Identification of the Wnt maturation complex
Date
2024
Authors
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Publisher
University of Delaware
Abstract
Wnt signal transduction is fundamental to human development and is responsible for maintaining cellular homeostasis later in life. During development, reduced or absent Wnt signaling results in a wide range of developmental defects and aberrant signaling in adults is recognized as a primary driver of cancers. Strikingly, Wnt signaling is so indispensable to bodily function that any perturbation or disruption throughout the pathway can lead to developmental defect or cancer; prominent examples include focal dermal hyperplasia, osteogenesis imperfecta, and colorectal cancer. While the numerous Wnt ligands and target receptors in receiving cells have been extensively studied, the maturation of virtually all Wnt ligands within sending cells is relegated to highly conserved yet understudied components, Porcupine (Por) and Wntless (Wls). Our initial characterization of Por and Wls function within the model organism Drosophila melanogaster to produce functional Wnt has informed much of our understanding of Wnt ligand maturation. Further characterization of the mechanisms by which these players interact to produce mature, active Wnt ligand is essential to better delineate how Wnt signaling is regulated. ☐ Herein we provide identification and initial characterization of the Wnt Maturation Complex. We discovered that Wls forms homo-dimers dependent upon intermolecular disulfide bridge formation, and these Wls dimers interact with both Wnt and Porcupine (2:1, Wls-Wnt, Wls-Por). Remarkably, this provides a universal mechanism for Wls dimerization and a possible explanation for the uncharacterized Wnt hand-off between Por and Wls in early ER Wnt processing. The overarching goals of this work are two-fold: to characterize disulfide bonded Wls dimer interactions in forming Wnt maturation complexes and to characterize disulfide bonded Wls dimer importance in Wnt post-translational modification, secretion, and signaling. We have generated various tagged forms of Drosophila Por and Wls wild-type (WT) and conserved cysteine mutants that are used in co-immunoprecipitation experiments to examine the importance of Wls disulfide bonds in forming early Wnt maturation complexes. To determine if Wls cysteine mutants affect post-translational modification, secretion, and signaling, we used readouts of Wnt N-glycosylation by western blot, secretion into media by western blot, and canonical Wnt signaling activity indicated by ß-catenin levels in western blot and by transcriptional reporter assays. This work will lead to an understanding of how Wls dimers function within a cell to produce functional Wnt ligand, impacting our comprehension of Wnt cellular dynamics, processing, and secretion mechanisms. Results from this work have the potential to identify targeted therapeutics for diseases fueled by aberrant Wnt signaling.
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Keywords
Wnt signal, Wntless, Drosophila melanogaster, Porcupine