Identification of a Sterol Δ7 Reductase Gene Involved in Desmosterol Biosynthesis in Mortierella alpina 1S-4

Molecular cloning of the gene encoding sterol Δ7 reductase from the filamentous fungus Mortierella alpina 1S-4, which accumulates cholesta-5,24-dienol (desmosterol) as the main sterol, revealed that the open reading frame of this gene, designated MoΔ7SR, consists of 1,404 bp and codes for 468 amino acids with a molecular weight of 53,965. The predicted amino acid sequence of MoΔ7SR showed highest homology of 51% with that of sterol Δ7 reductase (EC 1.3.1.21) from Xenopus laevis (African clawed frog). Heterologous expression of the MoΔ7SR gene in yeast Saccharomyces cerevisiae revealed that MoΔ7SR converts ergosta-5,7-dienol to ergosta-5-enol (campesterol) by the activity of Δ7 reductase. In addition, with gene silencing of MoΔ7SR gene by RNA interference, the transformant accumulated cholesta-5,7,24-trienol up to 10% of the total sterols with a decrease in desmosterol. Cholesta-5,7,24-trienol is not detected in the control strain. This indicates that MoΔ7SR is involved in desmosterol biosynthesis in M. alpina 1S-4. This study is the first report on characterization of sterol Δ7 reductase from a microorganism.     

Effect of Light on Sterol Changes in Medicago sativa

Most vascular plants contain Δ⁵-sterols as the predominant type; however, a few species such as Medicago sativa, have mainly Δ⁷-sterols. The Δ⁷-sterols of alfalfa are isomers of the common Δ⁵-sterols and are generally assumed to be their immediate precursors. Light had a significant influence on the sterol status of M. sativa. High light intensity and a long day favored the accumulation of dihydrospinasterol; a short day and low light intensity, particularly darkness, favored spinasterol accumulation. These data for Δ⁷-sterol plants agree with those reported for Δ⁵-sterol plants; light favors the accumulation of the monounsaturated 29 carbon sterols and darkness favors the accumulation of the diunsaturated sterols. Proposed is a mechanism to explain the effect of light on the accumulation of Δ⁷- and Δ⁵-sterols.     

Recent Developments in Nematode Steroid Biochemistry

Current knowledge of steroid nutrition, metabolism, and function in free-living, plant-parasitic and animal-parasitic nematodes is reviewed, with emphasis upon recent investigation of Caenorhabditis elegans. A number of 4-desmethylsterols with a trans-A/B ring configuration can satisfy the steroid nutritional requirement in C. elegans, but sterols with a cis-A/B ring configuration or trans-A/B sterols with a 4-methyl group cannot. C. elegans removes methyl or ethyl substituents at C-24 of the plant sterols sitosterol, campesterol, stigmasterol, stigmastanol, and 24-methylene-cholesterol to produce various sterols with structures partially dependent upon that of the dietary sterol. Additional metabolic steps in C. elegans include reduction of Δ²²- and Δ⁵-bonds, C-7 dehydrogenation, isomerization of a Δ⁷-bond to a Δ⁸⁽¹⁴⁾-bond, and 4α-methylation. An azasteroid and several long-chain alkyl amines interfere with the dealkylation pathway in C. elegans by inhibiting the Δ²⁴-sterol reductase; these compounds also inhibit growth and reproduction in various plant-parasitic and animal-parasitic nematodes. A possible hormonal role for various steroids identified in nematodes is discussed.     

Stereochemical aspects of the biosynthesis of the side chain of 9beta, 19-cyclopropyl sterols in maize seedlings treated with tridemorph

9β, 19-Cyclopropyl sterols such as 24-methyl pollinastanol accumulate dramatically in maize (Zea mays L. var LG 11) seedlings treated with Tridemorph (2,6-dimethyl-N-tridecyl-morpholine), a systemic fungicide (M. Bladocha, P. Benveniste, Plant Physiol 1983 41: 756-762). In contrast to the situation in control plants where 24-ethyl sterols predominate largely, 24-methyl sterols were more than 98% of total cyclopropyl sterols. In addition, 24-methyl cyclopropyl sterols were a mixture of (24-R)- and (24-S)-24-methyl epimers and are similar in that respect to the 24-methyl cholesterol of control plants. The presence of two epimers at C-24 has been previously explained by the operation of two routes (M. Zakelj, L. J. Goad, Phytochemistry 1983 22: 1931-1936). One may proceed via Δ²⁴⁽²⁸⁾- and Δ²⁴⁽²⁵⁾-sterols to produce the (24-R)-24-methyl epimer. The other route may involve reduction of either a Δ²⁴⁽²⁸⁾-, a Δ²³-, or a Δ²⁵-sterol intermediate to give the (24-S)-24-methyl epimer. Such intermediates have been searched for in excised Zea mays axes grown aseptically in the presence of Tridemorph and either [5-¹⁴C]mevalonic acid, or [Me-¹⁴C]-l-methionine. Whereas Δ²⁴⁽²⁸⁾- and Δ²⁴⁽²⁵⁾-cyclopropyl sterols were found in relatively large amounts, only traces of radioactivity were associated with Δ²⁵-sterols. Gas chromatography/mass spectrometry analysis of the sterols from axes grown in the presence of [Me-²H₃]-l-methionine showed that Δ²⁴⁽²⁸⁾-cyclopropyl sterols contained only two ²H atoms at C-28 as expected and that the 24-methyl pollinastanol fraction contained species with two ²H atoms and no species with three ²H atoms. These results indicate that both (24-R)- and (24-S)-epimers originate from a common Δ²⁴⁽²⁸⁾ precursor. After incubation of the axis with [5-¹⁴C,(4-R)-4-³H₁]mevalonic acid, the 24-methyl pollinastanol had a ³H:¹⁴C atomic ratio of 4:6 which is consistent with the intermediacy of a Δ²⁴⁽²⁵⁾-sterol. All these data are in accordance with a pathway where Δ²⁴⁽²⁸⁾-cyclopropyl sterols are isomerized to give Δ²⁴⁽²⁵⁾-cyclopropyl sterols which in turn would be reduced nonregiospecifically to yield both (24-R)- and (24-S)-24-methyl pollinastanols. A plausible mechanism for the reduction step is discussed.     

Effect of Steric and Nuclear Changes in Steroids and Triterpenoids on Sexual Reproduction in Phytophthora cactorum

The comparative biological activity of 21 naturally occurring or synthetically derived steroids, 7 tetracyclic and pentacylic triterpenoids, and antheridiol incubated with cultures of Phytophthora cactorum has been examined. There was greater dependence on precise steric features of the sterol side chain than on the extent of nuclear unsaturation in inducing oospore formation. There was no significant effect on oospore formation by changing nuclear unsaturation in ring B from Δ⁵ to Δ⁷ or to Δ^5,7. Converting the unsaturated sterol to its corresponding stanol resulted in a significant reduction in the number of oospores produced. The effectiveness of sterols bearing different side chains in inducing oospores was found to be in the following relative order: 24α-ethyl = trans-Δ²²-24α-ethyl > trans-Δ²²-24β-ethyl = 24α-E-ethylidene = 24α-methyl > 24β-methyl = trans-Δ²²-24β-methyl = 26-methyl = saturated C₇ side chain and C-20 R (17-αH, 20-αH, right-handed conformer) = cis-Δ²²-C₇ side chain and C-20 R > saturated C₇ side chain and C-20 S (17-αH, 20-βH, right-handed conformer) > no sterol = 29-hydroxyporiferasterol = 20α-hydroxycholesterol = 24ξ-hydroxy-24-vinylcholesterol. Of the sterols examined the most significant stereochemical criterion for the induction of oospore formation was absence of bulk on the front face of C-20. This follows from the observation that 20-isocholesterol and 20α-hydroxycholesterol, in which a methyl and hydroxy group, respectively, project to the front in the right handed conformation, were inactive in stimulating production of oospores. None of the triterpenoids studied induced oospore formation to any significant degree. Oospore formation was not induced by antheridiol nor 29-hydroxyporiferasterol in combination or added separately to growing cultures of P. cactorum in the concentration range 0.01 - 10.0 milligrams per liter.     

Coxiella burnetii Expresses a Functional Δ24 Sterol Reductase

Coxiella burnetii, the etiological agent of human Q fever, occupies a unique niche inside the host cell, where it replicates in a modified acidic phagolysosome or parasitophorous vacuole (PV). The PV membrane is cholesterol-rich, and inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. The precise source(s) of PV membrane cholesterol is unknown, as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. C. burnetii lacks enzymes for de novo cholesterol biosynthesis; however, the organism encodes a eukaryote-like Δ24 sterol reductase homolog, CBU1206. Absent in other prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis. In the present study, we examined the functional activity of CBU1206. Amino acid alignments revealed the greatest sequence identity (51.7%) with a Δ24 sterol reductase from the soil amoeba Naegleria gruberi. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under the control of a galactose-inducible promoter. Erg4 is a yeast Δ24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-green fluorescent protein (GFP), a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum. Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during C. burnetii intracellular growth.The intracellular Gram-negative bacterium Coxiella burnetii is the causative agent of the zoonosis Q fever. Inside the host cell, C. burnetii thrives in a unique parasitophorous vacuole (PV) that is considered “phagolysosome-like” due to its moderate acidity (pH ∼5), the presence of active hydrolytic enzymes, and labeling with lysosomal markers (14, 21). Proteins secreted by a specialized Dot/Icm type IV secretion system (T4SS) are thought to modify the PV to support pathogen replication (21). The C. burnetii PV promiscuously fuses with endosomes and lysosomes; however, it does not appear to intercept Golgi body-derived vesicles or to closely associate with the endoplasmic reticulum (ER) (4, 12).The C. burnetii PV membrane is structurally strong and contains lipid raft proteins such as flotillin, characteristics that likely reflect its high cholesterol content (13). Cholesterol is a critical component of cellular membranes, where it provides structural stability and platforms for signaling proteins. Cholesterol is also a precursor for a variety of signaling molecules (8). Intracellular pathogens exploit cholesterol as sources of energy (6, 19) and membrane lipid (7) and interact with cholesterol to manipulate host cell trafficking (10, 25). Indeed, we have previously shown that pharmacological inhibition of host cell cholesterol biosynthesis or uptake blocks C. burnetii PV formation and growth (13), suggesting a critical role for sterols in C. burnetii pathogenesis.Cholesterol synthesis occurs in the ER through a complex series of enzymatic reactions, with the final and required step being the reduction of the carbon-24 bond by a Δ24 sterol reductase (Fig. ​(Fig.1).1). Mutations in the human Δ24 sterol reductase DHCR24 result in desmosterolosis, where the absence of cholesterol results in severe developmental and neurological problems (24). Interestingly, analysis of the C. burnetii genome revealed two genes encoding putative sterol reductases: CBU1158 and CBU1206 (3, 20). Here, we utilize heterologous expression in Saccharomyces cerevisiae to demonstrate that CBU1206 is an active Δ24 sterol reductase.Open in a separate windowFIG. 1.Schematic showing reduction of carbon-24 double bonds by Δ24 sterol reductases. In mammalian cells, the final step in cholesterol synthesis is reduction of the C24 bond in desmosterol by DHCR24, a Δ24 sterol reductase. Ergosterol is the final sterol in yeast, with the Erg4 Δ24 sterol reductase catalyzing the reduction of ergosta-5,7,22,24(28)-tetraen-3β-ol.     

Functional importance for developmental regulation of sterol biosynthesis in Acanthamoeba castellanii

The sterol metabolome of Acanthamoeba castellanii (Ac) yielded 25 sterols. Substrate screening of cloned AcCYP51 revealed obtusifoliol as the natural substrate which converts to ?^8,14-sterol (<95%). The combination of [²H₃-methyl]methionine incubation to intact cultures showing C₂₈-ergosterol incorporates 2-²H atoms and C₂₉-7-dehydroporiferasterol incorporates 5 ²H-atoms, the natural distribution of sterols, CYP51 and previously published sterol methyltransferase (SMT) data indicate separate ?²⁴⁽²⁸⁾- and ?²⁵⁽²⁷⁾-olefin pathways to C₂₈- and C₂₉-sterol products from the protosterol cycloartenol. In cell-based culture, we observed a marked change in sterol compositions during the growth and encystment phases monitored microscopically and by trypan blue staining; trophozoites possess C₂₈/C₂₉-?^5,7-sterols, viable encysted cells (mature cyst) possess mostly C₂₉-?⁵-sterol and non-viable encysted cells possess C₂₈/C₂₉-?^5,7-sterols that turnover variably from stress to 6-methyl aromatic sterols associated with changed membrane fluidity affording lysis. An incompatible fit of steroidal aromatics in membranes was confirmed using the yeast sterol auxotroph GL7. Only viable cysts, including those treated with inhibitor, can excyst into trophozoites. 25-Azacycloartanol or voriconazole that target SMT and CYP51, respectively, are potent enzyme inhibitors in the nanomolar range against the cloned enzymes and amoeba cells. At minimum amoebicidal concentration of inhibitor amoeboid cells rapidly convert to encysted cells unable to excyst. The correlation between stage-specific sterol compositions and the physiological effects of ergosterol biosynthesis inhibitors suggests that amoeba fitness is controlled mainly by developmentally-regulated changes in the phytosterol B-ring; paired interference in the ?^5,7-sterol biosynthesis (to ?^5,7) - metabolism (to ?⁵ or 6-methyl aromatic) congruence during cell proliferation and encystment could be a source of therapeutic intervention for Acanthamoeba infections.     

Induction and Characterization of Penicillium caseicolum Mutants Resistant to Ergosterol Biosynthesis Inhibitors

The isolation of Penicillium caseicolum mutants resistant to different fungicides which inhibit ergosterol biosynthesis is reported. Mutational frequencies for resistance were high (3 × 10⁻³ to 3 × 10⁻⁵). The levels of resistance toward the inhibitors of sterol C-14 demethylation were always low (<10), whereas high values were obtained with mutants resistant to inhibitors of sterol Δ14 reduction or Δ8→Δ7 isomerization, or both. Generally, there was a positive cross-resistance between fungicides showing the same biochemical mode of action but not between compounds of two different groups. Mycelial growth rate and sporulation were tested; several mutants were not affected for these characteristics. We conclude that resistance to ergosterol biosynthesis inhibitors may be used as a good marker for genetic studies through protoplast fusion.     

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed.     

Plant Oxidosqualene Metabolism: Cycloartenol Synthase–Dependent Sterol Biosynthesis in Nicotiana benthamiana

The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C₃₀H₅₀O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ⁵-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.     

Sterol Biosynthesis Is Required for Heat Resistance but Not Extracellular Survival in Leishmania

Sterol biosynthesis is a crucial pathway in eukaryotes leading to the production of cholesterol in animals and various C24-alkyl sterols (ergostane-based sterols) in fungi, plants, and trypanosomatid protozoa. Sterols are important membrane components and precursors for the synthesis of powerful bioactive molecules, including steroid hormones in mammals. Their functions in pathogenic protozoa are not well characterized, which limits the development of sterol synthesis inhibitors as drugs. Here we investigated the role of sterol C14α-demethylase (C14DM) in Leishmania parasites. C14DM is a cytochrome P450 enzyme and the primary target of azole drugs. In Leishmania, genetic or chemical inactivation of C14DM led to a complete loss of ergostane-based sterols and accumulation of 14-methylated sterols. Despite the drastic change in lipid composition, C14DM-null mutants (c14dm ⁻) were surprisingly viable and replicative in culture. They did exhibit remarkable defects including increased membrane fluidity, failure to maintain detergent resistant membrane fraction, and hypersensitivity to heat stress. These c14dm ⁻ mutants showed severely reduced virulence in mice but were highly resistant to itraconazole and amphotericin B, two drugs targeting sterol synthesis. Our findings suggest that the accumulation of toxic sterol intermediates in c14dm ⁻ causes strong membrane perturbation and significant vulnerability to stress. The new knowledge may help improve the efficacy of current drugs against pathogenic protozoa by exploiting the fitness loss associated with drug resistance.     

Plant sterols: Diversity,biosynthesis, and physiological functions

Sterols, which are isoprenoid derivatives, are structural components of biological membranes. Special attention is now being given not only to their structure and function, but also to their regulatory roles in plants. Plant sterols have diverse composition; they exist as free sterols, sterol esters with higher fatty acids, sterol glycosides, and acylsterol glycosides, which are absent in animal cells. This diversity of types of phytosterols determines a wide spectrum of functions they play in plant life. Sterols are precursors of a group of plant hormones, the brassinosteroids, which regulate plant growth and development. Furthermore, sterols participate in transmembrane signal transduction by forming lipid microdomains. The predominant sterols in plants are β-sitosterol, campesterol, and stigmasterol. These sterols differ in the presence of a methyl or an ethyl group in the side chain at the 24th carbon atom and are named methylsterols or ethylsterols, respectively. The balance between 24-methylsterols and 24-ethylsterols is specific for individual plant species. The present review focuses on the key stages of plant sterol biosynthesis that determine the ratios between the different types of sterols, and the crosstalk between the sterol and sphingolipid pathways. The main enzymes involved in plant sterol biosynthesis are 3-hydroxy-3methylglutaryl-CoA reductase, C24-sterol methyltransferase, and C22-sterol desaturase. These enzymes are responsible for maintaining the optimal balance between sterols. Regulation of the ratios between the different types of sterols and sterols/sphingolipids can be of crucial importance in the responses of plants to stresses.     

Sterol regulatory element-binding protein Sre1 regulates carotenogenesis in the red yeast Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous is a basidiomycete yeast that produces carotenoids, mainly astaxanthin. Astaxanthin is an organic pigment of commercial interest due to its antioxidant and coloring properties. X. dendrorhous has a functional SREBP pathway, and the Sre1 protein is the SREBP homolog in this yeast. However, how sterol regulatory element (Sre)1 promotes the biosynthesis of sterols and carotenoids in X. dendrorhous is unknown. In this work, comparative RNA-sequencing analysis between modified X. dendrorhous strains that have an active Sre1 protein and the WT was performed to identify Sre1-dependent genes. In addition, Sre1 direct target genes were identified through ChIP combined with lambda exonuclease digestion (ChIP-exo) assays. SRE motifs were detected in the promoter regions of several Sre1 direct target genes and were consistent with the SREs described in other yeast species. Sre1 directly regulates genes related to ergosterol biosynthesis as well as genes related to the mevalonate (MVA) pathway, which synthesizes the building blocks of isoprenoids, including carotenoids. Two carotenogenic genes, crtE and crtR, were also identified as Sre1 direct target genes. Thus, carotenogenesis in X. dendrorhous is regulated by Sre1 through the regulation of the MVA pathway and the regulation of the crtE and crtR genes. As the crtR gene encodes a cytochrome P450 reductase, Sre1 regulates pathways that include cytochrome P450 enzymes, such as the biosynthesis of carotenoids and sterols. These results demonstrate that Sre1 is a sterol master regulator that is conserved in X. dendrorhous.     

Sterols and related metabolites from five species of sponges

Free sterol fractions were isolated from the marine sponges Phyllospongia madagascarensis, Scalarispongia sp., Oceanapia sp., Monanchora clathrata and studied by GLC, GLC–MS, and spectroscopy NMR. P. madagascarensis and Scalarispongia sp. contained common Δ⁵-sterols; cholesterol was shown to be a main sterol of both the sponges. Oceanapia sp. contained stanols and minor Δ⁵-sterols with 24R-24,25-methylene-5α-cholestan-3β-ol as a main constituent. Many free sterols from M. clathrata were Δ⁷-series compounds, and latosterol was a main sterol. Δ⁴-3-Ketosteroids and Δ⁵-sterol esters were found in the Antarctic sponge Haliclona sp., but free sterols were practically absent except for trace amount of cholesterol. A chemotaxonomic application of sterols in relation to the genera Phyllospongia, Oceanapia and the family Crambeidae is provided. The known cases of the absence of sterols in sponges and probable reasons of the phenomenon are discussed.     

Effects of Abnormal-Sterol Accumulation on Ustilago maydis Plasma Membrane H+-ATPase Stoichiometry and Polypeptide Pattern

Accumulation of 14α-methylated sterols or Δ⁸-sterols in Ustilago maydis affected three aspects of the plasma membrane H⁺-ATPase. Proton transport was reduced in Δ⁸-sterol-accumulating samples, due to an altered H⁺/ATP stoichiometry. ATP hydrolytic activity was increased, but no direct correlation with the extent or type of abnormal sterol accumulated could be drawn. Finally, Western blot analysis with antibodies against yeast PMA1 revealed a second lighter band (99-kDa band) in all samples from abnormal-sterol-accumulating sporidia. The conclusions are that the 99-kDa band and a reduced stoichiometry are directly linked to the presence of abnormal sterols, while changes in hydrolytic activity are linked only indirectly.     

Cyclopropyl sterol and phospholipid composition of membrane fractions from maize roots treated with fenpropimorph

Maize (Zea mays L.) caryopses were grown in the presence of fenpropimorph, a systemic fungicide, for 7 days in the dark. Membrane fractions enriched, respectively, in endoplasmic reticulum, plasma membrane, and mitochondria were isolated from control and treated maize roots and analyzed for their free sterol, phospholipid, and fatty acid composition. In treated plants, the intracellular distribution of free sterols was dramatically modified both qualitatively and quantitatively. The normally occurring Δ⁵-sterols disappeared almost completely and were replaced by 9β, 19-cyclopropyl sterols, mainly cycloeucalenol and 24-methyl pollinastanol. These new compounds were found to accumulate in all the membrane fractions in such a way that the endoplasmic reticulum-rich fraction became the richest one in free sterols instead of the plasma membrane. In contrast, the fenpropimorph treatment of maize roots was shown not to affect either the relative proportions or the amounts of the individual phospholipids, but an increase in the unsaturation index of phospholipid-fatty acyl chains of the endoplasmic reticulum-rich fraction was observed. The present data suggest that, in higher plant membranes, cyclopropyl sterols could play a structural role similar to that of the bulk of Δ⁵-sterols.     

Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro

Haloperidol, a typical antipsychotic, has been shown to inhibit cholesterol biosynthesis by affecting Δ⁷-reductase, Δ^8,7-isomerase, and Δ¹⁴-reductase activities, which results in the accumulation of different sterol intermediates. In the present work, we investigated the effects of atypical or second-generation antipsychotics (SGA), such as clozapine, risperidone, and ziprasidone, on intracellular lipid metabolism in different cell lines. All the SGAs tested inhibited cholesterol biosynthesis. Ziprasidone and risperidone had the same targets as haloperidol at inhibiting cholesterol biosynthesis, although with different relative activities (ziprasidone > haloperidol > risperidone). In contrast, clozapine mainly affected Δ²⁴-reductase and Δ^8,7-isomerase activities. These amphiphilic drugs also interfered with the LDL-derived cholesterol egress from the endosome/lysosome compartment, thus further reducing the cholesterol content in the endoplasmic reticulum. This triggered a homeostatic response with the stimulation of sterol regulatory element-binding protein (SREBP)-regulated gene expression. Treatment with SGAs also increased the synthesis of complex lipids (phospholipids and triacylglycerides). Once the antipsychotics were removed from the medium, a rebound in the cholesterol biosynthesis rate was detected, and the complex-lipid synthesis further increased. In this condition, apolipoprotein B secretion was also stimulated as demonstrated in HepG2 cells. These effects of SGAs on lipid homeostasis may be relevant in the metabolic side effects of antipsychotics, especially hypertriglyceridemia.     

Triparanol Inhibition of Sterol Biosynthesis in Chlorella ellipsoidea

The sterol composition of C. ellipsoidea was markedly changed when this alga was grown in the presence of 1 μg/g triparanol. Triparanol appears to inhibit the removal of 14α-methyl group, the second alkylation at C-24, Δ⁷-reductase, and Δ⁸ → Δ⁷-isomerase. The effect of triparanol in Chlorella is much more diversified than the specific effect originally assigned to it in animals.     

Amo 1618 effects on incorporation of 14C-MVA and 14C-acetate into sterols in Nicotiana and Digitalis seedlings and cell-free preparations from Nicotiana

Incorporation of radioactivity from acetate-[¹⁴C] and MVA-[¹⁴C] into sterols and sterol precursors in tobacco was inhibited by Amo 1618; differing patterns of accumulation were obtained with the two precursors, suggesting more than one point of inhibition. This was borne out with cell-free preparations with which it was demonstrated that both HMG-CoA reductase and squalene-2,3-epoxide cyclase were inhibited, the latter more strongly than the former. GLC analysis of gross sterol and hydrocarbon fractions confirmed previous indications that incorporation of radioactivity into individual sterols was inhibited by Amo 1618. Finally, incorporation of MVA-[¹⁴C] into sterols and sterol precursors of Digitalis was significantly altered by the retardant, thus expanding the generality of the relationship between sterol (particularly 4-desmethylsterol) biosynthesis inhibition and retardant effect.     

The Role of Glucosidase I (Cwh41p) in the Biosynthesis of Cell Wall β-1,6-Glucan Is Indirect

CWH41, a gene involved in the assembly of cell wall β-1,6-glucan, has recently been shown to be the structural gene for Saccharomyces cerevisiae glucosidase I that is responsible for initiating the trimming of terminal α-1,2-glucose residue in the N-glycan processing pathway. To distinguish between a direct or indirect role of Cwh41p in the biosynthesis of β-1,6-glucan, we constructed a double mutant, alg5Δ (lacking dolichol-P-glucose synthase) cwh41Δ, and found that it has the same phenotype as the alg5Δ single mutant. It contains wild-type levels of cell wall β-1,6-glucan, shows moderate underglycosylation of N-linked glycoproteins, and grows at concentrations of Calcofluor White (which interferes with cell wall assembly) that are lethal to cwh41Δ single mutant. The strong genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in the β-1,6-glucan biosynthetic pathway, disappear in the absence of dolichol-P-glucose synthase (alg5Δ). The triple mutant alg5Δcwh41Δkre6Δ is viable, whereas the double mutant cwh41Δkre6Δ in the same genetic background is not. The severe slow growth phenotype and 75% reduction in cell wall β-1,6-glucan, characteristic of the cwh41Δkre1Δ double mutant, are not observed in the triple mutant alg5Δcwh41Δkre1Δ. Kre6p, a putative Golgi glucan synthase, is unstable in cwh41Δ strains, and its overexpression renders these cells Calcofluor White resistant. These results demonstrate that the role of glucosidase I (Cwh41p) in the biosynthesis of cell wall β-1,6-glucan is indirect and that dolichol-P-glucose is not an intermediate in this pathway.