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.     

Protein prenylation: a new mode of host-pathogen interaction

Post translational modifications are required for proteins to be fully functional. The three step process, prenylation, leads to farnesylation or geranylgeranylation, which increase the hydrophobicity of the prenylated protein for efficient anchoring into plasma membranes and/or organellar membranes. Prenylated proteins function in a number of signaling and regulatory pathways that are responsible for basic cell operations. Well characterized prenylated proteins include Ras, Rac and Rho. Recently, pathogenic prokaryotic proteins, such as SifA and AnkB, have been shown to be prenylated by eukaryotic host cell machinery, but their functions remain elusive. The identification of other bacterial proteins undergoing this type of host-directed post-translational modification shows promise in elucidating host-pathogen interactions to develop new therapeutics. This review incorporates new advances in the study of protein prenylation into a broader aspect of biology with a focus on host-pathogen interaction.     

Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose

Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their nonprenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography medium that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target (“CAAX box”) sequences were enzymatically prenylated in vitro with natural and nonnatural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction, farnesylation of an mCherry-CAAX fusion construct, was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a nonnatural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.     

RhoGDI facilitates geranylgeranyltransferase-I-mediated RhoA prenylation

Protein prenylation is a post-translational modification where farnesyl or geranylgeranyl groups are enzymatically attached to a C-terminal cysteine residue. This modification is essential for the activity of small cellular GTPases, as it allows them to associate with intracellular membranes. Dissociated from membranes, prenylated proteins need to be transported through the aqueous cytoplasm by protein carriers that shield the hydrophobic anchor from the solvent. One such carrier is Rho GDP dissociation inhibitor (RhoGDI). Recently, it was shown that prenylated Rho proteins that are not associated with RhoGDI are subjected to proteolysis in the cell. We hypothesized that the role of RhoGDI might be not only to associate with prenylated proteins but also to regulate the prenylation process in the cell. This idea is supported by the fact that RhoGDI binds both unprenylated and prenylated Rho proteins with high affinity in vitro, and hence, these interactions may affect the kinetics of prenylation. We addressed this question experimentally and found that RhoGDI increased the catalytic efficiency of geranylgeranyl transferase-I in RhoA prenylation. Nevertheless, we did not observe formation of a ternary RhoGDI∗RhoA∗GGTase-I complex, indicating sequential operation of geranylgeranyltransferase-I and RhoGDI. Our results suggest that RhoGDI accelerates Rho prenylation by kinetically trapping the reaction product, thereby increasing the rate of product release.     

Towards complete sets of farnesylated and geranylgeranylated proteins

Three different prenyltransferases attach isoprenyl anchors to C-terminal motifs in substrate proteins. These lipid anchors serve for membrane attachment or protein-protein interactions in many pathways. Although well-tolerated selective prenyltransferase inhibitors are clinically available, their mode of action remains unclear since the known substrate sets of the various prenyltransferases are incomplete. The Prenylation Prediction Suite (PrePS) has been applied for large-scale predictions of prenylated proteins. To prioritize targets for experimental verification, we rank the predictions by their functional importance estimated by evolutionary conservation of the prenylation motifs within protein families. The ranked lists of predictions are accessible as PRENbase (http://mendel.imp.univie.ac.at/sat/PrePS/PRENbase) and can be queried for verification status, type of modifying enzymes (anchor type), and taxonomic distribution. Our results highlight a large group of plant metal-binding chaperones as well as several newly predicted proteins involved in ubiquitin-mediated protein degradation, enriching the known functional repertoire of prenylated proteins. Furthermore, we identify two possibly prenylated proteins in Mimivirus. The section HumanPRENbase provides complete lists of predicted prenylated human proteins-for example, the list of farnesyltransferase targets that cannot become substrates of geranylgeranyltransferase 1 and, therefore, are especially affected by farnesyltransferase inhibitors (FTIs) used in cancer and anti-parasite therapy. We report direct experimental evidence verifying the prediction of the human proteins Prickle1, Prickle2, the BRO1 domain-containing FLJ32421 (termed BROFTI), and Rab28 (short isoform) as exclusive farnesyltransferase targets. We introduce PRENbase, a database of large-scale predictions of protein prenylation substrates ranked by evolutionary conservation of the motif. Experimental evidence is presented for the selective farnesylation of targets with an evolutionary conserved modification site.     

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.     

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.     

Double prenylation by RabGGTase can proceed without dissociation of the mono-prenylated intermediate

Rab geranylgeranyltransferase (RabGGTase) catalyzes the prenylation of Rab proteins. Despite possessing a single active site, RabGGTase is able to add geranylgeranyl moieties onto each of the two C-terminal cysteine residues of Rab. We have studied the kinetics of Rab double prenylation employing a combination of a novel high pressure liquid chromatography (HPLC)-based in vitro prenylation assay and fluorescence spectroscopy. Transfer of the first geranylgeranyl group proceeds with a k(1) = 0.16 s(-1), while the conversion from singly to double prenylated Rab is 4-fold slower (k(2) = 0.039 s(-1)). We found that following the first transfer reaction, the conjugated lipid is removed from the active site of RabGGTase but mono-prenylated Rab.REP complex remains bound to RabGGTase with a K(d) < 1 nm. In contrast to the doubly prenylated Rab7.REP dissociation of the mono-prenylated species from RabGGTase was only weakly stimulated by phosphoisoprenoid. Based on the obtained rate constants we calculated that at least 72% of mono-prenylated Rab molecules proceed to double prenylation without dissociating from RabGGTase. The obtained data provides an explanation of how RabGGTase discriminates between mono-prenylated intermediate and double prenylated reaction product. It also indicates that the phosphoisoprenoid acts both as a substrate and as a sensor governing the kinetics of protein.protein interactions in the double prenylation reaction.     

Recent advances in the study of prenylated proteins

Post-translational modification of proteins with isoprenoids was first recognized as a general phenomenon in 1984. In recent years, our understanding, including mechanistic studies, of the enzymatic reactions associated with these modifications and their physiological functions has increased dramatically. Of particular functional interest is the role of prenylation in facilitating protein-protein interactions and membrane-associated protein trafficking. The loss of proper localization of Ras proteins when their farnesylation is inhibited has also permitted a new target for anti-malignancy pharmaceuticals. Recent advances in the enzymology and function of protein prenylation are reviewed in this article.     

Sequence dependence and differential expression of Ggamma5 subunit isoforms of the heterotrimeric G proteins variably processed after prenylation in mammalian cells

Between 1 and 2% of proteins coded for in the human genome, including all G protein gamma subunits, are predicted to be prenylated. Subsequently, prenylated proteins are proteolytically cleaved at the C terminus and carboxymethylated. These reactions are generally obligatory events required for functional expression of prenylated proteins. The biological role of prenyl substrates has made these reactions significant targets for anticancer drug development. Understanding the enzymology of this pathway will be key to success for this strategy. When Ggamma1, -2, -4, -10, -11, -12, and -13 were expressed in HEK293 cells they were completely processed according to the current understanding of the prenylation reaction. In contrast, Ggamma5 was processed to two forms; a minor one, fully processed as predicted, and a major one that was prenylated without further processing. When the Ca(1)a(2)X motif of Ggamma5, CSFL, was exchanged for that of Ggamma2, CAIL, Ggamma5 was completely processed. Conversely, Ggamma2-SFL was incompletely processed. Differential processing of Ggamma5 was found due to the presence of an aromatic amino acid in its Ca(1)a(2)X motif. Retrieving endogenous Ggamma subunits from HEK293 or Neuro-2a cells with FLAG-Gbeta constructs identified multiple Ggamma subunits by mass spectrometry in either cell, but in both cases the most prominent one was Ggamma5 expressed without C-terminal processing after prenylation. This work indicates that post-prenylation reactions can generate multiple products determined by the C-terminal Ca(1)a(2)X motif. Within the human genome 10% of predicted prenylated proteins have aromatic amino acids in their Ca(1)a(2)X sequence and would likely generate the prenylation pattern described here.     

A cell-specific,prenylation-independent mechanism regulates targeting of type II RACs

The RHO proteins, which regulate numerous signaling cascades, undergo prenylation, facilitating their interaction with membranes and with proteins called RHO.GDP dissociation inhibitors. It has been suggested that prenylation is required for RHO function. Eleven RHO-related proteins were identified in Arabidopsis. Eight of them are putatively prenylated. We show that targeting of the remaining three proteins, AtRAC7, AtRAC8, and AtRAC10, is prenylation independent, requires palmitoylation, and occurs by a cell-specific mechanism. AtRAC8 and AtRAC10 could not be prenylated by either farnesyltransferase or geranylgeranyltransferase I, whereas AtRAC7 could be prenylated by both enzymes in yeast. The association of AtRAC7 with the plasma membrane in plants did not require farnesyltransferase or a functional CaaX box. Recombinant AtRAC8 was palmitoylated in vitro, and inhibition of protein palmitoylation relieved the association of all three proteins with the plasma membrane. Interestingly, AtRAC8 and a constitutively active mutant, Atrac7mV(15), were not associated with the plasma membrane in root hair cells, whose elongation requires the localization of prenylated RHOs in the plasma membrane at the cell tip. Moreover, Atrac7mV(15) did not induce root hair deformation, unlike its prenylated homologs. Thus, AtRAC7, AtRAC8, and AtRAC10 may represent a group of proteins that have evolved to fulfill unique functions.     

Role of prenylation in the interaction of Rho-family small GTPases with GTPase activating proteins

The role of prenylation in the interaction of Rho-family small GTPases with their GTPase activating proteins (GAPs) was investigated. Prenylated and nonprenylated small GTPases were expressed in Sf9 insect cells and Escherichia coli, respectively. Nucleotide binding to and hydrolysis by prenylated and nonprenylated proteins were identical, but three major differences were observed in their reactions with GAPs. (1) Membrane-associated GAPs accelerate GTP hydrolysis only on prenylated Rac1 and RhoA, but they are inactive on the nonprenylated form of these proteins. The difference is independent of the presence of detergents. In contrast to Rac1 and RhoA, nonprenylated Cdc42 is able to interact with membrane-localized GAPs. (2) Full-length p50RhoGAP and p190RhoGAP react less intensely with nonprenylated Rac1 than with the prenylated protein, whereas no difference was observed in the reaction of isolated GAP domains of either p50RhoGAP or Bcr with the different types of Rac1. (3) Fluoride exerts a significant inhibitory effect only on the interaction of prenylated Rac1 with the isolated GAP domains of p50RhoGAP or Bcr. The effect of fluoride is not influenced by addition or chelation of Al(3+). This is the first detailed study demonstrating that prenylation of the small GTPase is an important factor in determining its reaction with GAPs. It is suggested that both intramolecular interactions and membrane targeting of GAP proteins represent potential mechanisms regulating Rac signaling.     

Splice Variants of SmgGDS Control Small GTPase Prenylation and Membrane Localization

Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. There is little previous evidence that cellular signals can restrain nonprenylated GTPases from entering the prenylation pathway, leading to the general belief that PBR-possessing GTPases are prenylated as soon as they are synthesized. Here, we present evidence that challenges this belief. We demonstrate that insertion of the dominant negative mutation to inhibit GDP/GTP exchange diminishes prenylation of Rap1A and RhoA, enhances prenylation of Rac1, and does not detectably alter prenylation of K-Ras. Our results indicate that the entrance and passage of these small GTPases through the prenylation pathway is regulated by two splice variants of SmgGDS, a protein that has been reported to promote GDP/GTP exchange by PBR-possessing GTPases and to be up-regulated in several forms of cancer. We show that the previously characterized 558-residue SmgGDS splice variant (SmgGDS-558) selectively associates with prenylated small GTPases and facilitates trafficking of Rap1A to the plasma membrane, whereas the less well characterized 607-residue SmgGDS splice variant (SmgGDS-607) associates with nonprenylated GTPases and regulates the entry of Rap1A, RhoA, and Rac1 into the prenylation pathway. These results indicate that guanine nucleotide exchange and interactions with SmgGDS splice variants can regulate the entrance and passage of PBR-possessing small GTPases through the prenylation pathway.     

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.     

Modification of Rab5 with a photoactivatable analog of geranylgeranyl diphosphate

A photoprobe analog of geranylgeranyl diphosphate (2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate or DATFP-FPP) inhibits mevalonate-dependent prenylation of in vitro translated Rab5 in rabbit reticulocyte lysate, suggesting that it competes for lipid binding to the Rab geranylgeranyl transferase. Modification of Rab5 with DATFP-FPP, demonstrated by gel mobility shift and Triton X-114 phase separation experiments, confirms that the enzyme uses the analog as a substrate. The sedimentation of DATFP-modified Rab5 as a larger mass complex on sucrose density gradients indicates that it binds to other factors in rabbit reticulocyte lysate. Most importantly, DATFP-Rab5 cross-links to these soluble factors upon exposure to UV light. Immunoprecipitation with antibodies raised against proteins known to interact with Rab5 reveals that the cross-linked complexes contain Rab escort protein and GDI-1. DATFP-Rab5 also associates with membranes in a guanosine-5'-O-(3-thiotriphosphate)-stimulated manner. However, although prenylated Rab5 can be cross-linked to two unknown membrane-associated factors by the chemical cross-linker disuccinimidyl suberate, these proteins fail to be UV cross-linked to membrane-bound DATFP-Rab5. These results strongly suggest that membrane-associated factors bind Rab5 through protein-protein interactions rather than protein-prenyl interactions. The modification of Rab5 with DATFP-FPP establishes a novel photoaffinity technique for the characterization of prenyl-binding sites.     

Principles of protein-protein interactions

In the postgenomic era, one of the most interesting and important challenges is to understand protein interactions on a large scale. The physical interactions between protein domains are fundamental to the workings of a cell: in multi-domain polypeptide chains, in multi-subunit proteins and in transient complexes between proteins that also exist independently. Thus experimental investigation of protein-protein interactions has been extensive, including recent large-scale screens using mass spectrometry. The role of computational research on protein-protein interactions encompasses not only prediction, but also understanding the nature of the interactions and their three-dimensional structures. I will discuss properties such as sequence conservation and co-regulation of genes and proteins involved in different types of physical interactions. Given that all proteins consist of their evolutionary units, the domains, all interactions occur between these domains. The interactions between domains belonging to different protein families will be the second topic of my talk.     

Structures of RabGGTase-substrate/product complexes provide insights into the evolution of protein prenylation

Post-translational isoprenylation of proteins is carried out by three related enzymes: farnesyltransferase, geranylgeranyl transferase-I, and Rab geranylgeranyl transferase (RabGGTase). Despite the fact that the last one is responsible for the largest number of individual protein prenylation events in the cell, no structural information is available on its interaction with substrates and products. Here, we present structural and biophysical analyses of RabGGTase in complex with phosphoisoprenoids as well as with the prenylated peptides that mimic the C terminus of Rab7 GTPase. The data demonstrate that, unlike other protein prenyl transferases, both RabGGTase and its substrate RabGTPases completely 'outsource' their specificity for each other to an accessory subunit, the Rab escort protein (REP). REP mediates the placement of the C terminus of RabGTPase into the active site of RabGGTase through a series protein-protein interactions of decreasing strength and selectivity. This arrangement enables RabGGTase to prenylate any cysteine-containing sequence. On the basis of our structural and thermodynamic data, we propose that RabGGTase has evolved from a GGTase-I-like molecule that 'learned' to interact with a recycling factor (GDI) that, in turn, eventually gave rise to REP.     

Exploring the biochemistry of the prenylome and its role in disease through proteomics: progress and potential

Introduction: Protein prenylation is a ubiquitous covalent post-translational modification characterized by the addition of farnesyl or geranylgeranyl isoprenoid groups to a cysteine residue located near the carboxyl terminal of a protein. It is essential for the proper localization and cellular activity of numerous proteins, including Ras family GTPases and G-proteins. In addition to its roles in cellular physiology, the prenylation process has important implications in human diseases and in the recent years, it has become attractive target of inhibitors with therapeutic potential.

Areas covered: This review attempts to summarize the basic aspects of prenylation integrating them with biological functions in diseases and giving an account of the current status of prenylation inhibitors as potential therapeutics. We also summarize the methodologies for the characterization of this modification.

Expert commentary: The growing body of evidence suggesting an important role of prenylation in diseases and the subsequent development of inhibitors of the enzymes responsible for this modification lead to the urgent need to identify the full spectrum of prenylated proteins that are altered in the disease or affected by drugs. Proteomic tools to analyze prenylated proteins are recently emerging, thanks to the advancement in the field of mass spectrometry coupled to enrichment strategies.     

Saccharomyces cerevisiae Pra1p/Yip3p interacts with Yip1p and Rab proteins.

The regulation of membrane traffic involves the Rab family of Ras-related GTPases, of which there are a total of 11 members in the yeast Saccharomyces cerevisiae. Previous work has identified PRA1 as a dual prenylated Rab GTPase and VAMP2 interacting protein [Martinic et al. (1999) J. Biol. Chem. 272, 26991-26998]. In this study we demonstrate that the yeast counterpart of PRA1 interacts with Rab proteins and with Yip1p, a membrane protein of unknown function that has been reported to interact specifically with the Rab proteins Ypt1p and Ypt31p. Yeast Pra1p/Yip3p is a factor capable of biochemical interaction with a panel of different Rab proteins and does not show in vitro specificity for any particular Rab. The interactions between Pra1p/Yip3p and Rab proteins are dependent on the presence of the Rab protein C-terminal cysteines and require C-terminal prenylation.