rab GTP-binding proteins implicated in vesicular transport are isoprenylated in vitro at cysteines within a novel carboxyl-terminal motif.

Low molecular mass GTP-binding proteins encoded by the mammalian rab genes are found in membranes of the Golgi complex and endosomes, suggesting that they play a role in the movement of exocytic and endocytic vesicles. The basis for the membrane association of these proteins has not been defined. Herein, we demonstrate that terminal cysteine residues in the rab1B, rab2, and rab5 proteins undergo thioether modification by isoprenyl groups when these proteins are translated in vitro in the presence of a radiolabeled isoprenoid precursor, [3H]mevalonate. Results of gel permeation chromatography of the radiolabeled hydrocarbons suggest that these proteins are modified specifically by isoprenyl groups of the 20-carbon diterpene class, rather than the 15-carbon farnesyl class known to be involved in modification of ras proteins. The rab1 and rab2 proteins lack the carboxyl-terminal amino acid motif common to all previously identified isoprenylated proteins, i.e. CXXX, where X is an unspecified amino acid. Analysis of altered translation products generated by site-directed mutagenesis indicates that modification of rab1B protein requires an intact carboxyl-terminal sequence consisting of GGCC. This represents a new amino acid motif for isoprenylation.     

Protein prenylation and human diseases: a balance of protein farnesylation and geranylgeranylation

The protein prenylation is one of the essential post-translational protein modifications, which extensively exists in the eukaryocyte. It includes protein farnesylation and geranylgeranylation, using farnesyl pyrophosphate(FPP) or geranylgeranyl pyrophosphate(GGPP) as the substrate, respectively. The prenylation occurs by covalent addition of these two types of isoprenoids to cysteine residues at or near the carboxyl terminus of the proteins that possess Caa X motif, such as Ras small GTPase family. The attachment of hydrophobic prenyl groups can anchor the proteins to intracellular membranes and trigger downstream cell signaling pathway. Geranylgeranyl biphosphate synthase(GGPPS) catalyzes the synthesis of 20-carbon GGPP from 15-carbon FPP. The abnormal expression of this enzyme will affect the relative content of FPP and GGPP, and thus disrupts the balance between protein farnesylation and geranylgeranylation, which participates into various aspects of cellular physiology and pathology. In this paper, we mainly review the property of this important protein post-translational modification and research progress in its regulation of cigarette smoke induced pulmonary disease, adipocyte insulin sensitivity, the inflammation response of Sertoli cells, the hepatic lipogenesis and the cardiac hypertrophy.     

Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by rac1, rac2, and ralA

Membrane localization of p21ras is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid is linked to a cysteine located within a conserved carboxyl-terminal sequence termed the "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid). We now show that three GTP-binding proteins encoded by the recently identified rac1, rac2, and ralA genes also undergo isoprenoid modification. cDNAs coding for each protein were transcribed in vitro, and the RNAs were translated in reticulocyte lysates. Incorporation of isoprenoid precursors, [3H]mevalonate or [3H]farnesyl pyrophosphate, indicated that the translation products were modified by isoprenyl groups. A protein recognized by an antibody to rac1 also comigrated with a protein metabolically labeled by a product of [3H] mevalonate in cultured cells. Gel permeation chromatography of radiolabeled hydrocarbons released from the rac1, rac2, and ralA proteins by reaction with Raney nickel catalyst indicated that unlike p21Hras, which was modified by a 15-carbon moiety, the rac and ralA translation products were modified by 20-carbon isoprenyl groups. Site-directed mutagenesis established that the isoprenylated cysteines in the rac1, rac2, and ralA proteins were located in the fourth position from the carboxyl terminus. The three-amino acid extension distal to the cysteine was required for this modification. The isoprenylation of rac1 (CSLL), ralA (CCIL), and the site-directed mutants rac1 (CRLL) and ralA (CSIL), demonstrates that the amino acid adjacent to the cysteine need not be aliphatic. Therefore, proteins with carboxyl-terminal CXXX sequences that depart from the CAAX motif should be considered as potential targets for isoprenoid modification.     

Post-translational modification of proteins by 15-carbon and 20-carbon isoprenoids in three mammalian cell lines

Summary A number of cellular proteins, including p21^ras, lamin B, and the G-protein subunits, undeDanvillergo post-translational modification by 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid moieties derived from pyrophosphate intermediates of the cholesterol biosynthetic pathway. In this study, isoprenylated proteins in three mammalian cell lines (Hela cells, Rat-6 fibroblasts and COS cells) were radiolabeled with an isoprenoid precursor, [³H]mevalonate, and resolved by SDS gel electrophoresis. Groups of proteins with different molecular masses were eluted from the gels and the chain-lengths of the radiolabeled isoprenyl groups, released from the proteins by Raney-nickel-catalyzed desulfurization, were established by gel permeation chromatography. 15-Carbon and 20-carbon isoprenyl groups were found in separate classes of proteins within each cell line. With the exception of p21^ras, which incorporated a 15-carbon group when expressed in COS cells, the proteins in the region of the 21–28 kDa ras-related GTP binding proteins contained mostly 20-carbon isoprenyl chains. In contrast, proteins belonging to the 66–72 kDa nuclear lamin family, as well as unidentified proteins with molecular masses of 41–46 kDa and 53–55 kDa, contained predominantly 15-carbon isoprenyl chains. The chain-lengths of the isoprenoids associated with particular classes of proteins did not vary from one cell line to another, suggesting that the nature of the isoprenoid modification (farnesyl versus geranylgeranyl) is determined by intrinsic structural features of the proteins, rather than the cell type in which the proteins are expressed.Abbreviations MVA Mevalonolactone - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis     

Isoprenoid requirement for intracellular transport and processing of murine leukemia virus envelope protein.

Lovastatin blocks the biosynthesis of the isoprenoid precursor, mevalonate. When Friend murine erythroleukemia (MEL) cells are cultured in medium containing lovastatin, the precursor of murine leukemia virus envelope glycoprotein (gPr90env) fails to undergo proteolytic processing, which normally occurs in the Golgi complex. Consequently, newly synthesized envelope proteins are not incorporated into viral particles that are shed into the culture medium. gPr90env appears to be localized in a pre-Golgi membrane compartment, based on its enrichment in subcellular fractions containing NADPH-cytochrome c reductase activity and the sensitivity of its carbohydrate chains to digestion with endoglycosidase H. Arrest of gPr90env processing occurs at concentrations of lovastatin that are not cytostatic, and the effect of the inhibitor is prevented by addition of mevalonate to the medium. The low molecular mass GTP-binding proteins, rab1p and rab6p, which are believed to function in early steps of the exocytic pathway, are normally modified posttranslationally by geranylgeranyl isoprenoids. However, in MEL cells treated with 1 microM lovastatin, nonisoprenylated forms of these proteins accumulate in the cytosol prior to arrest of gPr90env processing. These observations suggest that lovastatin may prevent viral envelope precursors from reaching the Golgi compartment by blocking the isoprenylation of rab proteins required for ER to Golgi transport.     

Prenylation of Rho G-Proteins: a Novel Mechanism Regulating Gene Expression and Protein Stability in Human Trabecular Meshwork Cells

Endogenous prenylation with sesquiterpene or diterpene isoprenoids facilitates membrane localization and functional activation of small monomeric GTP-binding proteins. A direct effect of isoprenoids on regulation of gene expression and protein stability has also been proposed. In this study, we determined the role of sesquiterpene or diterpene isoprenoids on the regulation of Rho G-protein expression, activation, and stability in human trabecular meshwork (TM) cells. In both primary and transformed human TM cells, limiting endogenous isoprenoid synthesis with lovastatin, a potent HMG-CoA reductase inhibitor, elicited marked increases in RhoA and RhoB mRNA and protein content. The effect of lovastatin was dose-dependent with newly synthesized inactive protein accumulating in the cytosol. Supplementation with geranylgeranyl pyrophosphate (GGPP) prevented, while inhibition of geranylgeranyl transferase-I mimicked, the effects of lovastatin on RhoA and RhoB protein content. Similarly, lovastatin-dependent increases in RhoA and RhoB mRNA expression were mimicked by geranylgeranyl transferase-I inhibition. Interestingly, GGPP supplementation selectively promoted the degradation of newly synthesized Rho proteins which was mediated, in part, through the 20S proteasome. Functionally, GGPP supplementation prevented lovastatin-dependent decreases in actin stress fiber organization while selectively facilitating the subcellular redistribution of accumulated Rho proteins from the cytosol to the membrane and increasing RhoA activation. Post-translational prenylation with geranylgeranyl diterpenes selectively facilitates the expression, membrane translocation, functional activation, and turnover of newly synthesized Rho proteins. Geranylgeranyl prenylation represents a novel mechanism by which active Rho proteins are targeted to the 20S proteasome for degradation in human TM cells.     

Isoprenoids and protein prenylation: implications in the pathogenesis and therapeutic intervention of Alzheimer’s disease

The mevalonate–isoprenoid–cholesterol biosynthesis pathway plays a key role in human health and disease. The importance of this pathway is underscored by the discovery that two major isoprenoids, farnesyl and geranylgeranyl pyrophosphate, are required to modify an array of proteins through a process known as protein prenylation, catalyzed by prenyltransferases. The lipophilic prenyl group facilitates the anchoring of proteins in cell membranes, mediating protein–protein interactions and signal transduction. Numerous essential intracellular proteins undergo prenylation, including most members of the small GTPase superfamily as well as heterotrimeric G proteins and nuclear lamins, and are involved in regulating a plethora of cellular processes and functions. Dysregulation of isoprenoids and protein prenylation is implicated in various disorders, including cardiovascular and cerebrovascular diseases, cancers, bone diseases, infectious diseases, progeria, and neurodegenerative diseases including Alzheimer’s disease (AD). Therefore, isoprenoids and/or prenyltransferases have emerged as attractive targets for developing therapeutic agents. Here, we provide a general overview of isoprenoid synthesis, the process of protein prenylation and the complexity of prenylated proteins, and pharmacological agents that regulate isoprenoids and protein prenylation. Recent findings that connect isoprenoids/protein prenylation with AD are summarized and potential applications of new prenylomic technologies for uncovering the role of prenylated proteins in the pathogenesis of AD are discussed.     

Expression of mammalian geranylgeranyltransferase type-II in Escherichia coli and its application for in vitro prenylation of Rab proteins

Mammalian geranylgeranyltransferase type II (GGTase-II) is a 100-kDa heterodimer that catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab GTPases. This modification is essential for the biological activity of Rab proteins. Geranylgeranylation can be performed in vitro using recombinant GGTase-II but so far large-scale production of the enzyme was challenging. We report here the design of a two plasmid expression system that will produce GGTase-II at levels as high as 15 mg/L in Escherichia coli. The protein was produced as a heterodimer with the alpha subunit bearing a cleavable tandem 6His-glutathione S-transferase (GST) tag that was used for two-step purification of the enzyme. Purified enzyme was functionally active as determined by in vitro prenylation and phosphoisoprenoid binding assay. Furthermore, the GST-tagged GGTase-II was used for preparative in vitro prenylation of the Rab7:REP-1 complex. Using this procedure, 10 mg of doubly prenylated Rab7:REP-1 complex were obtained.     

Feedback inhibition of polyisoprenyl pyrophosphate synthesis from mevalonate in vitro. Implications for protein prenylation.

The prenylation of proteins utilizes the polyisoprenyl pyrophosphates (FPP) and geranylgeranyl pyrophosphate (GGPP) as prenyl donors. These polyisoprenoids are also precursors to ubiquinone and dolichol synthesis. We have previously described the geranylgeranylation of rab 1b from labeled mevalonate in rabbit reticulocyte lysates (Khosravi-Far, R., Lutz, R. J., Cox, A. D., Conroy, L., Bourne, J. R., Sinensky, M., Balch, W. E., Buss, J. C., and Der, C. J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6264-6268). We now directly demonstrate the incorporation of mevalonate into FPP and GGPP in rabbit reticulocyte cytosol. High pressure liquid chromatography analysis reveals that only all-trans-E,E,E-GGPP, the prenyl donor for in vivo protein geranylgeranylation, is synthesized. Incubations with recombinant H-ras and rab1b result in an increased synthesis of farnesyl and geranylgeranyl derivatives, respectively. The increase is wholly accounted for by protein-incorporated polyisoprenoids with no change in the polyisoprenyl pyrophosphate pools. Further, GGPP inhibits its own synthesis, without affecting FPP synthesis, with half-maximal inhibition at approximately 3 microM GGPP. Inhibition of FPP synthesis by the inhibition of isopentenyl isomerase causes a dramatic increase in isopentenyl pyrophosphate synthesis. FPP also inhibits conversion of mevalonate into FPP. These findings indicate that these polyisoprenyl pyrophosphates can down-regulate their own synthesis in vitro, and this regulation may control the levels of these polyisoprenoids in vivo.     

The prenylation of proteins.

The prenylated proteins represent a newly discovered class of post-translationally modified proteins. The known prenylated proteins include the oncogene product p21ras and other low molecular weight GTP-binding proteins, the nuclear lamins, and the gamma subunit of the heterotrimeric G proteins. The modification involves the covalent attachment of a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) isoprenoid moiety in a thioether linkage to carboxyl terminal cysteine. The nature of the attached substituent is dependent on specific sequence information in the carboxyl terminus of the protein. In addition, prenylation entrains other posttranslational modifications forming a reaction pathway. In this article, we review our current understanding of the biochemical reactions involved in prenylation and discuss the possible role of this modification in the control of cellular functions such as protein maturation and cell growth.     

Lipidation by the Host Prenyltransferase Machinery Facilitates Membrane Localization of Legionella pneumophila Effector Proteins

The intracellular human pathogen Legionella pneumophila translocates multiple proteins in the host cytosol known as effectors, which subvert host cellular processes to create a membrane-bound organelle that supports bacterial replication. It was observed that several Legionella effectors encode a prototypical eukaryotic prenylation CAAX motif (where C represents a cysteine residue and A denotes an aliphatic amino acid). These bacterial motifs mediated posttranslational modification of effector proteins resulting in the addition of either a farnesyl or geranylgeranyl isoprenyl lipid moiety to the cysteine residue of the CAAX tetrapeptide. Lipidation enhanced membrane affinity for most Legionella CAAX motif proteins and facilitated the localization of these effector proteins to host organelles. Host farnesyltransferase and class I geranylgeranyltransferase were both involved in the lipidation of the Legionella CAAX motif proteins. Perturbation of the host prenylation machinery during infection adversely affected the remodeling of the Legionella-containing vacuole. Thus, these data indicate that Legionella utilize the host prenylation machinery to facilitate targeting of effector proteins to membrane-bound organelles during intracellular infection.     

Genoprotective pathways. II. Attenuation of oxidative DNA damage by isopentenyl diphosphate

Oxidative stress is believed to play a role in the pathogenesis of many diseases. Here we report that isopentenyl diphosphate (IPP), the 5-carbon building unit of all isoprenoids, is a potent antioxidant that is capable of inhibiting oxidative DNA damage at picomolar concentrations (IC50 = 1.7 x 10(-11) M). The diphosphate moiety is essential, since isopentenyl monophosphate (IMP) is unable to trigger antioxidative signaling. The 20-carbon isoprenyl, geranylgeranyl diphosphate (GGPP), but not the 15-carbon farnesyl diphosphate, displays similar genoprotective effects. The pathway activated by IPP is distinct from that of 2-chloroadenosine (2CA). 2CA-mediated genoprotective signaling is transduced through an A2a or A2b adenosine receptor (AR) and can be blocked by the cyclic AMP (cAMP)-dependent protein kinase (PKA) inhibitor, H-89. In contrast, IPP signaling is independent of A2aAR, A2bAR, cAMP or PKA. Unlike the 2CA-mediated pathway, the effect of IPP is dependent on the mevalonate pathway, a geranylgeranylated protein and on intact proteasome activity. Thus, IPP is a potent activator of a novel genoprotective pathway. These findings shed new light on the role of isoprenoids in oxidative stress biology and may help to develop novel preventive strategies against oxidative damage.     

Protein prenylation in eukaryotic microorganisms: genetics, biology and biochemistry

Modrfication of proteins at C-terminal cysteine residue(s) by the isoprenoids farnesyl (C15) and geranylgeranyl (C20) is essential for the biological function of a number of eukaryotic proteins including fungal mating factors and the small, GTP-binding proteins of the Ras superfamily. Three distinct enzymes, conserved between yeast and mammals, have been identified that prenylate proteins: farnesyl protein transferase, geranylgeranyl protein transferase type I and geranylgeranyl protein transferase type II. Each prenyl protein transferase has its own protein substrate specificity. Much has been learned about the biology, genetics and biochemistry of protein prenylation and prenyl protein transferases through studies of eukaryotic microorganisms, particularly Saccharo-myces cerevisiae. The functional Importance of protein prenylation was first demonstrated with fungal mating factors. The initial genetic analysis of prenyl protein transferases was in S. cerewisiae with the isolation and subsequent characterization of mutations in the RAM1, RAM2, CDC43 and BET2 genes, each of which encodes a prenyl protein transferase subunit. We review here these and other studies on protein prenylation in eukaryotic microbes and how they relate to and have contributed to our knowledge about protein prenylation in all eukaryotic cells.     

Post-translational processing of Schizosaccharomyces pombe YPT proteins.

ras proteins are post-translationally processed at their carboxyl-terminal CAAX motif by a triplet of modifications: prenylation of C with farnesyl, proteolytic trimming of AAX, and carboxyl-methylation. These modifications co-operate with palmitoylation of nearby sites or a polybasic region to target plasma membrane localization. The related YPT/rab proteins in contrast are localized to compartments of the endo-membrane system and may be involved in directing membrane traffic. These proteins end in XCC or CXC motifs. We have analyzed the processing of members of this subfamily form the fission yeast Schizosaccharomyces pombe. We find using in vitro translation in reticulocyte lysates that YPT1, -3, and -5 are prenylated with geranylgeranyl and that they incorporate label from [3H]mevalonic acid when expressed in transfected COS cells in vivo. Furthermore, prenylation was necessary for membrane binding in vivo. The CXC protein YPT5, but neither of the two XCC proteins YPT1 and YPT3, was carboxyl-methylated in S. pombe and in COS cells in vivo. However, YPT5 was not carboxyl-methylated in vitro in lysates which were able to methylate ras protein. YPT3 was detectably palmitoylated when expressed in COS cells, though at a much lower level than ras.     

Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines.

Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue.     

Isoprenoids and Related Pharmacological Interventions: Potential Application in Alzheimer’s Disease

Two major isoprenoids, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, serve as lipid donors for the posttranslational modification (known as prenylation) of proteins that possess a characteristic C-terminal motif. The prenylation reaction is catalyzed by prenyltransferases. The lipid prenyl group facilitates to anchor the proteins in cell membranes and mediates protein-protein interactions. A variety of important intracellular proteins undergo prenylation, including almost all members of small GTPase superfamilies as well as heterotrimeric G protein subunits and nuclear lamins. These prenylated proteins are involved in regulating a wide range of cellular processes and functions, such as cell growth, differentiation, cytoskeletal organization, and vesicle trafficking. Prenylated proteins are also implicated in the pathogenesis of different types of diseases. Consequently, isoprenoids and/or prenyltransferases have emerged as attractive therapeutic targets for combating various disorders. This review attempts to summarize the pharmacological agents currently available or under development that control isoprenoid availability and/or the process of prenylation, mainly focusing on statins, bisphosphonates, and prenyltransferase inhibitors. Whereas statins and bisphosphonates deplete the production of isoprenoids by inhibiting the activity of upstream enzymes, prenyltransferase inhibitors directly block the prenylation of proteins. As the importance of isoprenoids and prenylated proteins in health and disease continues to emerge, the therapeutic potential of these pharmacological agents has expanded across multiple disciplines. This review mainly discusses their potential application in Alzheimer's disease.     

Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif

Posttranslational isoprenylation of some small GTP-binding proteins is required for their biological activity. Rab geranylgeranyl transferase (Rab GGTase) uses geranylgeranyl pyrophosphate to modify Rab proteins, its only known substrates. Geranylgeranylation of Rabs is believed to promote their association with target membranes and interaction with other proteins. Plants, like other eukaryotes, contain Rab-like proteins that are associated with intracellular membranes. However, to our knowledge, the geranylgeranylation of Rab proteins has not yet been characterized from any plant source. This report presents an activity assay that allows the characterization of prenylation of Rab-like proteins in vitro, by protein extracts prepared from plants. Tomato Rab1 proteins and mammalian Rab1a were modified by geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. This modification required a conserved cysteine-cysteine motif. A mutant form lacking the cysteine-cysteine motif could not be modified, but inhibited the geranylgeranylation of its wild-type homolog. The tomato Rab proteins were modified in vitro by protein extract prepared from yeast, but failed to become modified when the protein extract was prepared from a yeast strain containing a mutant allele for the [alpha] subunit of yeast Rab GGTase (bet4 ts). These results demonstrate that plant cells, like other eukaryotes, contain Rab GGTase-like activity.     

In vitro identification of a soluble protein:geranylgeranyl transferase from rat tissues

The gamma subunit of mammalian trimeric G proteins has been shown previously to be modified in vivo on a cysteine residue situated at the carboxyl-terminal sequence-Cys-Ala-Ile-Leu-COOH by a 20-carbon prenyl moiety geranylgeranyl (Mumby, S. M., Casey, P. J., Gilman, A. G., Gutowski, S., and Sternweis, P. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5873-5877; Yamane, H. K., Farnsworth, C. C., Xie, H., Howald, W., Fung, B. K-K., Clarke, S., Gelb, M. H., and Glomset, J. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5866-5872). A biotinylated peptide acceptor comprising the eight carboxyl-terminal amino acids of the gamma subunit and tritiated geranylgeranyl diphosphate were utilized to monitor a protein:prenyl transferase activity in rat organs of varying age. The transferase activity was dependent upon the presence of divalent metal ions and maximal activity was achieved with either 1 mM ZnCl2 or 20 mM MgCl2. Activity was shown to be linear with respect to time, protein concentration, substrate concentration, and the pH optimum was 7.5. Protein:geranylgeranyl transferase activity was detected in all rat organs studied with the highest specific activity in brain S100. No activity was detected in the membrane fraction. The specific activity in brain, liver, kidney, and heart increased with age. Radioactivity incorporated into the peptide acceptor from both [1-3H]geranylgeranyl diphosphate and [5-3H]mevalonate by 21-day-old rat brain S100 was released by treatment with methyl iodide, and in both cases, analysis of the cleavage products by reversed phase high performance liquid chromatography showed a peak of radioactivity co-eluting with a geranylgeraniol standard which was well resolved from a farnesol standard. This indicated that the rat brain S100 contained not only the protein:geranylgeranyl transferase but also geranylgeranyl synthetase activity and that the peptide acceptor was specific for geranylgeranyl under the conditions tested.     

Therapeutic intervention based on protein prenylation and associated modifications

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.     

In vitro assembly,purification, and crystallization of the rab geranylgeranyl transferase:substrate complex

Posttranslational modification with the geranygeranyl moiety is essential for the ability of Rab GTPases to control processes of membrane docking and fusion. This modification is conferred by Rab geranylgeranyltransferase (RabGGTase), which catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab proteins. The enzyme consists of a catalytic alpha/beta heterodimer and an accessory protein termed Rab escort protein (REP-1) that delivers the newly prenylated Rab proteins to their target membrane. In order to understand the structural basis of Rab prenylation, we have investigated in vitro assembly and crystallization of the RabGGTase:REP-1:Rab complex. In order to ensure maximal stability of the ternary complex, we generated its monoprenylated form, which corresponds to a reaction intermediate and displays the highest affinity between the components. This was achieved by expressing the individual components in baculovirus and Escherichia coli systems with subsequent purification followed by in vitro monoprenylation of Rab7 with immobilized recombinant RabGGTase. Purified monoprenylated REP-1:Rab7 was complexed with recombinant RabGGTase and crystallized in hanging drops. The crystals obtained initially diffract to 8 A on an in-house X-ray source.