Cellular sterol ester synthesis in plants is performed by an enzyme (phospholipid:sterol acyltransferase) different from the yeast and mammalian acyl-CoA:sterol acyltransferases

A gene encoding a sterol ester-synthesizing enzyme was identified in Arabidopsis. The cDNA of the Arabidopsis gene At1g04010 (AtPSAT) was overexpressed in Arabidopsis behind the cauliflower mosaic virus 35S promoter. Microsomal membranes from the leaves of overexpresser lines catalyzed the transacylation of acyl groups from phosphatidylethanolamine to sterols. This activity correlated with the expression level of the AtPSAT gene, thus demonstrating that this gene encodes a phospholipid:sterol acyltransferase (PSAT). Properties of the AtPSAT were examined in microsomal fractions from the tissues of an overexpresser. The enzyme did not utilize neutral lipids, had the highest activity with phosphatidylethanolamine, had a 5-fold preference for the sn-2 position, and utilized both saturated and unsaturated fatty acids. Various sterols and sterol intermediates, including triterpenic precursors, were acylated by the PSAT, whereas other triterpenes were not. Sterol selectivity studies showed that the enzyme is activated by end product sterols and that sterol intermediates are preferentially acylated by the activated enzyme. This indicates that PSAT both regulates the pool of free sterols as well as limits the amount of free sterol intermediates in the membranes. Two T-DNA insertion mutants in the AtPSAT gene, with strongly reduced (but still measurable) levels of sterol esters in their tissues, had no detectable PSAT activity in the microsomal fractions, suggesting that Arabidopsis possess other enzyme(s) capable of acylating sterols. The AtPSAT is the only intracellular enzyme found so far that catalyzes an acyl-CoA-independent sterol ester formation. Thus, PSAT has a similar physiological function in plant cells as the unrelated acyl-CoA:sterol acyltransferase has in animal cells.     

Modulation of membrane function by cholesterol.

The molecular basis for the essential role of cholesterol in mammalian (and other cholesterol-requiring) cells has long been the object of intense interest. Cholesterol has been found to modulate the function of membrane proteins critical to cellular function. Current literature supports two mechanisms for this modulation. In one mechanism, the requirement of 'free volume' by integral membrane proteins for conformational changes as part of their functional cycle is antagonized by the presence of high levels of cholesterol in the membrane. In the other mechanism, the sterol modulates membrane protein function through direct sterol-protein interactions. This mechanism provides an explanation for the stimulation of the activity of important membrane proteins and for the essential requirement of a structurally-specific sterol for cell viability. In some cases, these latter membrane proteins exhibit little or no activity in the absence of the specific sterol required for growth of that cell type. The specific sterol required varies from one cell type to another and is unrelated to the ability of that sterol to affect the bulk properties of the membrane.     

Predicting the effect of steroids on membrane biophysical properties based on the molecular structure

The relationship between sterol structure and the resulting effects on membrane physical properties is still unclear, owing to the conflicting results found in the current literature. This study presents a multivariate analysis describing the physical properties of 83 steroid membranes. This first structure-activity analysis supports the generally accepted physical effects of sterols in lipid bilayers. The sterol chemical substituents and the sterol/phospholipid membrane physical properties were encoded by defining binary variables for the presence/absence of those chemical substituents in the polycyclic ring system and physical parameters obtained from phospholipid mixtures containing those sterols. Utilizing Principal Coordinates Analysis, the steroid population was grouped into five well-defined clusters according to their chemical structures. An examination of the membrane activity of each sterol structural cluster revealed that a hydroxyl group at C3 and an 8-10 carbon isoalkyl side-chain at C17 are mainly present in membrane active sterols having rigidifying, molecular ordering/condensing effects and/or a raft promoting ability. In contrast, sterol chemical structures containing a keto group at C3, a C4-C5-double bond, and polar groups or a short alkyl side-chain at C17 (3 to 7 atoms) are mostly found in sterols having opposite effects. Using combined multivariate approaches, it was concluded that the most important structural determinants influencing the physical properties of sterol-containing mixtures were the presence of an 8-10 carbon C17 isoalkyl side-chain, followed by a hydroxyl group at C3 and a C5-C6 double bond. Finally, a simple Logistic Regression model predicting the dependence of membrane activity on sterol chemical structure is proposed.     

Effects of ethephon and norbornadiene on sterol and glycoalkaloid biosynthesis in potato tuber discs

The accumulation of glycoalkaloids that normally takes place in aerobically incubated potato ( Solanum tuberosum L.) tuber discs has been found to be inhibited by the ethylene-releasing substance ethephon. Using ethephon and the ethylene action inhibitor norborna-2,5-diene, the effect of ethylene on the synthesis of sterols and glycoalkaloids, which partly share their biosynthetic pathway, was investigated.
Control discs showed incorporation of (2-¹⁴C)mevalonic acid into free sterols, steryl esters, steryl glycosides and acylated steryl glycosides at 24 h, thereafter the radioactivity decreased in free sterols and steryl esters concomitant with the appearance of radioactivity in glycoalkaloids. Discs with ethephon additions contained more radioactivity in all sterol classes at all time-points, but no glycoalkaloids were formed.
The enzyme S-adenosyl- l -methionine:sterol C24 methyltransferase (SMT, EC 2. 1. 1. 41), located at one presumed branching point in the sterol and glycoalkaloid pathway, was characterized and found to exhibit similar characteristics as in other plants, but a lower specific activity. The activity of SMT increased in ageing tuber discs and this increase was further stimulated by ethephon, but inhibited by norborna-2,5-diene. The activity of the glycoalkaloid-specific enzyme UDP-glucose:solanidine glucosyltransferase (EC 2. 4. 1) also increased after slicing, but here ethephon additions counteracted the induction. The activity of the sterol-specific UDP-glucose:sterol glucosyltransferase (EC 2. 4. 1) was unaffected by either tuber slicing or ethephon additions.
The results indicate that ethylene stimulates sterol synthesis in wounded potato discs, and that the wound-induction of SMT is regulated by ethylene.     

Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism.

Organelles of the yeast Saccharomyces cerevisiae were isolated and analyzed for sterol composition and the activity of three enzymes involved in sterol metabolism. The plasma membrane and secretory vesicles, the fractions with the highest sterol contents, contain ergosterol as the major sterol. In other subcellular membranes, which exhibit lower sterol contents, intermediates of the sterol biosynthetic pathway were found at higher percentages. Lipid particles contain, in addition to ergosterol, large amounts of zymosterol, fecosterol, and episterol. These sterols are present esterified with long-chain fatty acids in this subcellular compartment, which also harbors practically all of the triacylglycerols present in the cell but very little phospholipids and proteins. Sterol delta 24-methyltransferase, an enzyme that catalyzes one of the late steps in sterol biosynthesis, was localized almost exclusively in lipid particles. Steryl ester formation is a microsomal process, whereas steryl ester hydrolysis occurs in the plasma membrane and in secretory vesicles. The fact that synthesis, storage, and hydrolysis of steryl esters occur in different subcellular compartments gives rise to the view that ergosteryl esters of lipid particles might serve as intermediates for the supply of ergosterol from internal membranes to the plasma membrane.     

Role of the intestinal brush border in the absorption of cholesterol in rats

1. Short-term incubation of the everted intestinal sacs of rats in media containing cholesterol oleate or cholesterol plus oleic acid resulted in rapid hydrolysis, but no synthesis, of the sterol ester. 2. On separation of the brush border from the rest of the mucosal cell, almost all of the hydrolytic activity and appreciable amounts of the synthetic activity of the whole cell were found to be present in the brush-border fraction. 3. The isolated brush-border fraction contained considerable amounts of cholesterol, which was always present in the unesterified state; the rest of the cell contained about an equal amount of unesterified cholesterol, but, in addition, small but definite amounts of the esterified sterol were also found in this fraction. 4. On feeding rats with [4-(14)C]cholesterol, which was diluted with 3mg. of cholesterol, it was found that the brush border very rapidly took up the fed sterol without changing its net content of cholesterol. No traces of radioactive cholesterol ester could ever be detected in the isolated brush border after feeding with (14)C-labelled esterified or unesterified cholesterol. 5. The appearance of the labelled sterol was quite rapid in the rest of the cell also, where small proportions were found in the esterified state. 6. Therefore the sequence of events in the absorption of cholesterol appears to be: the dietary cholesterol esters are hydrolysed by the cholesterol ester hydrolase of pancreas or of the mucosal brush border or both, after which the brush border rapidly absorbs the de-esterified sterol and transfers it into the mucosal cell, by a mechanism of displacement, where it is slowly re-esterified for transport through the lymph.     

Modulation of activities of steryl glucoside hydrolase and UDPG: Sterol glucosyltransferase from Sinapis alba by detergents and lipids

Interactions of detergents and lipid compounds on the activity of delipidated preparations of UDPG: sterol glucosyltransferase and steryl β-d-glucoside hydrolase (SG hydrolase) isolated from white mustard seedlings were studied. It has been found that various lipids exert diverse effects on the activity of SG hydrolase. This activity was distinctly stimulated by several neutral, relatively unpolar compounds such as phytol, tripalmitoylglycerol, methyl stearate or cholesteryl acetate and, to a lesser extent, by free fatty acids. On the other hand a number of phospho- and glycolipids were inhibitory. A particularly strong inhibition was observed with charged, zwitterionic phospholipids such as PC, PE or their 2-lyso derivatives. These results point to the possibility of in vivo regulation of the membrane-bound SG hydrolase by its lipid microenvironment. In contrast to SG hydrolase no evidence was found for a clear-cut effect of lipids on the activity of UDPG: sterol glucosyltransferase even after a pretreatment of the enzyme preparation with phospholipase C.     

Deficiency in the Lipid Exporter ABCA1 Impairs Retrograde Sterol Movement and Disrupts Sterol Sensing at the Endoplasmic Reticulum

Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process.     

Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase

To get some insight into the regulatory mechanisms controlling the sterol branch of the mevalonate pathway, tobacco (Nicotiana tabacum cv Bright Yellow-2) cell suspensions were treated with squalestatin-1 and terbinafine, two specific inhibitors of squalene synthase (SQS) and squalene epoxidase, respectively. These two enzymes catalyze the first two steps involved in sterol biosynthesis. In highly dividing cells, SQS was actively expressed concomitantly with 3-hydroxy-3-methylglutaryl coenzyme A reductase and both sterol methyltransferases. At nanomolar concentrations, squalestatin was found to inhibit efficiently sterol biosynthesis as attested by the rapid decrease in SQS activity and [(14)C]radioactivity from acetate incorporated into sterols. A parallel dose-dependent accumulation of farnesol, the dephosphorylated form of the SQS substrate, was observed without affecting farnesyl diphosphate synthase steady-state mRNA levels. Treatment of tobacco cells with terbinafine is also shown to inhibit sterol synthesis. In addition, this inhibitor induced an impressive accumulation of squalene and a dose-dependent stimulation of the triacylglycerol content and synthesis, suggesting the occurrence of regulatory relationships between sterol and triacylglycerol biosynthetic pathways. We demonstrate that squalene was stored in cytosolic lipid particles, but could be redirected toward sterol synthesis if required. Inhibition of either SQS or squalene epoxidase was found to trigger a severalfold increase in enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, giving first evidence for a positive feedback regulation of this key enzyme in response to a selective depletion of endogenous sterols. At the same time, no compensatory responses mediated by SQS were observed, in sharp contrast to the situation in mammalian cells.     

The G464S amino acid substitution in Candida albicans sterol 14alpha-demethylase causes fluconazole resistance in the clinic through reduced affinity.

Fluconazole selectively inhibits fungal sterol 14alpha-demethylase, a cytochrome P450 enzyme found in plants, animals, fungi, and Mycobacteria. The mutation G464S, observed in the heme-binding domain of sterol 14alpha-demethylase in clinical strains of fluconazole-resistant Candida albicans, is shown here to cause resistance through substantially reducing the inhibitory effect of fluconazole and is associated with perturbation of the heme environment as indicated by spectral data. The protein exhibits 42% of the maximal enzymatic rate of the wild-type protein allowing continued production of the end product of fungal sterol biosynthesis, ergosterol, in resistant strains. This mutation may cause these phenotypes through altering the heme location, thus changing the ability of residues above the heme to bind the drug effectively. This perturbation would also account for the observation of reduced sterol demethylase catalytic activity by changing the location of the 14alpha-methyl group in relation to oxygen-bound heme during the catalytic cycle.     

Steroid profiles of transgenic tobacco expressing an Actinomyces 3-hydroxysteroid oxidase gene

Previously, we have shown that the expression of a 3-hydroxysteroid-oxidase gene in transgenic tobacco initiated a series of biochemical events leading to the conversion of sterol to stanol. As a result, the plants maintained a diminished sterol pool and a modified relative sterol ratio but demonstrated no observable morphological abnormalities. The maintenance of normal higher plant physiology in the absence of particular sterols or in the presence of modified sterol ratios is controversial. In this report, we present additional biochemical and physiological characteristics of transgenic tobacco expressing an Actinomyces 3-hydroxysteroid-oxidase gene. The total steroid accumulated in the transgenic plants is 6-fold higher than in control plants and consists of sterol, 3-ketosteroid and stanol. The relative abundance of sterols within whole plant and individual organs is grossly altered as ethylated side chain sterols account for 99% of the total sterol pool in the transgenic tobacco. Stigmasterol is readily apparent in all tissues and cholesterol is found at measurable levels in specific organs, while campesterol and sitosterol are detected at trace levels in the transgenic plants. Stanols and 3-ketosteroids accumulate in all tissues and represent 77% of the measurable steroid pool in the transgenic plants. The sum of sterol, the respective 3-ketosteroid plus stanol provide a relative abundance of steroid, which is similar to the abundance of sterol accumulated in control tissue. In vitro photosynthetic electron transport measurements demonstrate altered activity of chloroplasts under a variety of reaction conditions, indicating a link between the modified steroid pool and a modulation of chloroplast membrane function.     

Structural requirements for substrate recognition of Mycobacterium tuberculosis 14 alpha-demethylase: implications for sterol biosynthesis

Sterol 14 alpha-demethylase (14DM) is a cytochrome P-450 involved in sterol biosynthesis in eukaryotes. It was reported that Mycobacterium smegmatis also makes cholesterol and that cholesterol is essential to Mycobacterium tuberculosis (MT) infection, although the origin of the cholesterol is unknown. A protein product from MT having about 30% sequence identity with eukaryotic 14 alpha-demethylases has been found to convert sterols to their 14-demethyl products indicating that a sterol pathway might exist in MT. To determine the optimal sterol structure recognized by MT 14DM, binding of 28 sterol and sterol-like (triterpenoids) molecules to the purified recombinant 14 alpha-demethylase was examined. Like eukaryotic forms, a 3 beta-hydroxy group and a 14 alpha-methyl group are essential for substrate acceptability by the bacterial 14 alpha-demethylase. The high affinity binding of 31-norcycloartenol without detectable activity indicates that the Delta(8)-bond is required for activity but not for binding. As for plant 14 alpha-demethylases, 31-nor-sterols show a binding preference for MT 14DM. Similar to enzymes from mammals and yeast, a C24-alkyl group is not required for MT 14DM binding and activity, whereas it is for plant 14 alpha-demethylases.Thus, substrate binding to MT 14DM seems to share common features with all eukaryotic 14 alpha-demethylases, the MT form seemingly having the broadest substrate recognition of all forms of 14 alpha-demethylase studied so far. - Bellamine, A., A. T. Mangla, A. L. Dennis, W. D. Nes, and M. R. Waterman. Structural requirements for substrate recognition of Mycobacterium tuberculosis 14 alpha-demethylase: implications for sterol biosynthesis. J. Lipid Res. 2001. 42: 128;-136.     

Isolation and characterization of an Arabidopsis thaliana C-8,7 sterol isomerase: functional and structural similarities to mammalian C-8,7 sterol isomerase/emopamil-binding protein

The yeast C-8,7 sterol isomerase contains a polyvalent high-affinity drug binding site similar to mammalian sigma receptors. Exogenously supplied sigma ligands inhibit sterol biosynthesis in yeast, demonstrating a pharmacological relationship between sigma ligand-binding and C-8,7 sterol isomerase activity. We report the isolation of an Arabidopsis thaliana C-8,7 sterol isomerase by functional complementation of the corresponding sterol mutant in yeast and its characterization by exposure to sigma ligands. The yeast erg2 mutant, which lacks the C-8,7 sterol isomerase gene and activity, was transformed with an Arabidopsis cDNA yeast expression library. Transformed colonies were selected for restoration of C-8,7 sterol isomerase activity (i.e. wild-type ergosterol production) by enhanced resistance to the antibiotic cycloheximide. Sterols produced in complemented lines were characterized by gas chromatography-mass spectroscopy (GC-MS). The full-length A. thaliana cDNA (pA.t.SI1) that complemented the erg2 mutation contains an open reading frame encoding a 21 kDa protein that shares 68% similarity and 35% amino acid identity to the recently isolated mouse C-8,7 sterol isomerase. The sigma ligands, haloperidol, ifenprodil and verapamil inhibited the production of ergosterol in wild-type Saccharomyces cerevisiae and in the erg2 mutant complemented with pA.t.SI1. Structural and biochemical similarities between the A. thaliana C-8,7 sterol isomerase and the mammalian emopamil-binding protein (EBP) are discussed.     

Effects of pravastatin sodium on mevalonate metabolism in common marmosets

In experimental animals and humans, the concentration of serum mevalonate (MVA), a direct product of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is considered to reflect the activity of whole-body sterol synthesis. The relationship between the concentration of serum MVA and the activity of sterol synthesis in tissues, however, has not been fully clarified. In the present study, we examined MVA metabolism by using pravastatin, a liver-selective inhibitor of HMG-CoA reductase, and common marmosets, a good model animal for studying lipid metabolism. In the time course study, the maximal reduction in the concentration of serum MVA was observed 2 h after a single oral administration of 30 mg/kg pravastatin to common marmosets. We, therefore, examined the relationship between the concentrations of serum and hepatic MVA, and sterol synthesis in some tissues at this time point. Sterol synthesis was determined ex vivo in tissue slices by measuring the incorporation of [14C]acetate into digitonin-precipitable [14C]sterols. Pravastatin at 0.03-30 mg/kg reduced dose-dependently the activity of hepatic sterol synthesis, whereas no significant reduction of sterol synthesis was observed in other tissues such as intestine, kidney, testis and spleen, even with the highest dose (30 mg/kg). The liver-specific inhibition of sterol synthesis caused parallel reductions in the concentrations of both serum and liver MVA. In addition, there were good correlations between the concentration of either serum or hepatic MVA and the activity of hepatic sterol synthesis. These data indicate that the major origin of serum MVA is the liver, and that the concentration of serum MVA reflects the concentration of hepatic MVA and the activity of hepatic sterol synthesis 2 h after a single oral administration of pravastatin in common marmosets.     

24-methylenepollinastanol and 24-dehydropollinastanol—two sterols of pollen

The esterified and unesterified sterol fractions of bee-gathered mixed pollens were examined, and total sterol composition was determined. Two new sterols of pollens, 14α-methyl-9β,19-cyclo-5α-cholest-24-en-3β-ol (24-dehydropollinastanol) and 14α-methyl-5α-ergost-24(28)-en-3β-ol (24-methylenepollinastanol) were isolated and identified. Both sterols were found primarily in the esterified sterol fraction, and 24-methylenepollinastanol accounted for 43% of the sterols of this fraction. 24-Dehydropollinastanol and four other sterols which also contain a 9β,19-cyclopropane ring were found only in the esterified sterol fraction. 24-Methylenecholesterol was the major sterol of the unesterified sterol fraction.     

The replacement of cholesterol by phytosterols and the increase of total sterol content in model erythrocyte membranes

The activity of phytosterols on human organism includes the ability of these compounds to incorporate into membranes. In the consequence the plant sterols are able to increase total sterol concentration in membrane or/and to replace cholesterol molecules. The aim of this work was to compare the influence of both these effects on the properties of model erythrocyte membranes. Moreover, the interactions between the plant sterols (β-sitosterol and stigmasterol) and saturated–monounsaturated phosphatidylcholine were investigated and the condensing and ordering potency of these phytocompounds on membrane phospholipids were thoroughly analyzed. It was found that the addition of the plant sterols into model membrane modifies the condensation, ordering and interactions in the system. Moreover, the replacement of mammalian sterol by phytosterol more strongly influences the model system than even a 10% increase of total sterol concentration induced by the incorporation of the plant sterol, at constant content of cholesterol. The investigated plant sterols at their lower concentration in the mixed system are of similar effect on its properties. At higher content stigmasterol was found to modify the properties of model membrane more strongly than β-sitosterol.     

Relation of Cholesterol to Astrocytic Differentiation in C-6 Glial Cells

Abstract: The relation of cellular cholesterol content to a biochemical expression of astrocytic differentiation was investigated in cultured C-6 glial cells. The astrocytic marker, glutamine synthetase, was studied. Cellular sterol content was perturbed with compactin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase and, thereby, cholesterol biosynthesis. Depletion of cellular sterol resulted in 72 h in a more than twofold increase in glutamine synthetase activity. Production of various degrees of sterol depletion with different concentrations of compactin demonstrated a striking inverse relationship between glutamine synthetase activity and the cellular sterol/phospholipid molar ratio. That the effect of compactin, in fact, is mediated by depletion of sterol was shown further by prevention of the compactin-induced increase in synthetase activity by simultaneous addition of exogenous cholesterol. Moreover, addition of cholesterol alone to the culture medium led to both a decrease in glutamine synthetase activity and an increase in the sterol/phospholipid molar ratio. The possibility that the compactin-induced increase in glutamine synthetase activity is caused by an increase in synthesis of the enzyme was suggested by prevention of the increase by cycloheximide. The data suggest that astrocytic differentiation is stimulated by a decrease in cellular sterol content. When considered with our previous observation that oli-godendroglial differentiation is inhibited by such a decrease, the findings suggest that cellular sterol content is a critical determinant of the direction of glial differentiation, i.e., whether along astrocytic or oligodendroglial lines.