Therapeutic intervention based on protein prenylation and associated modifications

Abstract

In eukaryotic cells, a specific set of proteins are modified by C-terminal attachment of 15-carbon farnesyl groups or 20-carbon geranylgeranyl groups that function both as anchors for fixing proteins to membranes and as molecular handles for facilitating binding of these lipidated proteins to other proteins. Additional modification of these prenylated proteins includes C-terminal proteolysis and methylation, and attachment of a 16-carbon palmitoyl group; these modifications augment membrane anchoring and alter the dynamics of movement of proteins between different cellular membrane compartments. The enzymes in the protein prenylation pathway have been isolated and characterized. Blocking protein prenylation is proving to be therapeutically useful for the treatment of certain cancers, infection by protozoan parasites and the rare genetic disease Hutchinson-Gilford progeria syndrome.

Figure 1

Structures of the C termini of prenylated proteins. Farnesylated protein (left); geranylgeranylated protein (middle); doubly geranylgeranylated protein (right).

Figure 2

Enzymatic pathway for the modification of prenylated proteins. PFT attaches a farnesyl group to the C-terminal CaaX motif of specific cytosolic proteins. The farnesylated protein undergoes C-terminal aaX removal by the endoprotease Rce1p, and this is followed by S-adenosylmethionine (SAM)-dependent methylation of the COOH terminus by Imct; both modifications occur in the endoplasmic reticulum. Some proteins, such as N-Ras and H-Ras, are further modified after transfer to Golgi membranes by palmitoylation on one or two cysteines near the prenylated cysteine in a reaction catalyzed by DHHC9 and GCP16 in mammals (Erf2 and Erf4 in yeast endoplasmic reticulum membranes) and using palmitoyl coenzyme A (CoA) as the fatty-acid donor. After palmitoylation, fully lipidated N-Ras and H-Ras are transferred to the plasma membrane probably by vesicle transport. Other proteins, such as KRas, contain a polybasic region (KKKKKKSKTK) next to the prenylated C terminus that is thought to bind the protein to the cytosolic face of the plasma membrane through electrostatic interactions with acidic phospholipids. PPO, pyrophosphate. Farnesyl groups are shown in blue and palmitoyl groups are red.

Figure 3

Model for the trafficking of H-Ras and N-Ras from the Golgi complex to the plasma membrane and back,. S-Palmitoylation of the two Ras isoforms occurs at the Golgi (1), followed by directed vesicular transport to the plasma membrane (2), where the protein can be released after enzymatic hydrolysis of the thioester (3) and transported back via a nonvesicular pathway to the Golgi (4). APT1, DHHC9 and GCP16 may be involved in palmitoylation on the Golgi.

Figure 4

Model for extraction of prenylated Rab proteins from membranes via GDI, as previously formulated. (1) The initial recognition occurs via the low-affinity interaction of the Rab protein with the C-terminal–binding region (CBR) of GDI. Then, the lipid-binding site of GDI is positioned over the prenyl functionalities of the Rab protein (2), the first geranylgeranyl moiety is transferred (3) and the second geranylgeranyl moiety is transferred (4). (5) This finally results in the dissociation of the complex from the membrane.

Figure 5

Schematic representation of methods used to introduce natural and modified lipidated C termini on Ras GTPases, and a collection of semisynthetic neo-Ras proteins synthesized via these methods,,,,. (a) EPL. H-Ras (1–180) denotes the first 180 amino acids of this protein. (b) MIC ligation. Far, farnesyl; Ger, geranyl.

Figure 6

FTI and GGTI compounds. Three FTI compounds used in clinical trials are shown. These compounds are highly selective inhibitors of PFT and may be effective in inhibiting cancer and metastasis arising from the overexpression or overactivation of H-Ras, Rheb and PRL-3 proteins. Two GGTI compounds are shown. They may be effective in inhibiting the function of RhoC, RalA/B and K-Ras4B.