RESUMEN

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.

Asunto(s)

Fermentación , Oxidación-Reducción , Oxígeno , Saccharomyces cerevisiae , Vino , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Oxígeno/metabolismo , Vino/microbiología , Vino/análisis , Anaerobiosis , Vitis/microbiología , Vitis/metabolismo , NAD/metabolismo , Etanol/metabolismo , NADP/metabolismo , Aerobiosis , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Coenzimas/metabolismo

RESUMEN

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.

Asunto(s)

Ingeniería Metabólica , Ácido Mevalónico , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ingeniería Metabólica/métodos , Ácido Mevalónico/metabolismo , Alquenos/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Diterpenos/metabolismo , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , NADP/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sesquiterpenos

RESUMEN

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.

Asunto(s)

Señalización del Calcio , Calcio , NADP , NADP/análogos & derivados , NADP/metabolismo , Animales , Humanos , Calcio/metabolismo , Ratones , Sistemas de Mensajero Secundario , Permeabilidad de la Membrana Celular , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Células Asesinas Naturales/metabolismo , Linfocitos T/metabolismo

RESUMEN

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.

Asunto(s)

Fermentación , Homeostasis , NADP , Oxidación-Reducción , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , NADP/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Glutatión/metabolismo , Vía de Pentosa Fosfato

RESUMEN

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.

Asunto(s)

NAD , NAD/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Oxigenasas/metabolismo , Oxigenasas/química , Oxigenasas/aislamiento & purificación , Cristalografía por Rayos X/métodos , Complejo III de Transporte de Electrones/metabolismo , Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/aislamiento & purificación , NADP/metabolismo

RESUMEN

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.

Asunto(s)

Acetilcoenzima A , Ácidos Grasos , Glioblastoma , Proto-Oncogenes Mas , Glioblastoma/metabolismo , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ácidos Grasos/metabolismo , Ácidos Grasos/biosíntesis , Línea Celular Tumoral , Fosforilación , Acetilcoenzima A/metabolismo , Animales , Proteína Tirosina Quinasa CSK/metabolismo , Proteína Tirosina Quinasa CSK/genética , Familia-src Quinasas/metabolismo , Familia-src Quinasas/genética , Progresión de la Enfermedad , Ratones , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , NADP/metabolismo , Ratones Desnudos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo

RESUMEN

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.

Asunto(s)

Ácido Abscísico , Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , NADP , Peroxisomas , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Arabidopsis/metabolismo , Arabidopsis/genética , Peroxisomas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Glicosilación , Ácido Abscísico/metabolismo , NADP/metabolismo , Peróxido de Hidrógeno/metabolismo , Retículo Endoplásmico/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Núcleo Celular/metabolismo , Aparato de Golgi/metabolismo , Vía de Pentosa Fosfato , Reguladores del Crecimiento de las Plantas/metabolismo

RESUMEN

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 2.5 Šresolution 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.

Asunto(s)

Isomerasas Aldosa-Cetosa , Fosfomicina , Complejos Multienzimáticos , Toxoplasma , Toxoplasma/enzimología , Toxoplasma/efectos de los fármacos , Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/metabolismo , Isomerasas Aldosa-Cetosa/genética , Fosfomicina/farmacología , Fosfomicina/análogos & derivados , Fosfomicina/química , Cristalografía por Rayos X , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , NADP/metabolismo , NADP/química , Humanos , Modelos Moleculares , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/química , Oxidorreductasas/metabolismo

RESUMEN

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.

Asunto(s)

Daño del ADN , NADP , Mononucleótido de Nicotinamida , Sirtuina 2 , Regulación hacia Arriba , Humanos , Sirtuina 2/metabolismo , Sirtuina 2/genética , Células HeLa , NADP/metabolismo , Daño del ADN/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Mononucleótido de Nicotinamida/farmacología , Mononucleótido de Nicotinamida/metabolismo , Cobre/farmacología , Cobre/metabolismo , Sirtuinas/metabolismo

RESUMEN

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.

Asunto(s)

Norisoprenoides , Norisoprenoides/química , Norisoprenoides/metabolismo , Deshidrogenasas-Reductasas de Cadena Corta/metabolismo , Deshidrogenasas-Reductasas de Cadena Corta/química , Sphingomonadaceae/enzimología , NADP/metabolismo , Oxidorreductasas/metabolismo

Asunto(s)

Lisosomas , Lisosomas/metabolismo , Humanos , Animales , Transducción de Señal , Mecanotransducción Celular , Anoctaminas/metabolismo , NADP/análogos & derivados

RESUMEN

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.

Asunto(s)

Enzimas Inmovilizadas , Estructuras Metalorgánicas , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Estructuras Metalorgánicas/química , Catálisis , NADP/química , NADP/metabolismo , Formiato Deshidrogenasas/química , Formiato Deshidrogenasas/metabolismo , NAD/química , Reactivos de Enlaces Cruzados/química , Biocatálisis

RESUMEN

Enzyme specificity towards cofactors like NAD(P)H is crucial for applications in bioremediation and eco-friendly chemical synthesis. Despite their role in converting pollutants and creating sustainable products, predicting enzyme specificity faces challenges due to sparse data and inadequate models. To bridge this gap, we developed the cutting-edge INSIGHT platform to enhance the prediction of coenzyme specificity in NAD(P)-dependent enzymes. INSIGHT integrates extensive data from principal bioinformatics resources, concentrating on both NADH and NADPH specificities, and utilizes advanced protein language models to refine the predictions. This integration not only strengthens computational predictions but also meets the practical demands of high-throughput screening and optimization. Experimental validation confirms INSIGHT's effectiveness, boosting our ability to engineer enzymes for efficient, sustainable industrial and environmental processes. This work advances the practical use of computational tools in enzyme research, addressing industrial needs and offering scalable solutions for environmental challenges.

Asunto(s)

NADP , NAD , Ingeniería de Proteínas , NADP/metabolismo , NADP/química , Especificidad por Sustrato , NAD/metabolismo , NAD/química , Ingeniería de Proteínas/métodos , Biología Computacional/métodos , Modelos Moleculares , Coenzimas/metabolismo , Coenzimas/química

RESUMEN

Glucose-6-phosphate dehydrogenase (G6PD) is a promising target in cancer therapy. However, poor cellular uptake and off-target toxicity have impeded the clinical translation of a canonical G6PD inhibitor (6-aminonicotinamide/6AN). Here, we report a prodrug strategy to address this issue. The tailored 6AN prodrug contains an azo-bearing protection moiety. The hydrophobic prodrug showed increased cellular uptake than 6AN and was vulnerable to hypoxia, resulting in NAD(P)H quinone dehydrogenase 1 (NQO1)-triggered cleavage of azo bonds. Intriguingly, the prodrug showed configuration-dependent anti-cancer potency. Despite the lower thermodynamic stability, the cis isomer showed enhanced cellular uptake compared to the trans counterpart due to the increased aqueous solubility. Moreover, the boosted potency of the cis isomer compared to the trans isomer arose from the enhancement of NOQ1-catalyzed 6AN release under hypoxia, a hallmark of solid tumors. The discovery of hypoxia-responsive 6AN prodrugs in the current work opens up new avenues for G6PD-targeting cancer medicines.

Asunto(s)

6-Aminonicotinamida , Antineoplásicos , NADP , Oxidación-Reducción , Profármacos , Profármacos/química , Profármacos/farmacología , Profármacos/síntesis química , Humanos , 6-Aminonicotinamida/farmacología , 6-Aminonicotinamida/química , NADP/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Proliferación Celular/efectos de los fármacos , Estructura Molecular , Glucosafosfato Deshidrogenasa/metabolismo , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Hipoxia de la Célula/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales

RESUMEN

NADPH, the primary source of reducing equivalents in the cytosol, is used in vertebrate rod photoreceptor outer segments to reduce the all-trans retinal released from photoactivated visual pigment to all-trans retinol. Light activation of the visual pigment isomerizes the 11-cis retinal chromophore to all-trans, thereby destroying it and necessitating its regeneration. Release and reduction of all-trans retinal are the first steps in the series of reactions that regenerate the visual pigment. Glucose and glutamine can both support the reduction of all-trans retinal to retinol, indicating that the NADPH used in rod photoreceptor outer segments can be generated by the pentose phosphate pathway as well as by mitochondria-linked pathways. We have used the conversion of all-trans retinal to all-trans retinol to examine whether amino acids other than glutamine can also support the generation of NADPH in rod photoreceptors. We have measured this conversion in single isolated mouse rod photoreceptors by imaging the fluorescence of the all-trans retinal and retinol generated after exposure of the cells to light. In agreement with previous work, we find that 5 mM glucose or 0.5 mM glutamine support the conversion of ∼70-80% of all-trans retinal to retinol, corresponding to a reduced NADP fraction of ∼10%. All other amino acids at 0.5 mM concentration support the conversion to a much lesser extent, indicating reduced NADP fractions of 1-2% at most. Taurine was also ineffective at supporting NADPH generation, while formic acid, the toxic metabolite of methanol, suppressed the generation of NADPH by either glucose or glutamine.

Asunto(s)

Glutamina , Ratones Endogámicos C57BL , NADP , Células Fotorreceptoras Retinianas Bastones , Vitamina A , Animales , NADP/metabolismo , Ratones , Glutamina/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Vitamina A/metabolismo , Retinaldehído/metabolismo , Glucosa/metabolismo

RESUMEN

The influence of weak radio-frequency electromagnetic field (RF-EMF) on living organisms raises new concern because of the Industrial, Scientific, and Medical (ISM) frequency band at 6.78 MHz being promoted by the AirFuel Alliance for mid-range wireless power transfer (WPT) applications and product development. Human exposure to the RF-EMF radiation is unavoidable. In this study, we employed in vitro cell culture and molecular biology approach coupled with integrated transcriptomic and proteomic analyses to uncover the effects of RF-EMF on cells at molecular and cellular levels. Our study has demonstrated that weak RF-EMF is sufficient to exert non-thermal effects on human umbilical vein endothelial cells (HUVEC). Exposure of weak RF-EMF promotes cell proliferation, inhibits apoptosis and deregulates ROS balance. Alteration of several signaling pathways and key enzymes involved in NADPH metabolism, cell proliferation and ferroptosis were identified. Our current study provide solid evidence for the first time that the present safety standards that solely considered the thermal effect of RF-EMF on cell tissue are inadequate, prompt response and modification of existing Guidelines, Standards and Regulation are warranted.

Asunto(s)

Apoptosis , Proliferación Celular , Campos Electromagnéticos , Células Endoteliales de la Vena Umbilical Humana , NADP , Ondas de Radio , Especies Reactivas de Oxígeno , Humanos , Especies Reactivas de Oxígeno/metabolismo , NADP/metabolismo , Ondas de Radio/efectos adversos , Campos Electromagnéticos/efectos adversos , Transducción de Señal

RESUMEN

Heterogeneity of tumor metabolism is an important, but still poorly understood aspect of tumor biology. Present work is focused on the visualization and quantification of cellular metabolic heterogeneity of colorectal cancer using fluorescence lifetime imaging (FLIM) of redox cofactor NAD(P)H. FLIM-microscopy of NAD(P)H was performed in vitro in four cancer cell lines (HT29, HCT116, CaCo2 and CT26), in vivo in the four types of colorectal tumors in mice and ex vivo in patients' tumor samples. The dispersion and bimodality of the decay parameters were evaluated to quantify the intercellular metabolic heterogeneity. Our results demonstrate that patients' colorectal tumors have significantly higher heterogeneity of energy metabolism compared with cultured cells and tumor xenografts, which was displayed as a wider and frequently bimodal distribution of a contribution of a free (glycolytic) fraction of NAD(P)H within a sample. Among patients' tumors, the dispersion was larger in the high-grade and early stage ones, without, however, any association with bimodality. These results indicate that cell-level metabolic heterogeneity assessed from NAD(P)H FLIM has a potential to become a clinical prognostic factor.

Asunto(s)

Neoplasias Colorrectales , NADP , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Humanos , Animales , Ratones , NADP/metabolismo , Línea Celular Tumoral , Imagen Óptica/métodos , Metabolismo Energético

RESUMEN

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a crucial cofactor in metabolic networks. The efficient regeneration of NADPH is one of the limiting factors for productivity in biotransformation processes. To date, many metabolic engineering tools and static regulation strategies have been developed to regulate NADPH regeneration. However, traditional static regulation methods often lead to the NADPH/NADP+ imbalance, causing disruptions in cell growth and production. These methods also fail to provide real-time monitoring of intracellular NADP(H) or NADPH/NADP+ levels. In recent years, various biosensors have been developed for the detection, monitoring, and dynamic regulate of the intracellular NADP(H) levels or the NADPH/NADP+ balance. These NADPH-related biosensors are mainly used in the cofactor engineering of bacteria, yeast, and mammalian cells. This review analyzes and summarizes the NADPH metabolic regulation strategies from both static and dynamic perspectives, highlighting current challenges and potential solutions, and discusses future directions for the advanced regulation of the NADPH/NADP+ balance.

Asunto(s)

Técnicas Biosensibles , Ingeniería Metabólica , NADP , NADP/metabolismo , Ingeniería Metabólica/métodos , Técnicas Biosensibles/métodos , Humanos , Animales , Redes y Vías Metabólicas

RESUMEN

Calcium ions (Ca2+) play a vital role as intracellular messengers, regulating essential cellular processes. Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as a potent second messenger, responsible for releasing Ca2+ in both mammals and echinoderms. Despite identification of two human NAADP receptor proteins, their counterparts in sea urchins remain elusive. Sea urchin NAADP binding proteins are important due to their unique identities and NAADP binding properties which may illuminate new signaling modalities in other species. Consequently, the development of new photoactive and clickable NAADP analogs with specificity for binding targets in sea urchin egg homogenates is a priority. We designed and synthesized diazirine-AIOC-NAADP, a photoactive and "clickable" NAADP analog, to specifically label and identify sea urchin NAADP receptors. This analog, synthesized using a chemo-enzymatic approach, induced Ca2+ release from sea urchin egg homogenates at low-micromolar concentrations. The ability of diazirine-AIOC-NAADP to mobilize Ca2+ in cultured human cells was investigated by microinjection of the probe into U2OS cells. Microinjected NAADP elicited a robust Ca2+ release, but even 6000-fold higher concentrations of diazirine-AIOC-NAADP were unable to release Ca2+. Our results indicate that our new probe is specifically recognized at low concentration by sea urchin egg NAADP receptors but not by the NAADP receptors in a human cultured cell line.

Asunto(s)

Química Clic , Diazometano , NADP , Erizos de Mar , Animales , NADP/análogos & derivados , NADP/metabolismo , Erizos de Mar/metabolismo , Diazometano/análogos & derivados , Diazometano/química , Calcio/metabolismo , Humanos , Unión Proteica

RESUMEN

1,4-diaminobutane is widely used in the industrial production of polymers, pharmaceuticals, agrochemicals and surfactants. Owing to economic and environmental concerns, there has been a growing interest in using microbes to produce 1,4-diaminobutane. However, there is lack of research on the influence of cofactors pyridoxal phosphate (PLP) and NADPH on the synthesis of 1,4-diaminobutane. PLP serves as a cofactor of ornithine decarboxylase in the synthesis of 1,4-diaminobutane. Additionally, the synthesis of 1 mol 1,4-diaminobutane requires 2 mol NADPH, thus necessitating consideration of NADPH balance in the efficient synthesis of 1,4-diaminobutane by Escherichia coli. The aim of this study was to enhance the synthesis efficiency of 1,4-diaminobutane through increasing production of PLP and NADPH. By optimizing the expression of the genes associated with synthesis of PLP and NADPH in E. coli, cellular PLP and NADPH levels increased, and the yield of 1,4-diaminobutane also increased accordingly. Ultimately, using glucose as the primary carbon source, the yield of 1,4-diaminobutane in the recombinant strain NAP19 reached 272 mg/L·DCW, by increased 79% compared with its chassis strain.

Asunto(s)

Escherichia coli , NADP , Fosfato de Piridoxal , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfato de Piridoxal/metabolismo , NADP/metabolismo , Glucosa/metabolismo , Ingeniería Metabólica/métodos