RESUMO
Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p-coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae (FjTAL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing FjTAL combined with 4CL from Arabidopsis thaliana (At4CL) and CHS from Cucurbita maxima (CmCHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa (MsCHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli. KEY POINTS: ⢠Best enzyme and strain combination were selected for de novo naringenin production. ⢠After genetic and operational optimizations, 765.9 mg/L of naringenin was produced. ⢠This de novo production is the highest reported so far in E. coli.
Assuntos
Aciltransferases , Amônia-Liases , Vias Biossintéticas , Coenzima A Ligases , Escherichia coli , Flavanonas , Flavanonas/biossíntese , Flavanonas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vias Biossintéticas/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Amônia-Liases/genética , Amônia-Liases/metabolismo , Engenharia Metabólica/métodos , Ácidos Cumáricos/metabolismo , Liases Intramoleculares/genética , Liases Intramoleculares/metabolismo , Tirosina/metabolismoRESUMO
3-Hydroxypropionic acid (3-HP) is a highly sought-after platform chemical serving as a precursor to a variety of high value-added chemical products. In this study, we designed and constructed a novel light-powered in vitro synthetic enzymatic biosystem comprising acetyl-CoA ligase, acetyl-CoA carboxylase, malonyl-CoA reductase, and phosphotransferase to efficiently produce 3-HP through CO2 fixation from acetate, a cost-effective and readily available substrate. The system employed natural thylakoid membranes (TMs) for the regeneration of adenosine triphosphate and nicotinamide adenine dinucleotide phosphate. Comprehensive investigations were conducted on the effects of buffer solutions, substrate concentrations, enzyme loading levels, and TMs loading levels to optimize the yield of 3-HP. Following optimization, a production of 0.46 mM 3-HP was achieved within 6 h from an initial 0.5 mM acetate, with a yield nearing 92%. This work underscores the simplicity of 3-HP production via an in vitro biomanufacturing platform and highlights the potential for incorporating TMs as a sustainable and environmentally friendly approach in biomanufacturing processes.
Assuntos
Acetil-CoA Carboxilase , Dióxido de Carbono , Ácido Láctico , Dióxido de Carbono/metabolismo , Acetil-CoA Carboxilase/metabolismo , Ácido Láctico/metabolismo , Ácido Láctico/análogos & derivados , Luz , Tilacoides/metabolismo , Trifosfato de Adenosina/metabolismo , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Acetatos/metabolismo , Acetatos/química , OxirredutasesRESUMO
OBJECTIVE: Mitochondrial fatty acid oxidation is a metabolic pathway whose dysregulation is recognized as a critical factor in various cancers, because it sustains cancer cell survival, proliferation, and metastasis. The acyl-CoA synthetase long-chain (ACSL) family is known to activate long-chain fatty acids, yet the specific role of ACSL3 in breast cancer has not been determined. METHODS: We assessed the prognostic value of ACSL3 in breast cancer by using data from tumor samples. Gain-of-function and loss-of-function assays were also conducted to determine the roles and downstream regulatory mechanisms of ACSL3 in vitro and in vivo. RESULTS: ACSL3 expression was notably downregulated in breast cancer tissues compared with normal tissues, and this phenotype correlated with improved survival outcomes. Functional experiments revealed that ACSL3 knockdown in breast cancer cells promoted cell proliferation, migration, and epithelial-mesenchymal transition. Mechanistically, ACSL3 was found to inhibit ß-oxidation and the formation of associated byproducts, thereby suppressing malignant behavior in breast cancer. Importantly, ACSL3 was found to interact with YES proto-oncogene 1, a member of the Src family of tyrosine kinases, and to suppress its activation through phosphorylation at Tyr419. The decrease in activated YES1 consequently inhibited YAP1 nuclear colocalization and transcriptional complex formation, and the expression of its downstream genes in breast cancer cell nuclei. CONCLUSIONS: ACSL3 suppresses breast cancer progression by impeding lipid metabolism reprogramming, and inhibiting malignant behaviors through phospho-YES1 mediated inhibition of YAP1 and its downstream pathways. These findings suggest that ACSL3 may serve as a potential biomarker and target for comprehensive therapeutic strategies for breast cancer.
Assuntos
Neoplasias da Mama , Proliferação de Células , Coenzima A Ligases , Progressão da Doença , Metabolismo dos Lipídeos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-yes , Fatores de Transcrição , Proteínas de Sinalização YAP , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Feminino , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Proteínas de Sinalização YAP/metabolismo , Animais , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Proto-Oncogênicas c-yes/metabolismo , Proteínas Proto-Oncogênicas c-yes/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Transição Epitelial-Mesenquimal , Camundongos Nus , Prognóstico , Movimento Celular , Transdução de Sinais , Reprogramação MetabólicaRESUMO
Intermittent hypoxia (IH) is an independent risk factor for metabolic dysfunction-associated fatty liver disease (MAFLD). Copper deficiency can disrupt redox homeostasis, iron, and lipid metabolism. Here, we investigated whether hepatic copper deficiency plays a role in IH-associated MAFLD and explored the underlying mechanism(s). Male C57BL/6 mice were fed a western-type diet with adequate copper (CuA) or marginally deficient copper (CuD) and were exposed separately to room air (RA) or IH. Hepatic histology, plasma biomarkers, copper-iron status, and oxidative stress were assessed. An in vitro HepG2 cell lipotoxicity model and proteomic analysis were used to elucidate the specific targets involved. We observed that there were no differences in hepatic phenotypes between CuA-fed and CuD-fed mice under RA. However, in IH exposure, CuD-fed mice showed more pronounced hepatic steatosis, liver injury, and oxidative stress than CuA-fed mice. IH induced copper accumulation in the brain and heart and exacerbated hepatic copper deficiency and secondary iron deposition. In vitro, CuD-treated cells with IH exposure showed elevated levels of lipid accumulation, oxidative stress, and ferroptosis susceptibility. Proteomic analysis identified 360 upregulated and 359 downregulated differentially expressed proteins between CuA and CuD groups under IH; these proteins were mainly enriched in citrate cycle, oxidative phosphorylation, fatty acid metabolism, the peroxisome proliferator-activated receptor (PPAR)α pathway, and ferroptosis. In IH exposure, CuD significantly upregulated the ferroptosis-promoting factor arachidonyl-CoA synthetase long chain family member (ACSL)4. ACSL4 knockdown markedly eliminated CuD-induced ferroptosis and lipid accumulation in IH exposure. In conculsion, IH can lead to reduced hepatic copper reserves and secondary iron deposition, thereby inducing ferroptosis and subsequent MAFLD progression. Insufficient dietary copper may worsen IH-associated MAFLD.
Assuntos
Cobre , Ferroptose , Hipóxia , Camundongos Endogâmicos C57BL , Animais , Cobre/metabolismo , Cobre/deficiência , Masculino , Camundongos , Hipóxia/metabolismo , Humanos , Células Hep G2 , Fígado/metabolismo , Fígado/patologia , Estresse Oxidativo , Metabolismo dos Lipídeos , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Fígado Gorduroso/etiologia , Ferro/metabolismo , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , PPAR alfa/metabolismo , PPAR alfa/genéticaRESUMO
Chemotherapeutic efficacy is seriously impeded by chemoresistance in more than half of hepatocellular carcinoma (HCC) patients. However, the mechanisms involved in chemotherapy-induced upregulation of chemoresistant genes are not fully understood. Here, this study unravels a novel mechanism controlling nuclear acetyl-CoA production to activate the transcription of chemoresistant genes in HCC. NAT10 is upregulated in HCC tissues and its upregulation is correlated with poor prognosis of HCC patients. NAT10 is also upregulated in chemoresistant HCC cells. Targeting NAT10 increases the cytotoxicity of chemotherapy in HCC cells and mouse xenografts. Upon chemotherapy, NAT10 translocates from the nucleolus to the nucleus to activate the transcription of CYP2C9 and PIK3R1. Additionally, nuclear acetyl-CoA is specifically upregulated by NAT10. Mechanistically, NAT10 binds with ACLY in the nucleus and acetylates ACLY at K468 to counteract the SQSTM1-mediated degradation upon chemotherapy. ACLY K468-Ac specifically accumulates in the nucleus and increases nuclear acetyl-CoA production to activate the transcription of CYP2C9 and PIK3R1 through enhancing H3K27ac. Importantly, K468 is required for nuclear localization of ACLY. Significantly, ACLY K468-Ac is upregulated in HCC tissues, and ablation of ACLY K468-Ac sensitizes HCC cells and mouse xenografts to chemotherapy. Collectively, these findings identify NAT10 as a novel chemoresistant driver and the blockage of NAT10-mediated ACLY K468-Ac possesses the potential to attenuate HCC chemoresistance.
Assuntos
Acetilcoenzima A , Carcinoma Hepatocelular , Núcleo Celular , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Acetilcoenzima A/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Animais , Acetilação , Camundongos , Núcleo Celular/metabolismo , Linhagem Celular Tumoral , Camundongos Nus , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Acetiltransferases N-Terminal/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Camundongos Endogâmicos BALB C , MasculinoRESUMO
Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.
Assuntos
Acetaldeído , Melanoma , Peixe-Zebra , Melanoma/metabolismo , Melanoma/genética , Melanoma/patologia , Melanoma/tratamento farmacológico , Acetaldeído/metabolismo , Acetaldeído/farmacologia , Animais , Humanos , Linhagem Celular Tumoral , Aldeído Oxirredutases/metabolismo , Aldeído Oxirredutases/genética , Histonas/metabolismo , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Transcrição Gênica/efeitos dos fármacos , Crista Neural/metabolismo , Crista Neural/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
To analyze the mechanism of how interfering with the cytokeratin 19 (CK19) pathway via the ferroptosis pathway affects tumor biological behaviors in the process of oral squamous cell carcinoma (OSCC) development. TCGA was used to analyze the expression of CK19 in pan-cancer and head and neck squamous cell carcinoma (HNSC) and to explore the ferroptosis-related genes related to HNSC. The effect of silencing CK19 on the migration ability of HSC-4 cells was verified by wound healing and migration assay. HSC-4 cells with silencing of CK19 and tumor-bearing nude mouse model were constructed. RT-qPCR, immunofluorescence and western blot were used to analyze the expression of ferroptosis-related genes. CK19 is highly expressed in human OSCC and nude mice. The migration ability of cells in the CK19-silenced group was lower than that of the control group. In vivo and in vitro, CK19 was negatively correlated with the expression of ACSL4 and positively correlated with the expression of GPX4. Compared with the control group, GPX4 expression was down-regulated and ACSL4 expression was up-regulated in the CK19-silenced group. Silencing CK19 also increased intracellular Fe2+ content and MDA content. Silencing CK19 can affect the expression of GPX4 and ACSL4 to regulate ferroptosis and at the same time increase the content of MDA, Fe2+ and ROS levels, thereby activating the regulation of ferroptosis pathway in the development of OSCC.
Assuntos
Coenzima A Ligases , Ferroptose , Regulação Neoplásica da Expressão Gênica , Queratina-19 , Camundongos Nus , Neoplasias Bucais , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Animais , Humanos , Camundongos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Ferroptose/genética , Inativação Gênica , Queratina-19/metabolismo , Queratina-19/genética , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Neoplasias Bucais/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genéticaRESUMO
Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver. As the global obesity rate rises, non-alcoholic fatty liver disease (NAFLD) has emerged as the most rapidly increasing cause of HCC. Consequently, the regulation of lipid metabolism has become a crucial target for the prevention and treatment of HCC. Liquidambaric acid (LDA), a pentacyclic triterpenoid compound derived from various plants, exhibits diverse biological activities. We found that LDA could inhibit HCC cell proliferation by arresting cell cycle and prompting apoptosis. Additionally, LDA can augment the therapeutic efficacy of Regorafenib in HCC in vitro and vivo. Our study utilized transcriptome analysis, luciferase reporter assays, and co-immunocoprecipitation experiments to elucidate the anti-HCC mechanism of LDA. We discovered that LDA disrupts the formation of the PPARα-RXRα heterodimer, leading to the down-regulation of the ACSL4 gene and subsequently impacting the fatty acid metabolism of HCC cells, ultimately inhibiting HCC proliferation. Our research contributes to the identification of novel therapeutic agents and targets for the treatment of HCC.
Assuntos
Carcinoma Hepatocelular , Proliferação de Células , Coenzima A Ligases , Regulação para Baixo , Ácidos Graxos , Neoplasias Hepáticas , PPAR alfa , Receptor X Retinoide alfa , PPAR alfa/metabolismo , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Receptor X Retinoide alfa/metabolismo , Receptor X Retinoide alfa/genética , Animais , Ácidos Graxos/metabolismo , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Células Hep G2 , Camundongos Nus , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Camundongos , Compostos de Fenilureia/farmacologia , Masculino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Metabolismo dos Lipídeos/efeitos dos fármacos , PiridinasRESUMO
Ferroptosis, an iron-dependent form of non-apoptotic cell death, plays a pivotal role in various diseases and is gaining considerable attention in the realm of endometriosis. Considering the classical pathomechanism theories, we hypothesized that ferroptosis, potentially driven by increased iron content at ectopic sites, may contribute to the progression of endometriosis. This retrospective case-control study provides a comprehensive immunohistochemical assessment of the expression and tissue distribution of established ferroptosis markers: GPX4, ACSL4, and TfR1 in endometriosis patients. The case group consisted of 38 women with laparoscopically and histologically confirmed endometriosis and the control group consisted of 18 women with other gynecological conditions. Our study revealed a significant downregulation of GPX4 in stromal cells of endometriosis patients (M = 59.7% ± 42.4 versus 90.0% ± 17.5 in the control group, t (54) = -2.90, p = 0.005). This finding aligned with slightly, but not significantly, higher iron levels detected in the blood of endometriosis patients, using hemoglobin as an indirect predictor (Hb 12.8 (12.2-13.5) g/dL versus 12.5 (12.2-13.4) g/dL in the control group; t (54) = -0.897, p = 0.374). Interestingly, there was no concurrent upregulation of TfR1 (M = 0.7 ± 1.2 versus 0.2 ± 0.4 for EM, t (54) = 2.552, p = 0.014), responsible for iron uptake into cells. Our empirical findings provide support for the involvement of ferroptosis in the context of endometriosis. However, variances in expression patterns within stromal and epithelial cellular subsets call for further in-depth investigations.
Assuntos
Coenzima A Ligases , Endometriose , Ferroptose , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Receptores da Transferrina , Humanos , Feminino , Endometriose/metabolismo , Endometriose/patologia , Receptores da Transferrina/metabolismo , Receptores da Transferrina/genética , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Adulto , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Estudos de Casos e Controles , Estudos Retrospectivos , Antígenos CD/metabolismo , Antígenos CD/genética , Ferro/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Pessoa de Meia-Idade , Biomarcadores/metabolismoRESUMO
Acute lung injury (ALI) is a serious disorder characterized by the release of pro-inflammatory cytokines and cascade activation of macrophages. Ferroptosis, a form of iron-dependent cell death triggered by intracellular phospholipid peroxidation, has been implicated as an internal mechanism underlying ALI. In this study, we investigated the effects of m6A demethylase fat mass and obesity-associated protein (FTO) on the inhibition of macrophage ferroptosis in ALI. Using a mouse model of lipopolysaccharide (LPS)-induced ALI, we observed the induction of ferroptosis and its co-localization with the macrophage marker F4/80, suggesting that ferroptosis might be induced in macrophages. Ferroptosis was promoted during LPS-induced inflammation in macrophages in vitro, and the inflammation was counteracted by the ferroptosis inhibitor ferrostatin-1 (fer-1). Given that FTO showed lower expression levels in the lung tissue of mice with ALI and inflammatory macrophages, we further dissected the regulatory capacity of FTO in ferroptosis. The results demonstrated that FTO alleviated macrophage inflammation by inhibiting ferroptosis. Mechanistically, FTO decreased the stability of ACSL4 mRNA via YTHDF1, subsequently inhibiting ferroptosis and inflammation by interrupting polyunsaturated fatty acid consumption. Moreover, FTO downregulated the synthesis and secretion of prostaglandin E2, thereby reducing ferroptosis and inflammation. In vivo, the FTO inhibitor FB23-2 aggravated lung injury, the inflammatory response, and ferroptosis in mice with ALI; however, fer-1 therapy mitigated these effects. Overall, our findings revealed that FTO may function as an inhibitor of the inflammatory response driven by ferroptosis, emphasizing its potential as a target for ALI treatment.
Assuntos
Lesão Pulmonar Aguda , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Coenzima A Ligases , Ferroptose , Inflamação , Lipopolissacarídeos , Macrófagos , Animais , Ferroptose/efeitos dos fármacos , Camundongos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Masculino , Camundongos Endogâmicos C57BL , Fenilenodiaminas/farmacologia , Modelos Animais de Doenças , Células RAW 264.7 , CicloexilaminasRESUMO
Laryngeal carcinoma (LC) is a common cancer of the respiratory tract. This study aims to investigate the role of RNA-binding motif protein 15 (RBM15) in the cisplatin (DDP) resistance of LC cells. LC-DDP-resistant cells were constructed. RBM15, lysine-specific demethylase 5B (KDM5B), lncRNA Fer-1 like family member 4 (FER1L4), lncRNA KCNQ1 overlapping transcript 1 (KCNQ1OT1), glutathione peroxidase 4 (GPX4), and Acyl-CoA synthetase long-chain family (ACSL4) was examined. Cell viability, IC50, and proliferation were assessed after RBM15 downregulation. The enrichment of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) and N6-methyladenosine (m6A) on KDM5B was analyzed. KDM5B mRNA stability was measured after actinomycin D treatment. A tumor xenograft assay was conducted to verify the role of RBM15 in LC. Results showed that RBM15 was upregulated in LC and its knockdown decreased IC50, cell viability, proliferation, glutathione, and upregulated iron ion content, ROS, malondialdehyde, ACSL4, and ferroptosis. Mechanistically, RBM15 improved KDM5B stability in an IGF2BP3-dependent manner, resulting in FER1L4 downregulation and GPX4 upregulation. KDM5B increased KCNQ1OT1 and inhibited ACSL4. KDM5B/KCNQ1OT1 overexpression or FER1L4 knockdown promoted DDP resistance in LC by inhibiting ferroptosis. In conclusion, RBM15 promoted KDM5B expression, and KDM5B upregulation inhibited ferroptosis and promoted DDP resistance in LC by downregulating FER1L4 and upregulating GPX4, as well as by upregulating KCNQ1OT1 and inhibiting ACSL4. Silencing RBM15 inhibited tumor growth in vivo.
Assuntos
Cisplatino , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Ferroptose , Neoplasias Laríngeas , Proteínas de Ligação a RNA , Ferroptose/genética , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular Tumoral , Camundongos , Animais , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/metabolismo , Camundongos Nus , Regulação Neoplásica da Expressão Gênica , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismoRESUMO
SIAH2 function as an oncogene in various cancer. However, the roles of SIAH2 in hepatocellular carcinoma (HCC) are still unknown. This study aimed to investigate the roles of SIAH2 in HCC. Immunohistochemistry was used determine SIAH2 and ACSL4 expression in clinical samples. RT-qPCR was used to determine mRNA expression. Western blot assay was applied for determining protein expression. Ubiquitination assay was conducted for determining ubiquitination of ACSL4. Xenograft experiment was applied for determining tumor growth. Flow cytometry was applied to determine the functions of CD4+ and CD8+ T cells. SIAH2 expression was overexpressed in HCC tumors. High levels of SIAH2 predicted poor outcomes. However, SIAH2 knockdown promoted the proliferation of CD8+ T cells as well as promoted the ferroptosis of tumor cells, inhibiting tumor growth in HCC. ACSL4 is required for CD8+ T cell-mediated ferroptosis of HCC cells. However, SIAH2 induced ubiquitination of ACSL4 and inhibited its expression. SIAH2 specific inhibitor menadione promoted the immune checkpoint blockade. Taken together, SIAH2-mediated inactivation of CD8+ T cells inhibits the ferroptosis of HCC via mediating ubiquitination of ACSL4. Therefore, targeting SIAH2 may be a promising strategy for HCC.
Assuntos
Linfócitos T CD8-Positivos , Carcinoma Hepatocelular , Coenzima A Ligases , Neoplasias Hepáticas , Ubiquitina-Proteína Ligases , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Animais , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Linhagem Celular Tumoral , Ubiquitinação , Masculino , Feminino , Proliferação de Células , Regulação Neoplásica da Expressão GênicaRESUMO
Older livers are more prone to hepatic ischaemia/reperfusion injury (HIRI), which severely limits their utilization in liver transplantation. The potential mechanism remains unclear. Here, we demonstrate older livers exhibit increased ferroptosis during HIRI. Inhibiting ferroptosis significantly attenuates older HIRI phenotypes. Mass spectrometry reveals that fat mass and obesity-associated gene (FTO) expression is downregulated in older livers, especially during HIRI. Overexpressing FTO improves older HIRI phenotypes by inhibiting ferroptosis. Mechanistically, acyl-CoA synthetase long chain family 4 (ACSL4) and transferrin receptor protein 1 (TFRC), two key positive contributors to ferroptosis, are FTO targets. For ameliorative effect, FTO requires the inhibition of Acsl4 and Tfrc mRNA stability in a m6A-dependent manner. Furthermore, we demonstrate nicotinamide mononucleotide can upregulate FTO demethylase activity, suppressing ferroptosis and decreasing older HIRI. Collectively, these findings reveal an FTO-ACSL4/TFRC regulatory pathway that contributes to the pathogenesis of older HIRI, providing insight into the clinical translation of strategies related to the demethylase activity of FTO to improve graft function after older donor liver transplantation.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato , Coenzima A Ligases , Ferroptose , Fígado , Receptores da Transferrina , Traumatismo por Reperfusão , Regulação para Cima , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Animais , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Ferroptose/genética , Fígado/metabolismo , Fígado/patologia , Camundongos , Receptores da Transferrina/metabolismo , Receptores da Transferrina/genética , Masculino , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Camundongos Endogâmicos C57BL , Humanos , Transplante de Fígado , Estabilidade de RNA/genética , Antígenos CDRESUMO
Diabetic cardiomyopathy is a specific type of cardiomyopathy. In DCM, glucose uptake and utilization are impaired due to insulin deficiency or resistance, and the heart relies more heavily on fatty acid oxidation for energy, resulting in myocardial lipid toxicity-related injury. MARK4 is a member of the AMPK-related kinase family, and improves ischaemic heart failure through microtubule detyrosination. However, the role of MARK4 in cardiac regulation of metabolism is unclear. In this study, after successful establishment of a diabetic cardiomyopathy model induced by streptozotocin and a high-fat diet, MARK4 expression was found to be significantly increased in STZ-induced DCM mice. After AAV9-shMARK4 was administered through the tail vein, decreased expression of MARK4 alleviated diabetic myocardial damage, reduced oxidative stress and apoptosis, and facilitated cardiomyocyte mitochondrial fusion, and promoted myocardial lipid oxidation metabolism. In addition, through the RNA-seq analysis of differentially expressed genes, we found that MARK4 deficiency promoted lipid decomposition and oxidative metabolism by downregulating the expression of ACSL4, thus reducing myocardial lipid accumulation in the STZ-induced DCM model.
Assuntos
Coenzima A Ligases , Cardiomiopatias Diabéticas , Metabolismo dos Lipídeos , Miocárdio , Animais , Masculino , Camundongos , Apoptose , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/complicações , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/patologia , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/etiologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , EstreptozocinaRESUMO
Neurofibromatosis Type II (NFII) is a genetic condition caused by loss of the NF2 gene, resulting in activation of the YAP/TAZ pathway and recurrent Schwann cell tumors, as well as meningiomas and ependymomas. Unfortunately, few pharmacological options are available for NFII. Here, we undertake a genome-wide CRISPR/Cas9 screen to search for synthetic-lethal genes that, when inhibited, cause death of NF2 mutant Schwann cells but not NF2 wildtype cells. We identify ACSL3 and G6PD as two synthetic-lethal partners for NF2, both involved in lipid biogenesis and cellular redox. We find that NF2 mutant Schwann cells are more oxidized than control cells, in part due to reduced expression of genes involved in NADPH generation such as ME1. Since G6PD and ME1 redundantly generate cytosolic NADPH, lack of either one is compatible with cell viability, but not down-regulation of both. Since genetic deficiency for G6PD is tolerated in the human population, G6PD could be a good pharmacological target for NFII.
Assuntos
Sistemas CRISPR-Cas , Coenzima A Ligases , Glucosefosfato Desidrogenase , Neurofibromina 2 , Células de Schwann , Mutações Sintéticas Letais , Células de Schwann/metabolismo , Humanos , Glucosefosfato Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/genética , Neurofibromina 2/metabolismo , Neurofibromina 2/genética , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Animais , Neurofibromatose 2/metabolismo , Neurofibromatose 2/genética , NADP/metabolismo , Camundongos , OxirreduçãoRESUMO
KEY MESSAGE: DzMYB2 functions as an MYB activator, while DzMYB3 acts as an MYB repressor. They bind to promoters, interact with DzbHLH1, and influence phenolic contents, revealing their roles in phenylpropanoid regulation in durian pulps. Durian fruit has a high nutritional value attributed to its enriched bioactive compounds, including phenolics, carotenoids, and vitamins. While various transcription factors (TFs) regulate phenylpropanoid biosynthesis, MYB (v-myb avian myeloblastosis viral oncogene homolog) TFs have emerged as pivotal players in regulating key genes within this pathway. This study aimed to identify additional candidate MYB TFs from the transcriptome database of the Monthong cultivar at five developmental/postharvest ripening stages. Candidate transcriptional activators were discerned among MYBs upregulated during the ripe stage based on the positive correlation observed between flavonoid biosynthetic genes and flavonoid contents in ripe durian pulps. Conversely, MYBs downregulated during the ripe stage were considered candidate repressors. This study focused on a candidate MYB activator (DzMYB2) and a candidate MYB repressor (DzMYB3) for functional characterization. LC-MS/MS analysis using Nicotiana benthamiana leaves transiently expressing DzMYB2 revealed increased phenolic compound contents compared with those in leaves expressing green fluorescence protein controls, while those transiently expressing DzMYB3 showed decreased phenolic compound contents. Furthermore, it was demonstrated that DzMYB2 controls phenylpropanoid biosynthesis in durian by regulating the promoters of various biosynthetic genes, including phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR). Meanwhile, DzMYB3 regulates the promoters of PAL, 4-coumaroyl-CoA ligase (4CL), CHS, and CHI, resulting in the activation and repression of gene expression. Moreover, it was discovered that DzMYB2 and DzMYB3 could bind to another TF, DzbHLH1, in the regulation of flavonoid biosynthesis. These findings enhance our understanding of the pivotal role of MYB proteins in regulating the phenylpropanoid pathway in durian pulps.
Assuntos
Flavonoides , Frutas , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Fatores de Transcrição , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Frutas/genética , Frutas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Flavonoides/metabolismo , Flavonoides/biossíntese , Aciltransferases/genética , Aciltransferases/metabolismo , Propanóis/metabolismo , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Fenóis/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Fenilalanina Amônia-Liase/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Liases Intramoleculares/genética , Liases Intramoleculares/metabolismoRESUMO
OBJECTIVE: Tetramethylpyrazine (TMP) has been demonstrated to alleviate neuronal ferroptosis following spinal cord injury (SCI), thereby promoting neural repair. However, the precise underlying mechanisms remain elusive. METHODS: The SCI model was established using a modified version of Allen's method. TMP (40, 80, 120, and 160 mg/kg) and ras-selective lethal 3 (RSL3) (5 mg/kg) were administered intraperitoneally once daily for 7 days. HE and Nissl staining were employed to examine histomorphology and neurons, respectively. Perls staining was used to identify the distribution of iron. A transmission electron microscope was used to observe the microcosmic morphology of mitochondria. Immunofluorescence staining and Western blot were used to analyze neuronal nuclear protein (NeuN) and glial fibrillary acidic protein (GFAP) surrounding injury sites. Additionally, glutathione peroxidase 4 (GPX4)/NeuN + cells and acyl-CoA synthetase long-chain family member 4 (ACSL4)/NeuN + cells were observed. RT-qPCR was conducted to examine the mRNA expression levels of GPX4 and ACSL4. ELISA were used to quantify the concentrations of GPX4, reactive oxygen species (ROS), L-glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and tissue iron. RESULTS: TMP had an inhibitory effect on the concentrations of tissue iron, ROS, GSH, MDA, and SOD. TMP improved the microcosmic morphology of mitochondria and increased GPX4 level while decreasing that of ACSL4. TMP reduced lesion sizes, enhanced neuronal survival, and inhibited glial scar formation. However, the effect of TMP can be effectively reversed by RSL3. CONCLUSION: TMP alleviates neuronal ferroptosis by regulating the GPX4/ACSL4 axis, thereby protecting the remaining neurons surrounding injury sites and reducing glial scar formation.
Assuntos
Coenzima A Ligases , Ferroptose , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Pirazinas , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal , Ferroptose/efeitos dos fármacos , Animais , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Recuperação de Função Fisiológica/efeitos dos fármacos , Masculino , Modelos Animais de Doenças , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêuticoRESUMO
Elevated lactate levels are a significant biomarker of sepsis and are positively associated with sepsis-related mortality. Sepsis-associated lung injury (ALI) is a leading cause of poor prognosis in clinical patients. However, the underlying mechanisms of lactate's involvement in sepsis-associated ALI remain unclear. In this study, we demonstrate that lactate regulates N6-methyladenosine (m6A) modification levels by facilitating p300-mediated H3K18la binding to the METTL3 promoter site. The METTL3-mediated m6A modification is enriched in ACSL4, and its mRNA stability is regulated through a YTHDC1-dependent pathway. Furthermore, short-term lactate stimulation upregulates ACSL4, which promotes mitochondria-associated ferroptosis. Inhibition of METTL3 through knockdown or targeted inhibition effectively suppresses septic hyper-lactate-induced ferroptosis in alveolar epithelial cells and mitigates lung injury in septic mice. Our findings suggest that lactate induces ferroptosis via the GPR81/H3K18la/METTL3/ACSL4 axis in alveolar epithelial cells during sepsis-associated ALI. These results reveal a histone lactylation-driven mechanism inducing ferroptosis through METTL3-mediated m6A modification. Targeting METTL3 represents a promising therapeutic strategy for patients with sepsis-associated ALI.
Assuntos
Coenzima A Ligases , Ferroptose , Metiltransferases , Sepse , Metiltransferases/metabolismo , Metiltransferases/genética , Animais , Sepse/metabolismo , Sepse/complicações , Camundongos , Humanos , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Lesão Pulmonar/metabolismo , Lesão Pulmonar/etiologia , Lesão Pulmonar/patologia , Lesão Pulmonar/genética , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/genética , Masculino , Modelos Animais de Doenças , Ácido Láctico/metabolismoRESUMO
Resveratrol, a phenylpropanoid compound, exhibits diverse pharmacological properties, making it a valuable candidate for health and disease management. However, the demand for resveratrol exceeds the capacity of plant extraction methods, necessitating alternative production strategies. Microbial synthesis offers several advantages over plant-based approaches and presents a promising alternative. Yarrowia lipolytica stands out among microbial hosts due to its safe nature, abundant acetyl-CoA and malonyl-CoA availability, and robust pentose phosphate pathway. This study aimed to engineer Y. lipolytica for resveratrol production. The resveratrol biosynthetic pathway was integrated into Y. lipolytica by adding genes encoding tyrosine ammonia lyase from Rhodotorula glutinis, 4-coumarate CoA ligase from Nicotiana tabacum, and stilbene synthase from Vitis vinifera. This resulted in the production of 14.3 mg/L resveratrol. A combination of endogenous and exogenous malonyl-CoA biosynthetic modules was introduced to enhance malonyl-CoA availability. This included genes encoding acetyl-CoA carboxylase 2 from Arabidopsis thaliana, malonyl-CoA synthase, and a malonate transporter protein from Bradyrhizobium diazoefficiens. These strategies increased resveratrol production to 51.8 mg/L. The further optimization of fermentation conditions and the utilization of sucrose as an effective carbon source in YP media enhanced the resveratrol concentration to 141 mg/L in flask fermentation. By combining these strategies, we achieved a titer of 400 mg/L resveratrol in a controlled fed-batch bioreactor. These findings demonstrate the efficacy of Y. lipolytica as a platform for the de novo production of resveratrol and highlight the importance of metabolic engineering, enhancing malonyl-CoA availability, and media optimization for improved resveratrol production.
Assuntos
Engenharia Metabólica , Resveratrol , Sacarose , Yarrowia , Resveratrol/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , Engenharia Metabólica/métodos , Sacarose/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Vitis/microbiologia , Vitis/genética , Vitis/metabolismo , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Malonil Coenzima A/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologia , Rhodotorula/genética , Rhodotorula/metabolismo , Fermentação , Arabidopsis/genética , Arabidopsis/metabolismo , Amônia-Liases , Proteínas de BactériasRESUMO
Gastric cancer remains among the deadliest neoplasms worldwide, with limited therapeutic options. Since efficacies of targeted therapies are unsatisfactory, drugs with broader mechanisms of action rather than a single oncogene inhibition are needed. Preclinical studies have identified histone deacetylases (HDAC) as potential therapeutic targets in gastric cancer. However, the mechanism(s) of action of HDAC inhibitors (HDACi) are only partially understood. This is particularly true with regard to ferroptosis as an emerging concept of cell death. In a panel of gastric cancer cell lines with different molecular characteristics, tumor cell inhibitory effects of different HDACi were studied. Lipid peroxidation levels were measured and proteome analysis was performed for the in-depth characterization of molecular alterations upon HDAC inhibition. HDACi effects on important ferroptosis genes were validated on the mRNA and protein level. Upon HDACi treatment, lipid peroxidation was found increased in all cell lines. Class I HDACi (VK1, entinostat) showed the same toxicity profile as the pan-HDACi vorinostat. Proteome analysis revealed significant and concordant alterations in the expression of proteins related to ferroptosis induction. Key enzymes like ACSL4, POR or SLC7A11 showed distinct alterations in their expression patterns, providing an explanation for the increased lipid peroxidation. Results were also confirmed in primary human gastric cancer tissue cultures as a relevant ex vivo model. We identify the induction of ferroptosis as new mechanism of action of class I HDACi in gastric cancer. Notably, these findings were independent of the genetic background of the cell lines, thus introducing HDAC inhibition as a more general therapeutic principle.