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
1
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 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
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 membranebound 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
2
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 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 [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-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 ~45kDa Hh precursor is
translocated, presumably by the conventional signal recognition particlemediated 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 Cterminal fragment (Hh-C) are subsequently yielded by autocatalytic cleavage
catalysed by Hh-C [14]. Concurrently, a cholesterol molecule is covalently
3
attached to the C-terminus of Hh-N, thus forming the mature form of Hh, HhNp [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 - Ptc
and Smoothened (Smo). Ptc, a 12-transmembrane protein, is the
receptor for Hh through its 2 large extracellular loops. Smo, a 7transmembrane 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
4
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 Nterminal 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].
5
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 many
developmental 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
6
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 lipidmodified 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.
7
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 ERlocalised 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
8
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 94residue 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
9
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.
10
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: 111112.
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.: Teratogenmediated 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-R241
18. 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.
11
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 glycosylphosphatidylinositollinked 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: 1403714045.
23. Chamoun, Z. et al. Skinny hedgehog, an acyltransferase required for
palmitoylation and activity of the Hedgehog signal. Science 2001 293: 20802084.
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 liganddependent manner in mammalian cells. Biochem J. 2008 Mar 15;410(3):46372.
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):47183.
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):64159.
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.
12
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 longrange 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):185561. 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 longrange 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.
13
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):51523.
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 longrange 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.
14