Rate-determining Enzyme Research Articles

Evidence for the Quercetin Binding Site of Glycogen Phosphorylase as a Target for Liver-Isoform-Selective Inhibitors against Glioblastoma: Investigation of Flavanols Epigallocatechin Gallate and Epigallocatechin.

Glycogen phosphorylase (GP) is the rate-determining enzyme in glycogenolysis, and its druggability has been extensively studied over the years for the development of therapeutics against type 2 diabetes (T2D) and, more recently, cancer. However, the conservation of binding sites between the liver and muscle isoforms makes the inhibitor selectivity challenging. Using a combination of kinetic, crystallographic, modeling, and cellular studies, we have probed the binding of dietary flavonoids epigallocatechin gallate (EGCG) and epigallocatechin (EGC) to GP isoforms. The structures of rmGPb-EGCG and rmGPb-EGC complexes were determined by X-ray crystallography, showing binding at the quercetin binding site (QBS) in agreement with kinetic studies that revealed both compounds as noncompetitive inhibitors of GP, with EGCG also causing a significant reduction in cell viability and migration of U87-MG glioblastoma cells. Interestingly, EGCG exhibits different binding modes to GP isoforms, revealing QBS as a promising site for GP targeting, offering new opportunities for the design of liver-selective GP inhibitors.

Conjugates of 3,5-Bis(arylidene)-4-piperidone and Sesquiterpene Lactones Have an Antitumor Effect via Resetting the Metabolic Phenotype of Cancer Cells.

In recent years, researchers have often encountered the significance of the aberrant metabolism of tumor cells in the pathogenesis of malignant neoplasms. This phenomenon, known as the Warburg effect, provides a number of advantages in the survival of neoplastic cells, and its application is considered a potential strategy in the search for antitumor agents. With the aim of developing a promising platform for designing antitumor therapeutics, we synthesized a library of conjugates of 3,5-bis(arylidene)-4-piperidone and sesquiterpene lactones. To gain insight into the determinants of the biological activity of the prepared compounds, we showed that the conjugates of 3,5-bis(arylidene)-4-piperidone and sesquiterpene lactones, which are cytotoxic agents, demonstrate selective activity toward a number of tumor cell lines with glycolysis-inhibiting ability. Moreover, the results of molecular and in silico screening allowed us to identify these compounds as potential inhibitors of the pyruvate kinase M2 oncoprotein, which is the rate-determining enzyme of glycolysis. Thus, the results of our work indicate that the synthesized conjugates of 3,5-bis(arylidene)-4-piperidone and sesquiterpene lactones can be considered a promising platform for designing selective cytotoxic agents against the glycolysis process, which opens new possibilities for researchers involved in the search for antitumor therapeutics among compounds containing piperidone platforms.

Exploring the Deoxy-D-xylulose-5-phosphate Synthase Gene Family in Tomato (Solanum lycopersicum).

Isoprenoids are a wide family of metabolites including high-value chemicals, flavors, pigments, and drugs. Isoprenoids are particularly abundant and diverse in plants. The methyl-D-erythritol 4-phosphate (MEP) pathway produces the universal isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate in plant plastids for the downstream production of monoterpenes, diterpenes, and photosynthesis-related isoprenoids such as carotenoids, chlorophylls, tocopherols, phylloquinone, and plastoquinone. The enzyme deoxy-D-xylulose 5-phosphate synthase (DXS) is the first and main rate-determining enzyme of the MEP pathway. In tomato (Solanum lycopersicum), a plant with an active isoprenoid metabolism in several tissues, three genes encode DXS-like proteins (SlDXS1 to 3). Here, we show that the expression patterns of the three genes suggest distinct physiological roles without excluding that they might function together in some tissues. We also confirm that SlDXS1 and 2 are true DXS enzymes, whereas SlDXS3 lacks DXS activity. We further show that SlDXS1 and 2 co-localize in plastidial speckles and that they can be immunoprecipitated together, suggesting that they might form heterodimers in vivo in at least some tissues. These results provide novel insights for the biotechnological use of DXS isoforms in metabolic engineering strategies to up-regulate the MEP pathway flux.

Size-Specific Modulation of a Multienzyme Glucosome Assembly during the Cell Cycle.

Enzymes in glucose metabolism have been subjected to numerous studies, revealing the importance of their biological roles during the cell cycle. However, due to the lack of viable experimental strategies for measuring enzymatic activities particularly in living human cells, it has been challenging to address whether their enzymatic activities and thus anticipated glucose flux are directly associated with cell cycle progression. It has remained largely elusive how human cells regulate glucose metabolism at a subcellular level to meet the metabolic demands during the cell cycle. Meanwhile, we have characterized that rate-determining enzymes in glucose metabolism are spatially organized into three different sizes of multienzyme metabolic assemblies, termed glucosomes, to regulate the glucose flux between energy metabolism and building block biosynthesis. In this work, we first determined using cell synchronization and flow cytometric techniques that enhanced green fluorescent protein-tagged phosphofructokinase is adequate as an intracellular biomarker to evaluate the state of glucose metabolism during the cell cycle. We then applied fluorescence single-cell imaging strategies and discovered that the percentage of Hs578T cells showing small-sized glucosomes is drastically changed during the cell cycle, whereas the percentage of cells with medium-sized glucosomes is significantly elevated only in the G1 phase, but the percentage of cells showing large-sized glucosomes is barely or minimally altered along the cell cycle. Should we consider our previous localization-function studies that showed assembly size-dependent metabolic roles of glucosomes, this work strongly suggests that glucosome sizes are modulated during the cell cycle to regulate glucose flux between glycolysis and building block biosynthesis. Therefore, we propose the size-specific modulation of glucosomes as a behind-the-scenes mechanism that may explain functional association of glucose metabolism with the cell cycle and, thereby, their metabolic significance in human cell biology.

High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells.

We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, termed glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism and thus contribute to human cell biology. We developed a high-content quantitative high-throughput screening (qHTS) assay to identify regulatory mechanisms that control PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs further confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling network that regulates the formation of PFK1-mediated glucosome assembly in human cells.

Multidisciplinary docking, kinetics and X-ray crystallography studies of baicalein acting as a glycogen phosphorylase inhibitor and determination of its’ potential against glioblastoma in cellular models

Glycogen phosphorylase (GP) is the rate-determining enzyme in the glycogenolysis pathway. Glioblastoma (GBM) is amongst the most aggressive cancers of the central nervous system. The role of GP and glycogen metabolism in the context of cancer cell metabolic reprogramming is recognised, so that GP inhibitors may have potential treatment benefits. Here, baicalein (5,6,7-trihydroxyflavone) is studied as a GP inhibitor, and for its effects on glycogenolysis and GBM at the cellular level. The compound is revealed as a potent GP inhibitor against human brain GPa (Ki = 32.54 μM), human liver GPa (Ki = 8.77 μM) and rabbit muscle GPb (Ki = 5.66 μM) isoforms. It is also an effective inhibitor of glycogenolysis (IC50 = 119.6 μM), measured in HepG2 cells. Most significantly, baicalein demonstrated anti-cancer potential through concentration- and time-dependent decrease in cell viability for three GBM cell-lines (U-251 MG, U-87 MG, T98-G) with IC50 values of ∼20–55 μM (48- and 72-h). Its effectiveness against T98-G suggests potential against GBM with resistance to temozolomide (the first-line therapy) due to a positive O6-methylguanine-DNA methyltransferase (MGMT) status. The solved X-ray structure of rabbit muscle GP–baicalein complex will facilitate structure-based design of GP inhibitors. Further exploration of baicalein and other GP inhibitors with different isoform specificities against GBM is suggested.

Oncogene-mediated nuclear accumulation of lactate promotes epigenetic alterations to induce cancer cell proliferation.

Homeobox gene families are associated with embryonic development and organogenesis. Pieces of evidence suggest that these Homeobox genes are also crucial in facilitating oncogenesis when mutated or overexpressed. Paired homeodomain transcription factor-2 (PITX2), one of the members of this family, is involved in oncogenic regulation apart from its different development regulatory functions. PITX2 has been earlier shown to induce ovarian cancer cell proliferation through the activation of different signaling cascades. Increased cancer cell proliferation requires a constant supply of nutrients for both adenosine triphosphate and biomass synthesis, which is facilitated by altered cancer cell metabolism that includes enhanced glucose uptake and increased glycolytic rate. This present study highlights the involvement of PITX2 in enhancing the cellular glycolysis pathway in ovarian cancer cells through protein kinase B-phosphorylation (phospho-AKT). PITX2 expression correlates positively with that of the glycolytic rate-determining enzyme, lactate dehydrogenase-A (LDHA), in both high-grade serous ovarian cancer tissues and common ovarian cancer cell lines. Interestingly, transient localization of enzymatically active LDHA in the nucleus was observed in PITX2-overexpressed ovarian cancer cells. This nuclear LDHA produces higher concentrations of the glycolytic end product, lactate, which accumulates in the nuclear compartment resulting in decreased histone deacetylase (HDAC1/2) expression and increased histone acetylation at H3/H4. However, the mechanistic details of lactate-HDAC interaction are still elusive in the earlier reports. Our in silico studies elaborated on the interaction dynamics of lactate in the HDAC catalytic core through ligand-binding studies and molecular dynamics simulation approaches. Blocking lactate production by silencing LDHA reduced cancer cell proliferation. Thus, PITX2-induced epigenetic changes can lead to high cellular proliferation and increase the size of tumors in syngeneic mice as well. Taken together, this is the first report of its kind to show that the developmental regulatory homeobox gene PITX2 could enhance oncogenesis through enhanced glycolysis of tumor cells followed by epigenetic modifications.

MTHFD1 is critical for the negative regulation of retinoic acid receptor signalling in anencephaly.

Neural tube defects are the most severe congenital malformations that result from failure of neural tube closure during early embryonic development, and the underlying molecular mechanisms remain elusive. Retinoic acid, an active derivative of vitamin A, is critical for neural system development, and retinoic acid receptor (RAR) signalling malfunctions have been observed in human neural tube defects. However, retinoic acid-retinoic acid receptor signalling regulation and mechanisms in neural tube defects are not fully understood. The mRNA expression of RARs and retinoid X receptors in the different human neural tube defect phenotypes, including 11 pairs of anencephaly foetuses, 10 pairs of hydrocephalus foetuses and nine pairs of encephalocele foetuses, was investigated by NanoString nCounter technology. Immunoprecipitation-mass spectrometry was performed to screen the potential interacting targets of retinoic acid receptor γ. The interactions between proteins were confirmed by co-immunoprecipitation and immunofluorescence laser confocal microscopy. Luciferase and chromatin immunoprecipitation with quantitative real-time polymerase chain reaction assays were used to clarify the underlying mechanism. Moreover, a neural tube defect animal model, constructed using excess retinoic acid, was used for further analysis with established molecular biology technologies. We report that level of retinoic acid receptor γ (RARγ) mRNA was significantly upregulated in the brain tissues of human foetuses with anencephaly. To further understand the actions of retinoic acid receptor γ in neural tube defects, methylenetetrahydrofolate dehydrogenase 1 was identified as a specific retinoic acid receptor γ target from IP-MS screening. Additionally, methylenetetrahydrofolate dehydrogenase 1 negatively regulated retinoic acid receptor γ transcription factor activity. Furthermore, low expression of methylenetetrahydrofolate dehydrogenase 1 and activation of retinoic acid receptor signalling were further determined in human anencephaly and a retinoic acid-induced neural tube defect mouse model. This study reveals that methylenetetrahydrofolate dehydrogenase 1, the rate-determining enzyme in the one-carbon cycle, might be a specific regulator of retinoic acid receptors; these findings provide new insights into the functional linkage between nuclear folate metabolism and retinoic acid receptor signalling in neural tube defect pathology.

Engineering the Substrate Specificity of Toluene Degrading Enzyme XylM Using Biosensor XylS and Machine Learning.

Enzyme engineering using machine learning has been developed in recent years. However, to obtain a large amount of data on enzyme activities for training data, it is necessary to develop a high-throughput and accurate method for evaluating enzyme activities. Here, we examined whether a biosensor-based enzyme engineering method can be applied to machine learning. As a model experiment, we aimed to modify the substrate specificity of XylM, a rate-determining enzyme in a multistep oxidation reaction catalyzed by XylMABC in Pseudomonas putida. XylMABC naturally converts toluene and xylene to benzoic acid and toluic acid, respectively. We aimed to engineer XylM to improve its conversion efficiency to a non-native substrate, 2,6-xylenol. Wild-type XylMABC slightly converted 2,6-xylenol to 3-methylsalicylic acid, which is the ligand of the transcriptional regulator XylS in P. putida. By locating a fluorescent protein gene under the control of the Pm promoter to which XylS binds, a XylS-producing Escherichia coli strain showed higher fluorescence intensity in a 3-methylsalicylic acid concentration-dependent manner. We evaluated the 3-methylsalicylic acid productivity of XylM variants using the fluorescence intensity of the sensor strain as an indicator. The obtained data provided the training data for machine learning for the directed evolution of XylM. Two cycles of machine learning-assisted directed evolution resulted in the acquisition of XylM-D140E-V144K-F243L-N244S with 15 times higher productivity than wild-type XylM. These results demonstrate that an indirect enzyme activity evaluation method using biosensors is sufficiently quantitative and high-throughput to be used as training data for machine learning. The findings expand the versatility of machine learning in enzyme engineering.

Increased expression of ELOVL7 contributes to production of inflammatory cytokines in THP-1 cell-derived M1-like macrophages.

The elevation of intracellular very long-chain fatty acids (VLCFAs) augments pro-inflammatory activity of macrophages. VLCFAs are considered to function as regulators in macrophage inflammatory responses; however, the precise mechanism of regulating the production of VLCFAs is unclear. In this study, we focused on elongation of the very‑long‑chain fatty acid protein (ELOVL) family, rate-determining enzymes for VLCFA synthesis, in macrophages. ELOVL7 mRNA was upregulated in human monocytic THP-1 cell-derived M1-like macrophages. Metascape analysis using the RNA-seq data set showed the involvement of NF-κB and STAT1 in transcriptional regulation of ELOVL7 highly correlated genes. Gene ontology (GO) enrichment analysis suggested that ELOVL7 highly correlated genes were closely associated with multiple pro-inflammatory responses, including response to virus and positive regulation of NF-κB signaling. Consistent with RNA-seq analysis, the NF-κB inhibitor BAY11-7082, but not the STAT1 inhibitor fludarabine, canceled ELOVL7 upregulation in M1-like macrophages. ELOVL7 knockdown decreased interleukin (IL)-6 and IL-12/IL-23 p40 production. Moreover, RNA-seq analysis of plasmacytoid dendritic cells (pDCs) revealed that ELOVL7 was upregulated in pDCs treated with TLR7 and TLR9 agonists. In conclusion, we propose that ELOVL7 is a novel pro-inflammatory gene that is upregulated by inflammatory stimuli, and regulates M1-like macrophage and pDC functions.

Antioxidant and 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitory activities of some plant samples

The antioxidant activity capacities of some plants, and their inhibitory effects on the HMG-CoA reductase enzyme, the rate-determining enzyme of cholesterol synthesis, were investigated in our study. Antioxidant activity capacity and inhibitory effect of the HMG-CoA reductase enzyme were detected in all plant extracts used in our research. The results obtained determined that both antioxidant activity and % inhibition values of HMG-CoA reductase enzyme increased as the plant extracts concentration increased. The strongest ABTS and DPPH radical scavenging activities were exhibited by pomegranate fruit extract (IC50= 1.07 ±0.04 mg/mL and IC50= 0.39 ±0.01 mg/mL, respectively). At the same time, lemon had the strongest DMPD radical scavenging activity (IC50= 9×10-4 ±6×10-5 mg/mL). The best HMG-CoA reductase inhibitory activity was observed in persimmon fruit extract (IC50= 0.71 ±0.18 µg/mL). The inhibitory power of this extract was much higher than that of the enzyme’s standard inhibitor, Atorvastatin (IC50 = 1.76 ±0.12 µg/mL). The extracts' potent antioxidant and inhibitory properties can be attributed to the rich phytochemical composition of plant extracts. Thus, it may be a potential source of new bioactive compounds effective against oxidative stress, hypercholesterolemia and cardiovascular complications.

PS-B02-14: DISCOVERY OF A NEW DRUG FOR PRIMARY ALDOSTERONISM TARGETING THE CYP11B2 AND KCNJ5 MUTANTS

Primary aldosteronism (PA) is a common cause of secondary hypertension. PA induces damage to cardiovascular and renal blood vessels. Therefore, PA is regarded as a problem because it increases the risk of stroke, cardiomyopathy, kidney disease, etc. Aldosterone-producing adenomas (APA) are one of the major causes of PA, and gene mutations are often identified in tumor cells. KCNJ5 mutation is well known as a gene mutation that causes the loss of ion selectivity in APA. KCNJ5 is an inwardly rectifying potassium channel. KCNJ5 induces sodium ion influx due to mutations in APA. The sodium ion influx induces depolarization, and which overexpresses CYP11B2. CYP11B2 is the rate-determining enzyme for aldosterone synthesis, overexpression of CYP11B2 induces aldosterone overproduction. Examples of therapeutic drugs for PA and APA include calcium channel blockers, aldosterone antagonist and β;-blockers. However, therapeutic drugs for PA and APA targeting cell membrane depolarization have not been developed yet. We aim to find compounds that suppress CYP11B2 expression by membrane depolarization to develop novel therapeutic drugs for PA and APA. By developing a therapeutic drug that targets depolarization, we hope to establish an effective therapeutic method for APA caused by gene mutations such as KCNJ5 mutation. We performed high-throughput screening (HTS) using Tohoku University Compound Library (6080 compounds). First, we generated H295R-CYP11B2 N-Luc cells in which the luciferase gene is inserted downstream of the CYP11B2 locus. The cells H295R-CYP11B2 N-Luc were stimulated with KCl to cause depolarization, after which candidate compounds were added and HTS was performed by Luc assay. As a result, 101 compounds were selected. Furthermore, 25 of 101 compounds were selected by examining the cytotoxicity and dose response. Also, 11 of the 25 compounds suppressed CYP11B2 mRNA expression in KCl stimulated H295R cells. Secondary, we generated KCNJ5 mutant cells that expressed the KCNJ5 mutant (L168R) in a Doxycycline-inducible manner. 3 of 11 compounds that suppressed the expression of CYP11B2 mRNA were selected by examination using KCNJ5 mutant cells. After that, 2 of 3 compounds decreased the aldosterone production. These compounds have a different structure from existing therapeutic drugs such as calcium channel blockers. Since these can be novel therapeutic drugs for PA and APA caused by depolarization such as KCNJ5, we are examing the detailed mechanism of action of the compounds. In addition, we would like to further experiment the effects of the compounds in vivo using hypertensive model mice, Task1 /Task3 double KO mice.

The dysfunction of hormone-sensitive lipase induces lipid deposition and reprogramming of nutrient metabolism in fish.

Hormone-sensitive lipase (HSL) is one of the rate-determining enzymes in the hydrolysis of TAG, playing a crucial role in lipid metabolism. However, the role of HSL-mediated lipolysis in systemic nutrient homoeostasis has not been intensively understood. Therefore, we used CRISPR/Cas9 technique and Hsl inhibitor (HSL-IN-1) to establish hsla-deficient (hsla-/-) and Hsl-inhibited zebrafish models, respectively. As a result, the hsla-/- zebrafish showed retarded growth and reduced oxygen consumption rate, accompanied with higher mRNA expression of the genes related to inflammation and apoptosis in liver and muscle. Furthermore, hsla-/- and HSL-IN-1-treated zebrafish both exhibited severe fat deposition, whereas their expressions of the genes related to lipolysis and fatty acid oxidation were markedly reduced. The TLC results also showed that the dysfunction of Hsl changed the whole-body lipid profile, including increasing the content of TG and decreasing the proportion of phospholipids. In addition, the systemic metabolic pattern was remodelled in hsla-/- and HSL-IN-1-treated zebrafish. The dysfunction of Hsl lowered the glycogen content in liver and muscle and enhanced the utilisation of glucose plus the expressions of glucose transporter and glycolysis genes. Besides, the whole-body protein content had significantly decreased in the hsla-/- and HSL-IN-1-treated zebrafish, accompanied with the lower activation of the mTOR pathway and enhanced protein and amino acid catabolism. Taken together, Hsl plays an essential role in energy homoeostasis, and its dysfunction would cause the disturbance of lipid catabolism but enhanced breakdown of glycogen and protein for energy compensation.

LBODP025 A Novel Drug Discovery For Primary Aldosteronism Targeting Aldosterone Synthase (cyp11b2) And Kcnj5 Mutant

Abstract Primary aldosteronism (PA) is a common cause of secondary hypertension. PA induces damage to cardiovascular and renal blood vessels. Therefore, PA is regarded as a problem because it increases the risk of stroke, cardiomyopathy, kidney disease, etc. Aldosterone-producing adenomas (APA) are one of the major causes of PA, and gene mutations are often identified in tumor cells. KCNJ5 mutation is well known as a gene mutation that causes the loss of ion selectivity in APA. KCNJ5 is an inwardly rectifying potassium channel. KCNJ5 induces sodium ion influx due to mutations in APA. The sodium ion influx induces depolarization, and which overexpresses CYP11B2. CYP11B2 is the rate-determining enzyme for aldosterone synthesis, overexpression of CYP11B2 induces aldosterone overproduction. Examples of therapeutic drugs for PA and APA include calcium channel blockers, aldosterone antagonist and β-blockers. However, therapeutic drugs for PA and APA targeting cell membrane depolarization have not been developed yet. We aim to find compounds that suppress CYP11B2 expression by membrane depolarization to develop novel therapeutic drugs for PA and APA. By developing a therapeutic drug that targets depolarization, we hope to establish an effective therapeutic method for APA caused by gene mutations such as KCNJ5 mutation. We performed high-throughput screening (HTS) using Tohoku University Compound Library (6080 compounds). First, we generated H295R-CYP11B2 N-Luc cells in which the luciferase gene is inserted downstream of the CYP11B2 locus. The cells H295R-CYP11B2 N-Luc were stimulated with KCl to cause depolarization, after which candidate compounds were added and HTS was performed by Luc assay. As a result, 101 compounds were selected. Furthermore, 25 of 101 compounds were selected by examining the cytotoxicity and dose response. Also, 11 of the 25 compounds suppressed CYP11B2 mRNA expression in KCl stimulated H295R cells. Secondary, we generated KCNJ5 mutant cells that expressed the KCNJ5 mutant (L168R) in a Doxycycline-inducible manner. 3 of 11 compounds that suppressed the expression of CYP11B2 mRNA were selected by examination using KCNJ5 mutant cells. After that, 2 of 3 compounds decreased the aldosterone production. These compounds have a different structure from existing therapeutic drugs such as calcium channel blockers. Since these can be novel therapeutic drugs for PA and APA caused by depolarization such as KCNJ5, we are examining the detailed mechanism of action of the compounds. Presentation: No date and time listed

PSUN84 Chronic Adrenergic Stimulation in Humans Increases Plasma Bile Acids and Expression of Bile Acid Synthesis Enzymes in White Adipose Tissue

Abstract Bile acids (BAs) are classically known as facilitators of fat digestion. Moreover, they are now known to be ligands for receptors involved in glucose and lipoprotein metabolism. BA synthesis was originally thought to be exclusive to hepatic tissue, but emerging evidence in rodents has demonstrated the expression of BA transporters and de novo synthetic enzymes in murine adipose tissue. We previously showed that chronic activation of brown adipose tissue (BAT) and white adipose tissue (WAT) in adult women via the β3-adrenergic receptor (ADRB3) agonist mirabegron increases total plasma BA levels. The largest increases were seen with primary unconjugated BAs. Overall, these data suggest that chronic adrenergic stimulation may increase the synthesis and activity of BA synthesis enzymes in human adipose tissue, which contributes to the increase in total plasma BAs. To confirm this hypothesis, we measured expression levels of BAT and BA synthesis enzymes in liver and adipose tissues collected from healthy patients and those with pheochromocytoma (pheo) who experienced chronic adrenergic activation. As expected, expression of thermogenic UCP1 and ADRB3 was higher in pheo peri-renal BAT than in liver tissue (76,000x, P<0.001; and 6,000x, P=0.02, respectively) or either healthy subcutaneous WAT or pheo subcutaneous WAT (≥1500x, P<0.001 for UCP1). Conversely, expression of the rate-determining enzyme in BA synthesis CYP7A1 was similar in liver and both WAT depots (P>0.05), and liver had slightly higher expression than BAT (3x, P=0.03). Otherwise, most of the principal BA synthesis genes’ expression levels demonstrated the following stepwise pattern: liver tissue > pheo WAT = subcutaneous WAT > BAT (CYP8B1, CYP27A1, AKR1D1, and BAAT). These results suggest that BA production in humans is not limited to the liver. Rather, human WAT in particular may contribute to the BA pool and could serve as a target for producing BA-mediated improvements in glucose and lipoprotein metabolism. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Integrated metabolite profiling and transcriptome analysis reveals tissue-specific regulation of terpenoid biosynthesis in Artemisia argyi

Artemisia argyi L. is a widely distributed medicinal plant in China. The major bioactive substances of essential oils extracted from leaves are terpenoids. Although many researches have studied the pharmacological effects of the essential oils, the tissue-specific accumulation of terpenoid biosynthesis and the regulatory networks in A. argyi are poorly understood. This study conducted an integrated metabolomic and transcriptomic analysis of roots, stems, and leaves to investigate the tissue-specific regulatory network of terpenoid biosynthesis in A. argyi. We identified 77 unigenes putatively involved in terpenoid backbone biosynthesis. Three rate-determining enzyme genes (DXS, DXR, and HDR) of the methylerythritol phosphate pathway were predominantly expressed in leaves, and strongly co-expressed with eight transcription factors (2 MYBs, 4 WRKYs, and 2 AP2s). An metabolite-transcript correlation analysis revealed 26 putative cytochrome P450s related to terpenoid metabolism in leaves. These results provide a foundation for the future metabolic engineering of useful terpenoids in A. argyi.

Limited Mechanistic Link Between the Monod Equation and Methanogen Growth: a Perspective from Metabolic Modeling.

ABSTRACTThe Monod equation has been widely applied as the general rate law of microbial growth, but its applications are not always successful. By drawing on the frameworks of kinetic and stoichiometric metabolic models and metabolic control analysis, the modeling reported here simulated the growth kinetics of a methanogenic microorganism and illustrated that different enzymes and metabolites control growth rate to various extents and that their controls peak at either very low, intermediate, or very high substrate concentrations. In comparison, with a single term and two parameters, the Monod equation only approximately accounts for the controls of rate-determining enzymes and metabolites at very high and very low substrate concentrations, but neglects the enzymes and metabolites whose controls are most notable at intermediate concentrations. These findings support a limited link between the Monod equation and methanogen growth, and unify the competing views regarding enzyme roles in shaping growth kinetics. The results also preclude a mechanistic derivation of the Monod equation from methanogen metabolic networks and highlight a fundamental challenge in microbiology: single-term expressions may not be sufficient for accurate prediction of microbial growth.IMPORTANCE The Monod equation has been widely applied to predict the rate of microbial growth, but its application is not always successful. Using a novel metabolic modeling approach, we simulated the growth of a methanogen and uncovered a limited mechanistic link between the Monod equation and the methanogen’s metabolic network. Specifically, the equation provides an approximation to the controls by rate-determining metabolites and enzymes at very low and very high substrate concentrations, but it is missing the remaining enzymes and metabolites whose controls are most notable at intermediate concentrations. These results support the Monod equation as a useful approximation of growth rates and highlight a fundamental challenge in microbial kinetics: single-term rate expressions may not be sufficient for accurate prediction of microbial growth.

Zeb1-induced metabolic reprogramming of glycolysis is essential for macrophage polarization in breast cancer

Aerobic glycolysis (the Warburg effect) has been demonstrated to facilitate tumor progression by producing lactate, which has important roles as a proinflammatory and immunosuppressive mediator. However, how aerobic glycolysis is directly regulated is largely unknown. Here, we show that ectopic Zeb1 directly increases the transcriptional expression of HK2, PFKP, and PKM2, which are glycolytic rate-determining enzymes, thus promoting the Warburg effect and breast cancer proliferation, migration, and chemoresistance in vitro and in vivo. In addition, Zeb1 exerts its biological effects to induce glycolytic activity in response to hypoxia via the PI3K/Akt/HIF-1α signaling axis, which contributes to fostering an immunosuppressive tumor microenvironment (TME). Mechanistically, breast cancer cells with ectopic Zeb1 expression produce lactate in the acidic tumor milieu to induce the alternatively activated (M2) macrophage phenotype through stimulation of the PKA/CREB signaling pathway. Clinically, the expression of Zeb1 is positively correlated with dysregulation of aerobic glycolysis, accumulation of M2-like tumor-associated macrophages (TAMs) and a poor prognosis in breast cancer patients. In conclusion, these findings identify a Zeb1-dependent mechanism as a driver of breast cancer progression that acts by stimulating tumor–macrophage interplay, which could be a viable therapeutic target for the treatment of advanced human cancers.

Heterologous production of ascofuranone and ilicicolin A in Aspergillus sojae.

Ascofuranone and its precursor, ilicicolin A, are secondary metabolites with various pharmacological activities that are produced by Acremonium egyptiacum. In particular, ascofuranone strongly inhibits trypanosome alternative oxidase and represents a potential drug candidate against African trypanosomiasis. However, difficulties associated with industrial production of ascofuranone by A. egyptiacum, specifically the co-production of ascochlorin, which inhibits mammalian respiratory chain complex III at low concentrations, has precluded its widespread application. Therefore, in this study, ascofuranone biosynthetic genes (ascA-E and H-J) were heterologously expressed in Aspergillus sojae, which produced very low-levels of endogenous secondary metabolites under conventional culture conditions. As a result, although we obtained transformants producing both ilicicolin A and ascofuranone, they were produced only when an adequate concentration of chloride ions was added to the medium. In addition, we succeeded in increasing the production of ilicicolin A, by enhancing the expression of the rate-determining enzyme AscD, using a multi-copy integration system. The heterologous expression approach described here afforded the production of both ascofuranone and ilicicolin A, allowing for their development as therapeutics.

In-silico HMG-CoA reductase-inhibitory and in-vivo anti-lipidaemic/anticancer effects of carotenoids from Spondias mombin.

Inhibition of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase, the rate rate-determining enzyme for the biogenesis of cholesterol is known to show antineoplastic effects. Therefore, this study investigates the in-silico HMG-CoA reductase (HMGCR)-inhibitory and in-vivo anti-lipidaemic/anticancer effects of carotenoids from Spondias mombin. Carotenoids from S. mombin leaves were characterized with the aid of liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The characterized phytochemicals were obtained from PubChem. They were docked into the orthosteric site of human HMGCR (Protein Data Bank code 1HW8) using AutoDock 4.0 suites. DMBA (7,12-dimethylbenz[a]anthracene) model of breast cancer was treated with the carotenoids extract from S. mombin (100 mg/kg and 200 mg/kg doses) to assess its anti-lipidaemic cum anticancer effects. Carotenoids from S. mombin; beta-carotene-15,15'-epoxide, astaxanthin and 7,7',8,8'-tetrahydro-β-β-carotene demonstrate HMGCR inhibition. They form hydrophobic interactions with key residues within the catalytic domain of HMGCR. The carotenoids extract exhibits anti-lipidaemic/anticancer effects, lowering serum triglyceride, LDL and cholesterol concentration. It increases HDL concentration and downregulates the expression of HMGR, AFP, CEACAM-3, BRCA-1 and HIF-1 mRNAs. Carotenoids from S. mombin demonstrate HMG-CoA reductase (HMGCR) inhibition, anti-lipidaemic, and anticancer effects. The inhibition of HMGCR by the carotenoids extract further poses it as a potential anti-hypercholesterolaemia compounds.