Acyltransferases for secreted signalling proteins (Review)

Abstract

Members of the MBOAT family of multispanning transmembrane enzymes catalyze 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. Characterization of these enzymes could lead to their identification as therapeutic targets for diverse human diseases such as cancers, obesity and diabetes.

• MBOAT
• hedgehog
• wingless/Wnt
• ghrelin
• palmitoylation
• acylation
• protein acyl transferase

Introduction

Post-translational modifications are known in several secreted signalling molecules, 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 the 16-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 and between cells through interactions with other proteins, but also by 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. Many intracellular proteins are acylated and this is catalyzed by the DHHC family of multispanning transmembrane enzymes. This family is extensively reviewed by other contributions in this issue of Molecular Membrane Biology, notably that from Planey and Zacharias (see Pages 14–31), so they will not be discussed here. It is becoming clear that acylation of extracellular 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]. Recent studies have shown that many MBOAT family members are lysophospholipid acyltransferases (reviewed in [7]). This review focuses on the members of the MBOAT family that have been shown to affect the palmitoylation of protein substrates, i.e. Hedgehog acyltransferase (Hhat), Porcupine (Porc) and ghrelin O-acyltransferase (GOAT). The MBOAT family 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 [8]. 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. However, Saccharomyces cerevisiae Gup1 does contain the histidine and has been implicated in the remodelling of GPI anchors, acting as an O-acyltransferase for long chain saturated fatty acids [9]. MBOAT proteins contain between 8–12 transmembrane domains based on structure prediction programmes, so the localization of the C-terminus to the cytoplasmic or extracytoplasmic side of cellular membranes is currently a matter of conjecture (Figure 1).

Figure 1.  MBOAT family members’ topology. MBOAT (membrane-bound O-acyltransferase) family members that promote protein acylation – Hhat, Porc and GOAT – contain multiple membrane-spanning domains. These palmitoylacyltransferases (PATs) are responsible for attaching fatty acids (originating in the cytoplasm in the form of activated CoA esters) to secreted signalling proteins (SSP), such as Hedgehogs, Wg/Wnts and ghrelin, within the lumen of the secretory pathway. The histidine shown is conserved in almost all members of the MBOAT family and may contribute to the active site of the putative acyltransferase. This Figure is reproduced in colour in Molecular Membrane Biology online.

Hedgehog proteins

Hedgehog proteins (Hh), acting as morphogens, were first discovered in the 1980s encoded by a gene family originally discovered through the Drosophila segmental pattern mutation hedgehog. In mammals, all three Hh homologues (Sonic (Shh), Indian (Ihh) and Desert (Dhh) Hedgehog) display a variety of roles in embryonic development, adult homeostasis, and cancer [10], [11]. 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 [12–14]. During differentiation and tumorigenesis, diverse targets of Hh signalling are involved in cell adhesion, signal transduction, cell cycle, apoptosis and angiogenesis. 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-translational modifications (Figure 2), including the unique N-terminal palmitoylation and C-terminal cholesterol attachment. Hhs are the best established examples of cholesteroylated proteins in nature. In Drosophila the ∼45 kDa 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 [15]. A ∼19 kDa N-terminal fragment (Hh-N) and a ∼25 kDa C-terminal fragment (Hh-C) are subsequently yielded by autocatalytic cleavage catalyzed by Hh-C [16]. Concurrently, a cholesterol molecule is covalently attached to the C-terminus of Hh-N, thus forming the mature form of Hh, Hh-Np [17]. Unlike Hh-C, Hh-N contains all the signalling functions. Processing of mammalian Hh proteins is probably highly analogous.

Figure 2.  Post-translational modifications of Hh. Drosophila and vertebrate Hedgehog proteins are synthesized as ∼45 kDa precursors that undergo dual lipidation as well as internal autoprocessing at a conserved sequence. After its signal peptide is removed co-translationally, the precursor is palmitoylated at its N-terminal cysteine using PalmitoylCoA. Palmitoylation is facilitated by Hhat and it is suggested that an intramolecular S-to-N acyl shift occurs, transferring the thioester-linked fatty acid to amide linkage. Autocatalytic cleavage occurs at the GCF sequence, catalyzed by the C-terminal domain of the precursor, and produces a ∼19 kDa signalling domain Hh-N and a ∼25 kDa Hh-C. During the autocleavage event, Hh-N is modified by cholesterol at its carboxy-terminal glycine to form a fully active Hh-Np which contains all known signalling activities. This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 3.  Model of active soluble Hh/Shh multimeric complex transport. After post-translational modifications in Hh-producing cells (palmitoylation, red; cholesteroylation, green), Hh-Np proteins are secreted to the extracellular matrix and form a multimeric complex with Heparan Sulphate Proteoglycans (HSPGs). In Hh-receiving cells, the receptor PTC is repressed by Hh and this releases SMO to activate downstream signalling. HSPGs can improve the solubility of Hh proteins during intercellular trafficking in the extracellular matrix and enhance Hh-PTC interaction on Hh-receiving cells, therefore promoting subsequent signalling activity.

For release from producing cells, cholesterol-modified Hhs require the activity of another multispanning membrane protein Dispatched (Disp) [18], [19], and hence this protein is required for long range Hh/Shh signalling. Disp contains twelve transmembrane domains, is related to the bacterial resistance nodulation-division (RND) family of proton-driven pumps and also contains a sterol-sensing domain which may play a role in binding cholesterol attached to Hhs [18], [20]. The mechanism of Disp action to release Hhs is not yet clear but it is interesting that it is related to Patched (Ptc), the Hh receptor on receiving cells, so the binding of the cholesterol moiety of Hhs may be a common theme both for their release and reception.

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 [21]. 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 Ptc [21], [22].

In Hh-receiving cells, Hh signaling is regulated by two proteins – Ptc and Smoothened (Smo). Ptc, a 12-transmembrane protein, binds 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 [22]. 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 organization 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 [23], [24].

Palmitoylation of Hh

Hhs are unusual in being dually lipid-modified to be fully active [25]. 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 [26]. 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 Hhs, 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 [27] and this is still a plausible mechanism of the N-terminal acylation. N-terminal palmitoylation of Hhs is facilitated by the product (Hhat) of the hedgehog acyltransferase gene (also known as skinny hedgehog, sightless, central missing or rasp) [22], [28], [29]. 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 [15]. 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 [30]. 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 [31]. These authors also confirmed the observation made previously by others that Hhat is localized in intracellular membranes of the secretory pathway, ER and Golgi. In cells, PalCoA 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 [32].

The MBOAT family member Gup1 is highly homologous to Hhat, with very similar gene organization, membrane topology and intracellular localization although the expression patterns differ somewhat between cell lines. It is interesting that exogenous overexpressed 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 [8]. 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. In addition, Hhat shares homology with Porc in Drosophila and its Caenorhabditis elegans homologue Mom-1 and with GOAT, putative acyltransferases that are also part of the MBOAT family and are responsible for the palmitoylation of Wg, a morphogen involved in embryonic patterning in Drosophila, and its human homologues Wnts (see below), and of ghrelin, respectively. 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 Hhs 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], [33]. Recently, it has been demonstrated that dual lipid modification is critical for the interactions between Hh, HSPGs and Ptc receptor [34]. 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 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 [34], [35].

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 [34] 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 [36]. In the same study, overexpressionv of an unacylated Shh mutant (ShhC25S) in transgenic mice caused reduced Shh protein activity in inducing Shh responses, and lacking both types of lipid modification (ShhNC25S) had lower levels of residual activity.

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

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 interactions between cells and their environment but also regulate the distribution of extracellular signalling molecules such as morphogens through binding to them [37–40]. This diversity of function is based on HSPGs’ enormous structural differences – partly via the additional modifications in HS chains through the repeating disaccharide chain elongation.

Hhs are known to act as major mediators in many developmental processes and require HSPGs for their proper distribution and signalling activity [41]. In the case of Hh/Shh long-range signalling in Drosophila and mice, activity is enhanced through forming a multimeric complex to increase Hh/Shh solubility, which is one critical criterion for protein stability [42]. The Hh/Shh multimeric complex is the major form responsible for activating Hh/Shh signalling [26]. In addition, it has been suggested that these multimers could form extracellular aggregates, called large punctuated structures, in the embryo [43], [44]. Both acylation and cholesteroylation are necessary for Hh/Shh to incorporate into this complex [26] and requires both the HSPG core proteins and their attached HS GAG [42], [45], [46]. There are several conjectural mechanisms for how HSPGs promote this signalling. Shh and Hh are secreted from cells as both monomeric and multimeric forms [20], [26], [47]. The soluble Shh multimeric complex with specific HSPGs – Perlecan and Glypican – is freely diffusive and can mediate Shh signalling [48], [49]. On the other hand, the interaction between HSPGs and growth factors could influence both their extracellular distribution and their ability to signal [50], e.g. Perlecan by directly binding to Shh as a co-receptor can affect Shh signalling [48], [51]. In addition, it has been found that formation of the polymeric lipid-modified Hh-HSPG complex is mediated by Shifted, a secreted Wnt Inhibitory Factor homologue [4], [52], indicating that lipid modifications of Hh are essential for Hh/HSPGs interaction in the extracellular matrix and long range diffusion and action. In contrast, lipid-unmodified Hh is poorly retained and stabilized by the ECM and tends to diffuse freely [53]. 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 [54], [55].

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 [56], [57]. 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 [58]. Recently, a second site of acylation has been identified as a serine some distance downstream (Ser209 in murine Wnt3a) [59] 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 [60], in contrast to an earlier report form the same laboratory [58]. 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.

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-localized member of the MBOAT family called Porc [61–63]. This appears highly unusual because if Porc catalyzes both acylations it would need to recognize 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 rationalization is that initial acylation at Ser209 may be with palmitate and this could subsequently be converted to palmitoleate by a desaturase [64], but there is as yet no evidence for this. It has not been definitively proven that Porc is the enzyme responsible for either of these acylations. Since cysteine acylation is dependent on previous serine acylation [55], [57] 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 characterize 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 [65], [66]. Hence, Porc is a potential therapeutic target in several human diseases.

The function of Wg/Wnt acylation, as for Hhs, 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 [67]. 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 [57]. The authors’ interpretation is that the overall level of acylation is important for signalling, suggesting that membrane association is crucial. However, they show that 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/Srt or with membrane microdomains (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 [68]). 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 [69]. WntD, which is not acylated, does not require the action of Evi/Wls/Srt for release and is secreted at higher rates than other Wg/Wnt proteins which is compatible with its more systemic role in the fly innate immune response to infection [60]. Finally, the dual acylation of Wg/Wnt proteins may also facilitate their interaction with membrane lipid microdomains/lipid rafts that could be the site of assembly of the lipoprotein transport packages [70], hence the dependence of Wg long-range secretion on the raft resident protein reggie-1/flotillin-2 [71].

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) [72], [73]. 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 [74]. 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 [72] 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 [75].

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 [76] 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 localization of GOAT and acylated ghrelin production to the stomach also makes it likely that orally administered agents may be effective if appropriately packaged and stabilized. 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 proteins.

The source of medium chain FAs for ghrelin acylation is of interest, as mammalian cells are not generally thought to synthesize them, although during fasting white adipose tissue might release FAs that could be used by GOAT [77]. GOAT has the ability to transfer a wide range of short and medium chain FAs onto ghrelin in vitro[66]. FAs for ghrelin acylation could come from the diet [78], 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[78], 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 localized in the cytoplasm and are bound to the high affinity ACBP, possibly to prevent these highly reactive thioesters from reacting non-specifically with cell components such as proteins [30]. 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 [73] and as early as 1996 for Porc [61], 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-catalyzed 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. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.