PMID- 28655768
OWN - NLM
STAT- MEDLINE
DCOM- 20170905
LR  - 20210205
IS  - 1083-351X (Electronic)
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 292
IP  - 33
DP  - 2017 Aug 18
TI  - An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the 
      acyltransferase Porcupine.
PG  - 13507-13513
LID - 10.1074/jbc.C117.800136 [doi]
AB  - 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 IC(50) 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.
CI  - (c) 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
FAU - Asciolla, James J
AU  - Asciolla JJ
AD  - From the Cell Biology Program and.
AD  - the Biochemistry, Cell Biology and Molecular Biology Graduate Program, Weill 
      Cornell Graduate School of Medical Sciences, New York, New York 10065.
FAU - Miele, Matthew M
AU  - Miele MM
AD  - Proteomics and Microchemistry Core Laboratory, Memorial Sloan Kettering Cancer 
      Center, New York, New York 10065 and.
FAU - Hendrickson, Ronald C
AU  - Hendrickson RC
AD  - Proteomics and Microchemistry Core Laboratory, Memorial Sloan Kettering Cancer 
      Center, New York, New York 10065 and.
FAU - Resh, Marilyn D
AU  - Resh MD
AD  - From the Cell Biology Program and reshm@mskcc.org.
AD  - the Biochemistry, Cell Biology and Molecular Biology Graduate Program, Weill 
      Cornell Graduate School of Medical Sciences, New York, New York 10065.
LA  - eng
GR  - P30 CA008748/CA/NCI NIH HHS/United States
GR  - R01 GM116860/GM/NIGMS NIH HHS/United States
GR  - T32 GM008539/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20170627
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Membrane Proteins)
RN  - 0 (Peptide Fragments)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (WNT3A protein, human)
RN  - 0 (Wnt3A Protein)
RN  - 48TCX9A1VT (Cystine)
RN  - EC 2.3.- (Acyltransferases)
RN  - EC 2.3.1.- (HHAT protein, human)
RN  - EC 2.3.1.- (PORCN protein, human)
RN  - EC 2.3.1.- (Porcn protein, mouse)
SB  - IM
MH  - Acylation/drug effects
MH  - Acyltransferases/antagonists & inhibitors/chemistry/genetics/*metabolism
MH  - Amino Acid Substitution
MH  - Animals
MH  - Cystine/chemistry/metabolism
MH  - Enzyme Inhibitors/pharmacology
MH  - Focal Dermal Hypoplasia/*genetics/metabolism
MH  - HEK293 Cells
MH  - Humans
MH  - Membrane Proteins/antagonists & inhibitors/chemistry/genetics/*metabolism
MH  - Mice
MH  - Mutagenesis, Site-Directed
MH  - Peptide Fragments/chemistry/metabolism
MH  - *Point Mutation
MH  - Protein Conformation
MH  - *Protein Processing, Post-Translational/drug effects
MH  - Recombinant Fusion Proteins/chemistry/metabolism
MH  - Substrate Specificity
MH  - Wnt Signaling Pathway/drug effects
MH  - Wnt3A Protein/chemistry/*metabolism
PMC - PMC5566510
OTO - NOTNLM
OT  - Wnt pathway
OT  - disulfide
OT  - enzyme mechanism
OT  - fatty acid
OT  - protein palmitoylation
COIS- The authors declare that they have no conflicts of interest with the contents of 
      this article
EDAT- 2017/06/29 06:00
MHDA- 2017/09/07 06:00
PMCR- 2018/08/18
CRDT- 2017/06/29 06:00
PHST- 2017/06/01 00:00 [received]
PHST- 2017/06/26 00:00 [revised]
PHST- 2017/06/29 06:00 [pubmed]
PHST- 2017/09/07 06:00 [medline]
PHST- 2017/06/29 06:00 [entrez]
PHST- 2018/08/18 00:00 [pmc-release]
AID - S0021-9258(20)34338-6 [pii]
AID - C117.800136 [pii]
AID - 10.1074/jbc.C117.800136 [doi]
PST - ppublish
SO  - J Biol Chem. 2017 Aug 18;292(33):13507-13513. doi: 10.1074/jbc.C117.800136. Epub 
      2017 Jun 27.