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1.
Mol Cell ; 81(18): 3848-3865.e19, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34547241

RESUMO

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP+. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.


Assuntos
Senescência Celular/fisiologia , NAD/metabolismo , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Animais , Linhagem Celular Tumoral , Senescência Celular/genética , Citosol , Glucose/metabolismo , Humanos , Hidrogênio/química , Hidrogênio/metabolismo , Malato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , NAD/fisiologia , Oxirredução , Piruvato Carboxilase/metabolismo , Ácido Pirúvico/metabolismo
2.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445270

RESUMO

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD+ on: (1) the Ca2+-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca2+-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca2+-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca2+-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD+ increased the CRC of liver, heart, and brain mitochondria 1.5-2.5 times, insignificantly affecting the rate of Ca2+-uptake and the free Ca2+ concentration in the medium. NAD(H) suppressed the Ca2+-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca2+-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.


Assuntos
Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , NAD/metabolismo , Animais , Cálcio/metabolismo , Fluoresceínas/metabolismo , Hepatócitos/metabolismo , Masculino , Ratos , Ratos Wistar
3.
Cell Physiol Biochem ; 55(4): 477-488, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34375044

RESUMO

BACKGROUND/AIMS: Cesium (Cs) is an alkali metal element that is of no essential use for humans; it has no known beneficial function that is verified by clinical research. When used as an alternative cancer therapy, it even causes toxicity in high doses. Thus, before using Cs as treatment in clinical settings, it is important to clearly determine its biological effects on cells. However, Cs was found to suppress the proliferation of human cervical cancer cells in a dose-dependent manner, and it was assumed that Cs inhibits the glycolysis pathway. In this study, we clearly determined the step of the glycolysis pathway that is affected by Cs. METHODS: The glycolytic enzyme expressions, activities, and metabolite concentrations in HeLa cells were measured by PCR, western blotting, and enzymatic methods, after treating the cells with Cs for 3 days. RESULTS: Cs treatment decreased transcriptional and expression levels of hexokinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase (PK), and lactate dehydrogenase and the activity of PK. Analysis of glycolysis pathway metabolites revealed that Cs treatment reduces lactate level and increases the level of nicotinamide adenine dinucleotide (oxidized form, NAD+); however, it did not affect the levels of pyruvate and nicotinamide adenine dinucleotide (reduced form, NADH). Increase of the [NAD+]/[NADH] ratio and decrease of the [lactate]/[pyruvate] ratio indicate that Cs treatment inhibits the aerobic glycolysis pathway. CONCLUSION: Cs treatment inhibits PK activity and increases the [NAD+]/[NADH] ratio. Hence, Cs has been determined to inhibit glycolysis, especially the aerobic glycolysis pathway. These results suggest that suppression of HeLa cell proliferation following Cs treatment was caused by inhibition of aerobic glycolysis by Cs.


Assuntos
Proliferação de Células/efeitos dos fármacos , Césio/farmacologia , Glicólise/efeitos dos fármacos , NAD/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HeLa , Humanos
5.
J Phys Chem B ; 125(34): 9692-9707, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34410128

RESUMO

The dynamics of polarized fluorescence in NADH in alcohol dehydrogenase (ADH) in buffer solution has been studied using the TCSPC spectroscopy. A global fit procedure was used for determination of the fluorescence parameters from experiment. The interpretation of the results obtained was supported by ab initio calculations of the NADH structure. A theoretical model was developed describing the polarized fluorescence decay in ADH-NADH complexes that considered several interaction scenarios. A comparative analysis of the polarization-insensitive fluorescence decay using multiexponential fitting models has been carried out. As shown, the origin of a significant enhancement of the decay time in the ADH-NADH complex can be attributed to the decrease of nonradiative relaxation rates in the nicotinamide ring in the conditions of the apolar binding site environment. The existence of a single decay time in the ADH-NADH complex in comparison with two decay times observed in free NADH was attributed to a single NADH unfolded conformation in the ADH binding site. Comparison of the experimental data with the theoretical model suggested the existence of an anisotropic relaxation time of about 1 ns that is related with the rotation of fluorescence transition dipole moment due to the rearrangement of the excited state NADH nuclear configuration.


Assuntos
Álcool Desidrogenase , NAD , Álcool Desidrogenase/metabolismo , Anisotropia , Sítios de Ligação , NAD/metabolismo , Espectrometria de Fluorescência
6.
Molecules ; 26(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34443607

RESUMO

Cellular conformation of reduced pyridine nucleotides NADH and NADPH sensed using autofluorescence spectroscopy is presented as a real-time metabolic indicator under pressurized conditions. The approach provides information on the role of pressure in energy metabolism and antioxidant defense with applications in agriculture and food technologies. Here, we use spectral phasor analysis on UV-excited autofluorescence from Saccharomyces cerevisiae (baker's yeast) to assess the involvement of one or multiple NADH- or NADPH-linked pathways based on the presence of two-component spectral behavior during a metabolic response. To demonstrate metabolic monitoring under pressure, we first present the autofluorescence response to cyanide (a respiratory inhibitor) at 32 MPa. Although ambient and high-pressure responses remain similar, pressure itself also induces a response that is consistent with a change in cellular redox state and ROS production. Next, as an example of an autofluorescence response altered by pressurization, we investigate the response to ethanol at ambient, 12 MPa, and 30 MPa pressure. Ethanol (another respiratory inhibitor) and cyanide induce similar responses at ambient pressure. The onset of non-two-component spectral behavior upon pressurization suggests a change in the mechanism of ethanol action. Overall, results point to new avenues of investigation in piezophysiology by providing a way of visualizing metabolism and mitochondrial function under pressurized conditions.


Assuntos
NADP/química , NADP/metabolismo , NAD/química , NAD/metabolismo , Pressão , Fluorescência , Conformação Molecular
7.
J Enzyme Inhib Med Chem ; 36(1): 1916-1921, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34461785

RESUMO

An earlier described three-component variant of the Castagnoli-Cushman reaction employing homophthalic anhydrides, carbonyl compound and ammonium acetate was applied towards the preparation of 1-oxo-3,4-dihydroisoquinoline-4-carboxamides with variable substituent in position 3. These compounds displayed inhibitory activity towards poly(ADP-ribose) polymerase (PARP), a clinically validated cancer target. The most potent compound (PARP1/2 IC50 = 22/4.0 nM) displayed the highest selectivity towards PARP2 in the series (selectivity index = 5.5), more advantageous ADME prameters compared to the clinically used PARP inhibitor Olaparib.


Assuntos
Acetatos/química , Antineoplásicos/química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Quinolonas/química , Acetatos/farmacologia , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Sítios de Ligação , Dano ao DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , NAD/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
8.
Plant Mol Biol ; 107(1-2): 63-84, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34460049

RESUMO

KEY MESSAGE: Overexpressing Nicotinamidase 3 gene, and the exogenous application of its metabolite nicotinic acid (NA), enhance drought stress tolerance and increase biomass in Arabidopsis thaliana. With progressive global climatic changes, plant productivity is threatened severely by drought stress. Deciphering the molecular mechanisms regarding genes responsible for balancing plant growth and stress amelioration could imply multiple possibilities for future sustainable goals. Nicotinamide adenine dinucleotide (NAD) biosynthesis and recycling/ distribution is a crucial feature for plant growth. The current study focuses on the functional characterization of nicotinamidase 3 (NIC3) gene, which is involved in the biochemical conversion of nicotinamide (NAM) to nicotinic acid (NA) in the salvage pathway of NAD biosynthesis. Our data show that overexpression of NIC3 gene enhances drought stress tolerance and increases plant growth. NIC3-OX plants accumulated more NA as compared to WT plants. Moreover, the upregulation of several genes related to plant growth/stress tolerance indicates that regulating the NAD salvage pathway could significantly enhance plant growth and drought stress tolerance. The exogenous application of nicotinic acid (NA) showed a similar phenotype as the effect of overexpressing NIC3 gene. In short, we contemplated the role of NIC3 gene and NA application in drought stress tolerance and plant growth. Our results would be helpful in engineering plants with enhanced drought stress tolerance and increased growth potential.


Assuntos
Adaptação Fisiológica/genética , Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Biomassa , Secas , Genes de Plantas , Niacina/farmacologia , Nicotinamidase/genética , Adaptação Fisiológica/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Modelos Biológicos , NAD/metabolismo , NADP/metabolismo , Nicotinamidase/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/fisiologia , Plantas Geneticamente Modificadas , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
9.
Elife ; 102021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34343089

RESUMO

Poly(ADP-ribose) polymerase (PARP) enzymes initiate (mt)DNA repair mechanisms and use nicotinamide adenine dinucleotide (NAD+) as energy source. Prolonged PARP activity can drain cellular NAD+ reserves, leading to de-regulation of important molecular processes. Here, we provide evidence of a pathophysiological mechanism that connects mtDNA damage to cardiac dysfunction via reduced NAD+ levels and loss of mitochondrial function and communication. Using a transgenic model, we demonstrate that high levels of mice cardiomyocyte mtDNA damage cause a reduction in NAD+ levels due to extreme DNA repair activity, causing impaired activation of NAD+-dependent SIRT3. In addition, we show that myocardial mtDNA damage in combination with high dosages of nicotinamideriboside (NR) causes an inhibition of sirtuin activity due to accumulation of nicotinamide (NAM), in addition to irregular cardiac mitochondrial morphology. Consequently, high doses of NR should be used with caution, especially when cardiomyopathic symptoms are caused by mitochondrial dysfunction and instability of mtDNA.


Assuntos
Reparo do DNA , DNA Mitocondrial/metabolismo , Cardiopatias/fisiopatologia , Coração/fisiopatologia , Miocárdio/metabolismo , NAD/metabolismo , Animais , Dano ao DNA , Células HeLa , Humanos , Camundongos , Mitocôndrias/metabolismo , Niacinamida/efeitos adversos , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Compostos de Piridínio/efeitos adversos , Sirtuínas/antagonistas & inibidores
10.
Nat Methods ; 18(9): 1091-1102, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34413523

RESUMO

Mitochondria display complex morphology and movements, which complicates their segmentation and tracking in time-lapse images. Here, we introduce Mitometer, an algorithm for fast, unbiased, and automated segmentation and tracking of mitochondria in live-cell two-dimensional and three-dimensional time-lapse images. Mitometer requires only the pixel size and the time between frames to identify mitochondrial motion and morphology, including fusion and fission events. The segmentation algorithm isolates individual mitochondria via a shape- and size-preserving background removal process. The tracking algorithm links mitochondria via differences in morphological features and displacement, followed by a gap-closing scheme. Using Mitometer, we show that mitochondria of triple-negative breast cancer cells are faster, more directional, and more elongated than those in their receptor-positive counterparts. Furthermore, we show that mitochondrial motility and morphology in breast cancer, but not in normal breast epithelia, correlate with metabolic activity. Mitometer is an unbiased and user-friendly tool that will help resolve fundamental questions regarding mitochondrial form and function.


Assuntos
Neoplasias da Mama/patologia , Imageamento Tridimensional/métodos , Mitocôndrias , Software , Imagem com Lapso de Tempo/métodos , Algoritmos , Neoplasias da Mama/metabolismo , Células Cultivadas , Feminino , Humanos , Glândulas Mamárias Humanas/citologia , Mitocôndrias/metabolismo , NAD/metabolismo , Reprodutibilidade dos Testes , Neoplasias de Mama Triplo Negativas/patologia
11.
Nat Commun ; 12(1): 5001, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408151

RESUMO

As a basic structure of most polypyridinal metal complexes, [Ru(bpy)3]2+, has the advantages of simple structure, facile synthesis and high yield, which has great potential for scientific research and application. However, sonodynamic therapy (SDT) performance of [Ru(bpy)3]2+ has not been investigated so far. SDT can overcome the tissue-penetration and phototoxicity problems compared to photodynamic therapy. Here, we report that [Ru(bpy)3]2+ is a highly potent sonosensitizer and sonocatalyst for sonotherapy in vitro and in vivo. [Ru(bpy)3]2+ can produce singlet oxygen (1O2) and sono-oxidize endogenous 1,4-dihydronicotinamide adenine dinucleotide (NADH) under ultrasound (US) stimulation in cancer cells. Furthermore, [Ru(bpy)3]2+ enables effective destruction of mice tumors, and the therapeutic effect can reach deep tissues over 10 cm under US irradiation. This work paves a way for polypyridinal metal complexes to be applied to the noninvasive precise sonotherapy of cancer.


Assuntos
Antineoplásicos/química , Neoplasias/terapia , Rutênio/química , Terapia por Ultrassom , Animais , Antineoplásicos/administração & dosagem , Humanos , Camundongos , Camundongos Endogâmicos BALB C , NAD/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Oxirredução/efeitos da radiação , Porfirinas/química , Rutênio/administração & dosagem , Oxigênio Singlete/metabolismo , Ondas Ultrassônicas
12.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445787

RESUMO

Mechanical stress is an important factor affecting bone tissue homeostasis. We focused on the interactions among mechanical stress, glucose uptake via glucose transporter 1 (Glut1), and the cellular energy sensor sirtuin 1 (SIRT1) in osteoblast energy metabolism, since it has been recognized that SIRT1, an NAD+-dependent deacetylase, may function as a master regulator of the mechanical stress response as well as of cellular energy metabolism (glucose metabolism). In addition, it has already been demonstrated that SIRT1 regulates the activity of the osteogenic transcription factor runt-related transcription factor 2 (Runx2). The effects of mechanical loading on cellular activities and the expressions of Glut1, SIRT1, and Runx2 were evaluated in osteoblasts and chondrocytes in a 3D cell-collagen sponge construct. Compressive mechanical loading increased osteoblast activity. Mechanical loading also significantly increased the expression of Glut1, significantly decreased the expression of SIRT1, and significantly increased the expression of Runx2 in osteoblasts in comparison with non-loaded osteoblasts. Incubation with a Glut1 inhibitor blocked mechanical stress-induced changes in SIRT1 and Runx2 in osteoblasts. In contrast with osteoblasts, the expressions of Glut1, SIRT1, and Runx2 in chondrocytes were not affected by loading. Our present study indicated that mechanical stress induced the upregulation of Glut1 following the downregulation of SIRT1 and the upregulation of Runx2 in osteoblasts but not in chondrocytes. Since SIRT1 is known to negatively regulate Runx2 activity, a mechanical stress-induced downregulation of SIRT1 may lead to the upregulation of Runx2, resulting in osteoblast differentiation. Incubation with a Glut1 inhibitor the blocked mechanical stress-induced downregulation of SIRT1 following the upregulation of Runx2, suggesting that Glut1 is necessary to mediate the responses of SIRT1 and Runx2 to mechanical loading in osteoblasts.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Transdução de Sinais/fisiologia , Sirtuína 1/metabolismo , Idoso , Diferenciação Celular/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , Humanos , NAD/metabolismo , Estresse Mecânico
13.
Int J Mol Sci ; 22(16)2021 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-34445751

RESUMO

Sarcopenia, the age-related decline in muscle mass and function, derives from multiple etiological mechanisms. Accumulative research suggests that reactive oxygen species (ROS) generation plays a critical role in the development of this pathophysiological disorder. In this communication, we review the various signaling pathways that control muscle metabolic and functional integrity such as protein turnover, cell death and regeneration, inflammation, organismic damage, and metabolic functions. Although no single pathway can be identified as the most crucial factor that causes sarcopenia, age-associated dysregulation of redox signaling appears to underlie many deteriorations at physiological, subcellular, and molecular levels. Furthermore, discord of mitochondrial homeostasis with aging affects most observed problems and requires our attention. The search for the primary suspect of the fundamental mechanism for sarcopenia will likely take more intense research for the secret of this health hazard to the elderly to be unlocked.


Assuntos
Proteínas Musculares/metabolismo , Estresse Oxidativo , Sarcopenia/etiologia , Sarcopenia/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose , Homeostase , Humanos , Inflamação/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Junção Neuromuscular/metabolismo , Oxirredução , Peroxirredoxinas/metabolismo , Regeneração
14.
Nat Metab ; 3(8): 1109-1124, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34385701

RESUMO

Zika virus (ZIKV) infection during pregnancy can cause microcephaly in newborns, yet the underlying mechanisms remain largely unexplored. Here, we reveal extensive and large-scale metabolic reprogramming events in ZIKV-infected mouse brains by performing a multi-omics study comprising transcriptomics, proteomics, phosphoproteomics and metabolomics approaches. Our proteomics and metabolomics analyses uncover dramatic alteration of nicotinamide adenine dinucleotide (NAD+)-related metabolic pathways, including oxidative phosphorylation, TCA cycle and tryptophan metabolism. Phosphoproteomics analysis indicates that MAPK and cyclic GMP-protein kinase G signaling may be associated with ZIKV-induced microcephaly. Notably, we demonstrate the utility of our rich multi-omics datasets with follow-up in vivo experiments, which confirm that boosting NAD+ by NAD+ or nicotinamide riboside supplementation alleviates cell death and increases cortex thickness in ZIKV-infected mouse brains. Nicotinamide riboside supplementation increases the brain and body weight as well as improves the survival in ZIKV-infected mice. Our study provides a comprehensive resource of biological data to support future investigations of ZIKV-induced microcephaly and demonstrates that metabolic alterations can be potentially exploited for developing therapeutic strategies.


Assuntos
Microcefalia/etiologia , Microcefalia/metabolismo , NAD/metabolismo , Infecção por Zika virus/complicações , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/virologia , Células Cultivadas , Cromatografia Líquida , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Metabolômica , Camundongos , Microcefalia/patologia , Neurônios/metabolismo , Gravidez , Proteômica/métodos , Espectrometria de Massas em Tandem
15.
Biosens Bioelectron ; 193: 113573, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34425520

RESUMO

NAD+-dependent dehydrogenase-based biosensors usually suffer from the low accuracy due to the interference of cofactors in the complex environment, such as fermentation samples. Herein, we demonstrate the example of an integrated biosensor device that can be applied for analyzing xylose fermentation samples. The device is composed of one chamber for the elimination of NAD+ and NADH in the fermentation broth and another chamber for the sample analysis. In the first chamber, a cyclic voltammetry method coupled with Ni foam as a working electrode was proven to be effective in removing NAD+ and NADH in the fermentation broth. In the other chamber, xylose dehydrogenase, as the recognition element, and diaphorase, used for the regeneration of bioactive NAD+ mediated by vitamin K3, were co-immobilized on the surface of the magnetic nanoparticles, which was further coated onto a magnetic glassy carbon electrode. The detection range of the constructed biosensor was from 0.5 to 10 g L-1 with a detection limit of 0.01 g L-1 at a signal-to-noise ratio of 3. Moreover, the biosensor achieved high selectivity, recovery, reproducibility, and good long-time stability when analyzing real xylose fermentation samples, suggesting its promising application potential.


Assuntos
Técnicas Biossensoriais , Fermentação , NAD/metabolismo , Oxirredutases , Reprodutibilidade dos Testes , Xilose
16.
Trends Plant Sci ; 26(10): 1072-1086, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34281784

RESUMO

The pyridine nucleotides nicotinamide adenine dinucleotide [NAD(H)] and nicotinamide adenine dinucleotide phosphate [NADP(H)] simultaneously act as energy transducers, signalling molecules, and redox couples. Recent research into photosynthetic optimisation, photorespiration, immunity, hypoxia/oxygen signalling, development, and post-harvest metabolism have all identified pyridine nucleotides as key metabolites. Further understanding will require accurate description of NAD(P)(H) metabolism, and genetically encoded fluorescent biosensors have recently become available for this purpose. Although these biosensors have begun to provide novel biological insights, their limitations must be considered and the information they provide appropriately interpreted. We provide a framework for understanding NAD(P)(H) metabolism and explore what fluorescent biosensors can, and cannot, tell us about plant biology, looking ahead to the pressing questions that could be answered with further development of these tools.


Assuntos
Metabolismo Energético , NADP , NAD , Plantas/metabolismo , NAD/metabolismo , NADP/metabolismo , Oxirredução , Transdução de Sinais
17.
Biomolecules ; 11(6)2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198503

RESUMO

Subcellular compartmentation is a fundamental property of eukaryotic cells. Communication and metabolic and regulatory interconnectivity between organelles require that solutes can be transported across their surrounding membranes. Indeed, in mammals, there are hundreds of genes encoding solute carriers (SLCs) which mediate the selective transport of molecules such as nucleotides, amino acids, and sugars across biological membranes. Research over many years has identified the localization and preferred substrates of a large variety of SLCs. Of particular interest has been the SLC25 family, which includes carriers embedded in the inner membrane of mitochondria to secure the supply of these organelles with major metabolic intermediates and coenzymes. The substrate specificity of many of these carriers has been established in the past. However, the route by which animal mitochondria are supplied with NAD+ had long remained obscure. Only just recently, the existence of a human mitochondrial NAD+ carrier was firmly established. With the realization that SLC25A51 (or MCART1) represents the major mitochondrial NAD+ carrier in mammals, a long-standing mystery in NAD+ biology has been resolved. Here, we summarize the functional importance and structural features of this carrier as well as the key observations leading to its discovery.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , NAD/metabolismo , Proteínas Carreadoras de Solutos/metabolismo , Transporte Biológico/genética , Humanos , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , NAD/genética , Proteínas Carreadoras de Solutos/genética
18.
Int J Mol Sci ; 22(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204595

RESUMO

Among all the proposed pathogenic mechanisms to understand the etiology of Alzheimer's disease (AD), increased oxidative stress seems to be a robust and early disease feature where many of those hypotheses converge. However, despite the significant lines of evidence accumulated, an effective diagnosis and treatment of AD are not yet available. This limitation might be partially explained by the use of cellular and animal models that recapitulate partial aspects of the disease and do not account for the particular biology of patients. As such, cultures of patient-derived cells of peripheral origin may provide a convenient solution for this problem. Peripheral cells of neuronal lineage such as olfactory neuronal precursors (ONPs) can be easily cultured through non-invasive isolation, reproducing AD-related oxidative stress. Interestingly, the autofluorescence of key metabolic cofactors such as reduced nicotinamide adenine dinucleotide (NADH) can be highly correlated with the oxidative state and antioxidant capacity of cells in a non-destructive and label-free manner. In particular, imaging NADH through fluorescence lifetime imaging microscopy (FLIM) has greatly improved the sensitivity in detecting oxidative shifts with minimal intervention to cell physiology. Here, we discuss the translational potential of analyzing patient-derived ONPs non-invasively isolated through NADH FLIM to reveal AD-related oxidative stress. We believe this approach may potentially accelerate the discovery of effective antioxidant therapies and contribute to early diagnosis and personalized monitoring of this devastating disease.


Assuntos
Doença de Alzheimer/patologia , Microscopia de Fluorescência/métodos , NAD/metabolismo , Neurônios Receptores Olfatórios/patologia , Estresse Oxidativo , Animais , Antioxidantes/metabolismo , Humanos
19.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200964

RESUMO

For osteosarcoma (OS), the most common primary malignant bone tumor, overall survival has hardly improved over the last four decades. Especially for metastatic OS, novel therapeutic targets are urgently needed. A hallmark of cancer is aberrant metabolism, which justifies targeting metabolic pathways as a promising therapeutic strategy. One of these metabolic pathways, the NAD+ synthesis pathway, can be considered as a potential target for OS treatment. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the classical salvage pathway for NAD+ synthesis, and NAMPT is overexpressed in OS. In this study, five OS cell lines were treated with the NAMPT inhibitor FK866, which was shown to decrease nuclei count in a 2D in vitro model without inducing caspase-driven apoptosis. The reduction in cell viability by FK866 was confirmed in a 3D model of OS cell lines (n = 3). Interestingly, only OS cells with low nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1) RNA expression were sensitive to NAMPT inhibition. Using a publicly available (Therapeutically Applicable Research to Generate Effective Treatments (TARGET)) and a previously published dataset, it was shown that in OS cell lines and primary tumors, low NAPRT1 RNA expression correlated with NAPRT1 methylation around the transcription start site. These results suggest that targeting NAMPT in osteosarcoma could be considered as a novel therapeutic strategy, where low NAPRT expression can serve as a biomarker for the selection of eligible patients.


Assuntos
Acrilamidas/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glioma/tratamento farmacológico , NAD/metabolismo , Osteossarcoma/tratamento farmacológico , Pentosiltransferases/antagonistas & inibidores , Piperidinas/farmacologia , Apoptose , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proliferação de Células , Glioma/metabolismo , Glioma/patologia , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Células Tumorais Cultivadas
20.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202251

RESUMO

Dysregulated cross-talk between immune cells and epithelial compartments is responsible for the onset and amplification of pathogenic auto-inflammatory circuits occurring in psoriasis. NAMPT-mediated NAD salvage pathway has been recently described as an immunometabolic route having inflammatory function in several disorders, including arthritis and inflammatory bowel diseases. To date, the role of NAD salvage pathway has not been explored in the skin of patients affected by psoriasis. Here, we show that NAD content is enhanced in lesional skin of psoriatic patients and is associated to high NAMPT transcriptional levels. The latter are drastically reduced in psoriatic skin following treatment with the anti-IL-17A biologics secukinumab. We provide evidence that NAMPT-mediated NAD+ metabolism fuels the immune responses executed by resident skin cells in psoriatic skin. In particular, intracellular NAMPT, strongly induced by Th1/Th17-cytokines, acts on keratinocytes by inducing hyper-proliferation and impairing their terminal differentiation. Furthermore, NAMPT-mediated NAD+ boosting synergizes with psoriasis-related cytokines in the upregulation of inflammatory chemokines important for neutrophil and Th1/Th17 cell recruitment. In addition, extracellular NAMPT, abundantly released by keratinocytes and dermal fibroblasts, acts in a paracrine manner on endothelial cells by inducing their proliferation and migration, as well as the expression of ICAM-1 membrane molecule and chemokines important for leukocyte recruitment into inflamed skin. In conclusion, our results showed that NAMPT-mediated NAD salvage pathway contributes to psoriasis pathogenic processes by amplifying epithelial auto-inflammatory responses in psoriasis.


Assuntos
Citocinas/genética , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Psoríase/etiologia , Psoríase/metabolismo , Transdução de Sinais , Adulto , Idoso , Biomarcadores , Citocinas/metabolismo , Suscetibilidade a Doenças , Células Endoteliais/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Queratinócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Nicotinamida Fosforribosiltransferase/metabolismo , Psoríase/patologia
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