Chemical tools for understanding protein lipidation in eukaryotes

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Chemical tools for understanding protein lipidation in eukaryotes Guillaume Charron, John Wilson and Howard C Hang Lipidation of proteins is an important mechanism to regulate protein trafficking and activity in cell and tissues. The targeting of proteins to membranes by lipidation plays key roles in many physiological processes and when not regulated properly can lead to cancer and neurological disorders. Dissecting the precise roles of protein lipidation in physiology and disease is a major challenge. Recent advances in chemical biology have now enabled the semisynthesis of lipidated proteins for fundamental biochemical and cellular studies. In addition, new chemical reporters of protein lipidation have improved the detection and enabled the proteomic analysis of lipidated proteins. The expanding efforts in chemical biology are therefore providing new tools to dissect the mechanisms and functions of protein lipidation as well as develop therapeutics targeted at protein lipidation pathways in disease.

they are regulated is only beginning to be appreciated. Herein, we describe the different classes of lipidated proteins found in eukaryotes and highlight the recent advances in chemical biology that are enabling more mechanistic studies of protein lipidation and improved methods for the visualization and proteomic analysis of lipidated proteins. We also provide a survey of small molecule inhibitors targeted at lipidation enzymes that may serve as useful chemical probes of cellular pathways and lead compounds for treating diseases associated with aberrant protein lipidation.

Introduction

The enzymatic modification of proteins with lipids provides important mechanisms to spatially and temporally control the activity of proteins in eukaryotes (Figure 1) [1,2]. The addition of hydrophobic lipids can directly modulate the activity of proteins or control their interactions with other proteins and membranes to facilitate signal transduction, protein trafficking and vesicle transport [2]. Protein lipidation in eukaryotes comes in several different forms that include S-prenylation, fatty-acylation, C-terminal cholesterylation and glycophosphatidylinositol (GPI) anchor modification (Figure 1) [2–4]. The identification of lipidated proteins and characterization of their biosynthetic pathways has revealed key roles for protein lipidation in many physiological processes. For example, Sprenylation and S-palmitoylation of Ras are essential for cell proliferation, whereas S-palmitoylation of adapter proteins such as LAT and PSD-95 are crucial for T cell activation and synaptic transmission, respectively [2]. In addition to intracellular signaling pathways, protein lipidation can control extracellular communication as highlighted by the GPI-modified protein CD14 in innate immunity [5] and cholesterol-modified sonic hedgehog, a secreted morphogen that controls tissue development [4].

The discovery of prominent lipidated proteins and the characterization of their biosynthetic pathways have firmly established the importance of protein lipidation in physiology. Indeed, the aberrant expression of lipidated proteins or their biosynthetic enzymes are associated with many diseases that range from cancer to neurological disorders [1,2]. Despite the remarkable advances in genomics, proteomics, bioinformatics and cellular imaging, much remains to be learned with regard to the functions and diversity of protein lipidation in physiology and disease. The mechanisms that control the dynamic trafficking of lipidated proteins to discrete membrane compartments are still not well understood. Moreover, the complete repertoire of lipidated proteins that are expressed in various cell types in vivo and how

Protein prenylation encompasses S-farnesylation and Sgeranylgeranylation (Figure 1), which are catalyzed by farnesyltransferase (FTase) and geranylgeranyltransferase I and II (GGTase I and GGTase II), respectively [6]. FTase and GGTase I transfer farnesyl pyrophosphate and geranylgeranyl pyrophosphate, respectively, onto the Cterminus of proteins that contain a C-terminal CaaX motif, where C is the modified cysteine, ‘a’ are often aliphatic amino acids and X represents an amino acid that determines specificity for the prenyltransferase in vivo. For example, X is frequently Met, Gln, or Ser for FTase, and Leu or Phe for GGTase I, whereas some CaaX motif proteins are substrates for both prenyltransferases. Following S-prenylation, the endopeptidase Ras converting

Address The Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065, USA Corresponding author: Hang, Howard C ([email protected])

Current Opinion in Chemical Biology 2009, 13:382–391 This review comes from a themed issue on Analytical Techniques Edited by Lara K. Mahal and Carlito B. Lebrilla Available online 19th August 2009 1367-5931/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2009.07.010

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Chemical tools for understanding protein lipidation in eukaryotes Charron, Wilson and Hang 383

Figure 1

Survey of protein lipidation in eukaryotes. N-, S- and O-prefixes describe the linkage of the lipid attachment to proteins.

enzyme 1 (RCE1) cleaves the tripeptide (aaX) to reveal a C-terminal cysteine that is subsequently methylated by isoprenylcysteine carboxyl methyltransferase (ICMT). On the contrary, GGTase II (also called RabGGTase), a structurally and functionally different prenyltransferase, dually S-geranylgeranylates the family of Rab proteins at their C-terminal cysteine residues. RabGGTase substrate specificity is not dictated by a consensus sequence but by Rab escort proteins (REPs) that bind Rab proteins and target them for enzymatic modification [6]. Fatty-acylated proteins are synthesized by discrete families of acyltransferases that utilize fatty acid–CoA substrates to yield cytoplasmic proteins that are N-myristoylated [7] or S-palmitoylated [8] as well as secreted proteins that can be S-, N-, or O-acylated [4,9] (Figure 1). N-Myristoyltransferases (NMTs, 2 in mammals, 1 in yeast) catalyze the transfer of myristic acid to N-terminal glycine residues of proteins bearing the GXXXS/T consensus sequence [7], whereas the DHHC–protein acyltransferases (DHHC–PATs, 23 in mammals, 7 in budding yeast) install palmitic acid onto cysteine residues of proteins [8]. In comparison to Nmyristoylation, DHHC–PAT-mediated S-palmitoylation has no defined amino acid consensus sequence and S-palmitoylation sites are often modified with a heterogeneous composition of fatty acids. Moreover, Spalmitoylation is reversible, modulated by cellular stimulation and regulated by yet uncharacterized lipases [8], which suggests the S-acylation cycle may be particularly important for regulating dynamic processes in cells. Indeed, S-palmitoylation of N-myristoylated or S-prenylated proteins is typically required for stable membrane targeting of many proteins [2]. www.sciencedirect.com

A family of membrane-bound O-acyltransferases (MBOATs) palmitoylates cysteine and serine residues on secreted proteins in the endoplasmic reticulum (ER) [9,10]. Further N-terminal proteolysis of S-palmitoylated cysteine residues can lead to spontaneous S- to N-acyl shifts that result in N-palmitoylation of sonic hedgehog [10] (Figure 1). In addition to N-palmitoylation, sonic hedgehog is modified with cholesterol at its C-terminus through an intrinsic intein-splicing activity [4]. The attachment of GPI anchors to proteins also occurs in the ER by the action of a GPI transamidase [3]. The fatty-acylation and cholesterylation of secreted proteins is often essential for their export and long range signaling properties [4], whereas GPI modification typically regulates the lateral mobility of proteins in membranes [3]. Synthesis of lipoproteins

The chemical synthesis of homogeneous lipidated peptides and proteins has been instrumental in dissecting the regulatory and effector functions of protein lipidation [11]. Notably, the development of solid-phase synthesis methods for generation of fatty-acylated and prenylated peptides have provided key substrates for determining the specificities of enzymes and building blocks for semisynthesis of lipidated proteins [11]. The semisynthetic approaches toward lipoproteins take advantage of recombinant protein expression methods and versatility of chemical synthesis to site-specifically incorporate lipid modifications and probes that are not available by either method alone. For example, the conjugation of fluorescent lipopeptides to proteins bearing C-terminal cysteines by maleimidocaproyl (MIC) ligation afforded fluorescent Ras lipoprotein variants to interrogate their partitioning into membranes in vitro [12–14] and Current Opinion in Chemical Biology 2009, 13:382–391

384 Analytical Techniques

Scheme 1

Semi-synthetic synthesis of lipidated proteins. (a) Conjugation of proteins with lipidated and/or fluorescently tagged peptides by MIC ligation on cysteine residues. (b) Lipidation by sortase-mediated trans-peptidation. (c) Lipidation and/or fluorescent tagging of proteins by EPL.

trafficking in living cells [15] (Scheme 1a). The analysis of S-palmitoylated and non-hydrolyzable thioether variants of lipidated Ras demonstrated that dynamic S-palmitoylation of proteins is an important feature for the distribution and trafficking of lipidated proteins in cells [15]. Photocrosslinkers such as the benzophenone group can also be installed by MIC ligations to investigate lipidated protein–protein interactions [16,17]. In addition to isoprenoid and fatty acid analogs, other lipids such as cholesterol can be conjugated to proteins by MIC ligation [18]. Sortase-catalyzed transpeptidation provides an alternative method for the semisynthesis of C-terminal lipid-modified proteins [19] (Scheme 1b). While these methods have provided access to lipoprotein variants, both approaches introduce non-native peptide or lipid– protein linkages that may not be ideal. Expressed protein ligation (EPL) has emerged as a powerful method for the semisynthesis of proteins bearing various posttranslational modifications and biophysical probes with native peptide bonds [20] (Scheme 1c). Indeed, EPL has enabled the production of various lipidated protein constructs for biochemical and biophysical studies [21–24]. Of note, the semisynthesis of fluorescently modified and prenylated isoforms of Rab7, a member of the RabGTPase family of proteins involved in vesicular transport and membrane fusion, has revealed geranylgeranylation dependent protein–protein interactions [25]. For example, synthetic unmodified or Current Opinion in Chemical Biology 2009, 13:382–391

monogeranylgeranylated Rab7 binds to REP for further RabGGTase-mediated geranylgeranylation and subsequent membrane targeting, whereas GDP dissociation inhibitor (GDI) preferentially binds mono-geranylgeranylated and dually geranylgeranylated Rab7 to modulate RabGTPase membrane activity [25]. These biochemical studies provide important mechanistic insight into the cyclic regulation of RabGTPases [25], which are further supported by structural analysis of a monoprenylated Rab protein, Ypt1 also generated by EPL, in complex with GDI [26]. The semisynthesis of GPI-modified protein variants by EPL has also begun to facilitate the analysis of their biochemical and biophysical properties [23,24]. Chemoenzymatic methods can complement semisynthetic approaches for generating functionalized lipoproteins. For example, fluorescent nitrobenzoxadiazole (NBD) analogs of farnesyl and geranylgeranyl pyrophosphates (1, 2) were shown to be efficient in vitro substrates for FTase, and GGTase I/II, respectively [27] (Figure 2a). These fluorescent substrates provide a rapid non-radioactive assay for protein prenylation in vitro that facilitated the high-throughput screening of selective FTase and RabGGTase inhibitors [27,28]. Prenyl pyrophosphate analogs bearing photoaffinity crosslinkers such as arylazides (3) can also be enzymatically installed by FTase to map interactions of prenylated proteins with other proteins and membranes [29] (Figure 2a). In addition, a biotinylated geranyl pyrophosphate derivative www.sciencedirect.com

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Figure 2

for both FTase and GGTase I in vitro (Scheme 2a and Figure 2a). In addition, NMT can be used to transfer azide/alkyne–myristic acid analogs (11, 14) onto recombinant proteins bearing an N-terminal glycine in vitro or in bacteria [39,40] (Scheme 2b). Chemical reporters for protein lipidation in cells

Panel of lipid analogs for protein lipidation studies. (a) Isoprenoid analogs functionalized with fluorophore, photo-crosslinker, biotin, azide or alkyne group for in vitro protein S-prenylation studies. (b) Azide and alkyne chemical reporters of S-prenylation and fatty-acylation studies in cells.

(BGPP, 4) can be utilized by RabGGTase for the sitespecific modification of recombinant proteins with an affinity tag [30,31]. While BGPP is not a substrate for wild-type FTase or GTTase I, mutants of both enzymes have been identified that can utilize this biotinylated isoprenoid analog [31]. Alternatively, lipid analogs bearing smaller chemical tags (azide or alkyne) can serve as substrates of protein lipidation enzymes (Scheme 2a and b) and subsequently functionalized with fluorophores or affinity tags by bioorthogonal labeling reactions [32], namely the Staudinger ligation [33] (Scheme 2c) or the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition (commonly referred to as ‘click chemistry’) [34,35] (Scheme 2d). For example, geranyl pyrophosphate derivatives with both azide (5, 6) [30,36] and alkyne (7, 9) [36,37] were shown to be efficient substrates for FTase, while alkynyl–farnesyl pyrophosphate (8) [38] was a substrate www.sciencedirect.com

Protein lipidation in cells has historically been visualized by metabolic labeling with radioactive (3H, 14C) lipids followed by autoradiography of labeled proteins [41]. While effective, radioactivity often requires long exposure times and is hazardous [10,41]. The development of bioorthogonal labeling reactions and chemical reporters (azide/alkyne-modified lipids) [32] has provided improved methods for the detection of protein lipidation in cells (Scheme 3a). For example, an azide-derivative of farnesol (10) (Figure 2b) can be utilized by mammalian cells, installed onto prenylated proteins and readily visualized by streptavidin blot after reaction with phosphine–biotin reagents via the Staudinger ligation [42]. Likewise, azidoand alkynyl-fatty acids can be metabolically incorporated onto N-myristoylated or S-palmitoylated proteins depending on their chain length and readily visualized after bioorthogonal labeling with secondary detection tags [43,44,45] (Scheme 3a). Shorter fatty acid chemical reporters (az-12 (11) and alk-12 (14)) preferentially label N-myristoylated proteins, while longer chain analogs (az-15 (13) and alk-16 (16)) selectively target S-palmitoylation [43,44,45,46–48] (Figure 2b). It should be noted that protein fatty-acylation in cells is heterogeneous, as some Spalmitoylated proteins are labeled with shorter and longer chain fatty acids. These fatty acid chemical reporters can also be utilized to detect the lipidation of secreted proteins such as Wnts [49]. With regard to the various modes of visualizing protein fatty-acylation, alkynyl-fatty acids in combination with click chemistry labeling and in-gel fluorescence detection affords the optimal conditions for detection [44] (Scheme 3a). In addition to gel-based methods, fatty-acylated proteins in cells can be visualized by fluorescence microscopy and flow cytometry (Scheme 3a) [44]. Proteomic analysis of lipidated proteins

Bioinformatic methods based on protein lipidation motifs (Prenbase, Myrbase, CSS-Palm and GPI-DB) and gene expression profiles can provide a survey of candidate lipidated proteins (S-prenylation, N-myristoylation, S-palmitoylation and GPI modification, respectively) that may be expressed in cells or tissues [2,50]. Nonetheless, experimental validation of protein lipidation is still needed to fully appreciate the diversity of lipidated proteins as well as their posttranscriptional and posttranslational mechanisms of regulation. For example, N-myristoylation typically occurs cotranslationally in cells, but can function posttranslationally during programmed cell death as a consequence of protease activation that reveals de novo N-terminal glycines on proteins [51]. Current Opinion in Chemical Biology 2009, 13:382–391

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Scheme 2

Inhibitors of protein lipidation. FTase inhibitors: Lonafarnib and tipifarnib [66]; FTI-276 [56]; BMS-214662 [57]; GGTase I inhibitor: GGTI-2418 [59]; RabGGTase inhibitor [28]; NMT inhibitor: 2HMA and 2HMA-CoA [62]; DHHC-PATs inhibitors: 2BP [64]; Other PAT inhibitors [64,65].

Unique biochemical features of lipidated proteins and lipid chemical reporters have been exploited for the largescale proteomic analysis of different lipoprotein classes. Proteomic studies of S-prenylated proteins have been performed with azido-farnesol (10) metabolic labeling, Staudinger ligation-mediated biotinylation of cell lysates, streptavidin affinity enrichment, on-bead protease digestion and LC–MS/MS sequence of recovered peptides (Scheme 3a) [42]. Alternatively, pharmacological inhibition of S-prenylation in cells, followed by in vitro biotinylation of cell lysates with RabGGTase/BGPP and streptavidin capture enabled the proteomic analysis of many RabGTPases with multi-dimensional protein identification technology (MudPIT) [31]. These two studies have provided access to unique subsets of Sprenylated proteins in mammalian cells and set the stage for more comprehensive proteomic studies in the future. Lipid chemical reporters and bioorthogonal labeling methods provide an alternative approach for large-scale analysis of S-palmitoylated as well as N-myristoylated proteins (Scheme 3a). After metabolic labeling of cells with azido- or alkynyl-fatty acids (11–16), cell lysates can be biotinylated with click chemistry reagents, subjected to streptavidin enrichment, analyzed by on-bead protease digestion and MudPIT proteomics [45] or selectively eluted and evaluated by gel-based proteomics (Wilson, J et al. unpublished). The analysis of membrane Current Opinion in Chemical Biology 2009, 13:382–391

fractions from 17-octadecynoic acid (ODYA or alk-16 (16))-labeled Jurkat T cells by the on-bead digestion/ MudPIT protocol selectively identified 125 proteins with high confidence, among which were known GTPases, G-protein-subunits, receptors as well as many new candidate fatty-acylated proteins [45]. Our own proteomic analysis of Jurkat T cells labeled with azidoor alkynyl-fatty acids by selective elution/gel-based proteomics retrieved 178 proteins with high confidence that also included known fatty-acylated proteins and many new candidate fatty-acylated proteins (Wilson, J et al. unpublished). The comparative analysis of fatty acid chemical reporters of different chain lengths revealed lipidated proteins that were selectively targeted by short or long chain analogs, as well as many proteins that were labeled by multiple analogs (Wilson, J et al. unpublished). It should be noted that even though ODYA (alk-16 (16)) was used to target S-palmitoylated proteins, many N-myristoylated proteins were also selectively retrieved using this chemical reporter [45]. These observations are also consistent with our experiments with longer chain fatty acid analogs (Wilson, J et al. unpublished), reinforcing the notion that protein fatty-acylation is heterogeneous in eukaryotic cells [41]. In the case of S-palmitoylated proteins, the acyl-biotin exchange (ABE) protocol that exploits the hydroxylamine (NH2OH) sensitivity of thioesters has provided a useful www.sciencedirect.com

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Scheme 3

Methods for robust detection and proteomic analysis of lipidated proteins. (a) Metabolic labeling of prenylated or fatty-acylated proteins in cells with azido or alkynyl chemical reporters followed by bioorthogonal labeling with a fluorophore for imaging and in-gel fluorescence detection or with biotin for streptavidin blotting and affinity enrichment followed by subsequent proteomic analysis. (b) Acyl–biotin exchange for the identification of Spalmitoylated proteins. Unmodified cysteines are capped followed by cleavage of S-acyl modifications (mostly palmitates) by hydroxylamine (NH2OH). Newly exposed cysteines (former S-palmitoylation sites) are biotinylated, enriched and analyzed by MudPIT (top panel) [53]. Alternatively, biotinylation of former S-palmitoylation sites with thiol-reactive ICAT probe followed by electrophoretic separation and in-gel trypsinization allow for fatty-acylation site identification (lower panel) [54].

method for enrichment of S-acylated peptides and proteins (Scheme 3b) [52,53,54]. ABE involves capping of free sulfhydryls in cell lysates with N-ethyl maleimide (NEM), cleavage of thioester bonds with NH2OH, labeling of the newly liberated thiols with HPDP–biotin (a cleavable thiol-specific biotinylation reagent), streptavidin enrichment and elution of the captured proteins for SDS-PAGE and MudPIT analysis [52,53]. ABE studies on different budding yeast strains revealed 25 new candidate S-palmitoylated proteins and demonstrated that individual DHHC–PATs exhibit differential and overlapping substrate specificity [52]. The application of ABE to rat neurons identified 50 known and 113 www.sciencedirect.com

new candidate S-palmitoylated proteins with high confidence, one of which was the brain-specific isoform of Cdc42, a small GTPase that was concentrated in dendrites and associated with post-synaptic structures [53]. ABE can also be performed with isotopically encoded thiol-alkylating reagents (ICAT) for comparative analysis of S-acylated peptides, which identified 30 potentially S-palmitoylated peptides in high confidence corresponding to 26 S-palmitoylated proteins and their potential sites of modification from HeLa cells, one of which was decreased upon siRNA knockdown of DHHC2 [54] (Scheme 3b). A comparative analysis of ABE and chemical reporter-based proteomics methods suggest that these Current Opinion in Chemical Biology 2009, 13:382–391

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methods are robust and provide complementary approaches toward large-scale profiling of fatty-acylated proteins. ABE provides useful insight into S-acylation, while chemical reporters can also access non-S-acylated proteins such as N-myristoylated substrates. It is important to note that chemical reporters require metabolic labeling of cells or organisms, whereas in vitro methods can be used directly on tissue or cell lysates. GPIanchored proteins can be selectively retrieved from membrane preparations by selective cleavage with lipases for proteomic analysis [55]. The use of phospholipase D enabled the identification of 35 GPI-anchored proteins from plants (Arabidopsis thaliana) and 11 from mammalian cells (HeLa) [55]. Small molecule inhibitors of protein lipidation

Small molecule inhibitors provide important tools to dissect protein lipidation pathways and their associated diseases [6]. The mutational activation of Ras isoforms in 20% of human cancers and discovery that Ras prenylation is necessary and sufficient for its transforming activity motivated the development of selective FTase inhibitors (FTIs) for cancer therapy [1] (Figure 3). Many FTIs were designed based on FTI-276, a peptidomimetic of the Cterminal CVIM of K-Ras4B that inhibited FTase 100fold selectively over GGTase I in vitro [56]. BMS-214662, a non-peptidic inhibitor was >1000-fold more selective for FTase over GGTase I [57]. Unfortunately, clinical trials of drugs that target mevalonate pathways (i.e. statins) and FTIs (lonafarnib and tipifarnib) revealed that their anti-tumor activity was lower than expected and that

their mechanism(s) of action are more complex than appreciated [1,6]. In particular, further studies demonstrated that K- and N-Ras were geranylgeranylated when FTase was inhibited [58], which prompted interest in the development of selective GGTase I inhibitors. These efforts lead to compounds such as GGTI-2418 that display high potency toward GGTase I and 5580-fold selectivity against FTase [59]. Although inhibitors selective for FTase versus GGTase I have been developed with assays using radioactive FPP and GPP, the selectivity of these inhibitors against RabGGTase has not been reported. New fluorescent isoprenoids (NBD–GPP (1) and NBD–FPP (2)) (Figure 2a) were important advances for the development of high-throughput non-radioactive prenyltransferase assays to determine the specificity of potential inhibitors [27,60]. Screening a library of 469 peptides modeled on the naturally occurring FTase inhibitor pepticinnamin E yielded selective RabGGTase inhibitors and the first crystal structure of RabGGTase– inhibitor complex, which offers new opportunities to design improved RabGGTase inhibitors and evaluate the role of RabGGTase in disease [28]. In addition to cancer, FTIs are being developed to treat malaria and African sleeping sickness caused by the parasites Plasmodium falciparum and Trypanosoma brucei, respectively [61]. Fatty acids analogs have served as useful probes and inhibitors of protein fatty-acylation. For example, myristic acid analogs provided key insight into the substrate specificity of NMT that were later supported by struc-

Figure 3

Inhibitors of protein lipidation. lonafarnib and tipifarnib [66]; FTI-276 [56]; BMS-214662 [57]; GGTI-2418 [59]; RabGGTase inhibitor [28]; NMT inhibitor: 2HMA and 2HMA-CoA [62]; DHHC-PATs inhibitors: 2BP [64]; Other PAT inhibitors [64,65]. Current Opinion in Chemical Biology 2009, 13:382–391

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tural studies [7]. Compounds such as 2-hydroxymyristic acid (2HMA) [62] and 2-bromopalmitic acid (2BP) [63] are commonly used to inhibit N-myristoylation and Spalmitoylation in cells, respectively (Figure 3). Although 2BP has recently been shown to directly inhibit DHHC– PATs in vitro [64], its mechanism(s) of action in cells is still unclear. 2BP also inhibits fatty acid CoA ligase and other enzymes involved in lipid metabolism [41]. Recent efforts to identify selective DHHC–PATs inhibitors by high-throughput cell-based screening have yielded a few compounds that appear to block S-palmitoylation in vitro and in cells [64,65]. Determining the mechanism(s) of action for the currently available S-palmitoylation inhibitors will be crucial for their utility in cell biology.

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Conclusions

11. Brunsveld L, Kuhlmann J, Alexandrov K, Wittinghofer A, Goody RS, Waldmann H: Lipidated ras and rab peptides and proteins—synthesis, structure, and function. Angew Chem Int Ed Engl 2006, 45:6622-6646.

Chemical approaches have provided important insight into the biosynthesis and functions of protein lipidation in biology. The advances in the semisynthesis of lipidated peptides and proteins have yielded homogeneous materials for fundamental biochemical and cellular studies. In addition, the development of functionalized lipid analogs has also afforded new assays to evaluate small molecule inhibitors of lipidation enzymes as well as new tools to visualize and identify lipidated proteins. The proteomic analyses of various lipidated protein classes have revealed a greater diversity of protein lipidation in cells than previously appreciated and afford many protein substrates to further mechanistic studies. Collectively, these new chemical tools should help dissect the functions of protein lipidation in physiology and disease as well as facilitate the development of effective therapeutics for cancer and neurological disorders.

Conflict of interest The authors have no conflict of interest.

Acknowledgements HCH acknowledges support from The Rockefeller University, Irma T Hirschl/Monique Weill-Caulier Trust and Ellison Medical Foundation. GC thanks the Rockefeller/Sloan-Kettering/Cornell Tri-Institutional Program in Chemical Biology.

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