RESUMO
The preparation of a hybrid nanomaterial is reported by covalently attaching 3,5-dinitrobenzoic acid groups to the surface of oxidized multi-walled carbon nanotubes using 1,6-diaminohexane as cross-linking agent. This nanomaterial, modified with the redox mediator, was used as transduction element to construct an amperometric sensor for the efficient indirect determination of glutathione reductase at a low working potential of - 0.05 V, through the oxidation of unconsumed nicotinamide adenine dinucleotide phosphate (NADPH) in the enzymatic reaction. The sensor exhibited an excellent linear response in the range 1.6 to 174 µU/µL, with high reproducibility and selectivity. The developed device was successfully validated in real samples, accurately determining the active enzyme in diluted human serum, making it a promising alternative for the determination of glutathione reductase and other related NADPH-dependent enzymes with relevance in clinical analysis.
Assuntos
Técnicas Eletroquímicas , Eletrodos , Glutationa Redutase , Nanotubos de Carbono , Nanotubos de Carbono/química , Humanos , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Glutationa Redutase/metabolismo , Técnicas Biossensoriais/métodos , NADP/química , NADP/metabolismo , Oxirredução , Nitrobenzoatos/química , Limite de DetecçãoRESUMO
Significance: Autofluorescence characteristics of the reduced nicotinamide adenine dinucleotide and oxidized flavin cofactors are important for the evaluation of the metabolic status of the cells. The approaches that involve a detailed analysis of both spectral and time characteristics of the autofluorescence signals may provide additional insights into the biochemical processes in the cells and biological tissues and facilitate the transition of spectral fluorescence lifetime imaging into clinical applications. Aim: We present the experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of the reduced nicotinamide adenine dinucleotide and oxidized flavin under a single excitation wavelength aimed at understanding whether the use of multispectral detection is helpful for metabolic imaging of cancer cells. Approach: We use two-photon spectral fluorescence lifetime imaging microscopy. Starting from model solutions, we switched to cell cultures treated by metabolic inhibitors and then studied the metabolism of cells within tumor spheroids. Results: The use of a multispectral detector in combination with an excitation at a single wavelength of 750 nm allows the identification of fluorescence signals from three components: free and bound NAD(P)H, and flavins based on the global fitting procedure. Multispectral data make it possible to assess not only the lifetime but also the spectral shifts of emission of flavins caused by chemical perturbations. Altogether, the informative parameters of the developed approach are the ratio of free and bound NAD(P)H amplitudes, the decay time of bound NAD(P)H, the amplitude of flavin fluorescence signal, the fluorescence decay time of flavins, and the spectral shift of the emission signal of flavins. Hence, with multispectral fluorescence lifetime imaging, we get five independent parameters, of which three are related to flavins. Conclusions: The approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750 nm and a fluorescence time-resolved spectral detection with the consequent global analysis of the data not only simplifies image acquisition protocol but also allows to disentangle the impacts of free and bound NAD(P)H, and flavin components evaluate changes in their fluorescence parameters (emission spectra and fluorescence lifetime) upon treating cells with metabolic inhibitors and sense metabolic heterogeneity within 3D tumor spheroids.
Assuntos
Flavinas , NADP , Humanos , NADP/metabolismo , Flavinas/química , Flavinas/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Linhagem Celular Tumoral , Esferoides Celulares/metabolismo , Microscopia de Fluorescência/métodos , NAD/metabolismo , NAD/químicaRESUMO
MAT2B works together with MAT2A to synthesize S-Adenosyl methionine (SAM) as the primary methyl donor. MAT2B, despite lacking catalytic activity, exerts regulatory control over the enzymatic activity of MAT2A. In addition to the enzymatic activity regulation, we find that, in an NADP+-dependent manner, MAT2B binds and stabilizes MAT2A. Disruption of the cellular NADP+ remodels the protein level of MAT2A. The pentose phosphatase pathway regulates the level of MAT2A protein through the interaction of NADP+ with MAT2B. Additionally, MAT2B-MAT2A interaction regulates the mRNA m6A modification and stability. In liver tumors, the Mat2a mRNA level is elevated but the protein level is decreased by the restricted NADP+. Blocking the interaction between MAT2B and MAT2A by the keto diet can suppress liver tumor growth. These findings reveal that MAT2B is essential for regulating the protein levels of MAT2A and connecting SAM synthesis to mRNA m6A.
Assuntos
Adenosina , Neoplasias Hepáticas , Metionina Adenosiltransferase , Metionina Adenosiltransferase/metabolismo , Metionina Adenosiltransferase/genética , Humanos , Adenosina/metabolismo , Adenosina/análogos & derivados , Animais , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , NADP/metabolismo , Camundongos , S-Adenosilmetionina/metabolismo , Linhagem Celular Tumoral , Ligação ProteicaRESUMO
The Rieske non-heme iron oxygenases (Rieske oxygenases) comprise a class of metalloenzymes that are involved in the biosynthesis of complex natural products and the biodegradation of aromatic pollutants. Despite this desirable catalytic repertoire, industrial implementation of Rieske oxygenases has been hindered by the multicomponent nature of these enzymes and their requirement for expensive reducing equivalents in the form of a reduced nicotinamide adenine dinucleotide cosubstrate (NAD(P)H). Fortunately, however, some Rieske oxygenases co-occur with accessory proteins, that through a downstream reaction, recycle the needed NAD(P)H for catalysis. As these pathways and accessory proteins are attractive for bioremediation applications and enzyme engineering campaigns, herein, we describe methods for assembling Rieske oxygenase pathways in vitro. Further, using the TsaMBCD pathway as a model system, in this chapter, we provide enzymatic, spectroscopic, and crystallographic methods that can be adapted to explore both Rieske oxygenases and their co-occurring accessory proteins.
Assuntos
NAD , NAD/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Oxigenases/metabolismo , Oxigenases/química , Oxigenases/isolamento & purificação , Cristalografia por Raios X/métodos , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/isolamento & purificação , NADP/metabolismoRESUMO
BACKGROUND: Metabolic engineering enables the sustainable and cost-efficient production of complex chemicals. Efficient production of terpenes in Saccharomyces cerevisiae can be achieved by recruiting an intermediate of the mevalonate pathway. The present study aimed to evaluate the engineering strategies of S. cerevisiae for the production of taxadiene, a precursor of taxol, an antineoplastic drug. RESULT: SCIGS22a, a previously engineered strain with modifications in the mevalonate pathway (MVA), was used as a background strain. This strain was engineered to enable a high flux towards farnesyl diphosphate (FPP) and the availability of NADPH. The strain MVA was generated from SCIGS22a by overexpressing all mevalonate pathway genes. Combining the background strains with 16 different episomal plasmids, which included the combination of 4 genes: tHMGR (3-hydroxy-3-methylglutaryl-CoA reductase), ERG20 (farnesyl pyrophosphate synthase), GGPPS (geranyl diphosphate synthase) and TS (taxadiene synthase) resulted in the highest taxadiene production in S. cerevisiae of 528 mg/L. CONCLUSION: Our study highlights the critical role of pathway balance in metabolic engineering, mainly when dealing with toxic molecules like taxadiene. We achieved significant improvements in taxadiene production by employing a combinatorial approach and focusing on balancing the downstream and upstream pathways. These findings emphasize the importance of minor gene expression modification levels to achieve a well-balanced pathway, ultimately leading to enhanced taxadiene accumulation.
Assuntos
Engenharia Metabólica , Ácido Mevalônico , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Engenharia Metabólica/métodos , Ácido Mevalônico/metabolismo , Alcenos/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Diterpenos/metabolismo , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , NADP/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SesquiterpenosRESUMO
Upon stimulation of membrane receptors, nicotinic acid adenine dinucleotide phosphate (NAADP) is formed as second messenger within seconds and evokes Ca2+ signaling in many different cell types. Here, to directly stimulate NAADP signaling, MASTER-NAADP, a Membrane permeAble, STabilized, bio-rEversibly pRotected precursor of NAADP is synthesized and release of its active NAADP mimetic, benzoic acid C-nucleoside, 2'-phospho-3'F-adenosine-diphosphate, by esterase digestion is confirmed. In the presence of NAADP receptor HN1L/JPT2 (hematological and neurological expressed 1-like protein, HN1L, also known as Jupiter microtubule-associated homolog 2, JPT2), this active NAADP mimetic releases Ca2+ and increases the open probability of type 1 ryanodine receptor. When added to intact cells, MASTER-NAADP initially evokes single local Ca2+ signals of low amplitude. Subsequently, also global Ca2+ signaling is observed in T cells, natural killer cells, and Neuro2A cells. In contrast, control compound MASTER-NADP does not stimulate Ca2+ signaling. Likewise, in cells devoid of HN1L/JPT2, MASTER-NAADP does not affect Ca2+ signaling, confirming that the product released from MASTER-NAADP is a bona fide NAADP mimetic.
Assuntos
Sinalização do Cálcio , Cálcio , NADP , NADP/análogos & derivados , NADP/metabolismo , Animais , Humanos , Cálcio/metabolismo , Camundongos , Sistemas do Segundo Mensageiro , Permeabilidade da Membrana Celular , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Células Matadoras Naturais/metabolismo , Linfócitos T/metabolismoRESUMO
Arabinoside and derived nucleoside analogs, a family of nucleoside analogs, exhibit diverse typically biological activities and are widely used as antibacterial, antiviral, anti-inflammatory, antitumor, and other drugs in clinical and preclinical trials. Although with a long and rich history in the field of medicinal chemistry, the biosynthesis of arabinoside has only been sporadically designed and studied, and it remains a challenge. Here, we constructed an in vitro multi-enzymatic cascade for the biosynthesis of arabinosides. This artificial biosystem was systematically optimized, involving an exquisite pathway design, NADP+ regeneration, meticulous enzyme selection, optimization of the key enzyme dosage, and the concentration of inorganic phosphate. Under the optimized conditions, we achieved 0.37 mM of vidarabine from 5 mM of sucrose and 2 mM of adenine, representing 18.7% of the theoretical yield. Furthermore, this biosystem also has the capability to produce other arabinosides, such as spongouridine, arabinofuranosylguanine, hypoxanthine arabinofuranoside, fludarabine, and 2-methoxyadenine arabinofuranoside, from sucrose, and corresponding nucleobase by introducing different nucleoside phosphorylases. Overall, our biosynthesis approach provides a pathway for the biosynthesis of arabinose-derived nucleoside analogs, offering potential applications in the pharmaceutical industry.
Assuntos
Sacarose , Sacarose/metabolismo , Sacarose/química , Vidarabina/análogos & derivados , Vidarabina/química , Vidarabina/metabolismo , Pentosiltransferases/metabolismo , Pentosiltransferases/genética , NADP/metabolismoRESUMO
Glioblastoma represents the predominant and a highly aggressive primary neoplasm of the central nervous system that has an abnormal metabolism. Our previous study showed that chrysomycin A (Chr-A) curbed glioblastoma progression in vitro and in vivo. However, whether Chr-A could inhibit orthotopic glioblastoma and how it reshapes metabolism are still unclear. In this study, Chr-A markedly suppressed the development of intracranial U87 gliomas. The results from airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) indicated that Chr-A improved the abnormal metabolism of mice with glioblastoma. Key enzymes including glutaminase (GLS), glutamate dehydrogenases 1 (GDH1), hexokinase 2 (HK2) and glucose-6-phosphate dehydrogenase (G6PD) were regulated by Chr-A. Chr-A further altered the level of nicotinamide adenine dinucleotide phosphate (NADPH), thus causing oxidative stress with the downregulation of Nrf-2 to inhibit glioblastoma. Our study offers a novel perspective for comprehending the anti-glioma mechanism of Chr-A, highlighting its potential as a promising chemotherapeutic agent for glioblastoma.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Estresse Oxidativo , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Antraquinonas/farmacologia , Glutaminase/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Progressão da Doença , Glutamato Desidrogenase/metabolismo , NADP/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Masculino , Camundongos NusRESUMO
Pyridine nucleotide-disulfide oxidoreductases are underexplored as drug targets, and thioredoxin reductases (TrxRs) stand out as compelling pharmacological targets. Selective TrxR inhibition is challenging primarily due to the reliance on covalent inhibition strategies. Recent studies identified a regulatory and druggable pocket in Schistosoma mansoni thioredoxin glutathione reductase (TGR), a TrxR-like enzyme, and an established drug target for schistosomiasis. This site is termed the "doorstop pocket" because compounds that bind there impede the movement of an aromatic side-chain necessary for the entry and exit of NADPH and NADP+ during enzymatic turnover. This discovery spearheaded the development of new TGR inhibitors with efficacies surpassing those of current schistosomiasis treatment. Targeting the "doorstop pocket" is a promising strategy, as the pocket is present in all members of the pyridine nucleotide-disulfide oxidoreductase family, opening new avenues for exploring therapeutic approaches in diseases where the importance of these enzymes is established, including cancer and inflammatory and infectious diseases.
Assuntos
Inibidores Enzimáticos , Schistosoma mansoni , Tiorredoxina Dissulfeto Redutase , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxina Dissulfeto Redutase/química , Animais , Schistosoma mansoni/enzimologia , Humanos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , NADP/metabolismo , Complexos Multienzimáticos , NADH NADPH OxirredutasesRESUMO
Redox homeostasis is the balance between oxidation and reduction reactions. Its maintenance depends on glutathione, including its reduced and oxidized form, GSH/GSSG, which is the main intracellular redox buffer, but also on the nicotinamide adenine dinucleotide phosphate, including its reduced and oxidized form, NADPH/NADP+. Under conditions that enable yeast cells to undergo fermentative metabolism, the main source of NADPH is the pentose phosphate pathway. The lack of enzymes responsible for the production of NADPH has a significant impact on yeast cells. However, cells may compensate in different ways for impairments in NADPH synthesis, and the choice of compensation strategy has several consequences for cell functioning. The present study of this issue was based on isogenic mutants: Δzwf1, Δgnd1, Δald6, and the wild strain, as well as a comprehensive panel of molecular analyses such as the level of gene expression, protein content, and enzyme activity. The obtained results indicate that yeast cells compensate for the lack of enzymes responsible for the production of cytosolic NADPH by changing the content of selected proteins and/or their enzymatic activity. In turn, the cellular strategy used to compensate for them may affect cellular efficiency, and thus, the ability to grow or sensitivity to environmental acidification.
Assuntos
Fermentação , Homeostase , NADP , Oxirredução , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , NADP/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Glutationa/metabolismo , Via de Pentose FosfatoRESUMO
Environmental conditions significantly impact the metabolism of Saccharomyces cerevisiae, a Crabtree-positive yeast that maintains a fermentative metabolism in high-sugar environments even in the presence of oxygen. Although the introduction of oxygen has been reported to induce alterations in yeast metabolism, knowledge of the mechanisms behind these metabolic adaptations in relation to redox cofactor metabolism and their implications in the context of wine fermentation remains limited. This study aimed to compare the intracellular redox cofactor levels, the cofactor ratios, and primary metabolite production in S. cerevisiae under aerobic and anaerobic conditions in synthetic grape juice. The molecular mechanisms underlying these metabolic differences were explored using a transcriptomic approach. Aerobic conditions resulted in an enhanced fermentation rate and biomass yield. Total NADP(H) levels were threefold higher during aerobiosis, while a decline in the total levels of NAD(H) was observed. However, there were stark differences in the ratio of NAD+/NADH between the treatments. Despite few changes in the differential expression of genes involved in redox cofactor metabolism, anaerobiosis resulted in an increased expression of genes involved in lipid biosynthesis pathways, while the presence of oxygen increased the expression of genes associated with thiamine, methionine, and sulfur metabolism. The production of fermentation by-products was linked with differences in the redox metabolism in each treatment. This study provides valuable insights that may help steer the production of metabolites of industrial interest during alcoholic fermentation (including winemaking) by using oxygen as a lever of redox metabolism.
Assuntos
Fermentação , Oxirredução , Oxigênio , Saccharomyces cerevisiae , Vinho , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Oxigênio/metabolismo , Vinho/microbiologia , Vinho/análise , Anaerobiose , Vitis/microbiologia , Vitis/metabolismo , NAD/metabolismo , Etanol/metabolismo , NADP/metabolismo , Aerobiose , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Coenzimas/metabolismoRESUMO
In the pentose phosphate pathway, dehydroepiandrosterone (DHEA) uncompetitively inhibits glucose-6-phosphate dehydrogenase (G6PD), reducing NADPH production and increasing oxidative stress, which can influence the onset and/or progression of several diseases, including cancer. 2-Deoxy-D-glucose (2-DG), a glucose mimetic, competes with glucose for cellular uptake, inhibiting glycolysis and competing with glucose-6-phosphate (G-6-P) for G6PD activity. In this study, we report that DHEA-α-2-DG (5), an α-covalent conjugate of DHEA and 2-DG, exhibits better anticancer activity than DHEA, 2-DG, DHEA +2-DG, and polydatin in MCF-7 cells, and reduces NADPH/NADP+ ratio in cellular assays. In vitro enzyme kinetics and molecular docking studies showed that 5 uncompetitively inhibits human G6PD activity and binds to the structural NADP+ site but not to the catalytic NADP+ site. Further combining 5 with the FDA-approved drug tamoxifen enhanced its cytotoxicity against MCF-7 cells, suggesting that it could serve as a candidate for combination of drug strategies.
Assuntos
Antineoplásicos , Desidroepiandrosterona , Desoxiglucose , Glucosefosfato Desidrogenase , Simulação de Acoplamento Molecular , Humanos , Glucosefosfato Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/antagonistas & inibidores , Desidroepiandrosterona/farmacologia , Desidroepiandrosterona/química , Células MCF-7 , Desoxiglucose/farmacologia , Desoxiglucose/química , Desoxiglucose/análogos & derivados , Desoxiglucose/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , NADP/metabolismo , Tamoxifeno/farmacologia , Tamoxifeno/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , CinéticaRESUMO
Dihydro-ß-ionone, a high-value compound with distinctive fragrance, is widely utilized in the flavor and fragrance industries. However, its low abundance in plant sources poses a significant challenge to its application through traditional extraction methods. Development of an enzyme cascade reaction with artificial design offers a promising alternative. Herein, a short-chain dehydrogenase NaSDR, was identified from Novosphingobium aromaticivorans DSM 12444, which exhibited a high activity in converting ß-ionol to ß-ionone. A novel biosynthesis route to produce dihydro-ß-ionone from ß-ionol was developed, by utilizing alcohol dehydrogenase NaSDR and enoate reductase AaDBR1. Under the optimized conditions (0.29 mg/mL NaSDR, 0.39 mg/mL AaDBR1, 1 mM NADP+ and 2.5 mM ß-ionol at 40 °C for 2 h), a maximum yield (173.11 mg/L) of dihydro-ß-ionone was achieved with a molar conversion rate of 35.6 %, which was 2.7-fold higher than that before optimization. Additionally, this cascade reaction achieved self-sufficient NADPH regeneration through the actions of NaSDR and AaDBR1. This study offered a fresh perspective for achieving a green and sustainable synthesis of dihydro-ß-ionone and could inspire on another natural products biosynthesis.
Assuntos
Norisoprenoides , Norisoprenoides/química , Norisoprenoides/metabolismo , Redutases-Desidrogenases de Cadeia Curta/metabolismo , Redutases-Desidrogenases de Cadeia Curta/química , Sphingomonadaceae/enzimologia , NADP/metabolismo , Oxirredutases/metabolismoRESUMO
Metastasizing cancer cells encounter a multitude of stresses throughout the metastatic cascade. Oxidative stress is known to be a major barrier for metastatic colonization, such that metastasizing cancer cells must rewire their metabolic pathways to increase their antioxidant capacity. NADPH is essential for regeneration of cellular antioxidants and several NADPH-regenerating pathways have been shown to play a role in metastasis. We have found that metastatic melanoma cells have increased levels of both NADPH and NADP+ suggesting increased de novo biosynthesis of NADP+. De novo biosynthesis of NADP+ occurs through a single enzymatic reaction catalyzed by NAD+ kinase (NADK). Here we show that different NADK isoforms are differentially expressed in metastatic melanoma cells, with Isoform 3 being specifically upregulated in metastasis. We find that Isoform 3 is more potent in expanding the NADP(H) pools, increasing oxidative stress resistance and promoting metastatic colonization compared to Isoform 1. We have found that Isoform 3 is transcriptionally upregulated by oxidative stress through the action of NRF2. Together, our work presents a previously uncharacterized role of NADK isoforms in oxidative stress resistance and metastasis and suggests that NADK Isoform 3 is a potential therapeutic target in metastatic disease.
Assuntos
Regulação Neoplásica da Expressão Gênica , Isoenzimas , Melanoma , Fator 2 Relacionado a NF-E2 , Metástase Neoplásica , Estresse Oxidativo , Fosfotransferases (Aceptor do Grupo Álcool) , Melanoma/metabolismo , Melanoma/patologia , Melanoma/genética , Humanos , Animais , Isoenzimas/metabolismo , Isoenzimas/genética , Camundongos , Linhagem Celular Tumoral , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , NADP/metabolismoRESUMO
Dihydrofolate reductase (DHFR) is ubiquitously present in all living organisms and plays a crucial role in the growth of the fungal pathogen R.solani. Sequence alignment confirmed the evolutionary conservation of the essential lid domain, with the amino acid 'P' within the PEKN lid domain appearing with a frequency of 89.5% in higher organisms and 11.8% in lower organisms. Consequently, a K65P variant was introduced into R.solani DHFR (rDHFR). Subsequent enzymatic kinetics assays were conducted for human DHFR (hDHFR), rDHFR, E. coli DHFR (eDHFR), and the K65P variant. hDHFR exhibited the highest kcat of 0.95 s-1, followed by rDHFR with 0.14 s-1, while eDHFR displayed the lowest kcat of 0.09 s-1. Remarkably, the K65P variant induced a significant reduction in Km, resulting in a 1.8-fold enhancement in catalytic efficiency (kcat/Km) relative to the wild type. Differential scanning fluorimetry and binding free energy calculations confirmed the enhanced substrate affinity for both folate and NADPH in the K65P variant. These results suggest that the K65P mutation enhances substrate affinity and catalytic efficiency in DHFR, highlighting the evolutionary and functional importance of the K65 residue.
Assuntos
Simulação de Dinâmica Molecular , Tetra-Hidrofolato Desidrogenase , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Cinética , Substituição de Aminoácidos , Especificidade por Substrato , Ácido Fólico/metabolismo , Ácido Fólico/química , NADP/metabolismo , NADP/químicaRESUMO
Cuproptosis is characterized by lipoylated protein aggregation and loss of iron-sulfur (Fe-S) proteins, which are crucial for a wide range of important cellular functions, including DNA replication and damage repair. Sirt2 and sirt4 are lipoamidases that remove the lipoyl moiety from lipoylated proteins using nicotinamide adenine dinucleotide (NAD+) as a cofactor. However, to date, it is not clear whether nicotinamide mononucleotide (NMN), a precursor of NAD+, affects cellular sensitivity to cuproptosis. Therefore, in the current study, cuproptosis was induced by the copper (Cu) ionophore elesclomol (Es) in HeLa cells. It was also found that Es/Cu treatment increased cellular DNA damage level. On the other hand, NMN treatment partially rescued cuproptosis in a dose-dependent manner, as well as reduced cellular DNA damage level. In addition, NMN upregulated the expression of Fe-S protein POLD1, without affecting the aggregation of lipoylated proteins. Mechanistic study revealed that NMN increased the expression of sirt2 and cellular reduced nicotinamide adenine dinucleotide phosphate (NADPH) level. Overexpression of sirt2 and sirt4 did not change the aggregation of lipoylated proteins, however, sirt2, but not sirt4, increased cellular NADPH levels and partially rescued cuproptosis. Inhibition of NAD+ kinase (NADK), which is responsible for generating NADPH, abolished the rescuing function of NMN and sirt2 for Es/Cu induced cell death. Taken together, our results suggested that DNA damage is a characteristic feature of cuproptosis. NMN can partially rescue cuproptosis by upregulating sirt2, increase intracellular NADPH content and maintain the level of Fe-S proteins, independent of the lipoamidase activity of sirt2.
Assuntos
Dano ao DNA , NADP , Mononucleotídeo de Nicotinamida , Sirtuína 2 , Regulação para Cima , Humanos , Sirtuína 2/metabolismo , Sirtuína 2/genética , Células HeLa , NADP/metabolismo , Dano ao DNA/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Mononucleotídeo de Nicotinamida/farmacologia , Mononucleotídeo de Nicotinamida/metabolismo , Cobre/farmacologia , Cobre/metabolismo , Sirtuínas/metabolismoRESUMO
Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5â Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-ß-thia and ß-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.
Assuntos
Aldose-Cetose Isomerases , Fosfomicina , Complexos Multienzimáticos , Toxoplasma , Toxoplasma/enzimologia , Toxoplasma/efeitos dos fármacos , Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Aldose-Cetose Isomerases/genética , Fosfomicina/farmacologia , Fosfomicina/análogos & derivados , Fosfomicina/química , Cristalografia por Raios X , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , NADP/metabolismo , NADP/química , Humanos , Modelos Moleculares , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Oxirredutases/metabolismoRESUMO
Organometallic catalyst is extensively applied for the non-enzymatic regeneration of nicotinamide adenine dinucleotide (phosphate) cofactors, but suffering from the mutual inactivation with the enzymes in one pot. The spatially separated immobilization of organometallic catalyst and enzymes on suitable carriers not only can reduce their mutual inhabitation but also can enhance their reusability. Here in this work, we present a hierarchical porous COFs (HP-TpBpy) that incorporated with [(Cp*RhCl2]2 to generate the metalized COF, Rh-HP-TpBpy. The obtained Rh-HP-TpBpy exhibited superior performance in nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) regeneration using formate as the hydride donor, significantly outperforming the natural formate dehydrogenases in cofactor preference toward NADP+. Subsequently, the Lactobacillus fermentum short-chain dehydrogenase/reductase 1 (LfSDR1) was then cross-linked into enzyme aggregates (CLEA) and immobilized on hierarchical Rh-HP-TpBpy, achieving the integrated chemoenzymatic catalyst, LfSDR1@Rh-HP-TpBpy, which can catalyze the chemoenzymatic reduction of halogenated aryl ketones and give the corresponding optically active halohydrins with high conversion and enantiomeric excess (ee) value up to 99 %. The LfSDR1@Rh-HP-TpBpy also exhibits largely enhanced stability compared with the free LfSDR1 and the CLEAs-LfSDR1, enabling its excellent reusability.
Assuntos
Enzimas Imobilizadas , Estruturas Metalorgânicas , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Estruturas Metalorgânicas/química , Catálise , NADP/química , NADP/metabolismo , Formiato Desidrogenases/química , Formiato Desidrogenases/metabolismo , NAD/química , Reagentes de Ligações Cruzadas/química , BiocatáliseRESUMO
Unfavourable conditions, such as prolonged drought and high salinity, pose a threat to the survival and agricultural yield of plants. The phytohormone ABA plays a key role in the regulation of plant stress adaptation and is often maintained at high levels for extended periods. While much is known about ABA signal perception and activation in the early signalling stage, the molecular mechanism underlying desensitization of ABA signalling remains largely unknown. Here we demonstrate that in the endoplasmic reticulum (ER)-Golgi network, the key regulators of ABA signalling, SnRK2.2/2.3, undergo N-glycosylation, which promotes their redistribution from the nucleus to the peroxisomes in Arabidopsis roots and influences the transcriptional response in the nucleus during prolonged ABA signalling. On the peroxisomal membrane, SnRK2s can interact with glucose-6-phosphate (G6P)/phosphate translocator 1 (GPT1) to maintain NADPH homeostasis through increased activity of the peroxisomal oxidative pentose phosphate pathway (OPPP). The resulting maintenance of NADPH is essential for the modulation of hydrogen peroxide (H2O2) accumulation, thereby relieving ABA-induced root growth inhibition. The subcellular dynamics of SnRK2s, mediated by N-glycosylation suggest that ABA responses transition from transcriptional regulation in the nucleus to metabolic processes in the peroxisomes, aiding plants in adapting to long-term environmental stress.
Assuntos
Ácido Abscísico , Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , NADP , Peroxissomos , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Arabidopsis/metabolismo , Arabidopsis/genética , Peroxissomos/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Glicosilação , Ácido Abscísico/metabolismo , NADP/metabolismo , Peróxido de Hidrogênio/metabolismo , Retículo Endoplasmático/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Núcleo Celular/metabolismo , Complexo de Golgi/metabolismo , Via de Pentose Fosfato , Reguladores de Crescimento de Plantas/metabolismoRESUMO
Increased fatty acid synthesis benefits glioblastoma malignancy. However, the coordinated regulation of cytosolic acetyl-CoA production, the exclusive substrate for fatty acid synthesis, remains unclear. Here, we show that proto-oncogene tyrosine kinase c-SRC is activated in glioblastoma and remodels cytosolic acetyl-CoA production for fatty acid synthesis. Firstly, acetate is an important substrate for fatty acid synthesis in glioblastoma. c-SRC phosphorylates acetyl-CoA synthetase ACSS2 at Tyr530 and Tyr562 to stimulate the conversion of acetate to acetyl-CoA in cytosol. Secondly, c-SRC inhibits citrate-derived acetyl-CoA synthesis by phosphorylating ATP-citrate lyase ACLY at Tyr682. ACLY phosphorylation shunts citrate to IDH1-catalyzed NADPH production to provide reducing equivalent for fatty acid synthesis. The c-SRC-unresponsive double-mutation of ACSS2 and ACLY significantly reduces fatty acid synthesis and hampers glioblastoma progression. In conclusion, this remodeling fulfills the dual needs of glioblastoma cells for both acetyl-CoA and NADPH in fatty acid synthesis and provides evidence for glioma treatment by c-SRC inhibition.