Cholesterol Biosynthesis In Humans Research Articles

Voriconazole Treatment Induces a Conserved Sterol/Pleiotropic Drug Resistance Regulatory Network, including an Alternative Ergosterol Biosynthesis Pathway, in the Clinically Important FSSC Species, Fusarium keratoplasticum.

Fusarium keratoplasticum is the Fusarium species most commonly associated with human infections (fusariosis). Antifungal treatment of fusariosis is often hampered by limited treatment options due to resistance towards azole antifungals. The mechanisms of antifungal resistance and sterol biosynthesis in fusaria are poorly understood. Therefore, in this study we assessed the transcriptional response of F. keratoplasticum when exposed to voriconazole. Our results revealed a group of dramatically upregulated ergosterol biosynthesis gene duplicates, most notably erg6A (912-fold), cyp51A (52-fold) and ebp1 (20-fold), which are likely part of an alternative ergosterol biosynthesis salvage pathway. The presence of human cholesterol biosynthesis gene homologs in F. keratoplasticum (ebp1, dhcr7 and dhcr24_1, dhcr24_2 and dhcr24_3) suggests that additional sterol biosynthesis pathways may be induced in fusaria under other growth conditions or during host invasion. Voriconazole also induced the expression of a number of ABC efflux pumps. Further investigations suggested that the highly conserved master regulator of ergosterol biosynthesis, FkSR, and the pleiotropic drug resistance network that induces zinc-cluster transcription factor FkAtrR coordinate the response of FSSC species to azole antifungal exposure. In-depth genome mining also helped clarify the ergosterol biosynthesis pathways of moulds and provided a better understanding of antifungal drug resistance mechanisms in fusaria.

Discovering Peptide Inhibitors of Human Squalene Synthase Through Screening the Phage-Displayed Cyclic Peptide c7c Library.

Many drugs for the treatment of hypercholesterolemia are targeting the enzymes involved in human cholesterol biosynthesis pathway. Squalene synthase, the rate-limiting enzyme located at the downstream of cholesterol synthesis pathway, has become a better candidate to develop next-generation hypocholesterolemia drugs. In the present study, we cloned and expressed the recombinant human squalene synthase (hSQS) as the lure to isolate potential peptide inhibitors from screening the conformation-constrained phage-displayed cyclic peptide c7c library. Their binding capabilities were further estimated by ELISA. Their pharmaceutical potentials were then analyzed through molecular modeling and the ADMET property evaluations. Four ennea-peptides and nine tetra-peptides were finally synthesized to evaluate their inhibitory potentials toward hSQS. The results indicate that the ennea-peptide CLSPHSMFC, tetra-peptides SMFC, CKTE, and WHQW can effectively inhibit hSQS activities (IC50 values equal to 64, 76, 87, and 90μM, respectively). These peptides may have potentials to develop future cholesterol-lowering therapeutics. The ligand-protein interaction analysis also reveals that the inner hydrophobic pocket could be a more critical site of hSQS.

Significant association of urokinase plasminogen activator Pro141Leu with serum lipid profiles in a Japanese population

Urokinase plasminogen activator (uPA) plays important physiological and pathological roles in fibrinolysis, cancer metastasis, and atherosclerosis. One study suggested that uPA also has a major role in cholesterol biosynthesis in humans via its receptor uPAR. Thus, we investigated the associations of functional uPA polymorphism (plasminogen activator, urokinase; PLAU Pro141Leu, rs2227564) with serum lipid profiles in a Japanese cohort. The study included 5152 participants (1465 male, 3687 female; age range, 35–69years) of the Daiko Study, a part of the Japan Multi-Institutional Collaborative Cohort Study (J-MICC Study). Subjects were enrolled at the Daiko Medical Center from June 2008 to May 2010. Low-density lipoprotein cholesterol (LDL-C) and non-HDL-C (subtraction of high-density lipoprotein cholesterol from total cholesterol) in fasting blood of participants were each classified into two groups, < or ≥140mg/dL, and < or ≥170mg/dL, respectively. Genotype frequencies of PLAU Pro141Leu (rs2227564) were 59.1% for ProPro, 35.6% for ProLeu, and 5.3% for LeuLeu, and were in Hardy–Weinberg equilibrium (p=0.789). The allele frequencies were 0.769 for Pro and 0.231 for Leu. The multivariate-adjusted odd ratios (ORs) and 95% confidence intervals (CIs) for high LDL-C and non-HDL-C were 1.11 (95%CI; 1.00–1.23) and 1.16 (95%CI; 1.03–1.30) for those with Leu allele relative to ProPro. This study suggested that PLAU Pro141Leu (rs2227564) is significantly associated with serum lipid levels in a Japanese population.

Fasting reduces plasma proprotein convertase, subtilisin/kexin type 9 and cholesterol biosynthesis in humans

Proprotein convertase, subtilisin/kexin type 9 (PCSK9), a key regulator of plasma LDL-cholesterol (LDL-c) and cardiovascular risk, is produced in liver and secreted into plasma where it binds hepatic LDL receptors (LDLR), leading to their degradation. PCSK9 is transcriptionally activated by sterol response element-binding protein (SREBP)-2, a transcription factor that also activates all genes for cholesterol synthesis as well as the LDLR. Here we investigated the relationship between plasma PCSK9 levels and the lathosterol-to-cholesterol ratio, a marker of cholesterol biosynthesis, in 18 healthy subjects during a 48 h fast. In all individuals, plasma PCSK9 levels declined steadily during the fasting period, reaching a nadir at 36 h that was ∼58% lower than levels measured in the fed state (P < 0.001). Similarly, the lathosterol-to-cholesterol ratio declined in parallel with plasma PCSK9 concentrations during the fast, reaching a nadir at 36 h that was ∼28% lower than that measured in the fed state (P = 0.024). In summary, fasting has a marked effect on plasma PCSK9 concentrations, which is mirrored by measures of cholesterol synthesis in humans. Inasmuch as cholesterol synthesis and PCSK9 are both regulated by SREBP-2, these results suggest that plasma PCSK9 levels may serve as a surrogate marker of hepatic SREBP-2 activity in humans.

Cardiolipin synthesis is required to support human cholesterol biosynthesis from palmitate upon serum removal in Hela cellsThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

We examined whether cardiolipin (CL) synthesis was required to support cholesterol (CH) production from palmitate in Hela cells. Knockdown of human cardiolipin synthase-1 (hCLS1) in Hela cells has been shown to reduce CL synthesis. Therefore Hela cells stably expressing shRNA for hCLS1 and mock control cells were incubated for 16 h with [14C(U)]palmitate bound to albumin (1:1 molar ratio) in the absence or presence of serum. Knockdown of hCLS1 in Hela cells resulted in a reduction in [14C(U)]palmitate incorporation into CL and CH. This reduction in [14C(U)]palmitate incorporation into CH was most pronounced during incubation under serum-free conditions. The reduction in [14C(U)]palmitate incorporation into CH was not due to alterations in total uptake of [14C(U)]palmitate into cells or altered palmitate metabolism, since [14C(U)]palmitate incorporation into phosphatidylcholine, the major [14C(U)]palmitate-containing lipid, and its immediate precursor, 1,2-diacyl-sn-glycerol, were unaffected by hCLS1 knockdown. In addition, knockdown of hCLS1 did not affect CH pool size, indicating that CH catabolism was unaltered. Hydroxymethylglutaryl coenzyme A reductase enzyme activity and its mRNA expression were reduced by knockdown of hCLS1 and this was most pronounced in Hela cells cultured under serum-free conditions. These data indicate that CL synthesis is required to support human de novo CH biosynthesis under conditions of increased demand for CH.

Sterol dependent regulation of human TM7SF2 gene expression: Role of the encoded 3β-hydroxysterol Δ 14-reductase in human cholesterol biosynthesis

3β-hydroxysterol Δ 14-reductase operates during the conversion of lanosterol to cholesterol in mammalian cells. Besides the endoplasmic reticulum 3β-hydroxysterol Δ 14-reductase (C14SR) encoded by TM7SF2 gene, the lamin B receptor (LBR) of the inner nuclear membrane possesses 3β-hydroxysterol Δ 14-reductase activity, based on its ability to complement C14SR-defective yeast strains. LBR was indicated as the primary 3β-hydroxysterol Δ 14-reductase in human cholesterol biosynthesis, since mutations in LBR gene were found in Greenberg skeletal dysplasia, characterized by accumulation of Δ 14-unsaturated sterols. This study addresses the issue of C14SR and LBR role in cholesterol biosynthesis. Both human C14SR and LBR expressed in COS-1 cells exhibit 3β-hydroxysterol Δ 14-reductase activity in vitro. TM7SF2 mRNA and C14SR protein expression in HepG2 cells grown in delipidated serum (LPDS) plus lovastatin (sterol starvation) were 4- and 8-fold higher, respectively, than in LPDS plus 25-hydroxycholesterol (sterol feeding), resulting in 4-fold higher 3β-hydroxysterol Δ 14-reductase activity. No variations in LBR mRNA and protein levels were detected in the same conditions. The induction of TM7SF2 gene expression is turned-on by promoter activation in response to low cell sterol levels and is mediated by SREBP-2. The results suggest a primary role of C14SR in human cholesterol biosynthesis, whereas LBR role in the pathway remains unclear.

3-hydroxy-3-methylglutaryl-CoA synthase intermediate complex observed in "real-time".

The formation of carbon-carbon bonds via an acyl-enzyme intermediate plays a central role in fatty acid, polyketide, and isoprenoid biosynthesis. Uniquely among condensing enzymes, 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) catalyzes the formation of a carbon-carbon bond by activating the methyl group of an acetylated cysteine. This reaction is essential in Gram-positive bacteria, and represents the first committed step in human cholesterol biosynthesis. Reaction kinetics, isotope exchange, and mass spectroscopy suggest surprisingly that HMGS is able to catalyze the "backwards" reaction in solution, where HMG-CoA is cleaved to form acetoacetyl-CoA (AcAc-CoA) and acetate. Here, we trap a complex of acetylated HMGS from Staphylococcus aureus and bound acetoacetyl-CoA by cryo-cooling enzyme crystals at three different times during the course of its back-reaction with its physiological product (HMG-CoA). This nonphysiological "backwards" reaction is used to understand the details of the physiological reaction with regards to individual residues involved in catalysis and substrate/product binding. The structures suggest that an active-site glutamic acid (Glu-79) acts as a general base both in the condensation between acetoacetyl-CoA and the acetylated enzyme, and the hydrolytic release of HMG-CoA from the enzyme. The ability to trap this enzyme-intermediate complex may suggest a role for protein dynamics and the interplay between protomers during the normal course of catalysis.

Peroxisome deficiency does not result in deficiency of enzymes involved in cholesterol biosynthesis.

To unravel conflicting literature data on the dependence of cholesterol biosynthesis on functional peroxisomes, we have determined activities and levels of selected cholesterol biosynthetic enzymes in livers of a PEX5 knock-out mouse, a well-characterized animal model for human Zellweger syndrome. All enzymes measured, including putative peroxisomal enzymes, were normally active in the peroxisome-deficient mice indicating that mislocalization of these enzymes to the cytosol does not lead to decreased activity or degradation. Since these findings were in apparent contrast to previously reported data in human livers, we re-examined cholesterol biosynthetic enzymes in livers of Zellweger patients and control subjects. We found that the previously reported deficient enzymes mevalonate kinase and phosphomevalonate kinase are inactivated rapidly in a time-and temperature-dependent manner. This inactivation is not due to degradation as a result of mislocalization since normal levels of protein can be detected. Therefore, the deficiencies of these enzymes in livers of Zellweger patients reflect the bad condition of the livers, rather than mislocalization to the cytosol. Our data explain the conflicting findings in literature and demonstrate that great care should be taken in the interpretation of data obtained in postmortem material (1). To exclude that genetic differences, resulting in different defects in peroxisomal biogenesis, might influence the activity of selected cholesterol biosynthetic enzymes, we also have determined activities and levels of these enzymes in human fibroblasts of patients belonging to various different complementation groups. We included fibroblasts of patients with mutations in genes encoding PEX1, PEX5, PEX7, PEX16, and PEX19, resulting in selective deficiencies in import of PTS1 or PTS2 containing proteins or in a more general defect in peroxisome biogenesis resulting in the complete absence of peroxisomes or peroxisomal ghosts. We measured normal protein levels and activities of enzymes involved in cholesterol biosynthesis in all cell lines. We conclude that peroxisomes are not a prerequisite for cholesterol biosynthesis in humans.

Absence of functional peroxisomes does not lead to deficiency of enzymes involved in cholesterol biosynthesis

To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on functional peroxisomes, we determined activities and levels of selected enzymes involved in cholesterol biosynthesis in livers of PEX5 knockout mice, a well-characterized model for human Zellweger syndrome. We found that all enzymes measured, including putative peroxisomal enzymes, are at least as active in the peroxisome-deficient Zellweger mice as in control mice, indicating that mislocalization of enzymes to the cytosol does not lead to decreased activity or degradation. Prompted by these results, we re-examined this aspect in human subjects by specific enzyme activity measurements and immunoblotting with highly specific antisera. Our results show that the previously reported deficiencies of mevalonate kinase and phosphomevalonate kinase activity in livers from human Zellweger patients reflect the bad condition of the livers, rather than mislocalization to the cytosol. Our data provide an explanation for the conflicting findings in the literature and show that great care should be taken in the interpretation of data obtained in postmortem material.

Comparison of deuterium incorporation and mass isotopomer distribution analysis for measurement of human cholesterol biosynthesis

To compare endogenous cholesterol biosynthesis measured by deuterium incorporation (DI) and mass isotopomer distribution analysis (MIDA), cholesterol fractional and absolute synthetic rates were measured simultaneously by both techniques under identical physiological conditions. Twelve subjects (22 to 39 years of age) underwent a dual stable isotope protocol, involving oral deuterium oxide administration and measurement of incorporation of deuterium into cholesterol coincident with constant infusion of sodium [1-(13)C]acetate and measurement of the mass isotopomer distribution pattern of newly synthesized cholesterol. Synthesis was determined over 24 h with a 7-h feeding period. Both methods yielded similar measurements of fractional cholesterol synthesis (7.8 +/- 2.5% day(-)(1) for DI vs. 6.9 +/- 2.2% day(-)(1) for MIDA). Correlation of fractional synthesis across techniques was strong (r = 0.84, P = 0.0007). Absolute synthesis rates were also not different at 24 h (13.4 +/- 4.3 mg kg(-)(1) day(-)(1) for DI vs. 11.9 +/- 3.6 mg kg(-)(1) day(-)(1) for MIDA, r = 0.79, P < 0.002). We conclude that despite different assumptions and analytical requirements, deuterium incorporation and MIDA yield similar rates of cholesterogenesis in humans when measurements are made over 24 h. The decision as to which method to adopt depends on available clinical and analytical facilities

Upregulation of cholesterol synthesis after acute myocardial infarction—is cholesterol a positive acute phase reactant?

Acute myocardial infarction is associated with profound alterations in the plasma lipoprotein profile. The mechanism of these alterations is not clear, and both cholesterol biosynthesis up- and downregulation could possibly be a consequence of acute myocardial infarction. We determined plasma lipids, lipoproteins, apolipoproteins, and lathosterol—which is regarded as an estimate of whole body cholesterol biosynthesis in humans—concentrations in 34 patients (age 68±10 years, 24 male, 10 female) admitted to our hospital with acute MI and with onset of symptoms within the last 12 h. Samples were taken immediately after admission to the hospital, and 1, 2, and 10 days after admission. On the first day after admission there was a decrease in total cholesterol (C) by 14.1% ( P=0.01), in LDL-C by 14.4% ( P=0.03), in HDL-C by 9.3% (NS), and in triglycerides by 19.5% (NS). Apolipoprotein B100 was reduced by 18.3% ( P=0.008), and apolipoprotein AI by 12.3% (NS). The lathosterol/cholesterol ratio was increased by 23.1% after 1 day, and by 28.7% after 2 days ( P=0.05). After 10 days, all variables except the apolipoproteins had essentially returned to baseline values. In conclusion, the changes in the plasma lipid profile after acute myocardial infarction are associated with a profound increase of whole body cholesterol biosynthesis as judged by the lathosterol/cholesterol ratio. These changes may possibly enhance the delivery of cholesterol to cells involved in tissue repair mechanisms after acute myocardial infarction.

Validation of deuterium incorporation against sterol balance for measurement of human cholesterol biosynthesis

To examine the validity of the deuterium (D) incorporation technique for measurement of human cholesterol synthesis rates, D uptake from D2O into cholesterol was compared to sterol balance in 13 subjects each under three controlled diet settings. Subjects (age 62 +/- 3.6 yr, body weight 74 +/- 4.0 kg, BMI 27 +/- 1.4) consumed weight maintenance diets enriched in either corn oil, beef tallow, or stick corn oil margarine over a 5-week period. During the final week of the study period, subjects were given 1.2 g/D2O per kg body water. D enrichment was measured in plasma water and total cholesterol over 24 h. Also, during the final week, dietary intake and fecal elimination rates of cholesterol were assessed over one 6-day period to calculate sterol balance. There was no significant difference (t = 0.858, P = 0.397) between D incorporation into cholesterol (1,183 +/- 92 mg/day) and sterol balance (1,316 +/- 125 mg/day). Among diets, net cholesterol biosynthesis measured by D incorporation agreed (r = 0.745, P = 0.0001) with values derived from sterol balance. The degree of association between methods was not influenced by the wide range of fatty acid composition of the diet fat. These data demonstrate the utility of the simple, non-restrictive deuterium incorporation method as a reliable means of determining cholesterol biosynthesis in free-living humans.

Diurnal variations in the plasma concentrations of mevalonic acid in patients with abetalipoproteinaemia

Previous studies have demonstrated that changes in the rates of cholesterol biosynthesis can be evaluated by the determination of plasma concentrations of sterol intermediates, including mevalonic acid and lathosterol and that, in normal human subjects, a diurnal rhythm exists in which the highest concentrations of sterol intermediates are observed at night. The factors responsible for this diurnal rhythm in cholesterol synthesis are, however, unknown. To test the hypothesis that the nocturnal increase in cholesterol biosynthesis is attributable to a reduced rate of hepatic uptake of chylomicron remnants at night as compared to higher rates of uptake during the daytime in response to alimentary lipaemia, we have examined the diurnal rhythm of mevalonic acid in six normal volunteers and three patients with phenotypic abetalipoproteinaemia. The latter patients do not absorb appreciable amounts of dietary cholesterol and are unable to synthesize chylomicron particles. Plasma concentrations of mevalonic acid exhibited a diurnal rhythm in the normal subjects, and the highest plasma concentrations were observed between 24.00 hours/04.00 hours. A similar rhythm was observed in the plasma of patients with abetalipoproteinaemia. These results suggest that the nocturnal increase in cholesterol biosynthesis which occurs in humans is not attributable to reduced hepatic uptake of chylomicron remnants at night; further studies are needed to better define those factors which influence the periodicity of cholesterol biosynthesis in humans.

Lanosterol 14 alpha-demethylase (P45014DM): effects of P45014DM inhibitors on sterol biosynthesis downstream of lanosterol.

Lanosterol 14 alpha-demethylase (P45014DM) is the cytochrome P450 enzyme complex responsible for an early step in cholesterol biosynthesis, namely the 14 alpha-demethylation of lanosterol. We have synthesized a novel series of steroidal substrate analogues, designed to be specific and potent inhibitors of P45014DM. We describe here the effects of these compounds on sterol biosynthesis downstream from lanosterol, focusing ultimately on their efficacy as inhibitors of cholesterol biosynthesis. Results using a radio-high performance liquid chromatography (HPLC) assay show that in rat liver microsomal preparations, with [24,25-3H]dihydrolanosterol as substrate, the compounds do indeed inhibit the biosynthesis of sterols downstream from lanosterol. A range of inhibitory potencies was observed, and the key enzyme being inhibited was believed to be P45014DM. Inhibitor efficacy was readily correlated with non-metabolized [24,25-3H]dihydrolanosterol, formation of 4,4-dimethyl-cholest-8-en-3 beta-ol, and formation of lathosterol, a sterol believed to be an excellent indicator of whole body cholesterol biosynthesis in humans.

The YeastSaccharomyces cerevisiaein Molecular and Cellular Biology: A Smaller but not Lower Eucaryote

The baker's yeast Saccharomyces cerevisiae is a single celled eucaryote that is gaining appeal for studies of fundamental processes in cellular and molecular biology. This article describes three examples of the use of yeast to study problems of importance to human medicine. Specifically, the enzymes involved in neuropeptide processing, the RAS oncogenes, and the regulation of cholesterol biosynthesis in humans are shown to have remarkable similarities to their equivalent processes in yeast. In several cases the yeast and human enzymes are functionally interchangeable, emphasizing the unity of basic principles in biology.

Increased urinary mevalonic acid excretion in patients with abetalipoproteinemia and homozygous hypobetalipoproteinemia

Previous reports in which cholesterol homeostasis has been examined in patients with phenotypic abetalipoproteinemia have shown an increase in whole body cholesterol synthesis when measured by sterol balance techniques but normal rates of cholesterol synthesis when measured by isotopic cholesterol turnover. Recent studies have indicated that increases in cholesterol biosynthesis are paralleled by increases in the plasma concentrations of mevalonic acid and by higher rates of excretion of mevalonic acid in the urine. In the present report we have measured the 24-h urinary excretion of mevalonic acid in 7 patients with phenotypic abetalipoproteinemia and compared this to control subjects. Urinary excretion of mevalonic acid was significantly higher in the patients with abetalipoproteinemia (57.2 ± 10.2 nmol/kg body weight per day, mean ± SEM) as compared to control subjects (23.1 ± 1.5 nmol/kg per day). The magnitude of the increase in urinary mevalonic acid excretion seen in patients with abetalipoproteinemia (148%) is greater than the increase in whole body cholesterol biosynthesis assessed by sterol balance techniques (57% increase). Our results serve to further validate the usefulness of urinary mevalonate as an indicator of relative rates of cholesterol biosynthesis in humans and suggest that this measurement provides a valuable means to potentially screen for disorders associated with an oversynthesis of cholesterol.