Sterol Methyltransferase Research Articles

Molecular characterization and functional analysis of Glycine max sterol methyl transferase 2 genes involved in plant membrane sterol biosynthesis

Sterol C24 methyltransferase (SMT2) genes governing the pattern of phytosterols synthesized in higher plants have been studied in Glycine seedlings and wild-type and engineered Arabidopsis thaliana plants. The SMT2 genes of soybean (SMT2-1 and SMT2-2) previously cloned and characterized (Neelakandan et al. 2009) were shown to complement the SMT deficient cvp1 mutant Arabidopsis plants, consistent with their role in regulation of 24-alkyl sterol-controlled plant physiology. Further analysis of these genes showed that environmental cues, including dehydration, cold, and abscisic acid induced differential changes in transcript levels of the SMT2 during soybean seedling growth. Sterol analyses of transgenic Arabidopsis seeds originating in variant constructs of AtHMGR1, GmSMT1, and GmSMT2 engineered in seeds showed relevant modifications in the ratio of 24-methyl to 24-ethyl sterol in the direction of sitosterol formation. To provide insight into the structural features of the sterol gene that affects transcript regulation, the upstream promoter sequences of soybean SMT2 genes were cloned and characterized. Sequence analysis revealed several important cis-elements and transcription factor binding sites. The analysis of promoter-GUS fusions in transgenic Arabidopsis plants revealed shared and distinct expression features in different developmental stages and tissues. The data are interpreted to imply that SMT2 is an important contributor to normal plant growth and development.

The Sterol Methyltransferases SMT1, SMT2, and SMT3 Influence Arabidopsis Development through Nonbrassinosteroid Products

Plant sterols are structural components of cell membranes that provide rigidity, permeability, and regional identity to membranes. Sterols are also the precursors to the brassinosteroid signaling molecules. Evidence is accumulating that specific sterols have roles in pattern formation during development. COTYLEDON VASCULAR PATTERNING1 (CVP1) encodes C-24 STEROL METHYLTRANSFERASE2 (SMT2), one of three SMTs in Arabidopsis (Arabidopsis thaliana). SMT2 and SMT3, which also encodes a C-24 SMT, catalyze the reaction that distinguishes the synthesis of structural sterols from signaling brassinosteroid derivatives and are highly regulated. The deficiency of SMT2 in the cvp1 mutant results in moderate developmental defects, including aberrant cotyledon vein patterning, serrated floral organs, and reduced stature, but plants are viable, suggesting that SMT3 activity can substitute for the loss of SMT2. To test the distinct developmental roles of SMT2 and SMT3, we identified a transcript null smt3 mutant. Although smt3 single mutants appear wild type, cvp1 smt3 double mutants show enhanced defects relative to cvp1 mutants, such as discontinuous cotyledon vein pattern, and produce novel phenotypes, including defective root growth, loss of apical dominance, sterility, and homeotic floral transformations. These phenotypes are correlated with major alterations in the profiles of specific sterols but without significant alterations to brassinosteroid profiles. The alterations to sterol profiles in cvp1 mutants affect auxin response, demonstrated by weak auxin insensitivity, enhanced axr1 auxin resistance, ectopically expressed DR5:beta-glucuronidase in developing embryos, and defective response to auxin-inhibited PIN2-green fluorescent protein endocytosis. We discuss the developmental roles of sterols implied by these results.

Side chain azasteroids and thiasteroids as sterol methyltransferase inhibitors in ergosterol biosynthesis

The synthesis of some novel azasteroids and thiasteroids based on a pregnan nucleus with a Δ7 double bond in two to five steps from the key aldehyde (3 S,20 S)-20-formylpregn-7-en-3-yl acetate has been disclosed herein. These compounds were evaluated as potential inhibitors of the enzyme Δ24-sterol methyltransferase (24-SMT), which is a key enzyme in the biosynthesis of ergosterol, and for their effects on the growth of the yeast Yarrowia lipolytica. Most of the side chain modified analogues were recognized as 24-SMT inhibitors, and in particular the 23-azasteroids 5f– 5i and the 24-azasteroid 11 showed potent antifungal activity. The target enzyme could be identified unambiguously using an improved whole-cell assay combined with GC–MS analysis of the sterol pattern resulting upon incubation with the inhibitors.

Growth inhibition and ultrastructural alterations induced by Δ24(25)-sterol methyltransferase inhibitors in Candida spp. isolates, including non-albicans organisms

BackgroundAlthough Candida species are commensal microorganisms, they can cause many invasive fungal infections. In addition, antifungal resistance can contribute to failure of treatment.The purpose of this study was to evaluate the antifungal activity of inhibitors of Δ24(25)-sterol methyltransferase (24-SMTI), 20-piperidin-2-yl-5α-pregnan-3β-20(R)-diol (AZA), and 24(R,S),25-epiminolanosterol (EIL), against clinical isolates of Candida spp., analysing the ultrastructural changes.ResultsAZA and EIL were found to be potent growth inhibitors of Candida spp. isolates. The median MIC50 was 0.5 μg.ml-1 for AZA and 2 μg.ml-1 for EIL, and the MIC90 was 2 μg.ml-1 for both compounds. All strains used in this study were susceptible to amphotericin B; however, some isolates were fluconazole- and itraconazole-resistant. Most of the azole-resistant isolates were Candida non-albicans (CNA) species, but several of them, such as C. guilliermondii, C. zeylanoides, and C. lipolytica, were susceptible to 24-SMTI, indicating a lack of cross-resistance. Reference strain C. krusei (ATCC 6258, FLC-resistant) was consistently susceptible to AZA, although not to EIL. The fungicidal activity of 24-SMTI was particularly high against CNA isolates. Treatment with sub-inhibitory concentrations of AZA and EIL induced several ultrastructural alterations, including changes in the cell-wall shape and thickness, a pronounced disconnection between the cell wall and cytoplasm with an electron-lucent zone between them, mitochondrial swelling, and the presence of electron-dense vacuoles. Fluorescence microscopy analyses indicated an accumulation of lipid bodies and alterations in the cell cycle of the yeasts. The selectivity of 24-SMTI for fungal cells versus mammalian cells was assessed by the sulforhodamine B viability assay.ConclusionTaken together, these results suggest that inhibition of 24-SMT may be a novel approach to control Candida spp. infections, including those caused by azole-resistant strains.

Sterol Biosynthesis Pathway as Target for Anti-trypanosomatid Drugs

Sterols are constituents of the cellular membranes that are essential for their normal structure and function. In mammalian cells, cholesterol is the main sterol found in the various membranes. However, other sterols predominate in eukaryotic microorganisms such as fungi and protozoa. It is now well established that an important metabolic pathway in fungi and in members of the Trypanosomatidae family is one that produces a special class of sterols, including ergosterol, and other 24-methyl sterols, which are required for parasitic growth and viability, but are absent from mammalian host cells. Currently, there are several drugs that interfere with sterol biosynthesis (SB) that are in use to treat diseases such as high cholesterol in humans and fungal infections. In this review, we analyze the effects of drugs such as (a) statins, which act on the mevalonate pathway by inhibiting HMG-CoA reductase, (b) bisphosphonates, which interfere with the isoprenoid pathway in the step catalyzed by farnesyl diphosphate synthase, (c) zaragozic acids and quinuclidines, inhibitors of squalene synthase (SQS), which catalyzes the first committed step in sterol biosynthesis, (d) allylamines, inhibitors of squalene epoxidase, (e) azoles, which inhibit C14α-demethylase, and (f) azasterols, which inhibit Δ24(25)-sterol methyltransferase (SMT). Inhibition of this last step appears to have high selectivity for fungi and trypanosomatids, since this enzyme is not found in mammalian cells. We review here the IC50 values of these various inhibitors, their effects on the growth of trypanosomatids (both in axenic cultures and in cell cultures), and their effects on protozoan structural organization (as evaluted by light and electron microscopy) and lipid composition. The results show that the mitochondrial membrane as well as the membrane lining the protozoan cell body and flagellum are the main targets. Probably as a consequence of these primary effects, other important changes take place in the organization of the kinetoplast DNA network and on the protozoan cell cycle. In addition, apoptosis-like and autophagic processes induced by several of the inhibitors tested led to parasite death.

Cyclobranol: A substrate for C25-methyl sterol side chains and potent mechanism-based inactivator of plant sterol methyltransferase

Cyclobranol 8A, an analog of the cycloartenol substrate 1A for the plant sterol C24-methyltransferase (SMT), was shown to be an acceptor of the soybean SMT1 as well as an inhibitor of enzyme action. The K m and k cat for 8A was 37 μM and 0.006 min −1, respectively. The enzyme-generated product was identified by MS and 1H NMR to be a C24, C25-doubly alkylated Δ 24(28)-olefin 10A. Inhibitor treatment was concentration and time-dependent affording an apparent K i of 25 μM, a maximum rate of inactivation of 0.15 min −1 and a partition ratio ( k cat/ k inact) calculated to be 0.04.

Paracoccidioides brasiliensis: chemical and molecular tools for research on cell walls, antifungals, diagnosis, taxonomy

Paracoccidioides brasiliensis is a dimorphic fungus, a causative agent of paracoccidioidomycosis, one of the most frequent systemic mycoses that affect the rural population in Latin America, only geographical region in which this fungus is to be found. In this work, we discuss matters related to (a) cell wall studies based on the cloning and analysis of genes involved in the synthesis of cell wall components, and their possible roles in virulence and dimorphism in P. brasiliensis, (b) molecular taxonomy and the molecular classification of P. brasiliensis as an Ascomycete belonging in the Order Onygenales, (c) phylogeny of P. brasiliensis and the possible existence of cryptic species within the genus Paracoccidioides, and (d) new experimental antifungal drugs such as azasterols or sterol hydrazones, compounds that affect the activity of delta 24(28) sterol methyl reductase (SMR) and/or delta (24)-sterol methyl transferase (SMT), and (e) specific primers for the molecular detection of P. brasiliensis in vitro and in clinical samples.

Synthesis and characterization of platinum–sterol hydrazone complexes with biological activity against Leishmania (L.) mexicana

Leishmaniasis is a parasitic zoonosis caused by protozoans of the genus Leishmania transmitted by insects known as phlebotomines, which are found in wild or urban environments. The disease occurs in tropical and sub-tropical areas, mainly in Asia, Europe, Africa and the Americas. At present, there is no effective treatment for this disease. In the search for new rational chemotherapeutic alternatives, two novel trans [Pt(Hpy 1) 2(Cl) 2] ( 1) and trans [Pt(Hpy 2) 2 (Cl) 2] ( 2) complexes were synthesized by the reaction of K 2PtCl 4 with sterol hydrazone ligands 20-hydrazone-pyridin-2-yl-5α-pregnan-3β-ol (Hpy 1) and 22-hydrazone-pyridin-2-yl-chol-5-ene-3β-ol (Hpy 2). These organic compounds are specific inhibitors of sterol methyl transferase (SMT). The new platinum complexes were characterized by a combination of ESI–MS (electrospray ionization–mass spectroscopy), UV–vis, infrared and NMR spectroscopies; elemental analysis and molar conductivity. Promastigotes of Leishmania (L.) mexicana were treated for 48 h with 10 μM of the sterol hydrazones Hpy 1 or Hpy 2 alone or coordinated to Pt. Hpy 1 produced higher leishmanistatic activity than Hpy 2 (39% growth inhibition vs. 16%), which significatively increased (71%, p < 0.001) when the complex trans-[Pt(Hpy 1) 2(Cl) 2] was used. This complex represents a new chemotherapeutic alternative to be evaluated in depth in experimental models of leishmaniasis.

Cholesterol import fails to prevent catalyst-based inhibition of ergosterol synthesis and cell proliferation of Trypanosoma brucei

Trypanosoma brucei (TB) cultured in rat blood, bovine serum, or lipid-depleted serum generated distinct differences in cholesterol availability. Whereas cell proliferation of the parasite was relatively unaffected by cholesterol availability, the ratios of cellular ergostenols to cholesterol varied from close to unity to 3 orders of magnitude different with cholesterol as the major sterol (>99%) of bloodstream form cells. In the procyclic form cultured with lipid-depleted serum, 15 sterols at 52 fg/cell were identified by GC-MS. The structures of these sterols reveal a nonconventional ergosterol pathway consistent with the novel product diversity catalyzed by the recently cloned sterol methyltransferase (SMT). A potent transition state analog of the TB SMT C24 alkylation reaction, 25-azalanosterol (25-AL; inhibition constant Ki = 39 nM), was found to inhibit the growth of the procyclic and bloodstream forms at an IC(50) of approximately 1 microM. This previously unrecognized catalyst-specific inhibition of cell growth was unmasked further using the 25-AL-treated procyclic form, which, compared with control cultures, caused a change in cellular sterol content from ergostenols to cholesterol. However, growth of the bloodstream form disrupted by 25-AL was not rescued by cholesterol absorption from the host, suggesting an essential role for ergosterol (24-methyl sterol) in cell proliferation and that the SMT can be a new enzyme target for drug design.

Sterol Biosynthesis Inhibitors: Potential for Transition State Analogs and Mechanism‐Based Inactivators Targeted at Sterol Methyltransferase

Sterol biosynthesis inhibitors (SBIs), discovered in the late 1960s and subsequently used commercially to treat ergosterol-dependent fungal diseases, represent a unique drug class targeted at an enzyme in a biosynthetic pathway. To date, few drugs have been commercialized as enzyme inhibitors; yet, prescription of SBIs has emerged as the gold standard for some cases of non-life-threatening antifungal chemotherapy and in crop protection. SBIs are not designed for their structural resemblance to the sterol molecule; they nonetheless can engender a curative effect by interfering with sterol production and homeostasis in the pathogenic organism. The increased use of SBIs in recent years, particularly the azole antifungals, has resulted in the development of resistance to those drugs, necessitating additional work to further our understanding of antifungal resistance and to explore opportunities to develop new enzyme inhibitors and uncover new enzyme targets that can regulate carbon flux in the post-lanosterol/cycloartenol pathway. This article reports general considerations for enzyme mechanism and active-site probes using inhibitors of the C-methylation reaction, including a potential new class of antifungal/antiparasitic agents of phytosterol synthesis tailored as mechanism-based inactivators. These steroid-based compounds prepared with different sterol side chain functionalities are designed to reversibly or irreversibly impair the sterol methyltransferase, an enzyme expressed in pathogenic microbes and plants but not in the human host. The salient aspects of these and related topics directed toward the enzyme recognition of sterol structure, and the inhibitory properties and catalytic competence of a series of specifically modified substrate analogs that affect sterol methyltransferase action are discussed.

Effects of diet and metamorphosis upon the sterol composition of the butterfly Morpho peleides

Whole body sterol metabolism in insects has seldom been studied. We were able to design an appropriate study at a butterfly farm in Belize. We collected six larvas of butterfly (Morpho peleides), their food (leaves of Pterocarpus bayessii), and their excretions. In addition, six adult butterflies were collected. The sterols of the diet, the larva, and adult butterfly were analyzed by gas-liquid chromatography. The structures of these sterols were identified by digitonin precipitation, GC-MS, and NMR. Four sterols (cholesterol, campesterol, stigmasterol, and sitosterol) and a sterol mixture were found in the food, the body, and the excreta of the larva. The tissue sterol content of the larva was 326 microg. They consumed 276 microg of sterols per day. Their excretion was 185 microg per day as sterols. The total tissue sterol contents of the larva and butterfly were similar, but they had different sterol compositions, which indicated interconversion of sterols during development. There was a progressive increase in the cholesterol content from larva to butterfly and a decrease in the content of sitosterol and other plant sterols, which were likely converted to cholesterol. Our data indicated an active sterol metabolism in butterfly larva. Diet played an important role in determining its sterol composition. During metamorphosis, there was an interconversion of sterols. This is the first paper documenting the fecal sterol excretion in insects as related to dietary intakes.

Mechanistic Analysis of a Multiple Product Sterol Methyltransferase Implicated in Ergosterol Biosynthesis in Trypanosoma brucei

Sterol methyltransferase (SMT) plays a key role in sterol biosynthesis in different pathogenic organisms by setting the pattern of the side chain structure of the final product. This catalyst, absent in humans, provides critical pathway-specific enzymatic steps in the production of ergosterol in fungi or phytosterols in plants. The new SMT gene was isolated from Trypanosoma brucei genomic DNA and cloned into an Escherichia coli expression system. The recombinant SMT was purified to homogeneity to give a band at 40.0 kDa upon SDS-PAGE and showed a tetrameric subunit organization by gel chromatography. It has a pH optimum of 7.5, an apparent kcat value of 0.01 s(-1), and a Km of 47 +/- 4 microm for zymosterol. The products of the reaction were a mixture of C24-monoalkylated sterols, ergosta-8,24 (25)-dienol, ergosta-8,25 (27)-dienol, and ergosta-8,24 (28)-dienol (fecosterol), and an unusual double C24-alkylated sterol, 24,24-dimethyl ergosta-8,25 (27)-dienol, typically found in plants. Inhibitory profile studies with 25-azalanosterol (Ki value of 39 nm) or 24(R,S), 25-epiminolanosterol (Ki value of 49 nm), ergosterol (Ki value of 27 microm) and 26,27-dehydrozymosterol (Ki and kinact values of 29 microm and 0.26 min(-1), respectively) and data showing zymosterol as the preferred acceptor strongly suggest that the protozoan SMT has an active site topography combining properties of the SMT1 from plants and yeast (37-47% identity). The enzymatic activation of this and other SMTs reveals that the catalytic requirements for the C-methyl reaction are remarkably versatile, whereas the inhibition studies provide a powerful approach to rational design of new anti-sleeping sickness chemotherapeutic drugs.

Effects of Inhibitors of Δ24(25)-Sterol Methyl Transferase on the Ultrastructure of Epimastigotes ofTrypanosoma cruzi

Trypanosoma cruzi is the ethiological agent of Chagas disease. New compounds are being developed based on the biosynthesis and function of sterols, because T. cruzi has a requirement for specific endogenous sterols for growth and survival. Sterol biosynthesis inhibitors (SBIs) are drugs commonly used against fungal diseases. These drugs act by depleting essential and specific membrane components and/or inducing the accumulation of toxic intermediary or lateral products of the biosynthetic pathway. In this work we present the effects of WSP488, WSP501, and WSP561, specific inhibitors of Delta24(25)-sterol methyl transferase, on the ultrastructure of T. cruzi epimastigotes. All three drugs inhibited parasite multiplication at low concentrations, with IC50 values of 0.48, 0.44, and 0.48 muM, respectively, and induced marked morphological changes including (a) blockage of cell division; (b) swelling of the mitochondrion, with several projections and depressions; (c) swelling of the perinuclear space; (d) presence of autophagosomes and myelin-like figures; (e) enlargement of the flagellar pocket and of a cytoplasmic vacuole located in close association with the flagellar pocket; (f) detachment of the membrane of the cell body; and (g) formation of a vesicle at the surface of the parasite between the flagellar pocket and the cytostome. Our results show that these drugs are potent in vitro inhibitors of growth of T. cruzi.

Enzyme redesign and interactions of substrate analogues with sterol methyltransferase to understand phytosterol diversity, reaction mechanism and the nature of the active site

Several STM (sterol methyltransferase) genes have been cloned, sequenced and expressed in bacteria recently, making it possible to address questions of the relationship between sterol structure and function. The active site and mechanism of action of a set of phylogenetically diverse SMTs have been probed by site-directed mutagenesis as well as by using substrate and related analogues of the SMT-catalysed reaction. An active-site model has been developed that is in accord with the results presented, which is consistent with the hypothesis that SMTs are bifunctional enzymes kinetically responsible to bind Δ24-acceptor sterols of specific steric and electronic character and rigid orientation imposed by multiple hydrophobic active site contacts exacted from a common waxy core. Functional divergence influenced by the architectural role of sterols in membranes is considered to govern the evolution of product distribution and specificity of individual SMTs as discussed.

Probing the sterol binding site of soybean sterol methyltransferase by site-directed mutagenesis: Functional analysis of conserved aromatic amino acids in Region 1

Soybean sterol methyltransferase (SMT) in the presence of AdoMet catalyzes the transmethylation of the Δ 24-bond of the sterol side chain to produce phytosterols with a methyl(lene) or ethyl(idene) group at C-24. The function of six aromatic amino acids associated with the putative active center of the SMT, i.e., Region 1 that extends from Phe82 to Phe93 in soybean SMT, was studied by site-directed mutagenesis and heterologous expression in BL21(DE3) bacterial cells. The enzyme-generated products were characterized kinetically and by GC–MS analysis. Substitution of the aromatic amino acids at positions 82, 83, 85, 87, 91, and 93 with a leucine residue produced mutant SMTs with varying activities. The mutants converted cycloartenol to 24(28)-methylene cycloartanol [C 1-activity] from a few percent to as much as 95% of the control activity. In contrast, none of the leucine mutants were found to catalyze 24(28)-methylene lophenol [C 2-activity], suggesting a loss of function associated with the second C 1-transfer activity. In contrast to the loss of the second C 1-transfer activity of the Phe82Leu, replacement of the Phe82 residue to isoleucine had minimal effect on the first or second C 1-transfer activities, suggesting that the increased bulk (branching) in the leucine side chain contributes to significant perturbations in the active site that generate inaccurate positioning of the substrate side chain disfavoring the second C 1-transfer activity. Replacement of Tyr83 to phenylalanine resulted in an increase of the specificity constant ( k cat/ K m) for the substrate of the second C 1-transfer activity by a factor of 5 compared to control and an increase of Δ 24(28)Z-ethylidene sterol formation in the 24-ethyl sterol product set, suggesting that loss of steric bulk from the phenolic hydroxyl group on tyrosine generates a less precise fit of the Δ 24(28) sterol side chain into the active site favoring the second C 1-transfer activity and prompting reaction channeling during catalysis. Circular dichroism spectra, equilibrium dialysis studies of AdoMet, and chromatographic information of the wild-type and Tyr83 mutants confirmed retention of the overall conformation of the enzyme during the experiments. Together, these findings suggest that the amino acids of Region 1 provide a tight substrate orientation imposed by hydrophobic interactions between the sterol side chain and the SMT active site contacts and control the production and processing of the transmethylation pathways governed by the first and second C 1-transfer activities.

Enzyme redesign and interactions of substrate analogues with sterol methyltransferase to understand phytosterol diversity, reaction mechanism and the nature of the active site

Several STM (sterol methyltransferase) genes have been cloned, sequenced and expressed in bacteria recently, making it possible to address questions of the relationship between sterol structure and function. The active site and mechanism of action of a set of phylogenetically diverse SMTs have been probed by site-directed mutagenesis as well as by using substrate and related analogues of the SMT-catalysed reaction. An active-site model has been developed that is in accord with the results presented, which is consistent with the hypothesis that SMTs are bifunctional enzymes kinetically responsible to bind Delta24-acceptor sterols of specific steric and electronic character and rigid orientation imposed by multiple hydrophobic active site contacts exacted from a common waxy core. Functional divergence influenced by the architectural role of sterols in membranes is considered to govern the evolution of product distribution and specificity of individual SMTs as discussed.

Paracoccidioides brasiliensis, Paracoccidioidomycosis, and Antifungal Antibiotics

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), a human systemic, chronic and progressive mycosis. Preferred antifungals are sulfamethoxazol-trimethoprim, itraconazole, amphotericin B. Treatment is lengthy, the drugs may have undesirable side effects, and some are costly. Occasional resistant strains have been reported. Therefore, the search for more selective and efficient antifungals to treat this and other mycoses continues. Ajoene, chemically derived from garlic, behaves as an antifungal agent against P. brasiliensis and other fungi. Its antiproliferative effects in P. brasiliensis are associated with a reduction of phosphatidyl choline, a concomitant increase in its precursor phosphatidyl ethanolamine, and a large increase in unsaturated fatty acids in the pathogenic yeast phase. The sterol biosynthetic pathway has been largely studied for the search of antifungals. Azoles and allilamines act on differents steps of this pathway. However, they may interfere with similar steps in the host. Hence, the search for drugs that may act on more specific steps is ongoing. One such step focuses on the sterol C-methylations catalyzed by the enzyme (S)-adenosyl-L-methionine: Delta(24) - sterol methyl transferase (SMT). SMT inhibitors such as azasterols and derivatives (AZA1, AZA2, AZA3) have proven highly effective as antiproliferative agents against protozoa and some fungi, among them, P. brasiliensis. Their chemical synthesis and structure, and their molecular electrostatic potential are discussed in order to understand their mechanism of action, and derive rationally designed improvements on these molecules, that would favour a higher efficacy and selectivity.

Ectopic endoreduplication caused by sterol alteration results in serrated petals in Arabidopsis

The Arabidopsis frill1 (frl1) mutant, that has serrated petals and sepals but no other large changes in plant morphology, was studied. The frl1 had a mutation in STEROL METHYLTRANSFERASE 2 and an altered sterol composition. It was found that the frl1 mutation causes ectopic endoreduplication in petal tips that do not normally endoreduplicate. The rosette leaves of frl1 also showed an enhanced level of endoreduplication, but their morphology was hardly affected. These facts suggest that the suppression of endoreduplication is important for petal morphogenesis and the normal sterol composition is required for this suppression.

Antiproliferative synergism of azasterols and antifolates against Toxoplasma gondii

The antiproliferative effects of two azasterols, 22,26-azasterol (20-piperidin-2-yl-5α-pregnan-3β–20(R,S)-diol, AZA) and 24,25(R,S)-epiminolanosterol (EIL), in combination with sulphadiazine (SDZ) and pyrimethamine (PYR) were studied against Toxoplasma gondii tachyzoites growing in cultured mammalian cells. We had previously shown that AZA and EIL, two known inhibitors of Δ 24(25)sterol methyl transferase in fungi and protozoa, have a potent and selective anti- T. gondii activity, although no 24-alkyl sterols have been detected in this parasite. We now report that when these sterol analogues were used in combination with the conventional SDZ/PYR treatment, potent synergistic effects were observed, ranging from 10- to 100-fold reductions of the IC 50 values in the presence of sub-optimal doses of azasterols. When exposed to these drug combinations, intracellular T. gondii parasites displayed diverse subcellular alterations, including mitochondrial swelling, the arrest of the endodiogeny process with fragmented nuclei and subsequent cell lysis. These results suggest a potential new approach for the treatment of toxoplasmosis, which could significantly lower the required levels of antifolates and thus their adverse side effects.

The ERG28-encoded protein, Erg28p, interacts with both the sterol C-4 demethylation enzyme complex as well as the late biosynthetic protein, the C-24 sterol methyltransferase (Erg6p)

In Saccharomyces cerevisiae, the C-24 sterol methyltransferase (Erg6p) converts zymosterol to fecosterol, an enzymatic step following C-4 demethylation of 4,4-dimethylzymosterol. Our previous study showed that an endoplasmic reticulum (ER) transmembrane protein, Erg28p, functions as a scaffold to tether the C-4 demethylation enzymatic complex (Erg25p–Erg26p–Erg27p) to the ER. To determine whether Erg28p also interacts with other ergosterol biosynthetic proteins, we compared protein levels of Erg3p, Erg6p, Erg7p, Erg11p and Erg25p in three pairs of erg28 and ERG28 strains. In erg28 strains, the Erg6p level in the ER fraction was decreased by about 50% relative to the wild-type strain, while ER protein levels of the four other ergosterol proteins showed no significant differences. Co-immunoprecipitation experiments, using an erg28 strain transformed with the epitope-tagged plasmid pERG28-HA and proteins detected with anti-HA and anti-Erg6p antibodies, indicated that Erg6p and Erg28p reciprocally co-immunoprecipitate. Further, the split ubiquitin yeast membrane two-hybrid system designed to detect protein interactions between membrane bound proteins also indicated an Erg28p–Erg6p interaction when pERG6-Cub was used as the bait and pERG28-NubG was used as the prey. We conclude that Erg28p may not only anchor the C-4 demethylation enzyme complex to the ER but also acts as a protein bridge to the Erg6p enzyme required for the next ergosterol biosynthetic step.