RESUMEN
ß-Aspartyl compounds, such as ß-aspartyl hydroxamate (serine racemase inhibitor), ß-aspartyl-l-lysine (moisture retention), and ß-aspartyl-l-tryptophan (immunomodulator) are physiologically active compounds. There is limited literature on the development of effective methods of production of ß-aspartyl compounds. In this study, we describe the biochemical characterization of asparagine synthetase (AS) from Streptococcus thermophilus NBRC 13957 (StAS) and the enzymatic synthesis of ß-aspartyl compounds using StAS. Recombinant StAS was expressed in Escherichia coli BL21(DE3) and it displayed activity towards hydroxylamine, methylamine, ethylamine, and ammonia, as acceptors of the ß-aspartyl moiety. StAS exhibited higher activity toward hydroxylamine and ethylamine as acceptor substrates compared with the enzymes from Lactobacillus delbrueckii NBRC 13953, Lactobacillus reuteri NBRC 15892, and E. coli. The coupling of the synthesis of ß-aspartyl compounds by StAS with an ATP-regeneration system using polyphosphate kinase from Deinococcus proteoliticus NBRC 101906 displayed an approximately 2.5-fold increase in the production of ß-aspartylhydroxamate from 1.06 mM to 2.53 mM after a 76-h reaction.
Asunto(s)
Aspartatoamoníaco Ligasa , Escherichia coli , Proteínas Recombinantes , Streptococcus thermophilus , Streptococcus thermophilus/enzimología , Streptococcus thermophilus/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Aspartatoamoníaco Ligasa/metabolismo , Aspartatoamoníaco Ligasa/genética , Aspartatoamoníaco Ligasa/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Ácido Aspártico/metabolismo , Ácido Aspártico/biosíntesis , Especificidad por Sustrato , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/químicaRESUMEN
Uncoupling proteins (UCPs) form a distinct subfamily of the mitochondrial carrier family (MCF) SLC25. Four UCPs, DmUCP4A-C and DmUCP5, have been identified in Drosophila melanogaster on the basis of their sequence homology with mammalian UCP4 and UCP5. In a Parkinson's disease model, DmUCP4A showed a protective role against mitochondrial dysfunction, by increasing mitochondrial membrane potential and ATP synthesis. To date, DmUCP4A is still an orphan of a biochemical function, although its possible involvement in mitochondrial uncoupling has been ruled out. Here, we show that DmUCP4A expressed in bacteria and reconstituted in phospholipid vesicles catalyzes a unidirectional transport of aspartate, which is saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. Swelling experiments carried out in yeast mitochondria have demonstrated that the unidirectional transport of aspartate catalyzed by DmUCP4 is not proton-coupled. The biochemical function of DmUCP4A has been further confirmed in a yeast cell model, in which growth has required an efflux of aspartate from mitochondria. Notably, DmUCP4A is the first UCP4 homolog from any species to be biochemically characterized. In Drosophila melanogaster, DmUCP4A could be involved in the transport of aspartate from mitochondria to the cytosol, in which it could be used for protein and nucleotide synthesis, as well as in the biosynthesis of ß-alanine and N-acetylaspartate, which play key roles in signal transmission in the central nervous system.
Asunto(s)
Ácido Aspártico/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Desacopladoras Mitocondriales/genética , Proteínas Desacopladoras Mitocondriales/metabolismo , Animales , Ácido Aspártico/análogos & derivados , Ácido Aspártico/biosíntesis , Transporte Biológico Activo , Clonación Molecular , Citosol/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mitocondrias/metabolismo , beta-Alanina/biosíntesisRESUMEN
Homoserine dehydrogenase (HSD), encoded by the hom gene, is a key enzyme in the aspartate pathway, which reversibly catalyzes the conversion of l-aspartate ß-semialdehyde to l-homoserine (l-Hse), using either NAD(H) or NADP(H) as a coenzyme. In this work, we presented the first characterization of the HSD from the symbiotic Polynucleobacter necessaries subsp. necessarius (PnHSD) produced in Escherichia coli. Sequence analysis showed that PnHSD is an ACT domain-containing monofunctional HSD with 436 amnio acid residues. SDS-PAGE and Western blot demonstrated that PnHSD could be overexpressed in E. coli BL21(DE3) cell as a soluble form by using SUMO fusion technique. It could be purified to apparent homogeneity for biochemical characterization. Size-exclusion chromatography revealed that the purified PnHSD has a native molecular mass of â¼160 kDa, indicating a homotetrameric structure. The oxidation activity of PnHSD was studied in this work. Kinetic analysis revealed that PnHSD displayed an up to 1460-fold preference for NAD+ over NADP+, in contrast to its homologs. The purified PnHSD displayed maximal activity at 35 °C and pH 11. Similar to its NAD+-dependent homolog, neither NaCl and KCl activation nor L-Thr inhibition on the enzymatic activity of PnHSD was observed. These results will contribute to a better understanding of the coenzyme specificity of the HSD family and the aspartate pathway of P. necessarius.
Asunto(s)
Ácido Aspártico/biosíntesis , Proteínas Bacterianas/genética , Burkholderiaceae/enzimología , Homoserina Deshidrogenasa/genética , NAD/metabolismo , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Burkholderiaceae/química , Burkholderiaceae/genética , Cromatografía en Gel , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Euplotes/microbiología , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Homoserina/metabolismo , Homoserina Deshidrogenasa/biosíntesis , Homoserina Deshidrogenasa/aislamiento & purificación , Cinética , Peso Molecular , NADP/metabolismo , Multimerización de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Simbiosis/fisiologíaRESUMEN
Autophagy catabolizes cellular constituents to promote survival during nutrient deprivation. Yet, a metabolic comprehension of this recycling operation, despite its crucial importance, remains incomplete. Here, we uncover a specific metabolic function of autophagy that exquisitely adjusts cellular metabolism according to nitrogen availability in the budding yeast Saccharomyces cerevisiae. Autophagy enables metabolic plasticity to promote glutamate and aspartate synthesis, which empowers nitrogen-starved cells to replenish their nitrogen currency and sustain macromolecule synthesis. Our findings provide critical insights into the metabolic basis by which autophagy recycles cellular components and may also have important implications in understanding the role of autophagy in diseases such as cancer.
Asunto(s)
Ácido Aspártico/biosíntesis , Autofagia , Ácido Glutámico/biosíntesis , Nitrógeno/deficiencia , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Compuestos de Amonio/metabolismo , Autofagia/efectos de los fármacos , Glutamato-Sintasa (NADH)/metabolismo , Sustancias Macromoleculares/metabolismo , Modelos Biológicos , Mutación/genética , Ácidos Nucleicos/biosíntesis , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirolimus/farmacologíaRESUMEN
The bacterial pathogen Staphylococcus aureus is capable of infecting a broad spectrum of host tissues, in part due to flexibility of metabolic programs. S. aureus, like all organisms, requires essential biosynthetic intermediates to synthesize macromolecules. We therefore sought to determine the metabolic pathways contributing to synthesis of essential precursors during invasive S. aureus infection. We focused specifically on staphylococcal infection of bone, one of the most common sites of invasive S. aureus infection and a unique environment characterized by dynamic substrate accessibility, infection-induced hypoxia, and a metabolic profile skewed toward aerobic glycolysis. Using a murine model of osteomyelitis, we examined survival of S. aureus mutants deficient in central metabolic pathways, including glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, and amino acid synthesis/catabolism. Despite the high glycolytic demand of skeletal cells, we discovered that S. aureus requires glycolysis for survival in bone. Furthermore, the TCA cycle is dispensable for survival during osteomyelitis, and S. aureus instead has a critical need for anaplerosis. Bacterial synthesis of aspartate in particular is absolutely essential for staphylococcal survival in bone, despite the presence of an aspartate transporter, which we identified as GltT and confirmed biochemically. This dependence on endogenous aspartate synthesis derives from the presence of excess glutamate in infected tissue, which inhibits aspartate acquisition by S. aureus Together, these data elucidate the metabolic pathways required for staphylococcal infection within bone and demonstrate that the host nutrient milieu can determine essentiality of bacterial nutrient biosynthesis pathways despite the presence of dedicated transporters.
Asunto(s)
Ácido Aspártico/biosíntesis , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Nutrientes/metabolismo , Osteomielitis/metabolismo , Osteomielitis/microbiología , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/genéticaRESUMEN
Marked elevation in the brain concentration of N-acetyl-L-aspartate (NAA) is a characteristic feature of Canavan disease, a vacuolar leukodystrophy resulting from deficiency of the oligodendroglial NAA-cleaving enzyme aspartoacylase. We now demonstrate that inhibiting NAA synthesis by intracisternal administration of a locked nucleic acid antisense oligonucleotide to young-adult aspartoacylase-deficient mice reverses their pre-existing ataxia and diminishes cerebellar and thalamic vacuolation and Purkinje cell dendritic atrophy. Ann Neurol 2020;87:480-485.
Asunto(s)
Ácido Aspártico/análogos & derivados , Enfermedad de Canavan/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Acetiltransferasas/antagonistas & inhibidores , Amidohidrolasas/deficiencia , Amidohidrolasas/genética , Animales , Ácido Aspártico/biosíntesis , Ataxia/complicaciones , Ataxia/tratamiento farmacológico , Atrofia/complicaciones , Atrofia/tratamiento farmacológico , Enfermedad de Canavan/complicaciones , Enfermedad de Canavan/patología , Cerebelo/patología , Femenino , Técnicas de Silenciamiento del Gen , Infusiones Intraventriculares , Masculino , Ratones , Mutación , Oligonucleótidos Antisentido/administración & dosificación , Células de Purkinje/patología , Prueba de Desempeño de Rotación con Aceleración Constante , Tálamo/patología , Vacuolas/efectos de los fármacos , Vacuolas/patologíaRESUMEN
RATIONALE: Hypertrophied hearts switch from mainly using fatty acids (FAs) to an increased reliance on glucose for energy production. It has been shown that preserving FA oxidation (FAO) prevents the pathological shift of substrate preference, preserves cardiac function and energetics, and reduces cardiomyocyte hypertrophy during cardiac stresses. However, it remains elusive whether substrate metabolism regulates cardiomyocyte hypertrophy directly or via a secondary effect of improving cardiac energetics. OBJECTIVE: The goal of this study was to determine the mechanisms of how preservation of FAO prevents the hypertrophic growth of cardiomyocytes. METHODS AND RESULTS: We cultured adult rat cardiomyocytes in a medium containing glucose and mixed-chain FAs and induced pathological hypertrophy by phenylephrine. Phenylephrine-induced hypertrophy was associated with increased glucose consumption and higher intracellular aspartate levels, resulting in increased synthesis of nucleotides, RNA, and proteins. These changes could be prevented by increasing FAO via deletion of ACC2 (acetyl-CoA-carboxylase 2) in phenylephrine-stimulated cardiomyocytes and in pressure overload-induced cardiac hypertrophy in vivo. Furthermore, aspartate supplementation was sufficient to reverse the antihypertrophic effect of ACC2 deletion demonstrating a causal role of elevated aspartate level in cardiomyocyte hypertrophy. 15N and 13C stable isotope tracing revealed that glucose but not glutamine contributed to increased biosynthesis of aspartate, which supplied nitrogen for nucleotide synthesis during cardiomyocyte hypertrophy. CONCLUSIONS: Our data show that increased glucose consumption is required to support aspartate synthesis that drives the increase of biomass during cardiac hypertrophy. Preservation of FAO prevents the shift of metabolic flux into the anabolic pathway and maintains catabolic metabolism for energy production, thus preventing cardiac hypertrophy and improving myocardial energetics.
Asunto(s)
Ácido Aspártico/biosíntesis , Cardiomegalia/metabolismo , Glucosa/metabolismo , Miocitos Cardíacos/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Animales , Ácido Aspártico/farmacología , Cardiomegalia/etiología , Células Cultivadas , Ácidos Grasos/metabolismo , Masculino , Ratones , Miocitos Cardíacos/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
BACKGROUND: To assess whether patients with Persistent Depressive Disorder (PDD) have abnormal levels of N-acetyl-aspartate (NAA) and whether those levels normalize following treatment with the antidepressant medication duloxetine. Furthermore, we conducted post hoc analyses of other important brain metabolites to understand better the cellular and physiological determinants for changes in NAA levels. METHODS: We acquired proton (1H) magnetic resonance spectroscopic imaging (MRSI) data on a 3 Tesla (3T), GE Magnetic Resonance Imaging (MRI) scanner in 41 patients (39.9±10.4 years, 22 males) with PDD at two time points: before the start and at the end of a 10-week, placebo-controlled, double-blind, randomized controlled trial (RCT) of the antidepressant medication duloxetine. Patients were randomized such that 21 patients received the active medication and 20 patients received placebo during the 10 week period of the trial. In addition, we acquire 1H MRSI data once in 29 healthy controls (37.7±11.2 years, 17 males). FINDINGS: Patients had significantly higher baseline concentrations of NAA across white matter (WM) pathways and subcortical gray matter, and in direct proportion to the severity of depressive symptoms. NAA concentrations declined in duloxetine-treated patients over the duration of the trial in the direction toward healthy values, whereas concentrations increased in placebo-treated patients, deviating even further away from healthy values. Changes in NAA concentration did not mediate medication effects on reducing symptom severity, however; instead, changes in symptom severity partially mediated the effects of medication on NAA concentration, especially in the caudate and putamen. INTERPRETATION: These findings, taken together, suggest that PDD is not a direct consequence of elevated NAA concentrations, but that a more fundamental pathophysiological process likely causes PDD and determines the severity of its symptoms. The findings also suggest that although duloxetine normalized NAA concentrations in patients, it did so by modulating the severity of depressive symptoms. Medication presumably reduced depressive symptoms through other, as yet unidentified, brain processes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00360724.
Asunto(s)
Antidepresivos/farmacología , Encéfalo/efectos de los fármacos , Trastorno Depresivo Mayor/tratamiento farmacológico , Clorhidrato de Duloxetina/farmacología , Adulto , Ácido Aspártico/análogos & derivados , Ácido Aspártico/biosíntesis , Encéfalo/metabolismo , Trastorno Depresivo Mayor/diagnóstico por imagen , Método Doble Ciego , Femenino , Humanos , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
l-Aspartate has been widely used in medicine and the food and chemical industries. In this study, Serratia marcescens maleate cis-trans isomerase (MaiA) and Escherichia coli aspartase (AspA) were coupled and coexpressed in an engineered E. coli strain in which the byproduct metabolic pathway was inactivated. The engineered E. coli strain containing the dual-enzyme system (pMA) was employed to bioproduce l-aspartate from maleate with a conversion of 98%. We optimized the activity ratio of double enzymes through ribosome binding site (RBS) regulation and molecular modification of MaiA, resulting in an engineered strain: pMA-RBS4-G27A/G171A. The conversion of l-aspartate biotransformed from maleate using the pMA-RBS4-G27A/G171A strain was almost 100%. It required 40 min to complete the whole-cell catalysis, without the intermediate product and byproduct, compared to 120 min before optimization. The induction timing and the amount of inducer in a 5-liter fermentor were optimized for scale-up of the production of l-aspartate. The amount of produced l-aspartate using the cells obtained by fermentation reached 419.8 g/liter (3.15 M), and the conversion was 98.4%. Our study demonstrated an environmentally responsible and efficient method to bioproduce l-aspartate from maleate and provided an available pathway for the industrial production of l-aspartate. This work should greatly improve the economic benefits of l-aspartate, which can now be simply produced from maleate by the engineered strain constructed based on dual-enzyme coupling.IMPORTANCE l-Aspartate is currently produced from fumarate by biological methods, and fumarate is synthesized from maleate by chemical methods in industry. We established a biosynthesis method to produce l-aspartate from maleate that is environmentally responsible, convenient, and efficient. Compared to conventional l-aspartate production, no separation and purification of intermediate products is required, which could greatly improve production efficiency and reduce costs. As environmental issues are attracting increasing attention, conventional chemical methods gradually will be replaced by biological methods. Our results lay an important foundation for the industrialization of l-aspartate biosynthesis from maleate.
Asunto(s)
Ácido Aspártico/biosíntesis , Escherichia coli/metabolismo , Maleatos/metabolismo , Serratia marcescens/enzimología , Proteínas Bacterianas/metabolismo , Catálisis , Escherichia coli/genética , Fermentación , Ingeniería Metabólica , Serratia marcescens/genética , cis-trans-Isomerasas/metabolismoRESUMEN
L-aspartate is an important 4-carbon platform compound that can be used as the precursor of numerous chemical products. The bioproduction of L-aspartate directly from biomass resources is expected to provide a more cost-competitive technique route. Yet little metabolic engineering work on this matter has been carried out. In this study, we designed a shortcut pathway of L-aspartate biosynthesis in Escherichia coli, with a maximized stoichiometric yield of 2â¯mol/mol glucose. L-aspartate aminotransferase (AspC) was overexpressed for producing L-aspartate and coexpressed with L-aspartate-a-decarboxylase (PanD) for producing L-aspartate's derivative ß-alanine. L-aspartate could only be detected after directing carbon flux towards oxaloacetate and blocking the "futile cycle" with TCA cycle. A cofactor self-sufficient system successfully improved the efficiency of AspC-catalyzing L-aspartate biosynthesis reaction, and the glucose uptake remolding capably decreased byproducts from pyruvate. More targets were modified for relieving the bottleneck during fed-batch bioconversion. As a result, 1.01â¯mol L-aspartate/mol glucose and 1.52â¯mol ß-alanine/mol glucose were produced in corresponding strains respectively. Fed-batch bioconversion allowed 249â¯mM (33.1â¯g/L) L-aspartate or 424â¯mM (37.7â¯g/L) ß-alanine production, respectively. The study provides a novel promising metabolic engineering route for the production of L-aspartate and its derivate chemicals using biomass resources. These results also represent the first report of the efficient bioproduction of L-aspartate directly from glucose in E. coli and the highest yield of ß-alanine reported so far.
Asunto(s)
Ácido Aspártico , Carboxiliasas , Ciclo del Ácido Cítrico/genética , Proteínas de Escherichia coli , Escherichia coli , Ingeniería Metabólica , beta-Alanina , Ácido Aspártico/biosíntesis , Ácido Aspártico/genética , Carboxiliasas/genética , Carboxiliasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , beta-Alanina/genética , beta-Alanina/metabolismoRESUMEN
Cellular aspartate drives cancer cell proliferation, but signaling pathways that rewire aspartate biosynthesis to control cell growth remain largely unknown. Hypoxia-inducible factor-1α (HIF1α) can suppress tumor cell proliferation. Here, we discovered that HIF1α acts as a direct repressor of aspartate biosynthesis involving the suppression of several key aspartate-producing proteins, including cytosolic glutamic-oxaloacetic transaminase-1 (GOT1) and mitochondrial GOT2. Accordingly, HIF1α suppresses aspartate production from both glutamine oxidation as well as the glutamine reductive pathway. Strikingly, the addition of aspartate to the culture medium is sufficient to relieve HIF1α-dependent repression of tumor cell proliferation. Furthermore, these key aspartate-producing players are specifically repressed in VHL-deficient human renal carcinomas, a paradigmatic tumor type in which HIF1α acts as a tumor suppressor, highlighting the in vivo relevance of these findings. In conclusion, we show that HIF1α inhibits cytosolic and mitochondrial aspartate biosynthesis and that this mechanism is the molecular basis for HIF1α tumor suppressor activity.
Asunto(s)
Ácido Aspártico/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Neoplasias/metabolismo , Proteínas Supresoras de Tumor/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Aspartato Aminotransferasa Citoplasmática/metabolismo , Aspartato Aminotransferasa Mitocondrial/metabolismo , Ácido Aspártico/farmacología , Carcinoma de Células Renales/enzimología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Glutamina/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Renales/enzimología , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/antagonistas & inhibidores , Neoplasias/patología , Oxidación-Reducción , Proteínas Supresoras de Tumor/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
Breast cancer susceptibility gene 1 (BRCA1) has been implicated in modulating metabolism via transcriptional regulation. However, direct metabolic targets of BRCA1 and the underlying regulatory mechanisms are still unknown. Here, we identified several metabolic genes, including the gene which encodes glutamate-oxaloacetate transaminase 2 (GOT2), a key enzyme for aspartate biosynthesis, which are repressed by BRCA1. We report that BRCA1 forms a co-repressor complex with ZBRK1 that coordinately represses GOT2 expression via a ZBRK1 recognition element in the promoter of GOT2. Impairment of this complex results in upregulation of GOT2, which in turn increases aspartate and alpha ketoglutarate production, leading to rapid cell proliferation of breast cancer cells. Importantly, we found that GOT2 can serve as an independent prognostic factor for overall survival and disease-free survival of patients with breast cancer, especially triple-negative breast cancer. Interestingly, we also demonstrated that GOT2 overexpression sensitized breast cancer cells to methotrexate, suggesting a promising precision therapeutic strategy for breast cancer treatment. In summary, our findings reveal that BRCA1 modulates aspartate biosynthesis through transcriptional repression of GOT2, and provides a biological basis for treatment choices in breast cancer.
Asunto(s)
Aspartato Aminotransferasa Mitocondrial/genética , Ácido Aspártico/biosíntesis , Proteína BRCA1/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Animales , Aspartato Aminotransferasa Mitocondrial/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Ácidos Cetoglutáricos/metabolismo , Metotrexato/farmacología , Ratones , Persona de Mediana Edad , Modelos Biológicos , Fenotipo , Unión Proteica/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Proliferación Celular , Glucólisis , L-Lactato Deshidrogenasa/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Factor de Transcripción Activador 4/genética , Sistema de Transporte de Aminoácidos ASC/genética , Sistema de Transporte de Aminoácidos ASC/metabolismo , Ácido Aspártico/biosíntesis , Ácido Aspártico/genética , Línea Celular Tumoral , Supervivencia Celular , Humanos , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Melanoma/genética , Melanoma/patología , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Serina/biosíntesis , Serina/genéticaRESUMEN
A growing number of siderophores are found to contain ß-hydroxyaspartic acid (ß-OH-Asp) as a functional group for Fe(III) coordination, along with the more common catechol and hydroxamic acid groups. This review covers the structures, biosynthesis, and reactions of peptidic ß-OH-Asp siderophores. Hydroxylation of Asp in siderophore biosynthesis is predicted to be carried out either through discrete aspartyl ß-hydroxylating enzymes or through hydroxylating domains within non-ribosomal peptide synthetases, both of which display sequence homology to known non-heme iron(II), α-ketoglutarate-dependent dioxygenases. Ferric complexes of ß-OH-Asp siderophores are photoreactive, resulting in reduction of Fe(III) and oxidative cleavage of the siderophore to yield distinct types of photoproducts. Probing the photoreactivity of synthetic Fe(III)-α-hydroxycarboxylate clusters yields mechanistic insights into the different photoproducts observed for ß-OH-Asp and other α-hydroxycarboxylate siderophore Fe(III) complexes.
Asunto(s)
Ácido Aspártico/análogos & derivados , Sideróforos/biosíntesis , Ácido Aspártico/biosíntesis , Ácido Aspártico/química , Estructura Molecular , Sideróforos/químicaRESUMEN
Zinc (Zn) deficiency remarkably depresses the protein concentration in the grain of winter wheat. Cultivar 'Pingan 8' showed lower Zn concentrations in the grain than did cultivar 'Yangao 006' after nitrogen (N) combined with Zn application. However, little is known about how amino acids are influenced by Zn combined with N application or about the differences in amino acid accumulation between the two winter wheat cultivars. A pot experiment was conducted to characterize amino acid accumulation in the low Zn-accumulating cultivar 'Pingan 8' and the high Zn-accumulating cultivar 'Yangao 006' at various growth stages (seedling, jointing, grain filling and maturity) as influenced by N and Zn supply. The N (N0.2) combined with Zn (Zn10) application significantly increased grain yields and the concentrations of N, Zn and crude protein in the grain of both wheat cultivars. N combined with Zn application significantly increased the concentrations of glutamate (Glu) and asparagine (Asn) but decreased the concentrations of glutamine (Gln) and aspartate (Asp) in cultivar 'Yangao 006'; the N combined with Zn application decreased the concentrations of Glu and Gln but increased the concentrations of Asp and Asn in cultivar 'Pingan 8' at the jointing, grain filling and mature stages. Correlation analysis results showed that there were significant relationships between grain yields, spike number, grain number and Zn, N, crude protein, Glu, Gln, Asp and Asn concentrations in the shoots and grain of winter wheat at different growth stages. These results demonstrate that N combined with Zn application enhanced protein synthesis by altering amino acid accumulation in both winter wheat cultivars. Cultivar 'Pingan 8' had lower Gln, Asp and Asn concentrations and higher Glu concentrations than did cultivar 'Yangao 006' after the N0.05 treatment but had higher Glu, Gln, Asp, and Asn concentrations and lower Glu concentrations than did cultivar 'Yangao 006' after the N0.2 treatment. These results revealed that the difference in amino acid concentrations between the two cultivars was related to the N application level.
Asunto(s)
Asparagina/biosíntesis , Ácido Aspártico/biosíntesis , Ácido Glutámico/biosíntesis , Glutamina/biosíntesis , Nitrógeno/farmacología , Triticum/metabolismo , Zinc/farmacologíaRESUMEN
Cyclic di-3',5'-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism in Listeria monocytogenes by inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition of Lactococcus lactis PC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. In L. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool in L. lactis is negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP-insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.
Asunto(s)
Fosfatos de Dinucleósidos/metabolismo , Piruvato Carboxilasa/metabolismo , Piruvato Carboxilasa/fisiología , Adenosina Monofosfato/metabolismo , Ácido Aspártico/biosíntesis , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X/métodos , AMP Cíclico/metabolismo , Fosfatos de Dinucleósidos/fisiología , Lactobacillales/metabolismo , Lactococcus lactis/metabolismo , Conformación Proteica , Sistemas de Mensajero Secundario/fisiología , Relación Estructura-ActividadRESUMEN
Two Aureobasidium pullulans strains (L1 and L8), able to prevent postharvest fruit decay, were evaluated in order to elucidate how the competition for nutrients and space was involved in their activity against Monilinia laxa, the causal agent of peach brown rot. The competition for nutrients was studied by co-culturing pathogen conidia and antagonists in different conditions of nutrient availability and avoiding contact between them. Both antagonists prevented the germination of conidia of M. laxa in water, reducing germination rate by >35%. However, L1 and L8 showed the lowest inhibition of conidial germination in peach juice at 5%, with a reduction of 12.6% and 13.9% respectively. HPLC amino acid analysis of peach juice revealed that the addition of the yeast suspension greatly modified their composition: asparagine was completely depleted soon after 12h of incubation and was probably hydrolyzed to aspartic acid by the yeasts, as aspartic acid content markedly increased. Pure asparagine and aspartic acid were tested by in vitro trials at the concentrations found in peach juice: both influenced M. laxa growth, but in opposite ways. Asparagine stimulated pathogen growth; conversely, amended medium with aspartic acid significantly inhibited the conidia germination and mycelial development of M. laxa. Scanning Electron Microscopy revealed that both strains showed a great capability to compete with M. laxa for space (starting 8h after treatment), colonizing the wound surface and inhibiting pathogen growth. This study clearly showed that A. pullulans L1 and L8 strains could compete with M. laxa for nutrients and space; this mode of action may play an important role in the antagonistic activity, especially in the first hours of tritrophic host-pathogen-antagonist interaction, although several other mechanisms can interact each other.
Asunto(s)
Antibiosis/fisiología , Ascomicetos/fisiología , Asparagina/metabolismo , Ácido Aspártico/biosíntesis , Jugos de Frutas y Vegetales/microbiología , Frutas/microbiología , Prunus persica/microbiología , Saccharomycetales/crecimiento & desarrollo , Germinación/fisiología , Glucanos , Micelio/crecimiento & desarrollo , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Esporas Fúngicas/crecimiento & desarrolloRESUMEN
O-Linked ß-N-acetylglucosamine transferase (OGT) is an essential human enzyme that glycosylates numerous nuclear and cytoplasmic proteins on serine and threonine. It also cleaves Host cell factor 1 (HCF-1) by a mechanism in which the first step involves glycosylation on glutamate. Replacing glutamate with aspartate in an HCF-1 proteolytic repeat was shown to prevent peptide backbone cleavage, but whether aspartate glycosylation occurred was not examined. We report here that OGT glycosylates aspartate much faster than it glycosylates glutamate in an otherwise identical model peptide substrate; moreover, once formed, the glycosyl aspartate reacts further to form a succinimide intermediate that hydrolyzes to produce the corresponding isoaspartyl peptide. Aspartate-to-isoaspartate isomerization in proteins occurs in cells but was previously thought to be exclusively non-enzymatic. Our findings suggest it may also be enzyme-catalyzed. In addition to OGT, enzymes that may catalyze aspartate to isoaspartate isomerization include PARPs, enzymes known to ribosylate aspartate residues in the process of poly(ADP-ribosyl)ation.
Asunto(s)
Ácido Aspártico/biosíntesis , Factor C1 de la Célula Huésped/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Ácido Aspártico/química , Biocatálisis , Glicosilación , Factor C1 de la Célula Huésped/química , Humanos , Conformación MolecularRESUMEN
The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca2+-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.
Asunto(s)
Sistemas de Transporte de Aminoácidos/biosíntesis , Ácido Aspártico/análogos & derivados , Proliferación Celular , Regulación hacia Abajo , Proteínas Mitocondriales/biosíntesis , Neuronas/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Antiportadores/deficiencia , Antiportadores/genética , Antiportadores/metabolismo , Ácido Aspártico/biosíntesis , Línea Celular , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Humanos , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Neuronas/patología , Trastornos Psicomotores/genética , Trastornos Psicomotores/metabolismo , Trastornos Psicomotores/patologíaRESUMEN
Canavan disease is a leukodystrophy caused by aspartoacylase (ASPA) deficiency. The lack of functional ASPA, an enzyme enriched in oligodendroglia that cleaves N-acetyl-l-aspartate (NAA) to acetate and l-aspartic acid, elevates brain NAA and causes "spongiform" vacuolation of superficial brain white matter and neighboring gray matter. In children with Canavan disease, neuroimaging shows early-onset dysmyelination and progressive brain atrophy. Neuron loss has been documented at autopsy in some cases. Prior studies have shown that mice homozygous for the Aspa nonsense mutation Nur7 also develop brain vacuolation. We now report that numbers of cerebral cortical and cerebellar neurons are decreased and that cerebral cortex progressively thins in AspaNur7/Nur7 mice. This neuronal pathology is prevented by constitutive disruption of Nat8l, which encodes the neuronal NAA-synthetic enzyme N-acetyltransferase-8-like. SIGNIFICANCE STATEMENT: This is the first demonstration of cortical and cerebellar neuron depletion and progressive cerebral cortical thinning in an animal model of Canavan disease. Genetic suppression of N-acetyl-l-aspartate (NAA) synthesis, previously shown to block brain vacuolation in aspartoacylase-deficient mice, also prevents neuron loss and cerebral cortical atrophy in these mice. These results suggest that lowering the concentration of NAA in the brains of children with Canavan disease would prevent or slow progression of neurological deficits.