Tuladhar R et al. (2019), Stereoselective fatty acylation is essential fo...

XB-ART-56364

J Biol Chem 2019 Apr 19;29416:6273-6282. doi: 10.1074/jbc.RA118.007268.

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Stereoselective fatty acylation is essential for the release of lipidated WNT proteins from the acyltransferase Porcupine (PORCN).

Tuladhar R , Yarravarapu N , Ma Y , Zhang C , Herbert J , Kim J , Chen C , Lum L .

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The maintenance of adult animal tissues depends upon highly conserved intercellular signaling molecules that include the secreted WNT proteins. Although it is generally accepted that lipidation of WNTs by the acyltransferase Porcupine (PORCN) and their subsequent recognition by the Wntless (WLS) protein is essential for their cellular secretion, the molecular understanding of this process remains limited. Using structurally diverse fatty acyl donor analogs and mouse embryonic fibroblasts expressing PORCN protein from different metazoan phyla, we demonstrate here that PORCN active-site features, which are conserved across the animal kingdom, enforce cis-Δ9 fatty acylation of WNTs. Aberrant acylation of a WNT with an exogenously supplied trans-Δ9 fatty acid induced the accumulation of WNT-PORCN complexes, suggesting that the fatty acyl species is critical for the extrication of lipidated WNTs from PORCN. Our findings reveal a previously unrecognized fatty acyl-selective checkpoint in the manufacturing of a lipoprotein that forms a basis for WNT signaling sensitivity to trans fats and to PORCN inhibitors in clinical development.

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Species referenced: Xenopus
Genes referenced: ctrl dvl2 fas hap1 hhat myc porcn scd shh tbl1x wls wnt3a wnt7b wnt9b
GO keywords: cell-cell signaling [+]

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	Figure 2. The active-site features of PORCN enforce cis-9 palmitoleation on WNT proteins. A, an SCD inhibitor does not consistently block cell autonomous WNT signaling in different cell lines. The lung cancer– derived H23 and cervical cancer– derived HeLa cell lines were treated with either IWP2 (5M) or A939572 (5M). Although in H23 cells both compounds (cmpds) were able to block WNT activity, as indicated by the reduction of phosphorylated DVL2 protein, A939572 was only active in H23 cells. The accumulation of SCD1 is presumably due to the pharmacoperone-like activity of the SCD inhibitors. The experiment was repeated twice with similar results. B, a collection of alkynylated FAs differing in carbon chain length and desaturation position. C, characterization of PORCN fatty acyl donor preferences using a click chemistry approach. Cells transfected with cDNA encoding either WNT3A or the N-terminal signaling domain of SHH fused with the IgG Fc domain (WNT3A-Fc and SHHN-Fc, respectively) were treated with various alkynylated FAs (illustrated in B) and subjected to a cycloaddition reaction as described previously. SHHN-Fc labeling served as a control for alkynyl probe cellular availability. The experiment was repeated twice with similar results. D, active-site models of HHAT and PORCN. The PORCN active site differs from HHAT in its ability to recognize the position of desaturation within the fatty acyl donor.
	Figure 3. WNT molecules labeled with trans palmitoleic acid fail to leave the secretory pathway. A, PORCN exhibits stereoselectivity for its fatty acyl donor. Cis and trans alkynylated palmitoleic acids (C16:1n-7) were used to label WNT-Fc or SHHN-Fc proteins. A stearoyl-CoA desaturase inhibitor (SCDi, A939572, 5M) was used to prevent cellular isomerization of alkynylated probes. SHHN-Fc labeling served as a control for alkynyl probe cellular availability. The experiment was repeated three times with similar results. B, a click chemistry– based strategy for investigating the influence of palmitoleic acid isomerization on WNT cellular release. IP, immunoprecipitation. C, cell medium was collected 24 h following pulse-labeling of HEK293 cells with either cis ortrans alkynylated palmitoleic acid. WNT proteins from the culture medium were enriched using ConA-Sepharose beads and subjected to a cycloaddition reaction. A baseline labeling efficiency associated with each palmitoleate isomer was determined using a similar analysis of WNT protein isolated from total lysate. The experiment (Exp) was repeated twice with similar results. Ctrl, control. D, the values for total and released click chemistry–labeled WNT proteins were used to calculate the normalized WNT secretion value for each palmitoleate isomer. In two separate experiments, WNT protein release is compromised when it is adducted to a trans palmitoleate.

	Figure 5. Fatty acyl species-selectivity checkpoints in WNT production. A, PORCN active-site features conserved across animals favor cis-9 fatty acyl CoA substrates, an adduct on WNTs that is essential for their extrication from PORCN, presumably by WLS. ER, endoplasmic reticulum. B, active-site features that govern PORCN-mediated fatty acyl selectivity are likely attacked by PORCN inhibitors. C, addition of a trans fat to unacylated WNTs result in accumulation of PORCN–WNT complexes, presumably because of their inability to transfer to WLS.
	SI 1. PORCN sequence alignment across metazoan phyla. Amino acid sequence alignment of PORCN proteins from human (H. sapiens), mouse (M. musculus), frog (X. laevis), zebrafish (D. rerio), roundworm (C. elegans), fruitfly (D. melanogaster), flatworm (S. mansoni), and chicken (G. gallus). Predicted transmembrane regions are shown as blue cylinders. A highly conserved histidine residue is highlighted in yellow.
	SI 2. SCD is essential for WNT acylation. WNT3A-Fc labeling with a saturated alkynyl palmitic acid (C16:0) probe was abolished in the presence of a PORCN inhibitor (IWP2, 5 uM) or SCD inhibitor (A939572, 5 uM). RNAi-mediated gene knockdown of PORCN and SCD also prevented WNT3A-Fc labeling.

References [+] :

Abrami, Palmitoylation and ubiquitination regulate exit of the Wnt signaling protein LRP6 from the endoplasmic reticulum. 2008, Pubmed