Prenylated proteins. A convenient synthesis of farnesyl cysteinyl thioethers

Brown, Mark J.; Milano, P. D.; Lever, David C.; Epstein, William W.; Poulter, C. Dale

doi:10.1021/ja00008a058

Cited by 75 publications

(41 citation statements)

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“…In a similar fashion, the sulfhydryl groups of ␥Glu-Cys (Sigma) or Cys-Gly (Sigma) were alkylated with farnesyl bromide (Aldrich) (25). The carboxyl groups of farnesylated ␥Glu-Cys, Cys-Gly, and farnesylthioacetic acid (LC Laboratories, Woburn, MA) were converted to methyl esters by treatment with diazomethane (26), prepared using the Mini Diazald kit (Aldrich) according to manufacture's instructions.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Prenylcysteines That Interact with P-glycoprotein and Inhibit Drug Transport in Tumor Cells

Zhang

Sachs

et al. 1995

Journal of Biological Chemistry

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Prenylcysteine methyl esters that represent the C-terminal structures of prenylated proteins demonstrate specific substrate-like interactions with P-glycoprotein (Zhang, L., Sachs, C. W., Fine, R. L., and Casey, P. J. (1994) J. Biol. Chem. 269, 15973-15976). The simplicity of these compounds provides a unique system for probing the structural specificity of P-glycoprotein substrates. We have further assessed the structural elements of prenylcysteines involved in the interaction with P-glycoprotein. Carboxyl group methylation, a modification in many prenylated proteins, plays an essential role of blocking the negative charge at the free carboxylate. Substitution of the methyl ester with a methyl amide or simple amide does not change the ability of the molecule to stimulate P-glycoprotein ATPase activity, but substitution with a glycine is not tolerated unless the carboxyl group of glycine is methylated. The presence of a nitrogen atom, which is found in many P-glycoprotein substrates and modifiers, is also essential for prenylcysteines to interact with P-glycoprotein. The structure at the nitrogen atom can, however, influence the type of interaction. Acetylation of the free amino group of prenylcysteine results in a significant loss in the ability of prenylcysteines to stimulate P-glycoprotein ATPase activity. Instead, certain acetylated prenylcysteines behave as inhibitors of this activity. In studies using MDR1-transfected human breast cancer cells, the acetylated prenylcysteine analogs inhibit P-glycoprotein-mediated drug transport and enhance the

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Section: Methodsmentioning

confidence: 99%

Characterization of Prenylcysteines That Interact with P-glycoprotein and Inhibit Drug Transport in Tumor Cells

Zhang

Sachs

et al. 1995

Journal of Biological Chemistry

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“…Radiolabeled prenylated peptides were produced enzymatically using FTase and [ 3 H]FPP substrates and purified as described (24). Unlabeled farnesyl-CVIM was produced by chemical farnesylation of the peptide using farnesyl bromide, and the product was purified by HPLC (26). Silica G60 TLC plates were from EM Separations.…”

Section: Materials-the Isoprenoid Substrates Fpp and Itsmentioning

confidence: 99%

Substrate Binding Is Required for Release of Product from Mammalian Protein Farnesyltransferase

Tschantz

Furfine

Casey

1997

Journal of Biological Chemistry

109

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Protein farnesyltransferase (FTase) catalyzes the modification by a farnesyl lipid of Ras and several other key proteins involved in cellular regulation. Previous studies on this important enzyme have indicated that product dissociation is the rate-limiting step in catalysis. A detailed examination of this has now been performed, and the results provide surprising insights into the mechanism of the enzyme. Examination of the binding of a farnesylated peptide product to free enzyme revealed a binding affinity of ϳ1 M. However, analysis of the product release step under single turnover conditions led to the surprising observation that the peptide product did not dissociate from the enzyme unless additional substrate was provided. Once additional substrate was provided, the enzyme released the farnesylated peptide product with rates comparable with that of overall catalysis by FTase. Additionally, stable FTasefarnesylated product complexes were formed using Ras proteins as substrates, and these complexes also require additional substrate for product release. These data have major implications in both our understanding of overall mechanism of this enzyme and in design of inhibitors against this therapeutic target. Protein farnesyltransferase (FTase)1 catalyzes the S-farnesylation of a number of key cellular regulatory proteins. Farnesylation is directed by a C-terminal CAAX motif, where C is cysteine, A is usually an aliphatic residue, and X is typically methionine, serine, glutamine, or alanine (1, 2). The farnesyl lipid is attached to the substrate protein via a thioether linkage to the cysteine residue using farnesyl diphosphate (FPP) as the prenyl donor. Among the substrates for FTase are the Ras family of proto-oncogenes, several ␥ subunits of heterotrimeric G proteins, and nuclear lamins (1, 3). Farnesylation of these proteins is required for their proper membrane localization and activity. In the case of oncogenic forms of Ras proteins, the finding that farnesylation is required for expression of their transforming activity has led to FTase becoming an important target for anticancer drug design (4). Both in cell culture (5, 6) and in animal models (7), specific inhibitors of FTase have been shown to reverse the oncogenic phenotype induced by mutationally activated Ras.FTase has been purified to homogeneity from both rat and bovine brain by affinity purification on immobilized CAAX peptide substrates (8, 9). The enzyme is a Zn 2ϩ metalloenzyme that consists of ␣ and ␤ subunits that migrate on SDS-PAGE with apparent molecular masses of 48 and 46 kDa, respectively (8). Both subunits of the enzyme have been cloned (10 -12), and their co-expression in either Sf9 (13) or E. coli (14) results in production of quantities of the enzyme required for detailed biochemical and structural analyses. Cross-linking experiments have provided strong evidence that the ␤ subunit is involved in recognition of both the isoprenoid and protein substrates (15-17), although there is also evidence that the ␣ subunit may participate (15...

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“…The reaction proceeds well in liquid ammonia according to a procedure described by Poulter and co-workers. [21] However, in our hands, the use of a 1 m solution of ammonia in methanol [22] proved to be more practical. S-alkylated cysteines 7 were isolated in high yields; N-alkylation was not observed.…”

Section: Synthesis Of Fluorescently Labeled Ras Peptidesmentioning

confidence: 68%

Synthesis and Application of Fluorescent Ras Proteins for Live‐Cell Imaging

et al. 2005

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Semisynthetic Ras proteins are efficient probes for cell-biology experiments. With a Bodipy FL fluorophore introduced at an appropriate site on the Ras peptide by solid-phase synthesis, the resulting Ras chimera is processed by the cellular machinery and the intracellular localization of the protein can then be visualized by means of confocal laser fluorescence microscopy at relatively low concentrations. The absence of a large N-terminal protein tag overcomes possible interferences in the interaction with cellular partner proteins. The fluorescence emission from Bodipy FL is continuous and disappears only after irreversible bleaching. These characteristics make Ras proteins with nonprotein fluorophores suitable for biophysical analysis. The easy accessibility of the lipopeptide moiety by chemical synthesis opens up numerous options for further biological investigations.

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Prenylated proteins. A convenient synthesis of farnesyl cysteinyl thioethers

Cited by 75 publications

References 1 publication

Characterization of Prenylcysteines That Interact with P-glycoprotein and Inhibit Drug Transport in Tumor Cells

Characterization of Prenylcysteines That Interact with P-glycoprotein and Inhibit Drug Transport in Tumor Cells

Substrate Binding Is Required for Release of Product from Mammalian Protein Farnesyltransferase

Synthesis and Application of Fluorescent Ras Proteins for Live‐Cell Imaging

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