RESUMEN
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.
Asunto(s)
Brassicaceae , Clonación Molecular , Ácidos Erucicos , Elongasas de Ácidos Grasos , Brassicaceae/genética , Brassicaceae/metabolismo , Clonación Molecular/métodos , Ácidos Erucicos/metabolismo , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Secuencia de Aminoácidos , Semillas/genética , Semillas/metabolismo , Modelos Moleculares , Regulación de la Expresión Génica de las Plantas , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Genes de PlantasRESUMEN
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.
Asunto(s)
Linfoma de Células B Grandes Difuso , Humanos , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Carcinogénesis/genética , Carcinogénesis/metabolismo , Línea Celular Tumoral , Citidina/análogos & derivados , Citidina/farmacología , Citidina/metabolismo , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Acetiltransferasas N-Terminal , Transducción de Señal/genética , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
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.
Asunto(s)
Elongasas de Ácidos Grasos , Lipogénesis , Ratones Endogámicos C57BL , MicroARNs , Enfermedad del Hígado Graso no Alcohólico , Animales , MicroARNs/genética , MicroARNs/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Lipogénesis/genética , Lipogénesis/fisiología , Ratones , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Masculino , Dieta Alta en Grasa/efectos adversos , Hígado/metabolismo , Hígado/patología , Línea Celular , Acetiltransferasas/genética , Acetiltransferasas/metabolismoRESUMEN
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.
Asunto(s)
Efecto Espectador , Dependovirus , Vesículas Extracelulares , Terapia Genética , ARN Mensajero , Humanos , Terapia Genética/métodos , Dependovirus/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Vesículas Extracelulares/metabolismo , Mucopolisacaridosis III/terapia , Mucopolisacaridosis III/metabolismo , Mucopolisacaridosis III/genética , Vectores Genéticos , Acetiltransferasas/metabolismo , Acetiltransferasas/genéticaRESUMEN
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.
Asunto(s)
Apoptosis , Dexmedetomidina , Elongasas de Ácidos Grasos , Ácidos Grasos , Daño por Reperfusión Miocárdica , Miocitos Cardíacos , Animales , Dexmedetomidina/farmacología , Dexmedetomidina/uso terapéutico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/metabolismo , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Ratas , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Ácidos Grasos/metabolismo , Masculino , Línea Celular , Apoptosis/efectos de los fármacos , Ratas Sprague-Dawley , Acetiltransferasas/metabolismo , Acetiltransferasas/genética , Supervivencia Celular/efectos de los fármacosRESUMEN
Baccatin III is a crucial precursor in the biosynthesis pathway of paclitaxel. Its main sources are extraction from Taxus or chemical synthesis using 10-deacetylbaccatin III (10-DAB) as substrate. However, these preparation approaches exhibit serious limitations, including the low content of baccatin III in Taxus and the complicated steps of chemical synthesis. Heterologous expression of 10-deacetylbaccatin III-10-O-acetyltransferase (TcDBAT) in microbial strains for biotransformation of 10-DAB is a promising alternative strategy for baccatin III production. Here, the promotion effects of glycerol supply and slightly acidic conditions with a low-temperature on the catalysis of recombinant TcDBAT strain were clarified using 10-DAB as substrate. Taxus needles is renewable and the content of 10-DAB is relatively high, it can be used as an effective source of the catalytic substrate 10-DAB. Baccatin III was synthesized by integrating the extraction of 10-DAB from renewable Taxus needles and in situ whole-cell catalysis in this study. 40 g/L needles were converted into 20.66 mg/L baccatin III by optimizing and establishing a whole-cell catalytic bioprocess. The method used in this study can shorten the production process of Taxus extraction for baccatin III synthesis and provide a reliable strategy for the efficient production of baccatin III by recombinant strains and the improvement of resource utilization rate of Taxus needles.
Asunto(s)
Biotransformación , Taxoides , Taxus , Taxus/metabolismo , Taxus/química , Taxoides/metabolismo , Alcaloides/biosíntesis , Alcaloides/metabolismo , Alcaloides/química , Hojas de la Planta/metabolismo , Hojas de la Planta/química , Acetiltransferasas/metabolismo , Acetiltransferasas/genéticaRESUMEN
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which remains a significant global health challenge. The emergence of multidrug-resistant (MDR) Mtb strains imposes the development of new therapeutic strategies. This study focuses on the identification and evaluation of potential inhibitors against Mtb H37Ra through a comprehensive screening of an in-house chemolibrary. Subsequently, a promising pyrimidine derivative (LQM495) was identified as promising and then further investigated by experimental and in silico approaches. In this context, computational techniques were used to elucidate the potential molecular target underlying the inhibitory action of LQM495. Then, a consensus reverse docking (CRD) protocol was used to investigate the interactions between this compound and several Mtb targets. Out of 98 Mtb targets investigated, the enhanced intracellular survival (Eis) protein emerged as a target for LQM495. To gain insights into the stability of the LQM495-Eis complex, molecular dynamics (MD) simulations were conducted over a 400 ns trajectory. Further insights into its binding modes within the Eis binding site were obtained through a Quantum mechanics (QM) approach, using density functional theory (DFT), with B3LYP/D3 basis set. These calculations shed light on the electronic properties and reactivity of LQM495. Subsequently, inhibition assays and kinetic studies of the Eis activity were used to investigate the activity of LQM495. Then, an IC50 value of 11.0 ± 1.4 µM was found for LQM495 upon Eis protein. Additionally, its Vmax, Km, and Ki parameters indicated that it is a competitive inhibitor. Lastly, this study presents LQM495 as a promising inhibitor of Mtb Eis protein, which could be further explored for developing novel anti-TB drugs in the future.
Asunto(s)
Antituberculosos , Proteínas Bacterianas , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Antituberculosos/farmacología , Antituberculosos/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Relación Estructura-Actividad , Pruebas de Sensibilidad Microbiana , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Estructura Molecular , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/metabolismo , Relación Dosis-Respuesta a Droga , Simulación de Dinámica Molecular , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinas/síntesis químicaRESUMEN
Upon infection by an intracellular pathogen, host cells activate apoptotic pathways to limit pathogen replication. Consequently, efficient proliferation of the obligate intracellular pathogen Chlamydia trachomatis, a major cause of trachoma and sexually transmitted diseases, depends on the suppression of host cell apoptosis. C. trachomatis secretes deubiquitinase ChlaDUB1 into the host cell, leading among other interactions to the stabilization of antiapoptotic proteins and, thus, suppression of host cell apoptosis. Targeting the bacterial effector protein may, therefore, lead to new therapeutic possibilities. To explore the active site of ChlaDUB1, an iterative cycle of computational docking, synthesis, and enzymatic screening was applied with the aim of lead structure development. Hereby, covalent inhibitors were developed, which show enhanced inhibition with a 22-fold increase in IC50 values compared to previous work. Comprehensive insights into the binding prerequisites to ChlaDUB1 are provided, establishing the foundation for an additional specific antichlamydial therapy by small molecules.
Asunto(s)
Chlamydia trachomatis , Diseño de Fármacos , Chlamydia trachomatis/efectos de los fármacos , Chlamydia trachomatis/enzimología , Relación Estructura-Actividad , Simulación del Acoplamiento Molecular , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/metabolismo , Humanos , Antibacterianos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Enzimas Desubicuitinizantes/antagonistas & inhibidores , Enzimas Desubicuitinizantes/metabolismo , Estructura Molecular , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismoRESUMEN
Ferroptosis, a type of iron-dependent non-apoptotic cell death, plays a vital role in both tumor proliferation and resistance to chemotherapy. Here, our study demonstrates that MAX's Next Tango (MNT), by involving itself in the spermidine/spermine N1-acetyltransferase 1 (SAT1)-related ferroptosis pathway, promotes the proliferation of lung adenocarcinoma (LUAD) cells and diminishes their sensitivity to chemotherapy. Initially, an RNA-sequence screen of LUAD cells treated with ferroptosis inducers (FINs) reveals a significant increase in MNT expression, suggesting a potential link between MNT and ferroptosis. Overexpression of MNT in LUAD cells hinders changes associated with ferroptosis. Moreover, the upregulation of MNT promotes cell proliferation and suppresses chemotherapy sensitivity, while the knockdown of MNT has the opposite effect. Through the intersection of ChIP-Seq and ferroptosis-associated gene sets, and validation by qPCR and western blot, SAT1 is identified as a potential target of MNT. Subsequently, we demonstrate that MNT binds to the promoter sequence of SAT1 and suppresses its transcription by ChIP-qPCR and dual luciferase assays. Restoration of SAT1 levels antagonizes the efficacy of MNT to inhibit ferroptosis and chemosensitivity and promote cell growth in vitro as well as in vivo. In the clinical context, MNT expression is elevated in LUAD and is inversely connected with SAT1 expression. High MNT expression is also associated with poor patient survival. Our research reveals that MNT inhibits ferroptosis, and impairing chemotherapy effectiveness of LUAD.
Asunto(s)
Acetiltransferasas , Adenocarcinoma del Pulmón , Ferroptosis , Neoplasias Pulmonares , Ferroptosis/genética , Ferroptosis/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/tratamiento farmacológico , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Ratones , Línea Celular Tumoral , Animales , Resistencia a Antineoplásicos/genética , Ratones Desnudos , Regulación Neoplásica de la Expresión Génica , Proliferación Celular , Antineoplásicos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Femenino , Ratones Endogámicos BALB C , MasculinoRESUMEN
The compound 15-deacetylcalonectrin (15-deCAL) is a common pathway intermediate in the biosynthesis of Fusarium trichothecenes. This tricyclic intermediate is metabolized to calonectrin (CAL) by trichothecene 15-O-acetyltransferase encoded by Tri3. Unlike other trichothecene pathway Tri gene mutants, the Δtri3 mutant produces lower amounts of the knocked-out enzyme's substrate 15-deCAL, and instead, accumulates higher quantities of earlier bicyclic intermediate and shunt metabolites. Furthermore, evolutionary studies suggest that Tri3 may play a role in shaping the chemotypes of trichothecene-producing Fusarium strains. To better understand the functional role of Tri3p in biosynthesis and evolution, we aimed to develop a method to produce 15-deCAL by using transgenic Fusarium graminearum strains derived from a trichothecene overproducer. Unfortunately, introducing mutant Tri3, encoding a catalytically impaired but structurally intact acetylase, did not improve the low 15-deCAL production level of the ΔFgtri3 deletion strain, and the bicyclic products continued to accumulate as the major metabolites of the active-site mutant. These findings are discussed in light of the enzyme responsible for 15-deCAL production in trichothecene biosynthesis machinery. To efficiently produce 15-deCAL, we tested an alternative strategy of using a CAL-overproducing transformant. By feeding a crude CAL extract to a Fusarium commune strain that was isolated in this study and capable of specifically deacetylating C-15 acetyl, 15-deCAL was efficiently recovered. The substrate produced in this manner can be used for kinetic investigations of this enzyme and its possible role in chemotype diversification.
Asunto(s)
Fusarium , Mutación , Tricotecenos , Fusarium/genética , Fusarium/metabolismo , Tricotecenos/metabolismo , Acetiltransferasas/metabolismo , Acetiltransferasas/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Vías Biosintéticas/genéticaRESUMEN
Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.
Asunto(s)
Acetiltransferasas , Microscopía por Crioelectrón , Lisosomas , Mucopolisacaridosis III , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/metabolismo , Mucopolisacaridosis III/enzimología , Humanos , Lisosomas/metabolismo , Lisosomas/enzimología , Acetiltransferasas/metabolismo , Acetiltransferasas/química , Acetiltransferasas/genética , Dominio Catalítico , Mutación , Heparitina Sulfato/metabolismo , Acetilcoenzima A/metabolismo , Acetilcoenzima A/química , Modelos Moleculares , Glucosamina/metabolismo , Glucosamina/química , Acetilación , Membranas Intracelulares/metabolismoRESUMEN
Microtubule acetylation has been shown to regulate actin filament dynamics by modulating signaling pathways that control actin organization, although the precise mechanisms remain unknown. In this study, we found that the downregulation of microtubule acetylation via the disruption ATAT1 (which encodes α-tubulin N-acetyltransferase 1) inhibited the expression of RhoA, a small GTPase involved in regulating the organization of actin filaments and the formation of stress fibers. Analysis of RHOA promoter and chromatin immunoprecipitation assays revealed that C/EBPß is a major regulator of RHOA expression. Interestingly, the majority of C/EBPß in ATAT1 knockout (KO) cells was found in the nucleus as a 27-kDa fragment (referred to as C/EBPßp27) lacking the N-terminus of C/EBPß. Overexpression of a gene encoding a C/EBPßp27-mimicking protein via an N-terminal deletion in C/EBPß led to competitive binding with wild-type C/EBPß at the C/EBPß binding site in the RHOA promoter, resulting in a significant decrease of RHOA expression. We also found that cathepsin L (CTSL), which is overexpressed in ATAT1 KO cells, is responsible for C/EBPßp27 formation in the nucleus. Treatment with a CTSL inhibitor led to the restoration of RHOA expression by downregulation of C/EBPßp27 and the invasive ability of ATAT1 KO MDA-MB-231 breast cancer cells. Collectively, our findings suggest that the downregulation of microtubule acetylation associated with ATAT1 deficiency suppresses RHOA expression by forming C/EBPßp27 in the nucleus through CTSL. We propose that CTSL and C/EBPßp27 may represent a novel therapeutic target for breast cancer treatment. [BMB Reports 2024; 57(6): 293-298].
Asunto(s)
Acetiltransferasas , Proteína beta Potenciadora de Unión a CCAAT , Proteína de Unión al GTP rhoA , Humanos , Acetilación , Acetiltransferasas/metabolismo , Acetiltransferasas/genética , Catepsina L/metabolismo , Catepsina L/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Línea Celular Tumoral , Regulación hacia Abajo , Microtúbulos/metabolismo , Regiones Promotoras Genéticas/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteínas de Microtúbulos/genética , Proteínas de Microtúbulos/metabolismoRESUMEN
Elongation of very long-chain fatty acid (Elovl) proteins plays pivotal functions in the biosynthesis of the physiologically essential long-chain polyunsaturated fatty acids (LC-PUFA). Polychaetes have important roles in marine ecosystems, contributing not only to nutrient recycling but also exhibiting a distinctive capacity for biosynthesizing LC-PUFA. To expand our understanding of the LC-PUFA biosynthesis in polychaetes, this study conducted a thorough molecular and functional characterization of Elovl occurring in the model organism Platynereis dumerilii. We identify six Elovl in the genome of P. dumerilii. The sequence and phylogenetic analyses established that four Elovl, identified as Elovl2/5, Elovl4 (two genes) and Elovl1/7, have putative functions in LC-PUFA biosynthesis. Functional characterization confirmed the roles of these elongases in LC-PUFA biosynthesis, demonstrating that P. dumerilii possesses a varied and functionally diverse complement of Elovl that, along with the enzymatic specificities of previously characterized desaturases, enables P. dumerilii to perform all the reactions required for the biosynthesis of the LC-PUFA. Importantly, we uncovered that one of the two Elovl4-encoding genes is remarkably long in comparison with any other animals' Elovl, which contains a C terminal KH domain unique among Elovl. The distinctive expression pattern of this protein in photoreceptors strongly suggests a central role in vision.
Asunto(s)
Elongasas de Ácidos Grasos , Ácidos Grasos Insaturados , Filogenia , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Animales , Elongasas de Ácidos Grasos/metabolismo , Elongasas de Ácidos Grasos/genética , Poliquetos/metabolismo , Poliquetos/genética , Acetiltransferasas/metabolismo , Acetiltransferasas/genética , Anélidos/genética , Anélidos/metabolismoRESUMEN
KEY MESSAGE: Identification of selenium stress-responsive expression and molecular docking of serine acetyltransferase (SAT) and O-acetyl serine (thiol) lyase (OASTL) in Cardamine hupingshanensis. A complex coupled with serine acetyltransferase (SAT) and O-acetyl serine (thiol) lyase (OASTL) is the key enzyme that catalyzes selenocysteine (Sec) synthesis in plants. The functions of SAT and OASTL genes were identified in some plants, but it is still unclear whether SAT and OASTL are involved in the selenium metabolic pathway in Cardamine hupingshanensis. In this study, genome-wide identification and comparative analysis of ChSATs and ChOASTLs were performed. The eight ChSAT genes were divided into three branches, and the thirteen ChOASTL genes were divided into four branches by phylogenetic analysis and sequence alignment, indicating the evolutionary conservation of the gene structure and its association with other plant species. qRT-PCR analysis showed that the ChSAT and ChOASTL genes were differentially expressed in different tissues under various selenium levels, suggesting their important roles in Sec synthesis. The ChSAT1;2 and ChOASTLA1;2 were silenced by the VIGS system to investigate their involvement in selenium metabolites in C. hupingshanensis. The findings contribute to understanding the gene functions of ChSATs and ChOASTLs in the selenium stress and provide a reference for further exploration of the selenium metabolic pathway in plants.
Asunto(s)
Cardamine , Regulación de la Expresión Génica de las Plantas , Simulación del Acoplamiento Molecular , Filogenia , Proteínas de Plantas , Selenio , Selenio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Cardamine/genética , Cardamine/metabolismo , Redes y Vías Metabólicas/genética , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Liasas/metabolismo , Liasas/genéticaRESUMEN
The biosynthetic capability of the long-chain polyunsaturated fatty acids (LC-PUFA) in teleosts are highly diversified due to evolutionary events such as gene loss and subsequent neo- and/or sub-functionalisation of enzymes encoded by existing genes. In the present study, we have comprehensively characterised genes potentially involved in LC-PUFA biosynthesis, namely one front-end desaturase (fads2) and eight fatty acid elongases (elovl1a, elovl1b, elovl4a, elovl4b, elovl5, elovl7, elovl8a and elovl8b) from an amphidromous teleost, Ayu sweetfish, Plecoglossus altivelis. Functional analysis confirmed Fads2 with Δ6, Δ5 and Δ8 desaturase activities towards multiple PUFA substrates and several Elovl enzymes exhibited elongation capacities towards C18-20 or C18-22 PUFA substrates. Consequently, P. altivelis possesses a complete enzymatic capability to synthesise physiologically important LC-PUFA including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) from their C18 precursors. Interestingly, the loss of elovl2 gene in P. altivelis was corroborated by genomic and phylogenetic analyses. However, this constraint would possibly be overcome by the function of alternative Elovl enzymes, such as Elovl1b, which has not hitherto been functionally characterised in teleosts. The present study contributes novel insights into LC-PUFA biosynthesis in the relatively understudied teleost group, Osmeriformes (Stomiati), thereby enhancing our understanding of the complement of LC-PUFA biosynthetic genes within teleosts.
Asunto(s)
Ácido Graso Desaturasas , Elongasas de Ácidos Grasos , Ácidos Grasos Insaturados , Osmeriformes , Animales , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Ácidos Grasos Insaturados/genética , Osmeriformes/metabolismo , Osmeriformes/genética , Ácido Graso Desaturasas/metabolismo , Ácido Graso Desaturasas/genética , Elongasas de Ácidos Grasos/metabolismo , Elongasas de Ácidos Grasos/genética , Filogenia , Proteínas de Peces/metabolismo , Proteínas de Peces/genética , Vías Biosintéticas/genética , Acetiltransferasas/metabolismo , Acetiltransferasas/genéticaRESUMEN
Agmatine N-acetyltransferase (AgmNAT), which catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine, is a member of the GCN5-related N-acetyltransferase family. So far, knowledge of the physiological roles of AgmNAT in insects is limited. Here, we identified one gene encoding protein homologous to that of Drosophila AgmNAT using sequence information from an activity-verified Drosophila AgmNAT in a BLAST search of the Bactrocera dorsalis genome. We expressed and purified B. dorsalis AgmNAT in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Our application of the screening strategy to BdorAgmNAT led to the identification of agmatine as the best amine substrate for this enzyme, with the highest kcat/Km value. We successfully obtained a BdorAgmNAT knockout strain based on a wild-type strain (WT) using the CRISPR/Cas9 technique. The ovary development of the BdorAgmNAT knockout mutants was delayed for 10 days compared with the WT specimens. Moreover, mutants had a much smaller mature ovary size and laid far fewer eggs than WT. Loss of function of BdorAgmNAT caused by RNAi with mature WT females did not affect their fecundity. These findings indicate that BdorAgmNAT is critical for oogenesis. Our data provide the first evidence for AgmNAT in regulating ovary development.
Asunto(s)
Acetiltransferasas , Ovario , Tephritidae , Animales , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Ovario/enzimología , Femenino , Tephritidae/genética , Tephritidae/enzimología , Tephritidae/crecimiento & desarrollo , Tephritidae/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Agmatina/metabolismoRESUMEN
In plant secondary metabolite biosynthesis, acylation is a diverse physiological process, with BAHD acyltransferases playing an essential role. Borneol acetyltransferase (BAT) is an alcohol acetyltransferase, which catalyzes borneol and acetyl-CoA to synthesize bornyl acetate (BA). However, the enzymes involved in the biosynthesis of BA have so far only been characterized in Wurfbainia villosa, the studies on the WvBATs have only been conducted in vitro, and the catalytic activity was relatively low. In this research, three genes (WlBAT1, WlBAT2, and WlBAT3) have been identified to encode BATs that are capable of acetylating borneol to synthesize BA in vitro. We also determined that WlBAT1 has the highest catalytic efficiency for borneol-type substrates, including (+)-borneol, (-)-borneol, and isoborneol. Furthermore, we found that BATs could catalyze a wide range of substrate types in vitro, but in vivo, they exclusively catalyzed borneol-type substrates. Through molecular simulations and site-directed mutagenesis, it was revealed that residues D32, N36, H168, N297, N355, and H384 are crucial for the catalytic activity of WlBAT1, while the R382I-D385R double mutant of WlBAT1 exhibited an increasing acylation efficiency for borneol-type substrates in vitro and in vivo. These findings offer key genetic elements for the metabolic engineering of plants and synthetic biology to produce BA.
Asunto(s)
Acetiltransferasas , Canfanos , Proteínas de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Acetiltransferasas/química , Canfanos/metabolismo , Canfanos/química , Biocatálisis , Especificidad por Sustrato , Cinética , Mutagénesis Sitio-DirigidaRESUMEN
Docosahexaenoic acid (DHA, 22:6n-3) must be consumed from the diet or synthesized from polyunsaturated fatty acid (PUFA) precursors, such as α-linolenic acid (ALA, 18:3n-3). Elongase 2 (encoded by Elovl2 gene) catalyzes two elongation reactions in the PUFA biosynthesis pathway and may be important in regulating the observed sex differences in n-3 PUFA levels. Our aim was to determine how targeted knockout of liver Elovl2 affects tissue and blood n-3 PUFA levels in male and female C57BL/6J mice. Twenty-eight-day old male and female liver Elovl2-KO and control mice were placed onto one of two dietary protocols for a total of 8 weeks (4-8 mice per genotype, per diet, per sex): 1) an 8-week 2 % ALA in total fat diet or 2) a 4-week 2 % ALA diet followed by a 4-week 2 % ALA + 2 % DHA diet. Following this 8-week feeding period, 12-week-old mice were sacrificed and serum, red blood cells (RBC), liver, heart and brain were collected and fatty acid levels measured. Significant interaction effects (p < 0.05, sex x genotype) for serum, RBC, liver and heart DHA levels were identified. In serum and liver, DHA levels were significantly different (p < 0.01) between all groups with male controls > female controls > female KO > male KO in serum and female controls > male controls > female KO > male KO in liver. In RBCs and the heart, female controls = male controls > female KO > male KO (p < 0.001). The addition of DHA to diet removed the interaction effects on DHA levels in the serum, liver and heart, yielding a significant sex effect in serum, liver (female > male, p < 0.01) and brain (male > female, p < 0.05) and genotype effect in serum and heart (control > KO, p < 0.05). Ablation of liver Elovl2 results in significantly lower blood and tissue DHA in a sex-dependent manner, suggesting a role for Elovl2 on sex differences in n-3 PUFA levels.
Asunto(s)
Acetiltransferasas , Ácidos Docosahexaenoicos , Elongasas de Ácidos Grasos , Hígado , Ratones Endogámicos C57BL , Ratones Noqueados , Ácido alfa-Linolénico , Animales , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Masculino , Femenino , Ácidos Docosahexaenoicos/metabolismo , Ácidos Docosahexaenoicos/sangre , Hígado/metabolismo , Ratones , Ácido alfa-Linolénico/metabolismo , Ácido alfa-Linolénico/administración & dosificación , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Ácidos Grasos Omega-3/sangre , Ácidos Grasos Omega-3/metabolismo , Caracteres Sexuales , Factores SexualesRESUMEN
Perinatal exposure to omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) can be characterized through biomarkers in maternal or cord blood or breast milk. Objectives were to describe perinatal PUFA status combining multiple biofluids and to investigate how it was influenced by dietary intake during pregnancy and maternal FADS and ELOVL gene polymorphisms. This study involved 1,901 mother-child pairs from the EDEN cohort, with PUFA levels measured in maternal and cord erythrocytes, and colostrum. Maternal dietary PUFA intake during the last trimester was derived from a food frequency questionnaire. Twelve single-nucleotide polymorphisms in FADS and ELOVL genes were genotyped from maternal DNA. Principal component analysis incorporating PUFA levels from the three biofluids identified patterns of perinatal PUFA status. Spearman's correlations explored associations between patterns and PUFA dietary intake, and linear regression models examined pattern associations with FADS or ELOVL haplotypes. Five patterns were retained: "High omega-3 LC-PUFAs, low omega-6 LC-PUFAs"; "Omega-6 LC-PUFAs"; "Colostrum LC-PUFAs"; "Omega-6 precursor (LA) and DGLA"; "Omega-6 precursor and colostrum ALA". Maternal omega-3 LC-PUFA intakes were correlated with "High omega-3 LC-PUFAs, low omega-6 LC-PUFAs" (r(DHA) = 0.33) and "Omega-6 LC-PUFAs" (r(DHA) = -0.19) patterns. Strong associations were found between FADS haplotypes and PUFA patterns except for "High omega-3 LC-PUFAs, low omega-6 LC-PUFAs". Lack of genetic association with the "High omega-3 LC-PUFAs, low omega-6 LC-PUFAs" pattern, highly correlated with maternal omega-3 LC-PUFA intake, emphasizes the importance of adequate omega-3 LC-PUFA intake during pregnancy and lactation. This study offers a more comprehensive assessment of perinatal PUFA status and its determinants.
Asunto(s)
Ácido Graso Desaturasas , Ácidos Grasos Insaturados , Polimorfismo de Nucleótido Simple , Humanos , Femenino , Embarazo , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Adulto , Ácidos Grasos Insaturados/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Ácidos Grasos Omega-6/metabolismo , delta-5 Desaturasa de Ácido Graso , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-3/administración & dosificación , Dieta , Calostro/química , Calostro/metabolismo , Sangre Fetal/metabolismo , Sangre Fetal/química , Recién NacidoRESUMEN
Elongation of very long-chain fatty acids protein 6 (ELOVL6) plays a pivotal role in the synthesis of endogenous fatty acids, influencing energy balance and metabolic diseases. The primary objective of this study was to discover the molecular attributes and regulatory roles of ELOVL6 in male Nile tilapia, Oreochromis niloticus. The full-length cDNA of elovl6 was cloned from male Nile tilapia, and was determined to be 2255-bp long, including a 5'-untranslated region of 193 bp, a 3'-untranslated region of 1252 bp, and an open reading frame of 810 bp encoding 269 amino acids. The putative protein had typical features of ELOVL proteins. The transcript levels of elovl6 differed among various tissues and among fish fed with different dietary lipid sources. Knockdown of elovl6 in Nile tilapia using antisense RNA technology resulted in significant alterations in hepatic morphology, long-chain fatty acid synthesis, and fatty acid oxidation, and led to increased fat deposition in the liver and disrupted glucose/lipid metabolism. A comparative transcriptomic analysis (elovl6 knockdown vs. the negative control) identified 5877 differentially expressed genes with significant involvement in key signaling pathways including the peroxisome proliferator-activated receptor signaling pathway, fatty acid degradation, glycolysis/gluconeogenesis, and the insulin signaling pathway, all of which are crucial for lipid and glucose metabolism. qRT-PCR analyses verified the transcript levels of 13 differentially expressed genes within these pathways. Our findings indicate that elovl6 knockdown in male tilapia impedes oleic acid synthesis, culminating in aberrant nutrient metabolism.