RESUMO
BACKGROUND: Sepsis-induced pulmonary injury (SPI) is a common complication of sepsis with a high rate of mortality. N4-acetylcytidine (ac4C) is mediated by the ac4C "writer", N-acetyltransferase (NAT)10, to regulate the stabilization of mRNA. This study aimed to investigate the role of NAT10 in SPI and the underlying mechanism. METHODS: Twenty-three acute respiratory distress syndrome (ARDS) patients and 27 non-ARDS volunteers were recruited. A sepsis rat model was established. Reverse transcription-quantitative polymerase chain reaction was used to detect the expression of NAT10 and transferrin receptor (TFRC). Cell viability was detected by cell counting kit-8. The levels of Fe2+, glutathione, and malondialdehyde were assessed by commercial kits. Lipid reactive oxygen species production was measured by flow cytometric analysis. Western blot was used to detect ferroptosis-related protein levels. Haematoxylin & eosin staining was performed to observe the pulmonary pathological symptoms. RESULTS: The results showed that NAT10 was increased in ARDS patients and lipopolysaccharide-treated human lung microvascular endothelial cell line-5a (HULEC-5a) cells. NAT10 inhibition increased cell viability and decreased ferroptosis in HULEC-5a cells. TFRC was a downstream regulatory target of NAT10-mediated ac4C acetylation. Overexpression of TFRC decreased cell viability and promoted ferroptosis. In in vivo study, NAT10 inhibition alleviated SPI. CONCLUSION: NAT10-mediated ac4C acetylation of TFRC aggravated SPI through promoting ferroptosis.
Assuntos
Ferroptose , Receptores da Transferrina , Sepse , Sepse/metabolismo , Sepse/complicações , Sepse/etiologia , Acetilação , Animais , Humanos , Ratos , Masculino , Receptores da Transferrina/metabolismo , Receptores da Transferrina/genética , Feminino , Lesão Pulmonar/metabolismo , Lesão Pulmonar/etiologia , Lesão Pulmonar/patologia , Modelos Animais de Doenças , Acetiltransferases/metabolismo , Acetiltransferases/genética , Pessoa de Meia-Idade , Antígenos CD/metabolismo , Antígenos CD/genética , Citidina/análogos & derivados , Citidina/farmacologia , Linhagem Celular , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/patologia , Ratos Sprague-Dawley , Sobrevivência CelularRESUMO
Millions of years of evolution have optimized many biosynthetic pathways by use of multi-step catalysis. In addition, multi-step metabolic pathways are commonly found in and on membrane-bound organelles in eukaryotic biochemistry. The fundamental mechanisms that facilitate these reaction processes provide strategies to bioengineer metabolic pathways in synthetic chemistry. Using Brownian dynamics simulations, here we modeled intermediate substrate transportation of colocalized yeast-ester biosynthesis enzymes on the membrane. The substrate acetate ion traveled from the pocket of aldehyde dehydrogenase to its target enzyme acetyl-CoA synthetase, then the substrate acetyl CoA diffused from Acs1 to the active site of the next enzyme, alcohol-O-acetyltransferase. Arranging two enzymes with the smallest inter-enzyme distance of 60 Å had the fastest average substrate association time as compared with anchoring enzymes with larger inter-enzyme distances. When the off-target side reactions were turned on, most substrates were lost, which suggests that native localization is necessary for efficient final product synthesis. We also evaluated the effects of intermolecular interactions, local substrate concentrations, and membrane environment to bring mechanistic insights into the colocalization pathways. The computation work demonstrates that creating spatially organized multi-enzymes on membranes can be an effective strategy to increase final product synthesis in bioengineering systems.
Assuntos
Simulação de Dinâmica Molecular , Acetiltransferases/metabolismo , Acetiltransferases/química , Aldeído Desidrogenase/metabolismo , Aldeído Desidrogenase/química , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Acetato-CoA Ligase/metabolismo , Acetato-CoA Ligase/química , Acetato-CoA Ligase/genética , Acetilcoenzima A/metabolismo , Acetilcoenzima A/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Domínio Catalítico , ProteínasRESUMO
Acute myeloid leukemia (AML) is a devastating disease initiated and maintained by a rare subset of cells called leukemia stem cells (LSCs). LSCs are responsible for driving disease relapse, making the development of new therapeutic strategies to target LSCs urgently needed. The use of mass spectrometry-based metabolomics profiling has enabled the discovery of unique and targetable metabolic properties in LSCs. However, we do not have a comprehensive understanding of metabolite differences between LSCs and their normal counterparts, hematopoietic stem and progenitor cells (HSPCs). In this study, we used an unbiased mass spectrometry-based metabolomics analysis to define differences in metabolites between primary human LSCs and HSPCs, which revealed that LSCs have a distinct metabolome. Spermidine was the most enriched metabolite in LSCs compared with HSPCs. Pharmacological reduction of spermidine concentrations decreased LSC function but spared normal HSPCs. Polyamine depletion also decreased leukemic burden in patient-derived xenografts. Mechanistically, spermidine depletion induced LSC myeloid differentiation by decreasing eIF5A-dependent protein synthesis, resulting in reduced expression of a select subset of proteins. KAT7, a histone acetyltransferase, was one of the top candidates identified to be down-regulated by spermidine depletion. Overexpression of KAT7 partially rescued polyamine depletion-induced decreased colony-forming ability, demonstrating that loss of KAT7 is an essential part of the mechanism by which spermidine depletion targets AML clonogenic potential. Together, we identified and mechanistically dissected a metabolic vulnerability of LSCs that has the potential to be rapidly translated into clinical trials to improve outcomes for patients with AML.
Assuntos
Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Espermidina , Animais , Humanos , Camundongos , Acetiltransferases , Diferenciação Celular , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Metaboloma , Metabolômica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Espermidina/metabolismoRESUMO
Although therapeutic targets for colorectal cancer (CRC) treatment have been developed, the treatment outcomes are not ideal and survival rates for CRC patients remain low. It is critical to identify a specific target and develop an effective CRC treatment system. The ZNF334 gene is a newly identified member of Zinc-finger proteins (ZNFs), which is essential for key biological processes associated with tumorigenesis. Abnormal epigenetic reprogramming of the ZNF334 gene promoter region decreases its expression in CRC and further induces the occurrence of CRC. Here, we clarified that P300 in CRC can regulate the H3K9/27 ac in the ZNF334 promoter. Furthermore, histone acetylation of the ZNF334 promoter region was increased by dCas9-P300 to normalize the deficiency of ZNF334 expression, thereby inhibiting the growth of CRC. Collectively, our findings enable a facile way to affect gene expression using CRISPR/Cas9-based epigenome editing and further determine the causal link between histone acetylation and gene activation, providing a promising gene therapy strategy for the CRC treatment.
Assuntos
Sistemas CRISPR-Cas , Neoplasias Colorretais , Epigênese Genética , Edição de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Edição de Genes/métodos , Acetilação , Proliferação de Células/genética , Regiões Promotoras Genéticas/genética , Animais , Linhagem Celular Tumoral , Histonas/metabolismo , Camundongos , Acetiltransferases/genética , Acetiltransferases/metabolismoRESUMO
Sepsis-associated encephalopathy (SAE) is a critical neurological complication of sepsis and represents a crucial factor contributing to high mortality and adverse prognosis in septic patients. This study explored the contribution of NAT10-mediated messenger RNA (mRNA) acetylation in cognitive dysfunction associated with SAE, utilizing a cecal ligation and puncture (CLP)-induced SAE mouse model. Our findings demonstrate that CLP significantly upregulates NAT10 expression and mRNA acetylation in the excitatory neurons of the hippocampal dentate gyrus (DG). Notably, neuronal-specific Nat10 knockdown improved cognitive function in septic mice, highlighting its critical role in SAE. Proteomic analysis, RNA immunoprecipitation, and real-time qPCR identified GABABR1 as a key downstream target of NAT10. Nat10 deletion reduced GABABR1 expression, and subsequently weakened inhibitory postsynaptic currents in hippocampal DG neurons. Further analysis revealed that microglia activation and the release of inflammatory mediators lead to the increased NAT10 expression in neurons. Microglia depletion with PLX3397 effectively reduced NAT10 and GABABR1 expression in neurons, and ameliorated cognitive dysfunction induced by SAE. In summary, our findings revealed that after CLP, NAT10 in hippocampal DG neurons promotes GABABR1 expression through mRNA acetylation, leading to cognitive dysfunction.
Assuntos
Disfunção Cognitiva , RNA Mensageiro , Encefalopatia Associada a Sepse , Animais , Masculino , Camundongos , Acetilação , Acetiltransferases/metabolismo , Acetiltransferases/genética , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/genética , Giro Denteado/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Sepse/metabolismo , Sepse/complicações , Sepse/genética , Encefalopatia Associada a Sepse/metabolismo , Encefalopatia Associada a Sepse/genética , Receptores de GABA-BRESUMO
Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of N-acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. Acetylation of the terminal non-reducing amino group of α-D-glucosamine of HS is essential for its complete breakdown into monosaccharides and free sulfate. Heparan-α-glucosaminide N-acetyltransferase (HGSNAT), a resident of the lysosomal membrane, catalyzes this essential acetylation reaction by accepting and transferring the acetyl group from cytosolic acetyl-CoA to terminal α-D-glucosamine of HS in the lysosomal lumen. Mutation-induced dysfunction in HGSNAT causes abnormal accumulation of HS within the lysosomes and leads to an autosomal recessive neurodegenerative lysosomal storage disorder called mucopolysaccharidosis IIIC (MPS IIIC). There are no approved drugs or treatment strategies to cure or manage the symptoms of, MPS IIIC. Here, we use cryo-electron microscopy (cryo-EM) to determine a high-resolution structure of the HGSNAT-acetyl-CoA complex, the first step in the HGSNAT-catalyzed acetyltransferase reaction. In addition, we map the known MPS IIIC mutations onto the structure and elucidate the molecular basis for mutation-induced HGSNAT dysfunction.
Assuntos
Microscopia Crioeletrônica , Humanos , Acetiltransferases/metabolismo , Acetiltransferases/química , Acetiltransferases/genética , Conformação Proteica , Lisossomos/enzimologia , Acetilação , MutaçãoRESUMO
ELO-like elongase is a condensing enzyme elongating long chain fatty acids in eukaryotes. Eranthis hyemalis ELO-like elongase (EhELO1) is the first higher plant ELO-type elongase that is highly active in elongating a wide range of polyunsaturated fatty acids (PUFAs) and some monounsaturated fatty acids (MUFAs). This study attempted using domain swapping and site-directed mutagenesis of EhELO1 and EhELO2, a close homologue of EhELO1 but with no apparent elongase activity, to elucidate the structural determinants critical for catalytic activity and substrate specificity. Domain swapping analysis of the two showed that subdomain B in the C-terminal half of EhELO1 is essential for MUFA elongation while subdomain C in the C-terminal half of EhELO1 is essential for both PUFA and MUFA elongations, implying these regions are critical in defining the architecture of the substrate tunnel for substrate specificity. Site-directed mutagenesis showed that the glycine at position 220 in the subdomain C plays a key role in differentiating the function of the two elongases. In addition, valine at 161 and cysteine at 165 in subdomain A also play critical roles in defining the architecture of the deep substrate tunnel, thereby contributing significantly to the acceptance of, and interaction with primer substrates.
Assuntos
Acetiltransferases , Elongases de Ácidos Graxos , Mutagênese Sítio-Dirigida , Elongases de Ácidos Graxos/metabolismo , Elongases de Ácidos Graxos/genética , Especificidade por Substrato , Acetiltransferases/metabolismo , Acetiltransferases/genética , Acetiltransferases/química , Ácidos Graxos Insaturados/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Sequência de Aminoácidos , Ácidos Graxos/metabolismo , Modelos MolecularesRESUMO
Oil seeds now make up the world's second-largest food source after cereals. In recent years, the medicinal- oil plant Camelina sativa has attracted much attention for its high levels of unsaturated fatty acids and low levels of saturated fatty acids as well as its resistance to abiotic stresses. Improvement of oil quality is considered an important trait in this plant. Erucic acid is one of the fatty acids affecting the quality of camelina oil. Altering the fatty acid composition in camelina oil through genetic manipulation requires the identification, isolation, and cloning of genes involved in fatty acid biosynthesis. The Fatty Acid Elongase 1 (FAE1) gene encodes the enzyme ß-ketoacyl CoA synthase (KCS), a crucial enzyme in the biosynthesis of erucic acid. In this study, the isolation and cloning of the FAE1 gene from Camelina sativa were conducted to construct an antisense structure. The molecular homology modeling of DFAE1 proteins using the SWISS-MODEL server on ExPASy led to the generation of the 3D structures of FAE1 and DFAE1 proteins. The GMQE values of 0.44 for FAE1 and 0.08 for DFAE1 suggest high accuracy in the structural estimation of these genes. The fragments were isolated from the DNA source of the genomic Soheil cultivar with an erucic acid content of about 3% (in matured seeds) using PCR. After cloning the FAE1 gene into the Bluescript II SK+ vector and sequencing, the resulting fragments were utilized to construct the antisense structure in the pBI121 plant expression vector. The approved antisense structure was introduced into the Camelina plant using the Agrobacterium-mediated method, with optimization of tissue culture and gene transfer conditions. This approach holds potential to advance our knowledge of fat biosynthesis, leading to potential improvements in oil quality in Camelina sativa.
Assuntos
Brassicaceae , Clonagem Molecular , Ácidos Erúcicos , Elongases de Ácidos Graxos , Brassicaceae/genética , Brassicaceae/metabolismo , Clonagem Molecular/métodos , Ácidos Erúcicos/metabolismo , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Sequência de Aminoácidos , Sementes/genética , Sementes/metabolismo , Modelos Moleculares , Regulação da Expressão Gênica de Plantas , Acetiltransferases/genética , Acetiltransferases/metabolismo , Genes de PlantasRESUMO
Sorafenib, an anticancer drug, has been shown to induce ferroptosis in cancer cells. However, resistance to sorafenib greatly limits its therapeutic efficacy, and the exact mechanism of resistance is not fully understood. This study investigated the role of N-Acetyltransferase 10 (NAT10) in influencing the anticancer activity of sorafenib in nasopharyngeal carcinoma (NPC) and its molecular mechanism. NAT10 expression was significantly upregulated in NPC. Mechanistically, NAT10 promotes proteins of solute carrier family 7 member 11 (SLC7A11) expression through ac4C acetylation, inhibiting sorafenib-induced ferroptosis in NPC cells. The combined application of sorafenib and the NAT10 inhibitor remodelin significantly inhibits SLC7A11 expression and promotes ferroptosis in NPC cells. In vivo knockout of NAT10 inhibited the growth of sorafenib-resistant NPC. Our findings suggest that NAT10 inhibition might be a promising therapeutic approach to enhance the anticancer activity of sorafenib.
Assuntos
Sistema y+ de Transporte de Aminoácidos , Resistencia a Medicamentos Antineoplásicos , Ferroptose , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Sorafenibe , Sorafenibe/farmacologia , Humanos , Carcinoma Nasofaríngeo/tratamento farmacológico , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patologia , Carcinoma Nasofaríngeo/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/genética , Animais , Camundongos , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Antineoplásicos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Acetiltransferases/metabolismo , Acetiltransferases/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos Nus , Masculino , Acetilação/efeitos dos fármacos , FemininoRESUMO
Long-chain polyunsaturated fatty acids (LCPUFAs) are essential for both fetal and placental development. We characterized the FA composition and gene expression levels of FA-metabolizing enzymes in rabbit placentas. Total FA compositions from term rabbit placentas (n = 7), livers, and plasma (both n = 4) were examined: among LCPUFAs with more than three double bonds, dihomo-γ-linolenic acid (DGLA) was the most abundant (11.4 ± 0.69 %, mean ± SE), while arachidonic acid was the second-most rich component (6.90 ± 0.56 %). DGLA was barely detectable (<1 %) in livers and plasma from term rabbits, which was significantly lower than in placentas (both p < 0.0001). Compared with the liver, transcript levels of the LCPUFA-metabolizing enzymes FADS2 and ELOVL5 were 7- and 4.5-fold higher in placentas (both p < 0.05), but levels of FADS1 and ELOVL2 were significantly lower (both p < 0.01). Our results suggest a placenta-specific enzyme expression pattern and LCPUFA profile in term rabbits, which may support a healthy pregnancy.
Assuntos
Ácido 8,11,14-Eicosatrienoico , Dessaturase de Ácido Graxo Delta-5 , Ácidos Graxos Dessaturases , Placenta , Animais , Coelhos , Feminino , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Dessaturases/genética , Gravidez , Placenta/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Elongases de Ácidos Graxos/metabolismo , Elongases de Ácidos Graxos/genética , Fígado/metabolismo , Ácido Araquidônico/metabolismo , Acetiltransferases/metabolismo , Acetiltransferases/genéticaRESUMO
The present study focused on exploring the clinical value and molecular mechanism of LncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) in sepsis and sepsis-induced myocardial dysfunction (SIMD). 122 sepsis patients and 90 healthy were included. Sepsis patients were categorized into SIMD and non-MD. The expression levels of MCM3AP-AS1 and miRNA were examined using RT-qPCR. Diagnostic value of MCM3AP-AS1 in sepsis assessed by ROC curves. Logistic regression to explore risk factors influencing the occurrence of SIMD. Cardiomyocytes were induced by LPS to construct cell models in vitro. CCK-8, flow cytometry, and ELISA to analyze cell viability, apoptosis, and inflammation levels. Serum MCM3AP-AS1 was upregulated in patients with sepsis. The sensitivity and specificity of MCM3AP-AS1 were 75.41% and 93.33%, for recognizing sepsis from healthy controls. Additionally, elevated MCM3AP-AS1 is a risk factor for SIMD and can predict SIMD development. Compared with the LPS-induced cardiomyocytes, inhibition of MCM3AP-AS1 significantly attenuated LPS-induced apoptosis and inflammation; however, this attenuation was partially reversed by lowered miR-28-5p, but this reversal was partially eliminated by CASP2. MCM3AP-AS1 may be a novel diagnostic biomarker for sepsis and can predict the development of SIMD. MCM3AP-AS1 probably participated in SIMD progression by regulating cardiomyocyte inflammation and apoptosis through the target miR-28-5p/CASP2 axis.
Assuntos
Apoptose , Miócitos Cardíacos , RNA Longo não Codificante , Sepse , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Acetiltransferases , Biomarcadores/sangue , Cardiomiopatias/diagnóstico , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intracelular , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/sangue , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Valor Preditivo dos Testes , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/sangue , Sepse/diagnóstico , Sepse/complicações , Transdução de SinaisRESUMO
BACKGROUND: The human SAT1 gene encodes spermidine/spermine N1-acetyltransferase 1 (SSAT1), a regulatory biological catalyst of polyamine catabolism. Numerous essential biological processes, such as cellular proliferation, differentiation, and survival, depend on polyamines like spermidine and spermine. Thus, SSAT1 is involved in key cellular activities such as proliferation and survival of cells and mediates various diseases including cancer. A plethora of studies established the involvement of missense single nucleotide polymorphisms (SNPs) in numerous pathological conditions due to their ability to adversely affect the structure and subsequent function of the protein. AIMS: To date, an in silico study to identify the pathogenic missense SNPs of the human SAT1 gene has not been accomplished yet. This study aimed to filter the missense SNPs that were functionally detrimental and pathogenic. METHODS AND RESULTS: The rs757435207 (I21N) was ascertained to be the most deleterious and pathogenic by all algorithmic tools. Stability and evolutionary conservation analysis tools also stated that I21N variant decreased the stability and was located in the highly conserved residue. Molecular dynamics simulation revealed that I21N caused substantial alterations in the conformational stability and dynamics of the SSAT1 protein. Consequently, the I21N variant could disrupt the native functional roles of the SSAT1 enzyme. CONCLUSION: Therefore, the I21N variant was identified and concluded to be an oncogenic missense variant of the human SAT1 gene. Overall, the findings of this study would be a great directory of future experimental research to develop personalized medicine.
Assuntos
Acetiltransferases , Algoritmos , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Humanos , Acetiltransferases/genética , Neoplasias/genética , Biologia Computacional/métodosRESUMO
Aggressive triple-negative breast cancer (TNBC) still lacks approved targeted therapies, requiring more exploration of its underlying mechanisms. Previous studies have suggested a potential role of SAT1 (Spermidine/Spermine N1-acetyltransferase 1) in cancer, which needs to be further elucidated in breast cancer. In this study, highly expressed SAT1 in TNBC signified worse patient prognoses. And SAT1 knockdown effectively inhibited the proliferation and migration abilities of TNBC cells in vitro and in vivo. In terms of mechanism, the transcription factor JUN enhanced SAT1 transcriptional activity by binding to its promoter region. Then, SAT1 protein in the cytoplasm engaged in directly binding with YBX1 for sustaining YBX1 protein stability via deubiquitylation mediated by the E3 ligase HERC5. Further, SAT1 was found to suppress autophagy remarkably via stabilization of mTOR mRNA with the accumulation of YBX1-mediated methyl-5-cytosine (m5C) modification. These findings proved that SAT1 drives TNBC progression through the SAT1/YBX1/mTOR axis, which may provide a potential candidate for targeted therapy in advanced TNBC.
Assuntos
Autofagia , Progressão da Doença , Neoplasias de Mama Triplo Negativas , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Humanos , Feminino , Autofagia/genética , Camundongos , Linhagem Celular Tumoral , Animais , Acetiltransferases/genética , Acetiltransferases/metabolismo , Modelos Animais de Doenças , Proliferação de Células/genética , Proteína 1 de Ligação a Y-Box/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Regulação Neoplásica da Expressão Gênica/genética , Camundongos NusRESUMO
Ferroptosis, an iron-dependent lipid peroxidation-driven cell death pathway, has been linked to the development of Alzheimer's disease (AD). However, the role of ferroptosis in the pathogenesis of AD remains unclear. Cerebroprotein hydrolysate-I (CH-I) is a mixture of peptides with neurotrophic effects that improves cognitive deficits and reduces amyloid burden. The present study investigated the ferroptosis-induced signalling pathways and the neuroprotective effects of CH-I in the brains of AD transgenic mice. Seven-month-old male APPswe/PS1dE9 (APP/PS1) transgenic mice were treated with intraperitoneal injections of CH-I and saline for 28 days. The Morris water maze test was used to assess cognitive function. CH-I significantly improved cognitive deficits and attenuated beta-amyloid (Aß) aggregation and tau phosphorylation in the hippocampus of APP/PS1 mice. RNA sequencing revealed that multiple genes and pathways, including ferroptosis-related pathways, were involved in the neuroprotective effects of CH-I. The increased levels of lipid peroxidation, ferrous ions, reactive oxygen species (ROS), and altered expression of ferroptosis-related genes (recombinant solute carrier family 7, member 11 (SLC7A11), spermidine/spermine N1-acetyltransferase 1 (SAT1) and glutathione peroxidase 4 (GPX4)) were significantly alleviated after CH-I treatment. Quantitative real-time PCR and western blotting were performed to investigate the expression of key ferroptosis-related genes and the p53/SAT1/arachidonic acid 15-lipoxygenase (ALOX15) signalling pathway. The p53/SAT1/ALOX15 signalling pathway was found to be involved in mediating ferroptosis, and the activation of this pathway was significantly suppressed in AD by CH-I. CH-I demonstrated neuroprotective effects against AD by attenuating ferroptosis and the p53/SAT1/ALOX15 signalling pathway, thus providing new targets for AD treatment.
Assuntos
Doença de Alzheimer , Araquidonato 15-Lipoxigenase , Disfunção Cognitiva , Ferroptose , Camundongos Transgênicos , Transdução de Sinais , Proteína Supressora de Tumor p53 , Animais , Ferroptose/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Transdução de Sinais/efeitos dos fármacos , Masculino , Camundongos , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Acetiltransferases/metabolismo , Acetiltransferases/genética , Modelos Animais de Doenças , Presenilina-1/genética , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Peptídeos beta-Amiloides/metabolismoRESUMO
Investigating and identifying pathogenic molecules of non-alcoholic fatty liver disease (NAFLD) has become imperative, which would serve as effective targets in the future. We established high-fat diet (HFD)-induced NAFLD model in mice and palmitic acid (PA)-induced model in mouse AML12 cells. The level of miR-218-5p was examined by qRT-PCR, and Elovl5 was identified as the potential target gene of miR-218-5p. The binding relationship between miR-218-5p and Elovl5 was validated by double luciferase reporter gene assay, and inhibition/overexpression of miR-218-5p in vitro. The functional mechanisms of miR-218-5p/Elovl5 in regulating lipogenesis in NAFLD were investigated in vivo and in vitro through gain- and loss-of-function studies. MiR-218-5p was significantly increased, and Elovl5 was decreased in model group. According to the double luciferase reporter and gene interference experiments in AML12 cells, Elovl5 was a target gene of miR-218-5p and its expression was regulated by miR-218-5p. The SREBP1-mediated lipogenesis signaling pathway regulated by Elovl5 was upregulated in model group. Moreover, silencing of miR-218-5p significantly upregulated Elovl5 expression, and suppressed SREBP1 signaling pathway in PA-induced AML-12 cells. Correspondingly, the cell injury, elevated TC, TG contents and lipid droplet accumulation were ameliorated. Furthermore, the effect of miR-218-5p on lipogenesis in vitro and in vivo was obstructed by si-Elovl5, implicating that miR-218-5p promotes lipogenesis by targeting ELOVL5 in NAFLD. miR-218-5p could promote fatty acid synthesis by targeting Elovl5, thereby accelerating the development of NAFLD, which is one of the key pathogenic mechanisms of NAFLD and provides a new molecular target for the management of NAFLD.
Assuntos
Elongases de Ácidos Graxos , Lipogênese , Camundongos Endogâmicos C57BL , MicroRNAs , Hepatopatia Gordurosa não Alcoólica , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Lipogênese/genética , Lipogênese/fisiologia , Camundongos , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Masculino , Dieta Hiperlipídica/efeitos adversos , Fígado/metabolismo , Fígado/patologia , Linhagem Celular , Acetiltransferases/genética , Acetiltransferases/metabolismoRESUMO
MPS IIIC is a lysosomal storage disease caused by mutations in heparan-α-glucosaminide N-acetyltransferase (HGSNAT), for which no treatment is available. Because HGSNAT is a trans-lysosomal-membrane protein, gene therapy for MPS IIIC needs to transduce as many cells as possible for maximal benefits. All cells continuously release extracellular vesicles (EVs) and communicate by exchanging biomolecules via EV trafficking. To address the unmet need, we developed a rAAV-hHGSNATEV vector with an EV-mRNA-packaging signal in the 3'UTR to facilitate bystander effects, and tested it in an in vitro MPS IIIC model. In human MPS IIIC cells, rAAV-hHGSNATEV enhanced HGSNAT mRNA and protein expression, EV-hHGSNAT-mRNA packaging, and cleared GAG storage. Importantly, incubation with EVs led to hHGSNAT protein expression and GAG contents clearance in recipient MPS IIIC cells. Further, rAAV-hHGSNATEV transduction led to the reduction of pathological EVs in MPS IIIC cells to normal levels, suggesting broader therapeutic benefits. These data demonstrate that incorporating the EV-mRNA-packaging signal into a rAAV-hHGSNAT vector enhances EV packaging of hHGSNAT-mRNA, which can be transported to non-transduced cells and translated into functional rHGSNAT protein, facilitating cross-correction of disease pathology. This study supports the therapeutic potential of rAAVEV for MPS IIIC, and broad diseases, without having to transduce every cell.
Assuntos
Efeito Espectador , Dependovirus , Vesículas Extracelulares , Terapia Genética , RNA Mensageiro , Humanos , Terapia Genética/métodos , Dependovirus/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Vesículas Extracelulares/metabolismo , Mucopolissacaridose III/terapia , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/genética , Vetores Genéticos , Acetiltransferases/metabolismo , Acetiltransferases/genéticaRESUMO
Dexmedetomidine (Dex) is widely used in the sedation in intensive care units and as an anesthetic adjunct. Considering the anti-inflammatory and antioxidant properties of Dex, we applied in vivo rat model as well as in vitro cardiomyocyte models (embryonic rat cardiomyocytes H9c2 cells and neonatal rat cardiomyocytes, NRCMs) to evaluate the effects of Dex against myocardial ischemia reperfusion (I/R) injury. Transcriptomic sequencing for gene expression in heart tissues from control rats and Dex-treated rats identified that genes related to fatty acid metabolism were significantly regulated by Dex. Among these genes, the elongation of long-chain fatty acids (ELOVL) family member 6 (Elovl6) was most increased upon Dex-treatment. By comparing the effects of Dex on both wild type and Elovl6-knockdown H9c2 cells and NRCMs under oxygen-glucose deprivation/reoxygenation (OGD/R) challenge, we found that Elovl6 knockdown attenuated the protection efficiency of Dex, which was supported by the cytotoxicity endpoints (cell viability and lactate dehydrogenase release) and apoptosis as well as key gene expressions. These results indicate that Dex exhibited the protective function against myocardial I/R injury via fatty acid metabolism pathways and Elovl6 plays a key role in the process, which was further confirmed using palmitate exposure in both cells, as well as in an in vivo rat model. Overall, this study systematically evaluates the protective effects of Dex on the myocardial I/R injury and provides better understanding on the fatty acid metabolism underlying the beneficial effects of Dex.
Assuntos
Apoptose , Dexmedetomidina , Elongases de Ácidos Graxos , Ácidos Graxos , Traumatismo por Reperfusão Miocárdica , Miócitos Cardíacos , Animais , Dexmedetomidina/farmacologia , Dexmedetomidina/uso terapêutico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Ratos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ácidos Graxos/metabolismo , Masculino , Linhagem Celular , Apoptose/efeitos dos fármacos , Ratos Sprague-Dawley , Acetiltransferases/metabolismo , Acetiltransferases/genética , Sobrevivência Celular/efeitos dos fármacosRESUMO
BACKGROUND: Accumulating studies suggested that posttranscriptional modifications exert a vital role in the tumorigenesis of diffuse large B-cell lymphoma (DLBCL). N4-acetylcytidine (ac4C) modification, catalyzed by the N-acetyltransferase 10 (NAT10), was a novel type of chemical modification that improves translation efficiency and mRNA stability. METHODS: GEO databases and clinical samples were used to explore the expression and clinical value of NAT10 in DLBCL. CRISPER/Cas9-mediated knockout of NAT10 was performed to determine the biological functions of NAT10 in DLBCL. RNA sequencing, acetylated RNA immunoprecipitation sequencing (acRIP-seq), LC-MS/MS, RNA immunoprecipitation (RIP)-qPCR and RNA stability assays were performed to explore the mechanism by which NAT10 contributed to DLBCL progression. RESULTS: Here, we demonstrated that NAT10-mediated ac4C modification regulated the occurrence and progression of DLBCL. Dysregulated N-acetyltransferases expression was found in DLBCL samples. High expression of NAT10 was associated with poor prognosis of DLBCL patients. Deletion of NAT10 expression inhibited cell proliferation and induced G0/G1 phase arrest. Furthermore, knockout of NAT10 increased the sensitivity of DLBCL cells to ibrutinib. AcRIP-seq identified solute carrier family 30 member 9 (SLC30A9) as a downstream target of NAT10 in DLBCL. NAT10 regulated the mRNA stability of SLC30A9 in an ac4C-dependent manner. Genetic silencing of SLC30A9 suppressed DLBCL cell growth via regulating the activation of AMP-activated protein kinase (AMPK) pathway. CONCLUSION: Collectively, these findings highlighted the essential role of ac4C RNA modification mediated by NAT10 in DLBCL, and provided insights into novel epigenetic-based therapeutic strategies.
Assuntos
Linfoma Difuso de Grandes Células B , Humanos , Acetiltransferases/genética , Acetiltransferases/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Citidina/análogos & derivados , Citidina/farmacologia , Citidina/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Acetiltransferases N-Terminal , Transdução de Sinais/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
N4-acetylcytidine (ac4C), a conserved but recently rediscovered RNA modification on tRNAs, rRNAs and mRNAs, is catalyzed by N-acetyltransferase 10 (NAT10). Lysine acylation is a ubiquitous protein modification that controls protein functions. Our latest study demonstrates a NAT10-dependent ac4C modification, which occurs on the polyadenylated nuclear RNA (PAN) encoded by oncogenic DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV), can induce KSHV reactivation from latency and activate inflammasome. However, it remains unclear whether a novel lysine acylation occurs in NAT10 during KSHV reactivation and how this acylation of NAT10 regulates tRNAs ac4C modification. Here, we showed that NAT10 was lactylated by α-tubulin acetyltransferase 1 (ATAT1), as a writer at the critical domain, to exert RNA acetyltransferase function and thus increase the ac4C level of tRNASer-CGA-1-1. Mutagenesis at the ac4C site in tRNASer-CGA-1-1 inhibited its ac4C modifications, translation efficiency of viral lytic genes, and virion production. Mechanistically, KSHV PAN orchestrated NAT10 and ATAT1 to enhance NAT10 lactylation, resulting in tRNASer-CGA-1-1 ac4C modification, eventually boosting KSHV reactivation. Our findings reveal a novel post-translational modification in NAT10, as well as expand the understanding about tRNA-related ac4C modification during KSHV replication, which may be exploited to design therapeutic strategies for KSHV-related diseases.
Assuntos
Acetiltransferases , Citidina , Herpesvirus Humano 8 , Ativação Viral , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Humanos , Acetiltransferases/metabolismo , Acetiltransferases/genética , Citidina/análogos & derivados , Citidina/metabolismo , Células HEK293 , Acetiltransferases N-Terminal/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/genética , AcilaçãoRESUMO
Members of the domain of unknown function 231/trichome birefringence-like (TBL) family have been shown to be O-acetyltransferases catalyzing the acetylation of plant cell wall polysaccharides, including pectins, mannan, xyloglucan and xylan. However, little is known about the origin and evolution of plant cell wall polysaccharide acetyltransferases. Here, we investigated the biochemical functions of TBL homologs from Klebsormidium nitens, a representative of an early divergent class of charophyte green algae that are considered to be the closest living relatives of land plants, and Marchantia polymorpha, a liverwort that is an extant representative of an ancient lineage of land plants. The genomes of K. nitens and Marchantia polymorpha harbor two and six TBL homologs, respectively. Biochemical characterization of their recombinant proteins expressed in human embryonic kidney 293 cells demonstrated that the two K. nitens TBLs exhibited acetyltransferase activities acetylating the pectin homogalacturonan (HG) and hence were named KnPOAT1 and KnPOAT2. Among the six M. polymorpha TBLs, five (MpPOAT1 to 5) possessed acetyltransferase activities toward pectins and the remaining one (MpMOAT1) catalyzed 2-O- and 3-O-acetylation of mannan. While MpPOAT1,2 specifically acetylated HG, MpPOAT3,4,5 could acetylate both HG and rhamnogalacturonan-I. Consistent with the acetyltransferase activities of these TBLs, pectins isolated from K. nitens and both pectins and mannan from M. polymorpha were shown to be acetylated. These findings indicate that the TBL genes were recruited as cell wall polysaccharide O-acetyltransferases as early as in charophyte green algae with activities toward pectins and they underwent expansion and functional diversification to acetylate various cell wall polysaccharides during evolution of land plants.