Molecular Mechanisms of Wntless in Zaki Syndrome and Wnt secretion

Molecular Mechanisms of Wntless in Zaki Syndrome and Wnt secretion Approximately 3% of live births are affected by structural birth defects. The underlying pathological mechanisms and effective pharmacologic prevention, however, remain largely elusive. Zaki Syndrome is an inborn error marked by severe developmental abnormalities, including brain malformations, facial dysmorphisms, limb deformities, and growth retardation, posing a significant threat to children's health. This syndrome was identified in 2021 and is caused by homozygous mutations in the WLS (Wntless) gene, leading to disturbances in the Wnt signaling pathway, which is critical for embryonic and fetal development. WLS is a transmembrane protein essential for the maturation and secretion of Wnt proteins, which play a crucial role in cell differentiation during developmental process. WLS binds tightly to the modified Wnt proteins, facilitating their intracellular trafficking and extracellular secretion. Moreover, a Glycogen Synthase Kinase 3 inhibitor named CHIR99021 has been shown to restore development in the Zaki disease models. Interestingly, missing body parts in mouse embryos were regenerated, and the growth of organs resumed normally. Since it was identified not long ago, how Zaki syndrome mutations affect WLS for reduced Wnt production remain unclear, moreover, the exact mechanisms of Wnt release from WLS, an indispensable step in the human development, is not yet understood. In this project, we will use multi-faceted approaches to elucidate how Zaki syndrome-causing mutations affect the activity of WLS and whether these mutations specifically alter the secretion of certain Wnt proteins more than others. We will study all six mutations and assess how they influence WLS. Additionally, we will test our hypothesis that phospholipids mediate Wnt release from WL using synthetic probes and artificial lipid environments. Furthermore, three Zaki syndrome-causing mutations are in the C-terminus of WLS. We hypothesize that these mutations disrupt the interaction between WLS and its intracellular binding partners, interfering with WLS trafficking. To investigate this, we will delineate the trafficking step each mutation disrupts and identify novel WLS binding partners and post- translational modifications involved in its trafficking. Overall, our results will provide significant insights into the mechanisms underlying Wnt secretion and Zaki syndrome in the embryonic development. Our findings may also contribute to the design of therapeutic strategies to target WLS for the treatment of Zaki syndrome and thus improve children’s health.