Acyltransferases for secreted signalling proteins (Review)

Shu-Chun Chang and Anthony I. Magee

Address:

Section of Molecular Medicine

National Heart & Lung Institute

Sir Alexander Fleming Building

South Kensington

London SW7 2AZ

UK

Corresponding author: Anthony I. Magee

E-mail: t.magee@imperial.ac.uk

Telephone: +44 20 7594 3135

Keywords

MBOAT

Hedgehog

Wingless/Wnt

Ghrelin

Palmitoylation

Acylation

Protein acyl transferase

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Abstract

Members of the MBOAT family of multispanning transmembrane enzymes catalyse the acylation of important secreted signalling proteins of the Hedgehog, Wg/Wnt and ghrelin families. Acylation of these substrates occurs during transport through the secretory pathway and plays key roles in their biological activity and spread from producing cells, contributing to the formation of appropriate extracellular concentration gradients. Characterisation of these enzymes could lead to their identification as therapeutic targets for diverse human diseases such as cancers, obesity and diabetes.

Introduction

Post-translational modifications are known in several secreted signallingmolecules, i.e. the Hedgehog family that is conserved in vertebrates and invertebrates, the Wg/Wnt protein family, as well as the Epidermal Growth Factor Receptor (EGFR) ligand Spitz [1]. Palmitoylation is the attachment of the16-carbon saturated fatty acid palmitate from its coenzyme A ester (PalCoA) as a lipid donor, usually as a thioester to cysteine (S-acylation or thioacylation) residues of proteins (but sometimes as an oxyester to serine). Unlike myristoylation and farnesylation, palmitoylation provides modified cytoplasmic proteins accurate trafficking from the secretory pathway to the plasma membrane [2] and controls their targeting to membranes or membrane subdomains, affects protein–protein interactions, or influences the stability of proteins [3]. In addition, studies demonstrate that palmitoylation can facilitate the efficiency and specificity of signalling through not only correctly guiding a signalling molecule to its target within the cell but also membrane-anchoring at specific cell surface microdomains/lipid rafts [4, 5]. The more general term protein “acylation” can be used as fatty acids other than palmitate can also be used. It is becoming clear that acylation of secreted signalling proteins is carried out by members of the membrane-bound O-acyltransferases (MBOAT) family.

Membrane-bound O-acyltransferase (MBOAT) family

Members of the MBOAT family are multispanning transmembrane enzymes that usually catalyse the addition of a fatty acid to a hydroxyl group, typically of membrane-embedded substrates such as lipids [6]. They contain a characteristic histidine residue in one of the transmembrane domains that is conserved in almost all members of the family, one exception being mouse Gup1 which has a leucine in the equivalent position [7]. This histidine is thought to be involved in the acyltransferase activity of MBOAT proteins, so its absence in Gup1 calls into question whether this protein is an acyltransferase or rather has another activity that does not require this histidine. Gup1 is highly homologous to Hhat, with very similar gene organisation, membrane topology and intracellular localisation although the expression patterns differ somewhat between cell lines . It is interesting that exogenous overexpressed

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Gup1 interferes with the palmitoylation of Shh by endogenous Hhat (as judged by an indirect assay based on antibody recognition of palmitoylated Shh) suggesting that Gup1 may be a negative regulator of Shh palmitoylation [7]. The evidence available so far suggests that Gup1 can interact directly with both Shh and Hhat, and that it may reduce Shh palmitoylation by competiton with Hhat, although competition for available PalCoA is another possible mechanism. Whether these opposing roles of Gup1 and Hhat operate under physiological conditions and how they are regulated remains to be seen.

MBOAT proteins contain between 8-12 transmembrane domains based on structure prediction programmes, so the localisation of the C-terminus to the cytoplasmic or extracytoplasmic side of cellular membranes is currently a matter of conjecture (Figure 1).

(Insert Figure 1 near here)

Hedgehog proteins

Hedgehog proteins (Hh), acting as morphogens, were first discovered in the1980s encoded by a gene family originally discovered through the Drosophilasegmental pattern mutation hedgehog. In mammals, all three Hh homologues (Sonic (Shh), Indian (Ihh) and Desert (Dhh) Hedgehog) display a variety ofroles in embryonic development, adult homeostasis, and cancer [8]. Although the term “Hh” strictly only applies to Drosophila we use the term “Hhs” here for simplicity to also encompass vertebrate hedgehog proteins, unless the distinction is crucial. The Hh signalling pathway is one of the most critical signalling pathways in both vertebrates and invertebrates [8, 9]. Perturbations to this pathway manifest themselves in disease; for instance, over-activity of the pathway can lead to oncogenesis and lower activity of the pathway can result in developmental malformations [10, 11]. During differentiation and tumorigenesis, diverse targets of Hh signalling are involved in cell adhesion, signal transduction, cell cycle, apoptosis and angiogenesis [12]. In addition, it has been estimated that 25% of all human tumours require Hh signalling to maintain tumour cell viability, so potent Hh pathway inhibitors have therapeutic potential for diverse human tumours.

The most atypical feature of Hh proteins is their post-translationalmodifications (Figure 2), including the unique N-terminal palmitoylation andC-terminal cholesterol attachment. Hhs are the best established examples of cholesteroylated proteins in nature. In Drosophila the ~45kDa Hh precursor is translocated, presumably by the conventional signal recognition particle-mediated mechanism, into the endoplasmic reticulum (ER) and has its signal sequence removed co-translationally. It appears that Hhs are then palmitoylated on their highly conserved N-terminal Cys residue in the ER or Golgi complex [13]. A ~19kDa N-terminal fragment (Hh-N) and a ~25kDa C-terminal fragment (Hh-C) are subsequently yielded by autocatalytic cleavage catalysed by Hh-C [14]. Concurrently, a cholesterol molecule is covalently

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attached to the C-terminus of Hh-N, thus forming the mature form of Hh, Hh-Np [15]. Unlike Hh-C, Hh-N contains all the signalling functions. Processing of mammalian Hh proteins is probably highly analogous.

(Insert Figure 2 near here)

There is evidence that the role of N-terminal acylation of Hh-N is to enhance the affinity of Hh to biological membranes and to regulate the distribution of the Hh signal [15, 16]. Hence, these lipid modifications are significant for Hh intracellular trafficking and to its extracellular concentration regulation. In addition, according to mammalian studies, cholesterol covalently attached to Hh might improve target biological activity by facilitating the interaction between Hh and its receptor Patched (Ptc) [16].

In Hh-receiving cells, Hh signaling is regulated by two proteins - Ptcand Smoothened (Smo). Ptc, a 12-transmembrane protein, is thereceptor for Hh through its 2 large extracellular loops. Smo, a 7-transmembrane protein, is a positive transducer of Hh signaling, and it is believed that Ptc directly inhibits its biological activity [17]. The mechanism of Ptc inhibition of Smo activity is not entirely clear; one model for the lack of signalling in the absence of Hh is that Smo is impeded from signaling by Ptc. In contrast, in the presence of active Hh, Hh binds to Ptc and this releases Smo to activate downstream signalling. Interestingly, the organisation of the transmembrane domains of Ptc is similar to several cholesterol-binding proteins. This suggests that Ptc is not only a cholesterol-binding protein, but also a potential key to restrain unmodified Hh from interfering with signalling [18, 19].

Palmitoylation of Hh

Hhs are unusual in being dually lipid-modified to be fully active [20]. Moreover, it has been shown that dual lipidation is critical not only for the interactions between Hh and Ptc but also for forming a suitable complex of Hh with heparan sulphate proteoglycans (HSPGs) to target at the Hh-receiving cell [4]. It is now appreciated that Ptc might be located in lipid rafts/microdomains which provide platforms for signal transduction and intracellular sorting [21]. Hence, the interactions between particular HSPGs, Hh and Ptc are significant to Hh spreading through the epithelium surface, as well as Hh signal transduction.

During post-translational modification of Hh, N-palmitoylation occurs in the amino-terminal signalling domain of both Drosophila Hh and human Shh via amide linkage. This N-terminal palmitate is added to a highly conserved cysteine in a CGPGP motif exposed by signal peptide cleavage. It has been suggested that S-acylation of the cysteine sulphydryl could initially occur followed by a rapid and efficient intramolecular S- to N-acyl shift [22] and this is still a plausible mechanism of the N-terminal acylation. N-terminal

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palmitoylation of Hhs is facilitated by the product (Hhat) of the hedgehog acyltransferase gene (also known as skinny hedgehog, sightless, central missing or rasp) [20, 23, 24]. This multi-spanning transmembrane acyltransferase is directly and specifically required for the N-terminal addition of palmitate to Hhs. Hhat has recently been definitively shown by Buglino and Resh to be a specific acyltransferase for Shh using an in vitro assay with purified components [13]. The reaction is clearly enzymatic and requires a free N-terminal cysteine and PalCoA as cosubstrate, although the concentration of PalCoA used is much higher than that found in cells. This could be explained by the presence of acylCoA binding protein (ACBP) in cells which may present PalCoA to Hhat [25]. The authors favour the interpretation that Hhat is an N-palmitoyltransferase but their data are equally consistent with the S-acylation followed by acyl shift mechanism mentioned above, which would explain why an N-terminal cysteine is required and cannot be substituted with a residue that lacks a sulphydryl group. Buglino and Resh made the important observation that a peptide consisting of the N-terminal 11 amino acids of Shh is an effective substrate for Hhat, which could form the basis for a high throughput assay that could be used in the screening of Hhat inhibitors. Blocking Hhat enzymatic activity would prevent formation of active palmitoylated Hhs and down-regulate the Hh pathway in tumour cells which depend on active Hhs for their proliferation [26]. These authors also confirmed the observation made previously by others that Hhat is localised in intracellular membranes of the secretory pathway, ER and Golgi. In cells, Pal-CoA is not free in the cytoplasm but is bound to ACBP and therefore may require a transporter that facilitates its entry into the lumen of the secretory pathway where palmitoylation of Hhs presumably occurs [27].

Hhat shares homology with Porcupine (Porc) in Drosophila and its C. elegans homologue Mom-1, two putative acyltransferases that are also part of the MBOAT protein family and are responsible for the palmitoylation of Wg, a morphogen involved in embryonic patterning in Drosophila, and its human homologues Wnts (see below). This homology includes the conserved histidine residue that may be involved in the active site of the putative acyltransferase.

Experiments using Drosophila Hh variants and cultured mammalian cells showed that palmitoylation of Hh is essential for effective production of the Hh signal and pattering in both imaginal discs and in embryos. Also it is suggested that neither solely cholesterol modification nor N-terminal acylation of Hhs are adequate for their stable membrane localization [3, 28]. Recently it has been demonstrated that dual lipid modification is critical to the interactions between Hh, HSPGs and Ptc receptor [29]. These results support the conclusion that Hh lipidation might enable Hh to form this complex to ensure targeting to the receiving cell for efficient signalling, combined with the fact that Ptc receptor might be located in lipid rafts/microdomains, which provide platforms for signal transduction and intracellular sorting. On the contrary, lipid-unmodified Hh would be delivered free into the extracellular space instead of remaining in the extracellular matrix. This type of transmission can promote the activation of low-threshold target genes far from Hh-producing cells [29, 30].

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Compared to fully modified Hh, a cholesterol-deficient form of Hh (HhN) has less potency to activate the Hh cascade. Moreover, HhC85S, a Drosophila variant that lacks palmitate due to mutation of the acylation site, is much less potent than HhN [29] indicating that the palmitoyl adduct may play a more essential role in Hh signalling than cholesteroylation. In this study, it was also suggested that acylation plays a major role in guiding modified Hh proteins to specific membrane domains. Consistent with this observation, knockout mice deficient in Hhat are neonatal lethals that show defects in the developing neural tube and limbs similar to a loss of palmitoylated Shh [31]. In the same study, overexpression of an unacylated Shh mutant (ShhC25S) in transgenic mice exhibited reduced Shh protein activity in inducing Shh responses and Shh protein lacking both types of lipid modification (ShhNC25S) contained poorer levels of residual activity.

Hh/Shh multimeric complex formation and Heparan Sulphate Proteoglycans (HSPGs) in Hedgehog Signaling

HSPGs including secreted forms and cell-associated forms play key roles in Hh signalling and transport. Structurally, HSPGs consist of a core protein classified into three distinct classes - the Syndecans with a single transmembrane domain, the Glypicans with a GPI-anchor and the Perlecans, a varied group of secreted proteoglycans - with one or more HS chains. Functionally, HSPGs not only mediate significant interactions between cells and their environment but also regulate the distribution of extracellular signalling molecules such as morphogens through binding to them [32 –35]. This great potential is based on HSPGs’ enormous structural differences - partly via the additional modifications in HS chains through the repeating disaccharide chain elongation.

(Insert Figure 3 near here)

To date, Hh signal molecules are known to act as major mediators in manydevelopmental processes and require HSPGs for their proper distribution and signalling activity [36]. Nevertheless, the mechanisms of this dependence in man are still unclear. In the case of Hh/Shh long-range signalling in Drosophila and mice, the activity is enhanced through forming a multimeric complex to increase Hh/Shh solubility, which is one critical criterion for protein stability [37]. The Hh/Shh multimeric complex is the major active form in activating Hh/Shh signalling [31]. In addition, it has been suggested that these multimers could form extracellular aggregates, called large punctuated structures, in the embryo [38, 39]. Both lipid modifications are necessary for Hh/Shh to incorporate into this complex [31], it is more signalling efficient than the monomer, and requires both the HSPG core proteins and their attached HS GAG [37, 40, 41]. Based on previous studies, there are several conjectural mechanisms for how HSPGs promote this signalling. On the one hand, both Shh and Hh are secreted from cells as both monomeric and

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multimeric forms [31, 42, 43]. This soluble Shh multimeric complex with specific HSPGs - Perlecan and Glypican - is freely diffusive and can regulate Shh signalling [44, 45]. On the other hand, the interaction between HSPGs and growth factors could influence both their extracellular distribution and their ability to signal, [46] e.g. Perlecan by directly binding to Shh as a co-receptor can affect Shh signalling [44, 47]. More recently, the finding that only lipid-modified Hh could form into a polymeric complex [4] to enhance its solubility for long-range transportation might be linked to Hh by Shifted, a secreted Wnt Inhibitory Factor homologue, indicating that lipid modifications of Hh are not only essential for Hh/HSPGs interaction [48] but also critical for proper Ptc receptor anchoring. In contrast, lipid-unmodified Hh is poorly retained and stabilised by the ECM and tends to diffuse freely [48]. Furthermore, HSPGs might participate in promoting association of Hhs with cell surface microdomains and/or lipid rafts in which the crucial molecules are assembled into functional complexes [49, 50].

As well as Hh proteins, Hhat is also responsible for the N-terminal acylation of the Drosophila EGFR ligand Spitz at a highly homologous cysteine residue [1]. This modification has little effect on Spitz EGFR signalling activity in vitro but reduces its secretion and enhances its plasma membrane association. However, in vivo Spitz activity is enhanced and its diffusion is restricted by palmitoylation, suggesting that acylation is important for allowing the local concentration of Spitz near the producing cells to reach the threshold needed for activation of its targets.

Wg/Wnt acylation by Porcupine

Proteins of the Wg/Wnt family are, like Hhs, secreted signalling molecules with widespread effects in animal development and tumourigenesis. Almost all members of the family appear to be dually acylated in the lumen of the secretory pathway [51, 52]. The most N-terminal cysteine residue after the signal sequence (e.g. Cys77 in murine Wnt3a and Cys93 in Drosophila Wg) is usually S-acylated with a long chain fatty acid which has been identified as palmitate (C16:0) in some cases [53]. Recently, a second site of acylation has been identified as a serine some distance downstream (Ser209 in murine Wnt3a) [54] which is O-esterified with the monounsaturated fatty acid palmitoleic acid (C16:1). Interestingly, acylation of Ser209 is required for secretion of Wnt3a and possibly for Cys77 acylation, but the converse is not true, i.e. Ser209 acylation is not dependent on acylation of Cys77. Acylation is not an universal modification in the Wg/Wnt family, however. Drosophila WntD has very recently been shown not to be acylated [55], in contrast to an earlier report form the same laboratory [53]. Although WntD contains the conserved N-terminal Cys residue it does not contain an equivalent residue to Ser209, again suggesting that cysteine acylation is dependent on prior serine acylation. WntD is secreted efficiently, albeit in an apparently different manner to other Wg/Wnts, so in this case acylation is not required for secretion. The presence of this Wnt serine O-acylation raises the question of whether other secreted acylated signalling proteins are similarly modified - however, the Shh sequence at least does not contain an obvious homologous serine.

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Both acylations of Wg/Wnts are dependent on the product of the porcupine gene in fly or mammals (mom-1 in C. elegans), which encodes an ER-localised member of the MBOAT family [56-58]. This appears highly unusual because if Porcupine (Porc) catalyses both acylations it would need to recognise both different amino acid acceptor residues (Cys and Ser) and different acylCoAs (palmitoyl and palmitoyleoyl) for the two acylation sites, as well as catalyse thioester and oxyester formation – a tall order for a single enzyme. One possible rationalisation is that initial acylation at Ser209 may be with palmitate and this could subsequently be converted to palmitoleate by a desaturase [59], but there is as yet no evidence for this. It has not been definitively proven that Porcupine is the enzyme responsible for either of these acylations. Since cysteine acylation is dependent on previous serine acylation [54, 55] it is possible that Porc is responsible for the serine acylation but that the cysteine acylation is subsequently performed by a different enzyme. In that case, serine acylation would be permissive for cysteine acylation. It is important to characterise the biochemical function of Porc, as multiple Wnts play key roles in tumour formation and maintenance and human Porc (encoded by the X-linked gene PORCN) itself has been found to be mutated in developmental disorders such as Focal Dermal Hypoplasia [60, 61]. Hence Porc is a potential therapeutic target in several human diseases.

The function of Wg/Wnt acylation, like for Hh, is related to interactions with receptor, membranes and lipoprotein particles. Firstly, palmitoylation of Wnt5a is required for binding to its receptor Fz5 and activation of intracellular signalling [62]. Also, for Drosophila Wg, Cys93 acylation is essential for signalling activity and transport to the cell surface whereas Ser293 acylation seems to be less important for secretion but is still required for maximal signalling activity [52]. The authors’ interpretation is that the overall level of acylation is important for signalling, suggesting that membrane association is crucial. However, simply tethering Wg to the cell surface with a transmembrane domain does not rescue activity, so acylation confers something unique to the function of Wg, perhaps being involved directly in receptor binding, interaction with the transport protein Evi/Wls/Sprinter or with membrane microdonmains (see below). Dual acylation of proteins is usually required to provide stable membrane binding [3] and in the case of most Wg/Wnt proteins it seems to mediate interaction with the multimeric complexes that are the vehicles for transport of Wg/Wnts between cells (reviewed by Bartscherer and Boutros [63]). In order to release Wg/Wnts from producing cells the involvement of Evenness interrupted (Evi)/Wntless (Wls)/Sprinter (Srt) proteins is required - multispan membrane proteins that somehow promote Wg/Wnt release, possibly by mediating assembly into complexes with lipoproteins, lipids and HSPGs, analogously to Hhs and Disp. Once released, these packages promote transport of Wg/Wnts between cells but restrict their spread, thus contributing to the shape of the extracellular Wg/Wnt gradients that specify the effects of these signalling molecules on target cells, maintaining a high local concentration needed for activation of high-threshold target genes while also allowing transport to cells several microns away [58]. WntD, which is not acylated, does not require the action of Evi/Wls/Sprinter for release and is secreted at higher rates than other Wg/Wnt

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proteins which is compatible with its more systemic role in the fly innate immune response to infection [55]. Finally, the dual acylation of Wg/Wnt proteins may also facilitate their interaction with membrane lipid microdomains (MLMs, also called lipid rafts) that could be the site of assembly of the lipoprotein transport packages [64], hence the dependence of Wg long-range secretion on the MLM resident protein reggie-1/flotillin-2 [65].

Ghrelin acylation by GOAT Another MBOAT family member – ghrelin O-acyltransferase, GOAT - has very recently been implicated in metabolic activation of the 28-residue peptide hormone ghrelin by specifically acylating ghrelin on its critical serine-3 residue with medium chain fatty acids (FAs) [66, 67]. In vivo 20-30% of circulating ghrelin is acylated. This so far unique modification initially occurs on the 94-residue proghrelin precursor usually with octanoic acid, although decanoic, undecanoic and decenoic acids may also be used physiologically [68]. GOAT contains the Asn and His putative catalytic residues typical of the MBOAT family and these were shown to be required for activity. A GOAT knockout mouse fails to produce acylated ghrelin [66] and will undoubtedly be a useful resource for studying the physiological effects of acylated and unacylated ghrelin. These findings are highly topical because ghrelin causes growth hormone release and is orexigenic, i.e. it boosts appetite, so inhibitors of ghrelin acylation – which would be highly selective due to exquisite substrate specificity of GOAT - could be used to control appetite. Unacylated ghrelin, originally thought to be inactive, is now known to modulate the growth of some cell types and also have effects on appetite, although this is controversial [69].

The substrate specificity of GOAT is under intense study, with a view to designing specific inhibitors. Intriguingly, ghrelin with Ser replaced by Thr at position 3 (as found in bullfrog ghrelin) is still acylated by GOAT. Using a novel in vitro assay for proghrelin acylation by GOAT, Yang and colleagues [70] have identified key residues in the N-terminal five amino acids of ghrelin that determine GOAT activity, providing promising evidence that stable peptidomimetic agents can be designed to inhibit GOAT. An analogue acylated in amide linkage at position 3, [Dap3]octanoyl-ghrelin(1-5)-amide, is particularly effective as an end-product inhibitor. It would be interesting to find out if the acylated serine 3 could be replaced by a large hydrophobic amino acid residue such as leucine or isoleucine. The predominant localisation of GOAT and acylated ghrelin production to the stomach also makes it likely that orally administered agents may be effective if appropriately packaged and stabilised. In addition, these studies may provide information that is applicable to the design of inhibitors for Porc and Hhat, for example taking advantage of the highly conserved N-terminal acylation site CysGlyProGlyArgGlyPhe of mammalian Hedgehog proteins.

The source of medium chain FAs for ghrelin acylation is of interest, as mammalian cells are not generally thought to synthesise them, although during fasting white adipose tissue might release FAs that could be used by GOAT [71]. GOAT has the ability to transfer a wide range of short and

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medium chain FAs onto ghrelin in vitro [66]. FAs for ghrelin acylation could come from the diet [72], which might explain the very restricted expression pattern of GOAT and ghrelin to the stomach and pancreas. If ghrelin acylation is controlled, at least partially, by the availability of medium chain FAs it could act as a “sensor” of these in the diet and thus modify the animal’s appetite accordingly. The reason for the physiological choice of medium chain FAs for ghrelin acylation is a matter of speculation, but it could be related to such a sensing function. Modification of dietary medium chain FAs can modulate ghrelin acylation and activity in vivo [72], so reduction in these could suppress appetite thus having applications in obesity and type II diabetes, whereas supplementation could promote appetite and have applications in eating disorders such as anorexia, although the substantial psychological component of these disorders cannot be underestimated.

As for Hhat and Porc, GOAT presumably requires as its cosubstrate a fatty acyl coenzyme A (FACoA). This creates a topological problem as FACoAs are predominantly localised in the cytoplasm and are bound to a high affinity acyl CoA binding protein (ACBP), possibly to prevent these highly reactive thioesters from reacting non-specifically with cell components such as proteins [25]. Thus, to react with the luminal substrates (Hhs, Wg/Wnts and proghrelin) the FACoA would need to be transported across the ER membrane. The multispanning topology of Hhat, Porc and GOAT could facilitate this transport in as yet unknown ways, as suggested recently for GOAT [67] and as early as 1996 for Porc [56], and if so this could provide another therapeutic target activity.

Future perspectives

Focusing on studies of the mechanism by which MBOAT proteins add fatty acids to secreted signalling proteins and the effect of acylation on activity should shed light on the mechanisms of these intriguing enzymes. It will also answer questions about the functional effects of post-translational modifications of Hh, Wg/Wnt and ghrelin proteins/peptides, including their intracellular trafficking and signalling activity in vivo. Also, since the Shh and Wg/Wnt signalling pathways are involved in oncogenesis, future studies might provide new molecules that could be focused on as therapeutic targets for human tumour treatment. Similarly, targeting GOAT could provide pharmacological agents with applicability in obesity and type II diabetes. It will be interesting to find whether other secreted signalling molecules are also acylated and the mechanistic information currently being obtained, e.g. concensus sequences for MBOAT-catalysed acylation, will aid that search.

Acknowledgements

Work in the Magee laboratory is supported by the UK Medical Research Council and the Biotechnology and Biological Sciences Research Council.

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References:

1. Miura GI et al.: Palmitoylation of the EGFR ligand Spitz by Rasp increases Spitz activity by restricting its diffusion. Dev Cell. 2006 Feb;10(2):167-76.2. Smotrys, J. E. and Linder, M. E.: Palmitoylation of intracellular signalling proteins: regulation and function. Annu. Rev. of Biochem. 2004 73: 559-587. 3. Resh MD.: Trafficking and signaling by fatty-acylated and prenylated proteins. Nat Chem Biol. 2006 Nov;2(11):584-90.4. Gorfinkiel N, Sierra J, Callejo A, Ibañez C, Guerrero I.: The Drosophila ortholog of the human Wnt inhibitor factor Shifted controls the diffusion of lipid-modified Hedgehog. Dev Cell. 2005 Feb;8(2):241-53.

5. Zhai L et al.: Drosophila wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine. J Biol Chem. 2004 Aug 6; 279(32): 33220-7.

6. Hofmann, K.: A superfamily of membrane-bound O-acyltransferase with implications for Wnt signalling. Trends in Biochemical Science. 2000; 25: 111-112.

7. Abe Y et al.: Mammalian Gup1, a homolog of Saccharomyces cerevisiae glycerol uptake/transporter 1, acts as a negative regulator for N-terminal palmitoylation of Sonic hedgehog. FEBS J. 2008 Jan;275(2):318-31.8. Nybakken K, Perrimon N.: Hedgehog signal transduction: recent findings. Curr Opin Genet Dev. 2002 Oct;12(5):503-11. Review.9. Martí E, Bovolenta P.: Sonic hedgehog in CNS development: one signal, multiple outputs. Trends Neurosci. 2002 Feb;25(2):89-96. Review.10. Cooper, M. K., Porter, J. A., Young, K. E. and Beach, P. A.: Teratogen-mediated inhibition of target tissue response to Shh Signalling. 1998 Science. 280: 1603-1607. 11. Hahn H et al.: A mammalian patched homolog is expressed in target tissues of sonic hedgehog and maps to a region associated with developmental abnormalities. J Biol Chem. 1996 May 24;271(21):12125-8.12. Lauth M, Toftgard R: The Hedgehog pathway as a drug target in cancer therapy. Curr Poin Investig Drugs. 2007 Jun;8(6): 457-61.13. Buglino JA, Resh MD.: Hhat is a palmitoylacyltransferase with specificity for N-palmitoylation of Sonic Hedgehog. J Biol Chem. 2008 Aug 8;283(32):22076-88.14. Hall TM et al.: Crystal structure of a Hedgehog autoprocessing domain: homology between Hedgehog and self-splicing proteins. Cell. 1997 Oct 3; 91(1): 85-97.15. Randall K. Mann and Philip A. Beachy: Novel lipid modifications of secreted protein signals. Annu. Rev. Biochem. 2004 73: 891– 923

16. Lewis PM et al.: Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptc1. Cell. 2001 Jun 1;105(5): 599-612.

17. Ingham, P. Hedgehog signalling. Curr. Biol. 2007 18: R238-R24118. Incardona JP, Roelink H.: The role of cholesterol in Shh signaling and teratogen-induced holoprosencephaly. Cell Mol Life Sci. 2000 Nov;57(12):1709-19.

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19. Alcedo J, Noll M.: Hedgehog and its patched-smoothened receptor complex: a novel signalling mechanism at the cell surface. Biol Chem. 1997 Jul;378(7):583-90.20. Ingham PW.: Hedgehog signaling: a tale of two lipids. Science. 2001 Nov; 294(5548):1879-81.21. Rietveld A et al.: Association of sterol- and glycosylphosphatidylinositol-linked proteins with Drosophila raft lipid microdomains. J Biol Chem. 1999 Apr 23; 274(17): 12049-54.

22. Pepinsky, R. B. et al.: Identification of a palmitic acid-modified form of human Sonic hedgehog. Journal of Biological Chemistry. 1998; 273: 14037-14045.

23. Chamoun, Z. et al. Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the Hedgehog signal. Science 2001 293: 2080-2084.24. Micchelli, C.A. et al. rasp, a putative transmembrane acyltransferase, is required for Hedgehog signalling. Development 2002 129: 843-851.25. Hansen JS, Faergeman NJ, Kragelund BB, Knudsen J. Acyl-CoA-binding protein (ACBP) localizes to the endoplasmic reticulum and Golgi in a ligand-dependent manner in mammalian cells. Biochem J. 2008 Mar 15;410(3):463-72.26. Pasca di Magliano M, Hebrok M.: Hedgehog signalling in cancer formation and maintenance. Nat Rev Cancer. 2003 Dec;3(12):903-11. Review.27. Kümmel D, Heinemann U, Veit M.: Unique self-palmitoylation activity of the transport protein particle component Bet3: a mechanism required for protein stability. Proc Natl Acad Sci U S A. 2006 Aug 22;103(34):12701-6.

28. Gallet et al.: Cholesterol modification is necessary for controlled planar long-range activity of Hedgehog in Drosophila epithelia. Development. 2006 Feb;133(3):407-18.29. Callejo A et al.: Hedgehog lipid modifications are required for Hedgehog stabilization in the extracellular matrix. Development. 2006 Feb;133(3):471-83.

30. Gibson MC et al.: Lumenal transmission of decapentaplegic in Drosophila imaginal discs. Dev Cell. 2002 Sep;3(3):451-60.31. Chen MH, Li YJ, Kawakami T, Xu SM, Chuang PT.: Palmitoylation is required for the production of a soluble multimeric Hedgehog protein complex and long-range signaling in vertebrates. Genes Dev. 2004 Mar 15;18(6):641-59.32. Belting M.: Heparan sulfate proteoglycan as a plasma membrane carrier. Trends Biochem Sci. 2003 Mar;28(3):145-51. Review.

33. Esko JD, Selleck SB.: Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu Rev Biochem. 2002;71:435-71.

34. Nybakken K, Perrimon N.: Heparan sulfate proteoglycan modulation of developmental signaling in Drosophila. Biochim Biophys Acta. 2002 Dec 19;1573(3):280-91. Review.

35. Tsuda M et al.: The cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila. Nature. 1999 Jul 15;400(6741):276-80.

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36. Lin X and Perrimon N: Developmental roles of heparan sulfate proteoglycans in Drosophila. Glycoconj J. 2002 May-Jun;19(4-5):363-8. Review.

37. Carrasco H et al.: Heparan sulfate proteoglycans exert positive and negative effects in Shh activity. J Cell Biochem. 2005 Nov 1; 96(4): 831-8.

38. Gallet A et al.: Cholesterol modification of hedgehog is required for trafficking and movement, revealing an asymmetric cellular response to hedgehog. Dev Cell. 2003 Feb;4(2):191-204.39. Gallet A and Therond PP.: Temporal modulation of the Hedgehog morphogen gradient by a patched-dependent targeting to lysosomal compartment. Dev Biol. 2005 Jan 1;277(1):51-62.40. Desbordes SC, Sanson B.: The glypican Dally-like is required for Hedgehog signalling in the embryonic epidermis of Drosophila. Development. 2003 Dec;130(25):6245-55.

41. Perrimon N, Häcker U.: Wingless, hedgehog and heparan sulfate proteoglycans. Development. 2004 Jun;131(11):2509-11.42. Zeng X et al.: A freely diffusible form of Sonic hedgehog mediates long-range signalling. Nature. 2001 Jun 7;411(6838):716-20.43. Eaton, S. Multiple roles for lipids in the Hedgehog signalling pathway. Nat. Rev. Mol. Cell Biol. 2008 9: 437-445.44. Datta S, Pierce M, Datta MW.: Perlecan signaling: helping hedgehog stimulate prostate cancer growth. Int J Biochem Cell Biol. 2006;38(11):1855-61. Epub 2006 Apr 25. Review.45. Fico A, Maina F, Dono R.: Fine-tuning of cell signalling by glypicans. Cell Mol Life Sci. 2007 Dec 18.46. Kirkpatrick CA, Selleck SB.: Heparan sulfate proteoglycans at a glance. J Cell Sci. 2007 Jun 1;120(Pt 11):1829-32.47. Datta MW et al.: Perlecan, a candidate gene for the CAPB locus, regulates prostate cancer cell growth via the Sonic Hedgehog pathway. Mol Cancer. 2006 Mar 1;5:9.48. Torroja C, Gorfinkiel N, Guerrero I.: Mechanisms of Hedgehog gradient formation and interpretation. J Neurobiol. 2005 Sep 15;64(4):334-56. Review.

49. Couchman JR: Syndecans: proteoglycan regulators of cell-surface microdomains? Nat Rev Mol Cell Biol. 2003 Dec; 4(12): 926-37. Review.

50. Vyas, N. et al. Nanoscale organization of Hedgehog is essential for long-range signalling. Cell 2008 133: 1214.51. Takada R et al.: Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion. Dev Cell. 2006 Dec;11(6):791-801.52. Franch-Marro et al. In vivo role of lipid adducts on Wingless.J Cell Sci. 2008 May 15;121(Pt 10):1587-92. Epub 2008 Apr 22.53. Willert K et al.: Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature. 2003 May 22;423(6938):448-52.54. Takada R et al.: Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion. Dev Cell. 2006 Dec;11(6):791-801.55. Ching W et al.: Lipid-independent secretion of a Drosophila Wnt protein. J Biol Chem. 2008 Jun 20;283(25):17092-8.

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56. Kadowaki T et al.: The segment polarity gene porcupine encodes a putative multitransmembrane protein involved in Wingless processing. Genes Dev. 1996 Dec 15;10(24):3116-28.57. Tanaka K et al.: The evolutionarily conserved porcupine gene family is involved in the processing of the Wnt family. Eur J Biochem. 2000 Jul;267(13):4300-11.58. Galli LM et al.: Porcupine-mediated lipid-modification regulates the activity and distribution of Wnt proteins in the chick neural tube. Development. 2007 Sep;134(18):3339-48.59. Hausmann G and Basler K.: Wnt lipid modifications: not as saturated as we thought. Dev Cell. 2006 Dec;11(6):751-2.60. Chen Z et al.: Suppression of PPN/MG61 attenuates Wnt/beta-catenin signaling pathway and induces apoptosis in human lung cancer. Oncogene. 2008 May 29;27(24):3483-8.61. Paller AS.: Wnt signaling in focal dermal hypoplasia. Nat Genet. 2007 Jul;39(7):820-1.62. Kurayoshi M et al.: Post-translational palmitoylation and glycosylation of Wnt-5a are necessary for its signalling. Biochem J. 2007 Mar 15;402(3):515-23.63. Bartscherer K and Boutros M.: Regulation of Wnt protein secretion and its role in gradient formation. EMBO Rep. 2008 Oct;9(10):977-82.64. Zhai L et al.: Drosophila wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine. J Biol Chem. 2004 Aug 6;279(32):33220-7.65. Katanaev VL et al.: Reggie-1/flotillin-2 promotes secretion of the long-range signalling forms of Wingless and Hedgehog in Drosophila. EMBO J. 2008 Feb 6;27(3):509-21.66. Gutierrez JA et al.: Ghrelin octanoylation mediated by an orphan lipid transferase. Proc Natl Acad Sci U S A. 2008 Apr 29;105(17):6320-5.67. Yang J et al.: Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell. 2008 Feb 8;132(3):387-96.68. Zhu X et al.: On the processing of proghrelin to ghrelin. J Biol Chem. 2006 Dec 15;281(50):38867-70.69. Gualillo O et al.: One ancestor, several peptides post-translational modifications of preproghrelin generate several peptides with antithetical effects. Mol Cell Endocrinol. 2006 Aug 15;256(1-2):1-8.70. Yang J et al.: Inhibition of ghrelin O-acyltransferase (GOAT) by octanoylated pentapeptides. Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10750-5.71. Tong J et al.: Gastric O-acyl transferase activates hunger signal to the brain. Proc Natl Acad Sci U S A. 2008 Apr 29;105(17):6213-4.72. Nishi Y et al.: Ingested medium-chain fatty acids are directly utilized for the acyl modification of ghrelin. Endocrinology. 2005 May;146(5):2255-64.

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