Glycerol-3-phosphate Dehydrogenase Research Articles

Activities of Energy and Carbohydrate Metabolism Enzymes in Rainbow Trout Оncorhynchus mykiss Walb. upon Introduction of 24-hour Lighting in Aquaculture in Southern Russia

Abstract Activities of energy and carbohydrate metabolism enzymes (cytochrome c oxidase (COX), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PD), glycerol 1-phosphate dehydrogenase (G1PDH), lactate dehydrogenase (LDH), and aldolase) were studied in rainbow trout Oncorhynchus mykiss Walb. fish grown in aquaculture in North Ossetia–Alania after introducing a regime with 24-h lighting and night feeding. COX and PK activities in the liver of fish from the experimental group were found to be significantly higher than in control fish, indicating an increase in aerobic ATP synthesis. Aldolase activity in organs of fish grown with 24-h lighting was lower than in the control fish, indicating a decrease in carbohydrate utilization in glycolysis in muscles and a lower intensity of gluconeogenesis in the liver. The differences made it possible to assume that the introduction of 24-h lighting and night feeding changed energy and carbohydrate metabolism to facilitate biosynthetic processes and, therefore, weight gain in fish.

Transcriptome and Expression Analysis of Glycerol Biosynthesis-Related Genes in Glenea cantor Fabricius (Cerambycidae: Lamiinae)

Glenea cantor Fabricius (Cerambycidae: Lamiinae) is an important pest that damages kapok trees in Southeast Asia with a wide adaptability to temperature. Glycerol is a protectant and energy source for insects in low-temperature environments. However, glycerol biosynthesis-related genes at the molecular level are limited in G. cantor. In this study, the supercooling points and freezing points at different stages were measured, and the cold hardiness of male and female pupae significantly differed. Moreover, a full-length transcriptome of G. cantor was established; glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH) genes, which are related to glycerol metabolism, were identified, with a special focus on their expression profiles. A total of 24,476 isoforms stemmed from the full-length transcriptome, along with 568 lncRNAs, 56 transcription factor (TF) families, and 1467 alternative splicing (AS) events. The KEGG pathway enrichment analysis revealed that the isoforms associated with AS were enriched primarily in glycerolipid and glycerophospholipid metabolism. In total, three GK genes and one GPDH gene were identified, and GcGK1 and GcGK3 presented differential sex expression during the pupal stage, which may play a role in thermal adaptability. This study provides a valuable transcriptional database of G. cantor and helps to elucidate the function of glycerol in the thermal adaptation mechanism of longhorn beetles.

Fucosylated chondroitin sulfate alleviates diet-induced obesity by modulating intestinal lipid metabolism and colonic microflora

Fucosylated chondroitin sulfate from Pearsonothuria graeffei (FCS-Pg), a natural macromolecular polysaccharide, has been proven to prevent obesity, but its underlying molecular mechanism is still unclear. C57BL/6 J mice fed on high fat diet (HFD) were administered FCS-Pg lasting for ten weeks. The results demonstrated that FCS-Pg supplementation reduced body weight with dosage manner compared with HFD group. The expressions of intestinal lipid synthesis related proteins such as cluster of differentiation 36 (CD36) in FCS-Pg group were lower than those in the HFD group. Compared with the normal group, HFD caused gut microbiota disorder in the colon. FCS-Pg supplementation at high dosage restored the gut microbiota composition with higher abundance of Alistipes and Bacteroidetes and lower abundance of Colidextribacter, Bilophila and Firmicutes compared with HFD group. Moreover, FCS-Pg decreased the expression of proteins involved in triglyceride synthesis such as glycerol 3-phosphate dehydrogenase 1 (GPD1) and increased the expression of proteins involved in lipolysis and thermogenesis such as adipose triglyceride lipase (ATGL) and uncoupling protein 1 (UCP1) in the white adipose tissue (WAT) compared with HFD group. In conclusion, our study suggested that FCS-Pg significantly prevented obesity and improved WAT function in HFD-fed mice by regulating intestinal lipid metabolism and microflora composition.

Multiomics Studies on Metabolism Changes in Alcohol-Associated Liver Disease.

Metabolic dysfunction in the liver represents a predominant feature in the early stages of alcohol-associated liver disease (ALD). However, the mechanisms underlying this are only partially understood. To investigate the metabolic characteristics of the liver in ALD, we did a relative quantification of polar metabolites and lipids in the liver of mice with experimental ALD using untargeted metabolomics and untargeted lipidomics. A total of 99 polar metabolites had significant abundance alterations in the livers of alcohol-fed mice. Pathway analysis revealed that amino acid metabolism was the most affected by alcohol in the mouse liver. Metabolites involved in glycolysis and the TCA cycle were decreased, while glycerol 3-phosphate (G3P) and long-chain fatty acids were increased. Relative quantification of lipids unveiled an upregulation of multiple lipid classes, suggesting that alcohol consumption drives metabolism toward lipid synthesis. Results from enzyme expression and activity detection indicated that the decreased activity of mitochondrial glycerol 3-phosphate dehydrogenase contributed to the disordered metabolism.

MiR-210 as a therapeutic target in diabetes-associated endothelial dysfunction.

MicroRNA (miR)-210 function in endothelial cells and its role in diabetes-associated endothelial dysfunction are not fully understood. We aimed to characterize the miR-210 function in endothelial cells and study its therapeutic potential in diabetes. Two different diabetic mouse models (db/db and Western diet-induced), miR-210 knockout and transgenic mice, isolated vessels and human endothelial cells were used. miR-210 levels were lower in aortas isolated from db/db than in control mice. Endothelium-dependent relaxation (EDR) was impaired in aortas from miR-210 knockout mice, and this was restored by inhibiting miR-210 downstream protein tyrosine phosphatase 1B (PTP1B), mitochondrial glycerol-3-phosphate dehydrogenase 2 (GPD2), and mitochondrial oxidative stress. Inhibition of these pathways also improved EDR in both diabetic mouse models. High glucose reduced miR-210 levels in endothelial cells and impaired EDR in mouse aortas, effects that were reversed by overexpressing miR-210. However, plasma miR-210 levels were not affected in individuals with type 2 diabetes (T2D) following improved glycaemic status. Of note, genetic overexpression using miR-210 transgenic mice and pharmacological overexpression using miR-210 mimic in vivo ameliorated endothelial dysfunction in both diabetic mouse models by decreasing PTP1B, GPD2 and oxidative stress. Genetic overexpression of miR-210 altered the aortic transcriptome, decreasing genes in pathways involved in oxidative stress. miR-210 mimic restored decreased nitric oxide production by high glucose in endothelial cells. This study unravels the mechanisms by which down-regulated miR-210 by high glucose induces endothelial dysfunction in T2D and demonstrates that miR-210 serves as a novel therapeutic target.

Evaluation of glucocorticoid-related genes reveals GPD1 as a therapeutic target and regulator of sphingosine 1-phosphate metabolism in CRPC

Prostate cancer (PCa) is an androgen-dependent disease, with castration-resistant prostate cancer (CRPC) being an advanced stage that no longer responds to androgen deprivation therapy (ADT). Mounting evidence suggests that glucocorticoid receptors (GR) confer resistance to ADT in CRPC patients by bypassing androgen receptor (AR) blockade. GR, as a novel therapeutic target in CRPC, has attracted substantial attention worldwide. This study utilized bioinformatic analysis of publicly available CRPC single-cell data to develop a consensus glucocorticoid-related signature (Glu-sig) that can serve as an independent predictor for relapse-free survival. Our results revealed that the signature demonstrated consistent and robust performance across seven publicly accessible datasets and an internal cohort. Furthermore, our findings demonstrated that glycerol-3-phosphate dehydrogenase 1 (GPD1) in Glu-sig can significantly promote CRPC progression by mediating the cell cycle pathway. Additionally, GPD1 was shown to be regulated by GR, with the GR antagonist mifepristone enhancing the anti-tumorigenic effects of GPD1 in CRPC cells. Mechanistically, targeting GPD1 induced the production of sphingosine 1-phosphate (S1P) and enhanced histone acetylation, thereby inducing the transcription of p21 that involved in cell cycle regulation. In conclusion, Glu-sig could serve as a robust and promising tool to improve the clinical outcomes of PCa patients, and modulating the GR/GPD1 axis that promotes tumor growth may be a promising approach for delaying CRPC progression.

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.

Changes in primary metabolism and associated gene expression during host-pathogen interaction in clubroot resistance of Brassica napus.

The role of primary metabolism during Brassica napus-Plasmodiophora brassicae interaction leading to clubroot resistance has not yet been investigated thoroughly. In this study, we investigated some of the primary metabolites and their derivatives as well as expression of the genes involved in their biosynthesis to decipher this host-pathogen interaction. For this, two sets (clubroot resistant and susceptible) of canola lines were inoculated with P. brassicae pathotype 3A to investigate the endogenous levels of primary metabolites at 7-, 14-, and 21-days after inoculation (DAI). The associated pathways were curated, and expression of the selected genes was analyzed using qRT-PCR. Our results suggested the possible involvement of polyamines (spermidine and spermine) in clubroot susceptibility. Some of the amino acids were highly abundant at 7- or 14-DAI in both resistant and susceptible lines; however, glutamine and the amino acid derivative phenylethylamine showed higher endogenous levels in the resistant lines at later stages of infection. Organic acids such as malic, fumaric, succinic, lactic and citric acids were abundant in the susceptible lines. Conversely, the abundance of salicylic acid (SA) and the expression of benzoate/salicylate carboxyl methyltransferase (BSMT) were higher in the resistant lines at the secondary stage of infection. A reduced disease severity index and gall size were observed when exogenous SA (1.0 mM) was applied to susceptible B. napus; this further supported the role of SA in clubroot resistance. In addition, a higher accumulation of fatty acids and significant upregulation of the pathway genes, glycerol-3-phosphate dehydrogenase (GPD) and amino alcohol phosphotransferase (AAPT) were observed in the resistant lines at 14- and 21-DAI. In contrast, some of the fatty acid derivatives such as phosphatidylcholines represented a lower level in the resistant lines. In conclusion, our findings provided additional insights into the possible involvement of primary metabolites and their derivatives in clubroot resistance.

Engineering new-to-nature biochemical conversions by combining fermentative metabolism with respiratory modules

Anaerobic microbial fermentations provide high product yields and are a cornerstone of industrial bio-based processes. However, the need for redox balancing limits the array of fermentable substrate-product combinations. To overcome this limitation, here we design an aerobic fermentative metabolism that allows the introduction of selected respiratory modules. These can use oxygen to re-balance otherwise unbalanced fermentations, hence achieving controlled respiro-fermentative growth. Following this design, we engineer and characterize an obligate fermentative Escherichia coli strain that aerobically ferments glucose to stoichiometric amounts of lactate. We then re-integrate the quinone-dependent glycerol 3-phosphate dehydrogenase and demonstrate glycerol fermentation to lactate while selectively transferring the surplus of electrons to the respiratory chain. To showcase the potential of this fermentation mode, we direct fermentative flux from glycerol towards isobutanol production. In summary, our design permits using oxygen to selectively re-balance fermentations. This concept is an advance freeing highly efficient microbial fermentation from the limitations imposed by traditional redox balancing.

KM04416 suppressed lung adenocarcinoma progression by promoting immune infiltration

ObjectivesLung adenocarcinoma (LUAD) is a malignant tumor originating from the bronchial mucosa or glands of the lung, with the fastest increasing morbidity and mortality. Therefore, the prognosis of lung cancer remains poor. Glycerol-3-phosphate dehydrogenase 2 (GPD2) is a widely existing protein pattern sequence in biology and is closely related to tumor progression. The therapy values of GPD2 inhibitor in LUAD were unclear. Therefore, we aimed to analyze the therapy values of GPD2 inhibitor in LUAD.Materials and methodsThe Cancer Genome Atlas (TCGA)-LUAD database was used to analyze the expression levels of GPD2 in LUAD tissues. The relationship between GPD2 expression and LUAD patient survival was analyzed by Kaplan-Meier method. Moreover, KM04416 as a target inhibitor of GPD2 was used to further investigate the therapy value of GPD2 inhibitor in LUAD cells lines (A549 cell and H1299 cell). The TISIDB website was used to investigate the associations between GPD2 expression and immune cell infiltration in LUAD.ResultsThe results showed that GPD2 is overexpressed in LUAD tissues and significantly associated with poor survival. KM04416 can suppress the progression of LUAD cells by targeting GPD2. Low expression of GPD2 is related to high infiltration of immune cells.ConclusionsIn summary, our present study found that targeting inhibition of GPD2 by KM04416 can suppress LUAD progression via adjusting immune cell infiltration.

Modulation of adipogenesis and lipogenesis by indomethacin and pantoprazole

Endocrine disruptors are suggested to act as potential “obesogens” by interacting with various metabolic processes in adipose tissue. Besides industrial chemicals that are blamed for acting as endocrine disruptors as well as obesogens, pharmaceuticals can also cause obesogenic effects as unintended adverse effects. However, limited studies evaluated the obesogenic adverse effects of pharmaceuticals. Based on this information, the present study aimed to investigate the possible in vitro adipogenic/lipogenic potential of indomethacin and pantoprazole that are prescribed during pregnancy. Their effects on lipid accumulation, adiponectin level, glycerol-3-phosphate dehydrogenase (G3PDH) activity, and expression of adipogenic genes and proteins were investigated in 3 T3-L1 cell line. The range of concentrations of the pharmaceuticals was selected according to their Cmax values. Lipid accumulation was increased dependently with indomethacin dose and with pantoprazole at its highest concentration. Both pharmaceuticals also increased adiponectin levels, which was thought to play a role in stimulating the adipogenesis pathway. Moreover, both pharmaceuticals altered the gene and/or protein expression of some adipogenic/lipogenic transcriptional factors, which may lead to disruption of metabolic pathways during the fetal period. In conclusion, indomethacin and pantoprazole may have obesogenic effects through different mechanisms and their potential to cause obesity should be investigated by further in vivo and epidemiological studies.

Effect of 4 hydroxy fatty acids on lipid accumulation in the 3T3-L1 cells: a comparative study.

Notwithstanding the several investigations of the hydroxy fatty acids (hFAs)' physiological functions, studies focusing on their anti-obesity effects are limited. This study investigated the anti-obesity effects of 4 hFAs-10-hydroxy stearic acid (10-hSA), 12-hydroxy stearic acid (12-hSA), 9,12-hydroxy stearic acid (9,12-dhSA), and 12-hydroxy oleic acid (12-hOA)-on the 3T3-L1 cells. All hFAs suppressed lipid accumulation, with 10-hSA and 12-hOA exhibiting the strongest suppression, followed by 12-hSA and 9,12-hSA. This trend was similar to that observed for the glycerol-3-phosphate dehydrogenase (GPDH) activity level. Contrastingly, only 9,12-dhSA suppressed cell viability. The mRNA levels of HK1 and Aldoa were markedly suppressed by 10-hSA and 12-hSA compared to the control. Additionally, mRNA expression of Gyk was considerably suppressed by 12-hSA. Thus, all hFAs suppressed lipid accumulation by suppressing GPDH activity, although their molecular mechanisms were different. These findings will aid the application of hFAs in the food and medical industries.

Genistein and metformin regulate glycerol kinase and the enzymes of glycerol 3-phosphate shuttle in a differential manner in myocytes, hepatocytes and adipocytes

Glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (GPDH) are critical in glucose homeostasis. The role of genistein and metformin on these enzymes and glucose production was investigated in C2C12, HepG2, and 3T3-L1 cells. Enzyme kinetics, Real-Time PCR and western blots were performed to determine enzyme activities and expressions of mRNAs and proteins. Glucose production and uptake were also measured in these cells. siRNAs were used to assess their impact on the enzymes and glucose production. Ki values for the compounds were determined using purified GK and GPDH. Genistein decreased GK activity by ∼45 %, while metformin reduced cGPDH and mGPDH activities by ∼32 % and ∼43 %, respectively. Insignificant changes in expressions (mRNAs and proteins) of the enzymes were observed. The compounds showed dose-dependent alterations in glucose production and uptake in these cells. Genistein non-competitively inhibited His-GK activity (Ki 19.12 μM), while metformin non-competitively inhibited His-cGPDH (Ki 75.52 μM) and mGPDH (Ki 54.70 μM) activities. siRNAs transfection showed ∼50 % and ∼35 % decrease in activities of GK and mGPDH and a decrease in glucose production (0.38-fold and 0.42-fold) in 3T3-L1 cells. Considering the differential effects of the compounds, this study may provide insights into the potential therapeutic strategies for type II diabetes mellitus.

Post-Translational Regulation of a Bidomain Glycerol-3-Phosphate Dehydrogenase Catalyzing Glycerol Synthesis under Salinity Stress in Chlamydomonas reinhardtii

Core chlorophytes possess glycerol-3-phosphate dehydrogenases (GPDs) with an unusual bidomain structure, consisting of a glycerol-3-phosphate phosphatase (GPP) domain fused to canonical GPD domains. These plastid-localized enzymes have been implicated in stress responses, being required for the synthesis of glycerol under high salinity and triacylglycerols under nutrient deprivation. However, their regulation under varying environmental conditions is poorly understood. C. reinhardtii transgenic strains expressing constitutively bidomain GPD2 did not accumulate glycerol or triacylglycerols in the absence of any environmental stress. Although the glycerol contents of both wild type and transgenic strains increased significantly upon exposure to high salinity, cycloheximide, an inhibitor of cytoplasmic protein synthesis, abolished this response in the wild type. In contrast, GPD2 transgenic strains were still capable of glycerol accumulation when cultured in medium containing cycloheximide and NaCl. Thus, the pre-existing GPD2 protein appears to become activated for glycerol synthesis upon salt stress. Interestingly, staurosporine, a non-specific inhibitor of protein kinases, prevented this post-translational GPD2 protein activation. Structural modeling analyses suggested that substantial conformational rearrangements, possibly triggered by high salinity, may characterize an active GPD2 GPP domain. Understanding this mechanism(s) may provide insights into the rapid acclimation responses of microalgae to osmotic/salinity stress.

Aspergillus nidulans gfdB, Encoding the Hyperosmotic Stress Protein Glycerol-3-phosphate Dehydrogenase, Disrupts Osmoadaptation in Aspergillus wentii.

The genome of the osmophilic Aspergillus wentii, unlike that of the osmotolerant Aspergillus nidulans, contains only the gfdA, but not the gfdB, glycerol 3-phosphate dehydrogenase gene. Here, we studied transcriptomic changes of A. nidulans (reference strain and ΔgfdB gene deletion mutant) and A. wentii (reference strain and An-gfdB expressing mutant) elicited by high osmolarity. A. nidulans showed a canonic hyperosmotic stress response characterized by the upregulation of the trehalose and glycerol metabolism genes (including gfdB), as well as the genes of the high-osmolarity glycerol (HOG) map kinase pathway. The deletion of gfdB caused only negligible alterations in the transcriptome, suggesting that the glycerol metabolism was flexible enough to compensate for the missing GfdB activity in this species. A. wentii responded differently to increased osmolarity than did A. nidulans, e.g., the bulk upregulation of the glycerol and trehalose metabolism genes, along with the HOG pathway genes, was not detected. The expression of An-gfdB in A. wentii did not abolish osmophily, but it reduced growth and caused much bigger alterations in the transcriptome than did the missing gfdB gene in A. nidulans. Flexible glycerol metabolism and hence, two differently regulated gfd genes, may be more beneficial for osmotolerant (living under changing osmolarity) than for osmophilic (living under constantly high osmolarity) species.

SOME FEATURES OF CELLULAR ENERGY SUPPLY OF THE BODY IN PREMATURE INFANTS WITH SEVERE FORMS OF PERINATAL PATHOLOGY

The nature of postnatal adaptation of newborns, their further growth, development, health status and quality of life largelydepend on the well-being of intrauterine development and maturity at birth. Premature birth under hypoxia causes signifi cant metabolic disorders in the body, is accompanied by a high risk of severe organ dysfunction, which requires in-depth research to clarify the features of intracellular metabolism in order to further improve methods of diagnosis and treatment of newborns with various forms of perinatal pathology.Aim of the research. To study the trends of changes in energy metabolism in premature infants with severe forms of perinatal pathology to investigate the possibility of using them as additional criteria of hypoxic damage to the body in conditions of morphological and functional immaturity.Materials and methods. Clinical and laboratory examinations were performed on 68 newborns with severe forms of perinatal pathology at the gestational age of 32 to 34 weeks; the comparison group consisted of 27 conditionally healthy newborns at the gestational age of 34 to 37 weeks. Inclusion criteria for the main study group: gestational age at birth from 32 to 34 weeks, clinical signs of severe perinatal pathology. Exclusion criteria: gestational age at birth < 32 and ≥ 37 weeks, diagnosed congenital malformations and septic conditions. The list of diseases was determined according to the International Classifi cation of Diseases, X Revision. The following blood serum laboratory parameters were studied: lactate level, glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.99.5), succinate dehydrogenase (SDH), NADH dehydrogenase (NADH) (EC 1.6.5.3); the electron transport chain coeffi cient (ETC) was calculated. Laboratory studies were performed using micromethods. The study was conducted in accordance with the provisions of GCP (1996), the Convention of the Council of Europe on Human Rights and Biomedicine (April 4, 1997), the Declaration of Helsinki of the World Medical Association (1964-2008), Order of the Ministry of Health of Ukraine No. 690 dated September 23, 2009 (as amended by Order of the Ministry of Health of Ukraine No. 523 dated July 12, 2012). Protocol of scientifi c research of the Biomedical Ethics Committee of BSMU dated September 12, 2015. Informed written consent was obtained from the parents of the patients before the study with an explanationof the purpose, objectives and methods of laboratory testing. Statistical processing of the results was performed using STATISTICA software (StatSoft Inc., USA, version 10). Comparison of quantitative indicators with normal distribution was performed using Student’s t-test, and the probability of diff erences was considered statistically signifi cant at p<0.05. The complex of studies was conducted within the framework of the planned research topics of the Department of Pediatrics, Neonatology and Perinatal Medicine of the Bukovinian State Medical University: «Improvement of the areas of prognosis, diagnostics and treatment of perinatal pathology in newborns and infants, optimization of the schemes of catamenial observation and rehabilitation» (State registration No. 0115U002768, term of execution 01.2015-12.2019) and «Chronobiological and adaptive aspects and features of vegetative regulation in pathological conditions in children of diff erent age groups» (State registration No. 0122U002245, term of execution 01.2020-12.2024).Results. In newborns of the main group, in comparison with the control group, a signifi cant decrease in GFDH and SDHwas found, which indicates a signifi cant impairment of the activity of enzymes of the respiratory chain and explains the lack of oxygen absorption in the body at the cellular and tissue level, causing the severity of the condition of newborns. Determination of the level of GPDH and SDH in the dynamics of observation in children showed some improvement in these indicators. In the severe condition of newborns, a signifi cant increase in the NADPH index was found, with a further probable increase in its level in the dynamics of observation. Calculations of ETC coeffi cient in cord blood showed a signifi cant decrease of this indicator in comparison with the control group. The identifi ed disorders of intracellular energy metabolism under hypoxia indicate the expediency of conducting comprehensive scientifi c research to study the possibilities of appropriate pharmacological correction to improve the eff ectiveness of treatment of severe forms of perinatal pathology in premature infants.Conclusions: 1. The results of the study revealed a signifi cant decrease in GPDH and SDH at elevated levels of NADPH andETC coeffi cient, which confi rms the presence of signifi cant disturbances in intracellular energy metabolism and mitochondrial oxidation processes. 2. Timely detection of metabolic disorders with the determination of mitochondrial oxidation, in particular energy metabolism, is an important area for improving diagnostic measures in severe forms of perinatal pathology in preterm infants with hypoxic damage to the body in the pathogenesis.

Abstract SY21-02: Coenzyme Q-dependent metabolism: Lessons from structure-function studies of DHODH

Abstract Ubiquinone or coenzyme Q (CoQ) is perhaps best known as a key component of the mitochondrial electron transport chain and as an endogenous antioxidant. However, CoQ has another critical function in cells, acting as an essential cosubstrate for a number of important metabolic enzymes that are associated with the inner mitochondrial membrane, including dihydroorotate dehydrogenase (DHODH), choline dehydrogenase (CHDH), proline dehydrogenase 1 and 2 (PRODH1, PRODH2), and glycerol-3-phosphate dehydrogenase 2 (GPD2). These FAD-containing mitochondrial enzymes are all hypothesized to utilize a two-step ‘ping-pong’ mechanism of catalysis. In this kinetic model, the baseline conformation of the enzyme has specific affinity for its first cosubstrate (dihydroorotate, choline, proline, glycerol-3-phosphate) but not its second cosubstrate (CoQ). Binding of the first cosubstrate to the enzyme results in oxidation of the cosubstrate and reduction of FAD to FADH2. This reduced ‘intermediate’ form of the enzyme releases the byproduct of the first reaction and then binds CoQ. Upon binding of CoQ, FADH2 is oxidized back to FAD and CoQ is reduced to CoQH2. The oxidized form of the enzyme has a low affinity for CoQH2 and CoQH2 is therefore released, restoring the enzyme to its baseline ‘empty’ conformation. Although there is considerable biochemical evidence to support this ‘double-displacement’ model of CoQ-dependent catalysis, it has been challenging to study the structures of these enzymes in their catalytically-active states due to the profound insolubility of CoQ and due to technical challenges associated with producing highly purified recombinant forms of membrane-associated proteins. Perhaps the most well-studied CoQ-dependent metabolic enzyme is DHODH. DHODH catalyzes the fourth and rate-limiting step in the de novo pyrimidine biosynthesis pathway, oxidizing dihydroorotate to orotate, which is then converted to the nucleotide precursor UMP. Due to the critical requirement of rapidly dividing cells for pyrimidines, DHODH has been proposed as a therapeutic target in cancer, and a number of highly potent and highly specific DHODH inhibitors have been developed, all of which act as competitive inhibitors of CoQ. By binding and stabilizing the protein, these inhibitors have made it possible to solve the x-ray crystal structure of DHODH. These structural studies have revealed that the inhibitors (and presumably CoQ) bind in a narrow amphipathic tunnel that spans from the membrane-embedded surface of the protein to its FAD-containing catalytic core. These structural studies have also found that DHO or orotate can bind DHODH when the CoQ tunnel is occupied by drug. Given that the ‘ping-pong’ model of catalysis is predicated on the assumption that cosubstrates and byproducts cannot simultaneously bind enzyme, it would appear that either the ping-pong model does not accurately reflect the catalytic mechanism of DHODH or, alternatively, that the structure of DHODH bound to drug is not an accurate representation of the structure of DHODH bound to CoQ. In order to gain a greater understanding of the structure of the DHODH-CoQ complex, we undertook to solve the x-ray crystal structure of DHODH in complex with decylubiquinone (dUb), a CoQ analog that can act as an electron acceptor for DHODH in vitro. These studies revealed that the x-ray crystal structure of DHODH in its catalytically-active cofactor-bound state deviates in subtle but important ways from the known structure of drug-bound DHODH, providing novel insights into the catalytic mechanism of CoQ-dependent enzymes. Citation Format: Julie-Aurore Losman. Coenzyme Q-dependent metabolism: Lessons from structure-function studies of DHODH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY21-02.

Glycerol 3-Phosphate Dehydrogenase: Role of the Protein Conformational Change in Activation of a Readily Reversible Enzyme-Catalyzed Hydride Transfer Reaction.

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

Jasmonates and Ethylene Shape Floridoside Synthesis during Carposporogenesis in the Red Seaweed Grateloupia imbricata.

Floridoside is a galactosyl-glycerol compound that acts to supply UDP-galactose and functions as an organic osmolyte in response to salinity in Rhodophyta. Significantly, the UDP-galactose pool is shared for sulfated cell wall galactan synthesis, and, in turn, affected by thallus development alongside carposporogenesis induced by volatile growth regulators, such as ethylene and methyl jasmonate, in the red seaweed Grateloupia imbricata. In this study, we monitored changes in the floridoside reservoir through gene expression controlling both the galactose pool and glyceride pool under different reproductive stages of G. imbricata and we considered changing salinity conditions. Floridoside synthesis was followed by expression analysis of galactose-1-phosphate uridyltransferase (GALT) as UDP-galactose is obtained from UDP-glucose and glucose-1P, and through α-galactosidase gene expression as degradation of floridoside occurs through the cleavage of galactosyl residues. Meanwhile, glycerol 3-phosphate is connected with the galactoglyceride biosynthetic pathway by glycerol 3-phosphate dehydrogenase (G3PD), monogalactosyl diacylglyceride synthase (MGDGS), and digalactosyl diacylglyceride synthase (DGDGS). The results of our study confirm that low GALT transcripts are correlated with thalli softness to locate reproductive structures, as well as constricting the synthesis of UDP-hexoses for galactan backbone synthesis in the presence of two volatile regulators and methionine. Meanwhile, α-galactosidase modulates expression according to cystocarp maturation, and we found high transcripts in late development stages, as occurred in the presence of methyljasmonate, compared to early stages in ethylene. Regarding the acylglyceride pool, the upregulation of G3PD, MGDGS, and DGDGS gene expression in G. imbricata treated with MEJA supports lipid remodeling, as high levels of transcripts for MGDGS and DGDGS provide membrane stability during late development stages of cystocarps. Similar behavior is assumed in three naturally collected thalli development stages-namely, fertile, fertilized, and fertile-under 65 psu salinity conditions. Low transcripts for α-galactosidase and high for G3PD are reported in infertile and fertilized thalli, which is the opposite to high transcripts for α-galactosidase and low for G3PD encountered in fertile thalli within visible cystocarps compared to each of their corresponding stages in 35 psu. No significant changes are reported for MGDGS and DGDGS. It is concluded that cystocarp and thallus development stages affect galactose and glycerides pools with interwoven effects on cell wall polysaccharides.

LMO3 is a suppressor of the basal-like/squamous subtype and reduces disease aggressiveness of pancreatic cancer through glycerol 3-phosphate metabolism.

Pancreatic ductal adenocarcinoma (PDAC) encompasses diverse molecular subtypes, including the classical/progenitor and basal-like/squamous subtypes, each exhibiting distinct characteristics, with the latter known for its aggressiveness. We employed an integrative approach combining transcriptome and metabolome analyses to pinpoint potential genes contributing to the basal-like/squamous subtype differentiation. Applying this approach to our NCI-UMD-German and a validation cohort, we identified LIM Domain Only 3 (LMO3), a transcription co-factor, as a candidate suppressor of the basal-like/squamous subtype. Reduced LMO3 expression was significantly associated with higher pathological grade, advanced disease stage, induction of the basal-like/squamous subtype and decreased survival among PDAC patients. In vitro experiments demonstrated that LMO3 transgene expression inhibited PDAC cell proliferation and migration/invasion, concurrently downregulating the basal-like/squamous gene signature. Metabolome analysis of patient tumors and PDAC cells revealed a metabolic program linked to elevated LMO3 and the classical/progenitor subtype, characterized by enhanced lipogenesis and suppressed amino acid metabolism. Notably, glycerol 3-phosphate (G3P) levels positively correlated with LMO3 expression and associated with improved patient survival. Furthermore, glycerol-3-phosphate dehydrogenase 1 (GPD1), a crucial enzyme in G3P synthesis, showed upregulation in LMO3-high and classical/progenitor PDAC, suggesting its potential role in mitigating disease aggressiveness. Collectively, our findings suggest that heightened LMO3 expression reduces transcriptome and metabolome characteristics indicative of basal-like/squamous tumors with decreased disease aggressiveness in PDAC patients. The observations describe LMO3 as a candidate for diagnostic and therapeutic targeting in PDAC.