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Metabolic sources of NADP(H) and the cytosolic/mitochondrial NADPH shuttle. In the cytosol, NADPH is primarily produced by G6PD and 6PGD in the pentose phosphate pathway. ME1 also contributes to cytosolic NADPH production. Mitochondrial NADPH is generated by NADP +-dependent IDH2, GLUD, NNT, and ME3. The cytosolic and mitochondrial NADPH is exchanged through the isocitrate-a-KG shuttle, where cytosolic IDH1 and mitochondrial IDH2 catalyze the interconversion of isocitrate and a-KG in conjunction with the interconversion of NADP + and NADPH. The citrate carrier protein (encoded by SLC25A1 gene) and the a-KG/malate antiporter (encoded by SLC25A11 gene) mediate the transport of isocitrate and a-KG between cytosol and mitochondria, respectively. 6PG, 6-phosphogluconate; 6PGD, 6phosphogluconate dehydrogenase; G6P, glucose-6-phosphate; G6PD, glucose-6-phosphate dehydrogenase; NNT, nicotinamide nucleotide transhydrogenase; R5P, ribose-5-phosphate; SCL25A1, solute carrier family 25 member 1.
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Significance:
The NAD+/NADH and NADP+/NADPH redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly-identified biosynthetic enzy...
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Context 1
... generated from the PPP by glucose-6-phosphate dehydrogenase (G6PD) and 6- phosphogluconate dehydrogenase (6PGD) (87, 141). G6PD catalyzes the conversion of glucose-6-phosphate (G6P) into 6- phosphogluconate (6PG), which can be further metabolized into ribose-5-phosphate (R5P) by 6PGD. Both reactions are coupled to the reduction of NADP + to NADPH (Fig. 6). In addition, other enzymes also contribute to the cytosolic NADPH pool, such as IDHs and MEs (87, 139), all of which have both cytosolic and mitochondrial isozymes. Cytosolic IDH (IDH1) catalyzes the same reaction as mitochondrial IDH3 using NADP + rather than NAD + as a cofactor, forming NADPH. Cytosolic ME (ME1) catalyzes oxidative ...
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... mentioned earlier, the mitochondrial inner membrane is impermeable to NADP(H) (24,102,141). Communica- tion between cytosolic and mitochondrial NADP(H) pools is conducted by the isocitrate-a-KG shuttle (Fig. 6) (66,100). This NADPH shuttle functions through IDH1 and IDH2 isozymes. In the mitochondrial matrix, NADP + - dependent IDH2 converts a-KG into isocitrate by oxidizing NADPH to NADP + . Isocitrate is then pumped into the cytosol in exchange for malate by the citrate carrier protein (encoded by SLC25A1 gene). In the cytosol, IDH1 cata- ...
Citations
... Previous work has shown that MPC deletion or disruption diminishes NADPH production via the TCA cycle, which in turn promotes compensatory glutamine oxidation to sustain NADPH levels, directing glutamine away from glutathione (GSH) synthesis [6,52,53]. Additionally, NADPH is essential as a cofactor in converting oxidised glutathione (GSSG) via glutathione reductase [52,54]. Furthermore, disruption of the TCA cycle and mitochondrial dysfunction have been reported to activate the ISR through impaired redox and amino acid homeostasis [8,9]. ...
Unravelling how energy metabolism and stress responses are regulated in human scalp hair follicles could reveal novel insights into the controls of hair growth and provide new targets to manage hair loss disorders. The Mitochondrial Pyruvate Carrier (MPC) imports pyruvate, produced via glycolysis, into the mitochondria, fuelling the TCA cycle. Previous work has shown that MPC inhibition promotes lactate generation, which activates murine epithelial hair follicle stem cells (eHFSCs). However, by pharmacologically targeting the MPC in short-term human hair follicle ex vivo organ culture experiments using UK-5099, we induced metabolic stress-responsive proliferative arrest throughout the human hair follicle epithelium, including within Keratin 15+ eHFSCs. Through transcriptomics, MPC inhibition was shown to promote a gene expression signature indicative of disrupted FGF, IGF, TGFβ and WNT signalling, mitochondrial dysfunction, and activation of the integrated stress response (ISR), which can arrest cell cycle progression. The ISR, mediated by the transcription factor ATF4, is activated by stressors including amino acid deprivation and ER stress, consistent with MPC inhibition within our model. Using RNAScope, we confirmed the upregulation of both ATF4 and the highly upregulated ATF4-target gene ADM2 on human hair follicle tissue sections in situ. Moreover, treatment with the ISR inhibitor ISRIB attenuated both the upregulation of ADM2 and the proliferative block imposed via MPC inhibition. Together, this work reveals how the human hair follicle, as a complex and metabolically active human tissue system, can dynamically adapt to metabolic stress.
... Originally, NAD+ was identified for its role in enhancing fermentation in yeast, where researchers found evidence of a coenzyme whose presence was crucial for alcoholic fermentation [1]. Though this early research predates our current understanding of NAD+, it prompted diverse biological investigations into its role in cellular redox reactions [2], electron transfer [3], glycolysis [4], the Krebs cycle [5], and fatty acid β-oxidation [6]. ...
Recent years have seen a surge in research focused on NAD+ decline and potential interventions, and despite significant progress, new discoveries continue to highlight the complexity of NAD+ biology. Nicotinamide mononucleotide (NMN), a well-established NAD+ precursor, has garnered considerable interest due to its capacity to elevate NAD+ levels and induce promising health benefits in preclinical models. Clinical trials investigating NMN supplementation have yielded variable outcomes while shedding light on the intricacies of NMN metabolism and revealing the critical roles played by gut microbiota and specific cellular uptake pathways. Individual variability in factors such as lifestyle, health conditions, genetics, and gut microbiome composition likely contributes to the observed discrepancies in clinical trial results. Preliminary evidence suggests that NMN’s effects may be context-dependent, varying based on a person’s physiological state. Understanding these nuances is critical for definitively assessing the impact of manipulating NAD+ levels through NMN supplementation. Here, we review NMN metabolism, focusing on current knowledge, pinpointing key areas where further research is needed, and outlining future directions to advance our understanding of its potential clinical significance.
... LXC007989 encodes a subunit of NADH dehydrogenase and LXC000548 encodes ATP synthase subunit d. Both enzymes are related to energy metabolism [55]. In the X5-30 group, the low expression of genes in this pathway (Figure 8b) may have reduced energy metabolism so that life activities would be impaired compared to those in the Ctr-30 and X3-30 groups. ...
We conducted transcriptome sequencing on salt-tolerant mutants X5 and X3, and a control (Ctr) strain of Gracilariopsis lemaneiformis after treatment with artificial seawater at varying salinities (30‰, 45‰, and 60‰) for 3 weeks. Differentially expressed genes were identified and a weighted co-expression network analysis was conducted. The blue, red, and tan modules were most closely associated with salinity, while the black, cyan, light cyan, and yellow modules showed a close correlation with strain attributes. KEGG enrichment of genes from the aforementioned modules revealed that the key enrichment pathways for salinity attributes included the proteasome and carbon fixation in photosynthesis, whereas the key pathways for strain attributes consisted of lipid metabolism, oxidative phosphorylation, soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE) interactions in vesicular transport, and porphyrin and chlorophyll metabolism. Gene expression for the proteasome and carbon fixation in photosynthesis was higher in all strains at 60‰. In addition, gene expression in the proteasome pathway was higher in the X5-60 than Ctr-60 and X3-60. Based on the above data and relevant literature, we speculated that mutant X5 likely copes with high salt stress by upregulating genes related to lysosome and carbon fixation in photosynthesis. The proteasome may be reset to adjust the organism’s proteome composition to adapt to high-salt environments, while carbon fixation may aid in maintaining material and energy metabolism for normal life activities by enhancing carbon dioxide uptake via photosynthesis. The differences between the X5-30 and Ctr-30 expression of genes involved in the synthesis of secondary metabolites, oxidative phosphorylation, and SNARE interactions in vesicular transport suggested that the X5-30 may differ from Ctr-30 in lipid metabolism, energy metabolism, and vesicular transport. Finally, among the key pathways with good correlation with salinity and strain traits, the key genes with significant correlation with salinity and strain traits were identified by correlation analysis.
... Consequently, ferroptosis is significantly regulated by GPX4 and GSH, two of its substrates [46]. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione reductase simultaneously convert oxidized glutathione disulfide (GSSG) into GSH [47]. Additionally, GSH inhibits iron-dependent oxidation by binding to ferrous ions in the LIP [7,48,49]. ...
... The significance of Michaelis-Menten parameters is altered; for instance, the local concentration of the product of E2 in an extended cascade is probably well above its K M for the next enzyme E3. While the e-Leaf demonstrates the kinetic advantage of confining NAD(P)(H) cofactors, the degree to which the action of nicotinamide cofactors in vivo is localised or dispersed is of considerable interest 57,80 . Historically, central NAD(H) and NADP(H) pools have normally been assumed -the NAD(P) + /NAD(P)H ratio in cells is a measure of metabolic state. ...
An emerging concept and platform, the electrochemical Leaf (e-Leaf), offers a radical change in the way tandem (multi-step) catalysis by enzyme cascades is studied and exploited. The various enzymes are loaded into an electronically conducting porous material composed of metallic oxide nanoparticles, where they achieve high concentration and crowding – in the latter respect the environment resembles that found in living cells. By exploiting efficient electron tunneling between the nanoparticles and one of the enzymes, the e-Leaf enables the user to interact directly with complex networks, rendering simultaneous the abilities to energise, control and observe catalysis. Because dispersion of intermediates is physically suppressed, the output of the cascade – the rate of flow of chemical steps and information – is delivered in real time as electrical current. Myriad enzymes of all major classes now become effectively electroactive in a technology that offers scalability between micro-(analytical, multiplex) and macro-(synthesis) levels. This Perspective describes how the e-Leaf was discovered, the steps in its development so far, and the outlook for future research and applications.
... ME1 activity impacts cellular metabolism both via its role in NADPH generation and its production of pyruvate. Knockdown of ME1 has been shown to lower cytosolic NADPH levels, decreasing the availability of this important metabolite in cellular redox homeostasis [3][4][5]. Further, the NADPH produced by ME1 has been proposed to directly feed into fatty acid biosynthesis, specifically in the synthesis of long-chain saturated fatty acids, though the mechanism for selectivity remains unclear [6]. The production of pyruvate from ME1 furthers its metabolic impact on the cell, as the pyruvate produced is shuttled into the mitochondria for production of acetyl-CoA, linking cytosolic and mitochondrial metabolism [7,8]. ...
Malic Enzyme 1 (ME1) plays an integral role in fatty acid synthesis and cellular energetics through its production of NADPH and pyruvate. As such, it has been identified as a gene of interest in obesity, type 2 diabetes, and an array of epithelial cancers, with most work being performed in vitro. The current standard model for ME1 loss in vivo is the spontaneous Mod-1 null allele, which produces a canonically inactive form of ME1. Herein, we describe two new genetically engineered mouse models exhibiting ME1 loss at dynamic timepoints. Using murine embryonic stem cells and Flp/FRT and Cre/loxP class switch recombination, we established a germline Me1 knockout model (Me1 KO) and an inducible conditional knockout model (Me1 cKO), activated upon tamoxifen treatment in adulthood. Collectively, neither the Me1 KO nor Me1 cKO models exhibited deleterious phenotype under standard laboratory conditions. Knockout of ME1 was validated by immunohistochemistry and genotype confirmed by PCR. Transmission patterns favor Me1 loss in Me1 KO mice when maternally transmitted to male progeny. Hematological examination of these models through complete blood count and serum chemistry panels revealed no discrepancy with their wild-type counterparts. Orthotopic pancreatic tumors in Me1 cKO mice grow similarly to Me1 expressing mice. Similarly, no behavioral phenotype was observed in Me1 cKO mice when aged for 52 weeks. Histological analysis of several tissues revealed no pathological phenotype. These models provide a more modern approach to ME1 knockout in vivo while opening the door for further study into the role of ME1 loss under more biologically relevant, stressful conditions.
... NAD(P) binding domain superfamily proteins bind nicotinamide dinucleotide (NAD) or its phosphorylated form (NADP). These are co-factors that exist as redox couples (NAD + /NADH and NADP + /NADPH) in cells to serve distinct functions 35 . While NAD + /NADH is involved in cellular energy metabolism, NADP + /NADPH is involved in redox balance and fatty acid and nucleic acid synthesis 35 . ...
... These are co-factors that exist as redox couples (NAD + /NADH and NADP + /NADPH) in cells to serve distinct functions 35 . While NAD + /NADH is involved in cellular energy metabolism, NADP + /NADPH is involved in redox balance and fatty acid and nucleic acid synthesis 35 . On the other hand, Thiolase-domain proteins are involved in formation of acetoacetyl coA from acetyl-coA 36 . ...
Fructosamine-3-kinases (FN3Ks) are a conserved family of repair enzymes that phosphorylate reactive sugars attached to lysine residues in peptides and proteins. Although FN3Ks are present across the Tree of Life and share detectable sequence similarity to eukaryotic protein kinases, the biological processes regulated by these kinases are largely unknown. To address this knowledge gap, we leveraged the FN3K CRISPR Knock-Out (KO) HepG2 cell line alongside an integrative multi-omics study combining transcriptomics, metabolomics, and interactomics to place these enzymes in a pathway context. The integrative analyses revealed the enrichment of pathways related to oxidative stress response, lipid biosynthesis (cholesterol and fatty acids), and carbon and co-factor metabolism. Moreover, enrichment of nicotinamide adenine dinucleotide (NAD) binding proteins and localization of human FN3K (HsFN3K) to mitochondria suggests potential links between FN3K and NAD-mediated energy metabolism and redox balance. We report specific binding of HsFN3K to NAD compounds in a metal and concentration-dependent manner and provide insight into their binding mode using modeling and experimental site-directed mutagenesis. Our studies provide a framework for targeting these understudied kinases in diabetic complications and metabolic disorders where redox balance and NAD-dependent metabolic processes are altered.
... Nicotinamide is a precursor of the coenzyme's nicotinamide-adenine dinucleotide (NAD + ) and nicotinamide-adenine dinucleotide phosphate (NADP + ) [51]. However, NAD + and NADP + biosynthesis seem to require adenine and phosphate [75]. Thus, an alternative pathway for energy generation might occur in A. microphylla with HW 1:35 + N50. ...
Astronaut waste has the potential to be used for organic fertilizer to support waste recycling and bio-regenerative life support systems for future space exploration. Aquatic fern, Azolla, makes symbiosis with cyanobacteria that could assimilate free nitrogen from the atmosphere. The prospect of fermented human waste used as fertilizer to grow Azolla microphylla has been observed in this study. Fermented human waste (HW 1:35) supplemented with 50 mg/L NaNO3 may enhance A. microphylla fresh weight and green area index compared to other HW treatments. Lower biomass of A. microphylla with HW + 50 mg/L NaNO3 than fertilizer control might correlate with lower initial NO3− and PO43− in growth media. However, A. microphylla with HW 1:35 + NaNO3 50 mg/L showed a better green area index than A. microphylla with fertilizer. Metabolomics analysis revealed that A. microphylla with HW 1:35 + NaNO3 50 mg/L might perform alternative energy metabolism through up-regulation of adenosine and aspartic acid, resulting in a higher green index area. Limited PO43− in growth media might also highly induce chlorogenic acid and kaempferol expressions that might affect allelopathy and shift in energy use efficiency for biomass accumulation. Thus, sufficient N and P supplementation in HW is recommended for A. microphylla cultivation.
... Besides the direct effect of embryonic NAD deficiency as the cause of CNDD, it is possible that the low NAD levels observed in the yolk sac under NW3.5 diet also interfere with other crucial yolk sac functions that are linked to NAD, such as ATP-dependent active transport of essential nutrients (Chang, 2010, Cindrova-Davies et al., 2017, Dutta and Sinha, 2017 or NADP-dependent enzymes (Di Pietro et al., 2002, Xiao et al., 2018, thereby further impacting normal embryonic development. Likely, it is a combination of both. ...
Severe congenital malformations are a frequent cause of premature death and morbidity in children worldwide. Malformations can originate from numerous genetic or non-genetic factors but in most cases the underlying causes are unknown. Genetic disruption of nicotinamide adenine dinucleotide (NAD) de novo synthesis drives the formation of multiple congenital malformations, collectively termed Congenital NAD Deficiency Disorder (CNDD), highlighting the necessity of this pathway during embryogenesis. Previous work in mice shows that NAD deficiency perturbs embryonic development specifically during a critical period when organs are forming. While NAD de novo synthesis is predominantly active in the liver postnatally, the site of activity prior to and during organogenesis is unknown.
Here, we used a mouse model of human CNDD and applied gene expression, enzyme activity and metabolic analyses to assess pathway functionality in the embryonic liver and extraembryonic tissues. We found that the extra-embryonic visceral yolk sac endoderm exclusively performs NAD de novo synthesis during early organogenesis before the embryonic liver takes over this function. Furthermore, under CNDD-inducing conditions, mouse visceral yolk sacs had reduced NAD levels and altered NAD-related metabolic profiles which affected embryo metabolism. Expression of requisite genes for NAD de novo synthesis is conserved in the equivalent yolk sac cell type in humans.
Our findings show that visceral yolk sac-mediated NAD de novo synthesis activity is essential for mouse embryonic development and perturbation of this pathway results in CNDD. Given the functional homology between mouse and human yolk sacs, our data improve the understanding of human congenital malformation causation.
... PPP plays a pivotal role in the oxidative stress response. It generates the primary redox factor NADPH for the antioxidative machinery [27,28]. Furthermore, PPP functions as a metabolic redox sensor and regulator [29]. ...
Background
The production of succinic acid (SA) from biomass has attracted worldwide interest. Saccharomyces cerevisiae is preferred for SA production due to its strong tolerance to low pH conditions, ease of genetic manipulation, and extensive application in industrial processes. However, when compared with bacterial producers, the SA titers and productivities achieved by engineered S. cerevisiae strains were relatively low. To develop efficient SA-producing strains, it’s necessary to clearly understand how S. cerevisiae cells respond to SA.
Results
In this study, we cultivated five S. cerevisiae strains with different genetic backgrounds under different concentrations of SA. Among them, KF7 and NBRC1958 demonstrated high tolerance to SA, whereas NBRC2018 displayed the least tolerance. Therefore, these three strains were chosen to study how S. cerevisiae responds to SA. Under a concentration of 20 g/L SA, only a few differentially expressed genes were observed in three strains. At the higher concentration of 60 g/L SA, the response mechanisms of the three strains diverged notably. For KF7, genes involved in the glyoxylate cycle were significantly downregulated, whereas genes involved in gluconeogenesis, the pentose phosphate pathway, protein folding, and meiosis were significantly upregulated. For NBRC1958, genes related to the biosynthesis of vitamin B6, thiamin, and purine were significantly downregulated, whereas genes related to protein folding, toxin efflux, and cell wall remodeling were significantly upregulated. For NBRC2018, there was a significant upregulation of genes connected to the pentose phosphate pathway, gluconeogenesis, fatty acid utilization, and protein folding, except for the small heat shock protein gene HSP26. Overexpression of HSP26 and HSP42 notably enhanced the cell growth of NBRC1958 both in the presence and absence of SA.
Conclusions
The inherent activities of small heat shock proteins, the levels of acetyl-CoA and the strains’ potential capacity to consume SA all seem to affect the responses and tolerances of S. cerevisiae strains to SA. These factors should be taken into consideration when choosing host strains for SA production. This study provides a theoretical basis and identifies potential host strains for the development of robust and efficient SA-producing strains.
