Identification and preliminary characterization of protein-cysteine farnesyltransferase.

Manne, Veeraswamy; Roberts, Daniel M.; Tobin, Alyson K.; O'Rourke, Edward; Virgilio, M De; Meyers, Chester A.; Ahmed, Naseem; Kurz, Bodo; Resh, Marilyn D.; Kung, Hsiang‐Fu

doi:10.1073/pnas.87.19.7541

Cited by 133 publications

(81 citation statements)

References 23 publications

(38 reference statements)

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“…A protein farnesyltransferase (PFT) has recently been purified and characterized from several mammalian tissues (30)(31)(32). The PFT transfers a farnesyl moiety from farnesyl pyrophosphate to an unmodified form of c-Ha-ras p21 and appears to recognize the Cys-Ali-Ali-Xaa motif (30).…”

Section: Discussionmentioning

confidence: 99%

C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.

Farnsworth

Kawata

Yoshida

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

166

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smg p25A, also known as the rab3A protein, is a small GTP-binding protein that has been implicated in intracellular vesicle transport and the secretion of neurotransmitters. It has been shown to bind reversibly to membranes, though its cDNA-predicted sequence contains no obvious membrane-binding domains. However, smg p25A does contain a cDNA-predicted C-terminal Cys-Ala-Cys sequence at positions 218 through 220, which suggests that it may be posttranslationally modified. In the present study we used two different approaches to investigate this possibility. First, we incubated pheochromocytoma cells with [3H]mevalonolactone, examined the proteins that became labeled by two-dimensional gel electrophoresis, and demonstrated that two of these proteins exactly corresponded to smg p25A. Second, we purified smg p25A from bovine brain membranes and analyzed both the full-length protein and a proteolytically derived C-terminal peptide by a combination of high performance liquid chromatography and mass spectrometry. This approach revealed that the protein's C-terminal region is methyl-esterified and contains two geranylgeranyl groups linked via thioether bonds to Cys-218 and Cys-220. Since smg p25A is one of several small GTP-binding proteins that share a C-terminal Cys-Xaa-Cys consensus sequence (where Xaa is an unspecified amino acid), our results suggest that these proteins may be similarly geranylgeranylated and methyl-esterified.

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Section: Discussionmentioning

confidence: 99%

C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.

Farnsworth

Kawata

Yoshida

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

166

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“…These proteins include ras in crude extracts of yeast cells [2,18]. The requirement of two proteins, nuclear lamins and the yeast a-mating factor, which end genes, DPRI/RAMI (referred to here as DPRI) and RAM2 for with a unique C-terminal sequence, CysAAX (A is an aliphatic the yeast FTase activity suggested that the enzyme consisted of amino acid and X is the C-terminal amino acid) [1][2][3][4]. This two subunits encoded by these genes.…”

Section: Introductionmentioning

confidence: 99%

Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart

Gomez

Goodman

Tripathy

et al. 1993

Biochemical Journal

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Protein farnesyltransferase (FTase) catalyses the addition of a farnesyl group to a cysteine within the so-called ‘CAAX box’ at the C-terminus of various proteins. In the present paper we report purification of Saccharomyces cerevisiae FTase to near-homogeneity. This was accomplished by constructing a yeast strain overproducing FTase approx. 100-fold. The purified enzyme was a heterodimer of approx. 90 kDa and consisted of 43 kDa and 34 kDa subunits. The 43 kDa subunit was shown to be the product of the DPR1 gene by using antibody raised against baculovirus-produced DPR1 polypeptide. The purified enzyme required Mg2+, showed a pH optimum of 7.8 and was most active at 50 degrees C. The Km values for farnesyl pyrophosphate and GST-CIIS (glutathione S-transferase fused to the C-terminal 12 amino acids of yeast RAS2 protein), KmFpp and KmGST CIIS, were 8.1 and 5.1 microM respectively. The enzyme was capable of farnesylating GST-CIIL (the same as GST-CIIS, except that the C-terminal serine is changed to leucine), a substrate protein for the enzyme geranylgeranyltransferase, although with a higher apparent Km than for GST-CIIS. Like its mammalian counterpart, yeast FTase activity was inhibited by peptides containing the C-terminal CAAX sequence (that is, one where C = cysteine, A = aliphatic amino acid and X = any amino acid). These results provide direct evidence for the idea that the yeast and mammalian FTases are structurally and functionally very similar.

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“…Mammalian protein farnesyltransferase (PFT) 1 farnesylates the cysteine residue in carboxyl-terminal cysteine-aa aliphatic -aa aliphatic -aa x (CAAX) sequences, where aa x is methionine, glutamine, serine, threonine, or cysteine (13)(14)(15)(16). Protein geranylgeranyl transferase-I (PGGT-I) shows specificity for cysteine-aa aliphaticaa aliphatic -leucine sequences (13,(17)(18)(19)(20).…”

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confidence: 99%

Protein Farnesyltransferase and Protein Prenylation inPlasmodium falciparum

Chakrabarti¹,

Silva²,

Barger³

et al. 2002

Journal of Biological Chemistry

141

120

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Comparison of the malaria parasite and mammalian protein prenyltransferases and their cellular substrates is important for establishing this enzyme as a target for developing antimalarial agents. Nineteen heptapeptides differing only in their carboxyl-terminal amino acid were tested as alternative substrates of partially purified Plasmodium falciparum protein farnesyltransferase. Only NRSCAIM and NRSCAIQ serve as substrates, with NRSCAIM being the best. Peptidomimetics, FTI-276 and GGTI-287, inhibit the transferase with IC 50 values of 1 and 32 nM, respectively. Incubation of P. falciparum-infected erythrocytes with [ 3 H]farnesol labels 50-and 22-28-kDa proteins, whereas [ 3 H]geranylgeraniol labels only 22-28-kDa proteins. The 50-kDa protein is shown to be farnesylated, whereas the 22-28-kDa proteins are geranylgeranylated, irrespective of the labeling prenol. Protein labeling is inhibited more than 50% by either 5 M FTI-277 or GGTI-298. The same concentration of inhibitors also inhibits parasite growth from the ring stage by 50%, decreases expression of prenylated proteins as measured with prenyl-specific antibody, and inhibits parasite differentiation beyond the trophozoite stage. Furthermore, differentiation specific prenylation of P. falciparum proteins is demonstrated. Protein labeling is detected predominantly during the trophozoite to schizont and schizont to ring transitions. These results demonstrate unique properties of protein prenylation in P. falciparum: a limited specificity of the farnesyltransferase for peptide substrates compared with mammalian enzymes, the ability to use farnesol to label both farnesyl and geranylgeranyl moieties on proteins, differentiation specific protein prenylation, and the ability of peptidomimetic prenyltransferase inhibitors to block parasite differentiation.Malaria continues to be a major disease in tropical areas of the world. Parasite resistance to current drugs used in the treatment of malaria has lead investigators to seek out new drug targets. Among those targets are proteins and enzymes, which are necessary for cellular division and differentiation. Such targets include the protein prenyltransferases, which are necessary for the post-translational modification of proteins involved in the signal transduction pathways and in regulation of DNA replication and cell cycling (1-3). These enzymes are currently a major focus of efforts to design drugs that inhibit unregulated cell growth in cancer (4, 5). Several candidate compounds show great promise as antitumor drugs and are currently being tested in clinical trials. The potential for the application of such drugs to inhibit malarial parasite division and differentiation motivates one to examine the properties of the protozoan enzyme with the goal of identifying unique features of this enzyme that would make it a target for the development of parasite-specific drugs.A variety of proteins including small G-proteins, such as Ras, Rac, Rap, Rho, Rab (6), heterotrimeric G protein ␥ subunits (7), nuclear lamins (8), prot...

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Identification and preliminary characterization of protein-cysteine farnesyltransferase.

Abstract: Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus

Cited by 133 publications

References 23 publications

C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.

C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.

Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart

Protein Farnesyltransferase and Protein Prenylation inPlasmodium falciparum

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