Enzyme In Pathway Research Articles

Photothermal conditions and upwelling enhance very short-lived brominated halocarbons emissions in the western tropical Pacific Ocean

Sea-to-air emissions of very short-lived brominated halocarbons (VSLBrHs) are known to contribute to 30 % of stratospheric and tropospheric ozone depletion. However, empirical data on their occurrence in open ocean are scarce, which makes it difficult to estimate the significant contribution of open ocean releases to the global budget of halocarbons. This study was conducted in 2022 to explore the spatial variations of VSLBrHs and their controlling factors in the western tropical Pacific Ocean (WTPO). The findings highlighted that high biological productivity and the resulting dissolved organic matter (DOM) as well as upwelling dynamics significantly influenced the distribution and production of VSLBrHs in seawater, with atmospheric levels primarily governed by oceanic emissions. Based on the simultaneous observation of seawater and atmospheric concentrations, the mean sea-to-air fluxes of CH2Br2, CHBr3, CHBrCl2, and CHBr2Cl were estimated to be 1.01, 6.65, 9.31, and 7.25 nmol m−2 d−1, respectively. Sea-to-air fluxes of these gases in the upwelling regions were 9.0, 4.6, 2.9, and 6.8 times those in the non-upwelling regions, respectively. Additionally, in-situ incubation experiments revealed that the enzymatic mediated biosynthesis pathways of VSLBrHs were enhanced under temperature and light-induced stress and in waters rich in humus-like substances. Therefore, we tentatively concluded that abundant photothermal conditions and the existence of upwelling in the WTPO made it a potential hotspot for the emission of VSLBrHs. This study offers critical insights into the environmental dynamics of VSLBrHs emissions and underscores the importance of regional oceanic conditions in influencing atmospheric greenhouse gas compositions.

Effects of Walnut Septum on The Enzyme Pathways Associated with Plasma Cholesterol Level

Objective: Cholesterol is crucial compound that plays pivotal role in cellular function in living organisms. Its excess or deficiency in plasma can lead to destruction and disintegration of cell membrane structure. Maintaining balanced intake of cholesterol in diet and seeking medical treatment, if necessary, can help prevent these negative effects. Furthermore, people often resort to natural and herbal remedies, such as walnut septum. Due to dearth of scientific data regarding effects of walnut septum on cholesterol metabolism, this research was undertaken to explore its potential effects. Method: Analysis was begun by extracting septum using various solvents. Resulting extracts were then analyzed using GC-MS, and compounds were identified by using an integrated library database. To detect effects of extracts on cholesterol esterase and HMG-CoA reductase, a colorimetric method was employed. Result: Monophenol, 2,4-Di-tert-butylphenol, 2,6-Di-tert-butylphenol, ethyl linoleate, and butyl linoleate were some of compounds detected by GC-MS scanning. The highest inhibitions were observed in the enzymatic analysis, with a rate of 3.2% (acetone) in the HMG-CoA reductase analysis and 13.6% (water) in the cholesterol esterase analysis. Conclusion: Although the walnut septum extract contains various chemical compounds, our in vitro analysis data suggest that there is no inhibitory effect at therapeutic level on enzyme pathways that regulate plasma cholesterol levels, namely HMG-CoA reductase and cholesterol esterase. We believe that further research is necessary to comprehensively evaluate its effects on other pathways.

2 Another renaissance for bile acid gastrointestinal microbiology

Abstract The field of bile acid microbiology in the gastrointestinal tract is going through a current rebirth after a peak of activity in the late 1970s and early 1980s. This renewed activity is a result of many factors including the discovery near the turn of the century that bile acids are potent signaling molecules, technological advances in next generation sequencing, computation, culturomics, gnotobiology, and metabolomics. The current state of the field with particular emphasis on questions that have remained unanswered for many decades in both bile acid synthesis by the host, and metabolism by the gut microbiota will be addressed. Current knowledge of established enzymatic pathways including bile salt hydrolase, hydroxysteroid dehydrogenases involved in the oxidation and epimerization of bile acid hydroxy groups, the Hylemon-Bjӧrkhem Pathway (HBP) of bile acid C7-dehydroxylation, and the formation of secondary allo-bile acids are described. Aspects of bile acid conjugation and esterification, and evidence for bile acid C3- and C12-dehydroxylation that are less well understood, but potentially critical for our understanding of bile acid metabolism in the human gut will be covered. The physiological consequences of bile acid metabolism for human health, important caveats and cautionary notes on experimental design and interpretation of bile acid metabolism are also explored.

The expression of AcIDI1 reveals diterpenoid alkaloids’ allocation strategies in the roots of Aconitum carmichaelii Debx

Isopentenyl diphosphate isomerase (IDI), a key enzyme in the biosynthetic pathway of diterpenoid alkaloids (DAs), plays an essential regulatory role in the synthesis and accumulation of DAs. In this study, the coding sequence (CDS) of AcIDI1 was isolated from the mother roots of Aconitum carmichaelii Debx. (GeneBank accession number OR915879). Bioinformatics analysis showed that the CDS of AcIDI1 was 894 bp, encoding a protein with 297 amino acids and the putative protein localized in the chloroplast. AcIDI1 exhibited significant homology with sequences encoding IDI in other species, and was most closely related to Aconitum vilmorinianum. Furthermore, the fusion protein has been successfully expressed in Escherichia coli (E. coli), providing a basis for future functional studies of AcIDI1. The expression pattern of AcIDI1 was analyzed by real-time quantitative PCR (qPCR), which demonstrates that AcIDI1 is a tissue-specific gene in the roots of A. carmichaelii and exhibits high expression in both daughter and mother roots. By comparing the expression levels of AcIDI1 in three tissues of the roots of A. carmichaelii at different growth stages, we propose that the mother roots (MRs) are the centers of resources allocation. The roots of A. carmichaelii continuously absorb the energy from external environment, while resources transfer behavior from MRs to both daughter roots (DRs) and axillary buds (ABs) occurs as the plant grows. This study establishes a foundation for applying the IDI gene to regulate the biosynthesis and accumulation of DAs in A. carmichaelii.

Conformational Dynamics in Corynebacterium glutamicum Diaminopimelate Epimerase: Insights from Ligand Parameterization, Atomistic Simulation, and Markov State Modeling.

The microbial enzyme diaminopimelate epimerase (DapF), a vital enzyme in the lysine biosynthetic pathway, catalyzes the conversion of L, L-diaminopimelate (L, L-DAP) to D, L-diaminopimelate (D, L-DAP) using a catalytic cysteine dyad with one cysteine in thiol state and another in thiolate. Under oxidizing conditions, the catalytic cysteines of apo DapF form a disulfide bond that alters the structure and function of DapF. Given its potential as a target for antimicrobial resistance treatments, understanding DapF's functional dynamics is imperative. In the present work, we employ microsecond-scale all-atom molecular dynamics simulations of product-bound DapF and apo-DapF under oxidized and reduced conditions. We employ a polarized charge model for the ligand and the active site residues, which was necessary to preserve the electrostatic environment in the active site and retain the ligand in the active site. The product-bound DapF and apo-DapF in oxidized and reduced conditions exhibit a closed, semi-open, and open conformation, respectively, as identified using the internal coordinates of the dimeric enzyme and the principal component analysis. The conformational switch is guided by the dynamic catalytic (DC) loop, loop II, and loop III movements in the active site. The time scale of the close-to-open conformational transition is estimated to be 0.8 μs through Markov state modeling (MSM) and transition path theory (TPT). The present study explains the role of various active site residues and loops in ligand binding and protein dynamics in the DapF enzyme under different redox conditions. Such information will be helpful in future inhibitor design studies targeting the DapF enzyme.

Medium Chain Length Polyhydroxyalkanoate Production by Engineered Pseudomonas gessardii Using Acetate-formate as Carbon Sources.

Production of medium chain length polyhydroxyalkanoate (mcl-PHA) was attempted using Pseudomonas gessardii NIBRBAC000509957, which was isolated from Sunchang, Jeollabuk-do, Republic of Korea (35°24'27.7"N, 127°09'13.0"E) and effectively utilized acetate and formate as carbon sources. We first evaluated the utilization of acetate as a carbon source, revealing optimal growth at 5g/L acetate. Then, formate was supplied to the acetate minimal medium as a carbon source to enhance cell growth. After overexpressing the acetate and formate assimilation pathway enzymes, this strain grew at a significantly higher rate in the medium. As this strain naturally produces PHA, it was further engineered metabolically to enhance mcl-PHA production. The engineered strain produced 0.40g/L of mcl-PHA with a biomass content of 30.43% in fed-batch fermentation. Overall, this strain can be further developed to convert acetate and formate into valuable products.

Abstract PO3-25-12: Tigecycline-Induced Metabolic Reprogramming and Cytokine Modulation Suppress the Characteristics of Breast Cancer Cells

Abstract Objective: Patients with advanced breast cancer are prone to recurrence and metastasis. Hence, there is still a need to find new potential drugs or adjuvants to improve prognosis. The Warburg effect, which is highly dependent on glycolysis, has been found to be present in the energy metabolism of tumors, including breast cancer. The effect also promotes the production of cytokines, to adapt the inflammatory microenvironment of tumor. However, drugs targeting Warburg effect and inflammatory environment are rarely reported in the treatment of breast cancer. Tigecycline (Tige) is a commonly used glycylcycline antibiotic. Previous research has indicated it impede mitochondrial oxidative phosphorylation (OXPHOS) in various solid tumors. Based on the team's previous research, our study aimed to investigate whether Tige could inhibit breast cancer development by interfering with the Warburg effect and the inflammatory environment. Methods: MB231, MB468 and MCF7, T47D, which represent triple-negative and estrogen receptor positive breast cancers, were treated with Tige at different concentrations for different periods of time. CCK8, colony formation and sphere formation assays were used to evaluate the effects on proliferation and tumor stemness in vitro, respectively. The effects on migration and invasion were evaluated by wound healing assay and Transwell. RNA sequencing was used to explore the genes altered and analyze the pathways involved. After labeling with probes, the changes of reactive oxygen species (ROS) were detected by fluorescence microscope, microplate reader, and flow cytometry. Cell cycle and apoptosis were detected by flow cytometry. Liquid cytokine chip was used to analyze the secretion of cytokines. Electron microscopy was performed to observe the changes in mitochondrial morphology. RT-PCR was used to detect the expression of rate-limiting enzymes in glycolysis pathway. Glucose consumption, lactate and pyruvate production were measured using glucose, lactate and pyruvate detection kits, respectively. Western blot was used to detect the changes of respiratory chain protein complex and other related pathway proteins in OXPHOS. Results: Four breast cancer cells showed a significant decrease in cell proliferation, invasion and migratory ability after Tige treatment. Sphere-forming assays also showed that Tige had an effect on the stemness of breast cancer. RNA-sequencing showed that glycolysis, cell cycle, and immune-related pathways were inhibited after Tige treatment, and ROS production analysis revealed a decrease in ROS production. Flow cytometry also revealed that the cell cycle was arrested in S phase without significant apoptosis. Cytokine liquid chip analysis showed that the secretion of many cytokines, mainly interleukin (such as IL-1B), was inhibited after Tige intervention. RT-PCR results showed that the expression of several rate-limiting enzymes of the glycolytic pathway decreased. Glucose, lactate and pyruvate assay results showed that Tige reduced glucose uptake and lactate, pyruvate production. Electron microscopy results revealed that mitochondria in Tige treated cells showed significant contractures. WB results also showed a decrease in the expression of OXPHOS-related proteins. However, due to the heterogeneity of the four breast cancer cells, the degree of change in these capacities was not entirely consistent. Conclusions: Tige not only inhibits mitochondrial OXPHOS, but also inhibits the glycolysis pathway, which results in the inhibition of glucose uptake and the production of pyruvate and lactate. The secretion of early pro-inflammatory cytokines also affected. Therefore, this study suggests that Tige may change the glucose metabolism and energy metabolism, and also change the inflammatory environment of breast tumors, which provides new evidence for the anti-tumor effect of Tige in breast cancer. Citation Format: Haochen Yu, Lingfeng Tang, Shengchun Liu. Tigecycline-Induced Metabolic Reprogramming and Cytokine Modulation Suppress the Characteristics of Breast Cancer Cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-12.

Timing the evolution of phosphorus-cycling enzymes through geological time using phylogenomics

Phosphorus plays a crucial role in controlling biological productivity, but geological estimates of phosphate concentrations in the Precambrian ocean, during life’s origin and early evolution, vary over several orders of magnitude. While reduced phosphorus species may have served as alternative substrates to phosphate, their bioavailability on the early Earth remains unknown. Here, we reconstruct the phylogenomic record of life on Earth and find that phosphate transporting genes (pnas) evolved in the Paleoarchean (ca. 3.6-3.2 Ga) and are consistent with phosphate concentrations above modern levels ( > 3 µM). The first gene optimized for low phosphate levels (pstS; <1 µM) appeared around the same time or in the Mesoarchean depending on the reconstruction method. Most enzymatic pathways for metabolising reduced phosphorus emerged and expanded across the tree of life later. This includes phosphonate-catabolising CP-lyases, phosphite-oxidising pathways and hypophosphite-oxidising pathways. CP-lyases are particularly abundant in dissolved phosphate concentrations below 0.1 µM. Our results thus indicate at least local regions of declining phosphate levels through the Archean, possibly linked to phosphate-scavenging Fe(III), which may have limited productivity. However, reduced phosphorus species did not become widely used until after the Paleoproterozoic Great Oxidation Event (2.3 Ga), possibly linked to expansion of the biosphere at that time.

Isolation and functional characterization of (iso)eugenol O-methyltransferase (IEMT) gene in Asarum sieboldii

Methyleugenol is one of the main active constituents in the volatile oil of the traditional Chinese medicine Asari Radix et Rhizoma. It possesses various pharmacological effects such as analgesic, anesthetic, and anti-inflammatory properties. In biosynthesis, the initial precursor phenylalanine is finally converted into methyleugenol through a series of intermediate compounds including coniferyl acid, courmaryl acid, caffeic acid, ferulic acid/ferulic-CoA, coniferyl aldehyde, conferyl alcohol, cnfiferyl acetate, and eugenol/isoeugenol, which are produced through catalysis of a large number of enzymes. Eugenol O-methyltransferase(EOMT) is one of the key enzymes in the biosynthesis pathway, capable of methylating eugenol on the para-site hydroxyl group of the benzene ring, thereby generating methyleugenol. Here, an(iso)eugenol O-methyltransferase(IEMT) gene was cloned for the first time from Asarum siebo-ldii, holding an open reading frame that consisted of 1 113 bp and encoded a protein containing 370 amino acid residues. Bioinformatics analysis results showed that this protein was equipped with the characteristic structural domains of methyltransferases such as S-adenosylmethionine(SAM) binding sites and dimerization domains. The prokaryotic expression recombinant plasmid pET28a(+)-AsIEMT was constructed, and the candidate protein was induced and purified. In vitro enzyme assays confirmed that AsIEMT had dual functions. The enzyme could catalyze the production either of methyleugenol from eugenol or of methylisoeugenol from isoeugenol, although the latter was more prevalent. When isoeugenol was used as the substrate, the kinetics parameters K_m and V_(max) of catalytic reaction were(0.90±0.06) mmol·L~(-1) and(1.32±0.04)nmol·s~(-1)·mg~(-1), respectively. This study expanded our understandings of critical enzyme genes involved in phenylpropanoid metabolic pathways, and would facilitate the elucidation of quality formation mechanisms of the TCM Asari Radix et Rhizoma.

Assessing the ability of dihydrosterculic acid to activate PPARα

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor associated with the expression of specific target genes involved in fatty acid β-oxidation. Activation of PPARα could potentially be utilized to treat diseases associated with the accumulation of hepatic lipids such as non-alcoholic fatty liver disease (NAFLD), which currently impacts approximately 25% of the world’s population. At present, a class of synthetic ligands called fibrates can be prescribed to help manage NAFLD but are weak agonists for PPARα and therefore have limited effcacy. Consequently, alternative ligands for PPARα must be established and no natural endogenous ligands have been conclusively identified. This study expands on our previous work and aims to identify the direct effects of dihydrosterculic acid (DHSA), a cyclopropyl fatty acid found in cottonseed oil (CSO), on PPARα activity. To identify downstream effects of DHSA, male mice were placed on a CSO- (DHSA-containing) or isocaloric oil-enriched diet for 6 weeks and tissues were sent for genome-wide RNA sequencing analysis. CSO-fed mice demonstrated increased PPARα gene expression in the liver and concomitant decreased triglyceride in plasma (-75.1±11.5 milligrams/deciliter (mg/dL); p&lt;0.01) and liver (-86.7±71.7 mg/gram (g) protein; p=0.1) and decreased free fatty acid in liver (-16.5±3.4 micromolar (μM)/g protein; p&lt;0.01) relative to the oil-enriched control diet. Confirmational tissue analysis of liver samples show an increase in PPARα target gene expression in the DHSA containing groups compared to control diet groups, indicating that DHSA could potentially serve as an endogenous ligand for PPARα to increase its transcriptional activity to increase fat oxidation. To further test this hypothesis, female PPARα knockout mice were fed a DHSA containing diet or control diet for 4 weeks (n=8). Following the 4-week period, the chow-fed wild type females gained more weight (1.3±0.6 g; p&lt;0.05) than the PPARα knockout mice. Otherwise, there were no significant differences in body weight or food intake between diets or genotypes. Additional in vitro studies show increased PPARα target gene expression following treatments with DHSA, specifically for CPT1a (2.7±0.2 fold increase; p&lt;0.01), ACOX1 (1.3±0.1 fold increase; p&lt;0.05), and ACADVL (1.6±0.1 fold increase, p&lt;0.01), which encode key enzymes in lipid oxidation pathways. SCD1, the rate limiting enzyme in lipogenesis, was also downregulated (0.5±0.03 fold decrease, p&lt;0.01) in response to a 24-hour treatment with DHSA indicating a reduction in endogenous lipogenesis. From these results, we believe that DHSA could function through a PPARα-dependent mechanism. Further analysis of the tissues collected from the PPARα knockout mouse model will aid in determining the mechanism of DHSA as well as identifying its potential for the treatment of excess lipid accumulation. This project is funded by Cotton Incorporated. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Clinical outcomes of antithrombin III supplementation in an overt disseminated intravascular coagulation: a longitudinal single-institutional experience and retrospective analysis.

Antithrombin is a small plasma glycoprotein synthesized in the liver that belongs to the serpin family of serine protease inhibitors and inactivates several enzymes in the coagulation pathway. It plays a leading major factor on coagulation pathway, therefore administration of antithrombin is essential to treat serious clinical conditions such as disseminated intravascular coagulation (DIC). Despite the theoretical benefits of antithrombin supplementation, the optimal antithrombin activity for heparin efficacy and the benefits of antithrombin supplementation in various disease entities are not yet fully understood. The strict administration guidelines on antithrombin III in cases of DIC by the National Health Insurance Service and the Ministry of Food and Drug Safety complied as follows: antithrombin levels below 20 mg/dL in adults; antithrombin activity below 70% of normal in adults; total administration period of antithrombin must be carefully limited to within maximum 3 days, and the total administration dose must be below 7,000 international unit (IU), (loading dose, 1,000 IU in 1 hour: maintenance dose, 500 IU every 6 hours for 3 days). We identified 76 eligible for analysis according to the above-mentioned criteria in our institution (male/female, 59/17). Forty-four were identified to the non-survivor group and 32 patients were recognized as the survivor group. The baseline parameters in the non-survivor and survivor groups were comparable with no significant differences in age (66.5±18.1 vs. 66.0±16.2 years, P=0.90), sex (32/12 vs. 27/5, P=0.35), hospital length of stay (31.1±34.5 vs. 31.2±26.1 days, P=0.99), sequential organ failure assessment (SOFA) (7.3±2.5 vs. 6.6±2.0, P=0.22), simplified acute physiology score II (SAPS II) (46.0±8.8 vs. 43.5±9.2, P=0.23), cause for DIC (P=0.95), and underlying disease (P=0.38). The levels of antithrombin III on the day just before the administration significantly lower in the non-survivor groups than in the survivor groups (50.1%±13.6% vs. 57.6%±12.5%, P=0.01). The hemoglobin level in the 2nd day and 7th day after antithrombin III administration was significantly different between the non-survivor and survivor groups (9.9±1.9 vs. 11.0±2.0 g/dL, P=0.01, and 9.4±1.8 vs. 10.5±1.6 g/dL, P=0.006). The antithrombin III levels on the day of administration [area under the curve (AUC) =0.672] demonstrated significantly better prediction of mortality than the A antithrombin III levels on 1st day (AUC =0.552), the 2nd day (AUC =0.624), and 7th day (AUC =0.593). Our study suggests that the antithrombin administration may be effective tools for DIC treatment, and may be more positively considered, especially in the cases of DIC, which is a frequent complication of septic shock, sepsis, and other critical disease entities and which is associated with a high level of mortality. Furthermore, our study also suggests that the total doses and periods of antithrombin administration, which recommended by national guidelines, may be insufficient, therefore prolongation of period and increase of total dose of antithrombin supplement might be necessary.

Regulatory Effect of L-Cysteine on Testosterone Biosynthesis Genes in Leydig Cells and THP-1 Monocytes

Background: Spermatogenesis involves a series of complicated cellular events resulting in the production of mature spermatids. Leydig cells of the testis are essential to forming a spermatozoon. Leydig cells are the interstitial cells that reside between seminiferous tubules and their key role is in the biosynthesis of testosterone. Leydig cells also regulate testicular macrophage and are stocked with innate defense systems. CYP11A1 is a major enzyme in the biosynthesis pathway of testosterone which converts the cholesterol to pregnenolone in the mitochondria of Leydig cells. L-Cysteine (L-Cys) acts as a precursor of glutathione, an antioxidant that plays a vital role against oxidative stress damage. Previous animal studies report the protective effect of L-Cys against chemical/radiation-induced-testicular damage. This study examined the biochemical mechanisms by which L-Cys stimulates testosterone biosynthesis and relative expression of blood-testis barrier (BTB) genes in Leydig cells and THP-1 monocytes. Methodology: The THP-1 monocytes and Leydig cells were treated with L-Cys for 24 hours. Following treatment, RNA was extracted, and the expression of the testosterone biosynthesis gene and BTB genes was analyzed using quantitative RT-PCR. Data (n=4) is presented as mean ± SEM and p-values ≤0.05 are considered as significant. Results: Expression of CLDN1 (p=0.006), CLDN11 (p=0.03), and TJP1 (p=0.04) genes was significantly higher in Leydig cells than in the THP-1 monocytes. There is no significant change in the expression of testosterone-regulatory genes between Leydig cells and THP-1 monocytes. In THP-1 monocytes, L-Cys treatment significantly upregulated the expression of CYP11A1 (p=0.03), CYP19A1 (p=0.002), and BTB genes: CLDN1 (p=0.05), CLDN2 (p=0.001), CLDN4 (p=0.0006), CLDN11 (p=0.006), and TJP1 (p=0.03). Similarly, L-Cys (500uM) treatment significantly upregulated (p=0.03) the CYP11A1 gene expression in HLC, further, the expression of BTB genes: CLDN1 (p=0.03), CLDN11 (p=0.02), and TJP1 (p=0.02) was also upregulated. Conclusion: This study shows that L-Cys increases the expression of testosterone-related genes in both HLC and THP-1. The expression profile of the BTB genes was higher in HLC than in THP-1 monocytes. L-Cys supplementation upregulates the expression of the testosterone biosynthesis pathway gene which can increase spermatogenesis and reduce the male infertility risk. Funding source: NIH/NCCIH (5R33AT010637) and the Malcolm Feist Endowed Chair in Diabetes. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Design of Potential Inhibitors of Pf5-ALAS in Liver Stage Plasmodium falciparum: A Sustainable Chemotherapeutic Approach to Address Antimalarial Resistance

Plasmodium falciparum delta-aminolevulinate synthase (Pf5-ALAS) is the first enzyme in the heme biosynthetic pathway, and it is a liver stage specific enzyme in the developmental stages of Plasmodium falciparum. 8-amino quinoline derivatives have been reported to be active against liver stage parasite and hence was used as a template in the design of 12 derivatives as sustainable chemotherapeutics that were screened in this study designed as potential inhibitors of Pf5-ALAS. The target was modelled due to the unavailability of its experimentally validated 3-dimensional (3D) structure. The binding energy of all 12 designed compounds ranged from -7.9 to -9.1 Kcal/mol which all performed better than primaquine a known inhibitor of liver stage malaria. All twelve designed compounds had comparatively good pharmacokinetic profiles and did not present a toxicity risk, according to in silico ADMET prediction. The position and presence of a functional group that introduces a synergistic impact and subsequently raises the binding energy are highlighted in the qualitative structural assessment of the top three hits. This might pave way to highly economical new antimalarial therapeutic for sustainability health and wellbeing in sub-Saharan Africa and beyond.

Lactate Suppresses, While Glutamine or Branched-Chain Amino Acids Support, Cardiac Fibroblast Activation

Background: Although metabolic changes occur during activation of cardiac fibroblasts, it remains unclear how different metabolic pathways influence fibroblast phenotype and function. Substrates used for biosynthetic pathways could be particularly important to the fibrotic response because activated fibroblasts require higher anabolic output to provide biomolecular building blocks for cell division and ECM secretion. Hence, elucidating how metabolism influences fibroblast differentiation and activation could provide valuable information for understanding mechanisms of fibrosis. Hypothesis: We hypothesized that the availability of specific metabolic substrates regulate myofibroblast differentiation. Methods and Results: Naïve cardiac fibroblasts were isolated from wildtype, male C57BL/6J mice and treated with TGFβ or bFGF for 48 h. To examine metabolic genes that respond to profibrotic stimuli, we utilized RNA sequencing, which indicated that TGFβ significantly increases transcription of numerous glycolytic and glutamine metabolism genes as well as transporters for branched-chain amino acids (BCAAs), glutamine, and lactate. Guided by these results, we used an αSMA-reporter assay to examine how lactate, glutamine and BCAA availability influence TGFβ-induced myofibroblast differentiation. Fibroblasts were treated with deficient media supplemented with increasing concentrations of substrate and challenged with TGFβ for 48 hours, followed by assessment of αSMA positivity. In a dose-dependent manner, fibroblasts treated with lactate exhibited decreased αSMA reporter activity (EC50=6.17mM). Furthermore, extracellular flux analysis revealed that the presence of lactate (1mM–10mM) significantly reduced basal oxygen consumption rate and extracellular acidification rate in TGFβ-treated cells (n=2, p&lt;0.05). In contrast, αSMA expression was blunted with diminishing concentrations of glutamine (n=3, p&lt;0.05) and BCAAs, indicating suffcient supply of these substrates is essential for fibroblast differentiation. In addition, immunoblotting data of TGFβ-treated fibroblasts revealed that low extracellular BCAA levels (0 or 100μM) decreased abundance of col1a1 and periostin compared with treatment with BCAAs within the physiological range (400μM; n=4, p&lt;0.05). Conclusion: These preliminary findings provide insight into how specific alterations of glycolysis, glutaminolysis, and BCAA metabolism modulate fibroblast activation in vitro. Additional studies that include alterations to other critical substrates (glucose, fatty acids, ketone bodies, etc.) and enzymes of ancillary biosynthetic pathways of glucose metabolism could provide additional insights into how metabolism regulates fibroblast function. Ultimately, the goal of these studies is to discover potential genetic and pharmacological interventions that positively influence wound healing in the infarcted heart and limit the burden of cardiac fibrosis. NIH and AHA. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MTORC1-CTLH E3 ligase regulates the degradation of HMG-CoA synthase 1 through the Pro/N-degron pathway

Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis. In particular, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 (HMGCS1), the initial enzyme in the mevalonate pathway, exhibits the most significant half-life adaptation. Degradation of HMGCS1 is regulated by the C-terminal to LisH (CTLH) E3 ligase through the Pro/N-degron motif. HMGCS1 is ubiquitylated on two C-terminal lysines during mTORC1 inhibition, and efficient degradation of HMGCS1 in cells requires a muskelin adaptor. Importantly, modulating HMGCS1 abundance has a dose-dependent impact on cell proliferation, which is restored by adding a mevalonate intermediate. Overall, our unbiased degradomics study provides new insights into mTORC1 function in cellular metabolism: mTORC1 regulates the stability of limiting metabolic enzymes through the ubiquitin system.

HSP47 complements cholesterol synthesis-independent HMGCoA-reductase pathway-mediated fibroblast bioenergetics

Fibrosis, characterized by excessive extracellular matrix (ECM) components deposition, is a common pathological process underlying numerous chronic diseases. Differentiating fibroblasts into myofibroblasts leads to the deposition of fibrous proteins like collagen in the ECM. Heat shock protein 47 (HSP47) is a molecular chaperone protein that assists in the folding of collagen. Recent studies show that inhibition of cholesterol synthesis-independent HMGCoA-reductase pathway enzyme geranylgeranyl pyrophosphate synthase 1 (GGPS1) decreases fibroblast differentiation and its bioenergetics. However, the role of HSP47 in this pathway is unknown, although recent observations show that HSP47 is decreased when GGPS1 is inhibited. Hence, we tested the hypothesis that HSP47 promotes cellular respiration in myofibroblasts. Seahorse assays were performed on different fibroblasts/myofibroblasts repeatedly and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in the presence and absence of HSP47 inhibitor (HY124817). Complex V of the electron transport chain was measured by immunoblotting ATP-5A. HY124817 tends to decrease the OCR, basal respiration rate, ATP-linked, and spare respiratory capacity. Co-incubation with HY124817 and digeranyl bisphosphonate (DGBP), an inhibitor of GGPS1, significantly (P = 0.043) inhibited further the ATP-linked OCR compared to the TGF group. Immunoblotting showed that Complex V did not change significantly across treatments. These results suggest that HSP47 is involved in fibroblast bioenergetics related to ATP-linked OCR in both differentiated myofibroblasts and normal fibroblasts. Inhibition of HSP47 complements the decrease in myofibroblast cellular respiration caused by the GGPS1 inhibition. This finding is novel in that HSP47 plays a role in fibroblast bioenergetics regulated by the cholesterol synthesis-independent HMGCoA-reductase pathway. Further research is necessary to identify what HSP47 targets and regulatory elements involved in this process. This would provide more insight into the mechanisms that underlie critical fibrosis conditions like myocardial fibrosis and potential therapeutics. This experience was supported by the Medical College of Wisconsin's Advancing Student Potential for Inclusion with Research Experiences (ASPIRE) program. The program is funded by a federal grant from the National Heart, Lung, and Blood Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In silico analysis and characterization of potential inhibitors of MmaA3, a methoxy mycolic acid synthase from Mycobacterium tuberculosis

The emergence of the multi-and extensively drug-resistant (MDR and XDR) strains of Mycobacterium tuberculosis (M.tb), necessitates paradigm-shifting therapeutic approaches. The impermeable waxy lipid layer, primarily composed of mycolic acids, is a key factor in conferring resistance to conventional drugs. This study introduces a novel strategy to combat drug resistance by targeting Methoxy mycolic acid synthase 3 (MmaA3), a critical enzyme in the mycolic acid biosynthesis pathway. MmaA3 is responsible for the O-methylation of hydroxymycolate precursors and emerges as a promising therapeutic target. Through homology-based modeling, we generated a three-dimensional structure of MmaA3, providing crucial insights into its structural characteristics. High throughput virtual screening was performed against the MmaA3 model, using diverse sources: knowledge-based, FDA-approved Drugbank, and Asinex-Elite libraries. Through rigorous computational analyses, including binding affinity assessments, molecular interactions analysis, and binding free energy calculations, potential inhibitors of MmaA3 have been identified. Subsequent validation studies evaluated the stability of top protein-ligand complexes, and free energy calculations using molecular dynamics simulations. The stability of complexes within the catalytic site was confirmed through RMSD and RMSF profile analyses. Furthermore, binding free energy calculations using the MM-GBSA approach revealed significant binding affinity of identified ligands for MmaA3 target protein, comparable to its substrate/cofactors. These findings underscore the potential of the proposed molecules as candidates for further experimental exploration, offering promising avenues for the development of effective inhibitors against M.tb. Overall, our research contributes to significantly advancing the formulation of progressive therapeutic strategies in combating drug-resistant tuberculosis. Communicated by Ramaswamy H. Sarma

Effect of hydrothermal-acid pretreatment on methane yield and microbial community in anaerobic digestion of rice straw

Hydrothermal pretreatment has been proposed to enhance straw methane yield during anaerobic digestion recently. However, the combined effect of hydrothermal and organic acid pretreatment (HTOAP) needs further investigation. This study identified optimal pretreatment at 120 °C with 3 % acetic acid for 24 h by orthogonal design method. The HTOAP increased the reducing sugar content by destroying the lignocellulosic structure. A 79 % increment of methane production after HTOAP was observed compared to the untreated group. Microbial analysis showed that HTOAP enriched the relative abundance of lignocellulose-degraders, such as W5053, Thermanaerovibrio, Caldicoprobacter, as well as the syntrophic acetate oxidizing bacteria Syntrophaceticus. Moreover, Methanobacterium conducted hydrogenotrophic methanogenesis dominantly. Furthermore, the potential function analysis showed that HTOAP stimulated the expression of key enzymes in the hydrogenotrophic pathway, including carbon-monoxide dehydrogenase (EC 1.2.7.4) and coenzyme F420 hydrogenase (EC 1.12.98.1). This investigation illustrated the potential of HTOAP of rice straw to facilitate methane production.

Chromosome-level genome assembly of the yeast Lodderomyces beijingensis reveals the genetic nature of metabolic adaptations and identifies subtelomeres as hotspots for amplification of mating type loci.

Lodderomyces beijingensis is an ascosporic ascomycetous yeast. In contrast to related species Lodderomyces elongisporus, which is a recently emerging human pathogen, L. beijingensis is associated with insects. To provide an insight into its genetic makeup, we investigated the genome of its type strain, CBS 14171. We demonstrate that this yeast is diploid and describe the high contiguity nuclear genome assembly consisting of eight chromosome-sized contigs with a total size of about 15.1 Mbp. We find that the genome sequence contains multiple copies of the mating type loci and codes for essential components of the mating pheromone response pathway, however, the missing orthologs of several genes involved in the meiotic program raise questions about the mode of sexual reproduction. We also show that L. beijingensis genome codes for the 3-oxoadipate pathway enzymes, which allow the assimilation of protocatechuate. In contrast, the GAL gene cluster underwent a decay resulting in an inability of L. beijingensis to utilize galactose. Moreover, we find that the 56.5 kbp long mitochondrial DNA is structurally similar to known linear mitochondrial genomes terminating on both sides with covalently closed single-stranded hairpins. Finally, we discovered a new double-stranded RNA mycovirus from the Totiviridae family and characterized its genome sequence.

DszA Catalyzes C-S Bond Cleavage through N5-Hydroperoxyl Formation.

Due to its detrimental impact on human health and the environment, regulations demand ultralow sulfur levels on fossil fuels, in particular in diesel. However, current desulfurization techniques are expensive and cannot efficiently remove heteroaromatic sulfur compounds, which are abundant in crude oil and concentrate in the diesel fraction after distillation. Biodesulfurization via the four enzymes of the metabolic 4S pathway of the bacterium Rhodococcus erythropolis (DszA-D) is a possible solution. However, the 4S pathway needs to operate at least 500 times faster for industrial applicability, a goal currently pursued through enzyme engineering. In this work, we unveil the catalytic mechanism of the flavin monooxygenase DszA. Surprisingly, we found that this enzyme follows a recently proposed atypical mechanism that passes through the formation of an N5OOH intermediate at the re side of the cofactor, aided by a well-defined, predominantly hydrophobic O2 pocket. Besides clarifying the unusual chemical mechanism of the complex DszA enzyme, with obvious implications for understanding the puzzling chemistry of flavin-mediated catalysis, the result is crucial for the rational engineering of DszA, contributing to making biodesulfurization attractive for the oil refining industry.