Robert D. Simoni

Degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in endoplasmic reticulum membranes is accelerated as a result of increased susceptibility to proteolysis.

The endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is subject to regulated degradation when cells are presented with an excess of sterols or mevalonate. In this report, we demonstrate the degradation of HMG-CoA reductase in ER membranes prepared from cells which have been pretreated with mevalonate or sterols prior to membrane purification. Degradation of HMG-CoA reductase in membranes prepared from pretreated cells is more rapid than in membranes prepared from cells which have received no regulatory molecules. In vitro degradation is blocked by protease inhibitors previously shown to inhibit reductase degradation in vivo and is specific for intact HMG-CoA reductase. The lumenal contents of the ER membranes are dispensible for the regulated proteolysis and the proteases responsible for reductase degradation are stably associated with the ER membrane. Regulated proteolysis of HMG-CoA reductase is inhibited by lactacystin, a newly defined inhibitor of the multicatalytic protease, the proteasome, and in vitro degradation of reductase correlates with the presence of proteasome subunits in purified ER membranes. The ubiquitin system for protein degradation, which has recently been shown to be required for the degradation of several ER membrane proteins, is not required for the degradation of HMG-CoA reductase. Finally, we conclude that the regulated proteolysis of HMG-CoA reductase in response to regulatory molecules such as mevalonate or sterols is mediated by increased susceptibility of the reductase to ER proteases, rather than the induction of a new proteolytic activity.

Degradation of HMG-CoA Reductase in Vitro CLEAVAGE IN THE MEMBRANE DOMAIN BY A MEMBRANE-BOUND CYSTEINE PROTEASE

We have recently shown that the endoplasmic reticulum (ER) membrane protein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is cleaved in isolated membrane fractions enriched for endoplasmic reticulum. Importantly, the cleavage rate is accelerated when the membranes are prepared from cells that have been pretreated with mevalonate or sterols, physiological regulators of the degradation process in vivo (McGee, T. P., Cheng, H. H., Kumagai, H., Omura, S., and Simoni, R. D. (1996)J. Biol. Chem. 271, 25630–25638). In the current study, we further characterize this in vitro cleavage of HMG-CoA reductase. E64, a specific inhibitor of cysteine-proteases, inhibits HMG-CoA reductase cleavage in vitro. In contrast, lactacystin, an inhibitor of the proteasome, inhibits HMG-CoA reductase degradation in vivo but does not inhibit the in vitro cleavage. Purified ER fractions contain lactacystin-sensitive and E64-insensitive proteasome activity as measured by succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin hydrolysis. We removed the proteasome from purified ER fractions by solubilization with heptylthioglucoside and observed that the detergent extracted, proteasome-depleted membrane fractions retain regulated cleavage of HMG-CoA reductase. This indicates that ER-associated proteasome is not involved in degradation of HMG-CoA reductase in vitro. In order to determine the site(s) of proteolysis of HMG-CoA reductasein vitro, four antisera were prepared against peptide sequences representing various domains of HMG-CoA reductase and used for detection of proteolytic intermediates. The sizes and antibody reactivity of the intermediates suggest that HMG-CoA reductase is cleaved in the in vitro degradation system near the span 8 membrane region, which links the N-terminal membrane domain to the C-terminal catalytic domain of the protein. We conclude that HMG-CoA reductase can be cleaved in the membrane-span 8 region by a cysteine protease(s) tightly associated with ER membranes.

Oligomerization state influences the degradation rate of 3-hydroxy-3-methylglutaryl-CoA reductase.

The steady-state level of the resident endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), is regulated, in part, by accelerated degradation in response to excess sterols or mevalonate. Previous studies of a chimeric protein (HM-Gal) composed of the membrane domain of HMGR fused to Escherichia coli β-galactosidase, as a replacement of the normal HMGR cytosolic domain, have shown that the regulated degradation of this chimeric protein, HM-Gal, is identical to that of HMGR (Chun, K. T., Bar-Nun, S., and Simoni, R. D. (1990)J. Biol. Chem. 265, 22004–22010; Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836–6841). Since the cytosolic domain can be replaced with β-galactosidase without effect on regulated degradation, it has been assumed that the cytosolic domain was not important to this process and also that the membrane domain of HMGR was both necessary and sufficient for regulated degradation. In contrast to our previous results with HM-Gal, we observed in this study that replacement of the cytosolic domain of HMGR with various heterologous proteins can have an effect on the regulated degradation, and the effect correlates with the oligomeric state of the replacement cytosolic protein. Chimeric proteins that are oligomeric in structure are relatively stable, and those that are monomeric are unstable. To test the hypothesis that the oligomeric state of the cytosolic domain of HMGR influences degradation, we use an “inducible” system for altering the oligomeric state of a protein in vivo. Using a chimeric protein that contains the membrane domain of HMGR fused to three copies of FK506-binding protein 12, we were able to induce oligomerization by addition of a “double-headed” FK506-like “dimerizer” drug (AP1510) and to monitor the degradation rate of both the monomeric form and the drug-induced oligomeric form of the protein. We show that this chimeric protein, HM-3FKBP, is unstable in the monomeric state and is stabilized by AP1510-induced oligomerization. We also examined the degradation rate of HMGR as a function of concentrations within the cell. HMGR is a functional dimer; therefore, its oligomeric state and, we predict, its degradation rate should be concentration-dependent. We observed that it is degraded more rapidly at lower concentrations.

Effects of compactin on the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase in compactin-resistant C100 and wild-type cells☆

A cell line, C100, resistant to 225 microM compactin, has been isolated which overproduces 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase approximately 100-fold compared to the parental cell line [E. Hardeman, H. Jenke and R. Simoni (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1516-1520]. It is demonstrated that the overproduction of HMG-CoA reductase in these cells is the result of increased enzyme synthesis due to elevated levels of translatable mRNA. Furthermore, the apparent molecular weight of the in vitro translation product is 94,000, which agrees with the molecular weight of the in vivo synthesized HMG-CoA reductase protomer in C100 cells. However, a comparison of the Staphylococcus aureus V8 proteolysis patterns between the in vitro and in vivo translation products reveals structural differences which suggests in vivo post-translation modification(s). It is also demonstrated unequivocally, by comparing proteolytic cleavage patterns and pulse-chase experiments, that the previously reported 63,000-, 52,000-, and 38,000-Da polypeptides recognized by HMG-CoA reductase antiserum derive from the 94,000-Da protomer as a result of nonphysiological proteolysis. Finally, the types of regulatory mechanisms involved in both the induction and repression of the enzyme in the presence or absence of compactin were determined. Four biochemical parameters of HMG-CoA reductase were examined in variant and parental cells grown in the presence and absence of compactin: enzymatic activity, degradation rate, synthesis rate, and concentration of translatable mRNA. These studies revealed that changes in cellular HMG-CoA reductase content are a function of concurrent changes in the rates of enzyme degradation and synthesis. Changes in enzyme synthesis are due to alterations in the level of translatable mRNA.

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.

Activation of purified 3-hydroxy-3-methylglutaryl-CoA reductase by phospholipids.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), the enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis, has been purified by two previously reported procedures. Enzyme purified by the method of Heller, R. and Shrewsbury, M. (1976) J. Biol. Chem. 251, 3815-3822) shows up to 3-fold enhancement of activity by various types of lipid dispersions while the enzyme purified by the procedure of Tormanen et al. ((1976) Biochem. Biophys. Res. Commun. 68, 754-762) shows no activation. These results suggest that interaction with microsomal membrane lipids may be important in determining the activity of this enzyme. Analysis of bound lipid showed that enzyme prepared by the procedure of Tormanen contained at last 50 times as much phospholipid on a weight basis as enzyme prepared by Heller and Shrewsbury. Analysis of both preparations by gel-electrophoresis indicates that enzyme activities of the two comigrate, but in neither case does activity coincide with the major protein species.

Characterization of the genes coding for the F1F0 subunits of the sodium dependent ATPase of Propionigenium modestum

The DNA coding for the eight structural genes and uncI of the sodium dependent ATPase of Propionigenium modestum has been cloned and sequenced. Based on sequence homology, the genes were determined to appear in the order uncBEFHAGDC as in several other bacterial species. Minicell experiments revealed that plasmids containing the P. modestum DNA expressed those ATPase polypeptides in Escherichia coli. These were very similar in molecular mass to those obtained from the purified ATPase of P. modestum. No membrane-bound ATPase activity was observed in E. coli unc deletion strains containing the P. modestum ATPase genes. Amino acid alignments which were done with the Fo subunits revealed only a few conservative changes in the highly conserved regions of the polypeptides.

[34] Parinaric acid from Parinarium glaberrimum

Publisher Summary This chapter discusses the preparation of parinaric acid from Parinarium glaberrimum. α-Parinaric acid, is derived from the seed oil of P. glaberrimurn. cis-Parinaric acid and trans-parinaric acid and a number of their derivatives have been used in a wide range of applications as fluorescent probes. The freshly extracted oil is immediately dissolved in 300 ml of cool 5% potassium hydroxide in methanol containing BHT under an atmosphere of argon. Saponification of the oil is accomplished by gently reflexing the solution for 30 min with stirring. Stock solutions of parinaric acid in ethanol containing 0.001% BHT, stored at −20°, and flushed with argon each time they are opened, are stable for periods up to 2 years. Microliter aliquots of these solutions are appropriate for addition to aqueous biological preparations. For synthetic applications or long-term storage of large quantities of parinaric acid, one recommends that solutions of parinaric acid be frozen in benzene. It is found that pure, dry, parinaric acid is obtained by lyophilization of the solution.

[35] Preparation of parinaric acid derivatives

Publisher Summary This chapter describes the preparation of parinaric acid derivatives. The lysolecithin obtained from phospholipase A2 treatment of egg phosphatidylcholine is dried in a flask over night under vacuum over P 2 O 5 . Freshly distilled anhydrous chloroform, 5 ml, is added and the solution is treated with half of a solution, containing 469 mg of anhydrous parinaric acid and 274 mg of carbonyldiimidazole in 5 ml of chloroform. The reaction mixture is stirred under argon for 3 h at room temperature, and then the remaining half of the parinaroylimidazole solution is added. trans -Parinaroylhydroxysuccinimide ester, in 8.6 ml of tetrahydrofuran, is mixed with an aqueous solution containing glucosamine hydrochloride and NaHCO 3 . N -trans-parinaroylglucosamine is slightly soluble in methanol and more soluble in dimethyl sulfoxide (DMSO). A stock solution is prepared by dissolving the solid in DMSO and methanol. The Morgan-Elson method for testing of N -acylhexosamines is used, and the characteristic absorptions at 544 and 585 nm are observed. The Morgan-Elson test for N -acylhexosamine is positive. The heating time for this compound in the Morgan-Elson reaction is 6 min, and it gives the same extinction coefficients as N -acetylglucosamine.

Alanine transport by Chinese hamster ovary cells with altered phospholipid acyl chain composition

The Na+-dependent transport of alanine has been examined in Chinese hamster ovary (CHO) cells as a function of the fatty acid composition of their membrane lipids. Significant changes in the fatty acid composition of the CHO cell phospholipids were achieved by supplementation of the growth medium with specific saturated (palmitate) or monoenoic (oleate) free fatty acids. Arrhenius plots of the temperature-dependent uptake of alanine were constructed for cells of altered fatty acid composition. Alanine uptake was characterized by a single discontinuity in the Arrhenius plot. The temperature of this break was observed to be dependent upon the fatty acid composition of the cell phospholipids, ranging from 16 degrees C for cells enriched with oleate to 32 degrees C for cells enriched in palmitate. Calculation of the Km value for the uptake process showed no significant change with temperature or fatty acid supplementation. Correlations are made between the physical state of the membrane lipids and the temperature-dependence for alanine transport. The results are discussed in terms of membrane fatty acid composition, ordered in equilibrium fluid phase transitions and amino acid transport.