Membrane-bound O-acyltransferase Research Articles

The Membrane-bound O-Acyltransferase7 rs641738 Variant in Pediatric Nonalcoholic Fatty Liver Disease.

The rs641738 polymorphism in the membrane-bound O-acyltransferase domain containing protein 7 (MBOAT7) gene has been associated with increased risk of nonalcoholic fatty liver disease (NAFLD). To investigate the association between the MBOAT7 rs641738 polymorphism and both hepatic steatosis and biochemical markers of liver damage and to evaluate the potential additive effect of this variant and the I148M patatin-like phospholipase domain-containing 3 (PNPLA3) and the rs58542926 transmembrane 6 superfamily member 2 (TM6SF2) polymorphisms. One thousand and 2 obese children were genotyped for MBOAT7, PNPLA3, and TM6SF2 polymorphisms and underwent anthropometrical, ultrasonographic, and biochemical evaluation. Indirect measurement of liver fibrosis (Pediatric NAFLD Fibrosis Index [PNFI]) and a genetic risk score from these polymorphisms were calculated. Carriers of the MBOAT7 T allele showed both higher alanine transaminase (ALT) (P = 0.004) and PNFI values (P = 0.04) than noncarriers. These findings were confirmed also for the carriers of the MBOAT7 T allele polymorphism with hepatic steatosis compared with noncarriers. A higher genetic risk score was associated with higher ALT (P = 0.011) and with an odds ratio (OR) to show elevated ALT of 3.4 (95% CI 1.3-5.5, P = 0.003). Patients belonging to genetic risk score 3 group had an OR to present steatosis of 2.6 (95% CI 1.43-4.83, P = 0.0018) compared with those belonging to lower genetic risk score group. We first demonstrated in childhood obesity the role of the MBOAT7 rs641738 variant on serum ALT and the combined effect of the MBOAT7, PNPLA3, and TM6SF2 variants on NAFLD risk. We also provided the first pediatric association of the MBOAT7 polymorphism with indirect markers of liver fibrosis.

Discovery of Inhibitor of Wnt Production 2 (IWP-2) and Related Compounds As Selective ATP-Competitive Inhibitors of Casein Kinase 1 (CK1) δ/ε

Inhibitors of Wnt production (IWPs) are known antagonists of the Wnt pathway, targeting the membrane-bound O-acyltransferase porcupine (Porcn) and thus preventing a crucial Wnt ligand palmitoylation. Since IWPs show structural similarities to benzimidazole-based CK1 inhibitors, we hypothesized that IWPs could also inhibit CK1 isoforms. Molecular modeling revealed a plausible binding mode of IWP-2 in the ATP binding pocket of CK1δ which was confirmed by X-ray analysis. In vitro kinase assays demonstrated IWPs to be ATP-competitive inhibitors of wtCK1δ. IWPs also strongly inhibited the gatekeeper mutant M82FCK1δ. When profiled in a panel of 320 kinases, IWP-2 specifically inhibited CK1δ. IWP-2 and IWP-4 also inhibited the viability of various cancer cell lines. By a medicinal chemistry approach, we developed improved IWP-derived CK1 inhibitors. Our results suggest that the effects of IWPs are not limited to Porcn, but also might influence CK1δ/ε-related pathways.

Novel Role for Hedgehog Acyltransferase in the Uptake of Palmitoyl‐CoA into the Endoplasmic Reticulum

Post‐translational attachment of fatty acids to proteins plays a fundamental role in signaling pathways in normal and cancer cells. Most fatty acylated proteins are localized within cells, but a subset are secreted, including Hedgehog and Wnt proteins. Hedgehog proteins, of which Sonic Hedgehog (Shh) is the best studied, are modified by attachment of the 16‐carbon fatty acid palmitate during passage through the secretory pathway. Palmitoylation of Shh is mediated by Hedgehog Acyltransferase (Hhat) and is essential for short and long range Shh signaling in development and tumorigenesis. Hhat is localized to the endoplasmic reticulum (ER) and is a member of the membrane bound‐O‐acyltransferase (MBOAT) family of enzymes. The palmitoylation reaction occurs in the lumen of the ER, and thus palmitoyl‐CoA must be present in the ER lumen for Hhat to serve as a substrate. However, how palmitoyl‐CoA, which is not permeable across membranes, enters the ER lumen remains unknown. In mitochondria, a carnitine palmitoyltransferase (CPT1/2) system shuttles palmitoyl‐CoA across both the inner and outer mitochondrial membranes. We were interested in determining if a similar system exists in the ER or if the CPT1/2 system is shared at ER‐mitochondrial contact sites. Using cell‐based radiolabeling experiments, we showed that Etomoxir, a CPT1 inhibitor, strongly reduced palmitoylation of Shh but had no effect on palmitoylation of the Src family kinase Fyn, which is palmitoylated by cytoplasmic palmitoyl‐CoA. Etomoxir treatment had no effect on the ability of purified Hhat to palmitoylate Shh in vitro. We then developed in vitro assays to monitor palmitoyl‐CoA uptake into the lumen of microsomal membranes and to determine if Hhat plays a role in promoting this process. Using either fluorescent (NBD) or radiolabeled (125I) analogs of palmitoyl‐CoA, we observed a 3‐fold increase in uptake of palmitoyl‐CoA into the lumen of microsomal membranes prepared from cells overexpressing Hhat compared to membranes from cells transfected with empty vector. Increased palmitoyl‐CoA uptake was dependent on Hhat catalytic activity, as it was inhibited by a small molecule Hhat inhibitor and was not observed with H379A, an inactive Hhat mutant. Palmitoyl‐CoA uptake was also reduced after treatment of membranes with Etomoxir, and addition of the CPT1 substrate carnitine counteracted this inhibition. Microsomal membranes from Hhat −/− cells exhibited 2‐fold lower palmitoyl‐CoA uptake, compared to membranes from wild type cells, which supports the hypothesis that endogenous Hhat enhances uptake of this fatty acyl CoA. Taken together, this data suggests that a CPT1/2‐like system exists in the ER, or as part of an ER‐mitochondrial contact site. This system, along with Hhat, promotes uptake of palmitoyl CoA into the ER lumen to enable Hhat to catalyze fatty acylation of Shh. Understanding the mechanism by which fatty acids enter the ER lumen and are attached to proteins involved in cell signaling pathways can potentially reveal novel therapeutic targets in cancers characterized by aberrant signaling pathways.‐‐‐‐‐Support or Funding InformationNIH GM116860This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Discovery and characterization of a potent Wnt and hedgehog signaling pathways dual inhibitor

Embryonic stem cell pathways such as hedgehog and Wnt pathways are central to the tumorigenic properties of cancer stem cells (CSC). Since CSCs are characterized by their ability to self-renew, form differentiated progeny, and develop resistance to anticancer therapies, targeting the Wnt and hedgehog signaling pathways has been an important strategy for cancer treatment. Although molecules targeting either Wnt or hedgehog are common, to the best of our knowledge, those targeting both pathways have not been documented. Here we report a small molecule (compound 1) that inhibits both Wnt (IC50 = 0.5 nM) and hedgehog (IC50 = 71 nM) pathways based on reporter gene assays. We further identified that the molecular target of 1 for Wnt pathway inhibition was porcupine (a member of the membrane-bound O-acyltransferase family of proteins), a post-translational modification node in Wnt signaling; while the target of 1 mitigating hedgehog pathway was Smoothened, a key G protein coupled receptor (GPCR) mediating hedgehog signal transduction. Preliminary analysis of structure-activity-relationship identified key functional elements for hedgehog/Wnt inhibition. In in vivo studies, compound 1 demonstrated good oral exposure and bioavailability while eliciting no overt toxicity in mice. An important consideration in cancer treatment is the potential therapeutic escape through compensatory activation of an interconnected pathway when only one signaling pathway is inhibited. Toward this end, compound 1 may not only lead to the development of new therapeutics for Wnt and hedgehog related cancers, but may also help to develop potential cancer treatment which needs to target Wnt and hedgehog signaling simultaneously.

Genetic determinants of hepatic steatosis and serum cytokeratin-18 fragment levels in Taiwanese children.

There are substantial genetic components contributing to the susceptibility of nonalcoholic fatty liver disease (NAFLD). It has recently been reported that the rs641738 C>T variant in the membrane-bound O-acyltransferase domain-containing protein 7 (MBOAT7) gene increased severity of NAFLD in adults of European descent. We aimed to test the hypothesis that MBOAT7 rs641738 variant would increase hepatic steatosis and hepatocellular injury in obese children. A total of 831 obese children aged 7-15years were recruited. Hepatic steatosis was measured by ultrasonography. Because PNPLA3 rs738409, GCKR rs780094 and TM6SF2 rs58542926 variants are known to confer susceptibility to NAFLD, we assessed the influence of MBOAT7 rs641738 on hepatic steatosis, and serum levels of CK-18 fragment (a biomarker of hepatocellular injury and apoptosis for NAFLD) after adjusting the effects of PNPLA3, GCKR and TM6SF2 polymorphisms. Of the recruited obese children, 22.7% had hepatic steatosis. PNPLA3 rs738409, GCKR rs780094 and TM6SF2 rs58542926 variants were independent risk factors of hepatic steatosis and elevated ALT levels. In contrast, MBOAT7 rs641738 variants, neither heterozygous nor homozygous genotypes, were not associated with hepatic steatosis, insulin resistance, lipid levels and liver enzymes. The multiple linear regression model revealed that after adjusting for age, gender, body mass index z score, PNPLA3 rs738409 and GCKR rs780094 variants, but not MBOAT7 rs641738, were associated with serum levels of CK-18 fragment. The variant MBOAT7 rs641738 genotype is not associated with hepatic steatosis and serum levels of CK-18 fragment in obese Taiwanese children.

Porcupine-dependent Wnt activity within the uterine epithelium is essential for fertility.

The secretion of mammalian Wnt ligands within the cell is dependent on the activity of Porcupine, a gene located on the X-chromosome that encodes for a membrane-bound O-acyl transferase. Here, we report that postnatal ablation of Porcupine in the uterine luminal epithelium alone results in the decrease in endometrial gland number. Despite having uterine glands, mutant females are completely infertile. Epithelial ablation of Porcupine causes defects in timely apposition of the lumen, along with failure to respond to artificial decidual induction. Interestingly, progesterone supplementation was able to rescue the initiation of decidualization, but the decidua was not maintained and subsequently resorbed. Transcriptome analysis demonstrated that deletion of Porcupine in the epithelium resulted in the stromal dysregulation of members of the Wnt signaling pathway (Lef1, Wnt4, and Wnt16), dysregulation of receptors and ligands in the Notch signaling pathway (Notch1, Notch4, and Dll4) as well as Hoxa10. Our results demonstrate the crucial requirement of Wnt signaling in the epithelium for fertility and demonstrate that epithelial Wnts regulate stromal Wnt gene expression as well as regulating the expression of essential signaling factors and effectors required for successful embryo implantation.

Membrane topology and identification of key residues of EaDAcT, a plant MBOAT with unusual substrate specificity.

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol to form 3-acetyl-1,2-diacyl-sn-glycerol (acetyl-TAG). EaDAcT belongs to a small, plant-specific subfamily of the membrane bound O-acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed that EaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)-specific marker. By mapping the membrane topology of EaDAcT, we obtained an experimentally determined topology model for a plant MBOAT. The EaDAcT model contains four transmembrane domains (TMDs), with both the N- and C-termini orientated toward the lumen of the ER. In addition, there is a large cytoplasmic loop between the first and second TMDs, with the MBOAT signature region of the protein embedded in the third TMD close to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl-TAG-producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity invivo and invitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential for EaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity of EaDAcT, suggesting that multiple amino acids are important for substrate recognition.

An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine

Wnt proteins are a family of secreted signaling proteins that play key roles in regulating cell proliferation in both embryonic and adult tissues. Production of active Wnt depends on attachment of palmitoleate, a monounsaturated fatty acid, to a conserved serine by the acyltransferase Porcupine (PORCN). Studies of PORCN activity relied on cell-based fatty acylation and signaling assays as no direct enzyme assay had yet been developed. Here, we present the first in vitro assay that accurately recapitulates PORCN-mediated fatty acylation of a Wnt substrate. The critical feature is the use of a double disulfide-bonded Wnt peptide that mimics the two-dimensional structure surrounding the Wnt acylation site. PORCN-mediated Wnt acylation was abolished when the Wnt peptide was treated with DTT, and did not occur with a linear (non-disulfide-bonded) peptide, or when the double disulfide-bonded Wnt peptide contained Ala substituted for the Ser acylation site. We exploited this in vitro Wnt acylation assay to provide direct evidence that the small molecule LGK974, which is in clinical trials for managing Wnt-driven tumors, is a bona fide PORCN inhibitor whose IC50 for inhibition of Wnt fatty acylation in vitro closely matches that for inhibition of Wnt signaling. Side-by-side comparison of PORCN and Hedgehog acyltransferase (HHAT), two enzymes that attach 16-carbon fatty acids to secreted proteins, revealed that neither enzyme will accept the other's fatty acyl-CoA or peptide substrates. These findings illustrate the unique enzyme-substrate selectivity exhibited by members of the membrane-bound O-acyl transferase family.

Novel porcupine (PORCN) inhibitor RXC004: Evaluation in models of RNF43 loss of function cancers.

e14094 Background: Wnt signalling initiates oncogenic pathways involved in tumour initiation, growth, differentiation and metastasis.1 Targeting the Wnt pathway is an attractive approach to cancer treatment. Porcupine (PORCN) is a membrane-bound O-acyltransferase dedicated to the palmitoylation of Wnt ligands, an essential step in the processing of Wnt into active ligands.2 A PORCN inhibitor may benefit patients with cancers in which Wnt signalling is implicated. It has been shown that PORCN inhibition is efficacious in cell lines with upstream mutations in this pathway, eg RNF43 loss of function.3 Methods: Analysis of TCGA data was carried out to identify cancer types harbouring RNF43 mutations.4 Redx PORCN inhibitor RXC004 was evaluated in vitro for Wnt pathway inhibition and anti-proliferative effects using assays previously described for early lead compounds.5Anti-tumour effects of RXC004 were evaluated using a pancreatic CAPAN-2 xenograft in SCID Bg. mice and a gastric PDX model in nu/nu mice. Both models have RNF43 loss of function mutations. Results: TCGA data revealed that both gastric cancer and pancreatic cancer exhibit mutations in the RNF43 gene (18% and 4% respectively),4expected to result in loss of function. RXC004 showed activity in preclinical models of cancers with this genetic background. RXC004 significantly inhibited tumour growth in a CAPAN-2 pancreatic cancer xenograft and in a gastric cancer PDX model when dosed orally once or twice daily. Conclusions: Taken together, these data indicate that RXC004 is a potent inhibitor of tumour growth in RNF43 loss of function preclinical cancer models. RXC004 will be evaluated in a first in human Phase 1 study in 2017. Phase 1a aims are to characterise the safety profile and PK/PD relationship in cancer patients and establish a Phase 1b dose. The efficacy of RXC004 in gastric and pancreatic cancer patients prospectively selected for RNF43 loss of function mutations will be the focus of Phase 1b expansion groups. 1 . Nat. Rev. Cancer, 2013, 13 (1): 11-26.2. J. Biol. Chem., 2012, 287 (27): 23246-23254. 3. PNAS, 2013, 110 (31): 12649-12654 4. Sci. Signal, 2013, 269 (6): 1-34; Cancer Discov., 2012, 2 (5): 401-404 5. AACR Annual Meeting, 2015, 75 (15): abs. 5071.

MG56, A Membrane Bound O-Acyltransferase Protein, Regulates Lipid Composition and Membrane Vesicle Size in Skeletal Muscle

The human genome encodes 11 genes belonging to the membrane bound O-acyltransferase (MBOAT) family of proteins. MBOAT family proteins are known for containing a large number of transmembrane domains and exhibiting acyltransferase enzymatic activity. While collectively these proteins cover a broad-spectrum of acylation, substrates ranging from phospholipids to peptides, each member possesses specificity with regards to the target of acylation and the lipid species utilized. Mitsugumin 56 (MG56), also known as hedgehog acyltransferase-like protein, is an MBOAT protein predominantly expressed in cardiac and skeletal muscle. MG56 resides along the terminal cisternea of the Sarcoplasmic Reticulum (SR) in striated muscle. Ablation of MG56 in mice results in seemingly normal pups at birth, however severe growth retardation is observed around day 7, shortly followed by death under starvation conditions. Skeletal muscle from knockout mice displays swollen SR and dilated vacuoles within sarcomeres, however this phenomenon is not seen in cardiac tissue. Lipid analysis of skeletal muscle via matrix-assisted laser desorption/ionization fourier transform ion cyclotron resonance mass spectrometry (MALDI FT-ICR) shows a larger abundance of phospholipids in knockout muscle compared to wild type. Interestingly, knockout muscle also shows an increased abundance of high molecular mass phospholipids, indicative of tri-acyl phospholipids. Tri-acylated phospholipids, such as N-acyl phosphatidylethanolamine (NAPE), have an additional acyl chain attached to the phosphate group, and have been shown to alter membrane size and serve as precursors to N-acyl ethanolamine (NAE), a hormone shown to be an appetite suppressant in rodents. It is our hypothesis that MG56 functions to negatively regulate NAPE production, thereby fostering normal muscle development. We are working to identify the high molecular mass lipids seen in knockout skeletal muscle in hopes of determining the substrate of MG56.

Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase.

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. Invitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. Invitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols invitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants.

The Secreted Signaling Protein Wnt3 Is Associated with Membrane Domains In Vivo: A SPIM-FCS Study

Wnt3 is a morphogen that activates the Wnt signaling pathway and regulates a multitude of biological processes ranging from cell proliferation and cell fate specification to differentiation over embryonic induction to neural patterning. Recent studies have shown that the palmitoylation of Wnt3 by Porcupine, a membrane-bound O-acyltransferase, plays a significant role in the intracellular membrane trafficking of Wnt3 and subsequently, its secretion in live zebrafish embryos, where chemical inhibition of Porcupine reduced the membrane-bound and secreted fractions of Wnt3 and eventually led to defective brain development. However, the membrane distribution of Wnt3 in cells remains not fully understood. Here, we determine the membrane organization of functionally active Wnt3-EGFP in cerebellar cells of live transgenic zebrafish embryos and the role of palmitoylation in its organization using single plane illumination microscopy-fluorescence correlation spectroscopy (SPIM-FCS), a multiplexed modality of FCS, which generates maps of molecular dynamics, concentration, and interaction of biomolecules. The FCS diffusion law was applied to SPIM-FCS data to study the subresolution membrane organization of Wnt3. We find that at the plasma membrane in vivo, Wnt3 is associated with cholesterol-dependent domains. This association reduces with increasing concentrations of Porcupine inhibitor (C59), confirming the importance of palmitoylation of Wnt3 for its association with cholesterol-dependent domains. Reduction of membrane cholesterol also results in a decrease of Wnt3 association with cholesterol-dependent domains in live zebrafish. This demonstrates for the first time, to our knowledge, in live vertebrate embryos that Wnt3 is associated with cholesterol-dependent domains.

PNPLA3 p.I148M variant is associated with greater reduction of liver fat content after bariatric surgery

BackgroundObesity is the major trigger of nonalcoholic fatty liver disease (NAFLD). NAFLD is further favored by the patatin-like phospholipase domain-containing 3 (PNPLA3) p.I148M, transmembrane 6 superfamily member 2 (TM6SF2) p.E167K, and membrane-bound O-acyltransferase domain containing 7 (MBOAT7) rs641738 variants. ObjectivesTo investigate the relationship between the PNPLA3, TM6SF2, and MBOAT7 genotypes and the outcomes of bariatric surgery. SettingUniversity hospital. MethodsProspectively we monitored 84 obese individuals (body mass index 35–64 kg/m2) scheduled for bariatric surgery. The PNPLA3 p.I148M, TM6SF2 p.E167K, and MBOAT7 rs641738 variants were genotyped using restriction fragment length polymorphism analysis and TaqMan assays. Hepatic steatosis was determined before surgery using analysis of liver biopsy samples and a novel magnetic resonance imaging–based equation. One year later, steatosis was reevaluated by magnetic resonance imaging. ResultsThe presence of the PNPLA3 allle [M] was associated with increased hepatic triglyceride content (P = .03), steatosis detected by magnetic resonance imaging (P = 0.04), and decreased serum glucose concentrations (P = .04). Neither variant TM6SF2 nor MBOAT7 increased hepatic steatosis (all P>.05); however, the MBOAT7 polymorphism was associated with increased triglyceride, total cholesterol, low density lipoprotein, and serum glucose levels (all P<.05). Patients carrying the prosteatotic PNPLA3 allele [M] lost more weight (P<.01) and liver fat (P = .04) one year after surgery, as compared to individuals having the common genotype. The PNPLA3 genotype and initial grade of steatosis, but not the TM6SF2 or MBOAT7 variants, were independent predictors of NAFLD improvement (P = .03 and P<.01, respectively). ConclusionIn obese patients, the presence of the PNPLA3 p.I148M allele might be associated with greater improvement of hepatic steatosis after bariatric surgery in comparison to carriers of PNPLA3 wild-type alleles.

Discovery of Pyridinyl Acetamide Derivatives as Potent, Selective, and Orally Bioavailable Porcupine Inhibitors.

Blockade of aberrant Wnt signaling is an attractive therapeutic approach in multiple cancers. We developed and performed a cellular high-throughput screen for inhibitors of Wnt secretion and pathway activation. A lead structure (GNF-1331) was identified from the screen. Further studies identified the molecular target of GNF-1331 as Porcupine, a membrane bound O-acyl transferase. Structure-activity relationship studies led to the discovery of a novel series of potent and selective Porcupine inhibitors. Compound 19, GNF-6231, demonstrated excellent pathway inhibition and induced robust antitumor efficacy in a mouse MMTV-WNT1 xenograft tumor model.

Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons.

Glycerophospholipids, which are components of biomembranes, are formed de novo by the Kennedy pathway and subsequently mature through the Lands cycle. Lysophospholipid acyltransferases (LPLATs) are key enzymes in both pathways and influence the fatty acid composition of biomembranes. Neuronal differentiation is characterized by neurite outgrowth, which requires biomembrane biosynthesis. However, the role of LPLATs in neuronal differentiation remains unknown. In this study, we examined whether LPLATs are involved in neuronal differentiation using all-trans-retinoic acid (ATRA)-treated P19C6 cells. In these cells, mRNA levels of lysophosphatidylethanolamine acyltransferase (LPEAT)-1/membrane-bound O-acyltransferase (MBOAT)-1 were higher than those in undifferentiated cells. LPEAT enzymatic activity increased with 16:0- and 18:1-CoA as acyl donors. When LPEAT1/MBOAT1 was knocked down with small interfering RNA (siRNA), outgrowth of neurites and expression of neuronal markers decreased in ATRA-treated P19C6 cells. Voltage-dependent calcium channel activity was also suppressed in these cells transfected with LPEAT1/MBOAT1 siRNA. These results suggest that LPEAT1/MBOAT1 plays an important role in neurite outgrowth and function.-Tabe, S., Hikiji, H., Ariyoshi, W., Hashidate-Yoshida, T., Shindou, H., Okinaga, T., Shimizu, T., Tominaga, K., Nishihara, T. Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons.

Abstract B11: Porcupine inhibition stimulates osteosarcoma growth and non-canonical Wnt signaling

Abstract Purpose: The objective of this study is to characterize the function of porcupine inhibitors in osteosarcoma and to determine the contribution of the porcupine enzyme to osteosarcoma cell growth. Each year, approximately 1000 children and adults in the United States are diagnosed with osteosarcoma (OS), the most common primary bone malignancy among children. Although overall survival is relatively good in OS patients, the need for aggressive surgery (often involving limb amputation) to prevent relapse significantly impacts the quality of life of survivors. OS has been well characterized to display aberrantly activated Wnt signaling. The necessity for active Wnt signaling pathways in OS patient tissue and cultured cell lines has been well established. Recently, a group of Wnt pathway inhibitors targeting the porcupine enzyme have been developed and used in treatment of pancreatic adenocarcinoma, BRAF mutant colorectal cancer and other tumors reported to exhibit aberrant Wnt signaling. Porcupine is a membrane bound O-acyl transferase which lipid-modifies the Wnt proteins prior to their secretion. This processing step regulates their release and paracrine or autocrine activity in stimulating both canonical and non-canonical Wnt signaling. These drugs have proven effective in initial studies which have applied them in vitro, in mouse models of Wnt- dependent cancers, and a recent phase 1 clinical trial. Our data show the first report of increased porcupine enzyme expression in human OS in vitro cell lines, compared to primary human osteoblasts. These findings have been confirmed by quantitative real-time PCR (qRT-PCR) analysis of PPN mRNA expression. Surprisingly, we found that inhibition of the porcupine enzyme with newly developed porcupine inhibitors significantly increased OS cell viability. While we observed only slight decreases in canonical Wnt pathway signaling with PPN inhibitor treatment (as measured by TOPflash reporter assay), we found that both NFATc1 and Cyclin D1 mRNA expression was upregulated. These data indicate that treatment with PPN inhibitors may stimulate a pro-tumorigenic response that is mediated primarily through non-canonical Wnt signaling. Continuing work is being done to establish the mechanism of action of these drugs, as well as unique roles that PPN-mediated non-canonical Wnt signaling may play in promoting OS cell growth. In conclusion, this study is aimed to provide insight into the function of a group of novel Wnt-targeting therapies in osteosarcoma, a disease which is well established to exhibit aberrant Wnt signaling. We aim to utilize these insights to rationally exploit Wnt signaling as a therapeutic measure for osteosarcoma. Methods: Western blotting was performed according to accepted methods using BioRad Criterion™ 10% gel and an antibody polyclonal to known porcupine splice variants (anti-rabbit) obtained from GeneTex. Cell viability was determined by CellTiter 96 ® (Promega) proliferation assay. C59 (Novartis) was purchased from Cayman Chemical and reconstituted in DMSO. Quantitative PCR performed using an Applied Biosystems ABI 7900HT Fast Real Time PCR system. FuGENE® HD utilized for transfection of TOPflash components. Citation Format: Carl T. Gustafson, Avudai Maran, Michael J. Yaszemski. Porcupine inhibition stimulates osteosarcoma growth and non-canonical Wnt signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B11.

Progress in Small Molecule and Biologic Therapeutics Targeting Ghrelin Signaling.

Ghrelin is a circulating peptide hormone involved in regulation of a wide array of physiological processes. As an endogenous ligand for growth hormone secretagogue receptor (GHSR1a), ghrelin is responsible for signaling involved in energy homeostasis, including appetite stimulation, glucose metabolism, insulin signaling, and adiposity. Ghrelin has also been implicated in modulation of several neurological processes. Dysregulation of ghrelin signaling is implicated in diseases related to these pathways, including obesity, type II diabetes, and regulation of appetite and body weight in patients with Prader-Willi syndrome. Multiple steps in the ghrelin signaling pathway are available for targeting in the development of therapeutics for these diseases. Agonists and antagonists of GHS-R1a have been widely studied and have shown varying levels of effectiveness within ghrelin-related physiological pathways. Agents targeting ghrelin directly, either through depletion of ghrelin levels in circulation or inhibitors of ghrelin O-acyltransferase whose action is required for ghrelin to become biologically active, are receiving increasing attention as potential therapeutic options. We discuss the approaches utilized to target ghrelin signaling and highlight the current challenges toward developing small-molecule agents as potential therapeutics for ghrelin-related diseases.

Congenital protein losing enteropathy: an inborn error of lipid metabolism due to DGAT1 mutations.

Protein-losing enteropathy (PLE) is a clinical disorder of protein loss from the gastrointestinal system that results in hypoproteinemia and malnutrition. This condition is associated with a wide range of gastrointestinal disorders. Recently, a unique syndrome of congenital PLE associated with biallelic mutations in the DGAT1 gene has been reported in a single family. We hypothesize that mutations in this gene are responsible for undiagnosed cases of PLE in infancy. Here we investigated three children in two families presenting with severe diarrhea, hypoalbuminemia and PLE, using clinical studies, homozygosity mapping, and exome sequencing. In one family, homozygosity mapping using SNP arrays revealed the DGAT1 gene as the best candidate gene for the proband. Sequencing of all the exons including flanking regions and promoter regions of the gene identified a novel homozygous missense variant, p.(Leu295Pro), in the highly conserved membrane-bound O-acyl transferase (MBOAT) domain of the DGAT1 protein. Expression studies verified reduced amounts of DGAT1 in patient fibroblasts. In a second family, exome sequencing identified a previously reported splice site mutation in intron 8. These cases of DGAT1 deficiency extend the molecular and phenotypic spectrum of PLE, suggesting a re-evaluation of the use of DGAT1 inhibitors for metabolic disorders including obesity and diabetes.

Abstract IA35: Discovery of porcupine inhibitors targeting Wnt signaling in cancer

Abstract Wnt signaling is tightly controlled during cellular proliferation, differentiation and embryonic morphogenesis. Aberrant activation of this pathway plays a critical role in a variety of cancers. Blockade of Wnt signaling is therefore an attractive therapeutic approach for anticancer therapy. In this presentation, we will discuss our approach to search for inhibitors of Wnt ligand secretion. We developed and performed a cellular high-throughput screen using a co-culture system. Lead structure (GNF-1331) was identified and further target elucidation revealed Porcupine, a membrane bound O-acyl transferase, as its molecular target. Further structure-activity relationship studies led to the discovery of WNT974, a potent and specific Porcupine inhibitor. Treatment of WNT974 leads to tumor regression in a Wnt dependent MMTV-Wnt1 mouse model at well tolerated doses. WNT974 is currently in Phase 1 clinical trials. Citation Format: Shifeng Pan, Jun Liu, Dai Cheng, Dong Han, Guobao Zhang, Mindy Hsieh, Nicholas Ng, Chun Li, Shailaja Kasibhatla, Peter McNamara, H. Martin Seidel, Jennifer Harris. Discovery of porcupine inhibitors targeting Wnt signaling in cancer. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA35.

The Secreted Signaling Protein Wnt3 Resides in Plasma Membrane Lipid Domains in Vivo: A SPIM-FCS Study

Wnt3 is a signaling protein involved in development and disease (oncogenesis and tetraamelia syndrome). Recently, we have shown that Wnt3 regulates cerebellum development using zebrafish Wnt3 transgenics with either tissue-specific expression of an EGFP reporter or a functionally active fusion protein, Wnt3EGFP (Teh et al., Development 2015). We identified several fractions of Wnt3: i) an intracellular and ii) a secreted fraction, which comprises of monomeric Wnt3 as well as its complexes, and, iii) a membrane-bound fraction. The membrane-bound O-acyltransferase Porcupine is required for Wnt palmitoylation, secretion and biological activity. It was shown in vitro that for membrane localization in Wnt3-expressing cells Wnt3 undergoes two lipid modifications by palmitoylation at C77 and S209. Such posttranslational modifications have been linked with protein association with cholesterol-dependent lipid domains. This raises the question whether Wnt3 is targeted to these domains in live zebrafish. We therefore used SPIM-FCS and the FCS diffusion laws, which characterize the dependence of the diffusion coefficient on the observed area to deduce deviations from free diffusion, to characterize Wnt3 distribution on plasma membranes in zebrafish embryos.First, we demonstrate that the diffusion laws can be applied in a 3D environment of SPIM-FCS by characterizing lipid distributions in giant unilamellar vesicles (GUVs) of various compositions. Second, we applied this methodology to analyze Wnt3 distribution on plasma membranes in live Wnt3 transgenic embryos. This demonstrated that in vivo the Wnt3 membrane-bound fraction also shows consistent raft partitioning. Third, upon reduction of Wnt3 secretion by a specific inhibitor of Porcupine, Wnt3 partitioning into lipid domains is also reduced. Taken together this demonstrates that in developing zebrafish embryos the plasma membrane fraction of Wnt3 to be secreted needs to be compartmentalized into specialized lipid domains.