Metabolic engineering of Aspergillus niger for accelerated malic acid biosynthesis by improving NADPH availability.

Microbial production of L-malic acid from renewable carbon sources has attracted extensive attention. The reduced cofactor NADPH plays a key role in biotransformation because it participates in both biosynthetic reactions and cellular stress responses. In this study, NADPH or its precursors nicotinamide and nicotinic acid were added to the fermentation medium of Aspergillus niger RG0095, which significantly increased the yield of malic acid by 11%. To further improve the titer and productivity of L-malic acid, we increased the cytoplasmic NADPH levels of A. niger by upregulating the NAD kinases Utr1p and Yef1p. Biochemical analyses demonstrated that overexpression of Utr1p and Yef1p reduced oxidative stress, while also providing more NADPH to catalyze the conversion of glucose into malic acid. Notably, the strain overexpressing Utr1p reached a malate titer of 110.72 ± 1.91 g L-1 after 108 h, corresponding to a productivity of 1.03 ± 0.02 g L-1 h-1. Thus, the titer and productivity of malate were increased by 24.5% and 44.7%, respectively. The strategies developed in this study may also be useful for the metabolic engineering of fungi to produce other industrially relevant bulk chemicals.

Structural basis of human NOX5 activation.

NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which - in particular, how calcium triggers electron transfer from NADPH to FAD - is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.

Large enrichments in fatty acid 2H/1H ratios distinguish respiration from aerobic fermentation in yeast Saccharomyces cerevisiae.

Shifts in the hydrogen stable isotopic composition (2H/1H ratio) of lipids relative to water (lipid/water 2H-fractionation) at natural abundances reflect different sources of the central cellular reductant, NADPH, in bacteria. Here, we demonstrate that lipid/water 2H-fractionation (2εfattyacid/water) can also constrain the relative importance of key NADPH pathways in eukaryotes. We used the metabolically flexible yeast Saccharomyces cerevisiae, a microbial model for respiratory and fermentative metabolism in industry and medicine, to investigate 2εfattyacid/water. In chemostats, fatty acids from glycerol-respiring cells were >550‰ 2H-enriched compared to those from cells aerobically fermenting sugars via overflow metabolism, a hallmark feature in cancer. Faster growth decreased 2H/1H ratios, particularly in glycerol-respiring cells by 200‰. Variations in the activities and kinetic isotope effects among NADP+-reducing enzymes indicate cytosolic NADPH supply as the primary control on 2εfattyacid/water. Contributions of cytosolic isocitrate dehydrogenase (cIDH) to NAPDH production drive large 2H-enrichments with substrate metabolism (cIDH is absent during fermentation but contributes up to 20 percent NAPDH during respiration) and slower growth on glycerol (11 percent more NADPH from cIDH). Shifts in NADPH demand associated with cellular lipid abundance explain smaller 2εfattyacid/water variations (<30‰) with growth rate during fermentation. Consistent with these results, tests of murine liver cells had 2H-enriched lipids from slower-growing, healthy respiring cells relative to fast-growing, fermenting hepatocellular carcinoma. Our findings point to the broad potential of lipid 2H/1H ratios as a passive natural tracker of eukaryotic metabolism with applications to distinguish health and disease, complementing studies that rely on complex isotope-tracer addition methods.

mTORC1 regulates cell survival under glucose starvation through 4EBP1/2-mediated translational reprogramming of fatty acid metabolism.

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically, 4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1), thereby mitigating oxidative stress. This has important relevance for cancer, as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells.

Knockdown of NADK promotes LUAD ferroptosis via NADPH/FSP1 axis.

BACKGROUND: Lung cancer is a serious threat to human health and is the first leading cause of cancer death. Ferroptosis, a newly discovered form of programmed cell death associated with redox homeostasis, is of particular interest in the lung cancer, given the high oxygen environment of lung cancer. NADPH has reducing properties and therefore holds the potential to resist ferroptosis. Resistance to ferroptosis exists in lung cancer, but the role of NADK in regulating ferroptosis in lung cancer has not been reported yet. METHODS: Immunohistochemistry (IHC) was used to analyse the expression of NADK in 86 cases of lung adenocarcinoma(LUAD) and adjacent tissues, and a IHC score was assigned to each sample. Chi-square and kaplan-meier curve was performed to analyse the differences in metastasis and five-year survival between the two groups with NADK high or low scores. Proliferation of NADK-knockdown LUAD cell lines was detected in vivo and vitro. Furthermore, leves of ROS, MDA and Fe2+ were measured to validate the effect and mechanism of NADK on ferroptosis in LUAD. RESULTS: The expression of NADK was significantly evaluated in LUAD tissues as compared to adjacent non-cancerous tissues. The proliferation of NADK-knockdown cells was inhibited both in vivo and vitro, and increasing levels of intracellular ROS, Fe2+ and lipid peroxide products (MDA) were observed. Furthermore, NADK-knockdown promoted the ferroptosis of LUAD cells induced by Erastin/RSL3 by regulating the level of NADPH and the expression of FSP1. Knockdown of NADK enhanced the sensitivities of LUAD cells to Erastin/RSL3-induced ferroptosis by regulating NADPH level and FSP1 expression. CONCLUSIONS: NADK is over-expressed in LUAD patients. Knockdown of NADK inhibited the proliferation of LUAD cells both in vitro and in vivo and promotes the Erastin/RSL3-induced ferroptosis of LUAD cells by down-regulating the NADPH/FSP1 axis.

NADPH and NAC synergistically inhibits chronic ocular hypertension-induced neurodegeneration and neuroinflammation through regulating p38/MAPK pathway and peroxidation.

Glaucoma, the leading cause of irreversible blindness worldwide, is characterized by neurodegeneration and neuroinflammation with retinal NAD/NADP and GSH decline. Nicotinamide adenine dinucleotide (NAD)/NAD phosphate (NADP) and glutathione (GSH) are two redox reducers in neuronal and glial metabolism. However, therapeutic strategies targeting NAD/NADP or GSH do not exert ideal effects, and the underlying mechanisms are still poorly understood. We assessed morphological changes in retinal ganglion cells (RGCs), the affected neurons in glaucoma, and Müller cells, the major glial cells in the retina, as well as the levels of phosphorylated p38 (p-p38) and Caspase-3 in glaucoma patients. We constructed a modified chronic ocular hypertensive rat model and an oxygen-glucose deprivation (OGD) cell model. After applying NADPH and N-acetylcysteine (NAC), a precursor to cysteine, the rate-limiting substrate in GSH biosynthesis, to cells, apoptosis, axonal damage and peroxidation were reduced in the RGCs of the NAC group and p-p38 levels were decreased in the RGCs of the NADPH group, while in stimulated Müller cells cultured individually or cocultured with RGCs, gliosis and p38/MAPK, rather than JNK/MAPK, activation were inhibited. The results were more synergistic in the rat model, where either NADPH or NAC showed crossover effects on inhibiting peroxidation and p38/MAPK pathway activation. Moreover, the combination of NADPH and NAC ameliorated RGC electrophysiological function and prevented Müller cell gliosis to the greatest extent. These data illustrated conjoined mechanisms in glaucomatous RGC injury and Müller cell gliosis and suggested that NADPH and NAC collaborate as a neuroprotective and anti-inflammatory combination treatment for glaucoma and other underlying human neurodegenerative diseases.

Exogenous NADPH could mitigate pyroptosis-induced brain injury in fetal mice exposed to gestational intermittent hypoxia.

OBJECTIVE: Obstructive Sleep Apnea (OSA) during pregnancy is characterized by intermittent hypoxia (IH) during sleep and will lead to the rise of oxidative stress in the fetal body. Pyroptosis, a type of inflammatory and programmable cell death mediated by Gasdermin D (GSDMD), plays a substantial role in oxygen deprivation's contribution to neural system damage. Existing research shows that Nicotinamide Adenine Dinucleotide Phosphate (NADPH) plays a protective role in alleviating brain tissue pyroptosis. We speculate that exogenous NADPH may play a protective role in OSA during pregnancy. METHODS: A model of GIH group was established to simulate the pathophysiological mechanisms of OSA during pregnant and AIR group was established by giving the same frequency. Sham group was established by injecting NS and the NADPH group was established and given exogenous NADPH. We utilized the Morris Water Maze to assess cognitive function impairment, Luxol Fast Blue (LBF) staining to confirm myelin sheath formation, TUNEL staining to examine cell death in fetal mice brain tissue, and Western blotting to detect pertinent protein expressions. RESULTS: The GIH group offspring exhibited decreases in spatial learning and memory abilities, reduced numbers of oligodendrocytes and formed myelin, as well as increased expression of pyroptosis-related proteins. The NADPH group offspring showed restoration in spatial learning and memory abilities increased counts of oligodendrocytes and formed myelin sheaths, in addition to decreased expression of pyroptosis-related. CONCLUSIONS: This study demonstrates that early injection of exogenous NADPH can alleviate the damage to fetal brain development caused by gestational intermittent hypoxia (GIH).

Rice Origin Tracing Technology Based on Fluorescence Spectroscopy and Stoichiometry.

The origin of agricultural products is crucial to their quality and safety. This study explored the differences in chemical composition and structure of rice from different origins using fluorescence detection technology. These differences are mainly affected by climate, environment, geology and other factors. By identifying the fluorescence characteristic absorption peaks of the same rice seed varieties from different origins, and comparing them with known or standard samples, this study aims to authenticate rice, protect brands, and achieve traceability. The study selected the same variety of rice seed planted in different regions of Jilin Province in the same year as samples. Fluorescence spectroscopy was used to collect spectral data, which was preprocessed by normalization, smoothing, and wavelet transformation to remove noise, scattering, and burrs. The processed spectral data was used as input for the long short-term memory (LSTM) model. The study focused on the processing and analysis of rice spectra based on NZ-WT-processed data. To simplify the model, uninformative variable elimination (UVE) and successive projections algorithm (SPA) were used to screen the best wavelengths. These wavelengths were used as input for the support vector machine (SVM) prediction model to achieve efficient and accurate predictions. Within the fluorescence spectral range of 475-525 nm and 665-690 nm, absorption peaks of nicotinamide adenine dinucleotide (NADPH), riboflavin (B2), starch, and protein were observed. The origin tracing prediction model established using SVM exhibited stable performance with a classification accuracy of up to 99.5%.The experiment demonstrated that fluorescence spectroscopy technology has high discrimination accuracy in tracing the origin of rice, providing a new method for rapid identification of rice origin.

Cascade reactions with two non-physiological partners for NAD(P)H regeneration via renewable hydrogen.

An attractive application of hydrogenases, combined with the availability of cheap and renewable hydrogen (i.e., from solar and wind powered electrolysis or from recycled wastes), is the production of high-value electron-rich intermediates such as reduced nicotinamide adenine dinucleotides. Here, the capability of a very robust and oxygen-resilient [FeFe]-hydrogenase (CbA5H) from Clostridium beijerinckii SM10, previously identified in our group, combined with a reductase (BMR) from Bacillus megaterium (now reclassified as Priestia megaterium) was tested. The system shows a good stability and it was demonstrated to reach up to 28 ± 2 nmol NADPH regenerated s-1 mg of hydrogenase-1 (i.e., 1.68 ± 0.12 U mg-1, TOF: 126 ± 9 min-1) and 0.46 ± 0.04 nmol NADH regenerated s-1 mg of hydrogenase-1 (i.e., 0.028 ± 0.002 U mg-1, TOF: 2.1 ± 0.2 min-1), meaning up to 74 mg of NADPH and 1.23 mg of NADH produced per hour by a system involving 1 mg of CbA5H. The TOF is comparable with similar systems based on hydrogen as regenerating molecule for NADPH, but the system is first of its kind as for the [FeFe]-hydrogenase and the non-physiological partners used. As a proof of concept a cascade reaction involving CbA5H, BMR and a mutant BVMO from Acinetobacter radioresistens able to oxidize indole is presented. The data show how the cascade can be exploited for indigo production and multiple reaction cycles can be sustained using the regenerated NADPH.

ATP-free in vitro biotransformation of starch-derived maltodextrin into poly-3-hydroxybutyrate via acetyl-CoA.

In vitro biotransformation (ivBT) facilitated by in vitro synthetic enzymatic biosystems (ivSEBs) has emerged as a highly promising biosynthetic platform. Several ivSEBs have been constructed to produce poly-3-hydroxybutyrate (PHB) via acetyl-coenzyme A (acetyl-CoA). However, some systems are hindered by their reliance on costly ATP, limiting their practicality. This study presents the design of an ATP-free ivSEB for one-pot PHB biosynthesis via acetyl-CoA utilizing starch-derived maltodextrin as the sole substrate. Stoichiometric analysis indicates this ivSEB can self-maintain NADP+/NADPH balance and achieve a theoretical molar yield of 133.3%. Leveraging simple one-pot reactions, our ivSEBs achieved a near-theoretical molar yield of 125.5%, the highest PHB titer (208.3 mM, approximately 17.9 g/L) and the fastest PHB production rate (9.4 mM/h, approximately 0.8 g/L/h) among all the reported ivSEBs to date, and demonstrated easy scalability. This study unveils the promising potential of ivBT for the industrial-scale production of PHB and other acetyl-CoA-derived chemicals from starch.

Reprogramming the Metabolism of Yeast for High-Level Production of Miltiradiene.

Miltiradiene serves as a crucial precursor in the synthesis of various high-value abietane-type diterpenes, exhibiting diverse pharmacological activities. Previous efforts to enhance miltiradiene production have primarily focused on the mevalonate acetate (MVA) pathway. However, limited emphasis has been placed on optimizing the supply of acetyl-CoA and NADPH. In this study, we constructed a platform yeast strain for miltiradiene production by reinforcing the biosynthetic pathway of geranylgeranyl diphosphate (GGPP) and acetyl-CoA, and addressing the imbalance between the supply and demand of the redox cofactor NADPH within the cytoplasm, resulting in an increase in miltiradiene yield to 1.31 g/L. Furthermore, we conducted modifications to the miltiradiene synthase fusion protein tSmKSL1-CfTPS1. Finally, the comprehensive engineering strategies and protein modification strategies culminated in 1.43 g/L miltiradiene in the engineered yeast under shake flask culture conditions. Overall, our work established efficient yeast cell factories for miltiradiene production, providing a foothold for heterologous biosynthesis of abietane-type diterpenes.

Coordinating activation of endo-lysosomal two-pore channels and TRP mucolipins.

Two-pore channels and TRP mucolipins are ubiquitous endo-lysosomal cation channels of pathophysiological relevance. Both are Ca2+-permeable and regulated by phosphoinositides, principally PI(3,5)P2. Accumulating evidence has uncovered synergistic channel activation by PI(3,5)P2 and endogenous metabolites such as the Ca2+ mobilizing messenger NAADP, synthetic agonists including approved drugs and physical cues such as voltage and osmotic pressure. Here, we provide an overview of this coordination.

Specificity and dynamics of H2O2 detoxification by the cytosolic redox regulatory network as revealed by in vitro reconstitution.

The thiol redox state is a decisive functional characteristic of proteins in cell biology. Plasmatic cell compartments maintain a thiol-based redox regulatory network linked to the glutathione/glutathione disulfide couple (GSH/GSSG) and the NAD(P)H system. The basic network constituents are known and in vivo cell imaging with gene-encoded probes have revealed insight into the dynamics of the [GSH]2/[GSSG] redox potential, cellular H2O2 and NAD(P)H+H+ amounts in dependence on metabolic and environmental cues. Less understood is the contribution and interaction of the network components, also because of compensatory reactions in genetic approaches. Reconstituting the cytosolic network of Arabidopsis thaliana in vitro from fifteen recombinant proteins at in vivo concentrations, namely glutathione peroxidase-like (GPXL), peroxiredoxins (PRX), glutaredoxins (GRX), thioredoxins, NADPH-dependent thioredoxin reductase A and glutathione reductase and applying Grx1-roGFP2 or roGFP2-Orp1 as dynamic sensors, allowed for monitoring the response to a single H2O2 pulse. The major change in thiol oxidation as quantified by mass spectrometry-based proteomics occurred in relevant peptides of GPXL, and to a lesser extent of PRX, while other Cys-containing peptides only showed small changes in their redox state and protection. Titration of ascorbate peroxidase (APX) into the system together with dehydroascorbate reductase lowered the oxidation of the fluorescent sensors in the network but was unable to suppress it. The results demonstrate the power of the network to detoxify H2O2, the partially independent branches of electron flow with significance for specific cell signaling and the importance of APX to modulate the signaling without suppressing it and shifting the burden to glutathione oxidation.

Multi-modular metabolic engineering and efflux engineering for enhanced lycopene production in recombinant Saccharomyces cerevisiae.

Lycopene has been widely used in the food industry and medical field due to its antioxidant, anti-cancer, and anti-inflammatory properties. However, achieving efficient manufacture of lycopene using chassis cells on an industrial scale remains a major challenge. Herein, we attempted to integrate multiple metabolic engineering strategies to establish an efficient and balanced lycopene biosynthetic system in Saccharomyces cerevisiae. First, the lycopene synthesis pathway was modularized to sequentially enhance the metabolic flux of the mevalonate pathway, the acetyl-CoA supply module, and lycopene exogenous enzymatic module. The modular operation enabled the efficient conversion of acetyl-CoA to downstream pathway of lycopene synthesis, resulting in a 3.1-fold increase of lycopene yield. Second, we introduced acetate as an exogenous carbon source and utilized an acetate-repressible promoter to replace the natural ERG9 promoter. This approach not only enhanced the supply of acetyl-CoA but also concurrently diminished the flux toward the competitive ergosterol pathway. As a result, a further 42.3% increase in lycopene production was observed. Third, we optimized NADPH supply and mitigated cytotoxicity by overexpressing ABC transporters to promote lycopene efflux. The obtained strain YLY-PDR11 showed a 12.7-fold increase in extracellular lycopene level compared to the control strain. Finally, the total lycopene yield reached 343.7 mg/L, which was 4.3 times higher than that of the initial strain YLY-04. Our results demonstrate that combining multi-modular metabolic engineering with efflux engineering is an effective approach to improve the production of lycopene. This strategy can also be applied to the overproduction of other desirable isoprenoid compounds with similar synthesis and storage patterns in S. cerevisiae. ONE-SENTENCE SUMMARY: In this research, lycopene production in yeast was markedly enhanced by integrating a multi-modular approach, acetate signaling-based down-regulation of competitive pathways, and an efflux optimization strategy.

Systems Metabolic Engineering for Efficient Violaxanthin Production in Yeast.

Violaxanthin is a plant-derived orange xanthophyll with remarkable antioxidant activity that has wide applications in various industries, such as food, agriculture, and cosmetics. In addition, it is the key precursor of important substances such as abscisic acid and fucoxanthin. Saccharomyces cerevisiae, as a GRAS (generally regarded as safe) chassis, provides a good platform for producing violaxanthin production with a yield of 7.3 mg/g DCW, which is far away from commercialization. Herein, an integrated strategy involving zeaxanthin epoxidase (ZEP) source screening, cytosol redox state engineering, and nicotinamide adenine dinucleotide phosphate (NADPH) regeneration was implemented to enhance violaxanthin production in S. cerevisiae. 58aa-truncated ZEP from Vitis vinifera exhibited optimal efficiency in an efficient zeaxanthin-producing strain. The titer of violaxanthin gradually increased by 17.9-fold (up to 119.2 mg/L, 15.19 mg/g DCW) via cytosol redox state engineering and NADPH supplementation. Furthermore, balancing redox homeostasis considerably improved the zeaxanthin concentration by 139.3% (up to 143.9 mg/L, 22.06 mg/g DCW). Thus, the highest reported titers of violaxanthin and zeaxanthin in S. cerevisiae were eventually achieved. This study not only builds an efficient platform for violaxanthin biosynthesis but also serves as a useful reference for the microbial production of xanthophylls.

The role of the active site lysine residue on FAD reduction by NADPH in glutathione reductase.

Glutathione reductase (GR) is a two dinucleotide binding domain flavoprotein (tDBDF) that catalyzes the reduction of glutathione disulfide to glutathione coupled to the oxidation of NADPH to NADP+. An interesting feature of GR and other tDBDFs is the presence of a lysine residue (Lys-66 in human GR) at the active site, which interacts with the flavin group, but has an unknown function. To better understand the role of this residue, the dynamics of GR was studied using molecular dynamics simulations, and the reaction mechanism of FAD reduction by NADPH was studied using QM/MM molecular modeling. The two possible protonation states of Lys-66 were considered: neutral and protonated. Molecular dynamics results suggest that the active site is more structured for neutral Lys-66 than for protonated Lys-66. QM/MM modeling results suggest that Lys-66 should be in its neutral state for a thermodynamically favorable reduction of FAD by NADPH. Since the reaction is unfavorable with protonated Lys-66, the reverse reaction (the reduction of NADP+ by FADH-) is expected to take place. A phylogenetic analysis of various tDBDFs was performed, finding that an active site lysine is present in different the tDBDFs enzymes, suggesting that it has a conserved biological role. Overall, these results suggest that the protonation state of the active site lysine determines the energetics of the reaction, controlling its reversibility.

Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli.

5-Methyltetrahydrofolate (5-MTHF) is the sole active form of folate functioning in the human body and is widely used as a nutraceutical. Unlike the pollution from chemical synthesis, microbial synthesis enables green production of 5-MTHF. In this study, Escherichia coli BL21 (DE3) was selected as the host. Initially, by deleting 6-phosphofructokinase 1 and overexpressing glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, the glycolysis pathway flux decreased, while the pentose phosphate pathway flux enhanced. The ratios of NADH/NAD+ and NADPH/NADP+ increased, indicating elevated NAD(P)H supply. This led to more folate being reduced and the successful accumulation of 5-MTHF to 44.57 µg/L. Subsequently, formate dehydrogenases from Candida boidinii and Candida dubliniensis were expressed, which were capable of catalyzing the reaction of sodium formate oxidation for NAD(P)H regeneration. This further increased the NAD(P)H supply, leading to a rise in 5-MTHF production to 247.36 µg/L. Moreover, to maintain the balance between NADH and NADPH, pntAB and sthA, encoding transhydrogenase, were overexpressed. Finally, by overexpressing six key enzymes in the folate to 5-MTHF pathway and employing fed-batch cultivation in a 3 L fermenter, strain Z13 attained a peak 5-MTHF titer of 3009.03 µg/L, the highest level reported in E. coli so far. This research is a significant step toward industrial-scale microbial 5-MTHF production.

TIGAR reduces neuronal ferroptosis by inhibiting succinate dehydrogenase activity in cerebral ischemia.

Ischemia Stroke (IS) is an acute neurological condition with high morbidity, disability, and mortality due to a severe reduction in local cerebral blood flow to the brain and blockage of oxygen and glucose supply. Oxidative stress induced by IS predisposes neurons to ferroptosis. TP53-induced glycolysis and apoptosis regulator (TIGAR) inhibits the intracellular glycolytic pathway to increase pentose phosphate pathway (PPP) flux, promotes NADPH production and thus generates reduced glutathione (GSH) to scavenge reactive oxygen species (ROS), and thus shows strong antioxidant effects to ameliorate cerebral ischemia/reperfusion injury. However, in the current study, prolonged ischemia impaired the PPP, and TIGAR was unable to produce NADPH but was still able to reduce neuronal ferroptosis and attenuate ischemic brain injury. Ferroptosis is a form of cell death caused by free radical-driven lipid peroxidation, and the vast majority of ROS leading to oxidative stress are generated by mitochondrial succinate dehydrogenase (SDH) driving reverse electron transfer (RET) via the mitochondrial electron transport chain. Overexpression of TIGAR significantly inhibited hypoxia-induced enhancement of SDH activity, and TIGAR deficiency further enhanced SDH activity. We also found that the inhibitory effect of TIGAR on SDH activity was related to its mitochondrial translocation under hypoxic conditions. TIGAR may inhibit SDH activity by mediating post-translational modifications (acetylation and succinylation) of SDH A through interaction with SDH A. SDH activity inhibition reduces neuronal ferroptosis by decreasing ROS production, eliminating MitoROS levels and attenuating lipid peroxide accumulation. Notably, TIGAR-mediated inhibition of SDH activity and ferroptosis was not dependent on the PPP-NADPH-GPX4 pathways. In conclusion, mitochondrial translocation of TIGAR in prolonged ischemia is an important pathway to reduce neuronal ferroptosis and provide sustainable antioxidant defense for the brain under prolonged ischemia, further complementing the mechanism of TIGAR resistance to oxidative stress induced by IS.

The 2.5th generation enzymatic sensors based on the construction of quasi-direct electron transfer type NAD(P)-Dependent dehydrogenases.

We introduce a versatile method to convert NAD+ or NADP+ -dependent dehydrogenases into quasi-direct electron transfer (quasi-DET)-type dehydrogenases, by modifying with a mediator on the enzyme surface toward the development of 2.5th generation enzymatic sensors. In this study, we use ß-hydroxybutyrate (BHB) dehydrogenase (BHBDh) from Alcaligenes faecalis (AfBHBDh) as a representative NAD+ or NADP+ -dependent dehydrogenase. BHBDhs are important in ketone monitoring, especially for the diagnosis of diabetic ketoacidosis. We modified AfBHBDh with a thiol-reactive phenazine ethosulfate (trPES). We designed, constructed, and modified mutant BHBDhs harboring cysteine residues within 20 Å from the C4 nicotinamide in NAD+/NADH. Mutants Ser65Cys, Thr96Cys, and Lys106Cys showed indistinguishable catalytic activities from the wild-type enzyme, even after trPES modification. These trPES-modified mutants were immobilized on gold disk electrodes via amine coupling with succinimide-groups of dithiobis (succinimidyl hexanoate) self-assembled monolayers for electrochemical measurements. Considering there is a wide range of BHB concentrations, we exploited the linear regression in log scales. The linear range for the sensors with trPES-modified BHBDh mutants Ser65Cys, Thr96Cys, and Lys106Cys were 0.1-4.0 mM in both buffer solution and artificial interstitial fluid (ISF). They have limits of detection of 0.047 mM for Ser65Cys, 0.15 mM for Thr96Cys, and 0.060 mM for Lys106Cys in buffer solution, and 0.12 mM, 0.089 mM, and 0.044 mM in artificial ISF, respectively. These results indicate that redox mediator modification of NAD(P)-dependent dehydrogenases converts them into quasi-DET-type dehydrogenases, thereby enabling their utilization in 2.5th generation enzymatic sensors, which will facilitate the construction of enzymatic sensors suitable for continuous monitoring systems.

Distribution and Functional Analysis of Isocitrate Dehydrogenases across Kinetoplastids.

Isocitrate dehydrogenase is an enzyme converting isocitrate to α-ketoglutarate in the canonical tricarboxylic acid (TCA) cycle. There are three different types of isocitrate dehydrogenase documented in eukaryotes. Our study points out the complex evolutionary history of isocitrate dehydrogenases across kinetoplastids, where the common ancestor of Trypanosomatidae and Bodonidae was equipped with two isoforms of the isocitrate dehydrogenase enzyme: the NADP+-dependent isocitrate dehydrogenase 1 with possibly dual localization in the cytosol and mitochondrion and NADP+-dependent mitochondrial isocitrate dehydrogenase 2. In the extant trypanosomatids, isocitrate dehydrogenase 1 is present only in a few species suggesting that it was lost upon separation of Trypanosoma spp. and replaced by the mainly NADP+-dependent cytosolic isocitrate dehydrogenase 3 of bacterial origin in all the derived lineages. In this study, we experimentally demonstrate that the omnipresent isocitrate dehydrogenase 2 has a dual localization in both mitochondrion and cytosol in at least four species that possess only this isoform. The apparent lack of the NAD+-dependent isocitrate dehydrogenase activity in trypanosomatid mitochondrion provides further support to the existence of the noncanonical TCA cycle across trypanosomatids and the bidirectional activity of isocitrate dehydrogenase 3 when operating with NADP+ cofactor instead of NAD+. This observation can be extended to all 17 species analyzed in this study, except for Leishmania mexicana, which showed only low isocitrate dehydrogenase activity in the cytosol. The variability in isocitrate oxidation capacity among species may reflect the distinct metabolic strategies and needs for reduced cofactors in particular environments.