HHAT (Hedgehog acyltransferase) is a polytopic endoplasmic-reticulum membrane enzyme of the membrane-bound O-acyltransferase (MBOAT) superfamily, and the enzyme that lipidates the Hedgehog morphogens. It transfers palmitate from palmitoyl-CoA onto the alpha-amino group of the N-terminal cysteine exposed by autoprocessing of the Sonic, Desert and Indian Hedgehog precursors, forming an amide rather than a thioester bond, and it recognises the conserved Hedgehog N-terminal CGPGR motif with such selectivity that Hedgehog proteins are thought to be its only substrates. The protein comprises ten transmembrane helices and two reentrant loops, with the catalytic aspartate and histidine on opposite faces of the bilayer and a bound heme b that is required for activity; the active site is embedded in the membrane and receives palmitoyl-CoA from the cytosolic leaflet through an internal archway while the Hedgehog peptide enters from the ER lumen, so the enzyme brings together two chemically dissimilar substrates from opposite sides of the membrane. HHAT has also been reported to promote net movement of palmitoyl-CoA to the luminal side of the ER membrane, an activity separable from catalysis in reconstituted liposomes. The palmitate HHAT installs is not decoration: together with the C-terminal cholesterol added by Hedgehog autoprocessing it makes the dually lipidated ligand, and the palmitoyl chain is read directly by the receptor Patched, so palmitoylation is required for both short- and long-range Hedgehog signalling. HHAT is therefore a positive regulator of the pathway that acts entirely upstream of it, on the ligand rather than within the signalling cascade. Loss-of-function variants cause Nivelon-Nivelon-Mabille syndrome, an autosomal recessive disorder with progressive microcephaly, cerebellar vermis hypoplasia, skeletal dysplasia and, in some individuals, 46,XY gonadal dysgenesis; the enzyme is also a drug target in Hedgehog-dependent cancers, and small molecules such as IMP-1575 inhibit it by occluding the substrate site. Its closest paralog, HHATL, retains the fold but has lost acyltransferase activity, and its Drosophila counterpart is Rasp.
Definition: Catalysis of the transfer of a palmitoyl (systematic name, hexadecanoyl) group to the nitrogen atom of the alpha-amino group of an N-terminal cysteine residue of a protein, in the reaction hexadecanoyl-CoA + N-terminal L-cysteinyl-[protein] = CoA + N-terminal N-hexadecanoyl-L-cysteinyl-[protein] + H(+).
Justification: GO has no molecular-function term for N-terminal-cysteine N-palmitoylation, although it distinguishes the linkage on the process side (GO:0018009 N-terminal peptidyl-L-cysteine N-palmitoylation versus GO:0018230 peptidyl-L-cysteine S-palmitoylation). The only available MF term, GO:0019706 protein-cysteine S-palmitoyltransferase activity, is defined as transfer to a sulfur atom, which is not the reaction HHAT catalyses: HHAT forms an amide bond to the alpha-amino group, shown biochemically and resolved as density for the amide linkage in the product cryo-EM structure. The existing N-palmitoyltransferase terms (GO:0018031, GO:0140771, GO:0140772) are all peptidyl-lysine N6, and GO:0140186 protein N-acyltransferase activity is likewise defined on lysine N6. The proposed name is the one both UniProt and PANTHER already use for this protein - UniProt's recommended name is Protein-cysteine N-palmitoyltransferase HHAT and PANTHER's subfamily PTHR13285:SF20 is PROTEIN-CYSTEINE N-PALMITOYLTRANSFERASE HHAT - and the reaction is already encoded as RHEA:59528, with RHEA:59580 for the cholesterylated substrate. Adding the term would let HHAT, mouse Hhat and Drosophila Rasp be annotated to their actual chemistry, and would give GOAT and Porcupine, the other protein-acylating MBOATs, a correctly-typed sibling branch. Until it exists, GO:0019706 is retained on this record as the closest available term.
Parent term: palmitoyltransferase activity
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000139 Golgi membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Mapped from UniProt subcellular location SL-0134 (Golgi apparatus membrane). UniProt records that location for HHAT, but flags it as a curator inference rather than a direct observation (ECO:0000305 on PubMed:18534984), with the note that HHAT co-localizes with SHH in the ER and Golgi membrane. Reason: Kept, because the source annotation it derives from is a real UniProt assignment and the modification is known to happen as Hedgehog passes through the secretory system. Marked non-core because every characterised property of the enzyme - the topology work, the palmitoyl-CoA uptake work and both cryo-EM structures - places the catalytic machinery in the ER membrane, and the Golgi assignment rests on a single curator-inferred co-localisation. If HHAT has a distinct Golgi function, it has not been shown. Supporting Evidence: PMID:18534984 Both the Shh precursor and mature protein are N-palmitoylated by Hhat, and the reaction occurs during passage through the secretory pathway. file:human/HHAT/HHAT-uniprot.txt Golgi apparatus membrane {ECO:0000305|PubMed:18534984} file:human/HHAT/HHAT-uniprot.txt Note=Co-localizes with SHH in the ER and Golgi membrane. PMID:25505265 Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | REMOVE | Summary: A single interaction with SDCBP (syntenin-1, O00560), a PDZ-domain adaptor of the syndecan and exosome axis, detected in HuRI - the systematic human binary interactome map built from nine genome-scale yeast two-hybrid screens. No follow-up assay and no functional consequence is reported for the pair. Reason: Bare GO:0005515 carries no functional information. The policy is to modify such a row when the cited paper supports a more informative molecular function and otherwise to remove it; here there is nothing to modify to, because syntenin-1 appears nowhere in the described HHAT mechanism - HHAT's characterised partners are its substrates (Hedgehog proteins and palmitoyl-CoA) and its heme cofactor - and the HuRI authors state explicitly that function does not follow from detection. Removal is a judgement about the annotation's informativeness, not a claim that the interaction is false. HHAT's informative molecular function is captured by GO:0019706 and GO:0016409. Supporting Evidence: PMID:32296183 We screened this search space a total of nine times with a panel of three Y2H assay versions PMID:32296183 the cellular function of most individual PPIs remains to be elucidated PMID:34112694 The enzyme displays exquisite selectivity for Hedgehog proteins, which are thought to be its only substrates |
| GO:0005783 endoplasmic reticulum | IBA GO_REF:0000033 | ACCEPT | Summary: PANTHER IBD node PTN000328646, scoped to taxon:2759 (Eukaryota), places this MBOAT clade in the endoplasmic reticulum; the seeds are mouse Hhat and Hhatl, the yeast MBOATs GUP1 and GUP2, and human HHAT itself (interpro/panther/PTHR13285/PTHR13285-paint.tsv). Reason: The node scope matches the biology: ER residence is a property of the whole eukaryotic MBOAT group, including the yeast GUPs that have no Hedgehog to acylate, so placing it at a Eukaryota-level node rather than in the Hedgehog-specific clade is the right call. It is confirmed directly on the human protein, where catalysis occurs on the luminal face of the ER membrane. Human HHAT appearing in its own WITH/FROM is expected: its experimental rows are among the descendant evidences used to place the IBD, and the IBA adds the claim that the localisation is inherited. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000328646 · PTN000328646 SUPPORTS TRANSFER Eukaryota-scoped IBD node for GO:0005783 seeded by MGI:1922020, MGI:2444681, SGD:S000003052, SGD:S000006110 and UniProtKB:Q5VTY9; ER residence is shared by the yeast MBOATs, so the deep node placement is appropriate. Supporting Evidence: PMID:25505265 Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins PMID:31875564 Shh palmitoylation is catalyzed on the luminal side of the endoplasmic reticulum (ER) by Hedgehog acyltransferase (Hhat), an ER-resident enzyme. PMID:34112694 HHAT is an endoplasmic reticulum (ER) membrane protein that catalyzes the transfer of a palmitoyl lipid from palmitoyl-CoA to the N-terminal cysteine of Hedgehog precursor proteins |
| GO:0005783 endoplasmic reticulum | IDA PMID:18534984 Hhat is a palmitoylacyltransferase with specificity for N-pa... | ACCEPT | Summary: Buglino and Resh purified Hhat to homogeneity from an in vitro Shh palmitoylation assay and showed that the modification occurs as Shh transits the secretory pathway; UniProt cites this paper for the endoplasmic reticulum membrane location. Reason: Correct. The ER is where HHAT works, established independently by the topology study and by both cryo-EM structures. Only the abstract of this paper is cached, so the quoted support is abstract-level; the curator who made the annotation read the full text, and the conclusion is not in doubt. The more specific child GO:0005789 endoplasmic reticulum membrane is annotated separately with direct evidence. Supporting Evidence: PMID:18534984 Both the Shh precursor and mature protein are N-palmitoylated by Hhat, and the reaction occurs during passage through the secretory pathway. PMID:18534984 This study establishes Hhat as a bona fide Shh PAT PMID:25505265 Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins |
| GO:0005789 endoplasmic reticulum membrane | IDA PMID:25505265 Topological analysis of Hedgehog acyltransferase, a multipal... | ACCEPT | Summary: Konitsiotis et al. mapped HHAT's topology in the ER membrane: ten transmembrane domains and two reentrant loops, with the catalytic His and Asp on opposite faces of the bilayer, and showed that mutating the conserved His abolishes palmitoylation. Reason: The core location, with the right qualifier. is_active_in rather than merely located_in is justified because the catalytic residues are part of the membrane-embedded active site: the reaction happens inside the bilayer, not adjacent to it. Both later cryo-EM structures confirm a membrane-embedded reaction chamber that receives palmitoyl-CoA from the cytosolic leaflet and the Hedgehog peptide from the lumen. Supporting Evidence: PMID:25505265 We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. PMID:25505265 mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation PMID:34112694 The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. PMID:34890564 Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Combined electronic annotation from mouse Hhat (UniProtKB:Q8BMT9 / ensembl:ENSMUSP00000046686) together with the UniProt subcellular location SL-0097. Mouse Hhat has its own experimental rows for this term (EXP on PMID:15075292 and IDA on PMID:25488661 in the QuickGO record for Q8BMT9). Reason: The electronic assignment lands on exactly the compartment established directly for the human enzyme, and the ortholog source is itself experimental rather than another inference. Mouse and human HHAT are direct orthologs in PANTHER subfamily PTHR13285:SF20 with the same substrate and the same reaction. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q8BMT9 · Hhat (mouse) SUPPORTS TRANSFER Mouse source rows for GO:0005789 are experimental (EXP, IDA), not propagations; the human target also has its own IDA for the same term. UniProtKB-SubCell:SL-0097 · Endoplasmic reticulum membrane SUPPORTS TRANSFER UniProt subcellular-location assignment for HHAT, itself curated from PubMed:25505265 with ECO:0000269. Supporting Evidence: PMID:25505265 We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. file:human/HHAT/HHAT-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-5358343 | ACCEPT | Summary: Reactome reaction R-HSA-5358343 'HHAT palmitoylates Hh N-terminal fragment' places the reaction, and the enzyme, in the endoplasmic reticulum membrane. Reason: Correct location, correctly attributed. Reactome's author statement agrees with the primary literature and with the human IDA row for the same term. Supporting Evidence: Reactome:R-HSA-5358343 the N-terminal fragment of Hh (Hh-Np) is also palmitoylated by the O-acyltransferase HHAT PMID:31875564 Shh palmitoylation is catalyzed on the luminal side of the endoplasmic reticulum (ER) by Hedgehog acyltransferase (Hhat), an ER-resident enzyme. PMID:25505265 We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-5483229 | ACCEPT | Summary: Reactome reaction R-HSA-5483229 'HHAT G287V doesn't palmitoylate Hh-Np' models the disease allele found in syndromic 46,XY disorder of sex development; Reactome notes that the mutation does not change HHAT stability, expression or localization but abolishes palmitoyl transfer. The enzyme is placed in the ER membrane in that reaction too. Reason: Correct. The compartment assignment is the same as in the wild-type reaction and is independently supported; the fact that the row is exported from a loss-of-function reaction does not make the localisation claim any weaker, and Reactome explicitly states that the mutant's localization is unaffected. Supporting Evidence: Reactome:R-HSA-5483229 the mutant protein is unable to palmitoylate SHH or DHH in an in vitro assay PMID:25505265 We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. |
| GO:0005794 Golgi apparatus | IDA PMID:18534984 Hhat is a palmitoylacyltransferase with specificity for N-pa... | KEEP AS NON CORE | Summary: From Buglino and Resh, whose abstract reports that both the Shh precursor and the mature protein are N-palmitoylated and that the reaction occurs during passage through the secretory pathway. UniProt curates a Golgi apparatus membrane location from this paper but marks it ECO:0000305, a curator inference, and describes it as co-localization with SHH. Reason: Kept rather than removed: only the abstract of this paper is cached, the curator who made the annotation read the full text, and secretory-pathway transit is exactly the context in which HHAT works. Marked non-core because the enzyme's catalytic site has been placed in the ER membrane by topology mapping and by two independent cryo-EM structures, and no Golgi-specific activity of HHAT has been demonstrated; the Golgi pool is best read as enzyme in transit or trailing its substrate. Supporting Evidence: PMID:18534984 Both the Shh precursor and mature protein are N-palmitoylated by Hhat, and the reaction occurs during passage through the secretory pathway. file:human/HHAT/HHAT-uniprot.txt Note=Co-localizes with SHH in the ER and Golgi membrane. PMID:31875564 Shh palmitoylation is catalyzed on the luminal side of the endoplasmic reticulum (ER) by Hedgehog acyltransferase (Hhat), an ER-resident enzyme. |
| GO:0016409 palmitoyltransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PANTHER IBD node PTN000998821, scoped to taxon:6072 (Eumetazoa), asserts palmitoyltransferase activity for the Hedgehog-acylating clade, seeded by Drosophila rasp (FB:FBgn0024194), mouse Hhat (MGI:2444681) and human HHAT (interpro/panther/PTHR13285/PTHR13285-paint.tsv). Reason: A well-placed node, and in one respect a better-chosen term than its own child. The Eumetazoa scope matches where Hedgehog acylation exists; the three seeds span fly, mouse and human, each with experimental support; and the same PAINT slice records an IRD explicitly negating GO:0016409 and GO:0016746 in the HHATL branch (node PTN002665109, taxon:117571), which shows the curator separated HHAT from its catalytically compromised paralog rather than blanketing the family. Unlike GO:0019706, this term says nothing about the acceptor atom, so it is chemically accurate for an enzyme that forms an amide rather than a thioester. Accepted at the level PAINT asserts it. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000998821 · PTN000998821 SUPPORTS TRANSFER Eumetazoa-scoped IBD node for GO:0016409 seeded by FB:FBgn0024194, MGI:2444681 and UniProtKB:Q5VTY9; the same slice carries an IRD negating the activity in the HHATL branch, so the node is discriminating rather than family-wide. Supporting Evidence: PMID:34890564 A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). PMID:18534984 We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. PMID:34112694 HHAT is an endoplasmic reticulum (ER) membrane protein that catalyzes the transfer of a palmitoyl lipid from palmitoyl-CoA to the N-terminal cysteine of Hedgehog precursor proteins |
| GO:0016409 palmitoyltransferase activity | IEA GO_REF:0000107 | ACCEPT | Summary: Ensembl Compara transfer from mouse Hhat (UniProtKB:Q8BMT9 / ensembl:ENSMUSP00000046686), whose own rows for this term are experimental (IMP on PMID:15075292 in the QuickGO record for Q8BMT9). Reason: Sound ortholog transfer from an experimental source, landing on a term that is also asserted at the Eumetazoa PAINT node and demonstrated directly for the human enzyme. Mouse and human HHAT are one-to-one orthologs in PANTHER subfamily PTHR13285:SF20. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q8BMT9 · Hhat (mouse) SUPPORTS TRANSFER Mouse source rows for GO:0016409 are IMP, not propagations; human HHAT has direct biochemical and structural evidence for palmitoyl transfer. Supporting Evidence: PMID:34890564 A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). PMID:34112694 HHAT is an endoplasmic reticulum (ER) membrane protein that catalyzes the transfer of a palmitoyl lipid from palmitoyl-CoA to the N-terminal cysteine of Hedgehog precursor proteins |
| GO:0016747 acyltransferase activity, transferring groups other than amino-acyl groups | TAS Reactome:R-HSA-5358343 | MODIFY | Summary: Reactome exports HHAT's activity at the level of 'acyltransferase activity, transferring groups other than amino-acyl groups'. HHAT's acyl group is specifically palmitate, transferred from palmitoyl-CoA onto the Hedgehog N-terminal cysteine, and the enzyme acylates Hedgehog proteins and apparently nothing else. The exporting reaction, 'HHAT palmitoylates Hh N-terminal fragment', is itself specific about the acyl group and the substrate, so nothing is lost by using the palmitoyl-level term. Reason: Correct but three levels too broad: GO:0019706 is_a GO:0016409 is_a GO:0016747, so this row sits two steps above the palmitoyl-level term and says nothing that distinguishes HHAT from any acyltransferase. Replaced by GO:0016409 palmitoyltransferase activity, which is the most specific term in the current ontology that is chemically correct for HHAT - its own child GO:0019706 is used elsewhere on this record but specifies transfer to a sulfur atom, which HHAT does not do, so promoting a Reactome row into it would propagate that error. The missing N-linked term is requested in proposed_new_terms. Proposed replacements: palmitoyltransferase activity Supporting Evidence: Reactome:R-HSA-5358343 the N-terminal fragment of Hh (Hh-Np) is also palmitoylated by the O-acyltransferase HHAT PMID:18534984 We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. PMID:34112694 The enzyme displays exquisite selectivity for Hedgehog proteins, which are thought to be its only substrates |
| GO:0016747 acyltransferase activity, transferring groups other than amino-acyl groups | TAS Reactome:R-HSA-5483229 | MODIFY | Summary: Reactome exports HHAT's activity at the level of 'acyltransferase activity, transferring groups other than amino-acyl groups'. HHAT's acyl group is specifically palmitate, transferred from palmitoyl-CoA onto the Hedgehog N-terminal cysteine, and the enzyme acylates Hedgehog proteins and apparently nothing else. The exporting reaction, 'HHAT G287V doesn't palmitoylate Hh-Np', is about loss of palmitoyl transfer specifically, so the palmitoyl-level term is the one its own text supports. Reason: Correct but three levels too broad: GO:0019706 is_a GO:0016409 is_a GO:0016747, so this row sits two steps above the palmitoyl-level term and says nothing that distinguishes HHAT from any acyltransferase. Replaced by GO:0016409 palmitoyltransferase activity, which is the most specific term in the current ontology that is chemically correct for HHAT - its own child GO:0019706 is used elsewhere on this record but specifies transfer to a sulfur atom, which HHAT does not do, so promoting a Reactome row into it would propagate that error. The missing N-linked term is requested in proposed_new_terms. Proposed replacements: palmitoyltransferase activity Supporting Evidence: Reactome:R-HSA-5483229 the mutant protein is unable to palmitoylate SHH or DHH in an in vitro assay PMID:18534984 We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. PMID:34112694 The enzyme displays exquisite selectivity for Hedgehog proteins, which are thought to be its only substrates |
| GO:0019706 protein-cysteine S-palmitoyltransferase activity | IDA PMID:18534984 Hhat is a palmitoylacyltransferase with specificity for N-pa... | ACCEPT | Summary: Buglino and Resh developed an in vitro Shh palmitoylation assay, purified Hhat to homogeneity with it, and showed directly that the enzyme attaches palmitate via an amide linkage to the N-terminal cysteine of Shh, with kinetic parameters (KM 1.25 uM for SHH, 3 uM for hexadecanoyl-CoA) recorded in UniProt. Other palmitoylated proteins are not substrates. Reason: The defining molecular function of HHAT, demonstrated directly for the human enzyme by four independent studies using biochemistry, reconstitution and two cryo-EM structures, and encoded by UniProt as RHEA:59528. One caveat is recorded rather than acted on. GO:0019706 is defined as transfer of a palmitoyl group to a sulfur atom on a protein cysteine, whereas HHAT forms an amide bond to the alpha-amino group of the N-terminal cysteine - UniProt names the protein Protein-cysteine N-palmitoyltransferase HHAT and PANTHER names its subfamily PTHR13285:SF20 the same way. GO currently has no molecular-function term for N-terminal-cysteine N-palmitoylation (the N-palmitoyltransferase terms it does have, GO:0018031, GO:0140771 and GO:0140772, are all peptidyl-lysine N6), so GO:0019706 is the closest available term and the curators had no better option. The gap is recorded in proposed_new_terms instead of being papered over with a wrong MODIFY, and the correct-chemistry process term GO:0018009 is added as a NEW row. Supporting Evidence: PMID:18534984 We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. PMID:18534984 Other palmitoylated proteins (e.g. PSD95 and Wnt) are not substrates for Hhat, and Porcupine, a putative Wnt PAT, does not palmitoylate Shh. PMID:18534984 This study establishes Hhat as a bona fide Shh PAT |
| GO:0019706 protein-cysteine S-palmitoyltransferase activity | IDA PMID:31875564 Hedgehog Acyltransferase Promotes Uptake of Palmitoyl-CoA ac... | ACCEPT | Summary: Asciolla and Resh assayed purified Hhat in microsomes, semi-intact cells and liposomes, confirming palmitoyl transfer to Shh on the luminal face of the ER membrane while separating it from the enzyme's ability to take up palmitoyl-CoA. Reason: The defining molecular function of HHAT, demonstrated directly for the human enzyme by four independent studies using biochemistry, reconstitution and two cryo-EM structures, and encoded by UniProt as RHEA:59528. One caveat is recorded rather than acted on. GO:0019706 is defined as transfer of a palmitoyl group to a sulfur atom on a protein cysteine, whereas HHAT forms an amide bond to the alpha-amino group of the N-terminal cysteine - UniProt names the protein Protein-cysteine N-palmitoyltransferase HHAT and PANTHER names its subfamily PTHR13285:SF20 the same way. GO currently has no molecular-function term for N-terminal-cysteine N-palmitoylation (the N-palmitoyltransferase terms it does have, GO:0018031, GO:0140771 and GO:0140772, are all peptidyl-lysine N6), so GO:0019706 is the closest available term and the curators had no better option. The gap is recorded in proposed_new_terms instead of being papered over with a wrong MODIFY, and the correct-chemistry process term GO:0018009 is added as a NEW row. Supporting Evidence: PMID:31875564 Shh palmitoylation is catalyzed on the luminal side of the endoplasmic reticulum (ER) by Hedgehog acyltransferase (Hhat), an ER-resident enzyme. PMID:31875564 Reconstitution of purified Hhat into liposomes provided further evidence that palmitoyl-CoA uptake activity is an intrinsic property of Hhat. PMID:31875564 Attachment of palmitate to the N terminus of Sonic hedgehog (Shh) is essential for Shh signaling. |
| GO:0019706 protein-cysteine S-palmitoyltransferase activity | IDA PMID:34112694 Substrate and product complexes reveal mechanisms of Hedgeho... | ACCEPT | Summary: Jiang, Benz and Long determined cryo-EM structures of human HHAT bound to its palmitoyl-CoA substrate (2.7 A) and as a product complex with a palmitoylated Hedgehog peptide (3.2 A), with density for the amide bond itself, and validated catalytic residues Asp339 and His379 by mutagenesis. Reason: The defining molecular function of HHAT, demonstrated directly for the human enzyme by four independent studies using biochemistry, reconstitution and two cryo-EM structures, and encoded by UniProt as RHEA:59528. One caveat is recorded rather than acted on. GO:0019706 is defined as transfer of a palmitoyl group to a sulfur atom on a protein cysteine, whereas HHAT forms an amide bond to the alpha-amino group of the N-terminal cysteine - UniProt names the protein Protein-cysteine N-palmitoyltransferase HHAT and PANTHER names its subfamily PTHR13285:SF20 the same way. GO currently has no molecular-function term for N-terminal-cysteine N-palmitoylation (the N-palmitoyltransferase terms it does have, GO:0018031, GO:0140771 and GO:0140772, are all peptidyl-lysine N6), so GO:0019706 is the closest available term and the curators had no better option. The gap is recorded in proposed_new_terms instead of being papered over with a wrong MODIFY, and the correct-chemistry process term GO:0018009 is added as a NEW row. Supporting Evidence: PMID:34112694 HHAT is an endoplasmic reticulum (ER) membrane protein that catalyzes the transfer of a palmitoyl lipid from palmitoyl-CoA to the N-terminal cysteine of Hedgehog precursor proteins PMID:34112694 including density for the amide linkage to the N-terminal cysteine (C24) of Hedgehog, indicates that the palmitoylation reaction has occurred PMID:34112694 The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. |
| GO:0019706 protein-cysteine S-palmitoyltransferase activity | IDA PMID:34890564 Structure, mechanism, and inhibition of Hedgehog acyltransfe... | ACCEPT | Summary: Coupland et al. determined the structure of human HHAT bound to palmityl-CoA and a SHH-mimetic megabody, identified an essential bound heme b, and captured the enzyme with the inhibitor IMP-1575 occluding the substrate site. Reason: The defining molecular function of HHAT, demonstrated directly for the human enzyme by four independent studies using biochemistry, reconstitution and two cryo-EM structures, and encoded by UniProt as RHEA:59528. One caveat is recorded rather than acted on. GO:0019706 is defined as transfer of a palmitoyl group to a sulfur atom on a protein cysteine, whereas HHAT forms an amide bond to the alpha-amino group of the N-terminal cysteine - UniProt names the protein Protein-cysteine N-palmitoyltransferase HHAT and PANTHER names its subfamily PTHR13285:SF20 the same way. GO currently has no molecular-function term for N-terminal-cysteine N-palmitoylation (the N-palmitoyltransferase terms it does have, GO:0018031, GO:0140771 and GO:0140772, are all peptidyl-lysine N6), so GO:0019706 is the closest available term and the curators had no better option. The gap is recorded in proposed_new_terms instead of being papered over with a wrong MODIFY, and the correct-chemistry process term GO:0018009 is added as a NEW row. Supporting Evidence: PMID:34890564 A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). PMID:34890564 revealing a heme group bound to HHAT that is essential for HHAT function PMID:34890564 A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. |
| GO:0019706 protein-cysteine S-palmitoyltransferase activity | IEA GO_REF:0000107 | ACCEPT | Summary: Ensembl Compara transfer from mouse Hhat (UniProtKB:Q8BMT9), whose own rows for this term are experimental (three IDA rows, PMID:15075292, PMID:23112049 and PMID:25488661 in the QuickGO record for Q8BMT9). Reason: Sound transfer from an experimental source, and in any case superseded by four human IDA rows for the same term. One caveat is recorded rather than acted on. GO:0019706 is defined as transfer of a palmitoyl group to a sulfur atom on a protein cysteine, whereas HHAT forms an amide bond to the alpha-amino group of the N-terminal cysteine - UniProt names the protein Protein-cysteine N-palmitoyltransferase HHAT and PANTHER names its subfamily PTHR13285:SF20 the same way. GO currently has no molecular-function term for N-terminal-cysteine N-palmitoylation (the N-palmitoyltransferase terms it does have, GO:0018031, GO:0140771 and GO:0140772, are all peptidyl-lysine N6), so GO:0019706 is the closest available term and the curators had no better option. The gap is recorded in proposed_new_terms instead of being papered over with a wrong MODIFY, and the correct-chemistry process term GO:0018009 is added as a NEW row. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q8BMT9 · Hhat (mouse) SUPPORTS TRANSFER Mouse source rows for GO:0019706 are IDA from three studies, not propagations; the human target has four IDA rows of its own. Supporting Evidence: PMID:18534984 We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. PMID:34112694 HHAT is an endoplasmic reticulum (ER) membrane protein that catalyzes the transfer of a palmitoyl lipid from palmitoyl-CoA to the N-terminal cysteine of Hedgehog precursor proteins |
| GO:0045880 positive regulation of smoothened signaling pathway | IDA PMID:18534984 Hhat is a palmitoylacyltransferase with specificity for N-pa... | ACCEPT | Summary: Buglino and Resh established Hhat as the Shh palmitoyl acyltransferase in the same work that frames palmitoylation as critical for effective Shh signalling at both short and long range. Reason: Accepted as a core role, and the reasoning matters because HHAT is not a pathway component. HHAT never contacts the receptor and acts before the ligand is released, so a bare GO:0007224 smoothened signaling pathway row would be an over-annotation. But HHAT does perform the act that raises pathway output: it covalently converts the ligand into its high-activity form, and the palmitate it installs is read directly by Patched1. The regulation branch is exactly how GO expresses an entity that acts on a pathway from outside it, and the work of that regulation - the palmitoyl transfer - is HHAT's own chemistry, so the participation test is satisfied by the enzyme rather than by its substrate. Note that the human record's use of the regulation term is preferable to the mouse and fly records, which carry GO:0007224 and GO:0007225 respectively. Supporting Evidence: PMID:18534984 Palmitoylation of Sonic Hedgehog (Shh) is critical for effective long- and short-range signaling. PMID:18534984 We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. PMID:18534984 This study establishes Hhat as a bona fide Shh PAT |
| GO:0045880 positive regulation of smoothened signaling pathway | IDA PMID:31875564 Hedgehog Acyltransferase Promotes Uptake of Palmitoyl-CoA ac... | ACCEPT | Summary: Asciolla and Resh state the functional stake of the reaction directly: attachment of palmitate to the Shh N terminus is essential for Shh signalling, and Hhat is the enzyme that performs it on the luminal face of the ER. Reason: Accepted as a core role, and the reasoning matters because HHAT is not a pathway component. HHAT never contacts the receptor and acts before the ligand is released, so a bare GO:0007224 smoothened signaling pathway row would be an over-annotation. But HHAT does perform the act that raises pathway output: it covalently converts the ligand into its high-activity form, and the palmitate it installs is read directly by Patched1. The regulation branch is exactly how GO expresses an entity that acts on a pathway from outside it, and the work of that regulation - the palmitoyl transfer - is HHAT's own chemistry, so the participation test is satisfied by the enzyme rather than by its substrate. Note that the human record's use of the regulation term is preferable to the mouse and fly records, which carry GO:0007224 and GO:0007225 respectively. Supporting Evidence: PMID:31875564 Attachment of palmitate to the N terminus of Sonic hedgehog (Shh) is essential for Shh signaling. PMID:31875564 Shh palmitoylation is catalyzed on the luminal side of the endoplasmic reticulum (ER) by Hedgehog acyltransferase (Hhat), an ER-resident enzyme. |
| GO:0045880 positive regulation of smoothened signaling pathway | IDA PMID:34112694 Substrate and product complexes reveal mechanisms of Hedgeho... | ACCEPT | Summary: The structural work shows why the modification is required: Hedgehog precursors must undergo N-terminal palmitoylation before they can signal, and the attached lipid is what the receptor Patched1 structurally recognises. Reason: Accepted as a core role, and the reasoning matters because HHAT is not a pathway component. HHAT never contacts the receptor and acts before the ligand is released, so a bare GO:0007224 smoothened signaling pathway row would be an over-annotation. But HHAT does perform the act that raises pathway output: it covalently converts the ligand into its high-activity form, and the palmitate it installs is read directly by Patched1. The regulation branch is exactly how GO expresses an entity that acts on a pathway from outside it, and the work of that regulation - the palmitoyl transfer - is HHAT's own chemistry, so the participation test is satisfied by the enzyme rather than by its substrate. Note that the human record's use of the regulation term is preferable to the mouse and fly records, which carry GO:0007224 and GO:0007225 respectively. Supporting Evidence: PMID:34112694 Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). PMID:34112694 The attached lipid is required for downstream signaling and is structurally recognized by the Hedgehog receptor Patched1 |
| GO:0045880 positive regulation of smoothened signaling pathway | IEA GO_REF:0000107 | ACCEPT | Summary: Ensembl Compara transfer from mouse Hhat (UniProtKB:Q8BMT9), whose own rows for this term are experimental (IDA on PMID:15075292 and PMID:23112049 in the QuickGO record for Q8BMT9). Reason: Sound transfer from an experimental source onto a term the human protein independently carries by three IDA rows. Mouse Hhat also carries GO:0007224 smoothened signaling pathway by IMP; that deeper claim was not transferred here, which is the right restraint, since HHAT acts on the ligand and not within the pathway. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q8BMT9 · Hhat (mouse) SUPPORTS TRANSFER Mouse source rows for GO:0045880 are IDA from two studies; the human target has three IDA rows of its own. Supporting Evidence: PMID:34112694 Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). PMID:34112694 The attached lipid is required for downstream signaling and is structurally recognized by the Hedgehog receptor Patched1 |
| GO:0051604 protein maturation | IDA PMID:34112694 Substrate and product complexes reveal mechanisms of Hedgeho... | ACCEPT | Summary: The substrate and product structures capture the maturation step itself: Hedgehog precursor proteins must be N-terminally palmitoylated by HHAT before they can act as signalling molecules, and the product complex resolves the finished amide bond on the Hedgehog N-terminal cysteine. Reason: Core, and the most mechanistically direct process term on the record. HHAT catalyses one of the covalent steps by which the Hedgehog precursor becomes a functional ligand, alongside the autocleavage and autocholesterylation the ligand performs on itself - which is exactly how the curated GO-CAM models it (model 696022cd00000016, 'Protein maturation of SHH by autocleavage, autocholesterylation and palmitoylation by HHAT', where HHAT's GO:0019706 activity is part_of GO:0051604 and occurs_in GO:0005789). Supporting Evidence: PMID:34112694 Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). PMID:34112694 including density for the amide linkage to the N-terminal cysteine (C24) of Hedgehog, indicates that the palmitoylation reaction has occurred PMID:34112694 HHAT is an endoplasmic reticulum (ER) membrane protein that catalyzes the transfer of a palmitoyl lipid from palmitoyl-CoA to the N-terminal cysteine of Hedgehog precursor proteins |
| GO:0051604 protein maturation | IDA PMID:34890564 Structure, mechanism, and inhibition of Hedgehog acyltransfe... | ACCEPT | Summary: Coupland et al. describe the same step as the key modification of the SHH N terminus and show how HHAT transfers the acyl chain across the ER membrane to accomplish it. Reason: Core, and the most mechanistically direct process term on the record. HHAT catalyses one of the covalent steps by which the Hedgehog precursor becomes a functional ligand, alongside the autocleavage and autocholesterylation the ligand performs on itself - which is exactly how the curated GO-CAM models it (model 696022cd00000016, 'Protein maturation of SHH by autocleavage, autocholesterylation and palmitoylation by HHAT', where HHAT's GO:0019706 activity is part_of GO:0051604 and occurs_in GO:0005789). Supporting Evidence: PMID:34890564 A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). PMID:34890564 Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. |
| GO:0051604 protein maturation | IEA GO_REF:0000107 | ACCEPT | Summary: Ensembl Compara transfer from mouse Hhat (UniProtKB:Q8BMT9), whose own rows for this term are experimental (IDA on PMID:15075292 and PMID:23112049 in the QuickGO record for Q8BMT9). Reason: Sound transfer from an experimental source onto the term the human protein carries by two IDA rows and that the curated GO-CAM uses for HHAT's only activity. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q8BMT9 · Hhat (mouse) SUPPORTS TRANSFER Mouse source rows for GO:0051604 are IDA from two studies, not propagations. Supporting Evidence: PMID:34112694 Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). PMID:34890564 A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). |
| GO:0018009 N-terminal peptidyl-L-cysteine N-palmitoylation | IDA PMID:34112694 Substrate and product complexes reveal mechanisms of Hedgeho... | NEW | Summary: GO:0018009 names the exact chemistry HHAT performs: 'The covalent attachment of a palmitoyl group to a nitrogen (N) atom in an N-terminal cysteine residue to form N-palmitoyl-L-cysteine'. Human HHAT has no row for it. Its closest relatives both do: Drosophila rasp (UniProtKB:Q9VZU2, the other representative member of PANTHER family PTHR13285 in the Hedgehog module) carries it with two IDA rows and an IMP row, and mouse Hhat (UniProtKB:Q8BMT9) carries it by ISO with WITH/FROM UniProtKB:Q5VTY9 - that is, transferred from human HHAT. Reason: Proposed after both tests. Participation: HHAT catalyses the step, and the term's ancestors (GO:0018345 protein palmitoylation, GO:0006498 N-terminal protein lipidation, GO:0043543 protein acylation) are attachment processes, which name the enzyme that does the attaching rather than the substrate that receives it - the mirror image of the Drosophila hh case, where the ligand is the substrate and the acyltransferase activity is not the ligand's function. Comparator check: rather than a systematic absence signalling a convention, the term is present on rasp with three experimental rows and on mouse Hhat by transfer from human HHAT itself, so a curator has already judged that the human protein has this function and the human row is snapshot lag. Non-redundancy: GO:0018009 is neither an ancestor nor a descendant of GO:0051604 protein maturation, of GO:0045880, or of any molecular-function term on this record. It is the only NEW row proposed here; the reported palmitoyl-CoA conduit activity was deliberately not proposed, because no MBOAT in this family carries a transport term in any species and the 2021 structures recast the movement as substrate access to a membrane-embedded active site. Supporting Evidence: PMID:34112694 including density for the amide linkage to the N-terminal cysteine (C24) of Hedgehog, indicates that the palmitoylation reaction has occurred PMID:18534984 We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. PMID:34112694 Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). PMID:31875564 Shh palmitoylation is catalyzed on the luminal side of the endoplasmic reticulum (ER) by Hedgehog acyltransferase (Hhat), an ER-resident enzyme. |
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Download this section (compressed HTML)Q: Is HHAT's reported palmitoyl-CoA uptake activity a distinct transport function or simply substrate access to the membrane-embedded active site? Liposome reconstitution showed uptake is intrinsic to HHAT and separable from catalysis, yet the cryo-EM structures describe an internal archway feeding the reaction chamber, and no MBOAT in PANTHER family PTHR13285 carries a transport annotation in any species. Resolving this would determine whether a transporter-activity annotation is warranted, and whether GO needs a term for it - the nearest existing MF term, GO:0015607, is ABC-type and does not apply.
Q: Should GO gain a molecular-function term for N-terminal-cysteine N-palmitoylation? The process side already distinguishes GO:0018009 from GO:0018230, and the reaction is encoded as RHEA:59528, but the only available MF term (GO:0019706) specifies transfer to a sulfur atom. The same gap affects mouse Hhat, Drosophila Rasp and, by analogy, the other protein-acylating MBOATs GOAT and Porcupine.
Q: Does HHAT have any function in the Golgi, or is the Golgi pool simply enzyme in transit or following its substrate? The Golgi assignment rests on a curator-inferred co-localization with SHH (ECO:0000305 on PubMed:18534984), while all catalytic characterisation places the active site in the ER membrane.
Q: HHATL sits in the same PANTHER family and retains the fold, and the PAINT slice records an explicit IRD negating acyltransferase activity in its branch. What does HHATL actually do, and does it regulate HHAT - for instance by competing for palmitoyl-CoA or for Hedgehog substrate in the ER membrane?
Experiment: In reconstituted liposomes, compare wild-type HHAT with catalytically dead active-site mutants (D339A, H379A) and with archway mutants, measuring (i) palmitoyl-CoA accumulation in the lumen in the absence of any Hedgehog acceptor and (ii) release of intact palmitoyl-CoA from the luminal compartment. Net luminal accumulation of free palmitoyl-CoA without an acceptor would support a genuine transport function; accumulation only as acylated product would support substrate access.
Hypothesis: Palmitoyl-CoA movement across the ER membrane by HHAT is part of substrate delivery to the buried active site, not net transport of a cargo released into the lumen.
Type: liposome reconstitution with donor-tracking
Experiment: Retarget HHAT with ER-exit and Golgi-retention signals in HHAT-null cells and assay SHH palmitoylation stoichiometry by click chemistry or mass spectrometry, alongside Hedgehog reporter output. If Golgi-restricted HHAT cannot palmitoylate SHH, the Golgi annotation is transit rather than a site of action.
Hypothesis: The ER membrane is the only compartment in which HHAT is catalytically competent.
Type: compartment retargeting with quantitative palmitoylation readout
Experiment: Combine an unbiased palmitoyl-proteomics comparison of HHAT-null and rescued cells with a designed panel of reporter proteins in which the CGPGR consensus is grafted onto unrelated secreted proteins, and quantify N-palmitoylation of each. This would either confirm exclusive Hedgehog selectivity or identify substrates that current annotation misses.
Hypothesis: HHAT is dedicated to Hedgehog proteins, so the N-terminal CGPGR motif is a sufficient acylation determinant and no other cellular substrate exists.
Type: palmitoyl-proteomics plus designed substrate panel
Experiment: Test HHATL for Hedgehog palmitoylation with purified protein and in cells, and assay whether co-expression or co-reconstitution of HHATL changes HHAT kinetics, palmitoyl-CoA occupancy or Hedgehog output. This would put the PAINT IRD call on the HHATL branch on an experimental footing and clarify whether HHATL belongs in the Hedgehog module at all.
Hypothesis: HHATL modulates HHAT activity in the ER membrane rather than acting as an independent acyltransferase.
Type: paralog activity and interference assay
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