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Membrane bound O-acyltransferases and their inhibitors
Naoko Masumoto*†, Thomas Lanyon-Hogg*, Ursula R Rodgers‡, Antonios D Konitsiotis‡§,
Anthony I Magee†‡ and Edward W Tate*†
*Department of Chemistry, Imperial College London, South Kensington Campus, London,
SW7 2AZ, United Kingdom
†Institute of Chemical Biology, Department of Chemistry, Imperial College London
‡Molecular Medicine Section, National Lung & Heart Institute, Sir Alexander Fleming
Building, South Kensington Campus, Imperial College London, SW7 2AZ
§Current address: Max Planck Institute of Molecular Physiology, Department of Systemic
Cell Biology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
Joint corresponding Authors: Anthony I. Magee, Molecular Medicine Section, National Heart
& Lung Institute, Imperial College London, South Kensington Campus, London SW7 2AZ,
UK. Email: [email protected]
Edward W. Tate, Department of Chemistry, Imperial College London, South Kensington,
London SW7 2AZ, UK. Email: [email protected]
Abbreviations
Hh = Hedgehog, UAG = unacylated ghrelin, TMD = transmembrane domain, DAP = (S) 2,3diaminopropionic acid, cat-ELCCA = catalytic assay using enzyme-linked click chemistry,
Shh = Sonic Hedgehog
Key Words
Membrane bound O-acyltransferases (MBOATs), Octanoylation, Palmitoleoylation,
Palmitoylation, Ghrelin O-acyltransferase (GOAT), Porcupine (Porcn), Hedgehog
acyltransferase (HHAT)
Abstract
Since the identification of the Membrane Bound O-Acyltransferase (MBOATs) protein
family in the early 2000s, three distinct members (Porcupine, Hedgehog acyltransferase and
Ghrelin O-acyltransferase) have been shown to acylate specific proteins or peptides. In this
review, topology determination, development of assays to measure enzymatic activities, and
discovery of small molecule inhibitors are compared and discussed for each of these
enzymes.
Introduction
In 2000, Hoffmann [1] reported a family of multi-domain membrane spanning
acyltransferases responsible for O-acylation reactions called Membrane Bound O-Acyl
Transferases (MBOATs) during analysis of the conserved sequence of Porcupine (PORCN),
the activity of which is important in Wingless (Drosophila) /Wnt (vertebrates) signalling
pathways. Since this discovery, more than ten genes have been identified to encode
MBOATs in humans [2].
MBOATs are further categorised into three subgroups based on their biochemical
reactions: lipid biosynthesis, sterol acylation, and acylation of secreted proteins/peptides,
including the appetite stimulating peptide hormone ghrelin and the Hedgehog (Hh) and Wnt
morphogen families [3]. These acylated polypeptides are involved in cellular signalling or
subsequent protein-protein interactions which are dysregulated in a range of diseases, thus
making MBOATs attractive targets for novel drug discovery.
MBOATs are predicted to have 8-12 transmembrane domains and localise in the
protein secretory pathway (ER/Golgi Complex) [2]. Their acyl-CoA substrates are produced
during fatty acid beta-oxidation predominantly in the mitochondria and cytosol. In order for
catalysis to occur, acyl-CoAs must cross the ER membrane and MBOATs have been
suggested also to act as acyl-CoA transporters [4]. Analysis of MBOATs has proven
challenging due to their polytopic nature and limitations in available techniques and tools.
Investigating the activities of MBOATs using knock-out or transgenic mice has resulted in
developmental defects and embryonic lethality [5]. To date, five MBOAT family members
have been fully mapped topologically: human ACAT1 (Acyl-CoA:cholesterol acyltransferase,
also known as Sterol O-acyltransferase, SOAT), ACAT2, Ghrelin O-acyltransferase (GOAT),
Hedgehog acyltransferase (HHAT) and yeast Gup1p [6-11]. Common to all MBOATs are two
key residues: a highly conserved asparagine/aspartic acid and an invariant histidine residue
(Figure 1) [2]. These residues have been hypothesised to be involved in catalysis; however,
to date there is no conclusive evidence that defines the precise location of the catalytic
centre.
In the past few years rapid progress has been made in understanding the MBOATs
responsible for acylation of secreted polypeptides, aided by the development of methods to
study protein acylation such as bioorthogonal ligation techniques [12-14]. GOAT is involved
in octanoylation of ghrelin at Ser-3, which increases its potency as an appetite enhancing
hormone [7]. Porcupine (PORCN) catalyses primarily palmitoleoylation (C16:1) of Wnt-3a
proteins on Ser-209, which enhances Wnt secretion [15, 16]. Hh proteins are irreversibly
palmitoylated (C16:0) by HHAT at the N-terminal Cys-24 revealed by secretory signal
peptide cleavage [17], which is crucial for biological activity in vertebrates. In this review,
recent progress in the understanding of MBOATs and discovery of inhibitors of GOAT,
PORCN and HHAT are discussed.
GOAT
Ghrelin was identified by Kojima et al [18] during a search for a binding partner of an
orphan G protein coupled receptor (GHS-R1a) which stimulates secretion of growth
hormones in the pituitary gland. After cleavage of pro-ghrelin (117 amino acids), ghrelin (28
amino acids) undergoes post-translational octanoylation at Ser-3 in the ER lumen, which is
thought to be required for secretion. Although octanoylation of ghrelin augments its potency
1000 fold, the dominant form of ghrelin present in plasma is the unacylated form (unacylated
ghrelin, UAG). Initially, UAG was considered biologically non-functional due to its inability to
bind GHS-R1a. It was later revealed that non-endocrine activities are induced by UAG upon
binding to UAG receptors [19]. Ghrelin is also modified with different acyl chain lengths, such
as decanoate (C10:0) and decenoate (C10:1) [20], the function of which is unknown;
however, emerging evidence suggests these variations are partially due to nutrient intake.
As expected from its function, expression of GOAT is highly restricted to major ghrelin
releasing tissues, such as the stomach and intestine.
GOAT topology and key residues
Cole et al reported the first comprehensive study on GOAT topology [7],
demonstrating GOAT has 12 distinct hydrophobic regions with 11 transmembrane domains
(TMDs) and one re-entrant loop (RL), each separated by relatively short hydrophilic loops
(Figure 2A). It has short terminal tails, in the lumen at the N-terminus and the cytosolic Cterminus. The invariant histidine (His-338) is located on the luminal side and the conserved
asparagine (Asn-307) is on the cytosolic side. It is predicted that His-338 is likely to be
involved in the active site, whereas Asn-307 is unlikely to be involved in catalysis, although it
might be important for substrate interactions and transport or protein structural stability.
Photocrosslinkable acyl ghrelin analogues can bind at the C-terminal region of GOAT
indicating that the peptide and octanoyl-CoA interaction may occur near the C-terminus,
although the identification of the exact catalytic site was unsuccessful. Although MBOATs
are known to form oligomers in vitro and in cell culture, purified GOAT in detergent micelles
exists as monomers and ghrelin and its analogues bind to monomeric purified GOAT.
GOAT inhibitors
It has been previously demonstrated that GOAT is regulated by nutrient availability
and its activity mediates the impact on body adiposity [4, 21, 22]. So far, three different types
of GOAT inhibitors have been discovered: peptide-based analogues, bisubstrate analogues
and small molecules [23]. Yang et al exploited peptidomimetics by substituting the first
pentapeptide of the GOAT recognition motif of ghrelin (GSSFL) with different amino acids
[24]. Inhibition in vitro was significantly increased with amidated full-length ghrelin (2)
(IC50=0.2 µM) or a pentapeptide containing octanoylated (S)2,3-diaminopropionic acid (DAP)
in place of Ser-3 (IC50=1.0 µM) (3). However, these compounds pose pharmacologic
challenges for in vivo applications and are likely to act as potent agonists of GHS-1a. A
transition state mimic of Ser-3 octanoylation, BK-1114 (4), is effective in the micromolar
range on isolated enzyme and in intact cells [25]. Cole et al introduced compounds inspired
by bisubstrate GO-CoA-Tat inhibitors (5a-5c), on the premise that GOAT might form a
ternary complex with both substrates [26], consisting of octanoate, CoenzymeA (CoA) and
the first 10 amino acids of ghrelin linked irreversibly with an amide linkage (octanoate to the
ghrelin) and a thioether linkage (alpha-carbon of octanoate to CoA) (Figure 3). Validated in
vitro and in vivo, the outcome was especially encouraging in mice (reduction in weight gain
and improvement in glucose intolerance) despite their limited pharmacological utility in vivo
due to large size, polarity and cell penetration. Janda et al have developed and utilised catELCCA (catalytic assay using enzyme-linked click chemistry) to identify the first small
molecule inhibitors containing a naphthalene core structure (6) [27, 28]. Validation of these
compounds in cell based assays is still pending; however, this assay format has potential as
a platform to find new small molecule inhibitors for other MBOATs.
PORCN
Secreted Wnt proteins play key roles in embryonic development, tissue homeostasis
and stem cell self-renewal; among the 19 Wnt proteins encoded in humans, Wnt3a is the
most extensively studied isoform [29]. PORCN catalyses palmitoleoylation of Wnt signalling
proteins at a serine residue (209 in human Wnt3a). An early report that PORCN
palmitoylated Wnts at a cysteine residue in the N-terminal region (Cys-77 in Wnt3a) has
been shown to be erroneous [30]. The bent conformation of palmitoleate may provide the
appropriate three dimensional conformation for interaction of Wnt with Wntless (WIs) which
packages Wnt into exosomes for secretion [31]. The palmitoleate acts as an anchor to a
hydrophobic groove of the receptor Frizzled [32] where Wnt binds after travelling through the
extracellular space. Moreover, it appears that Wnt proteins can be modified with various
lengths of acyl chains (typically C13-16) with or without saturation [33], which may be
involved in gradient formation or differential regulation of Wnt ligand transport [16, 34].
PORCN topology and key residues
According to topology prediction by MEMSAT-SVM, Porcn has 11 TMDs with the
conserved Asn-306 in a cytosolic loop and the invariant His-341 embedded in TMD 9 (Figure
2B) [34]. Interestingly, PORCN itself is palmitoylated at cytosolic Cys-187[33]. Identification
of key residues and regions involved in catalysis were accomplished using alanine-scanning
mutagenesis. Within TMD 9, mutants N306A and W312A did not alter the activity of PORCN.
Therefore, the conserved Asn-306 is not involved in catalysis. W305A, Y316A and Y334A
showed moderate defects in activity (30-50%). S337A, L340A and H341A had little or no
activity (<20% WT), indicating that these residues are critical for acyltransferase activity.
Confocal imaging demonstrated that impaired enzymatic activity was not due to PORCN
misfolding or mislocalisation. Co-immunoprecipitation with Wnt-3a confirmed that mutants
Y334A, S337A and H341A were not able to bind the protein substrate; however, L340A was
capable of binding Wnt3a, suggesting that Leu-340 may be involved in fatty acid recognition
rather than Wnt binding.
PORCN inhibitors
Chen et al identified highly selective PORCN inhibitors termed ‘Inhibitors of Wnt
Production’ (e.g. IWP-2, (7)) which can be used in a variety of in vitro settings including
tissue engineering and stem cell biology [35]. Due to limited bioavailability, these are not
compatible with in vivo studies; however, recent studies show that PORCN can
accommodate chemically diverse scaffolds [36]. Among them, IWP12 (8) is effective in
zebrafish as evidenced by the loss of Wnt activity, and C59 (9), disclosed in a Novartis
patent [37], possesses nanomolar activity and was found to inhibit Wnt signalling and growth
of a Wnt-driven breast cancer cell line [37]. Similar to the C59 scaffold, LGK974 (Novartis)
(10) has entered phase I clinical trials (NCT01351103) for treatment of malignant cancers
[38]. Liu et al observed the regression of Wnt-driven tumours absent from the formation of
abnormal histopathological defects and delay in tumour growth [39].
HHAT
Hedgehog (Hh) proteins are involved in development and tissue homeostasis in adult
organisms and are unique in that they are post-translationally modified by two lipids during
their maturation process [40]. Addition of palmitate at the N-terminal cysteine is catalysed by
HHAT [41] and cholesterol attachment occurs at the C-terminus after autocatalytic cleavage
of a non-signalling domain [42]. It has been thought that dual lipidation of Hhs improves
membrane affinity and contributes to formation of extracellular multimeric complexes, which
translates to enhanced signalling activity [43]. A major role of palmitoylation is to direct Hh
proteins to specific membrane domains and to establish long-range signalling upon
formation of soluble multimeric complexes [5]. The importance of palmitate for Hh signalling
activity was demonstrated in rodent ventral forebrain formation [44] where removal of the
palmitoylation site abolished the induction of neuronal cell differentiation. On the other hand,
cholesterol provides affinity for cell membranes, regulates cell surface distribution, and
establishes the extracellular range and concentration gradient. Reports on the role of each
type of lipid modification and compositions of multimeric complexes frequently present
conflicting evidence [45-47]. Like PORCN, HHAT can accept various different lengths of
acyl-CoA as a substrate [41] in vitro and in cell-based assays in our lab (unpublished data).
In vitro, Hh proteins showed preference for modification by shorter acyl-CoAs [48]. The
dominant form of acyl-CoA in vivo is usually palmitoyl-CoA but other lengths are present and
may modify Hh depending on the local availability of different acyl-CoAs.
HHAT topology and key residues
The Tate/Magee and Resh groups recently reported the first detailed analyses of
HHAT topology [8, 9], which were independently obtained by somewhat different
experimental approaches but produced remarkably harmonious results. The current working
topology model consists of ten TMDs and two RLs (Figure 2C). The invariant His-379 is
between TM9 and 10 at the luminal side while the Asp-339 is within the cytosol. Point
mutagenesis experiments showed that HHAT activity was severely lost in the D339N mutant
whereas the H379A mutant retained ~50% activity, in agreement with observations from
Buglino et al [49]. In our study, it was demonstrated that HHAT itself is also palmitoylated,
similar to PORCN but at four distinct cytoplasmic sites. Mutagenesis experiments showed
that D339 is more important than H379 for HHAT palmitoylation [8], and that the state of
HHAT palmitoylation also affects HHAT activity.
HHAT Inhibitors
Resh et al identified the first candidate HHAT inhibitors (11-14) from a targetorientated high throughput screen [50, 51], opening a new avenue to explore the impact of
palmitoylation inhibition on Hh transport and signal transduction. Hh pathway inhibitors
targeting upstream components such as HHAT may also be useful to combat development
of resistance at the downstream components during chemotherapy [52, 53]. The compound
most used to date in the literature, RU-SKI-43, has been investigated in cell based assays
including various cancer cell lines; however, it showed limited utility in vivo (half-life in mouse
plasma of 17 minutes) [50]. To circumvent this issue, Panc-1 cells stably expressing shRNAs
against
HHAT,
Shh
and
a
control
scrambled
sequence
were
injected
into
immunocompromised mice [54] and over 72 hours of treatment tumour growth was inhibited
by 70% by both HHAT and Shh depletion. Moreover, in combination with the antiproliferative
compound Rapamycin, cell proliferation was inhibited further than with individual depletion.
These data should be treated with caution, based on two independent studies on Hh
signalling in pancreatic cancer that were reported recently in which tumour growth was
substantially enhanced after inhibition of Hh signalling, using a variety of mouse models [5557]. Although inhibition of Hh signalling leads to disruption in paracrine signalling and stroma
desmoplasia, it is not therapeutically beneficial as the stroma appears to physically restrain
tumour growth. In our lab, RU-SKI-43 showed a very narrow therapeutic window due to
significant off-target toxicity; however, modified RU-SKI compounds were more potent in
various in vitro and cell-based assays with lower cell toxicity (unpublished data).
Future Outlook
Progress to elucidate of MBOAT function so far represents only the start of a long
journey. Although topological analyses provided useful insights, there is lack of information
such as three-dimensional architecture of MBOATs, and mechanisms and key residues
involved in catalysis. Such information will be of great utility in drug discovery and
development in future. MBOATs are often involved in a small but highly significant part of a
more complex cell signalling network, and many unresolved questions will only be solved
through comprehensive analysis using multiple complementary techniques. Collaborative
efforts between academia and industry will be essential to continue fill the gaps in our
knowledge, and to develop more potent inhibitors against the MBOAT family.
Funding Sources
NM was supported by the Imperial College London Institute of Chemical Biology EPSRC
Centre for Doctoral Training (grant EP/F500416/1). TL-H and URR acknowledge funding by
Cancer Research UK (grant to AIM and EWT, C6433/A16402). AK was supported by a
Pancreatic Cancer Research Fund grant to AIM and EWT.
Q96T53|MBOA4_HUMAN
Q9H237|PORCN_HUMAN
Q5VTY9|HHAT_HUMAN
P35610|SOAT1_HUMAN
O75908|SOAT2_HUMAN
O75907|DGAT1_HUMAN
311
302
341
428
402
385
RKW---NQSTARWLRRLVFQH-----SRAWPL----LQTFAFSAWWHG
TSW---NLPMSYWLNNYVFKN-----ALRLGTFSAVLVTYAASALLHG
GMWRYFDVGLHNFLIRYVYIPVGGSQHGLLGTLFSTAMTFAFVSYWHG
NYYRTWNVVVHDWLYYYAYKDFLWFFSKRF-KSAAMLAVFAVSAVVHE
NYYRTWNVVVHDWLYSYVYQDGLRLLGARA-RGVAMLGVFLVSAVAHE
YFWQNWNIPVHKWCIRHFYKPMLRRGSS---KWMARTGVFLASAFFHE
339
342
380
461
435
416
Figure 1: Sequence Homology of MBOAT family in the putative catalytic region (alignment deduced
from Uniprot).
MBOA4 = GOAT; SOAT1/2 = Acyl-CoA:cholesterol acyltransferase; DGAT1 = Diglyceride
acyltransferase; HHAT = Hedgehog acyltransferase; PORCN = Porcupine (Wnt acyltransferase)
Highly conserved asparagine/aspartic acid (pink/yellow respectively) and histidine (red).
A: GOAT (empirical)
B: PORCN (predicted)
C: HHAT (empirical)
Figure 2: Topology of GOAT [7], PORCN (prediction adapted from [58]) and HHAT [8, 9]
(Blue arrows, conserved asparagine/aspartate and histidine; Red arrows, palmitoylation sites)
Figure 3: GOAT, PORCN and HHAT inhibitors.
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