Area of Science: 02 Wnt is a morphogen released from signaling cells to transduce signals to other cells over large distances. Wnt signaling is extensively involved in developmental processes, including cell polarity, fate specification, and organogenesis, and is conserved from Caenorhabditis elegans to humans. Aberrant Wnt signaling has been implicated in many types of cancers. Wnt proteins and receptors are promiscuous in binding, and each of the coexisting multiple Wnt pathways elicits distinct cell responses. Conventional methods are ineffective in studying Wnt signaling due to the functional redundancy of Wnt signaling members as well as the difficulty in working with the small and heavily modified Wnt protein in vitro. Despite intense efforts, the mechanisms of Wnt secretion, gradient formation, and Wnt signal specificity are poorly understood. We propose to genetically encode unnatural amino acids (UAAs) into proteins in C. elegans, and to tailor UAAs with novel chemical and physical properties for new and precise studies of the Wnt signaling pathway directly in vivo. Fluorescent UAAs will be encoded to image the localization and trafficking of Wnt proteins, which cannot be tagged by bulky fluorescent proteins. Photocrosslinking UAAs will be used to covalently lock interacting Wnt molecules in vivo with high sensitivity and specificity for identification. These strategies will enable us to visualize Wnt export and trafficking in vivo, to untangle the multiple Wnt pathways, and to identify novel ligands or receptors. This study represents the first attempt to genetically encode UAAs in a multicellular organism. The success of this approach will provide a new set of methodologies with the potential to revolutionize the investigation of various biological and biomedical problems directly in vivo. New insights into the functions of Wnt secretion and receptor activation may help identify and ameliorate Wnt-related pathologies as well as implicate this machinery in other known diseases.

Agency
National Institute of Health (NIH)
Institute
Office of The Director, National Institutes of Health (OD)
Type
NIH Director’s New Innovator Awards (DP2)
Project #
1DP2OD004744-01
Application #
7600418
Study Section
Special Emphasis Panel (ZGM1-NDIA-G (01))
Program Officer
Basavappa, Ravi
Project Start
2008-09-30
Project End
2013-06-30
Budget Start
2008-09-30
Budget End
2013-06-30
Support Year
1
Fiscal Year
2008
Total Cost
$2,841,000
Indirect Cost
Name
Salk Institute for Biological Studies
Department
Type
DUNS #
078731668
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Wang, Lei (2017) Engineering the Genetic Code in Cells and Animals: Biological Considerations and Impacts. Acc Chem Res 50:2767-2775
Kang, Ji-Yong; Kawaguchi, Daichi; Wang, Lei (2016) Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons. J Vis Exp :e53818
Hoppmann, Christian; Maslennikov, Innokentiy; Choe, Senyon et al. (2015) In Situ Formation of an Azo Bridge on Proteins Controllable by Visible Light. J Am Chem Soc 137:11218-21
Xiang, Zheng; Lacey, Vanessa K; Ren, Haiyan et al. (2014) Proximity-enabled protein crosslinking through genetically encoding haloalkane unnatural amino acids. Angew Chem Int Ed Engl 53:2190-3
Chen, Xiao-Hua; Xiang, Zheng; Hu, Ying S et al. (2014) Genetically encoding an electrophilic amino acid for protein stapling and covalent binding to native receptors. ACS Chem Biol 9:1956-61
Wang, Qian; Sun, Tingting; Xu, Jianfeng et al. (2014) Response and adaptation of Escherichia coli to suppression of the amber stop codon. Chembiochem 15:1744-9
Hoppmann, Christian; Lacey, Vanessa K; Louie, Gordon V et al. (2014) Genetically encoding photoswitchable click amino acids in Escherichia coli and mammalian cells. Angew Chem Int Ed Engl 53:3932-6
Kang, Ji-Yong; Kawaguchi, Daichi; Coin, Irene et al. (2013) In vivo expression of a light-activatable potassium channel using unnatural amino acids. Neuron 80:358-70
Xiang, Zheng; Ren, Haiyan; Hu, Ying S et al. (2013) Adding an unnatural covalent bond to proteins through proximity-enhanced bioreactivity. Nat Methods 10:885-8
Coin, Irene; Katritch, Vsevolod; Sun, Tingting et al. (2013) Genetically encoded chemical probes in cells reveal the binding path of urocortin-I to CRF class B GPCR. Cell 155:1258-69

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