Phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase

Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase has been purified to apparent homogeneity by a process involving the following steps: solubilization from microsomes and chromatography on Affi-Gel Blue, phosphocellulose, Bio-Gel A 1.5m, and agarose-hexane-ATP. The apparent M_r of the purified enzyme as judged by gel-filtration chromatography is 205,000 and by sodium dodecyl sulfate-gel electrophoresis is 105,000. Immunoprecipitation of homogeneous reductase phosphorylated by reductase kinase and [γ-³²P]ATP produces a unique band containing ³²P bound to protein which migrates at the same R_f as the reductase subunit. Incubation of ³²P-labeled HMG-CoA reductase with reductase phosphatase results in a time-dependent loss of protein-bound ³²P radioactivity, as well as an increase in enzymic activity. Reductase kinase, when incubated with ATP, undergoes autophosphorylation, and a simultaneous increase in its enzymatic activity is observed. Tryptic treatment of immunoprecipitated, ³²P-labeled HMG-CoA reductase phosphorylated with reductase kinase produces only one ³²P-labeled phosphopeptide with the same R_f as one of the two tryptic phosphopeptides that have been reported in a previous paper. The possible existence of a second microsomal reductase kinase is discussed.     

Molecular dynamics study of conformational changes in human serum albumin by binding of fatty acids

Human serum albumin (HSA) binds with fatty acids under normal physiologic conditions. To date, there is little published information on the tertiary structure of HSA-fatty acid complex in aqueous solution. In the present study, we used molecular dynamics (MD) simulations to elucidate possible structural changes of HSA brought about by the binding of fatty acids. Both unliganded HSA and HSA-fatty acid complex models for MD calculations were constructed based on the X-ray crystal structures. Five myristates (MYRs) were bound in the HSA-fatty acid complex model. In the present MD study, the motion of domains I and III caused by the binding of MYR molecules increased the radius of gyration of HSA. Root-mean-square fluctuations from the MD simulations revealed that the atomic fluctuations of the specific amino acids at drug-binding site I that can regulate the drug-binding affinity were increased by the binding of MYR molecules. Primary internal motions, characterized by the first three principal components, were observed mainly at domains I and III in the principal component analysis for trajectory data. The directional motion projected on the first principal component of unliganded HSA was conserved in HSA-MYR complex as the third principal directional motion with higher frequency. However, the third principal directional motion in unliganded HSA turned into the first principal directional motion with lower frequency in the HSA-MYR complex. Thus, the present MD study provides insights into the possible conformational changes of HSA caused by the binding of fatty acids.     

Isolation of a monocot 3-hydroxy-3-methylglutaryl coenzyme A reductase gene that is elicitor-inducible

The rice (Oryza sativa) phytoalexins, momilactones and oryzalexins, are synthesized by the isoprenoid pathway. An early step in this pathway, one that is rate-limiting in mammalian systems, is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). A gene that encodes this enzyme has been isolated from rice, and found to contain an open reading frame of 1527 bases. The encoded protein sequence of the rice HMGR appears to be conserved with respect to other HMGR proteins, and 1 or 2 membrane-spanning domains characteristic of plant HMGRs are predicted by a hydropathy plot of the amino acid sequence. The protein is truncated at its 5 end, and shows reduced sequence conservation in this region as compared to other plant sequences. The rice genome contains a small family of HMGR genes. The isolated gene, HMGR I, is expressed at low levels in both vegetative and floral organs of rice plants. It is not induced in plants by wounding, but is strongly and rapidly induced in suspension cells by a fungal cell wall elicitor from the pathogenMagnaporthe grisea, causal agent of rice blast disease. This suggests that HMGR I may be important in the induction of rice phytoalexin biosynthesis in response to pathogen attack, and therefore may play a key role as a component of the inducible defense mechanism in rice.     

The inhibition of degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by sterol regulatory element binding protein cleavage-activating protein requires four phenylalanine residues in span 6 of HMG-CoA reductase transmembrane domain

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme in the cholesterol biosynthetic pathway. This endoplasmic reticulum membrane protein contains a cytosolic catalytic domain and a transmembrane domain with eight membrane spans that are necessary for sterol-accelerated degradation. Competition experiments showed that wild-type transmembrane domains of HMGR and sterol regulatory element binding protein cleavage-activating protein (SCAP) blocked sterol-accelerated degradation of intact HMGR and HMGal, a model protein containing the membrane domain of HMGR linked to Escherichia coli beta-galactosidase. However, mutant transmembrane domains of HMGR and SCAP whose sterol-sensing functions were abolished did not inhibit sterol-accelerated degradation of HMGR and HMGal. In addition, our mutagenesis studies on HMGal indicated that four Phe residues conserved in span 6 of HMGR and the sterol-sensing domains of other sterol-related proteins are required for the regulated degradation of HMGR. These results suggest that HMGR and SCAP compete for binding to a sterol-regulated regulator protein, and this binding may need the four Phe residues.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels by L-triiodothyronine

In hypophysectomized rats, hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, immunoreactive 97-kilodalton (97-kDa) protein, and mRNA were all reduced to undetectable levels. Administration of triiodothyronine (T3) resulted in large increases in all three after a 36-h lag period. HMG-CoA reductase activity, immunoreactive 97-kDa protein levels, and reductase mRNA levels were tightly correlated. Feeding hypophysectomized rats diets containing the bile acid sequestrant colestipol, together with the potent reductase inhibitor mevinolin, resulted in an increase in HMG-CoA reductase activity similar to that seen with T3 but a lesser stimulation of reductase mRNA levels. These results suggest that agents which cause depletion of mevalonate-derived products may share in part with T3 a common mechanism for increasing levels of HMG-CoA reductase activity in order to satisfy cellular needs for these products. Dexamethasone treatment, which is known to prevent the T3-mediated stimulation of reductase activity, caused a marked decrease in 97-kDa immunoreactive material but had little effect on reductase mRNA levels.     

Regulation of luteal cell 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by estradiol

Recent studies have suggested that estradiol or androgen precursor may stimulate steroidogenesis in the luteal cell by modulating intracellular sterol availability and metabolism. This investigation was performed to examine the effect of estradiol on de novo synthesis of cholesterol. Pregnant rats hypophysectomized and hysterectomized on Day 12 were treated for 72 h with either estradiol or testosterone. De novo cholesterol synthesis was determined by measurement of the specific activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis, in microsome-enriched preparations of luteal tissue and incorporation of [14C] acetate into cholesterol by corpora lutea incubated in vitro. Estradiol or testosterone treatment caused a 4- to 5-fold stimulation of luteal cholesterol biosynthesis, as measured by these techniques. NaF, an inhibitor of phosphatase which blocks the conversion of the inactive enzyme to the active form, reduced the HMG CoA reductase activity to 30% in corpora lutea obtained from either steroid or vehicle-treated rats. However, an increase in enzyme activity of comparable magnitude by steroids was observed whether microsomes were isolated with or without NaF. The effect of estradiol appears to be enzyme-specific, since it failed to affect the microsomal marker, NADPH-cytochrome c reductase. Since the cholesteryl ester content of corpora lutea falls in response to steroid treatment, rats were treated with 4-aminopyrazolo-[3,4d]pyrimidine (4-APP) to deplete cellular cholesterol content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and immunological characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from human liver

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) has been isolated from human liver utilizing HMG-CoA affinity chromatography. The apparent monomer molecular weight of purified human HMG-CoA reductase by SDS-gel electrophoresis was 53,000, and the oligomeric molecular weight determined by sucrose density centrifugation was 104,000. A monospecific antibody prepared against rat liver HMG-CoA reductase inhibited the enzymic activity of microsomal and purified human liver enzyme and formed a single immunoprecipitin line by radial immunodiffusion. These results represent the initial isolation and characterization of human liver HMG-CoA reductase.     

Molecular dynamics investigations of structural and functional changes in Bcl-2 induced by the novel antagonist BDA-366

Apoptosis is a fundamental biological phenomenon, in which anti- or proapoptotic proteins of the Bcl-2 family regulate a committed step. Overexpression of Bcl-2, the prototypical antiapoptotic protein in this family, is associated with therapy resistance in various human cancers. Accordingly, Bcl-2 inhibitors intended for cancer therapy have been developed, typically against the BH3 domain. Recent experimental evidences have shown that the antiapoptotic function of Bcl-2 is not immutable, and that BDA-366, a novel antagonist of the BH4 domain, converts Bcl-2 from a survival molecule to an inducer of cell death. In this study, the underlying mechanisms of this functional conversion were investigated by accelerated molecular dynamics simulation. Results revealed that Pro127 and Trp30 in the BH4 domain rotate to stabilize BDA-366 via π-π interactions, and trigger a series of significant conformational changes of the α3 helix. This rearrangement blocks the hydrophobic binding site (HBS) in the BH3 domain and further prevents binding of BH3-only proteins, which consequently allows the BH3-only proteins to activate the proapoptotic proteins. Analysis of binding free energy confirmed that BDA-366 cross-inhibits BH3-only proteins, implying negative cooperative effects across separate binding sites. The newly identified blocked conformation of the HBS along with the open to closed transition pathway revealed by this study advances the understanding of the Bcl-2 transition from antiapoptotic to proapoptotic function, and yielded new structural insights for novel drug design against the BH4 domain.

Communicated by Ramaswamy H. Sarma     

Expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in maize tissues

The activity of the enzyme 3-hydroxy-3-methlglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34) is highly expressed in 4-day-old etiolated seedlings of normal (cv. DeKalb XL72AA), dwarf ( d ₅) and albino ( lw ₃) maize ( Zea mays L.). HMGR activity of maize seedlings appeared to be exclusively associated with the microsomal rather than the plastidic fraction of maize cells. Maize tissues with high meristematic activity such as germinating seeds, leaf bases, root tips and the site of origin of lateral roots contained high levels of microsomal HMGR activity. The activity of HMGR extracted from leaf tips of normal, dwarf and albino maize seedlings is regulated by light. Microsomal HMGR activity from leaf tips of 4-day-old maize seedlings was inhibited significantly following exposure to strong light (600 μmol m⁻² s⁻¹) for more than 10 h. By comparison, microsomal HMGR activity from leaf bases and root tips of maize was not inhibited by exposure to strong light. These results suggest that the microsomal HMGR which is highly expressed in maize may be related to sterol biosynthesis and membrane biogenesis rather than plastidic-associated isoprenoid synthesis and that light may regulate HMGR activity indirectly by increasing cell differentiation.     

Proteinase involvement in the solubilization of 3-hydroxy-3-methylglutaryl coenzyme a reductase

This paper demonstrates that a heavy particle fraction, which contains lysosomes, is required for the solubilization of HMG-CoA reductase from rat liver microsomes by the widely-used slow freeze-thaw procedure. This solubilization is effectively inhibited by the proteinase inhibitors, leupeptin and antipain, but not by phenylmethylsulfonyl fluoride, pepstatin A or N-α-p-tosyl-L-lysine methyl ester. These results suggest that a thiol proteinase, possibly derived from lysosomes, is responsible for solubilizing the reductase.     

Developmental pattern of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the rat

The activity, protein concentration and catalytic efficiency of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase was determined in rats aged 1 to 199 days. Microsomal enzyme total activity peaked on day 24, during weaning, and again on day 63, during the onset of puberty. Increased enzyme activity during weaning resulted primarily from an increase in the catalytic efficiency of the enzyme with a slight reduction in enzyme protein content. The rise in enzyme activity during the onset of puberty, however, was primarily the result of an increase in enzyme protein concentration. Thus, the activity of reductase in mammalian livers reflects, at different stages in development, the modulating influence of both the total number of reductase molecules and the catalytic efficiency of the enzyme.     

Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase in HeLa cells by glucocorticoids.

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) has been demonstrated both in homogenates and microsomes of the S3G strain of HeLa cells. It was increased 8- to 10-fold by the removal of serum from the growth medium. The presence of steroids, specifically of the glucocorticoid series, in the serum-less growth medium elicited an additional 100 to 345% increase over the serum-less control, whereas the addition of N6,O2'-dibutyryl adenosine 3':5'-monophosphate to the medium or dexamethasone to the assay mixture was without any stimulatory effect. Both inductions were blocked by cycloheximide and actinomycin D, suggesting a protein synthesis-dependent elevation of enzyme activity. Glucocorticoids were effective in the induction at concentrations ranging from 10(-6) to 10(-8) M and there was a demonstrated parallel between the magnitude of enzyme induction and glucocorticoid potency. The HMG-CoA reductase activities from steroid-induced and control cultures had identical assay characteristics (pH optima and apparent Km values for both NADPH and HMG-CoA). This induction of the rate-controlling enzyme of cholesterogenesis occurred despite the observation that glucocorticoids specifically depress the rate of acetate or water, but not mevalonate, incorporation into cholesterol.     

ATP-dependent degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in permeabilized cells

A system for the assay of 3-hydroxy-3-methyglutaryl (HMG) coenzyme A (CoA) reductase in digitonin-permeabilized Chinese hamster ovary cells is described. Under these conditions, HMG-CoA reductase remained intact and associated with the endoplasmic reticulum, and values for Km (HMG-CoA), Ki (mevinolin), and active/total activity were similar to those seen in sonicated cell preparations. However, the mechanism by which this rapidly turned over (half-life approximately 2 h) enzyme is degraded was disrupted. Addition of ATP at physiological concentrations to digitonin-permeabilized cells resulted in the rapid, irreversible loss of enzyme activity. Immunoblot analysis showed that this loss of activity was followed by cleavage of the intact 97-kilodalton enzyme to a 68-kilodalton fragment which was distinct from the catalytically active fragments generated by nonspecific proteolysis in sonicated cell homogenates. Assay of a lysosomal marker enzyme confirmed that ATP-mediated inactivation and cleavage of reductase was not due to release of lysosomal proteases. The possible role of ATP in phosphorylation, inactivation, and degradation of reductase is discussed.     

Coordinate regulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A synthase but not 3-hydroxy-3-methylglutaryl coenzyme A reductase in C-6 glia

The effects on cholesterol biosynthesis of growth of cultured C-6 glial cells in serumfree medium ± supplementation with linoleic or linolenic acid were studied. Markedly higher activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) were observed in cells grown in linoleate- or linolenate-supplemented versus nonsupplemented medium. After 48 h HMG-CoA reductase activities were two-and four-fold higher in cells supplemented with 20 and 100 μm linoleate, respectively. The increase in activity became apparent after 24 h and was marked after 48 h. Rates of incorporation of [¹⁴C]acetate or ³H₂O into sterols did not reflect the changes in reductase activity. Thus, in cells supplemented with 50 μm linoleate for 24 and 48 h rates of incorporation of [¹⁴C]acetate were 75–80% lower than rates in nonsupplemented cells. This difference resulted because over the first 24 h of the experiment a fivefold increase in the rate of sterol synthesis occurred in the nonsupplemented cells, whereas essentially no change occurred in the linoleate-supplemented cells; little further change occurred between 24 and 48 h in the nonsupplemented and the linoleate-supplemented cells. That the difference in sterol synthesis under these experimental conditions could be mediated at the level of HMG-CoA synthase (EC 4.1.3.5) was suggested by two series of findings, i.e., first, similar quantitative and temporal changes in the activity of this enzyme, and, second, no change in the activity of acetoacetyl-CoA thiolase (EC 2.3.1.9) or the incorporation of [¹⁴C]mevalonate into sterols. Thus, the data suggest that HMG-CoA synthase, and not HMG-CoA reductase, may direct the rate of cholesterol biosynthesis under these conditions of serum-free growth ± supplementation with polyunsaturated fatty acid.     

Infrared studies of water induced conformational changes in bacteriorhodopsin

Fourier transform infrared difference spectroscopy has been used to study the effect of water on the conformation of bacteriorhodopsin. The infrared spectra as a function of water content show a conformational change at about 0.06 g H₂O/g bacteriorhodopsin. By an interference method the thickness of the sample was measured and shows similar behavior as a function of water content. This study gives insight into the process of water absorption by purple membrane. The observations are in good agreement with those found for other proteins.Abbreviations IR infrared - FTIR Fourier transform IR