Zymosterol

The ATPase activity of ABCA1 is increased by cholesterol in the presence of anionic lipids.[Pubmed:38215730]

J Biochem. 2024 Jan 12:mvae003.

High-density lipoprotein (HDL) transports excess cholesterol from peripheral tissues back to the liver, and plasma HDL levels are inversely related to cardiovascular disease incidence. ATP-binding cassette A1 (ABCA1) is a member of the ABC protein superfamily, and generates nascent HDL, which consists of several hundreds of phospholipids and cholesterol wrapped by apolipoprotein A-I (apoA-I). However, it remains unclear whether cholesterol is a transport substrate of ABCA1. Since ATP hydrolysis of ABC proteins is typically increased by their transport substrates, we characterized the effects of cholesterol on the ATPase activity of purified ABCA1 using liposomes of various lipid compositions. ABCA1 showed substantial ATPase activity (20-30 nmol∙min-1∙mg-1) only in liposomes containing anionic lipids, including phosphatidylserine. Cholesterol increased the ATPase activity by 1.6- to 3-fold in the presence of anionic lipids. Moreover, phosphatidylserine addition to BHK/ABCA1 cells increased phosphatidylcholine and cholesterol efflux to apoA-I. Next, we investigated the sterol specificity of ABCA1. The ATPase activity of ABCA1 was strongly enhanced by desmosterol and Zymosterol, similar to cholesterol. In contrast, 7-dehydrocholesterol and lathosterol weakly increased the ATPase activity, and no increase was observed with stigmasterol or brassicasterol. These findings suggest that ABCA1 transports cholesterol, and prefers cholesterol over plant sterols as a transport substrate.

Effects of continuous intravenous infusion with propofol on intestinal metabolites in rats.[Pubmed:38169795]

Biomed Rep. 2023 Dec 20;20(2):25.

Microbial metabolites play an important role in regulating intestinal homeostasis and immune responses. Propofol is a common anesthetic in clinic, but it is not clear whether it affects intestinal metabolites in rats. Tail vein puncture was performed after adaptive feeding for 1 month in eight 2-month-old rats and they were given continuous intravenous infusion of propofol for 3 h. The feces of rats were divided into different groups based on time periods, with before and after anesthesia with propofol on days 1, 3 and 7 labeled as groups P, A1, A3 and A7, respectively. The effect of continuous intravenous infusion with propofol on rat fecal metabolites was determined using the non-targeted metabolomics technique gas chromatography coupled with a time-of-flight mass spectrometer analysis. The types and contents of metabolites in rat feces were changed after continuous intravenous infusion with propofol, but the changes were not statistically significant. The contents of the metabolites 3-hydroxyphenylacetic acid and palmitic acid increased from day 3 to 7, and it was shown that the two metabolites were positively correlated at a statistically significant level. Linoleic acid decreased to its lowest level on day 3, and it returned to pre-anesthesia level on day 7. At the same time, linoleic acid metabolism was a metabolic pathway that was co-enriched 7 days after infusion with propofol. Spearman correlation analysis showed that there was significant correlation between some differential metabolites and differential microorganisms. It was observed that Zymosterol 1, cytosin and elaidic acid were negatively correlated with Alloprevotella in the A3 vs. P group. In the A7 vs. P group, cortexolone 3 and coprostan-3-one were positively correlated with Faecalibacterium, whilst aconitic acid was negatively correlated with it. In conclusion, the present study revealed statistically insignificant effects of continuous intravenous propofol on the intestinal metabolites in rats.

Gut microbial metabolite deoxycholic acid facilitates Th17 differentiation through modulating cholesterol biosynthesis and participates in high-fat diet-associated colonic inflammation.[Pubmed:37789469]

Cell Biosci. 2023 Oct 3;13(1):186.

BACKGROUND: High-fat diet (HFD) is closely associated with the increased prevalence of inflammatory bowel disease (IBD). Excessive gut microbial metabolite deoxycholic acid (DCA) caused by HFD plays significant roles in eliciting intestinal inflammation, however, the mechanism underlining the induction of inflammatory response by DCA has not been fully elucidated. The purpose of this study was to investigate the role of DCA in the triggering of inflammation via affecting CD4(+) T cell differentiation. RESULTS: Murine CD4(+)T cells were cultured under Th1, Th2 or Th17-polarizing conditions treated with or without different dosage of DCA, and flowcytometry was conducted to detect the effect of DCA on CD4(+) T cell differentiation. Alteration of gene expression in CD4(+) T cells upon DCA treatment was determined by RNA-sequencing and qRT-PCR. Bioinformatic analysis, cholesterol metabolic profiling, ChIP assay and immuno-fluorescent staining were further applied to explore the DCA-regulated pathway that involved in CD4(+)T cell differentiation. The results showed that DCA could dose-dependently promote the differentiation of CD4(+) T cell into Th17 linage with pathogenic signature. Mechanistically, DCA stimulated the expression of cholesterol biosynthetic enzymes CYP51 and led to the increased generation of endogenous RORgammat agonists, including Zymosterol and desmosterol, therefore facilitating Th17 differentiation. Up-regulation of CYP51 by DCA was largely mediated via targeting transcription factor SREBP2 and at least partially through bile acid receptor TGR5. In addition, DCA-supplemented diet significantly increased intestinal Th17 cell infiltration and exacerbated TNBS-induced colitis. Administration of cholestyramine to eliminate fecal bile acid obviously alleviated colonic inflammation accompanied by decreased Th17 cells in HFD-fed mice. CONCLUSIONS: Our data establish a link between DCA-induced cholesterol biosynthesis in immune cells and gut inflammation. Modulation of bile acid level or targeting cholesterol metabolic pathway may be potential therapeutic measurements for HFD-related colitis.

COVID-19 and cholesterol biosynthesis: Towards innovative decision support systems.[Pubmed:37720097]

iScience. 2023 Aug 31;26(10):107799.

With COVID-19 becoming endemic, there is a continuing need to find biomarkers characterizing the disease and aiding in patient stratification. We studied the relation between COVID-19 and cholesterol biosynthesis by comparing 10 intermediates of cholesterol biosynthesis during the hospitalization of 164 patients (admission, disease deterioration, discharge) admitted to the University Medical Center of Ljubljana. The concentrations of Zymosterol, 24-dehydrolathosterol, desmosterol, and zymostenol were significantly altered in COVID-19 patients. We further developed a predictive model for disease severity based on clinical parameters alone and their combination with a subset of sterols. Our machine learning models applying 8 clinical parameters predicted disease severity with excellent accuracy (AUC = 0.96), showing substantial improvement over current clinical risk scores. After including sterols, model performance remained better than COVID-GRAM. This is the first study to examine cholesterol biosynthesis during COVID-19 and shows that a subset of cholesterol-related sterols is associated with the severity of COVID-19.

Improvement effect of biochar on soil microbial community structure and metabolites of decline disease bayberry.[Pubmed:37333636]

Front Microbiol. 2023 May 12;14:1154886.

Decline disease is a new disease that has recently caused severe damage in bayberry industry. The effect of biochar on decline disease was determined by investigating the changes in the vegetative growth and fruit quality of bayberry trees as well as soil physical and chemical properties, microbial community structure, and metabolites. Results indicated that the application of biochar could improve the vigor and fruit quality of diseased trees, and rhizosphere soil microbial diversity at the levels of phyla, orders, and genera. The relative abundance of Mycobacterium, Crossiella, Geminibasidium, and Fusarium were significantly increased, while Acidothermus, Bryobacter, Acidibacter, Cladophialophora, Mycena, and Rickenella were significantly decreased by biochar in rhizosphere soil of decline diseased bayberry. Analysis of redundancies (RDA) of microbial communities and soil characteristics revealed that the composition of bacterial and fungal communities was significantly affected by the pH, organic matter, alkali hydrolyzable nitrogen, available phosphorus, available potassium, exchangeable calcium and exchangeable magnesium in bayberry rhizosphere soil, and the contribution rates to fungi were larger than those to bacteria at the genus level. Biochar greatly influenced the metabolomics distribution of rhizosphere soils of decline disease bayberry. One hundred and nine different metabolites from both the presence and absence of biochar, mainly include acid, alcohol, ester, amine, amino acid, sterol, sugar, and other secondary metabolites, of which the contents of 52 metabolites were increased significantly such as aconitic acid, threonic acid, pimelic acid, epicatechin, and lyxose. The contents of 57 metabolites decreased significantly, such as conduritol beta-expoxide, Zymosterol, palatinitol, quinic acid, and isohexoic acid. There was a great difference between the absence and presence of biochar in 10 metabolic pathways, including thiamine metabolism, arginine and proline metabolism, glutathione metabolism, ATP-binding cassette (ABC) transporters, butanoate metabolism, cyanoamino acid metabolism, tyrosine metabolism, phenylalanine metabolism, phosphotransferase system (pts), and lysine degradation. There was a significant correlation between the relative content of microbial species and the content of secondary metabolites in rhizosphere soil at the levels of bacterial and fungal phyla, order, and genus. Overall, this study highlighted the significant influence of biochar in decline disease by regulating soil microbial community, physical and chemical properties, and secondary metabolites in rhizosphere soil, which provided a novel strategy for managing bayberry decline disease.

Characterization of an allosteric inhibitor of fungal-specific C-24 sterol methyltransferase to treat Candida albicans infections.[Pubmed:37160123]

Cell Chem Biol. 2023 May 18;30(5):553-568.e7.

Filamentation is an important virulence factor of the pathogenic fungus Candida albicans. The abolition of Candida albicans hyphal formation by disrupting sterol synthesis is an important concept for the development of antifungal drugs with high safety. Here, we conduct a high-throughput screen using a C. albicans strain expressing green fluorescent protein-labeled Dpp3 to identify anti-hypha agents by interfering with ergosterol synthesis. The antipyrine derivative H55 is characterized to have minimal cytotoxicity and potent inhibition of C. albicans hyphal formation in multiple cultural conditions. H55 monotherapy exhibits therapeutic efficacy in mouse models of azole-resistant candidiasis. H55 treatment increases the accumulation of Zymosterol, the substrate of C-24 sterol methyltransferase (Erg6). The results of enzyme assays, photoaffinity labeling, molecular simulation, mutagenesis, and cellular thermal shift assays support H55 as an allosteric inhibitor of Erg6. Collectively, H55, an inhibitor of the fungal-specific enzyme Erg6, holds potential to treat C. albicans infections.

Erg6 Acts as a Downstream Effector of the Transcription Factor Flo8 To Regulate Biofilm Formation in Candida albicans.[Pubmed:37098889]

Microbiol Spectr. 2023 Jun 15;11(3):e0039323.

The yeast-to-hyphal morphotype transition and subsequent biofilm formation are important virulence factors of Candida albicans and are closely associated with ergosterol biosynthesis. Flo8 is an important transcription factor that determines filamentous growth and biofilm formation in C. albicans. However, the relationship between Flo8 and regulation of the ergosterol biosynthesis pathway remains elusive. Here, we analyzed the sterol composition of a flo8-deficient C. albicans strain by gas chromatography-mass spectrometry and observed the accumulation of the sterol intermediate Zymosterol, the substrate of Erg6 (C-24 sterol methyltransferase). Accordingly, the transcription level of ERG6 was reduced in the flo8-deficient strain. Yeast one-hybrid experiments revealed that Flo8 physically interacted with the ERG6 promoter. Ectopic overexpression of ERG6 in the flo8-deficient strain partially restored biofilm formation and in vivo virulence in a Galleria mellonella infection model. These findings suggest that Erg6 is a downstream effector of the transcription factor Flo8 that mediates the cross talk between sterol synthesis and virulence factors in C. albicans. IMPORTANCE Biofilm formation by C. albicans hinders its eradication by immune cells and antifungal drugs. Flo8 is an important morphogenetic transcription factor that regulates the biofilm formation and in vivo virulence of C. albicans. However, little is known about how Flo8 regulates biofilm formation and fungal pathogenicity. Here, we determined that Flo8 directly binds to the promoter of ERG6 to positively regulate its transcriptional expression. Consistently, loss of flo8 results in the accumulation of the substrate of Erg6. Moreover, ectopic overexpression of ERG6 at least partially restores the biofilm formation and virulence of the flo8-deficient strain both in vitro and in vivo. This work provides a new perspective on the metabolic link between transcription factors and morphotypes in C. albicans.

Untargeted lipidomic analysis and network pharmacology for parthenolide treated papillary thyroid carcinoma cells.[Pubmed:37095470]

BMC Complement Med Ther. 2023 Apr 24;23(1):130.

BACKGROUND: With fast rising incidence, papillary thyroid carcinoma (PTC) is the most common head and neck cancer. Parthenolide, isolated from traditional Chinese medicine, inhibits various cancer cells, including PTC cells. The aim was to investigate the lipid profile and lipid changes of PTC cells when treated with parthenolide. METHODS: Comprehensive lipidomic analysis of parthenolide treated PTC cells was conducted using a UHPLC/Q-TOF-MS platform, and the changed lipid profile and specific altered lipid species were explored. Network pharmacology and molecular docking were performed to show the associations among parthenolide, changed lipid species, and potential target genes. RESULTS: With high stability and reproducibility, a total of 34 lipid classes and 1736 lipid species were identified. Lipid class analysis indicated that parthenolide treated PTC cells contained higher levels of fatty acid (FA), cholesterol ester (ChE), simple glc series 3 (CerG3) and lysophosphatidylglycerol (LPG), lower levels of Zymosterol (ZyE) and Monogalactosyldiacylglycerol (MGDG) than controlled ones, but with no significant differences. Several specific lipid species were changed significantly in PTC cells treated by parthenolide, including the increasing of phosphatidylcholine (PC) (12:0e/16:0), PC (18:0/20:4), CerG3 (d18:1/24:1), lysophosphatidylethanolamine (LPE) (18:0), phosphatidylinositol (PI) (19:0/20:4), lysophosphatidylcholine (LPC) (28:0), ChE (22:6), and the decreasing of phosphatidylethanolamine (PE) (16:1/17:0), PC (34:1) and PC (16:0p/18:0). Four key targets (PLA2G4A, LCAT, LRAT, and PLA2G2A) were discovered when combining network pharmacology and lipidomics. Among them, PLA2G2A and PLA2G4A were able to bind with parthenolide confirmed by molecular docking. CONCLUSIONS: The changed lipid profile and several significantly altered lipid species of parthenolide treated PTC cells were observed. These altered lipid species, such as PC (34:1), and PC (16:0p/18:0), may be involved in the antitumor mechanisms of parthenolide. PLA2G2A and PLA2G4A may play key roles when parthenolide treated PTC cells.

Anti-Obesity Effect of Auricularia delicate Involves Intestinal-Microbiota-Mediated Oxidative Stress Regulation in High-Fat-Diet-Fed Mice.[Pubmed:36839230]

Nutrients. 2023 Feb 8;15(4):872.

Auricularia delicate (ADe), an edible fungus belonging to the family Auriculariaceae and order Auriculariales, possesses antimicrobial, hepatoprotective, and antioxidant effects. In this study, after systematic analysis of its composition, ADe was administered to high-fat-diet (HFD)-fed mice to investigate its anti-obesity effect. ADe significantly controlled body weight; alleviated hepatic steatosis and adipocyte hypertrophy; reduced aspartate aminotransferase, total cholesterol, insulin, and resistin; and increased adiponectin levels in HFD-fed mice serum. Based on intestinal microbiota and lipidomics analysis, ADe treatment regulated the composition and abundance of 49 intestinal microorganisms and influenced the abundance of 8 lipid species compared with HFD-fed mice. Based on a correlation analysis of the intestinal microbiota and lipids, Coprococcus showed significant negative associations with ceramide (d18:0 20:0+O), phosphatidylserine (39:4), sphingomyelin (d38:4), and Zymosterol (20:2). Moreover, ADe treatment decreased the levels of ROS and MDA and increased the levels of Nrf2, HO-1, and three antioxidant enzymes in HFD-fed mice livers. Collectively, the anti-obesity effect of ADe involves the regulation of oxidative stress and is mediated by the intestinal microbiota. Hence, this study provides a reference for the application of ADe as a candidate food for obesity.

Role of Cholesterol and its Biosynthetic Precursors on Membrane Organization and Dynamics: A Fluorescence Approach.[Pubmed:36781437]

J Membr Biol. 2023 Apr;256(2):189-197.

Cholesterol is the most representative sterol present in membranes of higher eukaryotes, and is the end product of a long and multistep biosynthetic pathway. Lathosterol and Zymosterol are biosynthetic precursors of cholesterol in Kandutsch-Russell and Bloch pathways, respectively. Lathosterol differs with cholesterol merely in the position of the double bond in the sterol ring, whereas Zymosterol differs with cholesterol in position and number of double bonds. In this work, we have monitored the effect of cholesterol and its biosynthetic precursors (lathosterol and Zymosterol) on membrane organization and dynamics in fluid and gel phase membranes. Toward this goal, we have utilized two fluorescent membrane probes, DPH and its cationic derivative TMA-DPH. Our results using these probes show that cholesterol and its biosynthetic precursors (lathosterol and Zymosterol) exhibit similar trend in maintaining membrane organization and dynamics (as reported by fluorescence anisotropy and apparent rotational correlation time), in fluid phase POPC membranes. Notably, although lathosterol and Zymosterol show similar trend in maintaining membrane organization and dynamics, the corresponding change for cholesterol is different in gel phase DPPC membranes. These results demonstrate that the position and number of double bonds in sterols is an important determinant in maintaining membrane physical properties. Our results assume significance since accumulation of precursors of cholesterol have been reported to be associated with severe pathological conditions.

Optimizing accelerated solvent extraction combined with liquid chromatography-Orbitrap mass spectrometry for efficient lipid profile characterization of mozzarella cheese.[Pubmed:35759836]

Food Chem. 2022 Nov 15;394:133542.

In this study, a novel Accelerated Solvent Extraction (ASE) procedure combined with UHPLC-Q-Orbitrap-MS was developed for detailed untargeted lipid profile of mozzarella cheese. Response Surface Methodology and Pareto front, using a Central Composite Design (CCD), were employed to define the optimised combination of extraction temperature, number of extraction cycles and mix of solvents. LipidSearch software was used for a reliable and accurate lipid identification. A total of 13 subclasses, including ceramides, diacylglycerols, triacylglycerols, lysophosphatidylcholines, lysophosphatidylethanolamines, phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, sphingomyelins, bismethyl phosphatidic acids, cholesterol ester, Zymosterol ester, hexosyl ceramides were measured. The elaboration of the CCD showed that the solvents ratio was the main factor affecting the extraction efficiency. The optimised ASE method, together with the Folch extraction, synergistically contributed to a complete characterization of lipid profile of mozzarella cheese, confirming ASE technique, associated with high resolution mass spectrometry detection, as an efficient tool for Lipidomics in food science.

Cytostatic effects of structurally different ginsenosides on yeast cells with altered sterol biosynthesis and transport.[Pubmed:35724740]

Biochim Biophys Acta Biomembr. 2022 Oct 1;1864(10):183993.

Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene glycosides show membranolytic activity, and, therefore, are considered to be promising antimicrobial drugs. However, the interrelation between their structure, biological activities, and target membrane lipid composition remains elusive. Here we studied the antifungal effects of four Panax triterpene glycosides (ginsenosides) with sugar moieties at the C-3 (ginsenosides Rg3, Rh2), C-20 (compound K), and both (ginsenoside F2) positions in Saccharomyces cerevisiae mutants with altered sterol plasma membrane composition. We observed reduced cytostatic activity of the Rg3 and compound K in the UPC2-1 strain with high membrane sterol content. Moreover, LAM gene deletion reduced yeast resistance to Rg3 and digitonin, another saponin with glycosylated aglycon in the C-3 position. LAM genes encode plasma membrane-anchored StARkin superfamily-member sterol transporters. We also showed that the deletion of the ERG6 gene that inhibits ergosterol biosynthesis at the stage of Zymosterol increased the cytostatic effects of Rg3 and Rh2, but not the other two tested ginsenosides. At the same time, in silico simulation revealed that the substitution of ergosterol with Zymosterol in the membrane changes the spatial orientation of Rg3 and Rh2 in the membranes. These results imply that the plasma membrane sterol composition defines its interaction with triterpene glycoside depending on their glycoside group position. Our results also suggest that the biological role of membrane-anchored StARkin family protein is to protect eukaryotic cells from triterpenes glycosylated at the C-3 position.

Inhibiting C-4 Methyl Sterol Oxidase with Novel Diazaborines to Target Fungal Plant Pathogens.[Pubmed:35584803]

ACS Chem Biol. 2022 Jun 17;17(6):1343-1350.

With resistance to current agricultural fungicides rising, a great need has emerged for new antifungals with unexploited targets. In response, we report a novel series of diazaborines with potent activity against representative fungal plant pathogens. To identify their mode of action, we selected for resistant isolates using the model fungus Saccharomyces cerevisiae. Whole-genome sequencing of independent diazaborine-resistant lineages identified a recurring mutation in ERG25, which encodes a C-4 methyl sterol oxidase required for ergosterol biosynthesis in fungi. Haploinsufficiency and allele-swap experiments provided additional genetic evidence for Erg25 as the most biologically relevant target of our diazaborines. Confirming Erg25 as putative target, sterol profiling of compound-treated yeast revealed marked accumulation of the Erg25 substrate, 4,4-dimethylZymosterol and depletion of both its immediate product, Zymosterol, as well as ergosterol. Encouraged by these mechanistic insights, the potential utility of targeting Erg25 with a diazaborine was demonstrated in soybean-rust and grape-rot models of fungal plant disease.

Teriflunomide Promotes Oligodendroglial 8,9-Unsaturated Sterol Accumulation and CNS Remyelination.[Pubmed:34642237]

Neurol Neuroimmunol Neuroinflamm. 2021 Oct 12;8(6):e1091.

BACKGROUND AND OBJECTIVES: To test whether low concentrations of teriflunomide (TF) could promote remyelination, we investigate the effect of TF on oligodendrocyte in culture and on remyelination in vivo in 2 demyelinating models. METHODS: The effect of TF on oligodendrocyte precursor cell (OPC) proliferation and differentiation was assessed in vitro in glial cultures derived from neonatal mice and confirmed on fluorescence-activated cell sorting-sorted adult OPCs. The levels of the 8,9-unsaturated sterols lanosterol and Zymosterol were quantified in TF- and sham-treated cultures. In vivo, TF was administered orally, and remyelination was assessed both in myelin basic protein-GFP-nitroreductase (Mbp:GFP-NTR) transgenic Xenopus laevis demyelinated by metronidazole and in adult mice demyelinated by lysolecithin. RESULTS: In cultures, low concentrations of TF down to 10 nM decreased OPC proliferation and increased their differentiation, an effect that was also detected on adult OPCs. Oligodendrocyte differentiation induced by TF was abrogated by the oxidosqualene cyclase inhibitor Ro 48-8071 and was mediated by the accumulation of Zymosterol. In the demyelinated tadpole, TF enhanced the regeneration of mature oligodendrocytes up to 2.5-fold. In the mouse demyelinated spinal cord, TF promoted the differentiation of newly generated oligodendrocytes by a factor of 1.7-fold and significantly increased remyelination. DISCUSSION: TF enhances Zymosterol accumulation in oligodendrocytes and CNS myelin repair, a beneficial off-target effect that should be investigated in patients with multiple sclerosis.