Structural model of human PORCN illuminates disease-associated variants and drug-binding sites

Abstract

Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Based on homology to mammalian MBOAT proteins we developed and validated a structural model of PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model enhances our mechanistic understanding of PORCN substrate recognition and catalysis as well as the inhibition of its enzymatic activity and can facilitate the development of improved inhibitors and the understanding of disease relevant PORCN mutants.

Cellular Uptake of a Fluorescent Ligand Reveals Ghrelin O-Acyltransferase Interacts with Extracellular Peptides and Exhibits Unexpected Localization for a Secretory Pathway Enzyme

“…In contrast, mechanism 2 allows GOAT to act as a ligand transporter at the plasma membrane without enzyme internalization. Computational modeling of GOAT supports the presence of an internal transmembrane channel that could act as a pore, and similar channels are observed in the crystal structure of the bacterial MBOAT DltB, the cryo-EM structures of Hhat, and structural models of PORCN. ,− We note that our imaging studies do not support colocalization of ligand 15 with GOAT in either transfected HEK 293 cells or prostate cancer cells, which could potentially support ligand transport (mechanism 2) rather than enzyme–ligand internalization (mechanism 1). However, our studies were performed with fixed cells, and the short length and small number of amine groups within the peptide ligand may lead to inefficient ligand cross-linking during fixation, allowing intracellular dispersion during sample preparation.…”

The role of Evi/Wntless in exporting Wnt proteins

“…Porcupine belongs to the family of membrane-bound O-acyltransferases (MBOAT), members of which also lipid modify Hedgehog and Ghrelin. However, apart from Wnts, no other target of the catalytic activity of Porcupine has been discovered ( Galli et al, 2021 ; Yu et al, 2021b ). Despite the substantial interest in the molecular structure of Porcupine to better understand its catalytic mechanism and how its inhibitors function, structural insights had remained confined to comparisons with other MBOATs until recently ( Galli et al, 2021 ; Yu et al, 2021b ).…”

“…However, apart from Wnts, no other target of the catalytic activity of Porcupine has been discovered ( Galli et al, 2021 ; Yu et al, 2021b ). Despite the substantial interest in the molecular structure of Porcupine to better understand its catalytic mechanism and how its inhibitors function, structural insights had remained confined to comparisons with other MBOATs until recently ( Galli et al, 2021 ; Yu et al, 2021b ). Then, Liu and colleagues developed a novel antibody to aid cryo-EM particle alignment in order to determine the structure of Porcupine ( Liu et al, 2022 ).…”

“…Despite these insights, how the transfer of acylated Wnt from Porcupine to Evi/Wls occurs remains to be determined. Structural comparisons indicate that conformational changes and positional adjustments of the Wnt ligand within the lipid bilayer would be necessary to allow lateral transfer in a Porcupine-Evi/Wls-Wnt complex ( Liu et al, 2022 ; Nygaard et al, 2021 ; Yu et al, 2021b ; Zhong et al, 2021 ).…”

PORCN promotes hepatocellular carcinogenesis via Wnt/β-catenin-dependent manner

“…Porcupine O-acyltransferase (PORCN), a member of the membrane-bound O-acyltransferase (MBOAT) family, is a multichannel integral membrane enzyme expressed on the endoplasmic reticulum ( 6 , 7 ). PORCN palmitoylates Wnts and secretes them out of the cell membrane ( 8 ).…”