Inhibition of the class II HMG-CoA reductase of Pseudomonas mevalonii

There are two structural classes of HMG-CoA reductase, the third enzyme of the mevalonate pathway of isopentenyl diphosphate biosynthesis-the Class I enzymes of eukaryotes and the Class II enzymes of certain eubacteria. Structural requirements for ligand binding to the Class II HMG-CoA reductase of Pseudomonas mevalonii were investigated. For conversion of mevalonate to HMG-CoA the -CH(3), -OH, and -CH(2)COO(-) groups on carbon 3 of mevalonate were essential for ligand recognition. The statin drug Lovastatin inhibited both the conversion of HMG-CoA to mevalonate and the reverse of this reaction. Inhibition was competitive with respect to HMG-CoA or mevalonate and noncompetitive with respect to NADH or NAD(+). K(i) values were millimolar. The over 10(4)-fold difference in statin K(i) values that distinguishes the two classes of HMG-CoA reductase may result from differences in the specific contacts between the statin and residues present in the Class I enzymes but lacking in a Class II HMG-CoA reductase.     

Specific inhibitions of annonaceous acetogenins on class II 3-hydroxy-3-methylglutaryl coenzyme A reductase from Streptococcus pneumoniae

3-Hydroxy-3-methylglutaryl coenzyme A reductase (class II HMGR) could serve as a potential target to discover drugs fighting against the invasive diseases originated from Streptococcus pneumoniae, one of the major causes of bacterial disease in human. However, no strongly effective inhibitors of class II HMGR have been found so far. In the present study, for the first time, four annonaceous acetogenins (ACGs) were explored for the inhibition on S. pneumoniae HMGR. The results showed that the ACGs had higher inhibitory activities against S. pneumoniae HMGR with K(i) values in the range of 6.45-20.49 μM than the statin drug lovastatin (K(i)=116.25 μM), a classical inhibitor of class I HMGR. Then, three-dimensional modeling and docking simulations analyzed the possible binding mode of ACGs to S. pneumoniae HMGR and suggested a kind of novel structural and binding mode for designing promising inhibitor candidates of the targeted enzyme S. pneumoniae II HMGR.     

Lovastatin treatment inhibits sterol synthesis and induces HMG-CoA reductase activity in mononuclear leukocytes of normal subjects

The mechanism by which competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase decrease serum cholesterol is incompletely understood. The few available data in humans suggest that chronic administration of the competitive inhibitor, lovastatin, decreases serum cholesterol with little or no change in total body sterol synthesis. To further define the effect of lovastatin on cholesterol synthesis in normal subjects, we investigated the effect of a single oral dose of lovastatin and a 4-week treatment period of lovastatin on mononuclear leukocyte (ML) sterol synthesis as a reflection of total body sterol synthesis. In parallel, we measured serum lipid profiles and HMG-CoA reductase activity in ML microsomes that had been washed free of lovastatin. ML sterol synthesis did not significantly decrease (23 +/- 5%, mean +/- SEM) at 3 h after a single 40-mg dose of lovastatin. With a single oral 80-mg dose, ML sterol synthesis decreased by 57 +/- 10% (P less than 0.05) and remained low for the subsequent 6 h. With both doses, total HMG-CoA reductase enzyme activity in microsomes prepared from harvested mononuclear leukocytes was induced 4.8-fold (P less than 0.01) over baseline values. Both the 20-mg bid dose and the 40-mg bid dose of lovastatin administered for a 4-week period decreased serum cholesterol by 25-34%. Lovastatin at 20 mg bid decreased ML sterol synthesis by 23 +/- 6% (P less than 0.02) and increased ML HMG-CoA reductase 3.8 times (P less than 0.001) the baseline values. Twenty four hours after stopping lovastatin, ML sterol synthesis and HMG-CoA reductase enzyme activity had returned to the baseline values. The higher dose of lovastatin (40 mg bid) decreased ML sterol synthesis by 16 +/- 3% (P less than 0.05) and induced HMG-CoA reductase to 53.7 times (P less than 0.01) the baseline value at 4 weeks. Stopping this higher dose effected a rebound in ML sterol synthesis to 140 +/- 11% of baseline (P less than 0.01), while HMG-CoA reductase remained 12.5 times baseline (P less than 0.01) over the next 3 days. No rebound in serum cholesterol was observed. From these data we conclude that in normal subjects lovastatin lowers serum cholesterol with only a modest effect on sterol synthesis. The effect of lovastatin on sterol synthesis in mononuclear leukocytes is tempered by an induction of HMG-CoA reductase enzyme quantity, balancing the enzyme inhibition by lovastatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tissue-selective acute effects of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase on cholesterol biosynthesis in lens

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme that regulates cholesterol synthesis, lower serum cholesterol by increasing the activity of low density lipoprotein (LDL) receptors in the liver. In rat liver slices, the dose-response curves for inhibition of [14C]acetate incorporation into cholesterol were similar for the active acid forms of lovastatin, simvastatin, and pravastatin. The calculated IC50 values were approximately 20-50 nM for all three drugs. Interest in possible extrahepatic effects of reductase inhibitors is based on recent findings that some inhibitors of HMG-CoA reductase, lovastatin and simvastatin, can cause cataracts in dogs at high doses. To evaluate the effects of these drugs on cholesterol synthesis in the lens, we developed a facile, reproducible ex vivo assay using lenses from weanling rats explanted to tissue culture medium. [14C]Acetate incorporation into cholesterol was proportional to time and to the number of lenses in the incubation and was completely eliminated by high concentrations of inhibitors of HMG-CoA reductase. At the same time, incorporation into free fatty acids was not inhibited. In marked contrast to the liver, the dose-response curve for pravastatin in lens was shifted two orders of magnitude to the right of the curves for lovastatin acid and simvastatin acid. The calculated IC50 values were 4.5 +/- 0.7 nM, 5.2 +/- 1.5 nM, and 469 +/- 42 nM for lovastatin acid, simvastatin acid, and pravastatin, respectively. Thus, while equally active in the liver, pravastatin was 100-fold less inhibitory in the lens compared to lovastatin and simvastatin. Similar selectivity was observed with rabbit lens. Following oral dosing, ex vivo inhibition of [14C]acetate incorporation into cholesterol in rat liver was similar for lovastatin and pravastatin, but cholesterol synthesis in lens was inhibited by lovastatin by as much as 70%. This inhibition was dose-dependent and no inhibition in lens was observed with pravastatin even at very high doses. This tissue-selective inhibition of sterol synthesis by pravastatin was likely due to the inability of pravastatin to enter the intact lens since pravastatin and lovastatin acid were equally effective inhibitors of HMG-CoA reductase enzyme activity in whole lens homogenates. We conclude that pravastatin is tissue-selective with respect to lens and liver in its ability to inhibit cholesterol synthesis.     

Binding thermodynamics of statins to HMG-CoA reductase

The statins are powerful inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG-CoA reductase), the key enzyme in the cholesterol biosynthetic pathway, and are among the most widely prescribed drugs in the world. Despite their clinical importance, little is known about the binding thermodynamics of statins to HMG-CoA reductase. In this paper, we report the results of inhibition kinetics and microcalorimetric analysis of a representative type I statin (pravastatin) and four type II statins (fluvastatin, cerivastatin, atorvastatin, and rosuvastatin). Inhibition constants (K(i)) range from 2 to 250 nM for the different statins. Isothermal titration calorimetry (ITC) experiments yield binding enthalpies (DeltaH(binding)) ranging between zero and -9.3 kcal/mol at 25 degrees C. There is a clear correlation between binding affinity and binding enthalpy: the most powerful statins bind with the strongest enthalpies. The proportion by which the binding enthalpy contributes to the binding affinity is not the same for all statins, indicating that the balance among hydrogen bonding, van der Waals, and hydrophobic interactions is not the same for all of them. At 25 degrees C, the dominant contribution to the binding affinity of fluvastatin, pravastatin, cerivastatin, and atorvastatin is the entropy change. Only for rosuvastatin does the enthalpy change contribute more than 50% of the total binding energy (76%). Since the enthalpic and entropic contributions to binding originate from different types of interactions, the thermodynamic dissection presented here provides a way to identify interactions that are critical for affinity and specificity.     

Human hydroxymethylglutaryl-coenzyme A reductase (HMGCR) and statin sensitivity

While statins, hydroxymethylglutaryl-coenzyme A reductase (HMGCR) inhibitors, are clinically proven to reduce plasma cholesterol levels, a wide variation in inter-individual response to statin therapy has been observed. Pharmacogenetic studies have identified multiple loci that potentially contribute towards the statin response, including the HMGCR gene. To examine, if a statin-resistant, catalytically-active isoform of the human HMGCR could be generated, we have rationally altered the protein to include additional residues in the flap domain, which has a role in statin binding. Comparative enzyme assays with purified wild-type and mutant isoforms reveal the alteration imposes a slight (38%) decrease in the K(app)(M) for the substrate, a near 2-fold increase in turnover number, and a 480% increase in the Ki for lovastatin. Thus, alterations in HMGCR could contribute towards the synergistic effects of multiple loci in the statin response.     

Inhibition of cholesterol synthesis and esterification regulates high density lipoprotein interaction with isolated epithelial cells of human small intestine

The effect of two inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lovastatin and monacolin L, and an inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), Sandoz compound 58-035, on the interaction of 125I-labeled high density lipoprotein-3 (HDL3) with isolated human enterocytes was studied. Both HMG-CoA reductase inhibitors inhibited cholesterol synthesis and 125I-labeled HDL3 binding and degradation by enterocytes; a strong correlation between changes in cholesterol synthesis and interaction of 125I-labeled HDL3 with cells was observed. Lovastatin caused reduction of the apparent number of 125I-labeled HDL3 binding sites without affecting the binding affinity. No changes of cell cholesterol content were observed after incubation of cells with lovastatin. Mevalonic acid reversed the effect of lovastatin on 125I-labeled HDL3 binding. Lovastatin blocked up-regulation of the HDL receptor in response to loading of cells with nonlipoprotein cholesterol and modified cholesterol-induced changes of 125I-labeled HDL3 degradation. Lovastatin also reduced HDL-mediated efflux of endogenously synthesized cholesterol from enterocytes. The ACAT inhibitor caused a modest increase of 125I-labeled HDL3 binding to enterocytes and significantly decreased its degradation; both effects correlated with inhibition of cholesteryl ester synthesis. The results allow us to assume that the intracellular free cholesterol pool may play a key role in regulation of the HDL receptor.     

Mechanisms for cholesterol homeostasis in rat jejunal mucosa: effects of cholesterol, sitosterol, and lovastatin

The effects of feeding cholesterol, sitosterol, and lovastatin on cholesterol absorption, biosynthesis, esterification, and LDL receptor function were examined in the rat jejunal mucosa. Cholesterol absorption was measured by the dual-isotope plasma ratio method; the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, was measured as total and expressed enzyme activities (in the absence and presence of a phosphatase inhibitor, NaF, respectively); mucosal total and esterified cholesterol concentrations were determined by gas-liquid chromatography; LDL receptor function was assayed as receptor-mediated binding of (125)I-labeled LDL to mucosal membranes. Feeding 2% sitosterol or 0.04% lovastatin for 1 week significantly (P < 0.01) decreased the amounts of cholesterol absorbed per day (-85% and -63%, respectively). In contrast, feeding 2% cholesterol for 1 week increased the amounts of absorbed cholesterol 27-fold, even though the percent absorption significantly decreased. With all three treatments, there was a coordinate regulation of total HMG-CoA reductase activity and receptor-mediated LDL binding. Cholesterol feeding downregulated both total jejunal HMG-CoA reductase activity (P < 0.05) and receptor-mediated LDL binding (P < 0.01), whereas lovastatin- and sitosterol-supplemented diets significantly upregulated both of these parameters. In the control, cholesterol-fed, and sitosterol-fed animals, about half of the total jejunal HMG-CoA reductase activity was expressed (in functional dephosphorylated form). However, in the lovastatin-treated rats with 4-fold stimulation of HMG-CoA reductase, only 23% of the total enzyme activity was expressed. Changes in total HMG-CoA reductase activity and receptor-mediated LDL binding in all tested groups occurred with no change in total concentrations of mucosal cholesterol, and only cholesterol-fed animals had increased mucosal esterified cholesterol concentrations. Thus, in response to various fluxes of dietary or newly formed cholesterol, HMG-CoA reductase and receptor-mediated LDL binding are coordinately regulated to maintain constant cellular cholesterol concentrations in the jejunum.     

Lovastatin inhibits bone marrow-derived dendritic cell maturation and upregulates proinflammatory cytokine production

Statins are a group of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors which are most effective as lipid lowering agents, and are currently extensively used clinically. Recently, it was also shown that statins affect the immune response. We investigated the effects of lovastatin on the maturation and functional changes of bone marrow-derived dendritic cells (BM-DC). Lovastatin inhibited MHC class II and CD40 expression on DC in a dose-dependent manner, but had lesser effects on CD16, CD80, CD86, and CD11b expression. Nuclear extracts of lovastatin treated DC had decreased NF-kappaB DNA binding activity. Although antigen capture capacity of DC was not affected by lovastatin, the T-cell stimulatory activity of DC was inhibited. Lovastatin up-regulated DC pro-inflammatory cytokine production induced by LPS as measured by intracellular cytokine staining, ELISA and cDNA microarrays. Mevalonate, added in vitro, prevented these effects. These results indicate that lovastatin may inhibit BM-DC maturation and up-regulate cytokine production through a mevalonate dependent pathway, and may cause adverse effects on either innate or adaptive immunity.     

Functional expression of human HMG-CoA reductase in Saccharomyces cerevisiae: a system to analyse normal and mutated versions of the enzyme in the context of statin treatment

Aims:  Statins – inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase – are known to reduce blood cholesterol levels. In this paper, we present a Saccharomyces cerevisiae expression system, which enables quick evaluation of the sensitivity of the wild-type and/or mutant forms of human HMG-CoA reductase towards statins or other drugs.
Methods and results:  We analysed the sequence of the HMG-CoA reductase gene in DNA extracted from blood samples of 16 patients with cardiovascular disorders. We applied the yeast system to examine the sensitivity of the wild-type and mutated versions of the hHMG-CoA reductase to different types of statins.
Conclusion:  The yeast and mammalian HMG-CoA reductases demonstrate structural and functional conservation, and expression of human HMG-CoA reductase in yeast complements the lethal phenotype of strains lacking the HMG1 and HMG2 genes.
Significance and Impact of the Study:  These data indicate that a yeast expression system can serve to study the influence of selected mutations in human HMG-CoA reductase on the sensitivity of the enzyme to commonly prescribed statins. Our results suggest that this model system is suitable for the development and selection of lipid-lowering drugs as well as for the examination of DNA sequence variations in the context of statin therapy.     

Characterization of an HMG-CoA reductase from Listeria monocytogenes that exhibits dual coenzyme specificity

HMG-CoA reductase (HMGR) is an enzyme critical for cellular cholesterol synthesis in mammals and isoprenoid synthesis in certain eubacteria, catalyzing the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have isolated the gene encoding HMG-CoA reductase from Listeria monocytogenes and expressed the recombinant 6x-His-tagged form in Escherichia coli. Using NAD(P)(H), the enzyme catalyzes HMG-CoA reduction approximately 200-fold more efficiently than mevalonate oxidation in vitro. The purified enzyme exhibits dual coenzyme specificity, utilizing both NAD(H) and NADP(H) in catalysis; however, catalytic efficiency using NADP(H) is approximately 200 times greater than when using NAD(H). The statins mevinolin and mevastatin are weak inhibitors of L. monocytogenes HMG-CoA reductase, requiring micromolar concentrations for inhibition. Three-dimensional modeling reveals that the overall structure of L. monocytogenes HMG-CoA reductase is likely similar to the known structure of the class II enzyme from Pseudomonas mevalonii. It appears that the enzyme has catalytic amino acids in analogous positions that likely play similar roles and also has a flap domain that brings a catalytic histidine into the active site. However, in L. monocytogenes HMG-CoA reductase histidine 143 and methionine 186 are present in the putative NAD(P)(H)-selective site, possibly interacting with the 2' phosphate of NADP(H) or 2' hydroxyl of NAD(H) and providing the active site architecture necessary for dual coenzyme specificity.     

Enterococcus faecalis acetoacetyl-coenzyme A thiolase/3-hydroxy-3-methylglutaryl-coenzyme A reductase, a dual-function protein of isopentenyl diphosphate biosynthesis

Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, DeltaH(a) and K(m) values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar K(i) for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme.     

Mode of interaction of beta-hydroxy-beta-methylglutaryl coenzyme A reductase with strong binding inhibitors: compactin and related compounds

The sodium salts of compactin (1) and trans-6-[2-(2,4- dichloro-6-hydroxyphenyl)ethyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran- 2-one (3) are inhibitors of yeast beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase. The dissociation constants are 0.24 X 10(-9) and 0.28 X 10(-9) M, respectively. Similar values have been reported for HMG-CoA reductase from mammalian sources [Endo, A., Kuroda, M., & Tanzawa, K. (1976) FEBS Lett. 72, 323; Alberts, A. W., et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3957]. The structures of these compounds marginally resemble that of any substrates of HMG-CoA reductase. We, therefore, investigated the basis for the strong interaction between HMG-CoA reductase and these inhibitors. HMG-CoA and coenzyme A (CoASH), but not reduced nicotinamide adenine dinucleotide phosphate (NADPH), prevent binding of compactin to the enzyme. HMG-CoA, but not CoASH or NADPH, prevents binding of 3 to the enzyme. We also investigated the inhibitory activity of molecules that resemble structural components of compactin. Compactin consists of a moiety resembling 3,5-dihydroxyvaleric acid that is attached to a decalin structure. The sodium salt of DL-3,5-dihydroxyvaleric acid inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The dissociation constant for DL-3,5-dihydroxyvaleric acid, derived from protection against inactivation of enzyme by iodoacetic acid, is (2.1 +/- 0.9) X 10(-2) M. Two decalin derivatives (structurally identical with or closely related to the decalin moiety of compactin) showed no detectable inhibition. If the lack of inhibition is due to their limited solubility, the dissociation constant of these decalin derivatives may be conservatively estimated to be greater than or equal to 0.5 mM. Simultaneous addition of decalin derivatives and DL-3,5-dihydroxyvaleric acid does not lead to enhanced inhibition. The sodium salt of (E)-6-[2-(2-methoxy-1-naphthalenyl)ethenyl]-3,4,5,6- tetrahydro-4-hydroxy-2H-pyran-2-one (6) inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The inhibition constant (vs. HMG-CoA) is 0.8 microM. CoASH does not prevent binding of 6 to enzyme. Compound 6, therefore, behaves analogously to compound 3. We propose that these inhibitors occupy two sites on the enzyme: one site is the hydroxymethylglutaryl binding domain of the enzyme active site and the other site is a hydrophobic pocket located adjacent to the active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

The cytotoxicity of mevalonate, lovastatin and carminomycin with respect to MOLT-4 human malignant T-lymphoblasts in vitro]

It was shown in vitro that high concentrations of lovastatin, a competitive inhibitor of hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibited human malignant cells MOLT-4. The activity of lovastatin in doses of 50-250 microM was dose-dependent. Addition of mevalonate in a concentration of 3 mM to the growth medium completely prevented the cytotoxic effect of 100 microM of lovastatin. At the same time, exogenous mevalonate did not decrease the cytotoxicity of the anthracycline antibiotic carminomycin. Moreover, in a high concentration (7 mM) mevalonate slowly but significantly inhibited the growth of the malignant target cells and the effect was added to the cytotoxic effect of carminomycin low concentrations (0.08 to 0.175 microgram/ml). The results and the literature data suggested that combination of mevalonate, HMG-CoA reductase inhibitors and anthracyclines could be useful in tumor chemotherapy. The suggestion needs further investigation.     

Negative regulation of cell proliferation by mevalonate or one of the mevalonate phosphates

The role of mevalonate and its products in the regulation of cellular proliferation was examined using 6-fluoromevalonate (Fmev), a compound that blocks the conversion of mevalonate pyrophosphate to isopentenyl pyrophosphate. Fmev suppressed DNA synthesis by a variety of transformed and malignant T cell, B cell, and myeloid cell lines. In contrast to results previously reported with mitogen-stimulated human peripheral blood T cell DNA synthesis, low concentrations of low density lipoprotein (LDL) alone could not restore proliferation to these cell lines. The same concentrations of LDL were able to provide sufficient cholesterol and support the growth of all cell lines when mevalonate synthesis was blocked with a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, lovastatin. Fmev-mediated inhibition was totally prevented in some but not all cell lines when the concentration of exogenous LDL was increased 5-10-fold above that required to permit proliferation of lovastatin-blocked cells. Residual HMG-CoA reductase activity of cells cultured with LDL inversely correlated with the restoration of growth to Fmev-blocked cultures. Confirmation of the critical role of HMG-CoA reductase activity and mevalonate synthesis in the inhibition of cellular proliferation by Fmev was obtained by demonstrating that the specific inhibitor of this enzyme, lovastatin, restored proliferation of Fmev-blocked cells. Furthermore, supplementation of cultures with mevalonate, the product of HMG-CoA reductase activity, markedly inhibited proliferation of Fmev-blocked cells. These findings indicate that mevalonate or one of the mevalonate phosphates, which accumulates in Fmev-blocked cells, is a critical negative regulator of cellular proliferation.     

Quantitative proteomic analysis revealed lovastatin-induced perturbation of cellular pathways in HL-60 cells

Lovastatin, a member of the statin family of drugs, is widely prescribed for treating hypercholesterolemia. The statin family of drugs, however, also shows promise for cancer treatment and prevention. Although lovastatin is known to be an inhibitor for HMG-CoA reductase, the precise mechanisms underlying the drug's antiproliferative activity remain unclearly defined. Here we utilized mass spectrometry, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC), to analyze the perturbation of protein expression in HL-60 cells treated with lovastatin. We were able to quantify ～3200 proteins with both forward and reverse SILAC labeling experiments, among which ～120 exhibited significant alterations in expression levels upon lovastatin treatment. Apart from confirming the expected inhibition of the cholesterol biosynthesis pathway, our quantitative proteomic results revealed that lovastatin perturbed the estrogen receptor signaling pathway, which was manifested by the diminished expression of estrogen receptor α, steroid receptor RNA activator 1, and other related proteins. Lovastatin also altered glutamate metabolism through down-regulation of glutamine synthetase and γ-glutamylcysteine synthetase. Moreover, lovastatin treatment led to a marked down-regulation of carbonate dehydratase II (a.k.a. carbonic anhydrase II) and perturbed the protein ubiquitination pathway. Together, the results from the present study underscored several new cellular pathways perturbed by lovastatin.     

c-Jun N-terminal protein kinase signalling pathway mediates lovastatin-induced rat brain neuroblast apoptosis

We have previously shown that lovastatin, an HMG-CoA reductase inhibitor, induces apoptosis in rat brain neuroblasts. c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) are implicated in regulation of neuronal apoptosis. In this work, we investigated the role of JNK and p38 MAPK in neuroblast apoptosis induced by lovastatin. We found that lovastatin induced the activation of JNK, but not p38 MAPK. It also induced c-Jun phosphorylation with a subsequent increase in activator protein-1 (AP-1) binding, AP-1-mediated gene expression and BimEL protein levels. The effects of lovastatin were prevented by mevalonate. Pre-treatment with iJNK-I (a selective JNK inhibitor) prevented the effect of lovastatin on both neuroblast apoptosis and the activation of the JNK cascade. Furthermore, we found that the activation of the JNK signalling pathway triggered by lovastatin is accompanied by caspase-3 activation which is also inhibited by iJNK-I pre-treatment. Finally, a specific inhibitor of p38 MAPK, SB203580, had no effect on lovastatin-induced neuroblast apoptosis. Taken together, our data suggest that the activation of the JNK/c-Jun/BimEL signalling pathway plays a crucial role in lovastatin-induced neuroblast apoptosis. Our findings may also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy.     

Development of a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay for measurement of human leukocyte microsomal HMG-CoA reductase protein concentration

Methods were developed for determination of human mononuclear leukocyte HMG-CoA reductase protein concentration by a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay procedure. Leukocyte microsomal HMG-CoA reductase, first immobilized onto a nitrocellulose filter, is sequentially reacted with 1) monospecific, polyclonal rabbit anti-rat liver HMG-CoA reductase antiserum, which crossreacts with the human liver and leukocyte enzymes; 2) biotinylated donkey anti-rabbit immunoglobulin; 3) a streptavidin-horseradish peroxidase conjugate; and 4) 4-chloro-1-naphthol and H2O2 to visualize the quantity of horseradish peroxidase bound to the immunocomplex. Color development was proportional to the quantity of either purified liver or leukocyte microsomal HMG-CoA reductase applied to the nitrocellulose. Color development was not observed, however, when HMG-CoA reductase was omitted from the nitrocellulose, when one of the reactant species was omitted from the incubation reactions, or when anti-rat liver HMG-CoA reductase antiserum was pre-absorbed with either rat liver or human leukocyte HMG-CoA reductase. Immunoreactivity of microsomal HMG-CoA reductase was independent of the phosphorylation state of the enzyme, but was inversely related to the concentration of thiol-reducing agents present in the microsomal preparation up to 4 mM. Further increases in thiol-reductant failed to produce changes in immunoreactivity. Freshly isolated mononuclear leukocyte microsomal HMG-CoA reductase protein concentration in leukocytes from 31 healthy, normocholesterolemic subjects was a linear function of HMG-CoA reductase activity (R = 0.65; P less than 0.001). The catalytic efficiency of the freshly isolated mononuclear leukocyte enzyme was 313 +/- 34 pmol of mevalonate formed per min of incubation at 37 degrees C per mg immunoreactive protein. This methodology, in conjunction with that recently developed to measure human leukocyte HMG-CoA reductase activity (1984. J. Lipid Res. 25: 967-978), should prove useful in discriminating between HMG-CoA reductase regulatory mechanisms involving changes in enzyme protein concentration and those resulting from changes in enzyme catalytic efficiency.     

Suppression of statin effectiveness by copper and zinc in yeast and human cells

Lovastatin and other statins inhibit HMG-CoA reductase, which carries out an early step in the sterol biosynthesis pathway. Statins lower cholesterol and are widely prescribed to prevent heart disease, but like many drugs, they can interact with nutritionally acquired metabolites. To probe these interactions, we explored the effect of a diverse library of metabolites on statin effectiveness using a Saccharomyces cerevisiae model. In yeast, treatment with lovastatin results in reduced growth. We combined lovastatin with the library of metabolites, and found that copper and zinc ions impaired the ability of the statin to inhibit yeast growth. Using an integrated genomic and metabolomic approach, we found that lovastatin plus metal synergistically upregulated some sterol biosynthesis genes. This altered pattern of gene expression resulted in greater flux through the sterol biosynthesis pathway and an increase in ergosterol levels. Each sterol intermediate level was correlated with expression of the upstream gene. Thus, the ergosterol biosynthetic response induced by statin is enhanced by copper and zinc. In cultured mammalian cells, these metals also rescued statin growth inhibition. Because copper and zinc impair the ability of statin to reduce sterol biosynthesis, dietary intake of these metals could have clinical relevance for statin treatment in humans.     

Expression and characterization of the HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus

The thermostable class I HMG-CoA reductase of Sulfolobus solfataricus offers potential for industrial applications and for the initiation of crystallization trials of a biosynthetic HMG-CoA reductase. However, of the 15 arginine codons of the hmgA gene that encodes S. solfataricus HMG-CoA reductase, 14 (93%) are AGA or AGG, the arginine codons used least frequently by Escherichia coli. The presence of these rare codons in tandem or in the first 20 codons of a gene can complicate expression of that gene in E. coli. Problems include premature chain termination and misincorporation of lysine for arginine. We therefore sought to improve the expression and subsequent yield of S. solfataricus HMG-CoA reductase by expanding the pool size of tRNA(AGA,AGG), the tRNA that recognizes these two rare codons. Coexpression of the S. solfataricus hmgA gene with the argU gene that encodes tRNA(AGA,AGG) resulted in an over 10-fold increase in enzyme yield. This has provided significantly greater quantities of purified enzyme for potential industrial applications and for crystallographic characterization of a stable class I HMG-CoA reductase. It has, in addition, facilitated determination of kinetic parameters and of pH optima for all four catalyzed reactions, for determination of the K(i) for inhibition by the statin drug mevinolin, and for comparison of the properties of the HMG-CoA reductase of this thermophilic archaeon to those of other class I HMG-CoA reductases.