Defects in CYB5A and CYB5B Impact Sterol C4 Demethylation in Cholesterol Biosynthesis and Demonstrate Regulatory Roles of Dimethyl Sterols
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE278705 | GEO | 2024/11/05
REPOSITORIES: GEO
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Project description:Defects in CYB5A and CYB5B Impact Sterol C4 Demethylation in Cholesterol Biosynthesis and Demonstrate Regulatory Roles of Dimethyl Sterols
| PRJNA1168488 | ENA
Project description:Fusidane-type antibiotics represented by helvolic acid, fusidic acid and cephalosporin P1 are a class of bacteriostatic agents, which have drawn renewed attention because they have no cross-resistance to commonly used antibiotics. However, their biosynthesis is poorly understood. Here, we perform a stepwise introduction of the nine genes from the proposed gene cluster for helvolic acid into Aspergillus oryzae NSAR1, which enables us to isolate helvolic acid (~20 mg L-1) and its 21 derivatives. Anti-Staphylococcus aureus assay reveals that the antibacterial activity of three intermediates is even stronger than that of helvolic acid. Notably, we observe an unusual C-4 demethylation process mediated by a promiscuous short-chain dehydrogenase/reductase (HelC) and a cytochrome P450 enzyme (HelB1), which is distinct from the common sterol biosynthesis. These studies have set the stage for using biosynthetic approaches to expand chemical diversity of fusidane-type antibiotics.
| S-EPMC5696383 | biostudies-literature
Project description:The conversion of 4,4-dimethylcholest-7-enol into 4alpha-methylcholest-7-enol by rat liver microsomal preparations involves the decarboxylation of a sterol 3-oxo-4alpha-carboxylic acid. By using an (18)O-labelled substrate it was shown that this decarboxylation does not involve a Schiff-base intermediate.
| S-EPMC1165609 | biostudies-other
Project description:3-β-hydroxysteroid-Δ8, Δ7-isomerase, known as Emopamil-Binding Protein (EBP), is an endoplasmic reticulum membrane protein involved in cholesterol biosynthesis, autophagy, oligodendrocyte formation. The mutation on EBP can cause Conradi-Hunermann syndrome, an inborn error. Interestingly, EBP binds an abundance of structurally diverse pharmacologically active compounds, causing drug resistance. Here, we report two crystal structures of human EBP, one in complex with the anti-breast cancer drug tamoxifen and the other in complex with the cholesterol biosynthesis inhibitor U18666A. EBP adopts an unreported fold involving five transmembrane-helices (TMs) that creates a membrane cavity presenting a pharmacological binding site that accommodates multiple different ligands. The compounds exploit their positively-charged amine group to mimic the carbocationic sterol intermediate. Mutagenesis studies on specific residues abolish the isomerase activity and decrease the multidrug binding capacity. This work reveals the catalytic mechanism of EBP-mediated isomerization in cholesterol biosynthesis and how this protein may act as a multi-drug binder.
| S-EPMC6549186 | biostudies-literature
Project description:Dyslipidemia is considered an essential component of the pathological process of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disease. Although TAR DNA Binding Protein 43 kDa (TDP-43) links both familial and sporadic forms of ALS and cytoplasmic aggregates are a hallmark of most cases of ALS, the molecular mechanism and the in vivo relation of ALS dyslipidemia with TDP-43 have been unclear. To analyze the dyslipidemia-related gene expression by TDP-43, we performed expression microarray and RNA deep sequencing (RNA-Seq) using cell lines expressing high levels of TDP-43 and identified 434 significantly altered genes including sterol regulatory element-binding protein 2 (SREBP2), a master regulator of cholesterol homeostasis and its downstream genes. Elevated TDP-43 impaired SREBP2 transcriptional activity, leading to inhibition of cholesterol biosynthesis. The amount of cholesterol was significantly decreased in the spinal cords of TDP-43-overexpressed ALS model mice and in the cerebrospinal fluids of ALS patients. These results suggested that TDP-43 could play an essential role in cholesterol biosynthesis in relation to ALS dyslipidemia.
| S-EPMC9107471 | biostudies-literature
Project description:Sterols are essential eukaryotic lipids that are required for a variety of physiological roles. The diagenetic products of sterol lipids, sterane hydrocarbons, are preserved in ancient sedimentary rocks and are utilized as geological biomarkers, indicating the presence of both eukaryotes and oxic environments throughout Earth's history. However, a few bacterial species are also known to produce sterols, bringing into question the significance of bacterial sterol synthesis for our interpretation of sterane biomarkers. Recent studies suggest that bacterial sterol synthesis may be distinct from what is observed in eukaryotes. In particular, phylogenomic analyses of sterol-producing bacteria have failed to identify homologs of several key eukaryotic sterol synthesis enzymes, most notably those required for demethylation at the C-4 position. In this study, we identified two genes of previously unknown function in the aerobic methanotrophic ?-Proteobacterium Methylococcus capsulatus that encode sterol demethylase proteins (Sdm). We show that a Rieske-type oxygenase (SdmA) and an NAD(P)-dependent reductase (SdmB) are responsible for converting 4,4-dimethylsterols to 4?-methylsterols. Identification of intermediate products synthesized during heterologous expression of SdmA-SdmB along with 13C-labeling studies support a sterol C-4 demethylation mechanism distinct from that of eukaryotes. SdmA-SdmB homologs were identified in several other sterol-producing bacterial genomes but not in any eukaryotic genomes, indicating that these proteins are unrelated to the eukaryotic C-4 sterol demethylase enzymes. These findings reveal a separate pathway for sterol synthesis exclusive to bacteria and show that demethylation of sterols evolved at least twice-once in bacteria and once in eukaryotes.
| S-EPMC6003346 | biostudies-literature
Project description:We describe the results of a systematic search for a class of hitherto-overlooked chemical-genetic interactions in the Saccharomyces cerevisiae genome, which exists between a detrimental genetic mutation and a chemical/drug that can ameliorate, rather than exacerbate, that detriment. We refer to this type of interaction as "chemical suppression." Our work was driven by the hypothesis that genome instability in a certain class of mutants could be alleviated by mild replication inhibition using chemicals/drugs. We queried a collection of conditionally lethal, i.e., temperature-sensitive, alleles representing 40% of the yeast essential genes for those mutants whose growth defect can be suppressed by hydroxyurea (HU), known as a potent DNA replication inhibitor, at the restrictive temperature. Unexpectedly, we identified a number of mutants defective in diverse cellular pathways other than DNA replication. Here we report that HU suppresses selected mutants defective in the kinetochore-microtubule attachment pathway during mitotic chromosome segregation. HU also suppresses an ero1-1 mutant defective for a thiol oxidase of the endoplasmic reticulum by providing oxidation equivalents. Finally, we report that HU suppresses an erg26-1 mutant defective for a C-3 sterol dehydrogenase through regulating iron homeostasis and in turn impacting ergosterol biosynthesis. We further demonstrate that cells carrying the erg26-1 mutation show an increased rate of mitochondrial DNA loss and delayed G1 to S phase transition. We conclude that systematic gathering of a compendium of "chemical suppression" of yeast mutants by genotoxic drugs will not only enable the identification of novel functions of both chemicals and genes, but also have profound implications in cautionary measures of anticancer intervention in humans.
| S-EPMC3887538 | biostudies-literature
Project description:The formation of lipid electrophile-protein adducts is associated with many disorders that involve perturbations of cellular redox status. The identities of adducted proteins and the effects of adduction on protein function are mostly unknown and an increased understanding of these factors may help to define the pathogenesis of various human disorders involving oxidative stress. 7-Dehydrocholesterol (7-DHC), the immediate biosynthetic precursor to cholesterol, is highly oxidizable and gives electrophilic oxysterols that adduct proteins readily, a sequence of events proposed to occur in Smith-Lemli-Opitz syndrome (SLOS), a human disorder resulting from an error in cholesterol biosynthesis. Alkynyl lanosterol (a-Lan) was synthesized and studied in Neuro2a cells, Dhcr7-deficient Neuro2a cells and human fibroblasts. When incubated in control Neuro2a cells and control human fibroblasts, a-Lan completed the sequence of steps involved in cholesterol biosynthesis and alkynyl-cholesterol (a-Chol) was the major product formed. In Dhcr7-deficient Neuro2a cells or fibroblasts from SLOS patients, the biosynthetic transformation was interrupted at the penultimate step and alkynyl-7-DHC (a-7-DHC) was the major product formed. When a-Lan was incubated in Dhcr7-deficient Neuro2a cells and the alkynyl tag was used to ligate a biotin group to alkyne-containing products, protein-sterol adducts were isolated and identified. In parallel experiments with a-Lan and a-7-DHC in Dhcr7-deficient Neuro2a cells, a-7-DHC was found to adduct to a larger set of proteins (799) than a-Lan (457) with most of the a-Lan protein adducts (423) being common to the larger a-7-DHC set. Of the 423 proteins found common to both experiments, those formed from a-7-DHC were more highly enriched compared to a DMSO control than were those derived from a-Lan. The 423 common proteins were ranked according to the enrichment determined for each protein in the a-Lan and a-7-DHC experiments and there was a very strong correlation of protein ranks for the adducts formed in the parallel experiments.
| S-EPMC5333532 | biostudies-literature
Project description:Resistance to agricultural fungicides in the field has created a need for discovering fungicides with new modes of action. DNA microarrays, because they provide information on expression of many genes simultaneously, could help to identify the modes of action. To begin an expression pattern database for agricultural fungicides, transcriptional patterns of Saccharomyces cerevisiae strain S288C genes were analysed following 2-h treatments with I50 concentrations of ergosterol biosynthesis inhibitors commonly used against plant pathogenic fungi. Eight fungicides, representing three classes of ergosterol biosynthesis inhibitors, were tested. To compare gene expression in response to a fungicide with a completely different mode of action, a putative methionine biosynthesis inhibitor (MBI) was also tested. Expression patterns of ergosterol biosynthetic genes supported the roles of Class I and Class II inhibitors in affecting ergosterol biosynthesis, confirmed that the putative MBI did not affect ergosterol biosynthesis, and strongly suggested that in yeast, the Class III inhibitor did not affect ergosterol biosynthesis. The MBI affected transcription of three genes involved in methionine metabolism, whereas there were essentially no effects of ergosterol synthesis inhibitors on methionine metabolism genes. There were no consistent patterns in other up- or downregulated genes between fungicides. These results suggest that inspection of gene response patterns within a given pathway may serve as a useful first step in identifying possible modes of action of fungicides. agricultural sterol biosynthesis inhibitor fungicides. Keywords = agriculture Keywords = ergosterol Keywords = methionine Keywords = fungicide Keywords = Saccharomyces cerevisiae S288C Keywords = biosynthesis
2010-06-10 | E-GEOD-2412 | biostudies-arrayexpress
Project description:Resistance to agricultural fungicides in the field has created a need for discovering fungicides with new modes of action. DNA microarrays, because they provide information on expression of many genes simultaneously, could help to identify the modes of action. To begin an expression pattern database for agricultural fungicides, transcriptional patterns of Saccharomyces cerevisiae strain S288C genes were analysed following 2-h treatments with I50 concentrations of ergosterol biosynthesis inhibitors commonly used against plant pathogenic fungi. Eight fungicides, representing three classes of ergosterol biosynthesis inhibitors, were tested. To compare gene expression in response to a fungicide with a completely different mode of action, a putative methionine biosynthesis inhibitor (MBI) was also tested. Expression patterns of ergosterol biosynthetic genes supported the roles of Class I and Class II inhibitors in affecting ergosterol biosynthesis, confirmed that the putative MBI did not affect ergosterol biosynthesis, and strongly suggested that in yeast, the Class III inhibitor did not affect ergosterol biosynthesis. The MBI affected transcription of three genes involved in methionine metabolism, whereas there were essentially no effects of ergosterol synthesis inhibitors on methionine metabolism genes. There were no consistent patterns in other up- or downregulated genes between fungicides. These results suggest that inspection of gene response patterns within a given pathway may serve as a useful first step in identifying possible modes of action of fungicides. agricultural sterol biosynthesis inhibitor fungicides. Keywords = agriculture Keywords = ergosterol Keywords = methionine Keywords = fungicide Keywords = Saccharomyces cerevisiae S288C Keywords = biosynthesis
2005-05-01 | GSE2412 | GEO