Regulation of sterol biosynthesis and of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in cultured cells by progesterone

Treatment of rat intestinal epithelial cells in culture (IEC-6) with progesterone (10 micrograms/ml) caused a strong inhibition of cholesterol biosynthesis as indicated by a decreased incorporation of radiolabel from [3H]acetate. This inhibition was accompanied by an accumulation of radioactivity in an intermediate which coeluted with authentic desmosterol upon high performance liquid chromatography (HPLC). In addition, treatment of cells with progesterone caused lesser accumulation of radiolabel in products with retention times (RT) of 7.9 and 13.5 min on reverse-phase HPLC. The RT-13.5 compound was tentatively identified as cholesta-5,7,24-trien-3 beta-ol based on its relative retention and on its conversion to cholesterol upon incubation with untreated cells. The RT-7.9 compound was identified as 24 (S),25-epoxycholesterol (S-EC) based on its coelution with authentic S-EC and by its conversion to 25-hydroxycholesterol upon reduction with LiAlH4. Incubation of IEC-6 cells with chemically prepared S-EC resulted in dose-dependent suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity within 6 h (I50 = 0.3 microM). Pretreatment of cells with progesterone prevented this suppressive effect. No suppression of reductase activity was observed in progesterone-treated cells in spite of obvious accumulation of S-EC in amounts sufficient to effect regulation; instead, a 2-3-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity occurred within a 24-h period. Following the removal of progesterone from the culture medium, reductase activity declined rapidly over the next 6 h. However, IEC-6 cells could not metabolize S-EC, derived either endogenously or exogenously, during a similar time frame; nor did progesterone affect the uptake of exogenous S-EC by IEC-6 cells. These results show that although progesterone treatment of cultured cells promotes the synthesis of a natural oxysterol suppressor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the continued presence of progesterone prevents the regulatory action of S-EC. The unique nature of this interference is high-lighted by the observation that progesterone could not prevent the suppression of reductase activity by either 25-hydroxycholesterol or mevalonolactone.     

Subcellular localization of Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-microm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR-ER. Nevertheless, they represent a previously undescribed subcellular compartment likely capable of synthesizing mevalonate, which provides new evidence for multiorganelle compartmentalization of the isoprenoid biosynthetic pathways in plants.     

Slow binding inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase

The mechanism of slow binding inhibition of 3-hydroxy-3-methylglutaryl- coenzyme A reductase by lovastatin, fluindostatin, and related compounds was studied. Several of these compounds, including lovastatin, were found to be slow binding, while other less potent inhibitors were not. From a comparison of kinetic parameters obtained by steady-state measurements and progress curve analysis, it was concluded that the slow binding inhibitors bind by a mechanism which is more accurately described by biphasic binding than by single-step binding. The overall association rates of the slow binding inhibitors range from 1 x 10(6) to 4 x 10(-7) M-1 s-1, and the dissociation rates are in the range of 10(-3) s-1. The structures of slow binding and reversible inhibitors were compared by using molecular modeling methods. From these comparisons, it was proposed that the slow binding and very potent inhibition of, for instance, lovastatin, is not simply a result of binding of a transition state or reaction intermediate analogue. The various lipophilic groups of the inhibitors that do not seem to be related to structural features of the substrate may also play a crucial role in determining the mechanism of binding of HMGR inhibitors.     

Coordinate regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and prenyltransferase synthesis but not degradation in HepG2 cells

Human hepatoma HepG2 cells were used to demonstrate coordinate regulation of three enzymes of cholesterol synthesis under a variety of conditions. Addition of either delipidized serum and mevinolin or low density lipoprotein, 25-hydroxycholesterol, or mevalonic acid to HepG2 cells resulted in rapid changes both in the levels of the mRNAs and in the rates of synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and farnesyl pyrophosphate synthetase (prenyltranferase). In all cases, the changes in mRNA levels were paralleled by changes in the rates of specific protein synthesis. Pulse-chase techniques were used to determine the half-lives of all three proteins. Addition of low density lipoprotein to the media during the chase increased the rate of degradation of HMG-CoA reductase 4.6-fold but had no affect on the half-lives of HMG-CoA synthase or prenyltransferase. Therefore, we conclude that the coordinate regulation of these three enzymes under a variety of conditions occurs at the level of enzyme synthesis and not at the level of protein stability.     

Enzymes involved in the metabolism of 3-hydroxy-3-methylglutaryl-coenzyme A in Catharanthus roseus

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) is an important intermediate in various metabolic pathways, e.g. sterol biosynthesis, ketogenesis and leucine catabolism. The reactions and enzymes involved in the metabolism of HMG-CoA are briefly reviewed. These enzymes have been studied in Catharanthus roseus, a model system for studies on the regulation of secondary metabolic pathways, particularly those leading to terpenoidindole alkaloids. By using HPLC, three HMG-CoA catabolizing enzyme activities have been detected in protein extracts from suspension cultured C. roseus cells: HMG-CoA lyase, 3-nucleotidase and (tentatively identified) 3-methylglutaconyl-CoA hydratase (HMG-CoA hydrolyase). The enzymes have been partially purified. HMG-CoA is formed from three molecules of acetyl-CoA, via reactions which are catalyzed by two (as in yeast and animal cells, via intermediacy of acetoacetyl-CoA) or by just one enzyme (as in e.g. radish). It is yet not clear which process occurs in C. roseus.Abbreviations AACT acetoacetyl-CoA thiolase - AACT/HMGS acetoacetyl-COA thiolase/HMG-CoA synthase - CoASH coenzyme A (reduced form) - HMG-CoA 3-hydroxy-3-methylglutaryl-CoA - MG-CoA 3-methylglutaconyl-CoA     

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.     

Development of monoclonal antibodies to 3-hydroxy-3-methylglutaryl-coenzyme A reductase

This report describes the development of a series of monoclonal antibodies to rat liver 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR). Sera from hybridoma tumor-bearing mice were used to remove and characterize HMGR activity from a mixture of rat liver proteins. Two IgG₂ monoclonal antibodies removed separately greater than 80% HMGR activity while non-immune mouse or negative hybridoma-derived sera were ineffective. Radiolabeled immunoprecipitates of enzyme preparations resolved in one- and two-dimensional SDS-PAGE showed two predominant subunits at M_r 52,000 and 54,000. Our results indicate that in these preparations of rat liver proteins HMGR exists as a heteropolymer with at least two distinct subunits of different molecular weights.     

Regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in permeabilized cells.

We have studied the regulated degradation of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase within the endoplasmic reticulum in cells permeabilized with digitonin. Using Chinese hamster ovary cells transfected with a plasmid encoding HMGal, a chimeric protein containing the membrane domain of HMG-CoA reductase coupled to beta-galactosidase, we have demonstrated mevalonate and sterol-stimulated loss of beta-galactosidase activity. In pulse-chase experiments we have demonstrated mevalonate-stimulated degradation of both HMGal and HMG-CoA reductase. The rate of mevalonate-stimulated degradation observed in permeabilized cells tends to be slightly slower than that observed in intact cells treated with mevalonate and is dependent upon incubation of cells with mevalonate prior to permeabilization. The degradation process measured in this report extends a previous report of HMG-CoA reductase degradation in digitonin-permeabilized cells (Leonard, D. A., and Chen, H. W. (1987) J. Biol. Chem. 262, 7914-7919) by mimicking key physiological features of the in vivo process, including: stimulation by regulatory molecules, specifically mevalonate and sterols; inhibition by cycloheximide; and inhibition by an inhibitor of neutral cysteine proteases.     

Isolation and purification of 3-hydroxy-3-methylglutaryl-coenzyme A by ion-exchange chromatography

For precise determination of the catalytic activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34), the HMG-CoA employed as substrate must be free of HMG, CoA, and other inhibitors of HMG-CoA reductase activity. The standard purification of HMG-CoA by paper chromatography gives poor resolution of HMG-CoA from CoA and may be accompanied by some decomposition of HMG-CoA. We describe a simplified procedure for synthesis and for isolation from the reaction mixture of homogeneous, high specific activity [3(-14)C]HMG-CoA free of HMG, CoA, or nonpolar contaminants. Isolation of HMG-CoA utilizes ion-exchange chromatography in a gradient of ammonium formate, which is subsequently removed by lyophilization. The methods are proposed for use in the preparation or isolation of HMG0CoA.     

Characteristics of rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

A procedure for the preparation of rat liver microsomal fractions essentially devoid of contaminating lysosomes is described. When this preparation was examined by immunoblotting with a rabbit antiserum to rat 3-hydroxy-3-methylglutaryl-CoA reductase, a single band corresponding to an Mr of 100000 was observed. No evidence was found for glycosylation of rat liver-3-hydroxy-3-methylglutaryl-CoA reductase. Native rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase differs from the purified proteolytically modified species in that it displays allosteric kinetics towards NADPH.     

Identification of the catalytically important histidine of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

We identify His381 of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase as the basic residue functional in catalysis. The catalytic domain of 20 HMG-CoA reductases contains a single conserved histidine (His381 of the P. mevalonii enzyme). Diethyl pyrocarbonate inactivated the P. mevalonii enzyme, and hydroxylamine partially restored activity. We changed His381 to alanine, lysine, asparagine, and glutamine. The mutant proteins were overexpressed, purified to homogeneity, and characterized. His381 mutant enzymes were not inactivated by diethyl pyrocarbonate. All four mutant enzymes exhibited wild-type crystal morphology and chromatographed on substrate affinity supports like wild-type enzyme. The mutant enzymes had low catalytic activity (Vmax 0.06-0.5% that of wild-type enzyme), but Km values approximated those for wild-type enzyme. For wild-type enzyme and mutant enzymes H381A, H381N, and H381Q, Km values at pH 8.1 were 0.45, 0.27, 3.7, and 0.71 mM [(R,S)-mevalonate]; 0.05, 0.03, 0.20, and 0.11 mM [coenzyme A]; 0.22, 0.14, 0.81, and 0.62 mM [NAD+]. Km values at pH 11 for wild-type enzyme and mutant enzyme H381K were 0.32 and 0.75 mM [(R,S)-mevalonate]; 0.24 and 0.50 mM [coenzyme A]; 0.15 and 1.23 mM [NAD+]. Both pK values for the enzyme-substrate complex increased relative to wild-type enzyme (by 1-2.5 pH units for pK1 and by 0.5-1.3 pH units for pK2). For mutant enzyme H381K, the pK1 of 10.2 is consistent with lysine acting as a general base at high pH. His381 of P. mevalonii HMG-CoA reductase, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif of the catalytic domain of eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis.