Bryant A. Gilbert

Molecular and biochemical characterization of lecithin retinol acyltransferase.

The enzyme responsible for conversion of all-trans-retinol into retinyl esters, the lecithin retinol acyltransferase (LRAT) has been characterized at the molecular level. The cDNA coding for this protein was cloned and its amino acid sequence deduced. LRAT is composed of a polypeptide of 230 amino acid residues with a calculated mass of 25.3 kDa. Tissue distribution analysis by Northern blot showed expression of a 5.0-kilobase transcript in the human retinal pigment epithelium as well as in other tissues that are known for their high LRAT activity and vitamin A processing. Affinity labeling experiments using specific compounds with high affinity for LRAT and monospecific polyclonal antibodies raised in rabbits against two peptide sequences for LRAT confirmed the molecular mass of LRAT as a 25-kDa protein. High performance liquid chromatography analysis of the reaction product formed by HEK-293 cells transfected with LRAT cDNA confirmed the ability of the transfected cells to convert [3H]all-trans-retinol into authentic [3H]all-trans-retinyl palmitate as chemically determined.

RNA aptamers that specifically bind to a K ras-derived farnesylated peptide

RNA aptamers were selected against an affinity column containing a farnesylated peptide modeled after the carboxyl terminus of K ras, the major oncogenic form of this small G protein family. After 10-rounds of selection, 25% of the RNA applied to the column could be specifically eluted. Sequence analysis of the binding RNA aptamers revealed two consensus sequences--GGGUGGG and GGGAGG. Quantitative fluorescence binding studies on two of the high-affinity aptamers, showed a binding affinities of 139 nM and 0.93 microM, respectively for the farnesylated peptide. Binding to the nonfarnesylated peptide was at least 10-fold weaker, showing that the aptamers can recognize the hydrophobic farnesyl moiety. High affinity aptamers could be useful in specifically interfering with oncogenic ras function in particular, and G proteins in general.

Analogs of farnesylcysteine induce apoptosis in HL-60 cells

S‐Farnesyl‐thioacetic acid (FTA), a competitive inhibitor of isoprenylated protein methyltransferase, potently suppressed the growth of HL‐60 cells and induced apoptosis, as evidenced by the development of increased annexin‐V binding, decreased binding of DNA dyes and internucleosomal DNA degradation. FTA did not impair the membrane association of ras proteins, conversely, it brought about a decrease in the proportion of ras present in the cytosolic fraction. Farnesylated molecules which are weak inhibitors of the methyltransferase also induced DNA laddering and reduced the proportion of cytosolic ras. These findings suggest that neither inhibition of isoprenylated protein methylation nor impairment of ras membrane association are essential for apoptosis induced by farnesylcysteine analogs.

[19] Farnesylcysteine analogs to probe role of prenylated protein methyltransferase

Publisher Summary This chapter describes the development of new probes of methyltransferase function to ascertain the specificity of the effects of farnesylcysteine analogs. The S -adenosylmethionine (AdoMet)-linked methylation of proteins is considered to be of regulatory significance in a variety of organisms. Mammalian methyltransferases specific for prenylated proteins are described, which is of substantial regulatory significance. In the prenylation pathway, the cysteine residue is first farnesylated or geranylgeranylated, followed by endoproteolysis to generate the prenylated cysteine residue as the carboxyl-terminal residue. Methylation of the residue follows by an S -adenosylmethionine-linked methyltransferase. This methylation is the only reversible reaction in the pathway and the only reaction subject to dynamic regulation. On a functional level, the double posttranslational modifications of prenylation and methylation render the modified proteins membrane associated and thus, biochemically active. The L-farnesylcysteine (FC) analogs that are neither substrates nor inhibitors of the methyltransferase have sterically bulky moieties attached to the amino group of FC, suggesting a lack of bulk tolerance there. The inert FC analogs allow a direct test of whether the methyltransferase is the actual pharmacological target for N -acetyl- S -farnesyl- L -cysteine (AFC) and similar analogs.

[17] Inhibitors of prenylated protein endoprotease

Publisher Summary This chapter discusses the synthesis of potent inhibitors of the endoprotease, which processes substrates containing the CaaX motif. Proteins are subject to prenylation when they possess a carboxyl-terminal CaaX or a CXC or a CC(XX) sequence. The protein is first prenylated at the cysteine residue(s) with either all- trans -farnesyl (C15) or all- trans -geranylgeranyl (C20) pyrophosphate. In the case of modifications at a CaaX motif, proteolysis follows, to generate the prenylated cysteine residue as the new carboxyl terminus. This set of modifications is completed by the reversible carboxylmethylation of the isoprenylated cysteine residue. Proteolysis occurs in mammals primarily by endoproteolytic cleavage between the modified cysteine residue and the adjacent aliphatic amino acid to liberate the intact X 3 (or -aaX) tripeptide. The endoprotease substrates fall into the CaaX motif type. The chapter also discusses a method to determine whether N -acetyl-S-all-trans-farnesyl-L-cysteine (AFC)-based tetrapeptides containing hydrophilic amino acids are endoproteolyzed. The carboxylterminal sequence (Cys-Arg-Pro-Gln) of the Delta virus large antigen provides a pertinent example. The potent inhibitors of the endoproteolytic processing of prenylated CaaX-containing peptides described in the chapter do not interfere with the processing of the hydrophilic peptides, suggesting the presence of isoforms of the endoprotease.