Synthesis of fatty acid sterol esters using cholesterol esterase from Trichoderma sp. AS59

We recently reported the characterization of novel cholesterol esterase (EC. 3.1.1.13) from Trichoderma sp. and preliminary work on sterol ester synthesis. In the present study, we further examined the enzyme ability to synthesize cholesterol esters from cholesterol and free fatty acids of various chain lengths, and compared the fatty acid specificity in synthesis with that in hydrolysis. The enzyme catalyzed the synthesis of medium- and long-chain fatty acid cholesterol esters, but failed to synthesize short-chain fatty acid esters. The fatty acid specificities in the synthesis and hydrolysis of cholesterol esters were entirely different from each other. Unlike other lipolytic enzymes, the enzyme was largely independent of water content in the synthesis of cholesterol oleate, and it achieved near-complete esterification in the presence of an equimolar excess of oleic acid. Of additional interest is the finding that the addition of n-hexane markedly enhanced the esterification activities on all the medium- and long-chain saturated fatty acids used. Based on these findings, we attempted to synthesize stigmasterol stearate as a food additive to lower cholesterol levels in blood plasma, and found that the enzyme catalyzed effective synthesis of the ester without the need of dehydration during the reaction, indicating the potential utility of the enzyme in the food industry.     

Purification and characterization of <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. DS-1 cholesterol oxidase with thermal,organic solvent,and detergent tolerance

A new screening method for 6beta-hydroperoxycholest-4-en-3-one (HCEO)-forming cholesterol oxidase was devised in this study. As the result of the screening, a novel cholesterol oxidase producer (strain DS-1) was isolated and identified as Chromobacterium sp. Extracellular cholesterol oxidase of strain DS-1 was purified from the culture supernatant. The molecular mass of the purified enzyme was 58 kDa. This enzyme showed a visible adsorption spectrum having peaks at 355 and 450 nm, like a typical flavoprotein. The enzyme oxidized cholesterol to HCEO, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for every 1 mol of cholesterol oxidized. The enzyme oxidized 3beta-hydroxysteroids such as cholesterol, beta-cholestanol, and pregnenolone at high rates. The Km value for cholesterol was 26 microM. The enzyme was stable at pH 3 to 11 and most active at pH 7.0-7.5, showing optimal activity at pH 7.0 and 65 degrees C. The enzyme retained about 80% of its activity after incubation for 30 min at 85 degrees C. The thermal stability of the enzyme was the highest among the cholesterol oxidases tested. Moreover, the enzyme was more stable in the presence of various organic solvents and detergents than commercially available cholesterol oxidases.     

Burkholderia cepacia胆固醇酯酶及其分子伴侣基因的串联表达及酶学性质的研究

为实现胆固醇酯酶的可溶性表达,以Burkholderia cepacia ST-200胆固醇酯酶为研究对象,将酶及其分子伴侣通过人工添加的SD序列在大肠杆菌中进行串联表达。通过SDS-PAGE电泳分析,胆固醇酯酶成功获得可溶性表达,摇瓶水平的总酶量为1 077 U/L。经过镍柱纯化之后可以获得一条相对分子量约为37 kDa的单一条带。利用圆二色谱解析胆固醇酯酶的二级结构,并测定Tm值,从最适温度、最适pH、热稳定性、pH稳定性及有机溶剂耐受性等方面对胆固醇酯酶进行研究,结果表明该酶在pH为7.0,45℃条件下表现出最大活力。     

Endoplasmic reticulum stress-mediated inhibition of NSMase2 elevates plasma membrane cholesterol and attenuates NO production in endothelial cells

Chronic exposure of blood vessels to cardiovascular risk factors such as free fatty acids, LDL-cholesterol, homocysteine and hyperglycemia can give rise to endothelial dysfunction, partially due to decreased synthesis and bioavailability of nitric oxide (NO). Many of these same risk factors have been shown to induce endoplasmic reticulum (ER) stress in endothelial cells. The objective of this study was to examine the mechanisms responsible for endothelial dysfunction mediated by ER stress. ER stress elevated both intracellular and plasma membrane (PM) cholesterols in BAEC by ~ 3-fold, indicated by epifluorescence and cholesterol oxidase methods. Increases in cholesterol levels inversely correlated with neutral sphingomyelinase 2 (NSMase2) activity, endothelial nitric oxide synthase (eNOS) phospho-activation and NO-production. To confirm that ER stress-induced effects on PM cholesterol were a direct consequence of decreased NSMase2 activity, enzyme expression was either enhanced or knocked down in BAEC. NSMase2 over-expression did not significantly affect cholesterol levels or NO-production, but increased eNOS phosphorylation by ~ 1.7-fold. Molecular knock down of NSMase2 decreased eNOS phosphorylation and NO-production by 50% and 40%, respectively while increasing PM cholesterol by 1.7-fold and intracellular cholesterol by 2.7-fold. Furthermore, over-expression of NSMase2 in ER-stressed BAEC lowered cholesterol levels to within control levels as well as nearly doubled the NO production, restoring it to ~ 74% and 68% of controls using tunicamycin and palmitate, respectively. This study establishes NSMase2 as a pivotal enzyme in the onset of endothelial ER stress-mediated vascular dysfunction as its inactivation leads to the attenuation of NO production and the elevation of cellular cholesterol.     

Endoplasmic reticulum stress-mediated inhibition of NSMase2 elevates plasma membrane cholesterol and attenuates NO production in endothelial cells

Chronic exposure of blood vessels to cardiovascular risk factors such as free fatty acids, LDL-cholesterol, homocysteine and hyperglycemia can give rise to endothelial dysfunction, partially due to decreased synthesis and bioavailability of nitric oxide (NO). Many of these same risk factors have been shown to induce endoplasmic reticulum (ER) stress in endothelial cells. The objective of this study was to examine the mechanisms responsible for endothelial dysfunction mediated by ER stress. ER stress elevated both intracellular and plasma membrane (PM) cholesterols in BAEC by ~3-fold, indicated by epifluorescence and cholesterol oxidase methods. Increases in cholesterol levels inversely correlated with neutral sphingomyelinase 2 (NSMase2) activity, endothelial nitric oxide synthase (eNOS) phospho-activation and NO-production. To confirm that ER stress-induced effects on PM cholesterol were a direct consequence of decreased NSMase2 activity, enzyme expression was either enhanced or knocked down in BAEC. NSMase2 over-expression did not significantly affect cholesterol levels or NO-production, but increased eNOS phosphorylation by ~1.7-fold. Molecular knock down of NSMase2 decreased eNOS phosphorylation and NO-production by 50% and 40%, respectively while increasing PM cholesterol by 1.7-fold and intracellular cholesterol by 2.7-fold. Furthermore, over-expression of NSMase2 in ER-stressed BAEC lowered cholesterol levels to within control levels as well as nearly doubled the NO production, restoring it to ~74% and 68% of controls using tunicamycin and palmitate, respectively. This study establishes NSMase2 as a pivotal enzyme in the onset of endothelial ER stress-mediated vascular dysfunction as its inactivation leads to the attenuation of NO production and the elevation of cellular cholesterol.     

Regulation of hepatic cholesterol biosynthesis by berberine during hyperhomocysteinemia

Hyperhomocysteinemia, an elevation of blood homocysteine levels, is a metabolic disorder associated with dysfunction of multiple organs. We previously demonstrated that hyperhomocysteinemia stimulated hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase leading to hepatic lipid accumulation and liver injury. The liver plays an important role in cholesterol biosynthesis and overall homeostasis. HMG-CoA reductase catalyzes the rate-limiting step in cholesterol biosynthesis. Hepatic HMG-CoA reductase is a major target for lowering cholesterol levels in patients with hypercholesterolemia. The aim of the present study was to examine the effect of berberine, a plant-derived alkaloid, on hepatic cholesterol biosynthesis in hyperhomocysteinemic rats and to identify the underlying mechanism. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 4 wk. HMG-CoA reductase activity was markedly elevated in the liver of hyperhomocysteinemic rats, which was accompanied by hepatic lipid accumulation. Activation of HMG-CoA reductase was caused by an increase in its gene expression and a reduction in its phosphorylation (an inactive form of the enzyme). Treatment of hyperhomocysteinemic rats with berberine for 5 days inhibited HMG-CoA reductase activity and reduced hepatic cholesterol content. Such an inhibitory effect was mediated by increased phosphorylation of HMG-CoA reductase. Berberine treatment also improved liver function. These results suggest that berberine regulates hepatic cholesterol biosynthesis via increased phosphorylation of HMG-CoA reductase. Berberine may be therapeutically useful for the management of cholesterol homeostasis.     

In vitro activation of a soluble cholesterol esterase from bovine adrenals by a cAMP-dependent protein kinase.

Properties and partial purification of the bovine adrenal cholesterol esterase from the 100000 X g supernatant fraction were investigated. Variations of the enzyme activity with time-dependent (enzymatic) and time-dependent (non enzymatic) effects have been demonstrated. Mg2 has been proved to inhibit the enzyme activity by a non-enzymatic effect in 50mM Tris/HCl buffer, pH 7.4. A time-dependent inactivation of the cholesterol esterase has been observed in the same buffer. The enzyme could be protected from this enzymatic inactivation by its substrate, cholesterol oleate. cAMP, ATP and Mg2 cuase a time-dependent stimulation of the enzyme in 50mM Tris/HCl buffer, pH 7.4. This result suggests that corticotropin activates the soluble cholesterol esterase from bovine adrenals via cAMP-dependent protein kinase. This view is strengthened by the incorporation of 32P radioactivity from [gamma-32P] ATP into the protein fraction of the 100,000 X g supernatant. The protein-bound 32P radioactivity could be co-purified with the enzyme activity during the partial purification of the soluble cholesterol esterase.     

Short term regulation of acyl CoA: Cholesterol acyl transferase (ACAT) activity in the regenerating and perinatal liver

Acyl coenzyme A:cholesterol acyltransferase (ACAT), the enzyme catalyzing the hepatic cholesterol esterification could be involved in the modified availability of cholesterol detectable in proliferating systems. While no significant variations are detectable in the regenerating liver, the modified ACAT activity during liver development and its differential sensitivity to the in vitro stimulation of modulatory systems suggest an involvement of the enzyme in this proliferating process.     

Cholesterol effect on thermostability of the (Ca2+, Mg2+)-ATPase from cardiac muscle sarcolemma

When the cholesterol concentration in the sarcolemmal system is raised, the (Ca2+,Mg2+)-ATPase activity acquires an important degree of thermostability; phenomena that is completely lost if the experiment is carried out with cholesterol depleted sarcolemma. In this system, a gradual depletion of sarcolemmal cholesterol, renders the ATPase remarkably sensitive to temperature. At different concentrations of ATP, it is found that cholesterol affects the Vmax of the (Ca2+,Mg2+)-ATPase but not its Km. These results support our earlier suggestion of a direct effect of cholesterol upon the enzyme, and opens a possible mode of action of cholesterol on the enzyme. It is suggested that the inverse relationship between catalysis and thermostability is due to differences in the flexibility of the enzyme directly related to hydrophobicity changes caused by cholesterol.     

Homology modelling of human DHCR24 (seladin-1) and analysis of its binding properties through molecular docking and dynamics simulations

Recent biochemical and clinical evidences unveiled that DHCR24 enzyme (3-beta-hydoxysterol-Delta(24)-reductase, also named seladin-1), which catalyzes the last step of the cholesterol biosynthesis, is implicated in relevant neuroprotective processes by modulating the level of cholesterol in membrane. The present study was undertaken with a view to model the DHCR24 enzyme and its catalytic site, analyzing the substrate recognition at an atomic level. A homology model of the enzyme was obtained based on plant Cytokinin Dehydrogenase, and its active site was found to bind the desmosterol plus a set of post-squalenic intermediates of the cholesterol biosynthesis in a binding mode conducive to catalysis, even if the docking results suggested that the enzyme has a clear preference for the last intermediates of such biosynthetic pathway. Since DHCR24 possesses a putative transmembrane segment, the enzyme was, then, inserted in a suitable membrane model and the membrane-anchored structure in complex with desmosterol and cholesterol underwent 10ns MD simulations. Such simulations evidenced a clearly different behavior between substrate and product since the product only completely leaves the catalytic cavity whereas desmosterol firmly conserves its pivotal interactions during all simulation time. This is one of the first reports documenting the enzymatic product egress using simple MD simulations in which all atoms are free to move.     

Inhibition of hemolytic activity of Aeromonas salmonicida GCAT in rainbow trout red blood cells by a monoclonal antibody

The present study shows that a monoclonal antibody (MAb) directed to Aeromonas salmonicida glycerophospholipid:cholesterol acyltransferase (GCAT) is capable of protecting rainbow trout red blood cells from the cytotoxic effects of the enzyme in vitro.     

Regulation of cholesterol 7 alpha-hydroxylase by hepatic 7 alpha-hydroxylated bile acid flux and newly synthesized cholesterol supply

We measured hepatic cholesterol 7 alpha-hydroxylase activity, mass, and catalytic efficiency (activity/unit mass) in bile fistula rats infused intraduodenally with taurocholate and its 7 beta-hydroxy epimer, tauroursocholate, with or without mevalonolactone to supply newly synthesized cholesterol. Enzyme activity was measured by an isotope incorporation assay and enzyme mass by densitometric scanning of immunoblots using rabbit anti-rat liver cholesterol 7 alpha-hydroxylase antisera. Cholesterol 7 alpha-hydroxylase activity increased 6-fold, enzyme mass 34%, and catalytic efficiency 5-fold after interruption of the enterohepatic circulation for 48 h. When taurocholate was infused to the bile acid-depleted animals at a rate equivalent to the hepatic bile acid flux (27 mumol/100-g rat/h), cholesterol 7 alpha-hydroxylase activity and enzyme mass declined 60 and 61%, respectively. Tauroursocholate did not significantly decrease cholesterol 7 alpha-hydroxylase activity, mass and catalytic efficiency. The administration of mevalonolactone, which is converted to cholesterol, modestly increased cholesterol 7 alpha-hydroxylase activity and enzyme mass in the bile acid-depleted rats. However, when taurocholate was infused together with mevalonolactone, cholesterol 7 alpha-hydroxylase activity and catalytic efficiency were markedly depressed while enzyme mass did not change as compared with bile acid-depleted rats. These results show that (a) hepatic bile acid depletion increases bile acid synthesis mainly by activating cholesterol 7 alpha-hydroxylase with only a small rise in enzyme mass, (b) replacement with taurocholate for 24 h decreases both cholesterol 7 alpha-hydroxylase activity and mass proportionally, (c) when cholesterol is available (mevalonolactone supplementation), the infusion of taurocholate results in the formation of a catalytically less active cholesterol 7 alpha-hydroxylase, and (d) tauroursocholate, the 7 beta-hydroxy epimer of taurocholate, does not inhibit cholesterol 7 alpha-hydroxylase. Thus, bile acid synthesis is modulated by the catalytic efficiency and mass of cholesterol 7 alpha-hydroxylase. The enterohepatic flux of 7 alpha-hydroxylated bile acids and the formation of hepatic cholesterol apparently control cholesterol 7 alpha-hydroxylase by different mechanisms.     

A Method for the Increased Extraction of Cholesterol from Human Red Blood Cells by Modified Plasma Lipoprotein

The in vitro extraction of cholesterol from erythrocytes by plasma lipoproteins of reduced cholesterol content would be expected to be free of cholesterol-unrelated alterations of the cell membrane. The earlier application of this method utilized whole blood plasma in which the major part of the lipoprotein cholesterol was esterified by the plasma enzyme lecithin-cholesterol acyl transferase (LCAT) in a preliminary incubation. Because of the cholesterol remaining unesterified in the plasma, only 35% of the cell cholesterol could be removed. The method reported here uses HDL., a plasma lipoprotein which is the preferred substrate for LCAT, instead of whole plasma for the extraction. Multiple extractions with LCAT treated HDL, resulted in the removal of up to 77% of the erythrocyte cholesterol with only minor hemolysis.     

Circannual variations in mevalonate utilization in frog (Rana esculenta)

1. The fate of mevalonate, the product of HMGCoA reductase, was studied in male and female frogs (Rana esculenta) in order to explain the circannual variations of enzyme activity. 2. The incorporation of 2-14C MVA into unsaponifiable lipids, cholesterol and dolichol in liver, plasma and eggs was followed. 3. Labeled MVA shows a different utilization depending on season and sex. In spring and summer cholesterol synthesis is related to hepatic reserve storage in both sexes, while the peak of enzyme activity, present only in females in fall, seems committed to cholesterol export into the blood and uptake by the oocytes. 4. The presence of a MVA-derived protein identifiable with vitellogenin and labeled on the lipid moiety, suggests that HMGCoA reductase activity in fall is committed to the lipidation of this protein essential for oocyte maturation.     

The effect of blood serum on synthesis of lipids in rat thymocytes

The increase in the incorporation of [2-14C]sodium acetate into cholesterol, free fatty acids and rat thymocyte phospholipids during incubation of rat cells in the absence of blood serum was demonstrated. The label incorporation into the thymocytes was found to be activated in an insignificant degree. Rat blood sera obtained 1 hour after gamma-irradiation of animals with doses of 4 and 10 Gr contained the same concentrations of cholesterol and its esters as the intact rat sera. Irradiation of animals did not affect the ability of the sera to inhibit lipid synthesis in thymocytes or the levels of total phospholipids, cholesterol and its esters. Incubation of cells with the blood sera from irradiated rats led to an increase in the cholesterol/cholesterol ester ratio in the incubation mixture.     

Cholesterol is superior to 7-ketocholesterol or 7 alpha-hydroxycholesterol as an allosteric activator for acyl-coenzyme A:cholesterol acyltransferase 1

We compared the abilities of cholesterol versus various oxysterols as substrate and/or as activator for the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT), by monitoring the activity of purified human ACAT1 in response to sterols solubilized in mixed micelles or in reconstituted vesicles. The results showed that 5 alpha,6 alpha-epoxycholesterol and 7 alpha-hydroxycholesterol are comparable with cholesterol as the favored substrates, whereas 7-ketocholesterol, 7 beta-hydroxycholesterol, 5 beta,6 beta-epoxycholesterol, and 24(S),25-epoxycholesterol are very poor substrates for the enzyme. We then tested the ability of 7-ketocholesterol as an activator when cholesterol was measured as the substrate, and vice versa. When cholesterol was measured as the substrate, the addition of 7-ketocholesterol could not activate the enzyme. In contrast, when 7-ketocholesterol was measured as the substrate, the addition of cholesterol significantly activated the enzyme and changed the shape of the substrate saturation curve from sigmoidal to essentially hyperbolic. Additional results show that, as an activator, cholesterol is much better than all the oxysterols tested. These results suggest that ACAT1 contains two types of sterol binding sites; the structural requirement for the ACAT activator site is more stringent than it is for the ACAT substrate site. Upon activation by cholesterol, ACAT1 becomes promiscuous toward various sterols as its substrate.     

Cholesterol and its anionic derivatives inhibit 5-lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5-LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5-LO product synthesis.     

Purification of recombinant acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) from H293 cells and binding studies between the enzyme and substrates using difference intrinsic fluorescence spectroscopy

Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a membrane-bound enzyme utilizing long-chain fatty acyl-coenzyme A and cholesterol to form cholesteryl esters and coenzyme A. Previously, we had expressed tagged human ACAT1 (hACAT1) in CHO cells and purified it to homogeneity; however, only a sparse amount of purified protein could be obtained. Here we report that the hACAT1 expression level in H293 cells is 18-fold higher than that in CHO cells. We have developed a milder purification procedure to purify the enzyme to homogeneity. The abundance of the purified protein enabled us to conduct difference intrinsic fluorescence spectroscopy to study the binding between the enzyme and its substrates in CHAPS/phospholipid mixed micelles. The results show that oleoyl-CoA binds to ACAT1 with K(d) = 1.9 μM and elicits significant structural changes of the protein as manifested by the significantly positive changes in its fluorescence spectrum; stearoyl-CoA elicits a similar spectrum change but much lower in magnitude. Previously, kinetic studies had shown that cholesterol is an efficient substrate and an allosteric activator of ACAT1, while its diastereomer epicholesterol is neither a substrate nor an activator. Here we show that both cholesterol and epicholesterol induce positive changes in the ACAT1 fluorescence spectrum; however, the magnitude of spectrum changes induced by cholesterol is much larger than epicholesterol. These results show that stereospecificity, governed by the 3β-OH moiety in steroid ring A, plays an important role in the binding of cholesterol to ACAT1.     

Serum proteins in ethanol intoxication and hypercholesteremia

Guinea pigs after 30, 60 and 90 days of cholesterol, ethanol and cholesterol + ethanol action have been studied for content of cholesterol, lipoproteins of certain classes, quantitative and qualitative composition of blood serum proteins. It has been found that cholesterol does not induce expressed hypercholesterinemia and does not hinder cholesterol accumulation in the blood serum and liver of animals. The specific activity of [3H] cholesterol in the liver under cholesterinosis and its combination with ethanol intoxication for the whole period of experiments is lower than in the control, which testifies to retardation of its renewal. This may stimulate development of pathological hypercholesterinemia-induced states. After 3-month ethanol intoxication the amount of alkaline serum proteins has grown and ethanol retains its action against a background of hypercholesterinemia. The found effect is supposed to reflect one of the compensatory mechanisms for hypercholesterinemia and atherogenesis prevention.