Hedgehog acyltransferase catalyzes a random sequential reaction and utilizes multiple fatty acyl-CoA substrates.

Sonic hedgehog (Shh) signaling is a key component of embryonic development and is a driving force in several cancers. Hedgehog acyltransferase (Hhat), a member of the membrane-bound O-acyltransferase family of enzymes, catalyzes the attachment of palmitate to the N-terminal cysteine of Shh, a posttranslation modification critical for Shh signaling. The activity of Hhat has been assayed in cells and in vitro, and cryo-EM structures of Hhat have been reported, yet several unanswered questions remain regarding the enzyme's reaction mechanism, substrate specificity, and the impact of the latter on Shh signaling. Here, we present an in vitro acylation assay with purified Hhat that directly monitors attachment of a fluorescently tagged fatty acyl chain to Shh. Our kinetic analyses revealed that the reaction catalyzed by Hhat proceeds through a random sequential mechanism. We also determined that Hhat can utilize multiple fatty acyl-CoA substrates for fatty acid transfer to Shh, with comparable affinities and turnover rates for myristoyl-CoA, palmitoyl-CoA, palmitoleoyl-CoA, and oleoyl-CoA. Furthermore, we investigated the functional consequence of differential fatty acylation of Shh in a luciferase-based Shh reporter system. We found that the potency of the signaling response in cells was higher for Shh acylated with saturated fatty acids compared to monounsaturated fatty acids. These findings demonstrate that Hhat can attach fatty acids other than palmitate to Shh and suggest that heterogeneous fatty acylation has the potential to impact Shh signaling in the developing embryo and/or cancer cells.

A Direct in vitro Fatty Acylation Assay for Hedgehog Acyltransferase.

Several assays have been developed to monitor the in vitro catalytic activity of Hedgehog acyltransferase (Hhat), an enzyme critical to the Hedgehog signaling pathway in cells. However, the majority of these previously reported assays involve radioactive fatty acyl donor substrates, multiple steps to achieve product readout, or specialized equipment. To increase safety, efficiency, and convenience, we developed a direct, fluorescent in vitro assay to monitor Hhat activity. Our assay utilizes purified Hhat, a fluorescently labeled fatty acyl-CoA donor substrate, and a Sonic hedgehog (Shh) peptide recipient substrate sufficient for fatty acylation. The protocol is a straightforward process that yields direct readout of fatty acylated Shh peptide via fluorescence detection of the transferred fatty acyl group. This protocol was validated in: J Biol Chem (2022), DOI: 10.1016/j.jbc.2022.102422 Graphical abstract Graphical abstract adapted from Schonbrun and Resh (2022).

Open Biology: overview for special issue on dynamics of protein fatty acylation.

Fatty acylation is a widespread form of protein modification that occurs on specific intracellular and secreted proteins. Beyond increasing hydrophobicity and the affinity of the modified protein for lipid bilayers, covalent attachment of a fatty acid exerts effects on protein localization, inter- and intramolecular interactions and signal transduction. As such, research into protein fatty acylation has been embraced by an extensive community of biologists. This special issue highlights advances at the forefront of the field, by focusing on two families of enzymes that catalyse post-translational protein fatty acylation, zDHHC palmitoyl acyltransferases and membrane-bound O-acyl transferases, and signalling pathways regulated by their fatty acylated protein substrates. The collected contributions catalogue the tremendous progress that has been made in enzyme and substrate identification. In addition, articles in this special issue provide insights into the pivotal functions of fatty acylated proteins in immune cell, insulin and EGF receptor-mediated signalling pathways. As selective inhibitors of protein fatty acyltransferases are generated, the future holds great promise for therapeutic targeting of fatty acyltransferases that play key roles in human disease.

Palmitoylation of Hedgehog proteins by Hedgehog acyltransferase: roles in signalling and disease.

Hedgehog acyltransferase (Hhat), a member of the membrane-bound O-acyltransferase (MBOAT) family, catalyses the covalent attachment of palmitate to the N-terminus of Hedgehog proteins. Palmitoylation is a post-translational modification essential for Hedgehog signalling. This review explores the mechanisms involved in Hhat acyltransferase enzymatic activity, similarities and differences between Hhat and other MBOAT enzymes, and the role of palmitoylation in Hedgehog signalling. In vitro and cell-based assays for Hhat activity have been developed, and residues within Hhat and Hedgehog essential for palmitoylation have been identified. In cells, Hhat promotes the transfer of palmitoyl-CoA from the cytoplasmic to the luminal side of the endoplasmic reticulum membrane, where Shh palmitoylation occurs. Palmitoylation is required for efficient delivery of secreted Hedgehog to its receptor Patched1, as well as for the deactivation of Patched1, which initiates the downstream Hedgehog signalling pathway. While Hhat loss is lethal during embryogenesis, mutations in Hhat have been linked to disease states or abnormalities in mice and humans. In adults, aberrant re-expression of Hedgehog ligands promotes tumorigenesis in an Hhat-dependent manner in a variety of different cancers, including pancreatic, breast and lung. Targeting hedgehog palmitoylation by inhibition of Hhat is thus a promising, potential intervention in human disease.

Hedgehog Acyltransferase Promotes Uptake of Palmitoyl-CoA across the Endoplasmic Reticulum Membrane.

Attachment of palmitate to the N terminus of Sonic hedgehog (Shh) is essential for Shh signaling. Shh palmitoylation is catalyzed on the luminal side of the endoplasmic reticulum (ER) by Hedgehog acyltransferase (Hhat), an ER-resident enzyme. Palmitoyl-coenzyme A (CoA), the palmitate donor, is produced in the cytosol and is not permeable across membrane bilayers. It is not known how palmitoyl-CoA crosses the ER membrane to access the active site of Hhat. Here, we use fluorescent and radiolabeled palmitoyl-CoA probes to demonstrate that Hhat promotes the uptake of palmitoyl-CoA across the ER membrane in microsomes and semi-intact cells. Reconstitution of purified Hhat into liposomes provided further evidence that palmitoyl-CoA uptake activity is an intrinsic property of Hhat. Palmitoyl-CoA uptake was regulated by and could be uncoupled from Hhat enzymatic activity, implying that Hhat serves a dual function as a palmitoyl acyltransferase and a conduit to supply palmitoyl-CoA to the luminal side of the ER.

In Vitro Analysis of Hedgehog Acyltransferase and Porcupine Fatty Acyltransferase Activities.

Hedgehog and Wnt proteins are modified by covalent attachment of the fatty acids palmitate and palmitoleate, respectively. These lipid modifications are essential for Hedgehog and Wnt protein signaling activities and are catalyzed by related, but distinct fatty acyltransferases: Hedgehog acyltransferase (Hedgehog) and Porcupine (Wnt). In this chapter, we provide detailed methods to directly monitor Hedgehog and Wnt protein fatty acylation in vitro. Palmitoylation of Sonic hedgehog (Shh), a representative Hedgehog family member, is assayed using purified Hedgehog acyltransferase (Hhat) or Hhat-enriched membranes, a recombinant 19 kDa Shh protein or C-terminally biotinylated Shh 10-mer peptide, and 125I-iodopalmitoyl CoA as the donor fatty acyl CoA substrate. The radiolabeled reaction products are quantified by SDS-PAGE and phosphorimaging or by γ-counting. To assay Wnt acylation, the reaction consists of a biotinylated, double disulfide-bonded Wnt peptide containing the sequence surrounding the Wnt3a acylation site, [125I] iodo-cis-9-pentadecenoyl CoA, and Porcupine-enriched membranes. Radiolabeled, biotinylated Wnt3a peptide is captured on streptavidin coated beads and the reaction product is quantified by γ-counting.

Specification of positional identity in forebrain organoids.

Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.

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.

Palmitoylation of proteins in cancer.

Post-translational modification of proteins by attachment of palmitate serves as a mechanism to regulate protein localization and function in both normal and malignant cells. Given the essential role that palmitoylation plays in cancer cell signaling, approaches that target palmitoylated proteins and palmitoyl acyltransferases (PATs) have the potential for therapeutic intervention in cancer. Highlighted here are recent advances in understanding the importance of protein palmitoylation in tumorigenic pathways. A new study has uncovered palmitoylation sites within the epidermal growth factor receptor that regulate receptor trafficking, signaling and sensitivity to tyrosine kinase inhibitors. Global data analysis from nearly 150 cancer studies reveals genomic alterations in several PATs that may account for their ability to function as tumor suppressors or oncogenes. Selective inhibitors have recently been developed that target hedgehog acyltransferase (Hhat) and Porcupine (Porcn), the acyltransferases that modify hedgehog and Wnt proteins, respectively. These inhibitors, coupled with targeted knockdown of Hhat and Porcn, reveal the essential functions of fatty acylation of secreted morphogens in a wide variety of human tumors.

HO-2 Pockets Myristoylated Gag.

Membrane binding of viral and cellular N-myristoylated proteins can be regulated by selectively sequestering myristate. In this issue of Cell Host & Microbe, Zhu et al. (2017) report on a myristate binding site within the cellular protein heme oxygenase-2 that acts as a trap to inhibit N-myristoylated protein function.

Fatty acylation of proteins: The long and the short of it.

Long, short and medium chain fatty acids are covalently attached to hundreds of proteins. Each fatty acid confers distinct biochemical properties, enabling fatty acylation to regulate intracellular trafficking, subcellular localization, protein-protein and protein-lipid interactions. Myristate and palmitate represent the most common fatty acid modifying groups. New insights into how fatty acylation reactions are catalyzed, and how fatty acylation regulates protein structure and function continue to emerge. Myristate is typically linked to an N-terminal glycine, but recent studies reveal that lysines can also be myristoylated. Enzymes that remove N-terminal myristoyl-glycine or myristate from lysines have now been identified. DHHC proteins catalyze S-palmitoylation, but the mechanisms that regulate substrate recognition by individual DHHC family members remain to be determined. New studies continue to reveal thioesterases that remove palmitate from S-acylated proteins. Another area of rapid expansion is fatty acylation of the secreted proteins hedgehog, Wnt and Ghrelin, by Hhat, Porcupine and GOAT, respectively. Understanding how these membrane bound O-acyl transferases recognize their protein and fatty acyl CoA substrates is an active area of investigation, and is punctuated by the finding that these enzymes are potential drug targets in human diseases.

Hedgehog Acyltransferase as a target in estrogen receptor positive, HER2 amplified, and tamoxifen resistant breast cancer cells.

BACKGROUND: Hedgehog acyltransferase (Hhat) catalyzes the transfer of the fatty acid palmitate onto Sonic Hedgehog (Shh), a modification that is essential for Shh signaling activity. The Shh signaling pathway has been implicated in the progression of breast cancer. METHODS: To determine the functional significance of Hhat expression in breast cancer, we used a panel of breast cancer cell lines that included estrogen receptor (ER) positive, HER2 amplified, triple negative, and tamoxifen resistant cells. We monitored both anchorage dependent and independent proliferation of these cells following depletion of Hhat with lentiviral shRNA and inhibition of Hhat activity with RU-SKI 43, a small molecule inhibitor of Hhat. RESULTS: Depletion of Hhat decreased anchorage-dependent and anchorage-independent proliferation of ER positive, but not triple negative, breast cancer cells. Treatment with RU-SKI 43 also reduced ER positive cell proliferation, whereas a structurally related, inactive compound had no effect. Overexpression of Hhat in ER positive cells not only rescued the growth defect in the presence of RU-SKI 43 but also resulted in increased cell proliferation in the absence of drug. Furthermore, depletion or inhibition of Hhat reduced proliferation of HER2 amplified as well as tamoxifen resistant cells. Inhibition of Smoothened had no effect on proliferation, indicating that canonical Shh signaling was not operative. Moreover, Hhat regulated the proliferation of both Shh responsive and non-responsive ER positive cells, suggesting a Shh independent function for Hhat. CONCLUSIONS: These data suggest that Hhat plays a critical role in ER positive, HER2 amplified, and hormone resistant breast cancer proliferation and highlights the potential promise of Hhat inhibitors for therapeutic benefit in breast cancer.

Membrane topology of hedgehog acyltransferase.

Hedgehog acyltransferase (Hhat) is a multipass transmembrane enzyme that mediates the covalent attachment of the 16-carbon fatty acid palmitate to the N-terminal cysteine of Sonic Hedgehog (Shh). Palmitoylation of Shh by Hhat is critical for short and long range signaling. Knowledge of the topological organization of Hhat transmembrane helices would enhance our understanding of Hhat-mediated Shh palmitoylation. Bioinformatics analysis of transmembrane domains within human Hhat using 10 different algorithms resulted in highly consistent predictions in the C-terminal, but not in the N-terminal, region of Hhat. To empirically determine the topology of Hhat, we designed and exploited Hhat constructs containing either terminal or 12 different internal epitope tags. We used selective permeabilization coupled with immunofluorescence as well as a protease protection assay to demonstrate that Hhat contains 10 transmembrane domains and 2 re-entrant loops. The invariant His and highly conserved Asp residues within the membrane-bound O-acyltransferase (MBOAT) homology domain are segregated on opposite sides of the endoplasmic reticulum membrane. The localization of His-379 on the lumenal membrane surface is consistent with a role for this invariant residue in catalysis. Analysis of the activity and stability of the Hhat constructs revealed that the C-terminal MBOAT domain is especially sensitive to manipulation. Moreover, there was remarkable similarity in the overall topological organization of Hhat and ghrelin O-acyltransferase, another MBOAT family member. Knowledge of the topological organization of Hhat could serve as an important tool for further design of selective Hhat inhibitors.

Identification of key residues and regions important for porcupine-mediated Wnt acylation.

Wnts comprise a family of lipid-modified, secreted signaling proteins that control embryogenesis, as well as tissue homeostasis in adults. Post-translational attachment of palmitoleate (C16:1) to a conserved Ser in Wnt proteins is catalyzed by Porcupine (Porcn), a member of the membrane bound O-acyltransferase (MBOAT) family, and is required for Wnt secretion and signaling. Moreover, genetic alterations in the PORCN gene lead to focal dermal hypoplasia, an X-linked developmental disorder. Despite its physiological importance, the biochemical mechanism governing Wnt acylation by Porcn is poorly understood. Here, we use a cell-based fatty acylation assay that is a direct readout of Porcn acyltransferase activity to perform structure-function analysis of highly conserved residues in Porcn and Wnt3a. In total, 16-point mutations in Porcn and 13 mutations in Wnt3a were generated and analyzed. We identified key residues within Porcn required for enzymatic activity, stability, and Wnt3a binding and mapped these active site residues to predicted transmembrane domain 9. Analysis of focal dermal hypoplasia-associated mutations in Porcn revealed that loss of enzymatic activity arises from altered stability. A consensus sequence within Wnt3a was identified (CXCHGXSXXCXXKXC) that contains residues that mediate Porcn binding, fatty acid transfer, and Wnt signaling. We also showed that Ser or Thr, but not Cys, can serve as a fatty acylation site in Wnt, establishing Porcn as an O-acyltransferase. This analysis sheds light into the mechanism by which Porcn transfers fatty acids to Wnt proteins and provides insight into the mechanisms of fatty acid transfer by MBOAT family members.

Loss of function mutation in the palmitoyl-transferase HHAT leads to syndromic 46,XY disorder of sex development by impeding Hedgehog protein palmitoylation and signaling.

The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.

Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins.

Wnt proteins contain palmitoleic acid, an unusual lipid modification. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on the attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here, we show that stearoyl desaturase (SCD) generates a monounsaturated fatty acid substrate that is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation.

Inhibitors of Hedgehog acyltransferase block Sonic Hedgehog signaling.

Inhibition of Sonic hedgehog (Shh) signaling is of great clinical interest. Here we exploit Hedgehog acyltransferase (Hhat)-mediated Shh palmitoylation, a modification critical for Shh signaling, as a new target for Shh pathway inhibition. A target-oriented high-throughput screen was used to identify small-molecule inhibitors of Hhat. In cells, these Hhat inhibitors specifically block Shh palmitoylation and inhibit autocrine and paracrine Shh signaling.

Identification of N-terminal residues of Sonic Hedgehog important for palmitoylation by Hedgehog acyltransferase.

Sonic Hedgehog (Shh) is a secreted morphogen that regulates embryonic development. After removal of the signal peptide, Shh is processed to the mature, active form through autocleavage and a series of lipid modifications, including the attachment of palmitate. Covalent attachment of palmitate to the N-terminal cysteine of Shh is catalyzed by Hedgehog acyltransferase (Hhat) and is critical for proper signaling. The sequences within Shh that are responsible for palmitoylation by Hhat are not known. Here we show that the first six amino acids of mature Shh (CGPGRG) are sufficient for Hhat-mediated palmitoylation. Alanine scanning mutagenesis was used to determine the role of each amino acid and the positional sequence requirement in a cell-based Shh palmitoylation assay. Mutation of residues in the GPGR sequence to Ala had no effect on palmitoylation, provided that a positively charged residue was present within the first seven residues. The N-terminal position exhibited a strong but not exclusive requirement for Cys. Constructs with an N-terminal Ala were not palmitoylated. However, an N-terminal Ser served as a substrate for Hhat, but not the Drosophila melanogaster ortholog Rasp, highlighting a critical difference between the mammalian and fly enzymes. These findings define residues and regions within Shh that are necessary for its recognition as a substrate for Hhat-mediated palmitoylation. Finally, we report the results of a bioinformatics screen to identify other potential Hhat substrates encoded in the human genome.

Palmitoylation of Hedgehog proteins.

Hedgehog (Hh) proteins are secreted signaling proteins that contain amide-linked palmitate at the N-terminus and cholesterol at the C-terminus. Palmitoylation of Hh proteins is critical for effective long- and short-range signaling. The palmitoylation reaction occurs during transit of Hh through the secretory pathway, most likely in the lumen of the ER. Attachment of palmitate to Hh proteins is independent of cholesterol modification and autoprocessing and is catalyzed by Hhat (Hedgehog acyltransferase). Hhat is a member of the membrane bound O-acyltransferase (MBOAT) family, a subgroup of multipass membrane proteins that catalyze transfer of fatty acyl groups to lipids and proteins. Several classes of secreted proteins have recently been shown to be substrates for MBOAT acyltransferases, including Hh proteins and Spitz (palmitoylated by Hhat), Wg/Wnt proteins (modified with palmitate and/or palmitoleate by Porcupine) and ghrelin (octanoylated by ghrelin O-acyltransferase). These findings highlight protein fatty acylation as a mechanism that not only influences membrane binding of intracellular proteins but also regulates the signaling range and efficacy of secreted proteins.