The regulated degradation of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase reporter construct occurs in the endoplasmic reticulum

1994 ◽  
Vol 107 (9) ◽  
pp. 2635-2642
Author(s):  
L.W. Lecureux ◽  
B.W. Wattenberg
Keyword(s):  
Endoplasmic Reticulum ◽  
Coenzyme A ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Dna Encoding ◽  
Hmg Coa Reductase ◽  
Steady State Levels ◽  
Coa Reductase ◽  
Membrane Anchoring ◽  
Beta Galactosidase

The rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase, is regulated at a number of levels. One important mechanism is regulation of the half-life of the protein by a controlled proteolytic system. This comes about in response to downstream products of the sterol biosynthetic pathway. Little is known about this system, including where in the cell this regulated degradation occurs. HMG CoA reductase resides in the endoplasmic reticulum. To localize the site of regulated degradation of HMG CoA reductase, we used a construct that fuses the N-terminal membrane-anchoring domain of HMG CoA reductase in-frame with beta-galactosidase as a reporter domain (HM-Gal). HM-Gal has previously been shown to reproduce faithfully the degradative properties of native HMG CoA reductase (Chun et al. (1990) J. Biol. Chem. 265, 22004–22010). CHO cells transfected with DNA encoding HM-Gal were exposed to mevalonic acid, which enhances the rate of HMG CoA reductase degradation several fold, and leads to the reduction of the steady state levels of HM-Gal by 80–90%. To accumulate HMG CoA reductase at the site of degradation, cells were simultaneously treated with N-acetyl-leucyl-leucyl-norleucinal (ALLN), which inhibits the protease responsible for reductase degradation. HM-Gal was localized morphologically by immunofluorescence and biochemically by measuring beta-galactosidase activity in Percoll gradients of cellular homogenates. Using either technique HM-Gal localization was indistinguishable from that of ER markers in both control cells and in cells treated to accumulate HMG CoA reductase at the site of degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-Dependent Effect of Hydroxymethylglutaryl – Coenzyme A Reductase Inhibitor on Serum Cholesterol with Limited Dietary Restrictions: A Case Study

1993 ◽  
Vol 21 (2) ◽  
pp. 105-111
Author(s):  
S Okada ◽  
K Ichiki ◽  
S Tanokuchi ◽  
Z Ota
Keyword(s):  
Serum Cholesterol ◽  
Reductase Inhibitor ◽  
Coenzyme A ◽  
Cholesterol Biosynthesis ◽  
Dependent Diabetes Mellitus ◽  
Hmg Coa Reductase ◽  
Dietary Restrictions ◽  
Hmg Coa Reductase Inhibitor ◽  
Coa Reductase ◽  
Dose Dependent

Hydroxymethylglutaryl–coenzyme A (HMG–CoA) reductase inhibitor (pravastatin sodium) can selectively inhibit cholesterol biosynthesis in the liver and may lower serum cholesterol concentrations even where there are no particular dietary restrictions. A 72-year old housewife with non-insulin-dependent diabetes mellitus complicated by hyperlipaemia type IIb, who did not follow directions for diet therapy or kinesitherapy, was administered HMG–CoA reductase inhibitor. The initial dose of 10 mg/day HMG–CoA reductase inhibitor was increased by 10 mg/day every 4 weeks to 30 mg/day, maintained at 30 mg/day for 8 weeks and then reduced gradually until discontinuation after a further 27 weeks. Test results showed the changes in low-density lipoprotein cholesterol and apoprotein B to be dose-dependent. The findings represent the first clinical evidence that hypercholesterolaemia can be adequately managed by the use of HMG–CoA reductase inhibitor, even when no specific dietary restrictions are imposed, and may contribute to improvements in the quality of daily life for many patients suffering from hyperlipaemia type IIb.

scholarly journals Regulation of hepatic cholesterol biosynthesis. Effects of a cytochrome P-450 inhibitor on the formation and metabolism of oxygenated sterol products of lanosterol

1989 ◽  
Vol 264 (2) ◽  
pp. 495-502 ◽  
Author(s):  
J Iglesias ◽  
G F Gibbons
Keyword(s):  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Subcellular Fractions ◽  
Hmg Coa Reductase ◽  
Hepatocyte Cultures ◽  
C 32 ◽  
Lanosterol Demethylase ◽  
Cholesterol Precursor ◽  
Coa Reductase

The involvement of oxygenated cholesterol precursors in the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was studied by examining the effect of ketoconazole on the metabolism of mevalonic acid, lanosterol and the lanosterol metabolites, lanost-8-ene-3 beta,32-diol,3 beta-hydroxylanost-8-en-32-al and 4,4-dimethylcholesta-8,14-dien-3 beta-ol, in liver subcellular fractions and hepatocyte cultures. Inhibition of cholesterol synthesis from mevalonate by ketoconazole at concentrations up to 30 microM was due exclusively to a suppression of cytochrome P-450LDM (LDM = lanosterol demethylase) activity, resulting in a decreased rate of lanosterol 14 alpha-demethylation. No enzyme after the 14 alpha-demethylase step was affected. When [14C]mevalonate was the cholesterol precursor, inhibition of cytochrome P450LDM was accompanied by the accumulation of several labelled oxygenated sterols, quantitatively the most important of which was the C-32 aldehyde derivative of lanosterol. There was no accumulation of the 24,25-oxide derivative of lanosterol, nor of the C-32 alcohol. Under these conditions the activity of HMG-CoA reductase declined. The C-32 aldehyde accumulated to a far greater extent when lanost-8-ene-3 beta,32-diol rather than mevalonate was used as the cholesterol precursor in the presence of ketoconazole. With both precursors, this accumulation was reversed at higher concentrations of ketoconazole in liver subcellular fractions. A similar reversal was not observed in hepatocyte cultures.

Genetic and biochemical evaluation of eucaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl coenzyme A reductase

1990 ◽  
Vol 10 (2) ◽  
pp. 672-680
Author(s):  
C Sengstag ◽  
C Stirling ◽  
R Schekman ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Coenzyme A ◽  
Transmembrane Domains ◽  
Protein Domain ◽  
Yeast Cells ◽  
Endoplasmic Reticulum Membrane ◽  
Hmg Coa Reductase ◽  
Coa Reductase ◽  
Histidinol Dehydrogenase

Both 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase isozymes of the yeast Saccharomyces cerevisiae are predicted to contain seven membrane-spanning domains. Previous work had established the utility of the histidinol dehydrogenase protein domain, encoded by HIS4C, as a topologically sensitive monitor that can be used to distinguish between the lumen of the endoplasmic reticulum and the cytoplasm. This study directly tested the structural predictions for HMG-CoA reductase by fusing the HIS4C domain to specific sites in the HMG-CoA reductase isozymes. Yeast cells containing the HMG-CoA reductase-histidinol dehydrogenase fusion proteins grew on histidinol-containing medium if the HIS4C domain was present on the cytoplasmic side of the endoplasmic reticulum membrane but not if the HIS4C domain was targeted to the endoplasmic reticulum lumen. Systematic exchanges of transmembrane domains between the isozymes confirmed that both isozymes had equivalent membrane topologies. In general, deletion of an even number of putative transmembrane domains did not interfere with the topology of the protein, but deletion or duplication of an odd number of transmembrane domains inverted the orientation of the protein. The data confirmed the earlier proposed topology for yeast HMG-CoA reductase, demonstrated that the yeast enzymes are core glycosylated, and provided in vivo evidence that the properties of transmembrane domains were, in part, dependent upon their context within the protein.

scholarly journals 3-Hydroxy-3-methylglutaryl coenzyme A reductase localization in rat liver peroxisomes and microsomes of control and cholestyramine-treated animals: quantitative biochemical and immunoelectron microscopical analyses.

1986 ◽  
Vol 103 (3) ◽  
pp. 875-886 ◽  
Author(s):  
G A Keller ◽  
M Pazirandeh ◽  
S Krisans
Keyword(s):  
Rat Liver ◽  
Coenzyme A ◽  
Specific Activity ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Normal Liver ◽  
Hmg Coa Reductase ◽  
The Matrix ◽  
Coa Reductase ◽  
Liver Peroxisomes

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key regulatory enzyme involved in cholesterol biosynthesis, has recently been reported to be present in rat liver peroxisomes (Keller, G.A., M.C. Barton, D.J. Shapiro, and S.J. Singer, 1985, Proc. Natl. Acad. Sci. USA, 82:770-774). Immunoelectron labeling of ultrathin frozen sections of normal liver, using two monoclonal antibodies to purified rat liver microsomal HMG-CoA reductase, indicated that the enzyme is present in the matrix of peroxisomes. This study is a quantitative biochemical and immunoelectron microscopical analysis of HMG-CoA reductase in rat liver peroxisomes and microsomes of normal and cholestyramine-treated animals. Cholestyramine treatment produced a six- to sevenfold increase in the specific activity of peroxisomal HMG-CoA reductase, whereas the microsomal HMG-CoA reductase specific activity increased by about twofold. Using a computer program that calculates optimal linear combinations of marker enzymes, it was determined that between 20 and 30% of the total reductase activity was located in the peroxisomes of cholestyramine-treated animals. Less than 5% of the reductase activity was present in peroxisomes under control conditions. Quantitation of the immunoelectron microscopical data was in excellent agreement with the biochemical results. After cholestyramine treatment there was an eightfold increase in the density of gold particles per peroxisome, and we estimate about a threefold increase in the labeling of the ER.

scholarly journals Biogenesis of the crystalloid endoplasmic reticulum in UT-1 cells: evidence that newly formed endoplasmic reticulum emerges from the nuclear envelope.

1986 ◽  
Vol 102 (6) ◽  
pp. 2158-2168 ◽  
Author(s):  
R K Pathak ◽  
K L Luskey ◽  
R G Anderson
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Cholesterol Biosynthesis ◽  
Integral Membrane Protein ◽  
Enzyme Synthesis ◽  
Hmg Coa Reductase ◽  
Outer Nuclear Membrane ◽  
Sequence Of Events ◽  
Coa Reductase

The crystalloid endoplasmic reticulum (ER), a specialized smooth ER of the compactin-resistant UT-1 cell, is composed of multiple membrane tubules packed together in a hexagonal pattern. This membrane contains large amounts of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, an integral membrane protein that enzymatically regulates endogenous cholesterol biosynthesis. Using morphological and immunocytochemical techniques, we have traced the sequence of events in the biogenesis of this ER when compactin-withdrawn UT-1 cells, which do not have a crystalloid ER, are incubated in the presence of compactin. After 15 h of incubation in the presence of compactin, many cells had profiles of ER cisternae that were juxtaposed to the nuclear envelope and studded with ribosomes on their outer membrane. Both the outer nuclear membrane and the ER membrane contained HMG CoA reductase; however, there was little or no detectable enzyme in rough ER that was free in the cytoplasm. With longer times of incubation in the presence of compactin, these cells had lamellar stacks of smooth ER next to the nuclear envelope that contained HMG CoA reductase. Coordinate with the appearance of the smooth ER, crystalloid ER appeared in the same cell. Often regions of continuity were found between the membrane of the smooth ER and the membrane of the crystalloid ER tubules. These studies suggest that HMG CoA reductase is synthesized along the outer nuclear membrane and in response to increased enzyme synthesis, a membrane emerges from the outer nuclear membrane as smooth ER cisternae, which then transforms into crystalloid ER tubules.

scholarly journals Cholesterol and mevalonic acid are independent requirements for the in vitro proliferation of human bone marrow granulocyte progenitor cells: studies using ML-236B

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 667-671
Author(s):  
PC Hoffman ◽  
CM Richman ◽  
RA Larson ◽  
S Yachnin
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Human Bone ◽  
Hmg Coa Reductase ◽  
Coa Reductase

ML-236B is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the key regulatory enzyme in the sequence that catalyzes the conversion of acetate to mevalonic acid in cholesterol biosynthesis. This compound caused marked inhibition of human bone marrow granulocyte progenitor cell (CFU-C) proliferation, the 50% inhibitory concentration (IHD50) being 2.0 X 10(6)M. Inhibition of colony formation was reversed by mevalonic acid but not by cholesterol. ML-236B also inhibited DNA synthesis and acetate incorporation into cholesterol in marrow mononuclear cells (IHD50 = 5.6 x 10(6)M and 3.2 x 10(7)M, respectively). No inhibition of mevalonate incorporation into cholesterol was observed. These results differ from those observed with 25-hydroxycholesterol, another inhibitor of HMG CoA reductase. The latter compound also inhibited CFU-C proliferation and cholesterol biosynthesis from acetate; inhibition of colony formation was reversed by cholesterol but not by mevalonic acid. In addition, 25- hydroxycholesterol inhibited cholesterol synthesis from mevalonic acid precursor. We conclude that: (1) ML-236B is a potent inhibitor of CFU-C proliferation, DNA synthesis, and cholesterol biosynthesis from acetate precursor in marrow mononuclear cells; (2) the effects of ML-236B are completely reversed by mevalonic acid but not by cholesterol, suggesting that mevalonic acid per se or one or more of its nonsterol products are critical for cell growth; (3) the inhibitory effects of 25- hydroxycholesterol on CFU-C proliferation and cholesterol biosynthesis are not solely a result of its inhibition of HMG CoA reductase, but are due in part to inhibition of enzymatic steps distal to mevalonic acid in the sterol synthetic pathway; and (4) mevalonic acid and cholesterol are independent requirements for CFU-C proliferation and differentiation in vitro.

scholarly journals 3-Hydroxymethyl coenzyme A reductase inhibition attenuates spontaneous smooth muscle tone via RhoA/ROCK pathway regulated by RhoA prenylation

2010 ◽  
Vol 298 (6) ◽  
pp. G962-G969 ◽  
Author(s):  
Satish Rattan
Keyword(s):  
Smooth Muscle ◽  
Coenzyme A ◽  
Muscle Tone ◽  
Cholesterol Biosynthesis ◽  
Rho Kinase ◽  
Hmg Coa Reductase ◽  
Smooth Muscle Tone ◽  
Tonic Smooth Muscle ◽  
Coa Reductase

RhoA prenylation may play an important step in the translocation of RhoA in the basal internal anal sphincter (IAS) smooth muscle tone. Statins inhibit downstream posttranslational RhoA prenylation by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition (HMGCRI). The role of statins in relation to RhoA prenylation in the pathophysiology of the spontaneously tonic smooth muscle has not been investigated. In the present studies, we determined the effect of classical HMGCRI simvastatin on the basal IAS tone and RhoA prenylation and in the levels of RhoA/Rho kinase (ROCK) in the cytosolic vs. membrane fractions of the smooth muscle. Simvastatin produced concentration-dependent decrease in the IAS tone (via direct actions at the smooth muscle cells). The decrease in the IAS tone by simvastatin was associated with the decrease in the prenylation of RhoA, as well as RhoA/ROCK in the membrane fractions of the IAS, in the basal state. The inhibitory effects of the HMGCRI were completely reversible by geranylgeranyltransferase substrate geranylgeranyl pyrophosphate. Relaxation of the IAS smooth muscle via HMGCRI simvastatin is mediated via the downstream decrease in the levels of RhoA prenylation and ROCK activity. Studies support the concept that RhoA prenylation leading to RhoA/ROCK translocation followed by activation is important for the basal tone in the IAS. Data suggest that the role of HMG-CoA reductase may go beyond cholesterol biosynthesis, such as the regulation of the smooth muscle tone. The studies have important implications in the pathophysiological mechanisms and in the novel therapeutic approaches for anorectal motility disorders.

scholarly journals Genetic and biochemical evaluation of eucaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl coenzyme A reductase.

1990 ◽  
Vol 10 (2) ◽  
pp. 672-680 ◽  
Author(s):  
C Sengstag ◽  
C Stirling ◽  
R Schekman ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Coenzyme A ◽  
Transmembrane Domains ◽  
Protein Domain ◽  
Yeast Cells ◽  
Endoplasmic Reticulum Membrane ◽  
Hmg Coa Reductase ◽  
Coa Reductase ◽  
Histidinol Dehydrogenase

Both 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase isozymes of the yeast Saccharomyces cerevisiae are predicted to contain seven membrane-spanning domains. Previous work had established the utility of the histidinol dehydrogenase protein domain, encoded by HIS4C, as a topologically sensitive monitor that can be used to distinguish between the lumen of the endoplasmic reticulum and the cytoplasm. This study directly tested the structural predictions for HMG-CoA reductase by fusing the HIS4C domain to specific sites in the HMG-CoA reductase isozymes. Yeast cells containing the HMG-CoA reductase-histidinol dehydrogenase fusion proteins grew on histidinol-containing medium if the HIS4C domain was present on the cytoplasmic side of the endoplasmic reticulum membrane but not if the HIS4C domain was targeted to the endoplasmic reticulum lumen. Systematic exchanges of transmembrane domains between the isozymes confirmed that both isozymes had equivalent membrane topologies. In general, deletion of an even number of putative transmembrane domains did not interfere with the topology of the protein, but deletion or duplication of an odd number of transmembrane domains inverted the orientation of the protein. The data confirmed the earlier proposed topology for yeast HMG-CoA reductase, demonstrated that the yeast enzymes are core glycosylated, and provided in vivo evidence that the properties of transmembrane domains were, in part, dependent upon their context within the protein.

scholarly journals Cholesterol and mevalonic acid are independent requirements for the in vitro proliferation of human bone marrow granulocyte progenitor cells: studies using ML-236B

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 667-671 ◽  
Author(s):  
PC Hoffman ◽  
CM Richman ◽  
RA Larson ◽  
S Yachnin
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Human Bone ◽  
Hmg Coa Reductase ◽  
Coa Reductase

Abstract ML-236B is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the key regulatory enzyme in the sequence that catalyzes the conversion of acetate to mevalonic acid in cholesterol biosynthesis. This compound caused marked inhibition of human bone marrow granulocyte progenitor cell (CFU-C) proliferation, the 50% inhibitory concentration (IHD50) being 2.0 X 10(6)M. Inhibition of colony formation was reversed by mevalonic acid but not by cholesterol. ML-236B also inhibited DNA synthesis and acetate incorporation into cholesterol in marrow mononuclear cells (IHD50 = 5.6 x 10(6)M and 3.2 x 10(7)M, respectively). No inhibition of mevalonate incorporation into cholesterol was observed. These results differ from those observed with 25-hydroxycholesterol, another inhibitor of HMG CoA reductase. The latter compound also inhibited CFU-C proliferation and cholesterol biosynthesis from acetate; inhibition of colony formation was reversed by cholesterol but not by mevalonic acid. In addition, 25- hydroxycholesterol inhibited cholesterol synthesis from mevalonic acid precursor. We conclude that: (1) ML-236B is a potent inhibitor of CFU-C proliferation, DNA synthesis, and cholesterol biosynthesis from acetate precursor in marrow mononuclear cells; (2) the effects of ML-236B are completely reversed by mevalonic acid but not by cholesterol, suggesting that mevalonic acid per se or one or more of its nonsterol products are critical for cell growth; (3) the inhibitory effects of 25- hydroxycholesterol on CFU-C proliferation and cholesterol biosynthesis are not solely a result of its inhibition of HMG CoA reductase, but are due in part to inhibition of enzymatic steps distal to mevalonic acid in the sterol synthetic pathway; and (4) mevalonic acid and cholesterol are independent requirements for CFU-C proliferation and differentiation in vitro.

scholarly journals Immunological evidence for eight spans in the membrane domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum

1992 ◽  
Vol 117 (5) ◽  
pp. 959-973 ◽  
Author(s):  
J Roitelman ◽  
EH Olender ◽  
S Bar-Nun ◽  
WA Dunn ◽  
RD Simoni
Keyword(s):  
Endoplasmic Reticulum ◽  
Coenzyme A ◽  
Cultured Cells ◽  
Critical Role ◽  
Transmembrane Helix ◽  
Chimeric Protein ◽  
Membrane Domain ◽  
Hmg Coa Reductase ◽  
Coa Reductase ◽  
Peptide G

We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. Finer-Moore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli beta-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg224 through Leu242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr284 through Glu302 (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys248 and Asp276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.

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