Atg3-mediated lipidation of Atg8 is involved in encystation of Acanthamoeba

Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.     

Two glucosylated abscisic acid derivates from avocado seeds (Persea americana Mill. Lauraceae cv. Hass)

Phytochemical investigation of avocado seed material (Persea americana Mill., Lauraceae) resulted in the isolation of two glucosylated abscisic acid derivates. One of these was not known as a natural product and can be regarded as a potential 'missing link' in abscisic acid metabolism in plants. After fractionation by high-speed countercurrent chromatography, and multiple steps of column chromatography, structures were elucidated by 1D-, 2D-NMR, electrospray-MS to be the novel beta-d-glucoside of (1'S,6'R)-8'-hydroxyabscisic acid, and (1'R,3'R,5'R,8'S)-epi-dihydrophaseic acid beta-d-glucoside. Absolute configuration was determined by circulardichroism, optical rotation, and by NOE experiments.     

Deuterium-labeled phaseic acid and dihydrophaseic acids for internal standards

The concentration of abscisic acid in plants is regulated not only by biosynthesis, but also by metabolism. Abscisic acid is metabolized to phaseic acid via 8'-hydroxyabscisic acid, and phaseic acid is then converted to dihydrophaseic acid and its epimer. A quantitative analysis of these metabolites is important as well as that of abscisic acid to understand changes in the concentration of abscisic acid in plants. However, no internal standards of the metabolites suitable for quantitative analysis have been reported. We prepared 7'-deuterium-labeled phaseic acid with a deuterium content of 86%, using the equilibrium reaction between phaseic acid and 8'-hydroxyabscisic acid. 7'-Deuterium-labeled dihydrophaseic acids were obtained by reducing 7'-deuterium-labeled phaseic acid. The levels of the metabolites in plant organs were determined by using the deuterated metabolites as internal standards.     

Reversible phosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase in Morris hepatomas

The reversible phosphorylation of microsomal 3-hydroxy-3-methylglutaryl CoA reductase in host liver and hepatoma 5123C has been investigated. The percentage of the total enzyme activity in vivo was similar in the normal liver, host liver and hepatoma 5123C. The inclusion of 30 mM EDTA and 10 mM mevalonic acid in assays of 3-hydroxy-3-methylglutaryl CoA reductase inactivation in vitro eliminated artifacts generated by the presence of mevalonate kinase. Inactivation of 3-hydroxy-3-methylglutaryl CoA reductase from normal liver, host liver and hepatoma occurred at a similar rate with similar half-times. We conclude that phosphorylation/dephosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase occurs in hepatomas and that the lack of dietary cholesterol feedback inhibition in the hepatomas is not a result of a defect in this particular aspect of the reversible phosphorylation system.     

An unusual acylated malvidin 3-glucoside from flowers of Impatiens textori Miq. (Balsaminaceae)

Acylated malvidin 3-glucoside was isolated from the purple flowers of Impatiens textori Miq. as a major anthocyanin component along with malvidin 3-(6″-malonyl-glucoside). Its structure was elucidated to be malvidin 3-O-[6-O-(3-hydroxy-3-methylglutaryl)-β-glucopyranoside] by chemical and spectroscopic methods.     

A facile synthesis of lanost-8-en-3 beta-ol-24-one (24-ketolanosterol). An inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase

A facile chemical synthesis of lanost-8-en-3 beta-ol-24-one (24-ketolanosterol) is described. This compound was found to be a potent inhibitor of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity in cultured mouse L cells. The synthetic scheme developed in this study utilizes commercial lanosterol as a starting material and involves selective hydroboration of the C-24 double bond followed by oxidation of the carbon-boron bond at C-24 by pyridinium chlorochromate (PCC).     

Properties of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from Pisum sativum seedlings.

The properties of 3-hydroxy-3-methylglutaryl coenzyme A reductase from the microsomal fraction of Pisum sativum seedlings have been described. The enzyme requires NADPH for activity and NADH does not support the reaction. The presence of a thiol compound such as dithiothreitol, is required for activity and a concentration of 10 mmm is optimal. The pH optimum is 6.8 and the K_m (apparent) for dl-3-hydroxy-3-methylglutaryl coenzyme A is about 100 μm.Activity of the enzyme is not affected by mevalonic acid at the concentrations tested (up to 1.0 mm). 3-Hydroxy-3-methylglutaric acid and free CoA cause substantial inhibition, whereas gibberellic acid has no effect.The activity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase is twice as high in etiolated seedlings as in green seedlings. In green seedlings activity is highest in the apical bud, declines sharply in semimature leaves, and there is almost no activity in mature leaves.     

Interference with the determination of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and its properties by a microsomal enzymic activity.

Rat liver microsomes and microsomal extracts contain an enzymic activity which competes with 3-hydroxy-3-methylglutaryl coenzyme A reductase for 3-hydroxy-3-methylglutaryl coenzyme A. The presence of this activity in enzyme preparations causes errors in the determination of reductase activity and its properties. This contaminant can be removed by gel filtration using Bio-Gel A 1.5m, by washing the microsomes, or by incubating the microsomal extract at 37 °C. The K_m's of the reductase (free of this competing enzymic activity) for d-3-hydroxy-3-methylglutaryl coenzyme A and NADPH are 1.3 and 26 μm, respectively.     

Inhibitors of sterol synthesis. Chemical syntheses and spectral properties of 26-oxygenated derivatives of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

26-Oxygenated derivatives of delta 8(14)-15-ketosterols have been synthesized from (25R)-3 beta,26-diacetoxy-5 alpha-cholest-8(14)-en-15-one (IX) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Partial hydrolysis of IX gave a mixture, from which the 3 beta,26-diol II and the 26-acetate (XI) and 3 beta-acetate (X) monoesters were isolated. Mitsunobu reaction of XI followed by hydrolysis gave (25R)-3 alpha,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one (VI). Oxidation of XI with pyridinium chlorochromate followed by hydrolysis of the acetate gave (25R)-26-hydroxy-5 alpha-cholest-8(14)-ene-3,15-dione (VII). Oxidation of X with Jones reagent followed by hydrolysis of the acetate gave (25R)-3 beta-hydroxy-15-keto-5 alpha-cholest-8(14)-en-26-oic acid (IVa). Jones oxidation of II gave (25R)-3,15-diketo-5 alpha-cholest-8(14)-en-26-oic acid (VII). 1H and 13C nuclear magnetic resonance assignments and analyses of mass spectral fragmentation data are presented for each of the new compounds and their derivatives. The 3,15-diketone VII was found to be highly active in lowering the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells, with a potency comparable to that of I. In contrast, 3 alpha,26-diol VI was less potent than I or VII. The two carboxylic acid analogs IVa and VIII were considerably less potent than VI in lowering the levels of HMG-CoA reductase activity.     

Effects of compactin,a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor,on the growth of alfalfa (Medicago sativa) seedlings and the rhizogenesis of pepper (Capsicum annuum) explants

The effects of compactin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the growth of alfalfa seedlings in vivo and the rhizogenesis of pepper explants in vitro were investigated. Compactin added to the agar medium inhibited the elongation of roots and hypocotyls of etiolated alfalfa seedlings. The growth inhibition was accompanied by strict inhibition of sterol synthesis. Addition of mevalonic acid, the direct product of 3-hydroxy-3-methylglutaryl coenzyme A reductase, together with compactin relieved the growth inhibition. The sterol level in the seedlings was also protected against the lowering effect of compactin. Similarly, the rhizogenetic process of cultured explants of pepper was inhibited by compactin and relieved by mevalonic acid. Several isoprenoid end products were tested in combination with compactin to determine which compounds, if any, might be limiting for growth. Exogenously supplied isoprenoids failed to relieve the growth inhibition of seedlings. In contrast, they partly relieved the growth inhibition of explants, suggesting their important role in plant growth. During the course of these experiments, it was also found that brassinolide caused remarkable growth inhibition and twisting of alfalfa seedlings.     

Effect of an unnatural phospholipid base analog,N-isopropylethanolamine,on 3-hydroxy-3-methylglutaryl coenzyme a reductase in cultured glial and neuronal cells

Cultured C-6 glial and neuroblastoma cells were utilized to study the effect of the unnatural amino alcohol, N-isopropylethanolamine, on the microsomal enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase. Growth of both cell types in the presence of the compound was accompanied in 24 hr by a decrease in reductase activity to 25–35% of activity in control cells. The effect was accompanied by a comparable decrease in the rate of cholesterol synthesis. However, no comparable change occurred in cell growth, fatty acid synthetase activity, or in total protein synthesis from [³H]leucine. The data suggest that the polar head groups of microsomal membrane phospholipids play an important role in the regulation of reductase activity.     

Inhibitors of sterol synthesis. Chemical synthesis of 5 beta-cholest-8-ene-3 beta,15 alpha-diol and its effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells

5 beta-Cholest-8-ene-3 beta,15 alpha-diol, prepared by hydroboration of 5 beta-cholesta-8,14-dien-3 beta-ol, was determined to have the 14 alpha-H,15 alpha-OH configuration by comparisons of observed and calculated lanthanide-induced shifts for the 3-tertbutyldimethylsilyl derivative. The 3 beta,15 alpha-diol was found to exist partially in a conformation in which ring B is a 5 beta, 6 alpha-half chair and the axial-equatorial orientation of ring A substituents is reversed. This conformation has been observed previously for 3 beta-(p-bromobenzoyloxy)-5 beta-cholesta-8,14-diene and for some cis-decalin derivatives. 5 beta-Cholest-8-ene-3 beta,15 alpha-diol was found to be highly active in the lowering of the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells and only slightly less active than the corresponding sterol (5 alpha-cholest-8-ene-3 beta,15 alpha-diol) with the trans A-B ring junction.     

Comparison of movement and metabolism of indole-3-acetic acid and indole-3-butyric acid in mung bean cuttings

Indole-3-butyric acid (IBA) was much more effective than indole-3-acetic acid (IAA) in inducing adventitious root formation in mung bean ( Vigna radiata L.) cuttings. Prolonging the duration of treatment with both auxins from 24 to 96 h significantly increased the number of roots formed. Labelled IAA and IBA applied to the basal cut surface of the cuttings were transported acropetally. With both auxins, most radioactivity was detected in the hypocotyl, where roots were formed, but relatively more IBA was found in the upper sections of the cuttings. The rate of metabolism of IAA and IBA in these cuttings was similar. Both auxins were metabolized very rapidly and 24 h after application only a small fraction of the radioactivity corresponded to the free auxins. Hydrolysis with 7 M NaOH indicates that conjugation is the major pathway of IAA and IBA metabolism in mung bean tissues. The major conjugate of IAA was identified tentatively as indole-3-acetylaspartic acid, whereas IBA formed at least two major conjugates. The data indicate that the higher root-promoting activity of IBA was not due to a different transport pattern and/or a different rate of conjugation. It is suggested that the IBA conjugates may be a better source of free auxin than those of IAA and this may explain the higher activity of IBA.     

Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A

A new assay for 3-hydroxy-3-methylglutaryl CoA reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) is based upon the measurement of released coenzyme A (SH) during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate. Coenzyme A was measured in the presence of dithiothreitol, required for activity, by reaction with 5,5'-dithiobis(2-nitrobenzoic acid). Sodium arsenite forms a complex with the dithiol, but not with monothiols. Thus, reduced coenzyme A reacts instantaneously with the reagent and dithiothreitol reacts slowly. The absorbance due to the coenzyme A-5,5'-dithiobis(2-nitrobenzoic acid) reaction is determined by extrapolating the linear (dithiol) absorbance-time curve to the time of addition of the reagent. After subtraction of control absorbance (deletion of NADPH), the concentration of CoA-SH is calculated from epsilon(max) = 1.36 x 10(4) at 412 nm. The method of protein removal and reduction of sulfhydryl groups on the enzyme are critical. This method provides an immediate assay. Recovery of reduced coenzyme A was 98.7%. The assay is applicable for microsomes or purified enzyme and has an effective range of 0.5-50 nmoles of coenzyme A. It was applied to kinetic measurement of the pigeon liver microsomal enzyme reaction. The apparent K(m) value for 3-hydroxy-3-methylglutaryl CoA was 1.75 x 10(-5) M, and for NADPH the value was 6.81 x 10(-4) M. This method was compared with the dual-label method at high and low levels of activity. The data were not statistically different.     

Structure of the Atg12–Atg5 conjugate reveals a platform for stimulating Atg8–PE conjugation

Atg12 is conjugated to Atg5 through enzymatic reactions similar to ubiquitination. The Atg12–Atg5 conjugate functions as an E3‐like enzyme to promote lipidation of Atg8, whereas lipidated Atg8 has essential roles in both autophagosome formation and selective cargo recognition during autophagy. However, the molecular role of Atg12 modification in these processes has remained elusive. Here, we report the crystal structure of the Atg12–Atg5 conjugate. In addition to the isopeptide linkage, Atg12 forms hydrophobic and hydrophilic interactions with Atg5, thereby fixing its position on Atg5. Structural comparison with unmodified Atg5 and mutational analyses showed that Atg12 modification neither induces a conformational change in Atg5 nor creates a functionally important architecture. Rather, Atg12 functions as a binding module for Atg3, the E2 enzyme for Atg8, thus endowing Atg5 with the ability to interact with Atg3 to facilitate Atg8 lipidation.     

Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase

A detailed kinetic analysis of the recombinant soluble enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower proportion compared to the anabolic reaction. We report that the catalytically active species of HMGR in solution is the tetrameric form. Fluvastatin inhibited competitively the enzyme while cerivastatin binds by a mechanism which is more accurately described by a biphasic process characteristic of a class of ‘slow, tight-binding’ inhibitors.     

Crystal Structures of Human HMG-CoA Synthase Isoforms Provide Insights into Inherited Ketogenesis Disorders and Inhibitor Design

3-Hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (HMGCS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl CoA. It is ubiquitous across the phylogenetic tree and is broadly classified into three classes. The prokaryotic isoform is essential in Gram-positive bacteria for isoprenoid synthesis via the mevalonate pathway. The eukaryotic cytosolic isoform also participates in the mevalonate pathway but its end product is cholesterol. Mammals also contain a mitochondrial isoform; its deficiency results in an inherited disorder of ketone body formation. Here, we report high-resolution crystal structures of the human cytosolic (hHMGCS1) and mitochondrial (hHMGCS2) isoforms in binary product complexes. Our data represent the first structures solved for human HMGCS and the mitochondrial isoform, allowing for the first time structural comparison among the three isoforms. This serves as a starting point for the development of isoform-specific inhibitors that have potential cholesterol-reducing and antibiotic applications. In addition, missense mutations that cause mitochondrial HMGCS deficiency have been mapped onto the hHMGCS2 structure to rationalize the structural basis for the disease pathology.     

Chemical synthesis of 4,4'-dimethyl-7-oxygenated sterols. Inhibitors of 3-hydroxy-3-methylglutaryl reductase

The chemical syntheses of 4,4'-dimethylcholest-5-en-3 beta-ol-7-one, 4,4'-dimethylcholest-5-ene-3 beta, 7 beta-diol and 4,4'-dimethylcholest-5-ene-3 beta, 7 alpha-diol are described. All of these compounds were found to be potent inhibitors of 3-hydroxy-3-methylglutaryl (HMG-CoA) reductase activity in cultured mouse L cells. The synthetic scheme developed in this study utilizes commercial cholesterol as the starting material and provides a simplified method for the preparation of 4,4'-dimethyl-7-oxygenated steroids.     

Inhibitors of sterol synthesis. A highly efficient and specific side-chain oxidation of 3 beta-acetoxy-5 alpha-cholest-8(14)-en-15-one for construction of metabolites and analogs of the 15-ketosterol.

As part of a program directed towards the chemical syntheses of potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism, several routes have been explored for the preparation of 3 beta-hydroxy-15-keto-5 alpha-chol-8(14)-en-24-oic acid (IV). These investigations led to a remarkably specific and efficient side-chain oxidation of I. For example, treatment of the acetate of I with a mixture of trifluoroacetic anhydride, hydrogen peroxide, and sulfuric acid for 3.5 h at -2 degrees C gave a crude product consisting of 3 beta-acetoxy-24-trifluoroacetoxy-5 alpha-chol-8(14)-en-15-one (XI), 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (XII), and 3 beta, 24-diacetoxy-5 alpha-chol-8(14)-en-15-one (XIII) in yields of 58%, 8%, and 3%, respectively, by HPLC analysis. XI was readily hydrolyzed to XII upon treatment with triethylamine in methanol at room temperature. Oxidation of XII with Jones reagent gave 3 beta-acetoxy-15-keto-5 alpha-chol-8(14)-en-24-oic acid (XVIII) from which its methyl ester (IX) was prepared by treatment with diazomethane. Mild alkaline hydrolysis of XVIII gave the 3 beta-hydroxy-delta 8(14)-15-keto C24 acid (IV). Hydrolysis of the crude product of the side-chain oxidation with K2CO3 in methanol gave 3 beta,24-dihydroxy-5 alpha-chol-8(14)-en-15-one (XIV) which was oxidized with Jones reagent to yield 3,15-diketo-5 alpha-chol-8(14)-en-24-oic acid (XV). Treatment of XV with diazomethane gave its methyl ester (XVI) which, upon controlled reduction with NaBH4, yielded methyl 3 beta-hydroxy-15-keto-5 alpha-chol-8(14)-en-24-oate (XVII). Compound IX was also prepared by an independent route. Full 1H and 13C NMR assignments are presented for 12 new compounds. IV caused a approximately 56% reduction of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells at a concentration of 2.5 microM. In contrast, the corresponding 3,15-diketo acid XV had no detectable effect on reductase activity under the same conditions.