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Membrane bound O-acyltransferases and their inhibitors Naoko Masumoto*†, Thomas Lanyon-Hogg*, Ursula R. Rodgers‡, Antonios D. Konitsiotis‡1 , Anthony I Magee†‡2 and Edward W. Tate*†2 *Department of Chemistry, Imperial College London, South Kensington Campus, London, SW7 2AZ, U.K. †Institute of Chemical Biology, Department of Chemistry, Imperial College London, SW7 2AZ, U.K. ‡Molecular Medicine Section, National Lung & Heart Institute, Sir Alexander Fleming Building, South Kensington Campus, Imperial College London, SW7 2AZ, U.K.
Abstract Since the identification of the membrane-bound O-acyltransferase (MBOATs) protein family in the early 2000s, three distinct members [porcupine (PORCN), hedgehog (Hh) acyltransferase (HHAT) and ghrelin Oacyltransferase (GOAT)] 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 multidomain membrane spanning acyltransferases responsible for O-acylation reactions called membrane-bound Oacyltransferases (MBOATs) during analysis of the conserved sequence of porcupine (PORCN), the activity of which is important in wingless (Wnt; Drosophila vertebrates) signalling pathways. Since this discovery, more than 10 genes have been identified to encode MBOATs in humans [2]. MBOATs are further categorized 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 (TMDs) and localize in the protein secretory pathway [endoplasmic reticulum (ER)–Golgi complex; 2]. Their acyl-CoA substrates are produced during fatty acid βoxidation, predominantly in the mitochondria and cytosol. In order for catalysis to occur, acyl-CoAs must cross the ER membrane and MBOATs have also been suggested to
Key words: ghrelin O-acyltransferase (GOAT), hedgehog acyltransferase (HHAT), membranebound O-acyltransferases (MBOATs), octanoylation, palmitoleoylation, palmitoylation, porcupine (PORCN). Abbreviations: ACAT, acyl–coA:cholesterol acyltransferase; ER, endoplasmic reticulum; GOAT, ghrelin O-acyltransferase; Hh, hedgehog; HHAT, hedgehog acyltransferase; IWP, inhibitors of Wnt production; MBOAT, membrane-bound O-acyltransferase; Panc-1, pancreatic cancer; PORCN, porcupine; RL, re-entrant loop; Shh, sonic hedgehog; SOAT, sterol O-acyltransferase; TMD, transmembrane domain; UAG, unacylated ghrelin. 1 Current address: Max Planck Institute of Molecular Physiology, Department of Systemic Cell Biology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany. 2
Correspondence may be addressed to either author (email [email protected] or [email protected]).
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C 2015 Biochemical Society Authors Journal compilation
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 knockout 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 [acylCoA:cholesterol acyltransferase, also known as sterol Oacyltransferase (SOAT)], ACAT2, ghrelin O-acyltransferase (GOAT), Hh 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 hypothesized 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 bio-orthogonal ligation techniques [12–14]. GOAT is involved in octanoylation of ghrelin at Ser3 , which increases its potency as an appetite enhancing hormone [7]. PORCN catalyses primarily palmitoleoylation (C16:1) of Wnt-3a proteins on Ser209 , which enhances Wnt secretion [15,16]. Hh proteins are irreversibly palmitoylated (C16:0) by HHAT at the Nterminal Cys24 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 Biochem. Soc. Trans. (2015) 43, 246–252; doi:10.1042/BST20150018
Protein acylation: from mechanism to drug discovery
Figure 1 Sequence Homology of MBOAT family in the putative catalytic region (alignment deduced from Uniprot) MBOAT4 = GOAT; SOAT1/2 = ACAT1/2; DGAT1 = diglyceride acyltransferase; HHAT = hedgehog acyltransferase; PORCN = porcupine (Wnt acyltransferase). Highly conserved asparagine/aspartic acid (pink/yellow respectively); histidine (red).
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 posttranslational octanoylation at Ser3 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 GHSR1a. 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. [7] reported the first comprehensive study on GOAT topology, demonstrating GOAT has 12 distinct hydrophobic regions with 11 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 C-terminus. The invariant histidine (His338 ) is located on the luminal side and the conserved asparagine (Asn307 ) is on the cytosolic side. It is predicted that His338 is likely to be involved in the active site, whereas Asn307 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, bi-substrate analogues and small molecules [23]. Yang et al. [24] exploited peptidomimetics by substituting the first pentapeptide of the GOAT recognition motif of ghrelin (GSSFL) with different amino acids. 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 Ser3 (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-R1a. A transition state mimic of Ser3 octanoylation, BK-1114 (4), is effective in the micromolar range on isolated enzyme and in intact cells [25]. Cole and colleagues [26] introduced compounds inspired by bi-substrate GO–CoA–Tat inhibitors (5a–5c) on the premise that GOAT might form a ternary complex with both substrates, consisting of octanoate, CoA and the first 10 amino acids of ghrelin linked irreversibly with an amide linkage (octanoate to the ghrelin) and a thio-ether linkage (α-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. Garner and Janda [27,28] have developed and utilized cat-ELCCA (catalytic assay using enzyme-linked click chemistry) to identify the first small molecule inhibitors containing a naphthalene core structure (6). 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 homoeostasis and stem cell self-renewal; among the 19 Wnt proteins encoded in humans, Wnt3a is the most extensively studied isoform [29]. PORCN catalyses C The
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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)
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Protein acylation: from mechanism to drug discovery
Figure 3 GOAT, PORCN and HHAT inhibitors
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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 (Cys77 in Wnt3a) has been shown to be erroneous [30]. The bent conformation of palmitoleate may provide the appropriate 3D 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 between 9–11 TMDs with the conserved Asn306 in a cytosolic loop and the invariant His341 embedded in TMD 7 (Figure 2B) [34]. There is no comprehensive empirical analysis of the topology of PORCN reported to date. These key conserved residues Asn306 and His341 shown in Figure 2B are predicted to be located similarly to GOAT and HHAT. Interestingly, PORCN itself is palmitoylated at cytosolic Cys187 [33]. Identification of key residues and regions involved in catalysis were accomplished using alaninescanning mutagenesis. Within TMD 9, mutants N306A and W312A did not alter the activity of PORCN. Therefore, the conserved Asn306 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 [
Biochemical Society Transactions (2015) Volume 43, part 2
Membrane bound O-acyltransferases and their inhibitors Naoko Masumoto*†, Thomas Lanyon-Hogg*, Ursula R. Rodgers‡, Antonios D. Konitsiotis‡1 , Anthony I Magee†‡2 and Edward W. Tate*†2 *Department of Chemistry, Imperial College London, South Kensington Campus, London, SW7 2AZ, U.K. †Institute of Chemical Biology, Department of Chemistry, Imperial College London, SW7 2AZ, U.K. ‡Molecular Medicine Section, National Lung & Heart Institute, Sir Alexander Fleming Building, South Kensington Campus, Imperial College London, SW7 2AZ, U.K.
Abstract Since the identification of the membrane-bound O-acyltransferase (MBOATs) protein family in the early 2000s, three distinct members [porcupine (PORCN), hedgehog (Hh) acyltransferase (HHAT) and ghrelin Oacyltransferase (GOAT)] 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 multidomain membrane spanning acyltransferases responsible for O-acylation reactions called membrane-bound Oacyltransferases (MBOATs) during analysis of the conserved sequence of porcupine (PORCN), the activity of which is important in wingless (Wnt; Drosophila vertebrates) signalling pathways. Since this discovery, more than 10 genes have been identified to encode MBOATs in humans [2]. MBOATs are further categorized 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 (TMDs) and localize in the protein secretory pathway [endoplasmic reticulum (ER)–Golgi complex; 2]. Their acyl-CoA substrates are produced during fatty acid βoxidation, predominantly in the mitochondria and cytosol. In order for catalysis to occur, acyl-CoAs must cross the ER membrane and MBOATs have also been suggested to
Key words: ghrelin O-acyltransferase (GOAT), hedgehog acyltransferase (HHAT), membranebound O-acyltransferases (MBOATs), octanoylation, palmitoleoylation, palmitoylation, porcupine (PORCN). Abbreviations: ACAT, acyl–coA:cholesterol acyltransferase; ER, endoplasmic reticulum; GOAT, ghrelin O-acyltransferase; Hh, hedgehog; HHAT, hedgehog acyltransferase; IWP, inhibitors of Wnt production; MBOAT, membrane-bound O-acyltransferase; Panc-1, pancreatic cancer; PORCN, porcupine; RL, re-entrant loop; Shh, sonic hedgehog; SOAT, sterol O-acyltransferase; TMD, transmembrane domain; UAG, unacylated ghrelin. 1 Current address: Max Planck Institute of Molecular Physiology, Department of Systemic Cell Biology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany. 2
Correspondence may be addressed to either author (email [email protected] or [email protected]).
C The
C 2015 Biochemical Society Authors Journal compilation
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 knockout 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 [acylCoA:cholesterol acyltransferase, also known as sterol Oacyltransferase (SOAT)], ACAT2, ghrelin O-acyltransferase (GOAT), Hh 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 hypothesized 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 bio-orthogonal ligation techniques [12–14]. GOAT is involved in octanoylation of ghrelin at Ser3 , which increases its potency as an appetite enhancing hormone [7]. PORCN catalyses primarily palmitoleoylation (C16:1) of Wnt-3a proteins on Ser209 , which enhances Wnt secretion [15,16]. Hh proteins are irreversibly palmitoylated (C16:0) by HHAT at the Nterminal Cys24 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 Biochem. Soc. Trans. (2015) 43, 246–252; doi:10.1042/BST20150018
Protein acylation: from mechanism to drug discovery
Figure 1 Sequence Homology of MBOAT family in the putative catalytic region (alignment deduced from Uniprot) MBOAT4 = GOAT; SOAT1/2 = ACAT1/2; DGAT1 = diglyceride acyltransferase; HHAT = hedgehog acyltransferase; PORCN = porcupine (Wnt acyltransferase). Highly conserved asparagine/aspartic acid (pink/yellow respectively); histidine (red).
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 posttranslational octanoylation at Ser3 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 GHSR1a. 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. [7] reported the first comprehensive study on GOAT topology, demonstrating GOAT has 12 distinct hydrophobic regions with 11 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 C-terminus. The invariant histidine (His338 ) is located on the luminal side and the conserved asparagine (Asn307 ) is on the cytosolic side. It is predicted that His338 is likely to be involved in the active site, whereas Asn307 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, bi-substrate analogues and small molecules [23]. Yang et al. [24] exploited peptidomimetics by substituting the first pentapeptide of the GOAT recognition motif of ghrelin (GSSFL) with different amino acids. 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 Ser3 (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-R1a. A transition state mimic of Ser3 octanoylation, BK-1114 (4), is effective in the micromolar range on isolated enzyme and in intact cells [25]. Cole and colleagues [26] introduced compounds inspired by bi-substrate GO–CoA–Tat inhibitors (5a–5c) on the premise that GOAT might form a ternary complex with both substrates, consisting of octanoate, CoA and the first 10 amino acids of ghrelin linked irreversibly with an amide linkage (octanoate to the ghrelin) and a thio-ether linkage (α-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. Garner and Janda [27,28] have developed and utilized cat-ELCCA (catalytic assay using enzyme-linked click chemistry) to identify the first small molecule inhibitors containing a naphthalene core structure (6). 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 homoeostasis and stem cell self-renewal; among the 19 Wnt proteins encoded in humans, Wnt3a is the most extensively studied isoform [29]. PORCN catalyses C The
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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)
C The
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Protein acylation: from mechanism to drug discovery
Figure 3 GOAT, PORCN and HHAT inhibitors
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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 (Cys77 in Wnt3a) has been shown to be erroneous [30]. The bent conformation of palmitoleate may provide the appropriate 3D 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 between 9–11 TMDs with the conserved Asn306 in a cytosolic loop and the invariant His341 embedded in TMD 7 (Figure 2B) [34]. There is no comprehensive empirical analysis of the topology of PORCN reported to date. These key conserved residues Asn306 and His341 shown in Figure 2B are predicted to be located similarly to GOAT and HHAT. Interestingly, PORCN itself is palmitoylated at cytosolic Cys187 [33]. Identification of key residues and regions involved in catalysis were accomplished using alaninescanning mutagenesis. Within TMD 9, mutants N306A and W312A did not alter the activity of PORCN. Therefore, the conserved Asn306 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 [
