Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse peritoneal macrophages.

The lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl-(HMG-) CoA reductase in cultured mouse peritoneal macrophages has been investigated. In contrast to what has been reported for other cells, HMG-CoA reductase activity is not suppressed by normal serum or by normal low density lipoproteins (LDL) from humans or dogs. Suppression of reductase activity occurred when cells were cultured in the presence of beta-migrating very low density lipoproteins (beta-VLDL) or LDL from hypercholesterolaemic dogs, or LDL modified by acetoacetylation. Human beta-VLDL from an atypical type III hyperlipoproteinaemic patient was also effective, as was apolipoprotein (apo) E-containing high density lipoproteins (HDL) from cholesterol-fed dogs (apo-E HDLc). The results indicate that cholesterol biosynthesis in mouse peritoneal macrophages is regulated by lipoprotein cholesterol entering via receptor-mediated endocytosis. Normal LDL were not effective because of the poor binding and uptake of these lipoproteins by the apo-B, E (LDL) receptor. Only beta-VLDL, apo-E HDLc, and hypercholesterolaemic LDL were avidly taken up by this receptor and were able to suppress HMG-CoA reductase. Acetoacetylated LDL were internalized via the acetyl-LDL (scavenger) receptor. Thus, mouse macrophages differ from human fibroblasts and smooth muscle cells in their physiological regulation of cholesterogenesis.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse uterine epithelial cells.

The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase was studied in mouse uterine epithelium. The enzyme was rapidly inactivated during incubation with ATP/Mg2+ in vitro, and could be re-activated by incubation with partially purified rat liver phosphoprotein phosphatase. Enzyme activity was rapidly inhibited by mevalonate injection in vivo to approx. 30% of control. The percentage of total enzyme active in vivo was measured by inclusion of NaF in the isolation buffers. The percentage of enzyme active in vivo 18 h after stimulation by oestrogens remained at approx. 25% after inhibition of activity by mevalonate injection, cholesterol feeding or progesterone pretreatment. However, 9 h after oestrogen stimulation, cholesterol feeding inhibited enzyme activity to 57% of control, 94% of which was in the active form. We conclude that, although all components for a reversible phosphorylative regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity are present in uterine epithelial cells, a role in the rapid changes in epithelial enzyme activity has not been demonstrated.     

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.     

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.     

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.     

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.     

Inactive 3-hydroxy-3-methylglutaryl-coenzyme A reductase in broken cell preparations of various mammalian tissues and cell cultures.

Preincubation of broken cell preparations from a variety of tissues and cell cultures resulted in an apparent increase in the level of 3-hydroxy-3-methylglutaryl-CoA reductase activity. However, apparent activation of the reductase in mouse liver, hepatomas and primary liver cell cultures was attributed largely to the loss, during the preincubation period, of an interfering enzyme, 3-hydroxy-3-methylglutaryl-CoA lyase. Among non hepatic cells and tissues (which did not contain appreciable lyase activity) the proportion of latent reductase was high in sonicates of fetal brain and in L cells and was independent of the level of total enzyme activity present. Activation of the reductase was blocked by hydroxymethylglutaryl-CoA and NADPH as well as by KF so that activation did not occur under the conditions of the enzyme assay. The enzyme was activated slowly at 4 degrees C, so that partial activation of the latent form occurred during isolation of the microsomal fraction by differential centrifugation. The reductase present in sonicates of cells with either a high or low proportion of the latent enzyme was inactivated by incubation with ATP and Mg2+. Suppression of reductase activity in L cell cultures by treatment with 25-hydroxycholesterol and an age-related decline in brain enzyme activity did not involve reversible conversion of the reductase to an inactive form.     

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.     

Localization and regulation of epidermal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity by barrier requirements

Recent studies have shown that epidermal cholesterol synthesis is regulated by HMG CoA reductase activity and that this activity is modulated by changes in the cutaneous permeability barrier. Here, we quantitated HMG CoA reductase activity after acute and chronic barrier disruption in the upper and lower layers of murine epidermis. In unperturbed epidermis, 13 and 87% of enzyme activity localized to the upper and lower epidermis, respectively, with the majority of activity in the stratum basale. Acute barrier disruption with either acetone or sodium dodecylsulfate provoked an increase in HMG CoA reductase activity (54% and 30%) in the lower layers, but only a small change in the upper layers. However, the activation state of the enzyme was increased 50% in the upper epidermis. Correction of barrier function by occlusion with an impermeable Latex wrap prevented the increase both in enzyme activity and activation state. After chronic barrier disruption; i.e., essential fatty acid deficient (EFAD) diet, HMG CoA reductase activity was increased in the upper epidermis (161%); a change prevented by occlusion. These results show: (1) that HMG CoA reductase activity is present in both the upper and lower cell layers; (2) that acute insults to barrier integrity stimulate enzyme activity in both the upper and lower epidermis; and (3) that chronic insults provoke an increase in enzyme activity in the upper layers. These studies provide further insights into the linkage of the permeability barrier with epidermal cholesterol metabolism.     

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.     

Activation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in homogenates of developing rat brain.

The specific activity of 3-hydroxy-3-methylglutaryl-CoA reductase increases when homogenates of developing rat brain are incubated at 37 degrees C or kept on ice. This increase is completely blocked by the addition of F- to the homogenization medium and the assay mixture. The capacity for activation of the reductase is greatest during the early postnatal period and declines as brain maturation proceeds. The data suggest that catalytic modification of the reductase may play a role in the regulation of cholesterol synthesis in the developing brain.     

Effect of docosahexaenoic acid on brain 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in male ICR mice

We investigated the influence of docosahexaenoic acid ethyl ester (DHA-EE) on 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity in the brains of adult and aged mice. Male mice (Crlj:CD-1) were fed diets containing 3% lard plus 2% linoleic acid ethyl ester (LA-EE), or 2% DHA-EE, for 3 months. The brain HMG-CoA reductase activity of 8-month-old (adult) mice was not significantly influenced by dietary intake of DHA-EE. However, in 18-month-old (aged) mice, its activity was enhanced with dietary intake of DHA-EE. Brain HMG-CoA reductase activity and brain cholesterol content significantly increased with age. Hepatic HMG-CoA reductase activity and the cholesterol content of both adult and aged mice were reduced in DHA-EE diet groups, compared with LA-EE diet groups. The DHA percentages of brain and liver microsomal fractions increased with the intake of DHA-EE in adult and aged mice. These results suggest that DHA may enhance brain HMG-CoA reductase activity in aged mice.     

Novel and simple high-performance liquid chromatographic method for determination of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity

We present here a high-performance liquid chromatographic method for the evaluation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. The automated method was applied to fungal and mouse liver extracts and validated by the addition of mevastatin to the reaction mixture and by several intra- and inter-day assays. This method offers important advantages over those previously reported because no radiolabeled substrates or expensive techniques such as mass spectrometry are required, and the time of analysis is relatively short. Moreover, the method can be successfully applied to different biological samples; hence, it should be very useful in evaluating potential inhibitors of the HMG-CoA enzyme and investigating cholesterol metabolism, cell growth and differentiation processes.     

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.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by wounding and methyl jasmonate

HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase; E.C.1.1.1.34) supplies mevalonate for the synthesis of many plant primary and secondary metabolites, including the terpenoid component of indole alkaloids. Suspension cultures of Camptotheca acuminata and Catharanthus roseus, two species valued for their anticancer indole alkaloids, were treated with the elicitation signal transducer methyl jasmonate (MeJA). RNA gel blot analysis from MeJA treated cultures showed a transient suppression of HMGR mRNA, followed by an induction in HMGR message. Leaf disks from transgenic tobacco plants containing a chimeric hmgl::GUS construct were also treated with MeJA and showed a dose dependent suppression of wound-inducible GUS activity. The suppression of the wound response by MeJA was limited to the first 4 h post-wounding, after which time MeJA application had no effect. The results are discussed in relation to the differential regulation of HMGR isogenes in higher plants.Abbreviations GUS -glucuronidase - hmg gene of hmgr - HMGR 3-hydroxy-3-methylglutaryl-coenzyme A reductase - JA jasmonic acid - MeJA methyl jasmonate - MUG methylumbelliferyl--d-glucuronide - TDC tryptophan decarboxylase - SDS sodium dodecyl sulfate - SS strictosidine synthase