PMID- 30420431
OWN - NLM
STAT- MEDLINE
DCOM- 20190409
LR  - 20210314
IS  - 1083-351X (Electronic)
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 294
IP  - 1
DP  - 2019 Jan 4
TI  - In vitro reconstitution of Wnt acylation reveals structural determinants of 
      substrate recognition by the acyltransferase human Porcupine.
PG  - 231-245
LID - S0021-9258(20)36893-9 [pii]
LID - 10.1074/jbc.RA118.005746 [doi]
AB  - Wnt proteins regulate a large number of processes, including cellular growth, 
      differentiation, and tissue homeostasis, through the highly conserved Wnt 
      signaling pathway in metazoans. Porcupine (PORCN) is an endoplasmic reticulum 
      (ER)-resident integral membrane enzyme that catalyzes posttranslational 
      modification of Wnts with palmitoleic acid, an unsaturated lipid. This unique 
      form of lipidation with palmitoleic acid is a vital step in the biogenesis and 
      secretion of Wnt, and PORCN inhibitors are currently in clinical trials for 
      cancer treatment. However, PORCN-mediated Wnt lipidation has not been 
      reconstituted in vitro with purified enzyme. Here, we report the first successful 
      purification of human PORCN and confirm, through in vitro reconstitution with the 
      purified enzyme, that PORCN is necessary and sufficient for Wnt acylation. By 
      systematically examining a series of substrate variants, we show that PORCN 
      intimately recognizes the local structure of Wnt around the site of acylation. 
      Our in vitro assay enabled us to examine the activity of PORCN with a range of 
      fatty acyl-CoAs with varying length and unsaturation. The selectivity of human 
      PORCN across a spectrum of fatty acyl-CoAs suggested that the kink in the 
      unsaturated acyl chain is a key determinant of PORCN-mediated catalysis. Finally, 
      we show that two putative PORCN inhibitors that were discovered with cell-based 
      assays indeed target human PORCN. Together, these results provide discrete, 
      high-resolution biochemical insights into the mechanism of PORCN-mediated Wnt 
      acylation and pave the way for further detailed biochemical and structural 
      studies.
FAU - Lee, Chul-Jin
AU  - Lee CJ
AD  - Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute 
      of Child Health and Human Development, National Institutes of Health, Bethesda, 
      Maryland 20892.
FAU - Rana, Mitra S
AU  - Rana MS
AD  - Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute 
      of Child Health and Human Development, National Institutes of Health, Bethesda, 
      Maryland 20892.
FAU - Bae, Chanhyung
AU  - Bae C
AD  - Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, 
      NINDS, National Institutes of Health, Bethesda, Maryland 20892.
FAU - Li, Yan
AU  - Li Y
AD  - Protein/Peptide Sequencing Facility, NINDS, National Institutes of Health, 
      Bethesda, Maryland 20892.
FAU - Banerjee, Anirban
AU  - Banerjee A
AD  - Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute 
      of Child Health and Human Development, National Institutes of Health, Bethesda, 
      Maryland 20892. Electronic address: anirban.banerjee@nih.gov.
LA  - eng
SI  - PDB/4YMK
SI  - PDB/4UZQ
SI  - PDB/4F0A
GR  - ZIA HD008928/Intramural NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20181112
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Acyl Coenzyme A)
RN  - 0 (Membrane Proteins)
RN  - 0 (Wnt Proteins)
RN  - EC 2.3.- (Acyltransferases)
RN  - EC 2.3.1.- (PORCN protein, human)
SB  - IM
MH  - Acyl Coenzyme A/*chemistry/metabolism
MH  - Acylation
MH  - Acyltransferases/*chemistry/genetics/metabolism
MH  - Humans
MH  - *Lipoylation
MH  - Membrane Proteins/*chemistry/genetics/metabolism
MH  - Wnt Proteins/*chemistry/genetics/metabolism
PMC - PMC6322882
OTO - NOTNLM
OT  - MBOAT family
OT  - Wnt pathway
OT  - acyl selectivity
OT  - acyltransferase
OT  - enzyme mechanism
OT  - enzyme purification
OT  - membrane enzyme
OT  - post-translational modification (PTM)
OT  - protein acylation
OT  - protein lipidation
COIS- The authors declare that they have no conflicts of interest with the contents of 
      this article
EDAT- 2018/11/14 06:00
MHDA- 2019/04/10 06:00
PMCR- 2020/01/04
CRDT- 2018/11/14 06:00
PHST- 2018/09/06 00:00 [received]
PHST- 2018/10/30 00:00 [revised]
PHST- 2018/11/14 06:00 [pubmed]
PHST- 2019/04/10 06:00 [medline]
PHST- 2018/11/14 06:00 [entrez]
PHST- 2020/01/04 00:00 [pmc-release]
AID - S0021-9258(20)36893-9 [pii]
AID - RA118.005746 [pii]
AID - 10.1074/jbc.RA118.005746 [doi]
PST - ppublish
SO  - J Biol Chem. 2019 Jan 4;294(1):231-245. doi: 10.1074/jbc.RA118.005746. Epub 2018 
      Nov 12.