C-5 Sterol Desaturase Research Articles

Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus

Azole antifungals inhibit the sterol C14-demethylase (CYP51/Erg11) of the ergosterol biosynthesis pathway. Here we show that the azole-induced synthesis of fungicidal cell wall carbohydrate patches in the pathogenic mold Aspergillus fumigatus strictly correlates with the accumulation of the CYP51 substrate eburicol. A lack of other essential ergosterol biosynthesis enzymes, such as sterol C24-methyltransferase (Erg6A), squalene synthase (Erg9) or squalene epoxidase (Erg1) does not trigger comparable cell wall alterations. Partial repression of Erg6A, which converts lanosterol into eburicol, increases azole resistance. The sterol C5-desaturase (ERG3)-dependent conversion of eburicol into 14-methylergosta-8,24(28)-dien-3β,6α-diol, the “toxic diol” responsible for the fungistatic activity against yeasts, is not required for the fungicidal effects in A. fumigatus. While ERG3-lacking yeasts are azole resistant, ERG3-lacking A. fumigatus becomes more susceptible. Mutants lacking mitochondrial complex III functionality, which are much less effectively killed, but strongly inhibited in growth by azoles, convert eburicol more efficiently into the supposedly “toxic diol”. We propose that the mode of action of azoles against A. fumigatus relies on accumulation of eburicol which exerts fungicidal effects by triggering cell wall carbohydrate patch formation.

7-Dehydrocholesterol dictates ferroptosis sensitivity.

Ferroptosis, a form of regulated cell death that is driven by iron-dependent phospholipid peroxidation, has been implicated in multiple diseases, including cancer1-3, degenerative disorders4 and organ ischaemia-reperfusion injury (IRI)5,6. Here, using genome-wide CRISPR-Cas9 screening, we identified that the enzymes involved in distal cholesterol biosynthesis have pivotal yet opposing roles in regulating ferroptosis through dictating the level of 7-dehydrocholesterol (7-DHC)-an intermediate metabolite of distal cholesterol biosynthesis that is synthesized by sterol C5-desaturase (SC5D) and metabolized by 7-DHC reductase (DHCR7) for cholesterol synthesis. We found that the pathway components, including MSMO1, CYP51A1, EBP and SC5D, function as potential suppressors of ferroptosis, whereas DHCR7 functions as a pro-ferroptotic gene. Mechanistically, 7-DHC dictates ferroptosis surveillance by using the conjugated diene to exert its anti-phospholipid autoxidation function and shields plasma and mitochondria membranes from phospholipid autoxidation. Importantly, blocking the biosynthesis of endogenous 7-DHC by pharmacological targeting of EBP induces ferroptosis and inhibits tumour growth, whereas increasing the 7-DHC level by inhibiting DHCR7 effectively promotes cancer metastasis and attenuates the progression of kidney IRI, supporting a critical function of this axis in vivo. In conclusion, our data reveal a role of 7-DHC as a natural anti-ferroptotic metabolite and suggest that pharmacological manipulation of 7-DHC levels is a promising therapeutic strategy for cancer and IRI.

Long noncoding RNA lincsc5d regulates hepatic cholesterol synthesis by modulating sterol C5 desaturase in large yellow croaker.

Although long noncoding RNA (lncRNA) plays a vital role in cholesterol metabolism, very little information is available in fish. Thus, a 10-week feeding experiment was performed to estimate the effects of lncRNA on cholesterol metabolism in large yellow croaker fed with fish oil (FO), soybean oil (SO), olive oil (OO), and palm oil (PO) diets. Results showed that fish fed with OO and PO diets had higher liver total cholesterol (TC) and cholesterol ester (CE) contents compared with fish fed with FO diets. Analysis of the KEGG pathway showed that the steroid biosynthesis pathway was enriched in comparisons FO vs SO, FO vs OO, and FO vs PO. Meanwhile, sterol C5 desaturase (SC5D), a cholesterol synthase, was up-regulated in the steroid biosynthesis pathway. SC5D was widely expressed in all tissues examined, and the highest expression of SC5D was detected in brain. More importantly, a novel lncRNA associated with sc5d gene was identified by RNA sequencing and named as lincsc5d. The tissue distribution of lincsc5d was similar to that of sc5d. A nuclear/cytoplasmic RNA separation assay showed that lincsc5d was a nucleus-enriched lncRNA. qRT-PCR results demonstrated that lincsc5d was markedly up-regulated in the SO, OO, and PO groups. Furthermore, the results of TC content and the lincsc5d and sc5d expression in hepatocytes agreed with in vivo results. In conclusion, this study indicated that vegetable oils, especially OO and PO, increased hepatic cholesterol levels by promoting cholesterol synthesis, and lncRNA lincsc5d and sc5d might be involved in cholesterol synthesis.

Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response.

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Selection of suitable reference genes for quantitative real time PCR in different Tulasnella isolates and orchid-fungus symbiotic germination system.

Under natural conditions, mycorrhizal symbiosis accompanies nearly the entire life cycle of orchids from seed germination through to flowering and fruiting. Tulasnella-like orchid mycorrhizal fungi are the most common mycorrhizal fungi found in association with orchid species. Presently suitable reference genes have not been systematically selected for the quantification of gene expression via Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). We evaluated 12 candidate Tulasnella genes in nine different Tulasnella isolates and in the Dendrobium-fungal symbiotic germination associations followed by statistical analysis using the programs Bestkeeper, geNorm, and Normfinder to analyze the expression stability of the individual genes. The results showed that the EF2, UBC, and PP2A genes had the highest rankings with relatively stable expression levels across the different genotypes and during the symbiotic seed germination process by the three programs, and may be suitable for RT-qPCR normalization. Furthermore, the gene encoding C-5 Sterol desaturase (C5SD) was selected to verify the reliability of EF2, UBC, and PP2A expression during the Tulasnella-Dendrobium symbiotic seed germination process. This study is the first systematic exploration of optimal reference genes for gene expression studies during the colonization of orchid seeds by the mycorrhizal fungus Tulasnella.

Genomic instability at the locus of sterol C24-methyltransferase promotes amphotericin B resistance in Leishmania parasites.

Amphotericin B is an increasingly important tool in efforts to reduce the global disease burden posed by Leishmania parasites. With few other chemotherapeutic options available for the treatment of leishmaniasis, the potential for emergent resistance to this drug is a considerable threat. Here we characterised four novel amphotericin B-resistant Leishmania mexicana lines. All lines exhibited altered sterol biosynthesis, and hypersensitivity to pentamidine. Whole genome sequencing demonstrated resistance-associated mutation of the sterol biosynthesis gene sterol C5-desaturase in one line. However, in three out of four lines, RNA-seq revealed loss of expression of sterol C24-methyltransferase (SMT) responsible for drug resistance and altered sterol biosynthesis. Additional loss of the miltefosine transporter was associated with one of those lines. SMT is encoded by two tandem gene copies, which we found to have very different expression levels. In all cases, reduced overall expression was associated with loss of the 3’ untranslated region of the dominant gene copy, resulting from structural variations at this locus. Local regions of sequence homology, between the gene copies themselves, and also due to the presence of SIDER1 retrotransposon elements that promote multi-gene amplification, correlate to these structural variations. Moreover, in at least one case loss of SMT expression was not associated with loss of virulence in primary macrophages or in vivo. Whilst such repeat sequence-mediated instability is known in Leishmania genomes, its presence associated with resistance to a major antileishmanial drug, with no evidence of associated fitness costs, is a significant concern.

RNA Sequencing-Based Whole-Transcriptome Analysis of Friesian Cattle Fed with Grape Pomace-Supplemented Diet.

Simple SummaryGrape pomace (GPO) is an important source of polyphenols which are known to have antioxidant properties. In the past decade, GPO has received some attention as a bioactive dietary component in farm animals’ diet. In this study, we have analyzed the whole-transcriptome of Friesian calves fed with a GPO-supplemented diet using RNA-sequencing. We noted that the most affected pathway was the cholesterol lipid biosynthesis and this effect was consistent with a reduction in both serum cholesterol and lipid oxidation in the carcasses. This study provides evidence on the antioxidant property of GPO-supplemented diet, from a molecular biology standpoint.Grape pomace (GPO), the main by-product of the wine making process, is a rich source of polyphenols with potent antioxidant properties. Recently, GPO has emerged as a potential feed additive in livestock nutrition, with several reports describing its beneficial effects on animals’ overall health status or production traits. However, little is known about it from a molecular biology standpoint. In the present study, we report the first RNA sequencing-based whole-transcriptome profiling of Friesian calves fed with a GPO-supplemented diet. We identified 367 differentially expressed genes (p < 0.05) in the GPO-supplemented calves (n = 5), when compared with unsupplemented control group (n = 5). The pathway analysis showed that ‘cholesterol lipid biosynthesis’ was the most negatively-enriched (p < 0.001) pathway in the GPO-supplemented animals. In specific terms, five important genes coding for cholesterol biosynthesis enzymes, namely the Farnesyl-diphosphate Farnesyltransferase 1 (FDFT-1), Squalene Epoxidase (SQLE), NAD(P)-dependent Steroid Dehydrogenase-like (NSDHL), Methylsterol Monooxygenase (MSMO)-1, and Sterol-C5-desaturase (SC5D), two major transcription factors (the Sterol Regulatory Element-binding Transcription Factor 1 and 2), as well as the Low-Density Lipoprotein Receptor (LDLR), were all downregulated following GPO supplementation. Such an effect was mirrored by a reduction of blood cholesterol levels (p = 0.07) and a lowered (p < 0.001) Malondialdehyde (lipid oxidation marker) level in carcasses. We provide evidence on the effects of GPO-supplemented diets on the whole-transcriptome signature in veal calves, which mainly reflects an antioxidant activity.

Attenuation of UVR-induced vitamin D3 synthesis in a mouse model deleted for keratinocyte lathosterol 5-desaturase

The lower risk of some internal cancers at lower latitudes has been linked to greater sun exposure and consequent higher levels of ultraviolet radiation (UVR)-produced vitamin D3 (D3). To separate the experimental effects of sunlight and of all forms of D3, a mouse in which UVR does not produce D3 would be useful.To this end we have generated mice carrying a modified allele of sterol C5-desaturase (Sc5d), the gene encoding the enzyme that converts lathosterol to 7-dehydrocholesterol (7-DHC), such that Sc5d expression can be inactivated using the Cre/lox site-specific recombination system. By crossing to mice with tissue-specific expression of Cre or CreER2 (Cre/estrogen receptor), we generated two lines of transgenic mice. One line has constitutive keratinocyte-specific inactivation of Sc5d (Sc5dk14KO). The other line (Sc5dk14KOi) has tamoxifen-inducible keratinocyte-specific inactivation of Sc5d.Mice deleted for keratinocyte Sc5d lose the ability to increase circulating D3 following UVR exposure of the skin. Thus, unlike in control mice, acute UVR exposure did not affect circulating D3 level in inducible Sc5dk14KOi mice.Keratinocyte-specific inactivation of Sc5d was proven by sterol measurement in hair – in control animals lathosterol and cholesta-7,24-dien-3β-ol, the target molecules of SC5D in the sterol biosynthetic pathways, together constituted a mean of 10% of total sterols; in the conditional knockout mice these sterols constituted a mean of 56% of total sterols. The constitutive knockout mice had an even greater increase, with lathosterol and cholesta-7,24-dien-3β-ol accounting for 80% of total sterols.In conclusion, the dominant presence of the 7-DHC precursors in hair of conditional animals and the lack of increased circulating D3 following exposure to UVR reflect attenuated production of the D3 photochemical precursor 7-DHC and, consequently, of D3 itself. These animals provide a useful new tool for investigating the role of D3 in UVR-induced physiological effects and, more broadly, for investigations of the cholesterol synthetic pathway in the skin and other targeted tissues.

Electron Transfer Pathways in Cholesterol Synthesis

Cholesterol synthesis in the endoplasmic reticulum requires electron input at multiple steps and utilizes both NADH and NADPH as the electron source. Four enzymes catalyzing five steps in the pathway require electron input: squalene monooxygenase, lanosterol demethylase, sterol 4α-methyl oxidase, and sterol C5-desaturase. The electron-donor proteins for these enzymes include cytochrome P450 reductase and the cytochrome b5 pathway. Here I review the evidence for electron donor protein requirements with these enzymes, the evidence for additional electron donor pathways, and the effect of deletion of these redox enzymes on cholesterol and lipid metabolism.

Fungal sterol C22-desaturase is not an antimycotic target as shown by selective inhibitors and testing on clinical isolates

Inhibition of concise enzymes in ergosterol biosynthesis is one of the most prominent strategies for antifungal chemotherapy. Nevertheless, the enzymes sterol C5-desaturase and sterol C22-desaturase, which introduce double bonds into the sterol core and side chain, have not been fully investigated yet for their potential as antifungal drug targets. Lathosterol side chain amides bearing N-alkyl groups of proper length are known as potent inhibitors of the enzymes sterol C5-desaturase and sterol Δ24-reductase in mammalian cholesterol biosynthesis. Here we present the results of our evaluation of these amides for their ability to inhibit enzymes in fungal ergosterol biosynthesis. In the presence of inhibitor(s) an accumulation of sterols lacking a double bond at C22/23 (mainly ergosta-5,7-dien-3β-ol) was observed in Candida glabrata, Saccharomyces cerevisiae, and Yarrowia lipolytica. Hence, the lathosterol side chain amides were identified as selective inhibitors of the fungal sterol C22-desaturase, which was discussed as a specific target for novel antifungals. One representative inhibitor, (3S,20S)-20-N-butylcarbamoylpregn-7-en-3β-ol was subjected to antifungal susceptibility testing on patient isolates according to modified EUCAST guidelines. But, the test organisms showed no significant reduction of cell growth and/or viability up to an inhibitor concentration of 100μg/mL. This leads to the conclusion that sterol C22-desaturase is not an attractive target for the development of antifungals.

Inhibition of Human Sterol Δ7-Reductase and Other Postlanosterol Enzymes by LK-980, a Novel Inhibitor of Cholesterol Synthesis

Novel potential inhibitors of the postsqualene portion of cholesterol synthesis were screened in HepG2 cells. 2-(4-Phenethylpiperazin-1-yl)-1-(pyridine-3-yl)ethanol (LK-980) was identified as a prospective compound and was characterized further in cultures of human primary hepatocytes from seven donors. In vitro kinetic measurements show that the half-life of LK-980 is at least 4.3 h. LK-980 does not induce CYP3A4 mRNA nor enzyme activity. Target prediction was performed by gas chromatography-mass spectrometry, allowing simultaneous separation and quantification of nine late cholesterol intermediates. Experiments indicated that human sterol Δ(7)-reductase (DHCR7) is the major target of LK-980 (34-fold increase of 7-dehydrocholesterol), whereas human sterol Δ(14)-reductase (DHCR14), human sterol Δ(24)-reductase (DHCR24), and human sterol C5-desaturase (SC5DL) represent minor targets. In the absence of purified enzymes, we used the mathematical model of cholesterol synthesis to evaluate whether indeed more than a single enzyme is inhibited. In silico inhibition of only DHCR7 modifies the flux of cholesterol intermediates, resulting in a sterol profile that does not support experimental data. Partial inhibition of the DHCR14, DHCR24, and SC5DL steps, in addition to DHCR7, supports the experimental sterol profile. In conclusion, we provide experimental and computational evidence that LK-980, a novel inhibitor from the late portion of cholesterol synthesis, inhibits primarily DHCR7 and to a lesser extent three other enzymes from this pathway.

Mutations in Saccharomyces cerevisiae sterol C5-desaturase conferring resistance to the CYP51 inhibitor fluconazole

Understanding fluconazole resistance is important as it emerged as a serious clinical problem for this CYP51, sterol 14α-demethylase, inhibitor. One mechanism, observed first in Saccharomyces cerevisiae, was through defective sterol C5-desaturase (Erg3p) required to form the fungistatic sterol end-product resulting from CYP51 inhibition, 14α-methylergosta-8,24(28)-dien-3β,6α-diol. Here, we report molecular changes resulting in both blocked mutants and also leaky mutants in which reduced ergosterol levels were detected. Blocked mutants exhibited nonsense and frameshift mutations, while leaky mutants contained missense mutations that were generally in conserved positions based on the alignment of sterol C5-desaturases and located mainly between residues 250 and 282.

Nine of Ten Histidine Residues, Located in Three Conserved Clusters, are Required for Mammalian Sterol C5-Desaturase Activity

We previously reported three histidine-rich conserved motifs containing HX3H, HX2H and HX2HH in the deduced primary amino acid sequence of human sterol C-5 desaturase (SC5D), which introduces a double bond at the C5, 6 positions of A7-sterols to form A5,7-sterols (Nishi, S. et al. (2000) Biochim. Biophys. Acta 1490, 106-108). In the present study, the role of the 18 His residues contained in the enzyme was investigated by means of site-directed mutagenesis and expression of the mutated enzymes in an SC5D-deficient yeast strain (erg3). The activity of the recombinant SC5Ds to allow the yeast to convert ergosta-7,22-dien-3β-ol to ergosterol (ergosta-5,7,22-trien-3β-ol) was examined directly in the erg3 strain in vivo. Individual replacements of each of the ten conserved His residues with Arg completely eliminated the desaturase activity in each case, except for one, His190. The conserved His-containing clusters have a consistent positioning with respect to the putative membrane spanning domains, and we propose that they are located on the cytoplasmic face of the endoplasmic reticulum. In contrast, mutation of eight nonconserved flanking His residues to Arg did not affect the ability to complement the erg3 phenotype. Therefore, these non-conserved His residues are not essential for the catalysis, but may contribute to the activity through conformational or other effects. Western blot analysis confirmed that the steady-state expression levels were equivalent for the wild-type human SC5D and all the mutants, supporting the idea that the conserved His residues are essential for catalytic function. A possible role for these His residues could be to act as ligands for the iron atom(s) contained in the enzyme. Hydropathy analyses indicated that the enzyme contains at least four membrane-spanning domains. Topologically, SC5D is predicted to have a C-terminal cytoplasmic domain and an N-terminal region located in the luminal side of the endoplasmic reticulum, since the N-terminal sequence was resistant to proteinase K proteolysis, but the C-terminal sequence was not.

Molecular cloning and structural analysis of human sterol C5 desaturase

Sterol C5 desaturase (SC5D) converts lathosterol to 7-dehydrocholesterol in cholesterol biosynthesis. In this study, we investigated the genome structure of SC5D and transcription of the human SC5D gene to try to elucidate the mechanism by which cholesterol synthesis is regulated. The SC5D gene had a structural gene from a single copy from genome DNA that contained five exons and four introns. The human SC5D was found to be located in chromosome 11q24.2–24.3 by fluorescence in situ hybridization mapping. Human SC5D mRNA transcripts, of which the major transcript was a 2 kb and the minor transcripts were 8 kb and 1.4 kb mRNA transcripts, were detected in almost all of the tissues examined. The human SC5D gene contained a GC box instead of a TATA box upstream of the transcript start sites. Human SC5D transcription started from several transcription start sites, and the first start site was located 31 bp upstream of the translation start site (ATG). The expression level of SC5D mRNA extracted from human liver carcinoma cells decreased as the amount of cholesterol added to the culture medium was increased. Inhibition of SC5D transcription was ascribed to the suppression of promoter activity of SC5D.

Biochemical modifications and transcriptional alterations attendant to sterol feeding in Phytophthora parasitica.

Phytophthora species are eukaryotic sterol auxotrophs that possess the ability to grow, albeit poorly, in the complete absence of sterols. Growth of Phytophthora is often improved substantially when an exogenous source of sterol is provided. Additionally, sterols may be required for sexual and asexual sporulation in Phytophthora. Our research has been focused on identifying and characterizing the immediate physiological effects following sterol addition to cultures of P. parasitica. Through gas chromatographic analysis of extracts from P. parasitica cultures that were fed various sterols, we have obtained evidence for sterol C5 desaturase and delta7 reductase activities in this organism. Zoo blots were probed with DNA sequences encoding these enzymes, from Saccharomyces cerevisiae and Arabidopsis thaliana. Hybridization of a S. cerevisiae ERG3 probe to P. parasitica DNA was observed, implicating sequence similarity between the sterol C5 desaturase encoding genes. Differential display experiments, using RNA from P. parasitica, have demonstrated a pattern of altered gene expression between cultures grown in the presence and absence of sitosterol. Characterization of sterol-related metabolic effects and sterol functions in Phytophthora should lead to improved measures for control of this important group of plant pathogens.

CDNA cloning of the mammalian sterol C5-desaturase and the expression in yeast mutant

Sterol C5-desaturase (SC5D) cDNA is an enzyme that catalyzes the dehydrogenation to introduce C5–6 double bond into lathosterol in cholesterol biosynthesis. We have isolated and sequenced the cDNA clones encoding human and mouse SC5D. The functional complementation of a defective yeast mutant proves that the human and mouse cDNA clones encode SC5D. Mammalian SC5D was presumed as an integral membrane protein containing histidine residues conserved also in yeasts and plant.