Farnesyl thioether

By means of this method, a variety of Ras proteins with different lipidation patterns could be synthesized in multimilligram amounts. For instance, proteins were generated with the natural lipid combination, i.e. a farnesyl thioether and a palmitoyl thioester. Furthermore, analogous proteins were synthesized embodying only one lipid residue in which either the farnesyl- or the palmitoyl group was replaced by a stable hexadecyl thioether. In addition, proteins were built up containing a serine instead of a cysteine residue at the critical sites which normally are lipidated. In a further series of experiments, lipidated Ras proteins which carry a fluorescent Mant group incorporated into the farnesyl-type modification were synthesized.1251... 

FIGURE 9.19 Proteins containing the C-terminal sequence CAAX can undergo prenylation reactions that place thioether-linked farnesyl or geranylgeranyl groups at the cysteine side chain. Prenylation is accompanied by removal of the AAX peptide and methylation of the carboxyl group of the cysteine residue, which has become the C-terminal residue. 

The H- and N-isoforms of Ras support the first (isoprenoid) hydrophobic modification by additional thioester formation with palmitoylic acids [18]. At physiological temperature (37°C) the dissociation of doubly modified lipo-peptides with an isoprenyl thioether and a palmitoyl thioester is very slow and characterized by half-times in the order of 50 h. Here, the relative effect of the carboxymethylation is significantly reduced. Palmitoyl groups with their C16 alkane chain length contribute more efficiently to membrane anchoring than the farnesyl modification. 

Nuclear lamin proteins and the Ras proteins were later shown to be modified by this prenylation reaction [4-6]. The prenyl molecule involved was foimd to be a 15-carbon farnesyl moiety attached to a C-terminal cysteine by a thioether bond [5,6]. Since these farnesylated targets were involved in cell division and signal transduction associated with cell proliferation, the findings suggested an important role for this pathway in tumor cell... 

FTase catalyzes the covalent attachment of a farnesyl moiety via a thioether Unkage to the proteins bearing a C-terminal amino acid sequence known as the CAAX motif (Fig. 2) [12,21]. The farnesyl moiety is derived from farnesyl pyrophosphate (FPP), a 15-carbon isoprenyl intermediate in the mevalonate pathway of cholesterol biosynthesis. The binding of FPP to the enzyme has relatively high affinity (K = 1-lOnM), and FPP binding must precede the binding of the peptide substrate for successful catalysis [22,23]. 

Famesylation of the Ras protein occurs at the C-terminal CAAX sequence (A aliphatic amino acid, X Ser or Thr). The famesyl residue is attached, with the help of a farnesyl protein transferase, via a thioether bond to the Cys residue of the CAAX sequence. Next, the last three amino acids are cleaved off by proteases and the carboxyl group of the C-terminal cysteine residue undergoes a methylesterification (Fig. 9.6). In addition, the Ras proteins have a palmitinic acid anchor at different Cys residues in the vicinity of the C terminus. The membrane localization of the Ki-Ras protein is also supported by a polybasic sequence close to the C terminus (see 3.7 and Fig. 3.12). 

FIGURE 27-30 Farnesylation of a Cys residue. The thioether linkage is shown in red. The Ras protein is the product of the ras oncogene. 

A third lipid anchor is provided by the polyprenyl farnesyl (15-carbon) and geranylgeranyl (20-carbon) groups in thioether linkage to cysteine residues. These must be present in specific recognition sequences at the C termini of proteins, most often with the sequence CAAX.211-215 The prenylation (also called isoprenyla-tion) reaction is followed by proteolytic removal of the last three residues (AAX) and methylation of the new C-terminal carboxyl group as is discussed in Chapter 11, Section D,3. See also Chapter 22, Section A,4. 

Other proteins are transiently attached to the cytosolic face of the membrane either by amide linkage of a myristate (Cl 4 0) molecule to an N-terminal Gly residue (myristoylated proteins Fig. 2b), or by thioether linkage of a 15-carbon farnesyl or a 20-carbon geranylgeranyl polyunsaturated hydrocarbon to a C-terminal Cys residue (prenylated proteins Fig. 2c). Farnesyl and geranylgeranyl are synthesized from isopentenyl pyrophosphate, the precursor of cholesterol (see Topic K5). Some proteins are also modified on Cys residues with covalently attached palmitate (Cl 6 0) (palmitoylated proteins). These include some with membrane-spanning polypeptides (Fig. 2d), some prenylated proteins and some myristoylated proteins. 

Membrane attachment of Ras requires covalent addition of lipids. Virtually all members of the Ras superfamily are prenylated.io Farnesylation is essential. Defects in lipid modification make Ras non-functional. A 15-carbon farnesyl or 20-carbon geranyl-geranyl chain is attached to a cysteine in the COOH-terminus of Ras. The linkage is by a thioether bond and irreversible, and therefore not a point of regulatory control, n ... 

Icmt catalyzes the methyl esterification of the prenylated cysteine residue after Reel has proteolyzed the -CaaX-containing proteins. The first step in identification of the minimal substrate for Icmt was through identification of AFC (Figure 9.2) as described above. Interestingly, farnesylcys-teine (FC), which is devoid of the acetyl substitution, was not a substrate but did possess some activity as an inhibitor [51], suggesting that the free amine of FC requires modification for catalytic turnover. Alterations in the stereochemistry about the FC backbone also appeared to be detrimental to substrate activity. The stereoisomer, d-AFC, was not a substrate for Icmt but was a modest mixed-type inhibitor of the enzyme. AFC-methyl ester (AFC-Me) was also reported to be a mixed-type inhibitor with respect to both l-AFC and -adenosylmethionine (SAM), the methyl donor, with Ki values of 41 and 73 pM, respectively [52,53] The farnesyl homocysteine homolog of AFC is not a substrate for the enzyme however, the racemic DL-homocysteine farnesyl derivative is in fact a weak inhibitor [40]. Similar to the results with racemic prenylcysteine, these data demonstrate that the linker between the carboxylate and thioether moieties is critical for substrate activity. 

The farnesyl pyrophosphate generated in this pathway is also used in the farnesylation of proteins. The farnesyl group is attached to a protein via the thioether... 

Addition of isoprenyi Groups A number of eukaryotic proteins are modified by the addition of groups derived from isoprene (isoprenyl groups). A thioether bond is formed between the isoprenyl group and a Cys residue of the protein (see Fig. 11-14). The isoprenyl groups are derived from pyrophosphorylated intermediates of the cholesterol biosynthetic pathway (see Fig. 21-33), such as farnesyl pyrophosphate (Fig. 27-30). Proteins modified in this way include the Ras proteins, products of the ras oncogenes and proto-oncogenes, and G proteins (both discussed in Chapter 12), and lamins, pro-... 

Fig. 1. Structures of lipids covalently attached to proteins. Panel A shows proteins that are lipidated on cytoplasmi-cally exposed amino acids, whereas panel B shows lipidated proteins in the extracellular leaflet. (A) iV-myristoyl glycine, palmitate thioester-linked to cysteine, farnesyl, or geranylgeranyl (prenyl) thioether-linked to cysteine. (B) A/-palmitoyl cysteine, cholesterol ester-linked to glycine, and a minimal GPI anchor linked to the to amino acid in a GPI-anchored protein. The GPI structure is shown with a diacylglycerol moiety containing two ester-linked fatty acids. Other GPI anchors are based on ceramide, while yet others have monoacylglycerol, a fatty acid ether-linked to glycerol, and/or a fatty acid ester-linked to inositol.

Similar to Ras and Rho proteins, Rab proteins are posttranslationally modified at the C-terminus with prenyl (geranylgeranyl) groups that function as membrane anchors. Protein prenylation involves covalent attachment of the farnesyl (C-15 isoprenyl) or the geranylgeranyl (C-20 isoprenyl) moiety to one or two C-terminal cysteine residues of the protein substrate via a stable thioether linkage. 

The cysteine side chain of CaaX is the nucleophile that reacts with farnesyl pyrophosphate or geranylgeranyl pyrophosphate, forming a thioether and eliminating pyrophosphate. Once the protein has been prenylated, the C—aaX amide bond is hydrolyzed, causing cysteine to become the C-terminal amino acid. Cysteine s carboxyl group is then esterified with a methyl group. 

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