Plasma membrane rafts complete cholesterol synthesis by participating in retrograde movement of precursor sterols

Mammalian cells synthesize significant amounts of precursor sterols, in addition to cholesterol, at the endoplasmic reticulum (ER). The newly synthesized sterols rapidly move to the plasma membrane (PM). The mechanism by which precursor sterols move back to the ER for their enzymatic processing to cholesterol is essentially unknown. Here we performed pulse-chase experiments and showed that the C29/C30 sterols rapidly move from the PM to the ER and are converted to cholesterol. The retrograde precursor sterol transport is largely independent of the Niemann-Pick type C proteins, which play important roles in late endosomal cholesterol transport. In contrast, disrupting lipid rafts significantly retards the conversion of C29/C30 and C28 sterols to cholesterol, causing the accumulation of precursor sterols at the PM. Our results reveal a previously undisclosed function of the PM lipid rafts: they bring cholesterol biosynthesis to completion by participating in the retrograde movement of precursor sterols back to the ER.     

Sterol biosynthesis de nova via cycloartenol by the soil amoeba Acanthamoeba polyphaga.

The soil amoeba Acanthamoeba polyphaga is capable of synthesizing its sterols de novo from acetate. The major sterols are ergosterol and poriferasta-5,7,22-trienol. Furthermore C28 and C29 sterols of still unknown structure with an aromatic B-ring are also synthesized by the amoeba. The first cyclic sterol precursor is cycloartenol, which is the sterol precursor in all photosynthetic phyla. No trace of lanosterol, which is the sterol precursor in animals and fungi, could be detected. These results show that at least some of the biochemical processes of Acanthamoeba polyphaga might be phylogenetically related to those of unicellular algae. Addition of exogenous sterols to the culture medium does not influence the sterol biosynthesis and the sterol composition of the cells.     

Microsomal synthesis of cholesterol from squalene, Lanosterol, and desmosterol. Evidence for the presence of two noncatalytic activator proteins in the 105,000 g supernatant fraction from brain, heart, and kidney

The biosynthesis of C₂₇ sterols (used as a generic term for 3 β-hydroxysterols containing 27 carbon atoms) from squalene and lanosterol, of cholesterol from desmosterol, and of lanosterol from squalene by microsomal fractions from adult rat heart, kidney, and brain was investigated. These conversions required the presence of 105,000g supernatant fraction. Heat treatment of the supernatant fractions resulted in a significant loss of their capacity to stimulate the conversion of squalene to sterols, but the capacity to stimulate conversion of lanosterol to C₂₇ sterols and desmosterol to cholesterol was unaffected. The stimulatory activity (for the conversion of all three substrates) of both the heated and unheated supernatant fractions was lost on treatment with trypsin. Thus the soluble fraction appears to contribute at least two essential protein components for the overall conversion of squalene to cholesterol; one a heat labile protein, which functions in the squalene to lanosterol sequence, and the other a heat-stable protein, which is operative in the pathway between lanosterol and cholesterol. Hepatic supernatant factors required for cholesterol synthesis by liver microsomal enzymes function with heart, kidney, and brain microsomal enzymes in stimulating sterol synthesis from squalene and sterol precursors. Moreover, heart, kidney, and brain supernatant fractions prepared in 100 mm phosphate buffer stimulated cholesterol synthesis from squalene and other sterol precursors by liver microsomes. The supernatant fractions of the extrahepatic tissues prepared in 20 mm phosphate buffer lacked the ability to stimulate the biosynthesis of lanosterol from squalene by liver microsomes but were able to stimulate the conversion of lanosterol to C₂₇ sterols or conversion of desmosterol to cholesterol. These findings indicate that the heat-stable protein factor present in the supernatant fractions from extrahepatic tissues is perhaps identical to that in liver, but that the heat-labile factor in extrahepatic tissues, which catalyzes the cyclization of squalene to lanosterol, differs in some respect from that in liver.     

Gorgosterol biosynthesis: localization of squalene formation in the zooxanthellar component of various gorgonians

Four species of gorgonians: three related pseudoplexaurids Pseudoplexaura porosa, P. flagellosa and P. wagenaari; and the unrelated Pseudopterogorgia americana, are sources of zooxanthellae capable, in purified broken cell preparations, of converting [14C]labeled farnesyl pyrophosphate into squalene. More extensive studies with P. porosa and P. americana zooxanthellae preparations characterize the conversion as enzymatic and demonstrate farnesyl pyrophosphate and reduced pyridine nucleotide as substrates. NADPH and NADH are essentially equivalent. Anaerobic conditions are not required. Despite numerous attempts zooxanthellae formation of sterols, including gorgosterol, from radioactive substrates was unsuccessful. By enzyme studies, we can show only the conversion of mevalonate into squalene as a zooxanthellae capability.     

Inhibition of Sterol Biosynthesis in Monilinia fructigena by the Fungicide,S-1358

When Moniiinia fructigena was treated with S–1358 at a concentration of 10 μm, both quality and quantity of digitonin-precipitable sterols were markedly altered. The amount of ergosterol which is a major sterol in the control culture was reduced by S–1358 and the concomitant accumulation of obtusifoliol (one of 4α-methyl sterols) and 24-methylenedihy-drolanosterol (one of 4,4-dimethyl sterols) was observed. The time course study of acetate-U-¹⁴C incorporation into the digitonin-precipitable sterols revealed that 4α-methyl sterols accumulated slowly in the treated culture, while 4,4-dimethyl sterols accumulated rapidly. The accumulation of the sterols containing “extra1” methyl groups suggests that S–1358 blocks demethylation reactions in the conversion from lanosterol to ergosterol in M. fructigena.     

Possibilities of bioconversion of agricultural waste with the use of microalgae

A new methodology of biological treatment and conversion of farm waste (manure and wash water) with the use of intensively cultivated phototrophic microorganisms (microalgae) is reviewed. Criteria for selection of microalgae and peculiarities of their intensive cultivation for efficient removal of biogenic elements from and destruction of the organic components of the wastes as well as the possibilities of cost-effective utilization of the resulting microalgal biomass are considered. Advantages and drawbacks of the new methodology are compared with those of conventional anaerobic techniques. Special attention is paid to the integrated technologies combining the aerobic conversion methods with microalgal post-treatment.     

Conversion of DNA methyltransferases into azidonucleosidyl transferases via synthetic cofactors

Aziridine-based cofactor mimics have been synthesized and are shown to undergo methyltransferase-dependent DNA alkylation. Notably, each cofactor mimic possesses an azide functionality, to which can be attached an assortment of unnatural groups following methyltransferase-dependent DNA delivery. DNA duplexes modified with these cofactor mimics are capable of undergoing the Staudinger ligation with phosphines tethered to biological functionalities following enzymatic modification. This methodology provides a new tool by which to selectively modify DNA in a methyltransferase-dependent way. The conversion of biological methyltransferases into azidonucleosidyl transferases demonstrated here also holds tremendous promise as a means of identifying, as yet, unknown substrates of methylation.     

Sterols and the sensitivity of Pythium species to filipin

Schlosser, Eckart (University of Illinois, Urbana), and David Gottlieb. Sterols and the sensitivity of Pythium species to filipin. J. Bacteriol. 91:1080-1084. 1966.-The growth of several Pythium species was not affected by filipin. No leakage of inorganic phosphate was observed after treatment with the antibiotic. No sterol could be detected in 1 g (dry weight) of mycelium. Thus, the insensitivity of these fungi to the antibiotic may be explained by the lack of sterols, the postulated reaction site for filipin in the cell membrane. Though not capable of synthesizing sterols, Pythium species can incorporate exogeneous sterols, which renders them sensitive to filipin; such treatment causes a lag in growth and leakage of inorganic phosphate. The leakage after filipin treatment is indirect evidence that the sterols have been incorporated into the cell membrane. Induced sensitivity to filipin was reversible; it was lost when the sterols were diluted out by one transfer through a medium free from sterols. The hypothesis that the primary site of interaction of filipin is the sterol located in the cell membrane was strengthened by these studies. The experiments further demonstrated a change in sensitivity of a fungus to a toxic agent due to nutritional conditions.     

Dietary sterol preference in the silkworm, Bombyx mori

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that beta-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: beta-sitosterol > ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouth part might be different from the sterol recognition mechanism present in sterol metabolism.     

Characterization of ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid chromatography–tandem mass spectrometry

Ecdysteroids are polyhydroxylated steroids that function as molting hormones in insects. 20-Hydroxyecdysone (a 27C-ecdysteroid) is classically considered as the major steroid hormone of Drosophila melanogaster, but this insect also contains 28C-ecdysteroids. This arises from both the use of several dietary sterols as precursors for the synthesis of its steroid hormones, and its inability to dealkylate the 28C-phytosterols to produce cholesterol. The nature of Drosophila ecdysteroids has been re-investigated using both high-performance liquid chromatography coupled to enzyme immunoassay and a particularly sensitive nano-liquid chromatography–mass spectrometry methodology, while taking advantage of recently available ecdysteroid standards isolated from plants. In vitro incubations of the larval steroidogenic organ, the ring-gland, reveals the synthesis of ecdysone, 20-deoxy-makisterone A and a third less polar compound identified as the 24-epimer of the latter, while wandering larvae contain the three corresponding 20-hydroxylated ecdysteroids. This pattern results from the simultaneous use of higher plant sterols (from maize) and fungal sterols (from yeast). The physiological relevance of all these ecdysteroids, which display different affinities to the ecdysteroid receptors, is still a matter of debate.     

Inhibitors of sterol synthesis. Reverse phase high performance liquid chromatography for the separation of cholesterol, 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, and their fatty acid esters

A relatively simple and rapid method was required for the separation of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a potent inhibitor of sterol synthesis, from its major metabolites. Conditions have been determined which permit the resolution of the 15-ketosterol and cholesterol and fatty acid esters of the two sterols by reverse phase high performance chromatography. This methodology also permits the resolution of the major esters of the 15-ketosterol and of cholesterol.     

Role of sterol photoconversion products in the regulation of cholesterol biosynthesis in rat skin

In order to clarify the effect of products of photochemical conversion of sterols on cholesterol biosynthesis, rat skin samples were incubated with 2-(14)C-acetate in the presence of the antirachitic agent Dk and 7beta-hydroxycholesterol. The synthesis of sterols from acetate was activated in the presence of Dk. A correlation between the activation of sterol synthesis and the concentration of the antirachitic agent was found. An addition of 7beta-hydroxycholesterol to the incubation medium inhibited acetate incorporation into the sterols. The level of synthesis inhibition increased with an elevation of the 7beta-hydroxysterol concentration in the incubation medium. This indicates that both products of sterol photoconversion can be involved in the control of cholesterol biosynthesis.     

Sterol biosynthesis in the echinoderm Asterias rubens.

1. [2(-14)C]Mevalonic acid injected into the echinoderm Asterias rubens (Class Asteroidea) was effectively incorporated into the non-saponifiable lipid. 2. The most extensively labelled compounds were squalene and the 4,4-dimethyl sterols with much lower incorporations into the 4alpha-monomethyl and 4-demethyl sterol fractions. 3. Labelled compounds identified were squalene, lanosterol, 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol and 4alpha-methyl-5alpha-cholest-7-en-3beta-ol; these are all intermediates in sterol biosynthesis. 4. The major sterol in A. rubens, 5alpha-cholest-7-en-3beta-ol, was also labelled showing that this echinoderm is capable of sterol biosynthesis de novo. 5. No evidence was obtained for the incorporation of [2(-14)C]mevalonic acid into the C28 and C29 components of the 4-demethyl sterols or 9beta,19-cyclopropane sterols found in A. rubens and it is assumed that these sterols are of dietary origin. 6. Another starfish Henricia sanguinolenta also incorporated [2(-14)C]mevalonic acid into squalene and lanosterol. 7. Various isolated tissues of A. rubens were all capable of incorporation of [2(-14)C]mevalonic acid into the nonsaponifiable lipid. With the body-wall and stomach tissues radioactivity accumulated in squalene and the 4,4-dimethyl sterols, but with the gonads and pyloric caecae there was a more efficient incorporation of radioactivity into the 4-demethyl sterols, principally 5alpha-cholest-7-en-3beta-ol.     

The selective uptake of cholesterol by the rat tapeworm Hymenolepis diminuta (Cestoda)

1. The sterols of Hymenolepis diminuta are almost exclusively cholesterol or similar C-27 sterols; the free sterols of its environment (the lumen of the rat intestine) are cholesterol and various phytosterols. 2. During incubation of tapeworms with mixed micelles of taurocholate, glyceryl monooleate, and equimolar [3H]cholesterol and [14C]beta-sitosterol, the uptake of cholesterol is 40 times more rapid than the uptake of sitosterol. 3. Following uptake, the desorption of labeled sitosterol is six times more rapid than that of cholesterol. 4. We did not detect the esterification of absorbed sterols or the conversion of absorbed sitosterol of cholesterol. 5. The highly selective uptake of cholesterol and the moderately selective desorption of phytosterols can account for the selective accumulation of C-27 sterol by the tapeworm.     

Movement of plasma-membrane sterols to the endoplasmic reticulum in cultured cells.

The spontaneous turnover of plasma-membrane sterols, as measured by their transfer to the endoplasmic reticulum, was measured in quiescent cultured human skin fibroblasts and monkey arterial smooth-muscle cells. The plasma-membrane sterol pool was pulse-labelled with trace amounts of either [3H]desmosterol or [3H]cholesterol. We then measured the enzymic conversion of [3H]desmosterol into [3H]cholesterol and of [3H]cholesterol into [3H]cholesteryl esters in intact cells. Depending on the probe used, markedly different transfer or conversion rates were found in these cells. In quiescent human skin fibroblasts, incubated in a serum-free medium, about 1.1% of the plasma-membrane [3H]desmosterol was converted into [3H]cholesterol/h, whereas in monkey arterial smooth-muscle cells the corresponding rate was 0.4%. Under similar experimental conditions, these cells esterified less than 0.02% (fibroblasts) and 0.12% (smooth-muscle cells) of the plasma-membrane [3H]cholesterol/h. The movement of sterols from the plasma membrane to the endoplasmic reticulum, as measured by the conversion of [3H]desmosterol into [3H]cholesterol was not blocked by colchicine, but was markedly enhanced by 3% (w/v) dimethyl sulphoxide. In all, these results indicate that plasma-membrane sterols of cultured cells are continuously transferred to the interior of the cell at a rate substantially higher than previously appreciated. This turnover of plasma-membrane sterol molecules took place even when there was no mass transfer of sterols into the cells.     

Structural requirements of cholesterol for binding to Vibrio cholerae hemolysin

Cholesterol is necessary for the conversion of Vibrio cholerae hemolysin (VCH) monomers into oligomers in liposome membranes. Using different sterols, we determined the stereochemical structures of the VCH-binding active groups present in cholesterol. The VCH monomers are bound to cholesterol, diosgenin, campesterol, and ergosterol, which have a hydroxyl group at position C-3 (3betaOH) in the A ring and a C-C double bond between positions C-5 and C-6 (C-C Delta(5)) in the B ring. They are not bound to epicholesterol and dihydrocholesterol, which form a covalent link with a 3alphaOH group and a C-C single bond between positions C-5 and C-6, respectively. This result suggests that the 3betaOH group and the C-CDelta(5) bond in cholesterol are required for VCH monomer binding. We further examined VCH oligomer binding to cholesterol. However, this oligomer did not bind to cholesterol, suggesting that the disappearance of the cholesterol-binding potential of the VCH oligomer might be a result of the conformational change caused by the conversion of the monomer into the oligomer. VCH oligomer formation was observed in liposomes containing sterols with the 3betaOH group and the C-C Delta(5) bond, and it correlated with the binding affinity of the monomer to each sterol. Therefore, it seems likely that monomer binding to membrane sterol leads to the assembly of the monomer. However, since oligomer formation was induced by liposomes containing either epicholesterol or dihydrocholesterol, the 3betaOH group and the C-C Delta(5) bond were not essential for conversion into the oligomer.     

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.     

Sterol biosynthesis by a cell free extract of Dioscorea floribunda

The biosynthesis of sterols from mevalonate by a cell free extract prepared from actively growing tuber portions and leader shoots with young leaves of Dioscorea floribunda has been demonstrated. The preparation was capable of synthesizing 86.4 μg and 34.0 1£g of sterols from leader shoots with young leaves and actively growing tuber portions respectively. The cofactor requirement for the above system was also studied.     

Preimplantation mouse embryos synthesize membrane sterols

The total cholesterol content of preimplantation mouse embryos increases approximately threefold (to 1 pmole) during the development of a blastocyst from a fertilized egg. From the two-cell stage onwards embryos are capable of converting [³H]mevalonate into the membrane sterols lanosterol and cholesterol. However, activity of the ratelimiting enzyme in sterol synthesis, hydroxymethylglutaryl coenzyme A reductase, was only measurable in late expanded blastocysts. These estimates of cholesterol content and the amounts of ³H-sterol formed suggest that the preimplantation mouse embryo can synthesize membrane sterols from early cleavage stages onwards. Late compaction and early fluid accumulation (approx. 84 hr post-hCG) are associated with a transition from lanosterol to cholesterol synthesis. The possible relationship between this transition and changes in the properties of embryo membranes which occur at this time is discussed. The results, taken together with previous evidence for phospholipid synthesis in early embryos, demonstrate that the preimplantation mouse embryo is capable of synthesizing major membrane lipids and hence has the potential for assembling cell membranes and modulating their lipid-mediated properties.     

Dietary Sterol Preference in the Silkworm,Bombyx mori

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that β-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: β-sitosterol >> ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouthpart might be different from the sterol recognition mechanism present in sterol metabolism.