Farnesyl group

Some proteins can be posttranslationally modified by the addition of prenyl groups. Prenyl groups are long-chain, unsaturated hydrocarbons that are intermediates in isoprenoid synthesis. The farnesyl group has 15 carbons, and the geranylgeranyl has 20 carbons. They are attached to a cysteine residue near the end of the protein as a thiol ether (Protein-S-R). Other proteins can have a long-chain fatty acid (C14=myristoyl, C16=palmitoyl) attached to the amino terminus as an amide. These fatty acid modifications can increase the association of proteins with the membrane. 

Further investigations with bimanyl-labeled K-Ras4B peptides demonstrated that relatively small differences in membrane charging (approximately 10 mol %) are sufficient for an electrostatic enrichment in the more negative environment [230]. With the farnesyl group as a hydrophobic anchor, the peptide is still mobile and can swap between vesicles but may find its target membrane with the sensitive surface potential-sensing function of its lysine residues. 

In normal cells, the GDP/GTP-binding proteins, after protein synthesis, move to the cell membrane to which they become hooked by a hydrophobic farnesyl group. The y-subunit is anchored in the membrane by a post-translational modification of the C-terminal CAAX sequence (C - cystein, AA - aliphatic amino acids, X - methionine). This protein is first enzymatically farnesylated by a specific farnesyltransferase, then the AAX part is cleaved by specific proteases and finally the cystein residue is converted to a methyl ester. 

Acid- and base-sensitive lipidated peptides can be selectively deprotected by enzymatic hydrolysis of choline esters.[13al Choline esters of simple peptides, but also of sensitive peptide conjugates like phos-phorylated and glycosylated peptides,1141 nucleopep-tides1151 and lipidated peptides,113,1631 can be cleaved with acetyl choline esterase (AChE) and butyryl choline esterase (BChE) under virtually neutral conditions with complete chemoselectivity. Acid-labile farnesyl groups and base-sensitive thioesters are not attacked. 

Thus, lipoproteins could be injected over the surface of a lipid covered SPR sensor in a detergent free buffer solution and showed spontaneous insertion into the artificial membrane.171 Again two hydro-phobic modifications are necessary for stable insertion into the lipid layer, whereas lipoproteins with a farnesyl group only dissociate significantly faster out of the membrane. Therefore the isoprenylation of a protein is sufficient to allow interaction with membraneous structures, while trapping of the molecule at a particular location requires a second hydrophobic anchor. Interaction between the Ras protein and its effector Raf-kinase depends on complex formation of Ras with GTP (instead of the Ras GDP complex, present in the resting cell). If a synthetically modified Ras protein with a palmi-... 

In a related experiment CV-1 fibroblasts were incubated with fluorescent N-Ras lipopeptides bearing a free palmitoylation site. These peptides cause staining of the CV-1 plasma membrane and efficient S-acylation even if the farnesyl group was replaced by a n-octyl group.1271 The association of the N-Ras lipopeptides with the plasma membrane... 

The Farnesyl Group of H-Ras Facilitates the Activation of a Soluble Upstream Activator of Mitogen-activated Protein Kinase, P. McGeady, S. Kuroda, K. Shimizu, Y. Takai, M H. GelbJ. Biol Chem 1995,270,26347-26351. 

Replacement of the H-Ras Farnesyl Group by Lipid Analogues Implication for Downstream Processing and Effector Activation in Xenopus Oocytes, T. Dudler, M. H. Gelb, Biochemistry 1997, 36,12434-12441. 

In order for Ras to function properly, the protein must be localized to the plasma membrane. This is achieved by the addition of a farnesyl group to a cysteine residue near the carboxy terminus, which then acts as a tether to the cellular membrane. Farnesyl transferase, the enzyme that adds the farnesyl moiety to Ras, is also a target for small-molecule intervention. This class of inhibitors is the subject of another chapter (Angibaud et al, in this volume). 

To function, Ras must be attached to the plasma membrane. Translocation from the cytoplasm to membrane requires a series of posttranslational modifications that begin with farnesylation of the cysteine residue, the fourth amino acid residue from the C terminus of the protein, by famesyl protein transferase (FPTase) (64). Attachment of the hydrophobic 15-carbon lipid farnesyl group allows Ras molecule insertion into the plasma membrane and is crucial for Ras signaling activity and transformation properties. As farnesylation is required for oncogenic Ras function, FPTase inhibitors (FTIs) are obvious candidate antineoplastic agents. Several drugs that inhibit Ras farnesylation are at various stages of clinical development (65). 

Prenylation (covalent attachment of an isoprenoid see Fig. 27-30) is a common mechanism by which proteins are anchored to the inner surface of cellular membranes in mammals (see Fig. 11-14). In some of these proteins the attached lipid is the 15-carbon farnesyl group others have the 20-carbon geranylgeranyl group. Different enzymes attach the two types of lipids. It is possible that prenylation reactions target proteins to different membranes, depending on which lipid is attached. Protein prenylation is another important role for the isoprene derivatives of the pathway to cholesterol. 

Other covalent modifications These may be required for the functional activity of a protein. For example, additional carboxyl groups can be added to glutamate residues by vitamin Independent carboxylation (see p. 387). The resulting y-carboxy-glutamate resides are esssential for the activity of several of the blood-clotting proteins. Attachment of lipids, such as farnesyl groups, can help anchor proteins in membranes. In addition, many proteins are acetylated postranslationally. 

Familial adenomatous polyposis 574 Faraday, numerical value of 283 Farnesyl group 402, 559 Fat(s). See also Triacylglycerol (triglyceride) composition of 380 hydrolysis of 507 Fatty acid(s) 380-382 activation of 512 acyl CoA, derivatives of 507 biosynthesis of 722 branched chain 381 cyclopropane-containing 399 essential 721 in lipids 380 names of, table 380 oxidation 511 pKa values of 380 stability of 589... 

Sexual conjugation in yeast is also induced by pheromones (mating factors).325-327 Yeast cells of mating type a synthesize the 12-residue mating factor a which contains a C-terminal cysteine methyl ester S-alkylated with a frans,frans-farnesyl group (Table 30-5). Cells of type a synthesize a 13-residue factor ol 327a Cells are attracted to the pheromone produced by cells of the opposite type. The tremerogens, sex hormones of certain basidiomycetes, have related structures (Table 30-5)328... 

Prenylated proteins have characteristic C-terminal sequences. For example, the three allelic Ras proteins (H-Ras, K-Ras, and N-Ras) expressed in mammalian tissues contain a C-terminal tetrapeptide which begins with cysteine, and ends with either methionine or serine. This part of the molecule is referred to as the CaaX box where C = cysteine, a = an aliphatic amino acid, and X = a prenylation specificity residue. The first step in the posttranslational processing of Ras proteins utilizes FTase and farnesyl diphosphate (FPP) to covalently attach a farnesyl group to the cysteine thiol of the CaaX box. While subsequent processing events involve proteolytic removal of the aaX tripeptide and methylation of the resulting C-termi-nal carboxylate group, only the farnesyl modification is required for mutant Ras proteins to associate with the cell membrane and transform a cell.2-6... 

In plants and fungi, IPP and DMAPP are the precursors to many so-called isoprenoid compounds, including natural rubber. In animals, they are mainly precursors to sterols, such as cholesterol. The first step is condensation of one of each to geranyl pyrophosphate, which then condenses with another molecule of IPP to make farne-syl pyrophosphate. Some important membrane-bound proteins have a farnesyl group added on to them however, the primary fate of far-nesyl pyrophosphate is to accept a pair of electrons from NADPH and... 

Note that the formation of squalene results from the coupling of both of the farnesyl groups at the carbons attached to the pyrophosphate groups (head-to-head coupling) and that both pyrophosphate groups are lost during this process. The mechanism for this reaction differs dramatically from that shown in Figure 28.2 and is too complicated to present here. 

Fig. 3.15 FTase transfers the farnesyl group from farnesyl diphosphate (FPP) to the SH group of a cysteine near the COOH-terminus of the protein. The COOH-terminal tripeptide, AAX (A is an aliphatic amino acid and X is Val-Leu-Ser, or any other amino-acid residue), is removed by a protease. Subsequently, a methyl transferase donates the methyl (CH3) group from S-adenosylmethionine (SAM) to the COOH-terminal Sfarnesylated cysteine. The final step is the attachment of palmrtoyl groups to the cysteines near the farnesylated, carboxymethylated-terminus by a specific palmitoyl transferase.

Much attention has been focused on defining the transition state of FTase and the structural determinants of the chemical step. For FTase, there is evidence for both an electrophilic contribution to the transition state, obtained from studies with fluoromethyl FPP analogues, and a nucleophilic contribution, obtained from the metal-substitution and pH studies [31,40,41]. These results are supported by the inability to trap a carbocation intermediate, inversion of configuration at Cl of the farnesyl group during the reaction, and an a-secondary kinetic isotope effect near unity [31,42,43]. Taken together, the available data suggest that the transition state of FTase... 

The Agell laboratory has explored the relationship between PKC-mediated phosphorylation of K-Ras at S181 and binding of the second messenger CaM to this residue, and the consequences of these interactions for K-Ras localization and activity. They demonstrated that CaM interacts with GTP-bound K-Ras, but not with H-Ras or N-Ras [12], and, consistent with this selectivity, that the farnesyl group, polybasic residues, and S181 of K-Ras are all required for this interaction [13]. Phosphorylation of S181 and... 

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