Prenylated Protein Methyltransferase Research Articles

ES28.01 Biology of KRAS Targeting Agents

ES28.01 Biology of KRAS Targeting Agents

Ras as a therapeutic target in hematologic malignancies

The RAS gene product is normally a membrane-localized G protein (N-Ras, K-Ras and H-Ras) of 21 kDa classically described as a molecular off/on switch. It is inactive when bound to guanosine diphosphate and active when bound to GTP. When mutated, the gene produces an abnormal protein resistant to GTP hydrolysis by GTPase, resulting in a constitutively active GTP-bound protein that stimulates a critical network of signal transduction pathways that lead to cellular proliferation, survival and differentiation. At least three downstream effector pathways have been described, including Raf/MEK/ERK, PI3K/AKT and RalGDS, but they are not completely understood. Ras pathways are also important downstream effectors of several receptor tyrosine kinases localized in the cell membrane, most notably the BCR-ABL fusion protein seen in patients with Philadelphia chromosome positive chronic myelogenous leukemia. An important consideration in designing strategies to block Ras stimulatory effect is that Ras proteins are synthesized in the cytosol, but require post-translational modifications and attachment to anchor proteins or membrane binding sites in the cell membrane to be biologically active. Farnesyl transferase inhibitors (FTIs) are probably the best-studied class of Ras inhibitors in hematologic malignancies. They block the enzyme farnesyl-transferase (FTase), which is essential for post-translational modification. However, it has been observed that the Ras proteins also can be geranylgeranylated in the presence of FTIs, thus allowing membrane localization and activation, which limits their effectiveness. It is now hypothesized that their mechanism of action may be through FTase inhibition involving other signal transduction pathways. S-trans, trans-farnesylthiosalicylic acid, which was first designed as a prenylated protein methyltransferase inhibitor, has shown in vitro activity against all activated Ras proteins by dislodging them from their membrane-anchoring sites. Here, Ras biology, its signaling pathways and its implications as a therapeutic target in hematologic malignancies are reviewed.

Inhibition mechanism of S-adenosylmethionine-induced movement deficits by prenylcysteine analogs

We previously showed that S-adenosylmethionine (SAM) induces movement impairments similar to those observed in Parkinson's disease (PD) apparently by prenylated protein methylation; 5 kDa molecules being methylated and the symptoms being inhibited by prenylcysteine (PC) analogs. In the present study, we explore the biochemical mechanism of action of the PC analogs. N-acetylgeranylcysteine (AGC), N-acetylfarnesylcysteine (AFC), N-acetylgeranylgeranylcysteine (AGGC), farnesylthioacetic acid (FTA), farnesyl-2-ethanesulfonic acid (FTE) and farnesylsuccinic acid (FMS), but not farnesylthiotriazole (FTT) and farnesylthiolactic acid (FTL), inhibited the SAM-induced motor impairments. Incubation of the respective analogs with rat brain membranes containing prenylated protein methyltransferase (PPMTase) resulted in the methylation of AGC, AFC and AGGC. FTA, FTE, FMS and FTT, but not FTL, inhibited the enzyme activity. A single injection of the active analogs remained effective for at least 3 days against repeated injections of 1 μmol SAM. Amphetamine-induced hyperactivity in rats was inhibited by SAM but potentiated by FTE. During 60 min, the movement time for amphetamine-treated rats was 1477 s compared with 633 and 1664 s for amphetamine+SAM- and amphetamine+FTE-treated rats, respectively. The total distance for amphetamine+FTE-treated rats was 82% higher than for amphetamine. The horizontal activity was 30,728 (amphetamine), 15,430 (FTE), 18,526 (amphetamine+SAM), 41,736 (amphetamine+FTE) and 7004 (SAM) as compared to the PBS control (4726). The intricate relationship between the actions of SAM, which speeds up prenylated protein methylation and impairs movement, amphetamine, which increases synaptic dopamine levels and movement, and the PC analogs, which prevent the SAM-induced movement impairments, suggests a SAM-induced defect on dopamine signaling as the likely cause of the symptoms. The data reveal that interaction of PC analogs with PPMTase may not be an indicator of anti-PD-like activity.

Targeted Inactivation of the Isoprenylcysteine Carboxyl Methyltransferase Gene Causes Mislocalization of K-Ras in Mammalian Cells

After isoprenylation and endoproteolytic processing, the Ras proteins are methylated at the carboxyl-terminal isoprenylcysteine. The importance of isoprenylation for targeting of Ras proteins to the plasma membrane is well established, but the importance of carboxyl methylation, which is carried out by isoprenylcysteine carboxyl methyltransferase (Icmt), is less certain. We used gene targeting to produce homozygous Icmt knockout embryonic stem cells (Icmt-/-). Lysates from Icmt-/- cells lacked the ability to methylate farnesyl-K-Ras4B or small-molecule Icmt substrates such as N-acetyl-S-geranylgeranyl-L-cysteine. To assess the impact of absent Icmt activity on the localization of K-Ras within cells, wild-type and Icmt-/- cells were transfected with a green fluorescent protein (GFP)-K-Ras fusion construct. As expected, virtually all of the GFP-K-Ras fusion in wild-type cells was localized along the plasma membrane. In contrast, a large fraction of the fusion in Icmt-/- cells was trapped within the cytoplasm, and fluorescence at the plasma membrane was reduced. Also, cell fractionation/Western blot studies revealed that a smaller fraction of the K-Ras in Icmt-/- cells was associated with the membranes. We conclude that carboxyl methylation of the isoprenylcysteine is important for proper K-Ras localization in mammalian cells.

Characterization of prenylated protein methyltransferase in Leishmania

Prenylated protein methyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, has been characterized in a parasitic flagellated protozoan, Leishmania donovani. The activity of this enzyme was monitored by the methylation of an artificial substrate, an S-prenylated cysteine analogue, with S-adenosyl-L-[methyl-3H]methionine as methyl donor. More than 85% of the methyltransferase activity was associated with membranes. The enzyme methylates N-acetyl-S-trans,trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, but N-acetyl-S-trans,trans-geranyl-L-cysteine only very weakly. In contrast with the enzyme from mammals, the leishmanial enzyme had a greater affinity for the farnesylated substrate than for the geranylgeranylated one. Activity in vitro was not modulated by cAMP, protein kinase C activator or guanosine 5′-[γ-thio]triphosphate. An analysis of the endogenous substrates showed that the carboxymethylated proteins were also isoprenylated. The main carboxymethylated proteins have molecular masses of 95, 68, 55, 46, 34-23, 18 and less than 14 kDa. Treatment of cells with N-acetyl-S-trans,trans-farnesyl-L-cysteine decreased the carboxymethylation level, whereas treatment with guanosine 5′-[γ-thio]triphosphate increased the carboxymethylation of various proteins, particularly those of molecular masses 30-20 kDa.

Characterization of prenylated protein methyltransferase in Leishmania

Prenylated protein methyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, has been characterized in a parasitic flagellated protozoan, Leishmania donovani. The activity of this enzyme was monitored by the methylation of an artificial substrate, an S-prenylated cysteine analogue, with S-adenosyl-l-[methyl-(3)H]methionine as methyl donor. More than 85% of the methyltransferase activity was associated with membranes. The enzyme methylates N-acetyl-S-trans, trans-farnesyl-l-cysteine and N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, but N-acetyl-S-trans, trans-geranyl-l-cysteine only very weakly. In contrast with the enzyme from mammals, the leishmanial enzyme had a greater affinity for the farnesylated substrate than for the geranylgeranylated one. Activity in vitro was not modulated by cAMP, protein kinase C activator or guanosine 5'-[gamma-thio]triphosphate. An analysis of the endogenous substrates showed that the carboxymethylated proteins were also isoprenylated. The main carboxymethylated proteins have molecular masses of 95, 68, 55, 46, 34-23, 18 and less than 14 kDa. Treatment of cells with N-acetyl-S-trans,trans-farnesyl-l-cysteine decreased the carboxymethylation level, whereas treatment with guanosine 5'-[gamma-thio]triphosphate increased the carboxymethylation of various proteins, particularly those of molecular masses 30-20 kDa.

Guanosine 5′-(3-O-Thio)triphosphate Stimulates Protein Carboxyl Methylation in Cell Membranes

Using guanosine 5′-(3-O-thio)triphosphate (GTPγS), we previously reported that protein carboxyl methyltransferase activities in kidney brush border membranes were increased by the GTP analog (Arch. Biochem. Biophys. 351, 149–158, 1998). Here, we investigated the distribution and characterized the effect of GTPγS on protein carboxyl methylation activity. The analysis of species distribution of carboxyl methylation in kidney brush border membranes showed that the GTPγS strongly stimulated this activity in rat (15.9-fold), mouse (14.7-fold), human (2.9-fold), and rabbit (2.7-fold). Analysis of GTPγS-dependent carboxyl methylation in rat tissues and cell fractions indicated that the activity was mainly localized in membranes of intestine, lung, and kidney, with the highest activity found in liver. To characterize the methyltransferase activity modulated by GTPγS in liver membranes, their sensitivity to the detergent 3-[(3-cholamido)dimethylammonio]-1-propanesulfonic acid (Chaps) was used. Methylation of N-acetyl-S-farnesyl cysteine, a prenylated protein methyltransferase (PPMT) substrate was strongly inhibited (86%) in the presence of Chaps, while the methylation of bovine calmodulin and ovalbumin, both of which are substrates for the protein L-isoaspartyl/d-aspartyl methyltransferase (PIMT), was slightly reduced by the detergent (0–12%). The GTPγS-dependent carboxyl methylation of endogenous substrates in liver membranes was decreased by 35% in the presence of Chaps, suggesting that PPMT was not the predominant methyltransferase involved in the methylation stimulated by GTPγS in liver membranes. Electrophoretic analysis showed that radioactive methylation of several substrates induced by GTPγS in liver membranes was reduced by adding calmodulin. Interestingly, addition of GTPγS partially inhibited the methylation of two PIMT substrates, ovalbumin (24%) and bovine calmodulin (19%), when incubated with liver membranes. Immunoprecipitation of PIMT from liver and lung membranes strongly inhibited (88–94%) the methylation stimulated by GTPγS. Altogether, these data support the hypothesis that GTPγS could regulate PIMT activity and may provide new insights into the function of the methyltransferase.

Regulation by GTPγS of Protein Carboxylmethyltransferase Activity in Kidney Brush Border Membranes

The increase in carboxyl methylation induced by guanosine 5′,3-O-(thio)triphosphate (GTPγS) in brush border membranes from rat kidney cortex was studied, and the methyltransferase activities affected by this nucleotide analog were identified. Addition of GTPγS to brush border membranes stimulated the carboxyl methylation in a time-dependent manner while adenosine and guanine nucleotides were ineffective. The GTPγS-dependent carboxyl methylation was inhibited by the chelating agents EDTA (63%) and 1,10-phenanthroline (68%), suggesting that this activity required divalent cations. The methyl ester groups induced by the addition of GTPγS to brush border membranes were unstable, with about 80% of them hydrolyzed following 1 h incubation at 37°C. The GTPγS stimulation of the carboxyl methylation in brush border membranes was unaffected by the detergent 3-[(3cholamido)-dimethylammonio]-1-propanesulfonic acid up to a concentration of 0.4% (w/v). At this latter detergent concentration, the activity of prenylated protein methyltransferase (PPMT) was strongly inhibited and that ofl-isoaspartyl/d-aspartylmethyltransferase (PIMT) was increased twofold, as measured with their respective exogenous substrates,N-acetyl-S-farnesyl cysteine and ovalbumin. GTPγS increased the methylation of several substrates in brush border membranes. The induced methylation in substrates migrating between 20 and 36 kDa was strongly decreased by the competitive inhibitor farnesylthioacetic acid, a synthetic farnesylated substrate for PPMT, while a δ-sleep-inducing peptide containing anl-isoaspartyl residue inhibited that of substrates with molecular weights above 36 kDa, suggesting that PIMT activity was also involved. This interpretation was strengthened by the observation that the increased methylation induced by GTPγS in these membrane substrates was completely lost following their analysis by gel electrophoresis under alkaline conditions. Taken together, these results indicate that both PPMT and PIMT activities are regulated by guanine nucleotides in brush border membranes of rat kidney.

ChemInform Abstract: Novel Inhibitors of the Prenylated Protein Methyltransferase Reveal Distinctive Structural Requirements.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Novel inhibitors of the prenylated protein methyltransferase reveal distinctive structural requirements

Inhibitors of a prenylated protein methyltransferase were synthesized and evaluated. S-farnesyl-5-fluorothiosalicylic acid and the 5-chloro analog (but not the 4-fluoro, 4-chloro or 3-chloro analogs) were potent inhibitors, as was the parent compound S-farnesyl thiosalicylic acid (FTS), whose methyl ester was far less active. S-geranyl and S-geranylgeranyl thiosalicylic acids were more than ten times less potent than FTS.

Selective Inhibition of Ras-dependent Cell Growth by Farnesylthiosalisylic Acid

S-trans,trans-Farnesylthiosalicylic acid (FTS) is a novel farnesylated rigid carboxylic acid derivative. In cell-free systems, it acts as a potent competitive inhibitor (Ki = 2.6 microM) of the enzyme prenylated protein methyltransferase (PPMTase), which methylates the carboxyl-terminal S-prenylcysteine in a large number of prenylated proteins including Ras. In such systems, FTS inhibits Ras methylation but not Ras farnesylation. Inhibition of the PPMTase by FTS in homogenates or membranes of a variety of tissues and cell lines is inferred from a block in the methylation of exogenously added substrates such as N-acetyl-S-trans,trans-farnesyl-L-cysteine and of endogenous substrates including small GTP-binding proteins. FTS can also inhibit methylation of these proteins in intact cells (e.g. in Rat-1 fibroblasts, Ras-transformed Rat-1, and B16 melanoma cells). Unlike in cell-free systems, however, relatively high concentrations of FTS (50-100 microM) are required for partial blocking (10-40%) of protein methylation in the intact cells. Thus, FTS is a weak inhibitor of methylation in intact cells. Because methylation is the last step in the processing of Ras and related proteins, FTS is not likely to affect steps that precede it, e.g. protein prenylation. This may explain why the growth and gross morphology of a variety of cultured cell types (including Chinese hamster ovary, NIH3T3, Rat1, B16 melanoma, and PC12) is not affected by up to 25 microM FTS and is consistent with the observed lack of FTS-induced cytotoxicity. Nevertheless, FTS reduces the levels of Ras in cell membranes and can inhibit Ras-dependent cell growth in vitro, independently of methylation. It inhibits the growth of human Ha-ras-transformed cells (EJ cells) and reverses their transformed morphology in a dose-dependent manner (0.1-10 microM). The drug does not interfere with the growth of cells transformed by v-Raf or T-antigen but inhibits the growth of ErbB2-transformed cells and blocks the mitogenic effects of epidermal and basic fibroblast growth factors, thus implying its selectivity toward Ras growth signaling, possibly via modulation of Ras-Raf communication. Taken together, the results raise the possibility that FTS may specifically interfere with the interaction of Ras with a farnesylcysteine recognition domain in the cell membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Farnesyl derivatives of rigid carboxylic acids-inhibitors of ras-dependent cell growth.

Inhibitors of the enzyme that methylates ras proteins, the prenylated protein methyltransferase (PPMTase), are described. They are farnesyl derivatives of rigid carboxylic acids that recognize the farnesylcysteine recognition domain of the enzyme but do not serve as substrates. They also inhibit ras-dependent cell growth by a mechanism that is probably unrelated to inhibition of ras methylation, even though their potencies as PPMTase inhibitors and cell-growth inhibitors correlate well. The most potent inhibitor is S-trans,trans-farnesylthiosalicylic acid (FTS) (2). FTS (2) selectively inhibits the growth of human Ha-ras-transformed Rat1 cells in vitro (EC50 = 7.5 microM).

Characterization of the prenylated protein methyltransferase in human endometrial carcinoma

The processing or ras and of other GTP-binding proteins includes a final reversible step in which the carboxy terminal prenylated cysteine is methylated by the enzyme prenylated protein methyltransferase (PPMTase). The significance of this modification and of the role of PPMTase in human tumors has yet to be fully elucidated. Here we characterize the PPMTase of human endometrical carcinomas (tumors in which the frequency of ras gene mutations is relatively high) and compare it to the PPMTase of the normal endometrium. Our results show that in both types of tissues the enzyme is bound to the membranes. It can utilize synthetic substrate such as N- acetyl-S-farnesyl- l-cysteine (K m = 18–20 μ M) and is blocked by the PPMTase inhibitor S-farnesylthioacetic acid ( K i = 2 μM). In vitro methylation assays and [ α- 32P]GTP blot-overlay assays showed that the major endogenous PPMTase substrates are small GTP-binding proteins. Methylation of these proteins in vitro is blocked by farnesylthioacetic acid. The kinetic properties of PPMTase from the carcinomas and the normal tissues are very similar. however, levels of PPMTase activity (but not of its endogenous substrates) are higher in the carcinomatous endometrium than in the normal one. The elevated enzyme activity is restricted to the crude mitochondrial fraction (8.0 ± 0.4 vs. 5.4 ± 0.1 pmol N-acetyl farnesylcysteine methyl ester formed/min/mg protein by the carcinoma and by the normal endometrial preparations, respectively). As this fraction is enriched in plasma membranes, it appears that the elevated enzyme activity could be related to ras protein methylation; if so, selective methylation blockers might inhibit the growth of endometrial carcinomas.

Prenylated protein methyltransferase of rat cerebellum is developmentally co-expressed with its substrates

High levels of prenylated protein methyltransferase are expressed in the developing rat cerebellum and are responsible for methylation of endogenous G-proteins and 50–52 kDa synaptosomal proteins. Enzyme activity in cerebellar synaptosomes of 3 week postnatal rats is 2-fold higher than that found in adult rat cerebellum. A 10-fold rise in activity occurs at the end of the second and during the third postnatal weeks, followed by a subsequent decline. Expression of the enzymes' substrates follows the same pattern. The high methyltransferase activity in 3-week-old cerebellum coincides with the period of granule cell migration and synaptogenesis, suggesting a regulatory role for the enzyme and its substrates in cerebellar ontogenesis.