Protein Prenylation, Part A: 29 (The Enzymes)

Protein Prenylation, Part A, Volume 29 (The Enzymes): Medicine & Health Science Books @ leondumoulin.nl
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Rce1p has been a focus of biochemical studies because of its key role in the CaaX processing of Ras proteins. Using defined synthetic substrates and membrane-associated or partially purified Rce1p, investigators have shown that Rce1p activity depends on the prenylation status of its substrate, and that Rce1p has a preference for particular CaaX motifs 26 — Unfortunately, Rce1p has eluded purification, and neither its amino acid sequence nor its biochemical properties reveal straightforward clues about its mechanism of action.

Inhibitor and bioinformatic analyses suggest that Rce1p may be a serine protease or a metalloprotease, respectively 29 , However, mutagenesis of critical residues implicated by those studies does not affect enzyme activity 33 ; further mutational analysis of residues conserved among Rce1p orthologs may reveal new clues about the reaction mechanism of Rce1p. Studies of mouse fibroblasts with either null or conditional Rce1 mutant alleles have provided compelling evidence that the membrane association, plasma membrane targeting and transformation capacity of Ras are all substantially lower when Rce1p function is lacking; in fact cells lacking Rce1 function are sensitized to FTI treatment 34 — These findings have spurred renewed interest in developing Rce1 inhibitors.

So far, inhibitor studies have focused on substrate analogs, including prenylpeptide mimetics and related compounds that may act as competitive inhibitors 27 , Clearly, the identification of new Rce1p inhibitors presents a promising avenue for further research. Like the endoproteases, the methyltransferase Icmt is a multispanning endoplasmic reticulum membrane protein, with its active site presumably facing the cytosol Topology studies have shown that the yeast Icmt Ste14p has six trans-membrane spans, and two additional spans have been predicted for the mammalian enzyme Because of its membrane spans and because it lacks a classical methyltransferase consensus motif, Icmt is an atypical member of the methyltransferase family of enzymes However, the recent purification of Ste14p from yeast, and its reconstitution in liposomes in an enzymatically active form, have provided conclusive evidence that it is the sole component comprising Icmt activity 40 , Using the purified enzyme, researchers showed that yeast Ste14p recognizes the farnesylated and geranylgeranyl substrates N -acetyl- S -farnesylcysteine and N -acetyl- S -geranylgeranylcysteine equivalently.

As is the case for Rce1 -deficient fibroblasts, null and conditional Icmt mutant fibroblasts have defects in the membrane association and plasma membrane targeting of Ras, and also in Ras-induced transformation efficiency 43 , Notably, the analysis of Ras-transformed Icmt —deficient fibroblasts highlights an additional and important twist: As this example with RhoA illustrates, carboxyl methylation has diverse and sometimes unanticipated roles, affecting different prenylated proteins in different ways. Proposed roles for the carboxyl methylation of prenylated proteins, for which there is evidence in particular cases, include influencing intracellular protein localization, membrane attachment, metabolic stability and interactions with other proteins 19 , 20 , 45 , Although carboxyl methylation of prenylated proteins has been suggested to be reversible, and therefore could represent a regulatory event, evidence for this reversibility in cells has not been forthcoming.

Given the importance of carboxyl methylation for Ras-induced onco-genesis shown by the Icmt mouse studies mentioned above, the development of Icmt inhibitors as anticancer drugs is of obvious interest 19 , 41 , The well-known chemotherapeutic compound methotrexate an antifolate that blocks thymidine synthesis, which is necessary for tumor growth causes accumulation of S -adenosylhomocysteine, a potent inhibitor of nearly all cellular methyltransferases; it has been suggested that at least some of methotrexate's antitumor activity may stem from its ability to inhibit Icmt, though specificity for Icmt is lacking It is worth noting that there were initial reservations that specific Icmt inhibitors might be toxic to cells because they would affect all prenylated proteins; these concerns have been significantly mitigated by recent work showing that although the localization of farnesylated proteins is strongly affected when methylation is blocked, the localization of geranylgeranylated proteins the main class of prenylated proteins is relatively unaffected Efforts to identify specific Icmt inhibitors are currently underway.

Both the screening of compound libraries 50 and the search for mechanism-based and substrate-analog inhibitors 51 are beginning to yield candidate compounds. S -Palmitoylation of proteins entails connection of a C 16 fatty-acid chain to the thiol functionality of a cysteine via a thioester bond.

Palmitoylation of proteins has several possible functions, depending on the protein under scrutiny. Much like protein prenylation in general, protein palmitoylation enables or contributes to membrane binding and possibly localization 52 , Additionally, palmitoylation may modulate protein-protein or protein-lipid interactions and enzyme activity. In contrast to protein prenylation, palmitoylation is a reversible post-translational modification In the Ras superfamily, H-Ras and N-Ras are examples of proteins that are both prenylated and palmitoylated Palmitoylation of the C terminus of these Ras proteins occurs after the farnesylation of the C-terminal cysteine and at cysteine residues slightly upstream of the farnesylated cysteine Fig.

Although inhibitors of Ras palmitoylation are known 2-bromopalmitate 56 and cerulenin analogs 57 , 58 , for example , the mechanisms and selectivity steering this palmitoylation process are still unclear. Several palmitoylation motifs have been found in different classes of proteins and have been reviewed We focus on the recent progress in the elucidation of the enzymology and function of palmitoylation in Ras proteins. In yeast, the Erf2—Erf4 protein complex palmitoylates the yeast Ras homolog Ras2 refs.

Until recently no Erf2 orthologs or other proteins featuring Ras S -palmitoylation activity had been found in the genomes of eukaryotes, including the human genome. The eukaryotic acyl protein thioesterase 1 APT1 protein has been identified as participating in the removal of palmitate from proteins on the cytosolic face of membranes The relationship between Ras proteins and APT1 has therefore been further studied using a chemical biological approach In this investigation several semisynthetic Ras proteins, together with lipopeptides and newly developed APT1 inhibitors, were synthesized via combined chemical and biological methods and used as tools, without which the study would have been difficult The biological activity of an N-Ras lipoprotein having a complete but nonhydrolyzable membrane anchor was not reduced by the same inhibitors.

Additional in vitro experiments with these inhibitors and semisynthetic N-Ras lipopeptides and proteins has shown that the inhibitors may inhibit Ras palmitoylation and depalmitoylation. This observation led to the suggestion that APT1 functions in vitro as a bidirectional enzyme, promoting either Ras palmitoylation or Ras depalmitoylation, depending on the local environment and on substrate availability. However, the possible involvement of APT1 in Ras palmitoylation in vivo still needs to be proven. The complex also shows substrate selectivity for the C terminus of Ras proteins, which suggests that it is a human ortholog of the yeast palmitoyltransferase.

The localization of the protein complex in the Golgi suggests that it is an important contributor to the control of Ras palmitoylation in vivo. The importance of Ras palmitoylation has also been recently highlighted in studies on the regulation of its localization and activity 65 — Some of these studies used semisynthetic proteins, whereas others used fluorescently labeled proteins The specific subcellular distribution of palmitoylatable H-Ras and N-Ras isoforms is generated through a constitutive deacylation-reacylation cycle Fig. Palmitoylation status drives rapid exchange of Ras between the plasma membrane and the Golgi apparatus; depalmitoylated Ras protein exchanges rapidly between cytoplasm and membranes, and repalmitoylation occurs at the Golgi, where Ras signals or is redirected to the plasma membrane.

Therapeutic intervention based on protein prenylation and associated modifications

In addition, the two individual palmitoyl residues of H-Ras have a distinct role in protein trafficking, localization and signaling Model for the trafficking of H-Ras and N-Ras from the Golgi complex to the plasma membrane and back 67 , 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.

These results in regard to Ras palmitoylating enzymes and Ras processing in the cell are new starting points for the complete characterization of the Ras palmitoylation machinery and its functioning. Additional enzymes that are involved in protein palmitoylation are expected to be found, and the targeting mechanism of Ras via reversible post-translational modifications might be a paradigm for other types of proteins, palmitoylated or not.

On top of that, use of the palmitoylation process may open up a new opportunity to target oncogenic H-Ras and N-Ras The structure and lateral organization of lipids and proteins in biological membranes are under heavy scrutiny in the fields of membrane biochemistry and biophysics 71 , The existence of membrane subdomains with different lipid compositions and the relationship between lipid domain formation and the conformation and functional properties of membrane-anchored proteins are central topics in these fields.

As is true for other lipidated proteins, the microlocalization and signaling of Ras depends on its lipidation pattern, although other factors, such as the amino acid sequence of the backbone and conformation of specific domains, also have prominent roles In addition, the position of the lipid functionalities seems to determine the membrane specificity The different C-terminal amino acids and the concomitant lipidation motifs of Ras proteins are thought to target the different Ras iso-forms to different membrane microenvironments and thereby regulate their biological profiles.

Studies on fluorescently labeled N-Ras proteins and model membranes have shown that the complete protein has a preference for the liquid-disordered phase over the liquid-ordered and solid-ordered phases 76 , and NMR studies of this protein have shown a specific conformation of the lipidated C terminus when bound to the membrane Together with several studies on peptides, this indicates that the membrane localization of N-Ras is mainly governed by its lipidated C terminus, in contrast to H-Ras, for example, in which the GDP and GTP loading status also strongly regulates its localization 69 , The specific localization and accumulation of N-Ras proteins in interfaces of lipid bilayer domains may have a special biological relevance, as it might serve, for example, as a vehicle for protein association.

Recent computational modeling studies of immunogold spatial point patterns on intact plasma membrane sheets indicate that lipidated proteins on the inner plasma membrane are able to drive the formation of nanoclusters These findings are supported by independent studies on semisynthetic Ras proteins This indicates that Ras proteins are not passively targeted to microenvironments but may be actively involved in their generation The K-Ras protein contains eight lysine residues just upstream of the farnesylated C terminus Fig.

It is has been suggested that these cationic lysines form favorable electrostatic interactions with the cytosolic face of the plasma membrane; anionic phospholipids including phosphatidylserine and phosphatidylinositol are present at relatively high abundances on this membrane leaflet. An electrostatic basis for targeting of K-Ras to the plasma membrane rather than a mechanism involving the binding of the C terminus to a putative receptor protein located in the plasma membrane is consistent with the fact that mutation of all eight lysines to arginines 81 or to D -lysines 82 does not effect K-Ras targeting.

So far we have focused on the membrane anchoring ability of protein prenyl groups. Methylation of the prenylated cysteine is expected to increase the hydrophobicity of the protein's C terminus, thereby facilitating membrane anchoring. Studies also show that in some cases the prenyl group serves as a molecular handle to allow extraction of prenylated proteins from membranes by other proteins.

For example, the Rab3a GTP-binding protein cycles between the membrane of synaptic vesicles and the cytosol during regulated release of neurotransmitters The physiological significance of this cycling is not yet understood. Together with kinetic studies, this has resulted in a proposed two-step mechanism for the release of, for example, the Rho GTPase Cdc42 or of Rab proteins from membranes In the second step, the geranylgeranyl moiety is isomerized and inserted into the hydrophobic pocket, resulting in release from the membrane.

Back delivery of the GTPase into the membrane is thought to involve several factors such as phosphorylation status, phospholipid composition and protein displacement factors. Data obtained on the structural and kinetic properties of the GDI-Rab interaction have allowed investigators to formulate a comparable GDI-Rab membrane extraction model Fig.

Subsequent docking of the Rab C terminus onto GDI leads to stepwise extraction of the prenyl moieties from the membrane, a process that is driven by binding of the lipid groups in the highly hydrophobic GDI binding site. Logically, the mechanism for delivering Rab back into the membrane is a reversal of the extraction process, but it probably involves an additional membrane-bound factor termed GDI displacement factor having specificity for specific Rab molecules.

Rab escort proteins REPs that shuttle the Rab protein to and from the Rab geranylgeranyltransferase share structural features with GDIs, including a hydrophobic groove for binding geranylgeranyl groups. Model for extraction of prenylated Rab proteins from membranes via GDI, as previously formulated 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.

These models for extraction and delivery of prenylated proteins provide guidance for additional studies that will hopefully lead to an understanding of specific membrane targeting of these proteins. Progress in the area of protein ligation methods has given researchers access to a broad spectrum of methods for the semisynthetic production of post-translationally modified proteins, including lipidated proteins. These methods yield either native bonds for example, prior thiol capture, native chemical ligation and expressed protein ligation or non-native bonds for example, imine capture ligation, oxime ligation and maleimidocaproic acid ligation.

This combination of organic chemistry and molecular biology gives scientists access to modified proteins with both natural and non-natural modifications that are generally not accessible through other for example, enzymatic processes The first approach, expressed protein ligation EPL , connects a lipidated peptide having an N-terminal cysteine to the C terminus of a thioester-tagged Ras GTPase via a native peptide bond.

The second approach incorporates synthetic lipidated peptides into the GTPases using maleimidocaproyl MIC -controlled ligation. This ligation requires an accessible that is, C-terminal-free thiol group on the protein, usually on a cysteine, to connect the N-terminally MIC-modified peptide. 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 58 , 62 , 64 , 67 , H-Ras 1— denotes the first amino acids of this protein.

Far, farnesyl; Ger, geranyl. These include compounds that compete with CaaX peptide substrates or with farnesyl diphosphate, as well as compounds that coordinate to zinc, an essential metal involved in catalysis. Computer modeling has also aided in the improvement and design of FTIs.

This high specificity seems to be a result of selective stacking interactions between the FTI compound and aromatic residues in the binding site In addition to these highly specific compounds, there are FTI compounds that have dual specificity.


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Notably, this compound assumes two different modes of interaction with the two enzymes These compounds are strongly proapoptotic, which distinguishes them from other FTI 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. FTIs have been extensively examined as potential anticancer agents, and surprisingly they are well tolerated in clinical settings 90 , Clinical activity has been detected with hematologic malignancies including acute myeloid leukemia AML , chronic myeloid leukemia CML , multiple myeloma and myelodysplastic syndrome. Some clinical activity toward advanced breast cancer has been detected. However, effects of FTIs on solid tumors have been limited.

Combining FTIs with other chemotherapeutic agents has produced some promising results. In combination with paclitaxel, lonafarnib showed clinical activity in people with non—small cell lung cancer Although FTIs have clinical activities, the effects seem to be largely independent of the inhibition of Ras protein; no correlations with Ras mutation status or with Ras inhibition have been observed.

Because there are many farnesylated proteins in the cell, FTIs have the potential to inhibit many proteins. Thus, the anticancer action of FTIs may reflect combinatorial effects on several farnesylated target proteins. Recently, human disorders in which a particular farnesylated protein is overactivated or overexpressed have been reported. In these situations, the effects of FTIs may be a result of their inhibiting the function of these particular proteins.

H-Ras activation is observed in a small but substantial percentage of human cancer. Importantly, people with Costello syndrome have a predisposition to neoplasia, including transitional cell carcinoma and neuroblastoma. Tuberous sclerosis syndrome TSC is a genetic disorder that is associated with the appearance of benign tumors called hamartomas in a variety of organs, such as kidney, skin and brain Thus, overactivation of Rheb underlies the molecular basis of this disease. FTIs inhibit Rheb refs. The effects of FTIs, in this case, parallel the effects of rapamycin, as Rheb is an activator of mammalian target of rapamycin mTOR , TSC1 mutations also lead to lymphangioleiomyomatosis LAM disease, which is characterized by diffuse infiltration of the pulmonary parenchyma with benign smooth muscle—like cells A different farnesylated protein, PRL-3, is implicated in cancer metastasis.

This protein is overexpressed in metastatic colon cancer as a result of chromosomal amplification Expression of PRL-3 in colon cancer cell lines results in greater invasive property than in healthy colon cells, and this is associated with the decrease of Rac and increase of RhoC and RhoA proteins Thus, it is worth exploring the possibility that FTIs may influence the metastatic potential of human cancer cells.

This is mainly a result of three lines of investigation. First, the geranylgeranylated protein RalA acts downstream of Ras to transform cells in several cancers Third, another geranylgeranylated protein, RhoC, has emerged as a critical protein in cancer metastasis. Studies of RhoC knockout mice show that loss of RhoC results in inhibition of metastasis Replacing the central dipeptide portion with different scaffolds led to the development of a series of inhibitors.

Recently, further improvement has been made by using a benzoyleneurea scaffold GGTIs have shown cellular effects including inhibition of Rho signaling, cell cycle arrest at G0 and G1, and apoptosis induction. This compound is highly potent, with a half-maximal inhibitory concentration IC 50 of 8 nM, and requires more than fold higher concentration to inhibit PFT. Additional GGTIs are expected to be identified, and these will provide valuable reagents for studying and therapeutically inhibiting geranylgeranylation.

Children with HGPS have many characteristics of accelerated aging, including growth retardation, bone problems, hair loss and a receding mandible, and they die of heart disease, generally by about age The biogenesis pathway leading to mature lamin A involves several steps and provides a framework for understanding HGPS The prelamin A precursor undergoes CaaX prenylation, proteolysis and methylation. Then, an endoproteolytic cleavage step unusual among mammalian CaaX proteins removes the C-terminal 15 amino acids, including the newly generated carboxylmethylated farnesylcysteine.

The reason that cells go to the trouble of C-terminally modifying prelamin A and then removing these modifications is not understood. The endoproteolytic cleavage of prelamin A is mediated by the zinc metal-loprotease Zmpste24 refs. HGPS is commonly caused by a dominant de novo point mutation in LMNA that activates a cryptic splice site and results in the production of a mutant form of lamin A called progerin having a amino-acid deletion that includes the Zmpste24 site Thus, progerin is not a substrate for further endoproteolytic cleavage and instead remains persistently farnesylated and carboxylmethylated , Several lines of evidence indicate that accumulation of aberrantly CaaX-modified prelamin A accounts for the progeroid phenotypes characteristic of the HGPS mutation At the cellular level, the hallmark of fibroblasts from people with HGPS is their highly misshapen folded, lobed and severely blebbed nuclei , Mouse and human fibroblasts expressing wild-type lamin A but lacking Zmpste24 activity owing to mutational inactivation have a similar aberrant nuclear phenotype.

Evidence has recently been presented by several investigators showing that FTIs can block, and possibly even reverse, the aberrant nuclear morphology resulting from the expression of progerin in fibroblasts from people with HGPS, in mouse fibroblasts and in HeLa cells , , — These findings have generated considerable excitement because they provide proof of principle, at the cellular level, that FTIs may be a useful therapy for HGPS.

But what about at the organismal level? New results from Yang et al. Though caution is warranted, the minimal toxicity associated with FTIs, together with the encouraging results from the cellular and mouse studies discussed above, suggest the compelling possibility that FTIs may be beneficial for children with HGPS.

Protein Prenylation: Enzymes, Therapeutics, and Biotechnology Applications

A new use of FTIs is in the treatment of malaria and African sleeping sickness caused by the parasites Plasmodium falciparum and Trypanosoma brucei , respectively. They kill parasites in culture in the low nanomolar range and can cure rodents infected with malaria when dosed orally The attachment of prenyl groups to proteins in eukaryotic cells was discovered about two decades ago either through traditional natural-product isolation and structure elucidation of fungal pheromones or through careful observations centered on the timing of cholesterol biosynthesis during the mammalian cell cycle.

Interest in understanding protein farnesylation for the purpose of cancer therapy grew out of the finding that one of the most common cancer-promoting elements in cells, the Ras proteins, require farnesylation to transform cells into tumor progenitors. Early studies showing that PFT inhibitors are very effective at shrinking human tumor masses implanted into experimental animals have led to preclinical drug development of such inhibitors at many pharmaceutical companies.

After the first round of clinical trials, it is likely that one or two of these inhibitors will emerge as a well-tolerated agent for treating a subset of leukemias. Further anticancer clinical trials of PFT inhibitors, especially in combination with existing anticancer drugs, are expected in the near future. The possibility that PGGT-I inhibitors may have beneficial anticancer properties is currently under investigation. Also, future studies may reveal the true anticancer basis of these compounds, as the original view that they act against cancer simply by blocking Ras farnesylation cannot be the whole story.

The anticancer potential of inhibitors of other enzymes that are often required for processing prenylated proteins such as endoproteases, methyltransferases and palmitoyltransferases is a ripe area for new efforts in medicinal chemistry. Protein prenylation has revealed new features about the ways in which small GTP-binding proteins control many vital intracellular processes, including trafficking of membrane compartments within eukaryotic cells and cytoskeleton function.

The analysis of these prenyl groups has set the stage for the discovery of soluble protein factors that move GTP-binding proteins from membranes into the soluble cellular compartment. There is much to be learned about the ways in which this translocation is coupled to processes such as cell morphology changes and cell movement and to transport processes such as protein secretion and neurotransmitter release. Thus, there may be other unexplored roles of protein prenyl groups beyond the well-established membrane-anchoring and molecular-handle functions.

National Center for Biotechnology Information , U. Author manuscript; available in PMC Jun Find articles by Michael H Gelb. Find articles by Lucas Brunsveld. Find articles by Christine A Hrycyna. Find articles by Susan Michaelis. Find articles by Fuyuhiko Tamanoi. Find articles by Wesley C Van Voorhis. Find articles by Herbert Waldmann. Correspondence should be addressed to M.

Farnesyltransferase H264 - MOA

The publisher's final edited version of this article is available at Nat Chem Biol. See other articles in PMC that cite the published article. Abstract In eukaryotic cells, a specific set of proteins are modified by C-terminal attachment of carbon farnesyl groups or 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. Discovery of protein prenyl groups and structural varieties The first reports of prenylated proteins and peptides described the secreted pheromone peptides from jelly fungi 1 , 2 , whose structure resembles that of the well-known a-factor mating pheromone from baker's yeast Saccharomyces cerevisiae , which contains a cysteine methylester farnesylated at the C terminus 3.

Open in a separate window. Enzymatic processing of prenylated proteins So far, three distinct protein prenyltransferases that attach prenyl groups to proteins have been identified. Palmitoylation of prenylated proteins S -Palmitoylation of proteins entails connection of a C 16 fatty-acid chain to the thiol functionality of a cysteine via a thioester bond. Anchoring proteins to membranes: Semisynthetic tools for studying lipidated proteins Progress in the area of protein ligation methods has given researchers access to a broad spectrum of methods for the semisynthetic production of post-translationally modified proteins, including lipidated proteins.

FTIs have Ras-independent clinical activities FTIs have been extensively examined as potential anticancer agents, and surprisingly they are well tolerated in clinical settings 90 , Toward rational uses of FTIs Recently, human disorders in which a particular farnesylated protein is overactivated or overexpressed have been reported. Progeria therapeutics based on protein prenylation A new and unanticipated potential use for FTIs has emerged from recent studies of the rare premature-aging disease Hutchinson-Gilford progeria syndrome HGPS.

FTIs as tropical parasitic disease therapeutics A new use of FTIs is in the treatment of malaria and African sleeping sickness caused by the parasites Plasmodium falciparum and Trypanosoma brucei , respectively. Concluding remarks The attachment of prenyl groups to proteins in eukaryotic cells was discovered about two decades ago either through traditional natural-product isolation and structure elucidation of fungal pheromones or through careful observations centered on the timing of cholesterol biosynthesis during the mammalian cell cycle.

Sakagami Y, et al. Isolation of a novel sex hormone tremerogen A, controlling conjugation tube formation in Tremella mesenterica fries. Requirements of chemical structure for hormonal activity of lipo-peptidyl factors inducing sexual differentiation in vegetative cells of heterobasidiomycetous yeasts. Structure of Saccharomyces cerevisiae mating hormone a-factor.

Identification of S-farnesyl cysteine as a structural component. Prenyl proteins in eukaryotic cells: Human lamin B contains a farnesylated cysteine residue. Identification of geranylgeranyl-modified proteins in HeLa cells. Exploring the specificity of prenyl protein-specific methyltranferase with synthetic prenylated rab peptides. Structure of mammalian protein geranylgeranyltransferase type-I.

Reaction path of protein farnesyltransferase at atomic resolution. Crystal structure of Rab geranylgeranyltransferase at 2 angstrom resolution. Upstream polybasic region in peptides enhances dual specificity for prenylation by both farnesyltransferase and geranylgeranyltransferase type I. A protein geranylgeranyltransferase from bovine brain: Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity.

Waldmann and co-workers described the synthesis of fluorescent isoprenoid analogs based on NBD and BODIPY groups and demonstrated the use of compound 21 in flow cytometry and imaging for analysis of uptake of these analogs in mammalian cells and zebrafish embryos. As noted above, there is considerable interest in identifying proteins that are prenylated in a cellular context in order to determine which prenyltransferase protein substrates have their prenylation status affected by FTIs. In this approach, metabolic labeling of living cells is first carried out using isoprenoid analogs to tag prenylated proteins with a reporter group, such as an azide or alkyne.

These tagged proteins are then functionalized using bioorthogonal reactions to install either a fluorophore for gel-based proteomic studies or a biotin moiety for enrichment of tagged proteins. Tamanoi and co-workers incorporated an azide-modified analog of GGPP 23 into geranylgeranylated proteins and subsequently installed a fluorophore via Cu I -catalyzed click reaction on the azide-labeled proteins. Since alkyne-modified chemical reporters tend to give more sensitive and selective detection compared with their azido counterparts, the Distefano and Hang groups used alkyne-functionalized isoprenoid analogs for analysis of the prenylome.

Six prenylated proteins were identified upon MS analysis of some of the fluorescent protein spots. In another study, metabolic labeling with 25 was carried out in the presence and absence of a farnesyltransferase inhibitor. The two corresponding protein samples were then reacted with two spectrally orthogonal azide-functionalized dyes and mixed together.

In recent years, several reports indicated that intracellular human pathogens, which lack prenylation machinery, translocate several effector proteins containing CaaX-box motifs into host cell cytosol. Tandem mass spectrometric analysis of eluted proteins identified 17 prenylated proteins with high confidence and 5 proteins with medium confidence, along with many other candidate isoprenoid-modified proteins.

During this analysis, they discovered isoform-specific farnesylation of zinc-finger antiviral protein ZAP and found that farnesylation of this protein was essential for increasing the antiviral activity of this immune effector protein. Alternative approaches of prenylome analysis bypass the need for a bioorthogonal reaction. Alexandrov and colleagues employed a biotinylated isoprenoid, 26 , for in vitro prenylation of proteins using either wt GGTase-II or engineered FTase and GGTase-I, to allow for subsequent enrichment using streptavidin beads.

In a study carried out by Reuter and co-workers, anilinogeraniol, 27 , was used to tag the farnesylated proteome. Effects of FTI treatments on labeling with 27 were also visualized using this approach. Expression levels of all the genes in the presence and absence of FTI treatment were quantified by comparing the fluorescence intensity of the two color panels. Utilizing this method, the authors identified downstream effector proteins that get either up- or down-regulated as a result of FTI treatment.

The initial efforts to develop farnesyltransferase inhibitors FTIs targeted the inhibition of oncogenic Ras proteins. Later, potent inhibitors were identified from library screening efforts. Recently, experiments with a caged FTI demonstrated that such compounds may be useful for selective release of an FTI within a defined tissue location. Since K-Ras can be geranylgeranylated and other geranylgeranylated proteins may also be involved in cancer, inhibitors of GGTase-I GGTIs have been evaluated as an alternative strategy to achieve anticancer therapies.

It provided preliminary evidence that lonafarnib could potentially improve one or more disease measures related to HGPS. The pathogenic parasites causing diseases such as malaria, African sleeping sickness, and Chagas disease have their own farnesyltransferase enzyme.

In recent years, the properties of FTase to specifically modify a single cysteine residue located at the C-terminal CaaX motif and to incorporate isoprenoid analogs containing bioorthogonal functionalities have been exploited for site-specific modifications of proteins. This has been possible since the presence of a CaaX-box at its C-terminus is sufficient to render almost any protein an efficient FTase substrate.

Functionalization of the resulting proteins via bioorthogonal reactions provides a convenient methodology for preparing a wide of array of protein conjugates in a site-specific manner. Both the Poulter and Distefano groups have used azide- and alkyne-functionalized FPP analogs in FTase-catalyzed reactions followed by click reactions or Staudinger ligations for immobilization of proteins GFP or GST onto solid surfaces such as glass slides or agarose resin.

A simple and efficient method for the derivatization and purification of recombinant proteins such as YPT7, Rab7, GST was developed by Alexandrov and co-workers using a fluorescent analog of FPP and phase partitioning. One important application of the prenylation-based labeling strategy is the formation of site-specific protein modifications such as protein—DNA conjugates, PEGylated proteins, and dually labeled proteins.

Discovery of protein prenyl groups and structural varieties

In that case, a trifunctional FPP analog incorporating both aldehyde and alkyne functionality was used to create the key multifunctional fragment consisting of a cargo protein, fluorophore and protein dimerizer. Protein prenylation has emerged as an important post-translational modification responsible for the correct cellular localization, activity, and protein—protein interactions of a number of signaling proteins. Over the past 25 years, a large number of isoprenoid analogs have been synthesized and employed to probe the structural and mechanistic features of the prenyltransferase enzymes.

With the extensive studies using these analogs and peptide substrates and X-ray crystallographic analysis of the enzymes in complex with the substrates and product, the enzymology of prenyltransferases is now well understood. One of the early hypotheses in the field of prenylation was that prenyltransferase inhibitors could be used to suppress malignant activity of oncogenic Ras proteins.

Protein Prenylation: Enzymes, Therapeutics, and Biotechnology Applications

While those inhibitors gave early success in the laboratory, clinical trials proved less promising. Those results make it clear that much remains to be learned concerning the roles of prenylated proteins in living cells, and this remains an intense area of current investigation. Several peptide library screening efforts, proteomic studies, and yeast-based genomic experiments have provided preliminary results toward this end; however, completely defining the prenylated proteome is still an ongoing task; addressing this issue will be central for assessing which patients are the best candidates for treatment with prenylation inhibitors.

More work also needs to be carried out to explore the potential of FTIs and GGTIs for the treatment of other afflictions including progeria, multiple sclerosis, parasitic diseases, and bacterial and viral infections. Protein prenylation has also shown promise for site-specific modifications of proteins. While many interesting applications have been demonstrated in recent years, future work must focus on creating protein conjugates including antibody—drug conjugates, PEGylated proteins, and other constructs that are directly applicable to therapeutic studies so that they can be evaluated in clinical contexts.

Overall, these challenges suggest that investigation of protein prenylation and will remain an active and vibrant field of inquiry for some time to come. National Center for Biotechnology Information , U.

Published online Nov Received Oct 1; Accepted Nov This is an open access article published under an ACS AuthorChoice License , which permits copying and redistribution of the article or any adaptations for non-commercial purposes. This article has been cited by other articles in PMC. Open in a separate window. Protein Prenylation Protein prenylation was first discovered in fungi in , 1 and almost 10 years later, the first prenylated protein in mammalian cells, farnesylated lamin B, was detected. Peptide Substrate Specificity Early work with prenyltransferases suggested that the X residue in CaaX box determines whether the protein is farnesylated or geranylgeranylated and that CaaX sequences with the X residue being alanine, serine, methionine, or glutamine are preferred by FTase, whereas leucine, isoleucine, and phenylalanine are preferred by GGTase-I.

Isoprenoid Analogs A large number of isoprenoid analogs containing various functionalities have been synthesized to study a variety of aspects of the prenylation reaction and prenyltransferases. Analysis of the Prenylated Proteome As noted above, there is considerable interest in identifying proteins that are prenylated in a cellular context in order to determine which prenyltransferase protein substrates have their prenylation status affected by FTIs.

Chemical proteomic strategy for analysis of the prenylated proteome. Structures of isoprenoid analog used to analyze prenylated proteome. Inhibition and Therapeutic Applications The initial efforts to develop farnesyltransferase inhibitors FTIs targeted the inhibition of oncogenic Ras proteins. Biotechnological Applications In recent years, the properties of FTase to specifically modify a single cysteine residue located at the C-terminal CaaX motif and to incorporate isoprenoid analogs containing bioorthogonal functionalities have been exploited for site-specific modifications of proteins.

Concluding Remarks Protein prenylation has emerged as an important post-translational modification responsible for the correct cellular localization, activity, and protein—protein interactions of a number of signaling proteins. Notes The authors declare no competing financial interest. Molecular mechanisms and functional consequences. Biochemistry 34 , — Signalling 10 , — Enzymes 30 , 91— Cancer 11 , — Structure 8 , — Biochemistry 32 , — Steady-state kinetic studies of substrate binding.

Nature , — Biochemistry 42 , — Biochemistry 38 , — Substrate specificity, kinetic mechanism, metal requirements, and affinity labeling. Biochemistry 40 , — Biochemistry 45 , — Steady state kinetic studies of the recombinant enzyme. Biochemistry 35 , — MedChemComm 4 , — Biochemistry 37 , — Biochemistry 48 , — Enzymes 29 , — Cloning, expression, farnesyl diphosphate binding, and functional homology with yeast prenyl-protein transferases.

Synthesis, inhibition kinetics and photoinactivation of yeast protein farnesyltransferase. Biochemistry 51 , —