Ribose-5-phosphate Research Articles

A novel effect of sulforaphane on promoting mouse granulosa cells proliferation via the NRF2–TKT pathway

IntroductionGranulosa cells (GCs) is essential for maintaining follicular development. Follicle-stimulating Hormone (FSH) has been demonstrated to effectively promote GCs proliferation, driving the establishment of various superovulation techniques for animal husbandry. However, these techniques face challenges, such as high costs, hormonal imbalances, and an increased risk of early ovarian dysfunction. Therefore, it is important to investigate new methods to improve GCs proliferation. ObjectivesThis study aimed to investigate the effect of sulforaphane (SFN) on ovarian GCs proliferation and the underlying mechanisms. MethodsA comparative transcriptomic analysis of ovaries from the control, SFN, and FSH groups was conducted to identify the primary factors contributing to high proliferative capacity. The role of SFN in the regulation of cell proliferation has been examined in mouse ovarian GCs. Gene interference, overexpression, CUT&TAG technology, and transcriptome analyses were performed to elucidate the underlying mechanisms of the nuclear factor E2-related factor 2 (NRF2)–transketolase (TKT) axis in mediating GCs proliferation. ResultsOur research revealed a previously unknown function of SFN, an isothiocyanate of plant origin that is prevalent in cruciferous vegetables, in facilitating the proliferation of mouse ovarian GCs. The efficacy of SFN in enhancing GCs proliferation is similar to that of FSH. At the mechanistic level, SFN promotes NRF2 to transport to the nucleus, which subsequently activates the key enzyme of the non-oxidative pentose phosphate pathway TKT. This activation is instrumental in generating ribose 5-phosphate, a critical precursor for amino acid and nucleotide biosynthesis that underpins the proliferation of GCs. ConclusionCollectively, our findings delineate a novel pathway by which SFN, through the NRF2-TKT axis, enhances the nucleotide pool and thereby supports the proliferation of mouse GCs, presenting novel avenues for exploration in reproductive biology and agricultural sciences.

Glycerol 3-Phosphate Dehydrogenase Catalyzed Hydride Transfer: Enzyme Activation by Cofactor Pieces.

Glycerol 3-phosphate dehydrogenase catalyzes reversible hydride transfer from glycerol 3-phosphate (G3P) to NAD+ to form dihydroxyacetone phosphate; from the truncated substrate ethylene glycol to NAD+ in a reaction activated by the phosphite dianion substrate fragment; and from G3P to the truncated nicotinamide riboside cofactor in a reaction activated by adenosine 5'-diphosphate, adenosine 5'-monophosphate, and ribose 5-phosphate cofactor fragments. The sum of the stabilization of the transition state for GPDH-catalyzed hydride transfer reactions of the whole substrates by the phosphodianion fragment of G3P and the ADP fragment of NAD+ is 25 kcal/mol. Fourteen kcal/mol of this transition state stabilization is recovered as phosphite dianion and AMP activation of the reactions of the substrate and cofactor fragments. X-ray crystal structures for unliganded GPDH, for a binary GPDH·NAD+ complex, and for a nonproductive ternary GPDH·NAD+·DHAP complex show that the ligand binding energy is utilized to drive an extensive protein conformational change that creates a caged complex for these ligands. The phosphite dianion and AMP fragments are proposed to activate GPDH for the catalysis of hydride transfer by stabilization of this active caged complex. The closure of a conserved loop [292-LNGQKL-297] during substrate binding stabilizes the G3P and NAD+ complexes by interactions, respectively, with the Q295 and K296 loop side chains. The appearance and apparent conservation of two side chains that interact with the hydride donor and acceptor to stabilize the active closed enzyme are proposed to represent a significant improvement in the catalytic performance of GPDH.

Pentose phosphate pathway inhibition during in vitro maturation substantially affects the metabolism of bovine COCs and blastocysts

Glucose metabolism is widely examined in terms of its effect on oocytes and embryos quality. There are two main pathways of glucose metabolism – glycolysis and pentose phosphate pathway (PPP). The glycolytic pathway allows for energy production in the form of ATP and metabolites such as pyruvate and lactate, whereas PPP activity generates NADPH as well as ribose 5-phosphate, a precursor for the synthesis of nucleotides. The aim of the present experiment was the selective inhibition of either glycolysis or PPP during in vitro maturation of bovine cumulus-oocyte complexes (COCs) to demonstrate, how it affects COCs and further embryos with regard to selected lipidomic and metabolomic aspects. Inhibitors of glycolysis (IO) or PPP (DHEA) were applied during IVM, and the control group was matured under standard conditions. A set of COCs from each group was fertilized and obtained embryos were cultured to the blastocyst stage. ATP level was measured in oocytes, relative mRNA level of selected genes involved in energy metabolism was measured in cumulus cells (CC; real time PCR), lipid droplets parameters were evaluated in oocytes and CC whereas metabolome and lipidome (mass spectrometry) were evaluated in oocytes, CC and blastocysts as well. The experiment shows that glycolysis inhibition during IVM affects mainly CC with no effect in oocytes. It allows to maintain the good developmental potential of oocytes and no negative effect of blastocysts quality and quantity is observed. In contrary, PPP inhibition negatively affects metabolic and lipidomic parameters of both oocyte and CC, which further decreases blastocyst rate and quality. It is therefore concluded that PPP is the most crucial pathway of glucose metabolism for COC developmental potential.

Dysregulation of tetrahydrobiopterin metabolism in myalgic encephalomyelitis/chronic fatigue syndrome by pentose phosphate pathway.

Tetrahydrobiopterin (BH4) and its oxidized derivative dihydrobiopterin (BH2) were found to be strongly elevated in ME/CFS patients with orthostatic intolerance (ME + OI). However, the molecular mechanism of biopterin biogenesis is poorly understood in ME + OI subjects. Here, we report that the activation of the non-oxidative pentose phosphate pathway (PPP) plays a critical role in the biogenesis of biopterins (BH4 and BH2) in ME + OI subjects. Microarray-based gene screening followed by real-time PCR-based validation, ELISA assay, and finally enzyme kinetic studies of glucose-6-phosphate dehydrogenase (G6PDH), transaldolase (TALDO1), and transketolase (TK) enzymes revealed that the augmentation of anaerobic PPP is critical in the regulations of biopterins. To further investigate, we devised a novel cell culture strategy to induce non-oxidative PPP by treating human microglial cells with ribose-5-phosphate (R5P) under a hypoxic condition of 85%N2/10%CO2/5%O2 followed by the analysis of biopterin metabolism via ELISA, immunoblot, and dual immunocytochemical analyses. Moreover, the siRNA knocking down of the taldo1 gene strongly inhibited the bioavailability of phosphoribosyl pyrophosphate (PRPP), reduced the expressions of purine biosynthetic enzymes, attenuated GTP cyclohydrolase 1 (GTPCH1), and suppressed subsequent production of BH4 and its metabolic conversion to BH2 in R5P-treated and hypoxia-induced C20 human microglia cells. These results confirmed that the activation of non-oxidative PPP is indeed required for the upregulation of both BH4 and BH2 via the purine biosynthetic pathway. To test the functional role of ME + OI plasma-derived biopterins, exogenously added plasma samples of ME + OI plasma with high BH4 upregulated inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in human microglial cells indicating that the non-oxidative PPP-induced-biopterins could stimulate inflammatory response in ME + OI patients. Taken together, our current research highlights that the induction of non-oxidative PPP regulates the biogenesis of biopterins contributing to ME/CFS pathogenesis.

Design and Synthesis of Cyclic Dinucleotide Analogues Containing Triazolyl C-Nucleosides.

Natural cyclic dinucleotide (CDN) is the secondary messenger involved in bacterial hemostasis, human innate immunity, and bacterial antiphage immunity. Synthetic CDN and its analogues are key molecular probes and potential immunotherapeutic agents. Several CDN analogues are under clinical research for antitumor immunotherapy. A myriad of synthetic methods have been developed and reported for the preparation of CDN and its analogues. However, most of the protocols require multiple steps, and only one CDN or its analogue is prepared at a time. In this study, a strategy based on a macrocyclic ribose phosphate skeleton containing a 1'-alkynyl group was designed and developed to prepare CDN analogues containing triazolyl C-nucleosides by click chemistry. Combinatorial application of click chemistry and the sulfenylation cascade to the macrocyclic skeleton expanded the diversity of the CDN analogues. This macrocyclic skeleton strategy rapidly and efficiently provides CDN analogues to facilitate research on microbiology, immunology, and immunotherapy.

Integrated network pharmacology and metabolomics to reveal the mechanism of Pinellia ternata inhibiting non-small cell lung cancer cells

Lung cancer is a malignant tumor with highly heterogeneous characteristics. A classic Chinese medicine, Pinellia ternata (PT), was shown to exert therapeutic effects on lung cancer cells. However, its chemical and pharmacological profiles are not yet understood. In the present study, we aimed to reveal the mechanism of PT in treating lung cancer cells through metabolomics and network pharmacology. Metabolomic analysis of two strains of lung cancer cells treated with Pinellia ternata extracts (PTE) was used to identify differentially abundant metabolites, and the metabolic pathways associated with the DEGs were identified by MetaboAnalyst. Then, network pharmacology was applied to identify potential targets against PTE-induced lung cancer cells. The integrated network of metabolomics and network pharmacology was constructed based on Cytoscape. PTE obviously inhibited the proliferation, migration and invasion of A549 and NCI-H460 cells. The results of the cellular metabolomics analysis showed that 30 metabolites were differentially expressed in the lung cancer cells of the experimental and control groups. Through pathway enrichment analysis, 5 metabolites were found to be involved in purine metabolism, riboflavin metabolism and the pentose phosphate pathway, including D-ribose 5-phosphate, xanthosine, 5-amino-4-imidazolecarboxyamide, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Combined with network pharmacology, 11 bioactive compounds were found in PT, and networks of bioactive compound–target gene–metabolic enzyme–metabolite interactions were constructed. In conclusion, this study revealed the complicated mechanisms of PT against lung cancer. Our work provides a novel paradigm for identifying the potential mechanisms underlying the pharmacological effects of natural compounds.

Reconstruction and identification of the native PLP synthase complex from Methanosarcina acetivorans lysate.

Many protein-protein interactions behave differently in biochemically purified forms as compared to their in vivo states. As such, determining native protein structures may elucidate structural states previously unknown for even well-characterized proteins. Here we apply the bottom-up structural proteomics method, cryoID , toward a model methanogenic archaeon. While they are keystone organisms in the global carbon cycle and active members of the human microbiome, there is a general lack of characterization of methanogen enzyme structure and function. Through the cryoID approach, we successfully reconstructed and identified the native Methanosarcina acetivorans pyridoxal 5'-phosphate (PLP) synthase (PdxS) complex directly from cryogenic electron microscopy (cryoEM) images of fractionated cellular lysate. We found that the native PdxS complex exists as a homo-dodecamer of PdxS subunits, and the previously proposed supracomplex containing both the synthase (PdxS) and glutaminase (PdxT) was not observed in cellular lysate. Our structure shows that the native PdxS monomer fashions a single 8α/8β TIM-barrel domain, surrounded by seven additional helices to mediate solvent and interface contacts. A density is present at the active site in the cryoEM map and is interpreted as ribose 5-phosphate. In addition to being the first reconstruction of the PdxS enzyme from a heterogeneous cellular sample, our results reveal a departure from previously published archaeal PdxS crystal structures, lacking the 37 amino acid insertion present in these prior cases. This study demonstrates the potential of applying the cryoID workflow to capture native structural states at atomic resolution for archaeal systems, for which traditional biochemical sample preparation is nontrivial.

Glutaryl-CoA dehydrogenase suppresses tumor progression and shapes an anti-tumor microenvironment in hepatocellular carcinoma

Crotonylation, a crotonyl-CoA-based non-enzymatic protein translational modification, affects diverse biological processes, such as spermatogenesis, tissue injury, inflammation, and neuropsychiatric diseases. Crotonylation is decreased in hepatocellular carcinomas (HCCs), but the mechanism remains unknown. In this study, we aim to describe the role of glutaryl-CoA dehydrogenase (GCDH) in tumor suppression. Three cohorts containing 40, 248 and 17 pairs of samples were used to evaluate the link between GCDH expression levels and clinical characteristics of HCC, as well as responses to anti-programmed cell death protein 1 (PD-1) treatment. Subcutaneous xenograft, orthotopic xenograft, Trp53Δhep/Δhep; MYC- and Ctnnboe; METoe-driven mouse models were adopted to validate the effects of GCDH on HCC suppression. GCDH depletion promoted HCC growth and metastasis, whereas its overexpression reversed these processes. AsGCDH converts glutaryl-CoA to crotonyl-CoA to increase crotonylation levels, we performed lysine crotonylome analysisand identified the pentose phosphate pathway (PPP) and glycolysis-related proteins PGD, TKT, and ALDOC asGCDH-induced crotonylation targets. Crotonyl-bound targets showed allosteric effects that controlled their enzymatic activities, leading to decreases in ribose 5-phosphate and lactate production, further limiting the Warburg effect. PPPblockade also stimulated peroxidation, synergizing with senescent modulators to induce senescence in GCDHhigh cells.These cells induced the infiltration of immune cells by the SASP (senescence-associated secretory cell phenotype)toshape an anti-tumor immune microenvironment. Meanwhile, the GCDHlow population was sensitized to anti-PD-1 therapy. GCDH inhibits HCC progression via crotonylation-induced suppression of the PPP and glycolysis, resulting in HCC cell senescence. The senescent cell further shapes an anti-tumor microenvironment via the SASP. The GCDHlow population is responsive to anti-PD-1 therapy because of the increased presence of PD-1+CD8+ T cells. Glutaryl-CoA dehydrogenase (GCDH) is a favorable prognostic indicator in liver, lung, and renal cancers. In addition, most GCDH depletion-induced toxic metabolites originate from the liver, accumulate locally, and cannot cross the blood-brain barrier. Herein, we show that GCDH inhibits hepatocellular carcinoma (HCC) progression via crotonylation-induced suppression of the pentose phosphate pathway and glycolysis, resulting in HCC cell senescence. We also found that more PD-1+CD8+ T cells are present in the GCDHlow population, who are thus more responsive to anti-PD-1 therapy. Given that the GCDHlow and GCDHhigh HCC population can be distinguished based on serum glucose and ammonia levels, it will be worthwhile to evaluate the curative effects of pro-senescent and immune-therapeutic strategies based on the expression levels of GCDH.

Oleic acid stimulates proliferation of RMG-1 ovarian cancer cells by activating the pentose phosphate pathway and glutamine metabolism

Extracellular fatty acids (FAs) play an important role in regulating cellular functions such as cell proliferation, survival, and migration. The effects of oleic acid (OA) on cancer cells vary depending on the cell type. Our prior study showed that two distinct ovarian cancer cell lines, RMG-1 and HNOA, proliferate in response to OA, but they differ with respect to glucose utilization. Here, we aimed to elucidate the mechanism(s) by which OA stimulates proliferation of RMG-1 cells. We found that OA stimulates RMG-1 proliferation by activating the FA transporter CD36. OA also increases uptake of glucose and glutamine, which subsequently activate the pentose phosphate pathway (PPP) and glutamine metabolism, respectively. Given that ribose 5-phosphate derived from the PPP is utilized for glutamine metabolism and the subsequent de novo nucleotide synthesis, our findings suggest that OA affects the PPP associated with Gln metabolism, rather than glycolysis associated with glutaminolysis; this leads ultimately to activation of DNA synthesis, which is required for cell proliferation. This selective activation by OA contrasts with the mechanisms observed in HNOA cells, in which OA-induced cell proliferation is driven by transcriptional regulation of the GLUT gene. The diverse responses of cancer cells to OA may be attributed to distinct mechanisms of OA reception and/or different metabolic pathways activated by OA.

The immune response to RNA suppresses nucleic acid synthesis by limiting ribose 5-phosphate

During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.

Pyruvate kinase 2 from Synechocystis sp. PCC 6803 increased substrate affinity via glucose-6-phosphate and ribose-5-phosphate for phosphoenolpyruvate consumption

Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the Km values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.

Metabolic plasticity, essentiality and therapeutic potential of ribose-5-phosphate synthesis in Toxoplasma gondii

Ribose-5-phosphate (R5P) is a precursor for nucleic acid biogenesis; however, the importance and homeostasis of R5P in the intracellular parasite Toxoplasma gondii remain enigmatic. Here, we show that the cytoplasmic sedoheptulose-1,7-bisphosphatase (SBPase) is dispensable. Still, its co-deletion with transaldolase (TAL) impairs the double mutant’s growth and increases 13C-glucose-derived flux into pentose sugars via the transketolase (TKT) enzyme. Deletion of the latter protein affects the parasite’s fitness but is not lethal and is correlated with an increased carbon flux via the oxidative pentose phosphate pathway. Further, loss of TKT leads to a decline in 13C incorporation into glycolysis and the TCA cycle, resulting in a decrease in ATP levels and the inability of phosphoribosyl-pyrophosphate synthetase (PRPS) to convert R5P into 5′-phosphoribosyl-pyrophosphate and thereby contribute to the production of AMP and IMP. Likewise, PRPS is essential for the lytic cycle. Not least, we show that RuPE-mediated metabolic compensation is imperative for the survival of the ΔsbpaseΔtal strain. In conclusion, we demonstrate that multiple routes can flexibly supply R5P to enable parasite growth and identify catalysis by TKT and PRPS as critical enzymatic steps. Our work provides novel biological and therapeutic insights into the network design principles of intracellular parasitism in a clinically-relevant pathogen.

TREM1 disrupts myeloid bioenergetics and cognitive function in aging and Alzheimer disease mouse models.

Human genetics implicate defective myeloid responses in the development of late-onset Alzheimer disease. A decline in peripheral and brain myeloid metabolism, triggering maladaptive immune responses, is a feature of aging. The role of TREM1, a pro-inflammatory factor, in neurodegenerative diseases is unclear. Here we show that Trem1 deficiency prevents age-dependent changes in myeloid metabolism, inflammation and hippocampal memory function in mice. Trem1 deficiency rescues age-associated declines in ribose 5-phosphate. In vitro, Trem1-deficient microglia are resistant to amyloid-β42 oligomer-induced bioenergetic changes, suggesting that amyloid-β42 oligomer stimulation disrupts homeostatic microglial metabolism and immune function via TREM1. In the 5XFAD mouse model, Trem1 haploinsufficiency prevents spatial memory loss, preserves homeostatic microglial morphology, and reduces neuritic dystrophy and changes in the disease-associated microglial transcriptomic signature. In aging APPSwe mice, Trem1 deficiency prevents hippocampal memory decline while restoring synaptic mitochondrial function and cerebral glucose uptake. In postmortem Alzheimer disease brain, TREM1 colocalizes with Iba1+ cells around amyloid plaques and its expression is associated with Alzheimer disease clinical and neuropathological severity. Our results suggest that TREM1 promotes cognitive decline in aging and in the context of amyloid pathology.

Systematic Engineering of Escherichia coli for Efficient Production of Pseudouridine from Glucose and Uracil.

In this study, we proposed a biological approach to efficiently produce pseudouridine (Ψ) from glucose and uracil in vivo using engineered Escherichia coli. By screening host strains and core enzymes, E. coli MG1655 overexpressing Ψ monophosphate (ΨMP) glycosidase and ΨMP phosphatase was obtained, which displayed the highest Ψ concentration. Then, optimization of the RBS sequences, enhancement of ribose 5-phosphate supply in the cells, and overexpression of the membrane transport protein UraA were investigated. Finally, fed-batch fermentation of Ψ in a 5 L fermentor can reach 27.5 g/L with a yield of 89.2 mol % toward uracil and 25.6 mol % toward glucose within 48 h, both of which are the highest to date. In addition, the Ψ product with a high purity of 99.8% can be purified from the fermentation broth after crystallization. This work provides an efficient and environmentally friendly protocol for allowing for the possibility of Ψ bioproduction on an industrial scale.

The impact of some metals, molecular docking and molecular dynamic calculations on glucose 6-phosphate dehydrogenase activity in Capoeta trutta (Heckel, 1843) tissue

The first enzyme of the pentose phosphate metabolic pathway is glucose 6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP + oxidoreductase EC1.1.1.49; G6PD). G6PD has essential functions such as membrane lipid synthesis, ribose 5-phosphate, and NADPH production. In this study, the G6PD enzyme was purified from kidney, liver, and gill tissues of Capoeta trutta, one of the dominant fish species in the Euphrates-Tigris River System, and the in vitro effects of some metals (Ag+, Cd2+, Cu2+, Fe2+, Ni2+, Pb2+ and Zn2+) on the enzyme activity were investigated. For this purpose, firstly, the G6PD enzyme was purified from tissues using a 2′, 5′-ADP Sepharose 4B affinity column. The purity of the enzyme was checked by the SDS-PAGE method and a single band was seen in the gel. After the purity of the enzyme was determined, the effects of metals on the enzyme activity were determined using the spectrophotometric method. As a result of the study, it was determined that the Ag+ ion was the most potent inhibitor for C. trutta gill, kidney, and liver G6PD enzymes. Lastly, calculations were made to examine the activity of the glucose 6-phosphate dehydrogenase molecule against the G6PD enzymes. Afterwards, the interaction of the glucose 6-phosphate dehydrogenase molecule with the protein (PDB ID: 5JYU and 2BH9) with the highest activity was calculated in the range of 0–100 ns. Finally, ADME/T calculation was made to predict the effects and reactions of glucose 6-phosphate dehydrogenase molecule in human metabolism. This study explores the physiological functions and environmental sensitivities of G6PD in a dominant fish species, while also investigating its potential interactions and metabolic roles in humans. Understanding these aspects can contribute to environmental monitoring, fish health management, and even pharmaceutical development.

Mechanical abrasion stimulation: Altered epidermal mucus composition and microbial community in grass carp (Ctenopharyngodon idella)

In modern high-density farming, seasonal change, fishing, transportation, and other conditions easily cause mechanical abrasion of fish skin. This leads to alterations in the material balance of the epidermal mucus composition of fish, preventing normal immune function. Subsequently, the risk of disease increases. In this study, grass carp (Ctenopharyngodon idella) was used as an object to investigate immune-related enzyme activities, structural characteristics, and physiological changes of its epidermal mucous cells caused by mechanical abrasion. Furthermore, we explored the changes in body surface mucus composition and microbial community diversity and structure. Our results showed that mechanical abrasion stimulation, significantly changed the activities of immune-related enzymes, such as lysozyme (LZM), alkaline phosphatase (AKP), and acid phosphatase (ACP) were within 24 h (P < 0.05). Their levels gradually recovered after 48 h. In contrast, no significant change was observed in superoxide dismutase (SOD) levels. The number of mucous cells increased, especially those of type I and type II mucous cells in the dorsal skin, which were significantly increased (P < 0.05). Meanwhile, the stimulation changed the metabolites composition of the fish epidermal mucus in the short term, upregulating 12-OPDA, indole, quinic acid and GSH, which are beneficial to antibacterial and anti-inflammatory, and downregulating L-saccharopine, L-2-aminoadipic acid, spermine, GSSG and ribose 5-phosphate, which disrupt protein synthesis, energy production and DNA synthesis. Additionally, these metabolite changes further affect the composition of microorganism communities, rising the abundance of certain disease-associated microorganisms, such as Acidovorax, Rhizobiaceae, Aurantimicrobium, and Leifsonia, which increases the risk of disease in fish. The findings of the present study provide a deeper understanding of the role of mucus as a component of the immune system in fish and provide a scientific basis for effective grass carp culture management and disease control.

Comprehensive Analysis of Metabolites in Postmortem Brains of Patients with Alzheimer's Disease.

Disturbed metabolism has been proposed as being involved in the pathogenesis of Alzheimer's disease (AD), and more evidence from human AD brains is required. In this study, we attempted to identify or confirm modulations in the levels of metabolites associated with AD in postmortem AD brains. We performed metabolomics analyses using a gas chromatography mass spectrometry system in postmortem brains of patients with confirmed AD, patients with CERAD score B, and control subjects. Impaired phosphorylation of glucose and elevation of several tricarboxylic acid (TCA) metabolites, except citrate, were observed and the degree of impaired phosphorylation and elevation in the levels of the TCA cycle metabolites were negatively and positively correlated, respectively, with the clinical phenotypes of AD. The levels of uronic acid pathway metabolites were modulated in AD and correlated positively with the amyloid-β content. The associations of nucleic acid synthesis and amino acid metabolites with AD depended on the kinds of metabolites; in particular, the contents of ribose 5-phosphate, serine and glycine were negatively correlated, while those of ureidosuccinic acid and indole-3-acetic acid were positively modulated in AD. Comprehensive statistical analyses suggested that alterations in the inositol pathway were most closely associated with AD. The present study revealed many novel associations between metabolites and AD, suggesting that some of these might serve as novel potential therapeutic targets for AD.

The Emerging Roles of the Metabolic Regulator G6PD in Human Cancers.

Metabolic reprogramming, especially reprogrammed glucose metabolism, is a well-known cancer hallmark related to various characteristics of tumor cells, including proliferation, survival, metastasis, and drug resistance. Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway (PPP), a branch of glycolysis, that converts glucose-6-phosphate (G6P) into 6-phosphogluconolactone (6PGL). Furthermore, PPP produces ribose-5-phosphate (R5P), which provides sugar-phosphate backbones for nucleotide synthesis as well as nicotinamide adenine dinucleotide phosphate (NADPH), an important cellular reductant. Several studies have shown enhanced G6PD expression and PPP flux in various tumor cells, as well as their correlation with tumor progression through cancer hallmark regulation, especially reprogramming cellular metabolism, sustaining proliferative signaling, resisting cell death, and activating invasion and metastasis. Inhibiting G6PD could suppress tumor cell proliferation, promote cell death, reverse chemoresistance, and inhibit metastasis, suggesting the potential of G6PD as a target for anti-tumor therapeutic strategies. Indeed, while challenges-including side effects-still remain, small-molecule G6PD inhibitors showing potential anti-tumor effect either when used alone or in combination with other anti-tumor drugs have been developed. This review provides an overview of the structural significance of G6PD, its role in and regulation of tumor development and progression, and the strategies explored in relation to G6PD-targeted therapy.

Abstract B108: NUDT5 inhibition differentially affects IDH1-Wildtype and Mutant high-grade glioma to induce NAD+ independent radiosensitization and anti-proliferative effects

Abstract Adult-type diffuse gliomas are aggressive brain cancers. The widespread standard treatment for these high-grade gliomas (HGG) is the Stupp Protocol, consisting of maximal safe surgical resection followed by concurrent chemoradiotherapy. However, since the Stupp Protocol, no significant therapeutic advances have been as widely and easily adopted, halting improvements in HGG patient survival. Therefore, identifying new drug targets or treatments that enhance the Stupp Protocol would result in therapeutic benefit. HGG are classified by distinct genetic and metabolic alterations; the most well-known being somatic gain of function mutations in isocitrate dehydrogenase (IDH) 1/2 genes. These mutations distinguish the two most severe HGGs, namely Grade 4 IDH-Mutant Astrocytoma and IDH-Wildtype Glioblastoma (GBM). IDH-mutant tumors accumulate the oncometabolite D-2-Hydroxyglutarate, which alters metabolic pathways and DNA damage responses. One metabolic alteration is decreased nicotinamide adenine dinucleotide (NAD+) levels, a metabolite essential for cellular homeostasis, and crucial in IDH-mutants as targeting NAD+ homeostasis is effective against IDH-mutant tumors. Here, we investigated if additional metabolic vulnerabilities within NAD+ and purine biosynthetic pathways could be identified in HGG cells. For this, a panel of cell lines, including an isogenic IDH1-mutant cell line, was screened for their sensitivity to a range of metabolic inhibitors in combination or not with chemo- and radiotherapy. This screen identified NUDT5, a Nudix hydrolase that metabolizes ADP-Ribose into Ribose-5-Phosphate (R-5-P) to replenish NAD+ pools, and synthesizes nuclear ATP for hormone driven chromatin remodeling, as an important determinant in glioma cell survival. Cell viability assays determined NUDT5 inhibition (NUDT5i) sensitized IDH1-wildtype HGG cells to different radiotherapy modalities, specifically X-rays and alpha particles, whilst IDH1-mutant HGG were sensitive to NUDT5i alone. Live-cell imaging analysis indicated NUDT5i induced cytostasis followed by cytotoxicity in the IDH1-mutant, suggesting IDH1-mutations confer exploitable metabolic dependencies on NUDT5 for proliferation and survival. NUDT5i did not significantly deplete NAD+ levels and NAD+ precursor supplementation failed to rescue cell survival and proliferation, indicating effects of NUDT5i are NAD+ independent. Our results indicate NUDT5 plays a crucial role in HGG proliferation and survival. Given that NUDT5 produces R-5-P for downstream nucleotide synthesis, we hypothesize that NUDT5i impairs nucleotide homeostasis and leads to replication stress. Current investigations include nucleoside supplementation and characterization of replication stress signaling by western blotting. We anticipate our results to prelude further investigations into NUDT5 as a potential therapeutic target in HGG. We suggest NUDT5i could enhance the effectiveness of the Stupp Protocol regardless of IDH-Status and advance HGG therapies. Citation Format: Thomas J.R Cox, Eren Ozcagli, Giuseppe Schettino, Lisiane B Meira. NUDT5 inhibition differentially affects IDH1-Wildtype and Mutant high-grade glioma to induce NAD+ independent radiosensitization and anti-proliferative effects [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B108.

G6PDH as a key immunometabolic and redox trigger in arthropods.

The enzyme glucose-6-phosphate dehydrogenase (G6PDH) plays crucial roles in glucose homeostasis and the pentose phosphate pathway (PPP), being also involved in redox metabolism. The PPP is an important metabolic pathway that produces ribose and nicotinamide adenine dinucleotide phosphate (NADPH), which are essential for several physiologic and biochemical processes, such as the synthesis of fatty acids and nucleic acids. As a rate-limiting step in PPP, G6PDH is a highly conserved enzyme and its deficiency can lead to severe consequences for the organism, in particular for cell growth. Insufficient G6PDH activity can lead to cell growth arrest, impaired embryonic development, as well as a reduction in insulin sensitivity, inflammation, diabetes, and hypertension. While research on G6PDH and PPP has historically focused on mammalian models, particularly human disorders, recent studies have shed light on the regulation of this enzyme in arthropods, where new functions were discovered. This review will discuss the role of arthropod G6PDH in regulating redox homeostasis and immunometabolism and explore potential avenues for further research on this enzyme in various metabolic adaptations.