Control Of Cholesterol Synthesis Research Articles

The Cholesterol Synthesis Enzyme Lanosterol 14α‐Demethylase is Post‐Translationally Regulated by the E3 Ubiquitin Ligase MARCH6

Cholesterol synthesis is a complex process with over twenty enzymes involved. Each enzyme within the pathway undergoes distinct regulation to maintain cholesterol homeostasis. Typically, enzymes within the pathway are degraded in response to a high sterol status, but some are highly stable. In this study, we demonstrate that an enzyme at the branch point in the pathway, lanosterol 14α‐demethylase (LDM), undergoes unique regulation. We show that the preceding enzyme, lanosterol synthase (LSS) is stable, whilst LDM is rapidly degraded. Surprisingly, this degradation is not triggered by sterols but by nitric oxide. Here we identify that the important E3 ubiquitin ligase, MARCH6, is responsible for facilitating the ubiquitination and proteasomal degradation of LDM and also the terminal cholesterol synthesis enzyme, 24‐dehydrocholesterol reductase (DHCR24). Our work highlights MARCH6 as the first example of an E3 ubiquitin ligase which targets multiple steps in a single biochemical pathway and indicates new facets in the control of cholesterol synthesis.Support or Funding InformationThis work was supported by the Australian Research Council Grant DP170101178. The PhD students were funded by the Research Training Program from the Australian Government Department of Education.

The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.

Cholesterol synthesis is a tightly controlled pathway, with over 20 enzymes involved. Each of these enzymes can be distinctly regulated, helping to fine-tune the production of cholesterol and its functional intermediates. Several enzymes are degraded in response to increased sterol levels, whilst others remain stable. We hypothesised that an enzyme at a key branch point in the pathway, lanosterol 14α-demethylase (LDM) may be post-translationally regulated. Here, we show that the preceding enzyme, lanosterol synthase is stable, whilst LDM is rapidly degraded. Surprisingly, this degradation is not triggered by sterols. However, the E3 ubiquitin ligase membrane-associated ring-CH-type finger 6 (MARCH6), known to control earlier rate-limiting steps in cholesterol synthesis, also control levels of LDM and the terminal cholesterol synthesis enzyme, 24-dehydrocholesterol reductase. Our work highlights MARCH6 as the first example of an E3 ubiquitin ligase that targets multiple steps in a biochemical pathway and indicates new facets in the control of cholesterol synthesis.

The cytoplasmic and extracellular domains of LRP1 promote a convergent mechanism for feedback control of cholesterol synthesis and export, and protect against intracellular cholesterol accumulation

The cytoplasmic and extracellular domains of LRP1 promote a convergent mechanism for feedback control of cholesterol synthesis and export, and protect against intracellular cholesterol accumulation

Epigenetic regulation of cholesterol homeostasis.

Although best known as a risk factor for cardiovascular disease, cholesterol is a vital component of all mammalian cells. In addition to key structural roles, cholesterol is a vital biochemical precursor for numerous biologically important compounds including oxysterols and bile acids, as well as acting as an activator of critical morphogenic systems (e.g., the Hedgehog system). A variety of sophisticated regulatory mechanisms interact to coordinate the overall level of cholesterol in cells, tissues and the entire organism. Accumulating evidence indicates that in additional to the more “traditional” regulatory schemes, cholesterol homeostasis is also under the control of epigenetic mechanisms such as histone acetylation and DNA methylation. The available evidence supporting a role for these mechanisms in the control of cholesterol synthesis, elimination, transport and storage are the focus of this review.

Free Radical Attack on Cholesterol: Oxysterols as Markers of Oxidative Stress and as Bioactive Molecules

Oxysterols are oxygenated derivatives of cholesterol with a very short life-time relative to cholesterol. Oxysterols are present in nanomolar concentrations in biological fluids underscoring their role as important intermediates in a number of biochemical pathways including bile acid synthesis, reverse cholesterol transport, control of cholesterol synthesis in the brain, and oxidative stress. Most oxysterols are produced by enzymes of the cytochrome P450 family while others are produced by free radical reactions. This last group of oxysterols (stress oxysterols, or SOX) , which are mainly oxygenated in the C6 or C7 position, have attracted interest for mechanistic studies in the context of oxidative stress, and for probing oxidative stress in vivo . Sensitive and specific mass spectrometric methods have been prepared to measure SOX in a number of clinical settings, and to follow the changes induced by pharmacological treatments. Additional interest in oxidative stress oxysterols is linked to the increasing number of biological effects, obtained at cellular level and in animal models, implicated in the pathophysiological mechanisms that play a role in several diseases, including atherosclerosis, neurodegeneration, and cancer. Oxysterols have in fact been shown to induce apoptosis, cell differentiation, cytotoxicity, and impairment of endothelial function. This review is an evaluation of the recent literature on oxysterols, in particular on the role of oxysterols as bioactive compounds.

The production of oxysterols in bile by activated human leukocytes

Oxysterols are naturally occurring intermediates in the conversion of cholesterol to bile acids, the major route for elimination of cholesterol. Additionally, they are important signaling agents, particularly in control of cholesterol synthesis; however, some species also are cytotoxic and carcinogenic. Oxysterols in plasma, contained in oxidized low-density lipoprotein, are strongly correlated with atherosclerosis. Oxysterols are found in infected human bile and the oxysterol content in gallstones correlates with bacterial DNA in the stones. Here we demonstrate that human leukocytes, activated by the presence of bacterial lipopolysaccharide, are able to oxidize cholesterol to a variety of oxysterols, including species known to be carcinogenic.

Hypocholesterolemic Effect of Protein Isolated from Quinoa (Chenopodium quinoa Willd.) Seeds

We extracted the protein fraction (QP) from quinoa seeds. Mice were fed on 0.5% cholesterol diet, containing 0%, 2.5% and 5.0% of QP (control, QP 2.5% and QP 5.0%, respectively) for 4 weeks. Neither supplemental QP diet produced a difference in food intake and body weight gain. The QP supplementation significantly prevented the increase in plasma and liver total cholesterol levels. Fecal weight and excretion of bile acids in the QP groups was significantly higher than in the control group. QP showed bile acid-binding activity in vitro. Expression of mRNA of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the liver, a key enzyme for cholesterol biosynthesis, was suppressed by the QP diet, whereas the expression of cholesterol-7α-hydroxylase, a cholesterol catabolic enzyme, was found to be stimulated. These results suggest that the prevention of increases in plasma and liver cholesterol levels in mice fed the QP diet can be ascribed to the inhibition of the re-absorption of bile acids in the small intestine and control of cholesterol synthesis and catabolism.

Regulation of farnesyl diphosphate synthase gene expression by fatty acids.

Cholesterol biosynthesis depends on the activity of regulatory enzymes, including the peroxisomal Farnesyl Diphosphate Synthase (FPPS ). Cholesterol regulates its own synthesis rate. Hence, as a response to cholesterol depletion, a feed back mechanism is activated, whereby sterol regulatory binding proteins (SREBPla, 1c and 2 ) are subjected to sequential proteolytic activation, which permits their interaction with specific DNA response elements from responsive genes. In turn, the transcriptional activity of cholesterol biosynthesis genes is induced. Conversely, cholesterol accumulation decreases SREBP maturation and transcription of controlled genes. In addition, polyunsaturated fatty acids repress expression of genes from the lipogenesis pathway by preventing SREBP maturation, suggesting that fatty acids (FA ) could contribute to the control of cholesterol synthesis through a SREBP-dependent mechanism.

Dietary Cholesterol Inhibits Whole-Body but Not Cerebrum Cholesterol Synthesis In Young Pigs

We studied the effect of dietary cholesterol on tissue cholesterol synthesis in 10 genetically lean pigs using the 2H2O incorporation method. Five pigs were fed a diet containing 0% cholesterol (controls) and five were fed a diet containing 0.5% cholesterol (cholesterol-fed) from d 1 to d 54 of age. From d 30 to d 54, they received 2H2O and cholesterol synthesis was determined by measuring deuterium enrichment in erythrocyte cholesterol. All pigs were killed on d 54 and liver, ileum, jejunum, kidney, adipose tissue, muscle, and cerebrum were collected for measurement of free and esterified cholesterol and of the deuterium content of tissue free cholesterol. In vivo cholesterol synthesis was inhibited by dietary cholesterol as reflected by a decrease in fractional synthesis rate from 16.8 +/- 1.7%/d in controls to 2.3 +/- 1.1%/d in cholesterol-fed pigs. Cholesterol feeding raised cholesterol ester concentration in plasma, liver, and other tissues. Deuterium enrichment of free cholesterol in tissues, such as liver, was reduced from 11.98 +/- 2.82 milli atom percent excess in controls to 1.83 +/- 1.14 in cholesterol-fed pigs. Greater cholesterol intake did not inhibit cholesterol synthesis in the cerebrum. Deuterium enrichment levels of cholesterol in plasma and liver were similar to those in other tissues except for brain, suggesting an equilibration of cholesterol between the liver and most extrahepatic tissues. The control of cholesterol synthesis in brain appears to be independent of that in liver and other tissues of the body.

Control of cholesterol synthesis in cell proliferation

Control of cholesterol synthesis in cell proliferation

Feed-back control of cholesterol synthesis in partially hepatectomized rats

Partial hepatectomy caused a marked stimulation of cholesterol and fatty acid syntheses without affecting serum total cholesterol, total phospholipid and triacylglycerol concentrations of rats so far examined 48 h after the operation. Serum free cholesterol level, however, was increased by the treatment and the ratio of lysophosphatidylcholine to phosphatidylcholine was concomitantly decreased, suggesting the impairment of serum lecithin: cholesterol acyltransferase activity. The lipid content in the liver, especially triacylglycerol and ester cholesterol, was increased markedly by the operation. Feeding of a high cholesterol diet which elevated serum cholesterol and phospholipid levels to the partially hepatectomized rats, accelerated the accumulation of hepatic triacylglycerol and ester cholesterol by the partial hepatectomy. The weight of the regenerating liver was not influenced by cholesterol feeding, which suggested that cholesterol feeding did not inhibit the regeneration mechanism of the liver. The increase of cholesterol synthesis after partial hepatectomy was inhibited by cholesterol feeding. Therefore, it is conceivable that the negative feed-back control of cholesterol synthesis is induced by cholesterol feeding under the stimulated cell divisions of the liver after partial hepatectomy. It is suggested from the present data that a large amount of the cholesterol which is necessary for cell growth can be taken up from serum, when serum cholesterol concentration is high.

On the control of cholesterol synthesis

HE MORBID EXISTENCE of cholesterol within the walls of arteries represents the present prime threat to the physical well being of the Western world. Other modifying factors notwithstanding. its presence is the sine qua non of atherosclerosis. Extensive evidence from clinical, epidemiologic. and experimental derivations links cholesterol with this major form of occlusive vascular disease. Arterial and body cholesterol has its origins in both exogenous and endogenous sources. Several studies have indicated however that the bulk of arterial cholesterol in the natural disease state is of endogenous origin.‘,’ Accordingly then, knowledge of the mechanism of cholesterol formation is of profound importance. The Nobel award efforts of Lynen and Bloch as well as the work of many others have detailed precisely the biochemical steps required for the formation of cholesterol. In all. approximately 26 steps are known to be required to complete the biosynthesis of cholesterol from its precursor. acetyl CoA3,“. 18 acetyl CoA-cholesterol t 9 CO,. The early chemical reactions are part of pathways common to other metabolic end products. The fourth step however, the enzymatic reduction of betahydroxy-beta-methyl glutaryl CoA (HMG CoA) to mevalonic acid, is an irreversible reaction’ that leads nearly exclusively to cholesterol. It is at this step that one seeks and finds the prime locus of metabolic control for the biosynthesis of cholesterol. Studies utilizing isotopic mevalonate have shown that it and the intermediates beyond it are not subject to the same acute physiologic influences as are the compounds prior to this step.6 ’ It is of more than morphologic interest to observe that different enzymatic steps are separately packaged within the cell.9 ” Some reactions seem to have representation in two cellular compartments.‘~“~” The primal molecule, acetyl CoA, is the start of the synthetic sequence. Its successive condensation with itself is carried out initially in the cellsap, but HMG CoA reductase is found nearly exclusively in the microsomes. 9.” Following IV 14C acetate, over 90”,, ot the recovered 14C cholesterol is separated with the microsomal fraction after differential ultracentrifugation.9 In fact, most cellular cholesterol is associated with the microsomes.‘4 However, ketone body formation, which is actually ;I

Metabolic controls in precancerous liver—III. Further studies on the control of lipid synthesis during N-2-fluorenylacetamide feeding

We have examined, both in vivo and in vitro, various aspects of the control of cholesterol and fatty-acid synthesis in the livers of rats fed the hepatocarcinogen, N-2-fluorenylacetamide. Within 1–4 weeks of beginning carcinogen treatment “feedback” control of cholesterol synthesis was partially or completely lost. Other controls over lipogenesis, however—fasting inhibition of cholesterol synthesis and dietary control of fatty-acid synthesis (fasting, refeeding after fasting, high-fat diet)—were retained. The normal diurnal rhythm of hepatic cholesterol synthesis was missing from carcinogen-treated animals, but this was due to an alteration in their daily pattern of voluntary food intake. The loss of “feedback” control of cholesterol synthesis was found to be not due to the reduction in food intake that accompanied carcinogen feeding. This work is further evidence in support of the hypothesis that a breakdown in “feedback” control of cholesterol synthesis is an important feature in the development of liver cancer.

Control of cholesterol synthesis in hepatomas: Absence of diurnal rhythm in hepatomas 7794A and 9618A

We have examined the Morris hepatomas 7794A and 9618A for the presence of a diurnal variation in cholesterol synthesis such as is observed in the livers of normal rats. Whereas the livers of the tumour-bearing animals showed a 3–5 fold increase in rate of cholesterol synthesis from acetate between midday and midnight, no such increase occurred in the hepatomas. Furthermore, adrenalectomy resulted in a 3-fold increase in cholesterol synthesis (as measured at midday) in the host liver, but not in hepatoma 7794A. Work by others has indicated that diurnal variations in cholesterol synthesis in normal liver are due to variations in the amount rather than in the specific activity of the ratecontrolling enzyme of the cholesterogenic pathway, namely β-hydroxy-β-methylglutaryl co-enzyme A reductase. Our work has now demonstrated that at least two of the “minimal-deviation” hepatomas exhibit a defect in control over the synthesis and/or degradation of this enzyme, i.e. in addition to the defective “feedback” control of its activity that has been shown in many previous reports.

Control of cholesterol synthesis in hepatomas: The effect of bile salts

1. 1. Other authors have suggested that bile salts may be the cholesterol metabolite responsible, in vivo, for the “feed-back” regulation of cholesterol synthesis in liver. 2. 2. Since the liver tumour lacks this feed-back control, the role of bile salts has been examined in 2 transplantable hepatomas-5123C in Buffalo rats and BW7756 in C57 L/J mice. 3. 3. This paper reports: first, that when bile salts were removed in vivo, by feeding the bile-salt adsorbent, cholestyramine, then cholesterol synthesis was increased in the liver but not in the hepatomas; secondly, that when a bile salt (sodium deoxycholate, 0.5 mM) was added in vitro, to an homogenate preparation, then cholesterol synthesis was inhibited in both liver and hepatoma; and thirdly, that when sodium deoxycholate was added to an incubation medium with tissue slices, then by contrast to the results with homogenates, cholesterol synthesis was inhibited in liver but not hepatoma. 4. 4. This is further evidence in support of the hypothesis that the defective control of lipid synthesis shown by hepatomas, which defect has been suggested as perhaps fundamental to the neoplastic process, may be due to an inability of metabolic regulators to reach the active sites of enzyme action.

Time course of the negative feedback control of cholesterol synthesis in rats.

Time course of the negative feedback control of cholesterol synthesis in rats.