a major determinant of pulmonary artery smooth muscle cell (PASMC) proliferation in pulmonary hypertension (PH). Circular RNAs (circRNAs) are powerful regulators of glycolysis in multiple diseases; however, the role of circRNAs in glycolysis in PH has been poorly characterized. The aim of this study was to uncover the regulatory mechanism of a new circRNA, circNAP1L4, in human pulmonary artery smooth muscle cell (HPASMC) proliferation through the host protein NAP1L4 to regulate the super-enhancer-driven glycolysis gene hexokinase II (HK II). CircNAP1L4 was downregulated in hypoxic HPASMCs and plasma of PH patients. Functionally, circNAP1L4 overexpression inhibited glycolysis and proliferation in hypoxic HPASMCs. Mechanistically, circNAP1L4 directly bound to its host protein NAP1L4 and affected the ability of NAP1L4 to move into the nucleus to regulate the epigenomic signals of the super-enhancer of HK II. Intriguingly, circNAP1L4 overexpression inhibited the proliferation but not the migration of human pulmonary arterial endothelial cells (HPAECs) cocultured with HPASMCs. Furthermore, pre-mRNA-processing-splicing Factor 8 (PRP8) was found to regulate the production ratio of circNAP1L4 and linear NAP1L4. In vivo, targeting circNAP1L4 alleviates SU5416 combined with hypoxia (SuHx)-induced PH. Overall, these findings reveal a new circRNA that inhibits PASMC proliferation and serves as a therapeutic target for PH.
Assuntos
Proliferação de Células , Glicólise , Hexoquinase , Hipertensão Pulmonar , Miócitos de Músculo Liso , Artéria Pulmonar , RNA Circular , Humanos , Hexoquinase/metabolismo , Hexoquinase/genética , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/genética , Miócitos de Músculo Liso/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Animais , Camundongos , Masculino , Células Cultivadas , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologiaRESUMO
Clear cell renal cell carcinoma (ccRCC) demonstrates enhanced glycolysis, critically contributing to tumor development. Programmed death-ligand 1 (PD-L1) aids tumor cells in evading T-cell-mediated immune surveillance. Yet, the specific mechanism by which glycolysis influences PD-L1 expression in ccRCC is not fully understood. Our research identified that the glycolysis-related gene (GRG) HK3 has a unique correlation with PD-L1 expression. HK3 has been identified as a key regulator of O-GlcNAcylation in ccRCC. O-GlcNAcylation exists on the serine 900 (Ser900) site of EP300 and can enhance its stability and oncogenic activity by preventing ubiquitination. Stably expressed EP300 works together with TFAP2A as a co-transcription factor to promote PD-L1 transcription and as an acetyltransferase to stabilize PD-L1 protein. Furthermore, ccRCC exhibits interactive dynamics with tumor-associated macrophages (TAMs). The uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), which serves as a critical substrate for the O-GlcNAcylation process, facilitates TAMs polarization. In ccRCC cells, HK3 expression is influenced by IL-10 secreted by M2 TAMs. Our study elucidates that HK3-mediated O-GlcNAcylation of EP300 is involved in tumor immune evasion. This finding suggests potential strategies to enhance the efficacy of immune checkpoint blockade therapy.
Assuntos
Antígeno B7-H1 , Carcinoma de Células Renais , Proteína p300 Associada a E1A , Hexoquinase , Neoplasias Renais , Humanos , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/genética , Proteína p300 Associada a E1A/metabolismo , Antígeno B7-H1/metabolismo , Hexoquinase/metabolismo , Hexoquinase/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Neoplasias Renais/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Animais , Evasão da Resposta Imune , Macrófagos Associados a Tumor/metabolismo , Glicólise , CamundongosRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) are one of the most widely studied adult stem cells, while MSC replicative senescence occurs with serial expansion in vitro. We determined whether miR-34a can regulate MSC senescence by directly targeting glycolytic key enzymes to influence glycolysis. METHODS: Detected the effects of miR-34a on MSC senescence and glycolytic metabolism through gene manipulation. Bioinformatics prediction and luciferase reporter assay were applied to confirm that HK1 is a direct target of miR-34a. The underlying regulatory mechanism of miR-34a targeting HK1 in MSC senescence was further explored by a cellular function recovery experiment. RESULTS: In the current study, we revealed that miR-34a over-expression exacerbated senescence-associated characteristics and impaired glycolytic metabolism. Then we identified hexokinase1 (HK1) as a direct target gene of miR-34a. And HK1 replenishment reversed MSC senescence and reinforced glycolysis. In addition, miR-34a-mediated MSC senescence and lower glycolytic levels were evidently rescued following the co-treatment with HK1 over-expression. CONCLUSION: The miR-34a-HK1 signal axis can alleviate MSC senescence via enhancing glycolytic metabolism, which possibly provides a novel mechanism for MSC senescence and opens up new possibilities for delaying and suppressing the occurrence and development of aging and age-related diseases.
Assuntos
Senescência Celular , Glicólise , Hexoquinase , Células-Tronco Mesenquimais , MicroRNAs , Transdução de Sinais , MicroRNAs/metabolismo , MicroRNAs/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Hexoquinase/metabolismo , Hexoquinase/genética , HumanosRESUMO
N6-methyladenosine (m6A) serves as the most abundant posttranscription modification. However, the role of m6A in tumorigenesis and chemotherapeutic drugs sensitivity remains largely unclear. Present research focuses on the potential function of the m6A writer KIAA1429 in tumor development and sorafenib sensitivity in liver cancer. We found that the level of KIAA1429 was significantly elevated in liver cancer tissues and cells and was closely associated with poorer prognosis. Functionally, KIAA1429 promoted the proliferation and Warburg effect of liver cancer cells in vitro and in vivo. RNA-seq and MeRIP-seq analysis revealed the glycolysis was one of the most affected pathways by KIAA1429, and m6A-modified HK1 was the most likely targeted gene to regulate the Warburg effect. KIAA1429 depletion decreased Warburg effect and increased sorafenib sensitivity in liver cancer. Mechanistically, KIAA1429 could affect the m6A level of HK1 mRNA through directly binding with it. Moreover, KIAA1429 cooperated with the m6A reader HuR to enhance HK1 mRNA stability, thereby upregulating its expression. These findings demonstrated that KIAA1429/HK1 axis decreases the sensitivity of liver cancer cells to sorafenib by regulating the Warburg effect, which may provide a novel therapeutic target for liver cancer treatment.
Assuntos
Antineoplásicos , Hexoquinase , Neoplasias Hepáticas , Sorafenibe , Efeito Warburg em Oncologia , Animais , Humanos , Masculino , Camundongos , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Hexoquinase/metabolismo , Hexoquinase/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Sorafenibe/farmacologia , Efeito Warburg em Oncologia/efeitos dos fármacosRESUMO
The hepatocyte nuclear factor-1 (HNF1É) is a transcription factor that contributes to several kinds of cancer progression. However, very little is known regarding the mechanisms underlying the activity of HNF1É. We aimed to explore the role of HNF1É in the progress of colorectal cancer (CRC) and elucidate its molecular mechanism. HNF1É expression was upregulated in CRC samples and high expression of HNF1É was associated with poor prognosis of CRC patients. HNF1α knockdown and overexpression inhibited and promoted proliferation, migration and invasion of CRC cells both in vitro and in vivo respectively. Mechanistically, HNF1É increased the transcriptional activity of hexokinase domain component 1ï¼HKDC1ï¼promoter, thus activated AKT/AMPK signaling. Meanwhile, HKDC1 upregulation was important for the proliferation, migration and invasion of CRC cells and knockdown of HKDC1 significantly reversed the proliferation, migration and invasion induced by HNF1α overexpression. Taken together, HNF1É contributes to CRC progression and metastasis through binding to HKDC1 and activating AKT/AMPK signaling. Targeting HNF1É could be a potential therapeutic strategy for CRC patients.
Assuntos
Proteínas Quinases Ativadas por AMP , Movimento Celular , Proliferação de Células , Neoplasias Colorretais , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Linhagem Celular Tumoral , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Animais , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Camundongos , Masculino , Hexoquinase/metabolismo , Hexoquinase/genética , Feminino , Camundongos Nus , Invasividade Neoplásica , PrognósticoRESUMO
In brief: SORBS2, an RNA-binding protein, is identified as a regulator of aerobic glycolysis, which is essential for trophoblast migration and placental development. Reduced SORBS2 expression in preeclampsia may impair trophoblast migration by affecting mRNA stability and glycolysis, suggesting its role in the disease's pathogenesis. Abstract: Insufficient trophoblast migration and impaired uterine spiral artery remodeling are implicated in the pathogenesis of preeclampsia, contributing to inadequate placentation. However, the molecular mechanism underlying this process remains unclear. Aerobic glycolysis, which produces substantial lactate, is crucial for establishing a favorable microenvironment for early uterine preparation and supporting embryo implantation and trophoblast migration. In the present study, we have demonstrated that SORBS2, an RNA-binding protein, regulated aerobic glycolysis and significantly improved trophoblast migration in vitro. Our results showed that SORBS2 expression was significantly reduced in human PE placentas and trophoblasts during hypoxia. Overexpression of SORBS2 enhanced cell proliferation and migration, whereas knockdown of SORBS2 decreased these functions in HTR-8/SVneo cells. Mechanistic studies have demonstrated that SORBS2 directly interacts with the 3' untranslated regions (UTRs) of key glycolysis-related genes, specifically HK2. This interaction results in enhanced stability of HK2 and activation of glycolysis. Moreover, silencing HK2 abrogated the enhancement of proliferation and migration of HTR-8/SVneo cells induced by SORBS2. In conclusion, our findings suggest that the downregulation of SORBS2 may contribute to the pathogenesis of preeclampsia by regulating mRNA stability and inhibiting trophoblast migration during placentation.
Assuntos
Movimento Celular , Glicólise , Hexoquinase , Pré-Eclâmpsia , Estabilidade de RNA , RNA Mensageiro , Proteínas de Ligação a RNA , Trofoblastos , Humanos , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Trofoblastos/metabolismo , Trofoblastos/patologia , Feminino , Gravidez , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Hexoquinase/metabolismo , Hexoquinase/genética , Adulto , Proliferação de CélulasRESUMO
Endothelial cells (ECs) rely on glycolysis for energy production to maintain vascular homeostasis and the normalization of hyperglycolysis in tumor vessels has recently gained attention as a therapeutic target. We analyzed the TCGA database and found reduced Foxp1 expression in lung carcinoma. Immunostaining demonstrated reduced expression more restricted at tumor vascular ECs. Therefore, we investigated the function and mechanisms of Foxp1 in EC metabolism for tumor angiogenesis required for tumor growth. EC-Foxp1 deletion mice exhibited a significant increase of tumor and retinal developmental angiogenesis and Hif1α was identified as Foxp1 target gene, and Hk2 as Hif1α target gene. The Foxp1-Hif1α-Hk2 pathway in ECs is important in the regulation of glycolytic metabolism to govern tumor angiogenesis. Finally, we used genetic deletion of EC-Hif1α and RGD-peptide nanoparticles EC target delivery of Hif1α/Hk2-siRNAs to knockdown gene expression which reduced the tumor EC hyperglycolysis state and restricted angiogenesis for tumor growth. This study advances our understanding of EC metabolism for tumor angiogenesis, and meanwhile provides evidence for future therapeutic intervention of hyperglycolysis in tumor ECs for suppression of tumor growth.
Assuntos
Células Endoteliais , Fatores de Transcrição Forkhead , Glicólise , Hexoquinase , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neovascularização Patológica , Proteínas Repressoras , Animais , Humanos , Camundongos , Angiogênese/metabolismo , Células Endoteliais/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Hexoquinase/metabolismo , Hexoquinase/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/irrigação sanguínea , Camundongos Knockout , Neovascularização Patológica/metabolismo , Neovascularização Patológica/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Transdução de SinaisRESUMO
Neuroinflammation is a prominent feature of Alzheimer's disease (AD). Activated microglia undergo a reprogramming of cellular metabolism necessary to power their cellular activities during disease. Thus, selective targeting of microglial immunometabolism might be of therapeutic benefit for treating AD. In the AD brain, the levels of microglial hexokinase 2 (HK2), an enzyme that supports inflammatory responses by promoting glycolysis, are significantly increased. In addition, HK2 displays non-metabolic activities that extend its inflammatory role beyond glycolysis. The antagonism of HK2 affects microglial phenotypes and disease progression in a gene-dose-dependent manner. HK2 complete loss fails to improve pathology by exacerbating inflammation, while its haploinsufficiency reduces pathology in 5xFAD mice. We propose that the partial antagonism of HK2 is effective in slowing disease progression by modulating NF-κB signaling through its cytosolic target, IKBα. The complete loss of HK2 affects additional inflammatory mechanisms related to mitochondrial dysfunction.
Assuntos
Doença de Alzheimer , Progressão da Doença , Hexoquinase , Microglia , Hexoquinase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Doença de Alzheimer/metabolismo , Animais , Microglia/metabolismo , Microglia/efeitos dos fármacos , Microglia/patologia , Camundongos , Humanos , NF-kappa B/metabolismo , Camundongos Transgênicos , Transdução de Sinais , Inibidor de NF-kappaB alfa/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Inflamação/patologia , Inflamação/metabolismo , Encéfalo/patologia , Encéfalo/metabolismo , Glicólise/efeitos dos fármacos , Dosagem de GenesRESUMO
In this issue of Molecular Cell, Pilic et al.1 show that hexokinase, the first enzyme of glycolysis, forms perimitochondrial rings that prevent mitochondrial fragmentation when ATP levels drop.
Assuntos
Glucose , Hexoquinase , Mitocôndrias , Dinâmica Mitocondrial , Hexoquinase/metabolismo , Hexoquinase/genética , Mitocôndrias/metabolismo , Mitocôndrias/enzimologia , Glucose/metabolismo , Trifosfato de Adenosina/metabolismo , Humanos , Animais , GlicóliseRESUMO
Metabolic enzymes can adapt during energy stress, but the consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, forms rings around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and mitochondrial dynamics protein (MiD51). HK1-rings prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial fission factor (Mff) and mitochondrial fission 1 protein (Fis1). The disassembly of HK1-rings during energy restoration correlated with mitochondrial fission. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the formation of HK1-rings. Mutations that affect the formation of HK1-rings showed that HK1-rings rewire cellular metabolism toward increased TCA cycle activity. Our findings highlight that HK1 is an energy stress sensor that regulates the shape, connectivity, and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy-stress-related pathologies.
Assuntos
Dinaminas , Metabolismo Energético , Hexoquinase , Mitocôndrias , Dinâmica Mitocondrial , Proteínas Mitocondriais , Hexoquinase/metabolismo , Hexoquinase/genética , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mitocôndrias/enzimologia , Dinaminas/metabolismo , Dinaminas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Animais , Trifosfato de Adenosina/metabolismo , Estresse Fisiológico , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Ciclo do Ácido Cítrico , Glucose-6-Fosfato/metabolismo , Camundongos , Células HeLa , Células HEK293 , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , MutaçãoRESUMO
Rationale: Current treatments for ocular angiogenesis primarily focus on blocking the activity of vascular endothelial growth factor (VEGF), but unfavorable side effects and unsatisfactory efficacy remain issues. The identification of novel targets for anti-angiogenic treatment is still needed. Methods: We investigated the role of tsRNA-1599 in ocular angiogenesis using endothelial cells, a streptozotocin (STZ)-induced diabetic model, a laser-induced choroidal neovascularization model, and an oxygen-induced retinopathy model. CCK-8 assays, EdU assays, transwell assays, and matrigel assays were performed to assess the role of tsRNA-1599 in endothelial cells. Retinal digestion assays, Isolectin B4 (IB4) staining, and choroidal sprouting assays were conducted to evaluate the role of tsRNA-1599 in ocular angiogenesis. Transcriptomic analysis, metabolic analysis, RNA pull-down assays, and mass spectrometry were utilized to elucidate the mechanism underlying angiogenic effects mediated by tsRNA-1599. Results: tsRNA-1599 expression was up-regulated in experimental ocular angiogenesis models and endothelial cells in response to angiogenic stress. Silencing of tsRNA-1599 suppressed angiogenic effects in endothelial cells in vitro and inhibited pathological ocular angiogenesis in vivo. Mechanistically, tsRNA-1599 exhibited little effect on VEGF signaling but could cause reduced glycolysis and NAD+/NADH production in endothelial cells by regulating the expression of HK2 gene through interacting with YBX1, thus affecting endothelial effects. Conclusions: Targeting glycolytic reprogramming of endothelial cells by a tRNA-derived small RNA represents an exploitable therapeutic approach for ocular neovascular diseases.
Assuntos
Neovascularização de Coroide , Células Endoteliais , Glicólise , Animais , Glicólise/efeitos dos fármacos , Camundongos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/metabolismo , Humanos , Proteína 1 de Ligação a Y-Box/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Inibidores da Angiogênese/farmacologia , Hexoquinase/metabolismo , Hexoquinase/genética , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Modelos Animais de Doenças , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Retinopatia Diabética/genética , Células Endoteliais da Veia Umbilical Humana , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismoRESUMO
PURPOSE: Glycolysis and immune metabolism play important roles in acute myocardial infarction (AMI). Therefore, this study aimed to identify and experimentally validate the glycolysis-related hub genes in AMI as diagnostic biomarkers, and further explore the association between hub genes and immune infiltration. METHODS: Differentially expressed genes (DEGs) from AMI peripheral blood mononuclear cells (PBMCs) were analyzed using R software. Glycolysis-related DEGs (GRDEGs) were identified and analyzed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) for functional enrichment. A protein-protein interaction network was constructed using the STRING database and visualized using Cytoscape software. Immune infiltration analysis between patients with AMI and stable coronary artery disease (SCAD) controls was performed using CIBERSORT, and correlation analysis between GRDEGs and immune cell infiltration was performed. We also plotted nomograms and receiver operating characteristic (ROC) curves to assess the predictive accuracy of GRDEGs for AMI occurrence. Finally, key genes were experimentally validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting using PBMCs. RESULTS: A total of 132 GRDEGs and 56 GRDEGs were identified on the first day and 4-6 days after AMI, respectively. Enrichment analysis indicated that these GRDEGs were mainly clustered in the glycolysis/gluconeogenesis and metabolic pathways. Five hub genes (HK2, PFKL, PKM, G6PD, and ALDOA) were selected using the cytoHubba plugin. The link between immune cells and hub genes indicated that HK2, PFKL, PKM, and ALDOA were significantly positively correlated with monocytes and neutrophils, whereas G6PD was significantly positively correlated with neutrophils. The calibration curve, decision curve analysis, and ROC curves indicated that the five hub GRDEGs exhibited high predictive value for AMI. Furthermore, the five hub GRDEGs were validated by RT-qPCR and western blotting. CONCLUSION: We concluded that HK2, PFKL, PKM, G6PD, and ALDOA are hub GRDEGs in AMI and play important roles in AMI progression. This study provides a novel potential immunotherapeutic method for the treatment of AMI.
Assuntos
Biologia Computacional , Redes Reguladoras de Genes , Glicólise , Infarto do Miocárdio , Mapas de Interação de Proteínas , Humanos , Glicólise/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/diagnóstico , Perfilação da Expressão Gênica , Bases de Dados Genéticas , Transcriptoma , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Valor Preditivo dos Testes , Masculino , Pessoa de Meia-Idade , Hexoquinase/genética , Feminino , Estudos de Casos e Controles , Nomogramas , Reprodutibilidade dos TestesRESUMO
Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive sarcomas that can arise both sporadically and in patients with the genetic syndrome Neurofibromatosis type 1 (NF1). Prognosis is dismal, as large dimensions, risk of relapse, and anatomical localization make surgery poorly effective, and no therapy is known. Hence, the identification of MPNST molecular features that could be hit in an efficient and selective way is mandatory to envision treatment options. Here, we find that MPNSTs express high levels of the glycolytic enzyme Hexokinase 2 (HK2), which is known to shield cancer cells from noxious stimuli when it localizes at MAMs (mitochondria-associated membranes), contact sites between mitochondria and endoplasmic reticulum. A HK2-targeting peptide that dislodges HK2 from MAMs rapidly induces a massive death of MPNST cells. After identifying different matrix metalloproteases (MMPs) expressed in the MPNST microenvironment, we have designed HK2-targeting peptide variants that harbor cleavage sites for these MMPs, making such peptides activatable in the proximity of cancer cells. We find that the peptide carrying the MMP2/9 cleavage site is the most effective, both in inhibiting the in vitro tumorigenicity of MPNST cells and in hampering their growth in mice. Our data indicate that detaching HK2 from MAMs could pave the way for a novel anti-MPNST therapeutic strategy, which could be flexibly adapted to the protease expression features of the tumor microenvironment.
Assuntos
Hexoquinase , Peptídeos , Hexoquinase/metabolismo , Hexoquinase/genética , Humanos , Animais , Linhagem Celular Tumoral , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptídeos/química , Camundongos , Neoplasias de Bainha Neural/patologia , Neoplasias de Bainha Neural/genética , Neoplasias de Bainha Neural/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Proliferação de Células/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Microambiente TumoralRESUMO
Hexokinase 1 forms constricting rings around mitochondria that prevent fission induced by energy stress.
Assuntos
Hexoquinase , Mitocôndrias , Dinâmica Mitocondrial , Mitocôndrias/metabolismo , Hexoquinase/metabolismo , Hexoquinase/genética , Humanos , Dinâmica Mitocondrial/fisiologia , Animais , Metabolismo Energético , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genéticaRESUMO
Dysfunction of the ubiquitin-proteasome system (UPS) is involved in the pathogenesis of various malignancies including colorectal cancer (CRC). Ubiquitin domain containing 1 (UBTD1), a ubiquitin-like protein, regulates UPS-mediated protein degradation and tumor progression in some cancer types. However, the biological function and mechanism of UBTD1 are far from being well elucidated, and its role in CRC has not been explored yet. In our study, we analyzed CRC patients' clinical information and UBTD1 expression data, and found that the expression of UBTD1 in cancer tissue was significantly higher than that in adjacent normal tissue. Higher UBTD1 expression was significantly associated with poorer survival and more lymph node metastasis. Overexpression of UBTD1 could facilitate, while knockdown could inhibit CRC cell proliferation and migration, respectively. RNA-seq and proteomics indicated that c-Myc is an important downstream target of UBTD1. Metabolomics showed the products of the glycolysis pathway were significantly increased in UBTD1 overexpression cells. In vitro, we verified UBTD1 upregulating c-Myc protein and promoting CRC cell proliferation and migration via regulating c-Myc. UBTD1 promoted CRC cells' glycolysis, evidenced by the increased lactate production and glucose uptake following UBTD1 overexpression. Mechanistically, UBTD1 prolonged the half-life of the c-Myc protein by binding to E3 ligase ß-transducin repeat-containing protein (ß-TrCP), thereby upregulated the expression of glycolysis rate-limiting enzyme hexokinase II (HK2), and enhanced glycolysis and promoted CRC progression. In conclusion, our study revealed that UBTD1 promotes CRC progression by upregulating glycolysis via the ß-TrCP/c-Myc/HK2 pathway, suggesting its potential as a prognostic biomarker and therapeutic target in CRC.
Assuntos
Proliferação de Células , Neoplasias Colorretais , Progressão da Doença , Glicólise , Proteínas Proto-Oncogênicas c-myc , Regulação para Cima , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Hexoquinase/metabolismo , Hexoquinase/genética , Camundongos Nus , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Ubiquitinas/metabolismo , Ubiquitinas/genéticaRESUMO
Hexokinase (HK) catalyzes the first irreversible rate-limiting step in glycolysis that converts glucose to glucose-6-phosphate. HK1 is ubiquitously expressed in the brain, erythrocytes, and other tissues where glycolysis serves as the major source of ATP production. Spermatogenic cell-specific type 1 hexokinase (HK1S) is expressed in sperm but its physiological role in male mice is still unknown. In this study, we generate Hk1s knockout mice using the CRISPR/Cas9 system to study the gene function in vivo. Hk1s mRNA is exclusively expressed in testes starting from postnatal day 18 and continuing to adulthood. HK1S protein is specifically localized in the outer surface of the sperm fibrous sheath (FS). Depletion of Hk1s leads to infertility in male mice and reduces sperm glycolytic pathway activity, yet they have normal motile parameters and ATP levels. In addition, by using in vitro fertilization (IVF), Hk1s deficient sperms are unable to fertilize cumulus-intact or cumulus-free oocytes, but can normally fertilize zona pellucida-free oocytes. Moreover, Hk1s deficiency impairs sperm migration into the oviduct, reduces acrosome reaction, and prevents capacitation-associated increases in tyrosine phosphorylation, which are probable causes of infertility. Taken together, our results reveal that HK1S plays a critical role in sperm function and male fertility in mice.
Assuntos
Fertilidade , Hexoquinase , Infertilidade Masculina , Camundongos Knockout , Capacitação Espermática , Espermatozoides , Tirosina , Animais , Hexoquinase/genética , Hexoquinase/metabolismo , Masculino , Camundongos , Fosforilação , Espermatozoides/metabolismo , Capacitação Espermática/genética , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Fertilidade/genética , Tirosina/metabolismo , Feminino , Testículo/metabolismo , Motilidade dos Espermatozoides/genética , Glicólise , Espermatogênese/genéticaRESUMO
BACKGROUND: Gastric cancer (GC) is one of the most common cancers worldwide. Tumor microenvironment plays an important role in tumor progression. This study aims to explore the role of cancer-associated fibroblasts (CAFs) in GC and the underlying mechanism. METHODS: Cell viability, proliferation, invasion and migration were assessed by MTT, EdU, transwell and wound healing assays, respectively. Sphere formation assay was used to evaluate cell stemness. Glucose consumption, lactate production and ATP consumption were measured to assess glycolysis. In addition, The RNA and protein expression were detected by qRT-PCR and western blot. The interaction between wingless Type MMTV Integration Site Family, Member 5 A (WNT5A) and hexokinase 2 (HK2) was verified by Co-immunoprecipitation. The xenograft model was established to explore the function of CAFs on GC tumor growth in vivo. RESULTS: CAFs promoted the proliferation, metastasis, stemness and glycolysis of GC cells. WNT5A was upregulated in CAFs, and CAFs enhanced WNT5A expression in GC cells. Knockdown of WNT5A in either GC cells or CAFs repressed the progression of GC cells. In addition, WNT5A promoted HK2 expression, and overexpression of HK2 reversed the effect of WNT5A knockdown in CAFs on GC cells. Besides, knockdown of WNT5A in CAFs inhibits tumor growth in vivo. CONCLUSION: CAF-derived WNT5A facilitates the progression of GC via regulating HK2 expression.
Assuntos
Fibroblastos Associados a Câncer , Movimento Celular , Proliferação de Células , Glicólise , Hexoquinase , Neoplasias Gástricas , Proteína Wnt-5a , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Humanos , Proteína Wnt-5a/metabolismo , Proteína Wnt-5a/genética , Animais , Camundongos , Hexoquinase/metabolismo , Hexoquinase/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células Tumorais Cultivadas , Camundongos Nus , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Prognóstico , Camundongos Endogâmicos BALB C , Apoptose , Masculino , Linhagem Celular TumoralRESUMO
Energy status and nutrients regulate photosynthetic protein expression. The unicellular green alga Chromochloris zofingiensis switches off photosynthesis in the presence of exogenous glucose (+Glc) in a process that depends on hexokinase (HXK1). Here, we show that this response requires that cells lack sufficient iron (-Fe). Cells grown in -Fe+Glc accumulate triacylglycerol (TAG) while losing photosynthesis and thylakoid membranes. However, cells with an iron supplement (+Fe+Glc) maintain photosynthesis and thylakoids while still accumulating TAG. Proteomic analysis shows that known photosynthetic proteins are most depleted in heterotrophy, alongside hundreds of uncharacterized, conserved proteins. Photosynthesis repression is associated with enzyme and transporter regulation that redirects iron resources to (a) respiratory instead of photosynthetic complexes and (b) a ferredoxin-dependent desaturase pathway supporting TAG accumulation rather than thylakoid lipid synthesis. Combining insights from diverse organisms from green algae to vascular plants, we show how iron and trophic constraints on metabolism aid gene discovery for photosynthesis and biofuel production.