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1.
Biochemistry (Mosc) ; 89(2): 241-256, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38622093

RESUMO

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.


Assuntos
Oxirredutases , Vibrio , Oxirredutases/metabolismo , NAD/metabolismo , Cinamatos , Oxirredução , Vibrio/genética , Vibrio/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADH Desidrogenase/metabolismo , Flavinas/química , Transferases , Flavina-Adenina Dinucleotídeo/metabolismo
2.
FEMS Yeast Res ; 242024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38587863

RESUMO

Previously, we reported an engineered Saccharomyces cerevisiae CEN.PK113-1A derivative able to produce succinic acid (SA) from glycerol with net CO2 fixation. Apart from an engineered glycerol utilization pathway that generates NADH, the strain was equipped with the NADH-dependent reductive branch of the TCA cycle (rTCA) and a heterologous SA exporter. However, the results indicated that a significant amount of carbon still entered the CO2-releasing oxidative TCA cycle. The current study aimed to tune down the flux through the oxidative TCA cycle by targeting the mitochondrial uptake of pyruvate and cytosolic intermediates of the rTCA pathway, as well as the succinate dehydrogenase complex. Thus, we tested the effects of deletions of MPC1, MPC3, OAC1, DIC1, SFC1, and SDH1 on SA production. The highest improvement was achieved by the combined deletion of MPC3 and SDH1. The respective strain produced up to 45.5 g/L of SA, reached a maximum SA yield of 0.66 gSA/gglycerol, and accumulated the lowest amounts of byproducts when cultivated in shake-flasks. Based on the obtained data, we consider a further reduction of mitochondrial import of pyruvate and rTCA intermediates highly attractive. Moreover, the approaches presented in the current study might also be valuable for improving SA production when sugars (instead of glycerol) are the source of carbon.


Assuntos
Saccharomyces cerevisiae , Ácido Succínico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Succínico/metabolismo , Glicerol/metabolismo , Dióxido de Carbono/metabolismo , NAD/metabolismo , Ácido Pirúvico/metabolismo , Membranas Mitocondriais/metabolismo , Carbono/metabolismo , Engenharia Metabólica/métodos
3.
Commun Biol ; 7(1): 428, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38594590

RESUMO

NADH autofluorescence imaging is a promising approach for visualizing energy metabolism at the single-cell level. However, it is sensitive to the redox ratio and the total NAD(H) amount, which can change independently from each other, for example with aging. Here, we evaluate the potential of fluorescence lifetime imaging microscopy (FLIM) of NADH to differentiate between these modalities.We perform targeted modifications of the NAD(H) pool size and ratio in cells and mice and assess the impact on NADH FLIM. We show that NADH FLIM is sensitive to NAD(H) pool size, mimicking the effect of redox alterations. However, individual components of the fluorescence lifetime are differently impacted by redox versus pool size changes, allowing us to distinguish both modalities using only FLIM. Our results emphasize NADH FLIM's potential for evaluating cellular metabolism and relative NAD(H) levels with high spatial resolution, providing a crucial tool for our understanding of aging and metabolism.


Assuntos
Metabolismo Energético , NAD , Camundongos , Animais , NAD/metabolismo , Microscopia de Fluorescência , Oxirredução , Envelhecimento
4.
Sci Rep ; 14(1): 8355, 2024 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-38594438

RESUMO

Infections with dengue virus (DENV) remain a worldwide public health problem. A number of bona fide cellular targets of DENV have been identified including liver cells. Despite the many lines of evidence confirming the involvement of hepatocytes during DENV infection, only a few studies have used proteomic analysis to understand the modulation of the cellular proteome occurring upon DENV infection. We utilized a 2D-gel electrophoresis analysis to identify proteins that were differentially regulated by DENV 2 infection of liver (Hep3B) cells at 12 h post infection (hpi) and at 48 hpi. The analysis identifies 4 proteins differentially expressed at 12 hpi, and 14 differentially regulated at 48 hpi. One candidate protein identified as downregulated at 48 hpi in the proteomic analysis (GAPDH) was validated in western blotting in Hep3B cells, and subsequently in induced pluripotent stem cell (iPSC) derived human hepatocytes. The reduced expression of GAPDH was coupled with an increase in NADH, and a significantly reduced NAD + /NADH ratio, strongly suggesting that glycolysis is down regulated in response to DENV 2 infection. Metformin, a well characterized drug used in the treatment of diabetes mellitus, is an inhibitor of hepatic gluconeogenesis was shown to reduce the level of DENV 2 infection and new virus production. Collectively these results show that although glycolysis is reduced, glucose is still required, possibly for use by the pentose phosphate pathway to generate nucleosides required for viral replication.


Assuntos
Vírus da Dengue , Dengue , Humanos , Vírus da Dengue/fisiologia , Proteômica , NAD/metabolismo , Hepatócitos/metabolismo , Glicólise , Fígado/metabolismo , Replicação Viral , Proteoma/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo
5.
Endocrinology ; 165(5)2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38565429

RESUMO

NAD+ is one of the most important metabolites for cellular activities, and its biosynthesis mainly occurs through the salvage pathway using the nicotinamide phosphoribosyl transferase (NAMPT) enzyme. The main nicotinamide adenine dinucleotide (NAD) consumers, poly-ADP-ribose-polymerases and sirtuins enzymes, are heavily involved in DNA repair and chromatin remodeling. Since cancer cells shift their energy production pathway, NAD levels are significantly affected. NAD's roles in cell survival led to the use of NAD depletion in cancer therapies. NAMPT inhibition (alone or in combination with other cancer therapies, including endocrine therapy and chemotherapy) results in decreased cell viability and tumor burden for many cancer types. Many NAMPT inhibitors (NAMPTi) tested before were discontinued due to toxicity; however, a novel NAMPTi, KPT-9274, is a promising, low-toxicity option currently in clinical trials.


Assuntos
Neoplasias , Sirtuínas , Humanos , NAD/metabolismo , Citocinas/metabolismo , Neoplasias/tratamento farmacológico , Reparo do DNA , Sirtuínas/genética
6.
Elife ; 122024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38567911

RESUMO

The antibiotic heliomycin (resistomycin), which is generated from Streptomyces resistomycificus, has multiple activities, including anticancer effects. Heliomycin was first described in the 1960s, but its clinical applications have been hindered by extremely low solubility. A series of 4-aminomethyl derivatives of heliomycin were synthesized to increase water solubility; studies showed that they had anti-proliferative effects, but the drug targets remained unknown. In this study, we conducted cellular thermal shift assays (CETSA) and molecular docking simulations to identify and validate that heliomycin and its water-soluble derivative, 4-(dimethylaminomethyl)heliomycin (designated compound 4-dmH) engaged and targeted with sirtuin-1 (SIRT1) in p53-functional SAS and p53-mutated HSC-3 oral cancer cells. We further addressed the cellular outcome of SIRT1 inhibition by these compounds and found that, in addition to SIRT1, the water-soluble 4-dmH preferentially targeted a tumor-associated NADH oxidase (tNOX, ENOX2). The direct binding of 4-dmH to tNOX decreased the oxidation of NADH to NAD+ which diminished NAD+-dependent SIRT1 deacetylase activity, ultimately inducing apoptosis and significant cytotoxicity in both cell types, as opposed to the parental heliomycin-induced autophagy. We also observed that tNOX and SIRT1 were both upregulated in tumor tissues of oral cancer patients compared to adjacent normal tissues, suggesting their clinical relevance. Finally, the better therapeutic efficacy of 4-dmH was confirmed in tumor-bearing mice, which showed greater tNOX and SIRT1 downregulation and tumor volume reduction when treated with 4-dmH compared to heliomycin. Taken together, our in vitro and in vivo findings suggest that the multifaceted properties of water-soluble 4-dmH enable it to offer superior antitumor value compared to parental heliomycin, and indicated that it functions through targeting the tNOX-NAD+-SIRT1 axis to induce apoptosis in oral cancer cells.


Assuntos
Neoplasias Bucais , Compostos Policíclicos , Sirtuína 1 , Humanos , Animais , Camundongos , Sirtuína 1/metabolismo , Linhagem Celular Tumoral , NAD/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Simulação de Acoplamento Molecular , Apoptose , Neoplasias Bucais/tratamento farmacológico
7.
Microbiol Res ; 283: 127692, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38508088

RESUMO

NADP/NADPH plays an indispensable role in cellular metabolism, serving as a pivotal cofactor in numerous enzymatic processes involved in anabolic pathways, antioxidant defense, and the biosynthesis of essential cellular components. NAD/NADH kinases (NADKs) phosphorylate NAD/NADH, constituting the sole de novo synthetic pathway for NADP/NADPH generation. Despite the pivotal role of NADP/NADPH in cellular functions, the physiological role of NADK remains largely unexplored in filamentous fungi. In this study, we identified three putative NADKs in Fusarium graminearum-FgNadk1, FgNadk2, and FgNadk3-responsible for NAD/NADH phosphorylation. NADK-mediated formation of intracellular NADPH proved crucial for vegetative growth, sexual reproduction, and virulence. Specifically, FgNadk2, the mitochondrial NADK, played a role in oxidative stress resistance and the maintenance of mitochondrial reactive oxygen species levels. Moreover, the deletion of FgNADK2 resulted in arginine auxotrophy, contributing to the reduced fungal virulence. These findings underscore the necessity of mitochondrial NADK in fungal virulence in F. graminearum, revealing its involvement in mitochondrial redox homeostasis and the arginine biosynthetic pathway. This study provides critical insights into the interconnectedness of metabolic pathways essential for fungal growth, stress response, and pathogenicity.


Assuntos
Fusarium , NAD , Virulência , NAD/metabolismo , NADP/metabolismo , Estresse Oxidativo , Oxirredução , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo
8.
Biochemistry ; 63(8): 1016-1025, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38546289

RESUMO

Kinetic parameters are reported for glycerol 3-phosphate dehydrogenase (GPDH)-catalyzed hydride transfer from the whole substrate glycerol 3-phosphate (G3P) or truncated substrate ethylene glycol (EtG) to NAD, and for activation of the hydride transfer reaction of EtG by phosphite dianion. These kinetic parameters were combined with parameters for enzyme-catalyzed hydride transfer in the microscopic reverse direction to give the reaction equilibrium constants Keq. Hydride transfer from G3P is favored in comparison to EtG because the carbonyl product of the former reaction is stabilized by hyperconjugative electron donation from the -CH2R keto substituent. The kinetic data show that the phosphite dianion provides the same 7.6 ± 0.1 kcal/mol stabilization of the transition states for enzyme-catalyzed reactions in the forward [reduction of NAD by EtG] and reverse [oxidation of NADH by glycolaldehyde] directions. The experimental evidence that supports a role for phosphite dianion in stabilizing the active closed form of the GPDH (EC) relative to the ca. 6 kcal/mol more unstable open form (EO) is summarized.


Assuntos
Glicerolfosfato Desidrogenase , Glicerofosfatos , Fosfitos , Glicerolfosfato Desidrogenase/química , NAD/metabolismo , Catálise , Cinética
9.
J Physiol ; 602(5): 891-912, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38429930

RESUMO

Skeletal muscle cellular development requires the integrated assembly of mitochondria and other organelles adjacent to the sarcomere in support of muscle contractile performance. However, it remains unclear how interactions among organelles and with the sarcomere relates to the development of muscle cell function. Here, we combine 3D volume electron microscopy, proteomic analyses, and live cell functional imaging to investigate the postnatal reorganization of mitochondria-organelle interactions in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. Concomitantly, expression of proteins involved in mitochondria-organelle membrane contact sites decreases during postnatal development in tandem with a decrease in abundance of proteins associated with sarcomere assembly despite an overall increase in contractile protein abundance. Functionally, parallel measures of mitochondrial membrane potential, NADH redox status, and NADH flux within intact cells revealed that mitochondria in adult skeletal muscle fibres maintain a more activated electron transport chain compared with neonatal muscle mitochondria. These data demonstrate a developmental redesign reflecting a shift from muscle cell assembly and frequent inter-organelle communication toward a muscle fibre with mitochondrial structure, interactions, composition and function specialized to support contractile function. KEY POINTS: Mitochondrial network organization is remodelled during skeletal muscle postnatal development. The mitochondrial outer membrane is in frequent contact with other organelles at birth and transitions to more close associations with the contractile apparatus in mature muscles. Mitochondrial energy metabolism becomes more activated during postnatal development. Understanding the developmental redesign process within skeletal muscle cells may help pinpoint specific areas of deficit in muscles with developmental disorders.


Assuntos
NAD , Proteômica , Humanos , Adulto , Recém-Nascido , NAD/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Mitocôndrias Musculares/metabolismo , Gotículas Lipídicas/metabolismo
10.
PLoS One ; 19(3): e0300541, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38483875

RESUMO

Glycerol dehydrogenase (GDH) catalyzes glycerol oxidation to dihydroxyacetone in a NAD+-dependent manner. As an initiator of the oxidative pathway of glycerol metabolism, a variety of functional and structural studies of GDH have been conducted previously. Structural studies revealed intriguing features of GDH, like the flexible ß-hairpin and its significance. Another commonly reported structural feature is the enzyme's octameric oligomerization, though its structural details and functional significance remained unclear. Here, with a newly reported GDH structure, complexed with both NAD+ and glycerol, we analyzed the octamerization of GDH. Structural analyses revealed that octamerization reduces the structural dynamics of the N-domain, which contributes to more consistently maintaining a distance required for catalysis between the cofactor and substrate. This suggests that octamerization may play a key role in increasing the likelihood of the enzyme reaction by maintaining the ligands in an appropriate configuration for catalysis. These findings expand our understanding of the structure of GDH and its relation to the enzyme's activity.


Assuntos
NAD , Desidrogenase do Álcool de Açúcar , NAD/metabolismo , Glicerol/metabolismo , Desidrogenase do Álcool de Açúcar/metabolismo , Oxirredução , Glutamato Desidrogenase/metabolismo
11.
Commun Biol ; 7(1): 255, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38429435

RESUMO

Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) via the nicotinamide (NAM) salvage pathway. While the structural biochemistry of eukaryote NAMPT has been well studied, the catalysis mechanism of prokaryote NAMPT at the molecular level remains largely unclear. Here, we demonstrated the NAMPT-mediated salvage pathway is functional in the Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) for the synthesis of NAD+, and the enzyme activity of NAMPT in this bacterium is significantly higher than that of human NAMPT in vitro. Our structural analyses of Xcc NAMPT, both in isolation and in complex with either the substrate NAM or the product nicotinamide mononucleotide (NMN), uncovered significant details of substrate recognition. Specifically, we revealed the presence of a NAM binding tunnel that connects the active site, and this tunnel is essential for both catalysis and inhibitor binding. We further demonstrated that NAM binding in the tunnel has a positive cooperative effect with NAM binding in the catalytic site. Additionally, we discovered that phosphorylation of the His residue at position 229 enhances the substrate binding affinity of Xcc NAMPT and is important for its catalytic activity. This work reveals the importance of NAMPT in bacterial NAD+ synthesis and provides insights into the substrate recognition and the catalytic mechanism of bacterial type II phosphoribosyltransferases.


Assuntos
Niacinamida , Xanthomonas campestris , Humanos , Niacinamida/metabolismo , NAD/metabolismo , Mononucleotídeo de Nicotinamida/metabolismo , Mononucleotídeo de Nicotinamida/farmacologia , Xanthomonas campestris/metabolismo , Nicotinamida Fosforribosiltransferase/química , Nicotinamida Fosforribosiltransferase/metabolismo , Fosforilação
12.
Biotechnol J ; 19(3): e2300744, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38509791

RESUMO

NAD(P)H-dependent oxidoreductases are crucial biocatalysts for synthesizing chiral compounds. Yet, the industrial implementation of enzymatic redox reactions is often hampered by an insufficient supply of expensive nicotinamide cofactors. Here, a cofactor self-sufficient whole-cell biocatalyst was developed for the enzymatic asymmetric reduction of 2-oxo-4-[(hydroxy)(-methyl)phosphinyl] butyric acid (PPO) to L-phosphinothricin (L-PPT). The endogenous NADP+ pool was significantly enhanced by regulating Preiss-Handler pathway toward NAD(H) synthesis and, in the meantime, introducing NAD kinase to phosphorylate NAD(H) toward NADP+. The intracellular NADP(H) concentration displayed a 2.97-fold increase with the strategy compared with the wild-type strain. Furthermore, a recombinant multi-enzyme cascade biocatalytic system was constructed based on the Escherichia coli chassis. In order to balance multi-enzyme co-expression levels, the strategy of modulating rate-limiting enzyme PmGluDH by RBS strengths regulation successfully increased the catalytic efficiency of PPO conversion. Finally, the cofactor self-sufficient whole-cell biocatalyst effectively converted 300 mM PPO to L-PPT in 2 h without the need to add exogenous cofactors, resulting in a 2.3-fold increase in PPO conversion (%) from 43% to 100%, with a high space-time yield of 706.2 g L-1 d-1 and 99.9% ee. Overall, this work demonstrates a technological example for constructing a cofactor self-sufficient system for NADPH-dependent redox biocatalysis.


Assuntos
NADH NADPH Oxirredutases , NAD , NADP/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Oxirredução , Redes e Vias Metabólicas
13.
J Immunol ; 212(7): 1043-1050, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38498807

RESUMO

NAD+ biology is involved in controlling redox balance, functioning as a coenzyme in numerous enzymatic reactions, and is a cofactor for Sirtuin enzymes and a substrate for multiple regulatory enzyme reactions within and outside the cell. At the same time, NAD+ levels are diminished with aging and are consumed during the development of inflammatory and autoimmune diseases linked to aberrant immune activation. Direct NAD+ augmentation via the NAD+ salvage and Priess-Handler pathways is being investigated as a putative therapeutic intervention to improve the healthspan in inflammation-linked diseases. In this review, we survey NAD+ biology and its pivotal roles in the regulation of immunity and inflammation. Furthermore, we discuss emerging studies evaluate NAD+ boosting in murine models and in human diseases, and we highlight areas of research that remain unresolved in understanding the mechanisms of action of these nutritional supplementation strategies.


Assuntos
Doenças Autoimunes , NAD , Animais , Humanos , Camundongos , NAD/metabolismo , Autoimunidade , Oxirredução , Inflamação
14.
Appl Environ Microbiol ; 90(4): e0015024, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38551341

RESUMO

Avilamycins, which possess potent inhibitory activity against Gram-positive bacteria, are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes. Among these structurally related oligosaccharide antibiotics, avilamycin A serves as the main bioactive component in veterinary drugs and animal feed additives, which differs from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. However, the mechanisms underlying assembly and modification of the oligosaccharide chain to diversify individual avilamycins remain poorly understood. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C. Remarkably, the ratio of these two components produced by AviZ1 depends on the utilization of specific redox cofactors, namely NADH/NAD+ or NADPH/NADP+. These findings are inspired by gene disruption and complementation experiments and are further supported by in vitro enzymatic activity assays, kinetic analyses, and cofactor affinity studies on AviZ1-catalyzed redox reactions. Additionally, the results from sequence analysis, structure prediction, and site-directed mutagenesis of AviZ1 validate it as an NADH/NAD+-favored aldo-keto reductase that primarily oxidizes avilamycin C to form avilamycin A by utilizing abundant NAD+ in vivo. Building upon the biological function and catalytic activity of AviZ1, overexpressing AviZ1 in S. viridochromogenes is thus effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study represents, to our knowledge, the first characterization of biochemical reactions involved in avilamycin biosynthesis and contributes to the construction of high-performance strains with industrial value.IMPORTANCEAvilamycins are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes, which can be used as veterinary drugs and animal feed additives. Avilamycin A is the most bioactive component, differing from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. Currently, the biosynthetic pathway of avilamycins is not clear. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C. More importantly, AviZ1 exhibits a unique NADH/NAD+ preference, allowing it to efficiently catalyze the oxidation of avilamycin C to form avilamycin A using abundant NAD+ in cells. Thus, overexpressing AviZ1 in S. viridochromogenes is effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study serves as an enzymological guide for rational strain design, and the resulting high-performance strains have significant industrial value.


Assuntos
NAD , Streptomyces , Drogas Veterinárias , NAD/metabolismo , Aldo-Ceto Redutases/metabolismo , Oligossacarídeos , Oxirredução , Antibacterianos , Carbono/metabolismo , NADP/metabolismo , Aldeído Redutase/metabolismo
15.
Aging Cell ; 23(4): e14102, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38481042

RESUMO

Tryptophan catabolism is highly conserved and generates important bioactive metabolites, including kynurenines, and in some animals, NAD+. Aging and inflammation are associated with increased levels of kynurenine pathway (KP) metabolites and depleted NAD+, factors which are implicated as contributors to frailty and morbidity. Contrastingly, KP suppression and NAD+ supplementation are associated with increased life span in some animals. Here, we used DGRP_229 Drosophila to elucidate the effects of KP elevation, KP suppression, and NAD+ supplementation on physical performance and survivorship. Flies were chronically fed kynurenines, KP inhibitors, NAD+ precursors, or a combination of KP inhibitors with NAD+ precursors. Flies with elevated kynurenines had reduced climbing speed, endurance, and life span. Treatment with a combination of KP inhibitors and NAD+ precursors preserved physical function and synergistically increased maximum life span. We conclude that KP flux can regulate health span and life span in Drosophila and that targeting KP and NAD+ metabolism can synergistically increase life span.


Assuntos
Cinurenina , Triptofano , Animais , Cinurenina/metabolismo , Triptofano/metabolismo , Longevidade , NAD/metabolismo , Drosophila/metabolismo
16.
JCI Insight ; 9(7)2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38452174

RESUMO

Prior studies showed that polyglutamine-expanded androgen receptor (AR) is aberrantly acetylated and that deacetylation of the mutant AR by overexpression of nicotinamide adenine dinucleotide-dependent (NAD+-dependent) sirtuin 1 is protective in cell models of spinal and bulbar muscular atrophy (SBMA). Based on these observations and reduced NAD+ in muscles of SBMA mouse models, we tested the therapeutic potential of NAD+ restoration in vivo by treating postsymptomatic transgenic SBMA mice with the NAD+ precursor nicotinamide riboside (NR). NR supplementation failed to alter disease progression and had no effect on increasing NAD+ or ATP content in muscle, despite producing a modest increase of NAD+ in the spinal cords of SBMA mice. Metabolomic and proteomic profiles of SBMA quadriceps muscles indicated alterations in several important energy-related pathways that use NAD+, in addition to the NAD+ salvage pathway, which is critical for NAD+ regeneration for use in cellular energy production. We also observed decreased mRNA levels of nicotinamide riboside kinase 2 (Nmrk2), which encodes a key kinase responsible for NR phosphorylation, allowing its use by the NAD+ salvage pathway. Together, these data suggest a model in which NAD+ levels are significantly decreased in muscles of an SBMA mouse model and intransigent to NR supplementation because of decreased levels of Nmrk2.


Assuntos
Atrofia Bulboespinal Ligada ao X , Camundongos , Animais , Atrofia Bulboespinal Ligada ao X/genética , Atrofia Bulboespinal Ligada ao X/metabolismo , NAD/metabolismo , Proteômica , Músculos/metabolismo , Camundongos Transgênicos , Metabolismo Energético
17.
Nanoscale ; 16(13): 6585-6595, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38465774

RESUMO

Nicotinamide adenine dinucleotide (NADH) and its phosphorylated form, NADPH, are essential cofactors that play critical roles in cell functions, influencing antioxidation, reductive biosynthesis, and cellular pathways involved in tumor cell apoptosis and tumorigenesis. However, the use of nanomaterials to consume NAD(P)H and thus bring an impact on signaling pathways in cancer treatment remains understudied. In this study, we employed a salt template method to synthesize a carbon-coated-cobalt composite (C@Co) nanozyme, which exhibited excellent NAD(P)H oxidase (NOX)-like activity and mimicked the reaction mechanism of natural NOX. The C@Co nanozyme efficiently consumed NAD(P)H within cancer cells, leading to increased production of reactive oxygen species (ROS) and a reduction in mitochondrial membrane potential. Meanwhile, the generation of the biologically active cofactor NAD(P)+ promoted the expression of the deacetylase SIRT7, which in turn inhibited the serine/threonine kinase AKT signaling pathway, ultimately promoting apoptosis. This work sheds light on the influence of nanozymes with NOX-like activity on cellular signaling pathways in tumor therapy and demonstrates their promising antitumor effects in a tumor xenograft mouse model. These findings contribute to a better understanding of NAD(P)H manipulation in cancer treatment and suggest the potential of nanozymes as a therapeutic strategy for cancer therapy.


Assuntos
NADPH Oxidases , Nanoestruturas , Sirtuínas , Animais , Humanos , Camundongos , Glicogênio Sintase Quinase 3 beta/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , NAD/metabolismo , NADPH Oxidases/farmacologia , NADPH Oxidases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sirtuínas/efeitos dos fármacos , Sirtuínas/metabolismo , Nanoestruturas/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/terapia
18.
Cancer Lett ; 588: 216806, 2024 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-38467179

RESUMO

The aim of this study was to investigate the underlying molecular mechanism behind the promotion of cell survival under conditions of glucose deprivation by l-lactate. To accomplish this, we performed tissue microarray and immunohistochemistry staining to analyze the correlation between the abundance of pan-Lysine lactylation and prognosis. In vivo evaluations of tumor growth were conducted using the KPC and nude mice xenograft tumor model. For mechanistic studies, multi-omics analysis, RNA interference, and site-directed mutagenesis techniques were utilized. Our findings robustly confirmed that l-lactate promotes cell survival under glucose deprivation conditions, primarily by relying on GLS1-mediated glutaminolysis to support mitochondrial respiration. Mechanistically, we discovered that l-lactate enhances the NMNAT1-mediated NAD+ salvage pathway while concurrently inactivating p-38 MAPK signaling and suppressing DDIT3 transcription. Notably, Pan-Kla abundance was significantly upregulated in patients with Pancreatic adenocarcinoma (PAAD) and associated with poor prognosis. We identified the 128th Lysine residue of NMNAT1 as a critical site for lactylation and revealed EP300 as a key lactyltransferase responsible for catalyzing lactylation. Importantly, we elucidated that lactylation of NMNAT1 enhances its nuclear localization and maintains enzymatic activity, thereby supporting the nuclear NAD+ salvage pathway and facilitating cancer growth. Finally, we demonstrated that the NMNAT1-dependent NAD+ salvage pathway promotes cell survival under glucose deprivation conditions and is reliant on the activity of Sirt1. Collectively, our study has unraveled a novel molecular mechanism by which l-lactate promotes cell survival under glucose deprivation conditions, presenting a promising strategy for targeting lactate and NAD+ metabolism in the treatment of PAAD.


Assuntos
Adenocarcinoma , Nicotinamida-Nucleotídeo Adenililtransferase , Neoplasias Pancreáticas , Camundongos , Animais , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ácido Láctico , NAD/metabolismo , Glucose , Camundongos Nus , Lisina , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo
19.
Int J Biol Macromol ; 264(Pt 2): 130799, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38479663

RESUMO

The 3-quinuclidinone reductase plays an irreplaceable role in the biopreparation of (R)-3-quinuclidinol, an intermediate vital for synthesis of various pharmaceuticals. Thermal robustness is a critical factor for enzymatic synthesis in industrial applications. This study characterized a new 3-quinuclidinone reductase, named SaQR, with significant thermal stability. The SaQR was overexpressed in a GST-fused state, and substrate and cofactor screening were conducted. Additionally, three-dimensional structure prediction using AlphaFold and analysis were performed, along with relevant thermostability tests, and the evaluation of factors influencing enzyme activity. The findings highlight the remarkable thermostability of SaQR, retaining over 90% of its activity after 72 h at 50°C, with an optimal operational temperature of 85°C. SaQR showed typical structural traits of the SDR superfamily, with its cofactor-determining residue being aspartic acid, conferring nicotinamide adenine dinucleotide (NAD(H)) preference. Moreover, K+ and Na+, at a concentration of 400 mM, could significantly enhance the activity, while Mg2+ and Mn2+ only display inhibitory effects within the tested concentration range. The findings of molecular dynamics simulations suggest that high temperatures may disrupt the binding of enzyme to substrate by increasing the flexibility of residues 205-215. In conclusion, this study reports a novel 3-quinuclidinone reductase with remarkable thermostability.


Assuntos
Oxirredutases , Quinuclidinas , Oxirredutases/metabolismo , Quinuclidinas/farmacologia , Quinuclidinas/metabolismo , NAD/metabolismo , Simulação de Dinâmica Molecular , Estabilidade Enzimática
20.
Nature ; 627(8005): 847-853, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38480885

RESUMO

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.


Assuntos
Trifosfato de Adenosina , Arabidopsis , NAD , Tabaco , 60422 , Proteínas de Plantas , Domínios Proteicos , Trifosfato de Adenosina/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Morte Celular , Mutação , NAD/metabolismo , Tabaco/genética , Tabaco/imunologia , Tabaco/metabolismo , Proteínas NLR/química , Proteínas NLR/genética , Proteínas NLR/imunologia , Proteínas NLR/metabolismo , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Transdução de Sinais , Receptores Toll-Like/química , Receptores de Interleucina-1/química
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