title: Topological analysis of Hedgehog acyltransferase, a multipalmitoylated transmembrane protein.
abstract: Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. 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. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo.
authors.affiliation: From the Molecular Medicine Section and.
authors.forename: Antonio D
authors.initials: AD
authors.lastname: Konitsiotis
authors.affiliation: From the Molecular Medicine Section and.
authors.forename: Biljana
authors.initials: B
authors.lastname: Jovanović
authors.affiliation: Department of Chemistry, and Institute of Chemical Biology Imperial College London, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, United Kingdom.
authors.forename: Paulina
authors.initials: P
authors.lastname: Ciepla
authors.affiliation: FILM (Facility for Imaging by Light Microscopy), National Heart and Lung Institute.
authors.forename: Martin
authors.initials: M
authors.lastname: Spitaler
authors.affiliation: Department of Chemistry, and.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lanyon-Hogg
authors.affiliation: Department of Chemistry, and Institute of Chemical Biology Imperial College London, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, United Kingdom e.tate@imperial.ac.uk.
authors.forename: Edward W
authors.initials: EW
authors.lastname: Tate
authors.affiliation: From the Molecular Medicine Section and Institute of Chemical Biology Imperial College London, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, United Kingdom t.magee@imperial.ac.uk.
authors.forename: Anthony I
authors.initials: AI
authors.lastname: Magee
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D020134:Catalytic Domain; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D054878:Lipoylation; D009154:Mutation; D011499:Protein Processing, Post-Translational
chemical_list: D000217:Acyltransferases; C105599:HHAT protein, human
keywords: Acyltransferase; Cancer; Click Chemistry; HHAT; Hedgehog Signaling Pathway; MBOAT; Protein Palmitoylation; Topology; Transmembrane Domain