Uncovering the mechanisms of carrier-mediated WNT release and activity

Project Summary/Abstract The Wnt signaling pathway is essential for embryogenesis and regeneration, and its mis-regulation is involved in cancer and birth defects. The pathway is triggered by WNT ligands, a family of secreted proteins covalently modified with palmitoleate. While the palmitoleate moiety is essential for WNT binding to Frizzled (FZD) receptors and thus for signaling, it also makes WNTs stick strongly to membranes. This raised the fundamental question of how WNTs are released from producing cells and spread to distant target cells. In my postdoctoral work, I discovered that WNTs utilize a dedicated release pathway, forming soluble complexes with carrier proteins that shield the palmitoleate from the extracellular environment. In Aim 1 of the present proposal, I will extend these studies, to elucidate the unknown mechanism by which WNTs are transferred from the essential membrane protein Wntless (WLS) to carriers; this will involve cryo-EM structure determination, AlphaFold structural predictions, mutagenesis-guided experiments and mass spectrometry. In recent work, I identified a glypican (GPC)-mediated pathway for the reception of WNT-carrier complexes by target cells, which facilitates the transfer of WNTs to the FZD receptors. In Aim 2, I will determine the mechanism by which WNT-carrier complexes are unpacked to initiate signaling. Additionally, I will explore how carriers facilitate long-range WNT delivery to target cells, using advanced microscopy, and will investigate whether WNT- FZD and WNT-GPC interactions exhibit specificity for canonical or non-canonical WNTs. Given the absence of robust and quantitative reporter systems for non-canonical Wnt signaling (NWS), in Aim 3 I will first develop such reporters, based on NWS-specific genetic targets that I have identified and validated in preliminary work. I will then use the reporter cells above together with genome-wide CRISPR screening, to comprehensively discover novel components involved in NWS, which has remained poorly understood. I anticipate that this work will significantly advance our understanding of Wnt signaling, by deciphering the extracellular dynamics of WNT transfer between key binding partners. As these interactions occur extracellularly, they present opportunities for the development of protein-based therapeutics that target aberrant Wnt signaling, with greater specificity and fewer side effects compared to small molecules. To support this research, I have assembled a comprehensive training plan, with advisory committee members who are experts in mass spectrometry, structural biology, advanced microscopy, organoid culture, and antibody production. This proposal provides a strong foundation for my long-term objective of establishing an independent research lab focused on deciphering the molecular mechanisms of Wnt signaling and identifying novel therapeutic targets for correcting pathological signaling.