Quantitation of prenylcysteines by a selective cleavage reaction.

Abstract

The aflylic thioether bond of the prenylcysteines of prenylated proteins has been shown to be cleaved by 2-naphthol under alkaline conditions to yield substituted naphthopyrans. These products are readily resolved from interfering materials by HPLC and have a strongly absorbing chromophore. Thus, this reaction is suitable for quantitative analysis of prenyl substituents of proteins, and we have examined a number of tissues for their content of prenylcysteines. These amino acids are present in mammalian tissues at a concentration of 0.36-1.4 nmol/mg of protein, with a ratio of geranylgeranylcysteine to farnesylcysteine in the range of 4 to 10. Prenylcysteines were also found in the cytosolic fraction of two mouse tissues at about one-third the concentration of the whole organ. The level of these modified amino acids was found to be sigicantly less in a yeast, a fungus, a brown alga, a higher plant, and an insect. Again, geranylgeranylcysteine is predominant. Prenylcysteines were absent from Eschenchia coil but present in an archaebacterium. The prenylcysteine content of mmalan tissue is about 1% of that of cholesterol and about equal to that of ubiquinones and dolichols. Calculations indicate that about 0.5% of all proteins are prenylated.Prenylated proteins contain isoprenoid-modified cysteines in the carboxyl-terminal region (1-3). This posttranslational modification has usually been identified by metabolic labeling of proteins of cells in culture with radioisotopic mevalonate. Metabolic labeling is limiting because many organisms-e.g., yeasts and plants-do not incorporate this precursor readily. In addition, this technique may be impractical, since large doses of isotope would be required for studies with animals. The specific isoprenoid involved with this amino acid (4, 5) has been established by protein hydrolysis and isolation of the prenylcysteine (6) or by cleavage of the prenylcysteine with iodomethane (7) or Raney nickel (4, 5) followed by isolation of the cleavage product. Recently, fast atom bombardment mass spectrometry has been reported as a convenient method for qualitative analysis of prenylated peptides (8). Quantitative analysis with these techniques by direct determination of mass would be difficult if not impossible. Consequently, an alternative method for identifying prenyl modifications would be useful. In prenylated proteins the allylic thioether bond of the prenylcysteines is unique and provides an avenue for selective reactions for identification of prenyl groups. A good nucleophile should react preferentially with and cleave the isoprenoid from cysteine. Prenyl groups linked through oxygen or nitrogen would be expected to be unreactive. For example, a model study using radioactive farnesyl pyrophosphate and our naphthoxide conditions showed about 0.1% of a radioactive product. If the reagent possessed a strongly absorbing chromophoric group, then the derivative would provide a means for detection of the product. We have allowed prenylated proteins to react with 2-naphthol and hav...

Protein dolichylation in Plasmodium falciparum

“…Prenylation is a surprisingly frequent modification of proteins, and approximately 2% of all eukaryotic cell proteins are prenylated [15]. In addition to prenylation, others types of protein modification, such as the attachment of isoprenoid-derived compounds (for example diphytanylglyceryl [16] and phytol [17]), have been described in bacteria and plants, respectively.…”

Determinants of sensitivity to lovastatin-induced apoptosis in multiple myeloma

“…3A; Supplementary Table S1). 8 There were also no common differentially expressed transcripts among the three insensitive MM cell lines profiled. Only isopentenyl-diphosphate y isomerase was found to be commonly up-regulated in insensitive lines (LP1 and ANBL-6), but was also found to be up-regulated in the sensitive KMS11 cells (Supplementary Table S1).…”

“…One such group is the cytoskeleton gene ontology group (including KRT8, MYO5C, KIFAP3, VIM, TMSB4X, and ADD3), suggesting a role for cytoskeletal structure in MM cell lines sensitive to lovastatin-induced apoptosis. Furthermore, RhoB was significantly up-regulated in response to lovastatin in both sensitive cell lines and RhoA was 8 Supplementary material for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). also up-regulated in one line (H929).…”

“…Approximately 0.5% to 2% of cellular protein is posttranslationally modified by the addition of GGPP or farnesyl PPi (FPP) moieties at COOH-terminal motifs, broadly termed CAAX boxes, to ensure proper localization at cellular membranes (7,8). Of the known isoprenylated proteins, Rho and Ras GTPases are critical in promoting survival, proliferation, transformation, cell cycle control, and the regulation of cytoskeleton formation (9).…”

RhoA Prenylation Is Required for Promotion of Cell Growth and Transformation and Cytoskeleton Organization but Not for Induction of Serum Response Element Transcription

“…Ras and Rho proteins are post-translationally modified by the isoprenoid lipids, farnesyl, and geranylgeranyl (12). Two prenyltransferases, farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I), catalyze the covalent attachment of the farnesyl and geranylgeranyl groups, respectively, to the carboxyl-terminal cysteine of proteins ending in a CAAX motif (C is a cysteine, A usually aliphatic amino acid, and X any amino acid).…”