Synthesis of Lipidated Proteins - Bioconjugate Chemistry (ACS

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Synthesis of Lipidated Proteins Tom Mejuch, and Herbert Waldmann Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00261 • Publication Date (Web): 21 Jul 2016 Downloaded from http://pubs.acs.org on July 24, 2016

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Bioconjugate Chemistry

Synthesis of Lipidated Proteins Tom Mejuch,† Herbert Waldmann†,‡,* †

Department of Chemical Biology, Max-Planck Institute of Molecular Physiology, Otto-

Hahn-Strasse 11, 44227 Dortmund, Germany ‡

Department of Chemistry and Chemical Biology, Technical University of Dortmund, Otto-

Hahn-Strasse 6, 44227 Dortmund, Germany *

e-mail: [email protected], phone: +49 (231) 133-2400, fax: +49

(231) 133-2499.

Abstract Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signalling and identifying the 1 ACS Paragon Plus Environment


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protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins were developed. In this review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full length semisynthetic lipidated Ras, Rheb and LC3 proteins are presented.

1. Introduction Protein lipidation is a post- or co-translational covalent attachment of a lipid group to proteins. The attachment of the lipid group facilitates association of the lipidated proteins with particular membranes in eukaryotic cells. Protein lipidation is one of the most important protein modifications as it influences protein distribution, localization and activity. Lipid groups are responsible for the binding of the proteins to the target membrane. They promote various protein-protein interactions and actively participate in the regulation of signalling processes.1-3

Up to date several post- and co-translational lipid modifications were characterized, including N-myristoylation,

S-palmitoylation,

S-prenylation,

addition

of

cholesterol,

phosphatidylethanolamine and GPI anchor (Scheme 1). Proteins can undergo one or several different lipid modifications as in the cases of the Src-family kinase Lyn, which is myristoylated and palmitoylated, and H-Ras that is farnesylated and double plamitoylated.

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Scheme 1. Most common protein lipid modifications.

N-myristoylation refers to the irreversible addition of the myristate (C14) group to the Nterminal glycine via an amide bond. N-myristoylated proteins include non-receptor tyrosine kinases (members of Src family and Abl), G-proteins (transducin α-subunit), ADPribosylation factors (Arf-1, Arf-3, Arf-5 and Arf-6), Ca2+ binding proteins (Recoverin and Neurocalcin), viral proteins (Nef and Gag) and others.4 In eukaryotic cells, N-myristoylation is mediated by the enzyme N-myristoyltransferase (NMT) which transfers the acyl group from myristoyl-CoA to the N-terminal amine of proteins.5 This modification mostly occurs co-translationally but also in some cases post-translationally. During co-translational modification, the N-terminal glycine is modified following the cleavage of the N-terminal methionine residue by methionine aminopeptidases. Post-translational myristoylation typically occurs after a caspase cleavage, resulting in the exposure of an internal glycine residue.6 N-Myristoylation plays an essential role in protein-protein interactions and membrane targeting of proteins, which are involved in a wide range of signal transduction pathways.4

Palmitoylation in the majority of the cases refers to the covalent attachment of the palmitate (C16) moiety to cysteine residues via a thioester bond. This modification can be found in the proximity of the N-terminus and the C-terminus of proteins. Due to the lability of the thioester bond, palmitoylation is usually referred to as a reversible modification in contrast to 3 ACS Paragon Plus Environment


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the stable myristoylation and prenylation modifications.7,

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8

The palmitoylation process is

catalysed by palmitoyl transferase proteins.9-16 Various proteins can undergo multiple cycles of palmitoylation and depalmitoylation including the H-Ras and N-Ras proteins and the reversibility of these processes regulates the subcellular localization, membrane binding and activity of the proteins.17-19 Less frequently, palmitoylation can occur on the N-terminal cysteine of a protein via an amide bond (Hedgehog protein family).20 In such cases, the palmitoylation is stable and non-reversible.

Prenylation refers to the post-translational covalent attachment of the farnesyl (C15) or geranylgeranyl (C20) groups to a cysteine residue via a thioether bond. Proteins that undergo prenylation contain at their C-terminus a so-called “CAAX box” motif, where C is cysteine, A is an aliphatic amino acid and X is the residue that dictates whether the protein will be farnesylated or geranylgeranylated. The enzymes that catalyse the prenylation are protein farnesyl transferase (FTase) and protein geranylgeranyl transferase-I (GGTase-I).21 Prenylation of the proteins is followed by the proteolytic cleavage of the AAX residues by Ras converting enzyme I (RceI) and carboxymethylation of the newly generated C terminal cysteine residue by isoprenylcysteine carboxymethyltransferase (Icmt). Examples of farnesylated proteins include Ras, Rheb and Rho families of GTPase proteins, phosphodiesterase proteins (PDEα, PDEβ) and nuclear proteins (Lamin).22

For Rab proteins, the geranylgeranylation is accomplished by a different protein geranylgeranyl transferase (GGTase-II or RabGGTase). In contrast to GGTase-I, GGTase-II does not recognize directly its protein substrates, but requires a presence of an adaptor Rab escort protein (REP). Thus, Rab prenylation requires the formation of a ternary catalytic complex of Rab/REP/GGTase-II.23-25 Prior to the geranylgeranylation, REP preferentially interacts with the Rab protein in its GDP-bound form and mediates its recognition by GGTase-II. GGTase-II has no sequence preference since the specificity stems from the interactions with REP protein. Therefore, any of the possible C-terminal sequences found in Rab GTPases that include CC, CXC, CCX, CCXX, CCXXX and CXXX are prenylated.26, 27

Additional important lipid modifications of proteins include glycosylphosphatidylinositol (GPI) anchoring, addition of cholesterol to the C-terminus of Hedgehog family of proteins and addition of phosphatidylethanolamine. Proteins are anchored with the GPI moieties to the extracellular side of the plasma membrane. This posttranslational glycolipid modification is 4 ACS Paragon Plus Environment

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mediated by the GPI transamidase in the ER lumen. Proteins containing a GPI anchor are functionally diverse and play important roles in signal transduction, prion disease pathogenesis, immune response and the pathobiology of trypanosomal parasites.28 The glycolipid group can be cleaved by phospholipases and result in the release of the protein from the plasma membrane.

Cholesterol modification was found so far only in the Hedgehog family of proteins. Hedgehog proteins are synthesized as 45 kDa precursor, which undergo intramolecular processing to yield 20 kDa N-terminal fragments with a cholesterol moiety covalently attached to its carboxyl terminus via a thioester bond.29 The cholesterol modification causes association of Hedgehog protein with the cell membrane and is essential for its proper function. 30, 31

Phosphatidylethanolamine addition is also a rare post-translational modification that is observed in autophagy-related proteins Atg8 in yeast and the mammalian orthologue LC3.32

Studying trafficking, localization and function of palmitoylated proteins is challenging due to the fact that palmitoylation is a dynamic process in the cell and the state of protein lipidation is frequently dependent on the exact localization. Additionally, several plamitoyl transferase proteins are present in the cell that can rapidly palmitoylate and depalmitoylate the protein of interest.19 In contrast to palmitoyaltion, myristoylation and prenylation modifications are permanent and, therefore, easier to monitor and study. The access to myristoylated proteins is possible through bacterial co-expression of the desired protein with a recombinant NMT.33-39 Recently, a single dual-gene expression vector was introduced for the expression of the myristoylated viral protein Nef.40 The same strategy was later used for the expression of the lipidated BASP1 and Src proteins.41 In contrast, prenylated proteins cannot be accessed through bacterial co-expression. Recently developed bioconjugation techniques or so-called semi-synthetic approaches were developed to address this shortcoming. The chemical synthesis of the Rab proteins was previously reviewed in great details.42, 43 The semisynthetic methods to obtain proteins modified with GPI anchors were also extensively reviewed.44-49 Therefore, in this review, we focus on the synthesis of farnesylated Ras and Rheb protein conjugates and LC3 protein modified with phosphatidylethanolamine.

2. Synthesis of lipidated peptides 5 ACS Paragon Plus Environment


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Recent advances in bioconjugation methods allow access to sufficient quantities of the lipoproteins using semi-synthetic methods. This task is divided into three parts – (1) chemical synthesis of the lipidated peptide, (2) biochemical expression of the truncated protein and (3) ligation of the lipopeptide to the expressed protein to yield the desired full size lipidated protein.

Methods for the synthesis of the lipidated peptides involve various solution- or solid-phase synthesis strategies, different protection groups, e.g. 9-fluorenylmethoxycarbonyl (Fmoc) or the tert-butoxycarbonyl (Boc) strategy, and methods for the incorporation of the lipid groups.

2.1. Solution-phase synthesis of lipidated peptides Several solution-phase syntheses of Ras-derived peptides were reported earlier.50-56 However, in general solution phase synthesis is more challenging and time-demanding than the synthesis on solid support. The growing lipidated oligopeptide becomes increasingly insoluble in the reaction medium and causes problems with the subsequent coupling steps and the purification.57

2.2. Solid-phase synthesis of lipidated peptides Due to the difficulties involved in solution-phase synthesis of lipidated peptides, several solid-phase synthetic strategies were developed.58-62 Several parameters have to be taken into account while designing the strategy for the synthesis (Scheme 2). These parameters include the nature of the solid phase support that can allow easy cleavage of the peptide, the choice of the orthogonal protecting groups that can be selectively removed in the necessary stage of the synthesis, the possible point for the attachment of reporter groups (e.g. fluorophore) and the group that will allow ligation to the protein. All the aforementioned parameters have to allow introduction of the lipid group or lipidated peptide building blocks. Additionally, prenyl groups are acid-sensitive and are prone to protonation and isomerisation in the presence of strong acids. Therefore, solid supports or protecting groups that require high concentrations of acid for the cleavage can’t be used during the synthesis.43 Moreover, during posttranslational processing Ras proteins are converted into the C-terminal methyl ester. Hence, the prenylated peptides should be equipped with a C-terminal methyl ester group as well. This issue was successfully addressed by performing the synthesis on the hydrazide linker63 following the cleavage and trapping with methanol as nucleophile, on the Ellman sulfonamide solid support60, 64 following the cleavage and trapping with the amino group of 6 ACS Paragon Plus Environment

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farnesylated cysteine methyl ester as the nucleophile or by the cleavage of the peptide from the chlorotrityl solid support and subsequent methylation of the carboxylic acid using trimethylsilyl diazomethane.65 Alternatively, the second C-terminal amino acid can be attached to the solid support, followed by the coupling of the prenylated cysteine methyl ester.61

Scheme 2. Designing the synthesis of a lipidated peptide.

Typically, the synthesis of lipidated C-terminal peptides of the Ras protein family involves preparation of lipidated amino acid building blocks, and their incorporation into the elongating peptide chain. The prenylated cysteine building blocks can be easily prepared by an alkylation reaction of free cysteine thiolate with farnesyl66 or geranylgeranyl67 halides (Scheme 3a). Palmitoylated cysteine can be prepared from Fmoc-Cys(Trt)-OH by a deprotection of a trityl group and coupling with palmitoyl chloride (Scheme 3b).66 The palmitoyl thioester group is known to be unstable in presence of strong nucleophiles and can undergo undesired S,N-acyl shift. To overcome this shortcoming, the hexadecyl group was introduced as a stable analogue of the palmitoyl group. Hexadecylated cysteine can be conveniently prepared by a radical addition of the free thiol of the cysteine to 1-hexadecene (Scheme 3c).68



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Scheme 3. Synthesis of lipidated cysteine building blocks. a) farnesylated or geranylgeranylated cysteine; b) palmitoylated cysteine; c) hexadecylated analogue of palmitoylated cysteine.

Another issue that require attention is the conditions for the coupling of the lipidated cysteine residue. Cysteine is known as one of the amino acids prone to racemization. Coupling conditions that minimize this racemization were extensively studied.69 It was found that a combination of the coupling reagents as HBTU/HOBt or HCTU with trimethylpyridine (TMP) as a base in a 1:1 ratio in DCM/DMF (1:1) leads to minimal racemization. Alternatively, the lipid moiety can be introduced to the complete peptide.70-73 This strategy is less applicable when several lipidated cysteine moieties in the sequence require selective lipidation. To overcome this difficulty a protocol involving a selective deprotection and alkylation sequence was recently developed and applied in the synthesis of a double lipidated N-Ras-derived peptide.74

3. Protein expression The truncated protein, lacking the corresponding lipopetide can be produced by means of standard bacterial expression in E. coli.75 The advantages of the bacterial expression over other expression systems include easy handling of the cultures, rapid growth, and extended knowledge of E. coli on genetic and molecular level. The main disadvantage is in the lack of the post-translational modifications machinery present in the eukaryotic systems. In case that 8 ACS Paragon Plus Environment

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the protein can’t be successfully expressed in the prokaryotic system, several eukaryotic systems can be utilized that include yeast,76 Dictyostelium discoidum77, infected with baculovirus.

79

78

and insect cells

The main disadvantages of these methods include slow growth,

long development cycle under inconvenient conditions and a relatively high cost. In some cases it is necessary to introduce an amino acid that is not naturally present in the sequence in order to allow a site specific bioconjugation. For this purpose a site-directed mutagenesis technique is being used. This technique allows substitution of each amino acid in the sequence by any other naturally occurring amino acid to produce the desired mutant protein.80

4. Conjugation of the lipopetide to the protein Following the successful synthesis of the lipidated peptide and the expression of the truncated protein the stage is set for the coupling of building blocks. The conjugation of the peptide to the protein can be achieved using one of several reported ligation methods.81-83 In the context of the Ras and Rheb proteins, the three most commonly used ligation techniques are expressed protein ligation (EPL), maleimidocaproyl (MIC) ligation and sortase-mediated ligation.

4.1. Native chemical ligation and expressed protein ligation Currently, the most often used ligation chemistry technique is based on native chemical ligation (NCL) as an efficient method to couple two polypeptide fragments (Scheme 4).84

The NCL technique involves two unprotected peptides, equipped with a thioester at the Cterminus and an N-terminal cysteine residue respectively. The thiol group of the N-terminal cysteine attacks the thioester group resulting in a reversible transthioesterification reaction. Subsequently, the newly formed thioester undergoes attack by the N-terminal amino group resulting in an irreversible S,N-acyl shift and a formation of the native amide bond.



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Scheme 4. Native chemical ligation of the lipidated peptide with Ras protein. Protein thioester, suitable for ligation can be obtained by expressed protein ligation (EPL).85, 86

EPL takes advantage of the existing natural protein splicing process. In protein splicing,

two extein domains that are separated in the protein sequence by an additional intein domain are connected via a native amide bond following the cleavage of the intein domain. Initially, an N-terminal cysteine residue of the intein undergoes reversible N,S-acyl shift that results in the formation of a thioester between the intein and the N-terminal extein. In the next step, first cysteine residue from the C-extein participates in transthioesterification to form a new thioester. Following the intramolecular rearrangement with a C-terminal Asn residue the intein is cleaved off and the native amide bond between the N- and C-exteins through the S,N-acyl shift is established. In expressed protein ligation, the protein part of the bioconjugate can be genetically fused to an intein domain (Scheme 5). One of the exteins can serve as an affinity tag as for example a chitin-binding domain (CBD) facilitating the purification of the protein. This tag can be removed later before the final conjugation of the lipidated peptide. The resulting recombinant protein can be bound to a resin through the affinity tag, purified and released with an excess of a thiol such as sodium 2-mercaptoethanesulfonate (MESNA), leading to the formation of a new MESNA thioester. The new thioester can be ligated with the lipidated peptide containing an N-terminal cysteine residue through transthioesterification 10 ACS Paragon Plus Environment

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reaction. The S,N-acyl shift results in formation of a native amide bond between the expressed protein and the lipidated peptide similarly to the NCL.

Scheme 5. Expressed protein ligation of the lipidated peptide with Ras protein.

4.2. MIC ligation Another commonly used method for coupling two peptidic fragments utilizes the chemoselective Michael addition reaction of the cysteine thiol group to the corresponding Michael acceptor moiety present in the maleimide group (Scheme 6). This ligation proceeds 11 ACS Paragon Plus Environment


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smoothly at neutral pH and is commonly used to label surface-exposed cysteine residues with a fluorophore. Maleimidocaproic acid (MIC-OH) can be easily incorporated at the Nterminus of the lipidated peptide and used in the ligation reaction.

Scheme 6. MIC ligation of the lipidated peptide with truncated Ras protein.

4.3. Sortase-mediated ligation Another method for the conjugation of the lipidated Ras peptide to the expressed truncated protein is based on a sortase-mediated ligation.87,

88

Sortase A is a transpeptidase, isolated

from the Gram-positive bacterium Staphylococcus aureus that recognises the L-P-T-E-G motif on the processed protein and cleaves the sequence after the threonine residue leading to the formation of a thioester. The N-terminal glycine residue of the lipidated K-Ras peptide can then attack the thioester to form the native amide bond. This method, however, requires introduction of the non-native L-P-T-E-G motif at the C terminus of the expressed protein part.

Several additional ligation techniques were reported for the synthesis of other lipidated proteins as Rab proteins. These techniques include Diels-Alder ligation89,

90

and Click-

ligation.91 These types of ligation were not used so far in the context of the lipidated Ras and Rheb proteins.

5. Synthesis and applications of semisynthetic Ras proteins Ras proteins belong to the class of the small GTPases that are monomeric G proteins. The Ras family is involved in many key cellular processes including cell cycle regulation, nuclear import, vesicular transport and signal transduction.92 The Ras protein family includes four isoforms: H-Ras, N-Ras, K-Ras4A and K-Ras4B.. The four isoforms share approximately 12 ACS Paragon Plus Environment

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90% sequence identity in the first 168 residues but differ in the 20 C-terminal residues.93 They are anchored to the membrane through lipid moieties that are added post-translationally. All isoforms possess a farnesyl moiety at the C-terminal cysteine. N-Ras is additionally palmitoylated at the Cys-181. H-Ras is palmitoylated at the Cys-181 and Cys-184. Ras proteins serve as molecular switches that cycle between a GDP-bound and a GTP-bound state. Intrinsic nucleotide hydrolysis on Ras is a slow process. Therefore, the life time of both states is determined by the interactions with guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs).94 In the resting state Ras is found in a GDP-bound form. GEF catalyses the dissociation of GDP and causes the exchange to GTP, thus activating Ras. In the GTP-bound form Ras can interact with various effectors. These interactions are specific to the GTP-bound state and they are responsible for transmitting the signal. The signal transmission is terminated when GTP is hydrolysed to GDP. Activated forms of the human Ras proteins are present in 20-30% of human tumours. The constantly activated form of Ras is caused by a point mutation in the sequence that causes the inability to hydrolyse GTP.95

Initially, the majority of experiments with Ras proteins, such as structure determination and biochemical characterization, were carried out with soluble proteins, lacking the C terminus. The C-terminus of the Ras proteins is important since the different Ras members vary within this region of the proteins including the different lipidation patterns. Moreover, the Cterminal hypervariable region along with the different lipidation pattern dictates the localization of the Ras proteins. Therefore, reliable method to access the full length and fully modified proteins was required.

5.1. Synthesis and applications of semisynthetic N-Ras proteins Farnesylated and palmitoylated N-Ras C-terminal peptide, equipped with the MIC tag was synthesized on Ellman sulfonamide resin using standard Fmoc strategy.60 The farnesylated cysteine was attached to the resin using TFFH and DIPEA and the peptide was elongated using HBTU/HOBt mixture as coupling reagents and DIPEA as a base in DMF. Full length lipidated peptide was activated on the resin with iodoacetonitrile and cleaved from the resin using excess of methanol as nucleophile to obtain the desired carboxymethylated peptide. However, it was shown that the conditions for the loading and the cleavage of the peptide from the resin lead to high degree of racemization and modified conditions that result in minimal racemization were developed (Scheme 7).64 Fmoc-Pro-OH was loaded first on the 13 ACS Paragon Plus Environment


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resin, followed by the elongation of the peptide and the incorporation of the palmitoylated cysteine. The resulting peptide was activated with iodoacetonitrile and released from the resin following the reaction with the nucleophilic amino group of the farnesylated cysteine to furnish the desires lipidated N-Ras peptide.

Scheme 7. Synthesis of the farnesylated and palmitoylated N-Ras peptide on the Ellman sulfonamide linker. a) DIC, methylimidazole, DMF/CH2Cl2 (1:1); b) piperidine, DMF; c) Fmoc-AA-OH, DIC, HOBt, DMF; d) ICH2CN, DIPEA, NMP; e) H2N-Cys(Far)-CO2Me, CH2Cl2/THF (1:1). Truncated N-Ras protein with a C-terminal Cys-181, suitable for MIC ligation was expressed in E. coli. and purified.55 The peptide equipped with the MIC tag was ligated to the truncated protein to furnish the full length farnesylated and palmitoylated N-Ras protein. The resulting semi-synthetic N-Ras protein was used to study the palmitoylation cycle of the H- and N-Ras proteins in cells.17, 19

In another study, the same ligation technique was used to attach N-Ras peptide carrying a photoactivatable geranyl-benzophenone analogue of the farnesyl group (Scheme 8).96,

97

It

was shown that photoactive analogues of N-Ras retain their biological activity and are suitable to be used in photolabeling experiments to study protein-protein and protein-lipid interactions in cells. 14 ACS Paragon Plus Environment

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Scheme 8. Farnesyl cysteine (A) and photoactive geranyl benzophenone analogue (B).

In a further example, the synthesis of the lipidated N-Ras protein with a fluorescent tag at the C-terminus was described (Scheme 9).59, 98 In this case the synthesis of the farnesylated NRas peptide was carried out on solid support, using the Fmoc strategy. MIC ligation was used to attach the lipidated peptide to the truncated protein. This semi-synthetic construct was used to study the intracellular localization of Ras proteins by means of live-cell imaging. Similar N-Ras construct was used to investigate the partitioning of lipidated N-Ras proteins into lipid domains of canonical model raft mixtures99, 100 and to study the conformational changes of lipidated N-Ras protein under pressure modulation.101 Moreover, the dynamics of the lipidated membrane anchor of N-Ras protein were studied using solid-state NMR spectroscopy.102-104

Scheme 9. Fluorescently labelled N-Ras protein.

5.2. Synthesis and applications of semisynthetic K-Ras proteins The K-Ras4B protein (from now on simply K-Ras) was synthesized by using a combination of EPL and organic synthesis of the farnesylated peptide.61 In the original sequence K-Ras does not contain a suitable cysteine residue at its C terminus for protein ligation. Therefore, a cysteine residue was inserted at the N-terminal end of the polybasic amino acid sequence between Gly-174 and Lys-175. Truncated K-Ras (1-174) thioester was expressed in E. coli, purified using chitin-binding affinity chromatography and released from the column through treatment with MESNA to yield the K-Ras (1-174)-MESNA thioester as described in section 4.1. The remaining dodecapeptide fragment containing an N-terminal cysteine residue for the 15 ACS Paragon Plus Environment


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ligation and eight lysine residues was synthesized on the solid-phase, using the acid-sensitive 2-chlorotrityl resin (Scheme 10). The Lys-184 residue was attached to the resin through the side-chain amino group. After selective deprotection of the allyl ester, S-farnesylated cysteine methyl ester was attached using PyBOP as coupling reagent. The peptide chain was elongated by using standard Fmoc chemistry. The allyloxycarbonyl (Alloc) side-chain protecting groups of lysine were cleaved in the presence of a Pd[0] catalyst. The peptide was cleaved from the resin and the acid-labile trityl (Trt) groups of serine and threonine were removed under mild acidic conditions (1% TFA) without affecting the farnesyl group.

Scheme 10. Synthesis of the farnesylated K-Ras peptide. a) Fmoc-Lys-OAll, pyridine, DMF/CH2Cl2; b) Pd(PPh3)4, PhSiH3, CH2Cl2; c) H-Cys(Far)-OMe, PyBOP, NMM, DMF; d) piperidine, DMF; e) Fmoc-AA-OH, HCTU, DIPEA, DMF; f) Pd(PPh3)4, piperidine, DMF; g) TFA, TES, CH2Cl2.


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Subsequently, the N-terminal Cys-174 residue of the peptide was reduced with tris(2carboxyethyl)phosphane (TCEP) and the resulting peptide was ligated with the expressed KRas (1-174)-MESNA thioester (Scheme 11). After purification by cation exchangechromatography full-length K-Ras protein was obtained in 50-70% yield. Single ligation reaction can provide multi-milligram amounts of the farnesylated K-Ras protein.

Scheme 11. Ligation of the farnesylated peptide with truncated Rheb protein. a) TCEP, ligation buffer; b) Tris–HCl buffer, pH 7.4, MESNA.

The activity of the semi-synthetic farnesylated K-Ras protein was successfully shown, by investigating its ability to induce differentiation of the rat pheochromocytoma PC12 cell line. Additionally, interactions of farnesylated K-Ras protein with prenyl-binding protein PDEδ105, 106

were examined and the affinity was determined. Finally, the distribution of the

semisynthetic K-Ras lipoproteins into different membrane subdomains was studied. The sorting and the partitioning of K-Ras4B proteins into heterogeneous anionic model membranes were studied using the semi-synthetic farnesylated K-Ras construct.107

5.3. Synthesis and applications of semisynthetic Rheb proteins Important member of the Ras superfamily is Ras homologue enriched in brain (Rheb). Rheb plays an important role in the activation of mammalian target of rapamycin complex 1 (mTORC1) signalling pathway, which is crucial for the regulation of cell proliferation, growth and metabolism.108-111 Aberrant activation of Rheb causes misregulation of the mTORC1 pathway that contributes to tumour formation.112 Like in the case of the Ras proteins, Rheb has to be farnesylated and carboxymethylated at the C-terminus to be correctly localized to endomembranes to activate the mTOR kinase.113, 114



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For the study of the interactions of Rheb with its binding partners and its temporal and spatial organization, large amounts of full-length farnesylated and active protein equipped with suitable reporter groups and tags was essential. The synthesis of farnesylated Rheb protein was successfully addressed combining the EPL technique with the organic synthesis of lipidated peptides.61 The ligation site was chosen between the residues Ala-173 and Ala-174 in the flexible C-terminal region. The Ala-174 residue was replaced with Cys-174 to allow ligation with the truncated protein. The thioester corresponding to truncated Rheb (1-173) was expressed in E. coli, purified using chitin-binding affinity chromatography and released from the column through treatment with MESNA to yield the Rheb (1-173)-MESNA thioester. The farnesylated peptide corresponding to the fragment (174-181) of Rheb was synthesised on the solid-phase, using the acid-sensitive trityl resin (Scheme 12). Ser-180 was attached to the resin through the side-chain hydroxyl group. After selective removal of the allyl ester, S-farnesylated cysteine methyl ester was attached. The peptide chain was elongated by means of standard Fmoc chemistry. The side-chains of the amino acids used in the synthesis were masked with acid-labile protecting groups, such as methyl-trityl (Mtt) for lysine and trityl (Trt) for serine. The N-terminal Cys-174 residue was protected as an S-tertbutyl disulphide that can be cleaved by reduction directly before the ligation. The lipidated peptide was cleaved from the resin and fully deprotected under mild acidic conditions (1% TFA) without affecting the farnesyl group.


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Scheme 12. Synthesis of the farnesylated Rheb peptide (174-181). a) Fmoc-Ser-OAll, pyridine, DMF/CH2Cl2; b) Pd(PPh3)4, PhSiH3, CH2Cl2; c) H-Cys(Far)-OMe, HATU, TMP, DMF/CH2Cl2 ; d) piperidine, DMF; e) Fmoc-AA-OH, HCTU, DIPEA, DMF or d) piperidine, DMF; e) Fmoc-AA-OH, PyBOP, DIPEA, DMF; f) 1) piperidine, DMF; 2) Fmoc-Cys(StBu)OH, PyBOP, collidine, DMF/CH2Cl2; g) TFA, TES, CH2Cl2. Subsequently, the N-terminal Cys-174 residue of the peptide was reduced with tris(2carboxyethyl)phosphane (TCEP) and the peptide ligated with the expressed Rheb (1-173)MESNA thioester in the presence of the detergent cetyltrimethylammonium bromide (CTAB) (Scheme

13).

After

precipitation,

refolding

and

purification

by

size-exclusion

chromatography soluble protein was obtained in 20% yield. Single ligation reaction can provide multi-milligram amounts of the lipidated Rheb protein. Later, the ligation process was improved by replacing the CTAB with mercaptophenylacetic acid as a catalyst. This replacement significantly improved the yield of the ligation by circumventing the denaturing and refolding steps.115



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Scheme 13. Ligation of the farnesylated peptide with truncated Rheb protein. a) TCEP, ligation buffer; b) Tris–HCl buffer, pH 7.4, MESNA, CTAB.

The resulting protein was successfully characterized for integrity and functionality using CD spectroscopy and a fluorescence-based guanine nucleotide dissociation inhibition (GDI) assay.116 Like in the case of K-Ras protein, interactions of lipidated Rheb protein with the prenyl-binding protein PDEδ were studied. Moreover, the semi-synthetic farnesylated Rheb protein was crystallized in complex with PDEδ.115 Structural information obtained from the crystal structure led to the development of the small molecule inhibitors that block the interactions between PDEδ and KRas protein.117 The small molecule inhibitors were shown in vivo to interfere with the correct localization of the KRas protein and thus reduce the KRas oncogenic signalling.

6. Synthesis and applications of semisynthetic LC3 protein Light chain 3 (LC3) family of proteins is a mammalian orthologue of an autophagy-related 8 (Atg8) yeast protein. Atg8/LC3 proteins are required for the formation of autophagosomes in autophagy process in cells.118 LC3 proteins, as well as the yeast orthologue, have to be Cterminally conjugated to phosphatidylethanolamine (PE) for the correct localization and function.

The semisynthetic lipidated LC3 protein was obtained via chemical synthesis of the lipidated C-terminal peptide and the subsequent ligation to the truncated protein.119 The lipidated peptide was synthesized on the chlorotrityl resin using standard Fmoc strategy (Scheme 14). The cysteine was protected as an S-tert-butyl disulphide that can be cleaved prior to the ligation. The peptide was cleaved under mild acidic conditions from the resin and the PE group was attached. The truncated LC3 protein (1-114) was expressed in E. coli. In order to 20 ACS Paragon Plus Environment

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produce a soluble protein, N-terminal maltose-binding protein (MBP) was fused. The resulting protein was purified and treated with MESNA to yield MBP-LC3 thioester ready for the ligation with the lipidated peptide. The ligation in the presence of the TCEP was performed followed by the cleavage of the MBP domain with TEV protease to yield the fulllength lipidated LC3 protein.

Scheme 14. Synthesis of the lipidated LC3 peptide. a) DIPEA, CH2Cl2; b) piperidine, DMF, c) Fmoc-AA-OH, HCTU, DIPEA, DMF; d) 1% TFA, 3% TES, CH2Cl2; e) PFP-TFA, TEA, CH2Cl2; f) PE, TEA, CHCl3/CH3OH, g) 30% TFA, CH2Cl2. It was shown that the resulting LC3-PE construct can undergo proteolytic cleavage by Atg4B similarly to the native LC3 protein. The semisynthetic LC3-PE protein was used to study the induction of membrane tethering and fusion by using an in vitro liposome assay.

In another approach, the solubility problem during the ligation step was addressed by an introduction of a removable solubilizing side chain on the PE-modified lipopeptide.120 Photocleavable short arginine-rich peptide was introduced on the side chain of the glutamine. The peptide was synthesized on a chlorotrityl resin using standard Fmoc strategy and the modified glutamine residue was introduced during the synthesis (Scheme 15). The peptide was cleaved from the resin, modified with PE, fully deprotected and purified. The lipidated peptide was ligated to the truncated LC3 protein (1-114) equipped with MESNA thioester under detergent 21 ACS Paragon Plus Environment


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free conditions. The solubilizing tag was cleaved by UV irradiation to yield the full length lipidated LC3-PE construct that was used to study the co-localization of the protein with liposomes. O FmocHN

Cl

O

a FmocHN

OH

O

b-d

Pbf

Pbf

Pbf

Pbf

Arg

Arg

Arg

Arg

O Boc

Gly

HN

NO2

N H

NH Trt

Boc

Trt

tBu

Cys

Gln

Glu

Thr

Phe

Gly

OH

e, f

O Gly

Arg

Arg

Arg

Arg

HN

N H

Cys

NO2 NH

Gln

Glu

Thr

Phe

Gly

O

N H

O

O P

O

O

O O O

g, h

Cys

Gln

Glu

Thr

Phe

Gly

N H

O O

O

O P

O

O O O

Scheme 15. Synthesis of the lipidated LC3 peptide with solubilizing tag. a) DIPEA, CH2Cl2; b) piperidine, DMF, c) Fmoc-AA-OH, HBTU, HOBt DIPEA, DMF; d) TFE/CH2Cl2 (1:4); e) DE, DIC, HOAt, CHCl3; f) TFA/PhOH/H2O/TIPS (88:5:5:2); g) guanidinium chloride, NaH2PO4, 4-mercaptophenylacetic acid; h) hv = 365 nm.

7. Summary Lipidated proteins play key role in various cellular processes in eukaryotic cells, including cell growth, proliferation, metabolism, trafficking, signal transduction and immune response. 22 ACS Paragon Plus Environment

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To study the involvement of lipoproteins in these processes, an access to fully modified protein in large quantities is necessary. Such quantities usually are not readily accessible using existing prokaryotic or insect cell expression systems. The solution to this problem came in a combination of protein expression, organic synthesis and bioconjugation techniques. Recent advances in peptide chemistry and selective ligation methods allow easy access to lipidated peptides that can be subsequently ligated to expressed truncated protein. Such protein semi-synthesis can yield multi-milligram quantities of the desired lipoproteins. The resulting lipoproteins can be applied to study protein-protein interactions, proteinmembrane interactions, intracellular localization, and function of lipidated proteins in vitro and in cells to achieve a better understanding of eukaryotic cellular processes.

Acknowledgments T.M. acknowledges the Alexander von Humboldt Foundation for postdoctoral fellowship.

Abbreviations Abl - Abelson murine leukemia viral oncogene homolog ADP – adenosine diphosphate Alloc - allyloxycarbonyl Arf - ADP-ribosylation factors ATP – adenosine triphosphate Boc - tert-butoxycarbonyl CBD - chitin-binding domain CD – circular dichroism CTAB - cetyltrimethylammonium bromide DIC - N,N’diisopropylcarbodiimide DIPEA – diisopropylethylamine DMAP – 4-dimethylaminopyridine E. coli – Escherichia coli EPL – expressed protein ligation Fmoc - 9-fluorenylmethoxycarbonyl FTase - farnesyl transferase GAP - GTPase-activating proteins GDI - guanine nucleotide dissociation inhibition GDP – guanosine diphosphate 23 ACS Paragon Plus Environment


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GEF - guanine nucleotide exchange factors GGTase - geranylgeranyl transferase GPI - glycosylphosphatidylinositol GTP – guanosine triphosphate HBTU - 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaﬂuorophosphate HOBt - hydroxybenzotriazole Icmt - isoprenylcysteine carboxymethyltransferase LC3 - light chain 3 MBP – maltose-binding protein MESNA - sodium 2- mercaptoethanesulfonate MIC – maleimidocaproyl mTORC1 - mammalian target of rapamycin complex 1 Mtt – methyl trityl NCL – native chemical ligation NMT - N-myristoyltransferase PDE – phosphodiesterase PE - phosphatidylethanolamine PFP-TFA - pentafluorophenyl trifluoroacetate PTM - post-translational modifications PyBOP – benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Rab - Ras-related in brain Ras - rat sarcoma RceI - Ras converting enzyme I REP - Rab escort protein Rheb - Ras homologue enriched in brain Src - sarcoma proto-oncogene tyrosine-protein kinase TCEP - tris(2-carboxyethyl)phosphane TEA – triethylamine TEV – Tobacco etch virus TFA – trifluoroacetic acid TMP - 2,2,6,6-tetramethylpiperidine Trt – trityl

References 24 ACS Paragon Plus Environment

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Synthesis of Lipidated Proteins - Bioconjugate Chemistry (ACS

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