Julie L. Hutson

Effect of squalestatin 1 on the biosynthesis of the mevalonate pathway lipids

The effects of squalestatin 1 on rat brain and liver homogenates and on Chinese hamster ovary tissue culture cells have been investigated. This compound effectively inhibits squalene biosynthesis in a highly selective manner. Cytoplasmic farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthases are not affected, which is also the case for microsomal cis-prenyltransferase. In tissue culture cells, squalestatin 1 inhibits cholesterol biosynthesis completely, but does not alter dolichol synthesis or protein isoprenylation to a great extent. Incorporation of [3H]mevalonate into ubiquinone-9 and -10 increases 3-4-fold, probably as a result of increased synthesis of this lipid. Squalestatin 1 appears not only to be an effective inhibitor of cholesterol biosynthesis, but also to be more specific than other inhibitors used earlier in various in vitro and in vivo systems.

The squalestatins : potent inhibitors of squalene synthase : the role of the tricarboxylic acid moiety

In squalestatins possessing at C6 either a 4,6-dimethyloctenoate ester or a hydroxyl group, the 5-carboxylic acid is crucial for squalene synthase inhibitory activity. In the former seires, free carboxylic acids are not required at C3 or C4 for potent enzyme inhibitory activity whereas in the latter series esterification of the carboxylic acids at C3 or C4 results in a significant reduction in enzyme inhibitory activity.

Asymmetric synthesis and transmembrane movement of phosphatidylethanolamine synthesised by base-exchange in rat liver endoplasmic reticulum.

Using trinitrobenzenesulphonic acid (TNBS) as a probe we have observed that phosphatidylethanolamine (PE) formed by base-exchange is initially concentrated in the cytosolic leaflet of the membrane bilayer. At 2 min, the specific activity of the PE in this leaflet was 3-times that of the PE in the cisternal leaflet. After 30 min, the specific activities of the two pools of PE, determined with either phospholipase C or TNBS, were similar. Transbilayer movement of PE was slow at low temperature, prevented by EDTA and restored by the addition of calcium ions after EDTA treatment. Trypsin treatment of microsomes, under conditions in which the vesicles remained closed, inhibited the incorporation of ethanolamine into PE by 87%. The cytosolic location of the ethanolamine base-exchange enzyme is consistent with the initial concentration of newly synthesised PE at this site prior to its transmembrane movement to the cisternal leaflet.

The squalestatins: Potent inhibitors of squalene synthase, 3-hydroxymethyl derivatives.

A series of 3-hydroxymethyl derivatives of squalestatin 1 was prepared as inhibitors of squalene synthase. Potent in vitro inhibitory activity is retained in those analogues which possess C-6 and C-1 substituents analogous to those found in 1.

Structurally simplified squalestatins : monocyclic 1,3-dioxane analogues

Abstract Monocyclic analogues of squalestatin 1 based on a 1,3-dioxane ring were prepared and evaluated for their ability to inhibit squalene synthase in vitro. The compound 16a possessing a 4,6-dimethyloctenoyloxymethyl group at C4 and a carboxamide at C2 showed similar inhibitory activity to 1.

The squalestatins: novel inhibitors of squalene synthase. The optimal C1 chain-length requirements.

Abstract Analogues of squalestatin 1 modified in th C1 side-chain were prepared and evaluated for their ability to inhibit squalene synthase in vitro. An appropriately substituted 6-phenylhexyl chain was found to be optimal for effective enzyme inhibition.

Reversible Activation/Inactivation of the Deacylation/Acylation Cycle in Rat Liver Microsomes

Rat liver microsomes incorporate [14C]palmitoyl CoA into membrane phospholipids via the deacylation/acylation cycle. This activity is reversibly inactivated/activated by treatment of the microsomes with ATP, MgCl2, and 105,000g supernatant or with 105,000g supernatant alone. These observations suggest that the acylation cycle is controlled by a mechanism involving phosphorylation/dephosphorylation. As the pool of lysolecithin in the membranes is not altered by conditions increasing incorporation of palmitoyl CoA into phospholipid, it is probable that the site of regulation of deacylation/acylation is at the acyltransferase rather than the phospholipase.

Microsomal membranes contain phosphatidylcholine that equilibrates across the bilayer, and phosphatidylcholine that does not

Rat liver microsome Asymmetry Phosphatidylcholine Phospholipase C Phosphatidylcholine exchange protein Phosphatidylcholine pool