Mechanisms of Wnt protein secretion
Recent studies on how Wnt proteins are produced have revealed a tightly controlled system of trafficking factors that guide Wnts through the secretory pathway. Through genetic and biochemical approaches, our past research has contributed to a mechanistic understanding of the production and secretion of lipidated Wnt proteins. We identified and characterized the conserved multi-pass transmembrane protein Evi/Wls that acts as a cargo-receptor for Wnt proteins. Evi/Wls binds Wnt proteins in the ER, shuttles them to the plasma membrane and is recycled by the retromer complex back to the Golgi and ER. We further found that p24-proteins are required for sorting of Evi/Wls-Wnt complexes from the ER to the Golgi via COPIIvesicles and recently showed that ER-associated degradation (ERAD) controls the overall concentration of Evi/Wls, integrating the expression levels of different Wnt proteins. With B07, we have started to work towards a mechanistic and structural characterization of Evi/Wls, which is now continued as a joined project.
Structural studies in A01 are further supported for protein expression and purification provided by B07. In this funding period, we aim to identify novel Wnt secretion factors using a Clustered Regularly Interspersed Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas 9 and 12a) based in vivo screen and characterize their function. Expanding on the close collaboration between the Boutros and Sinning labs, we will dissect the interactions of Evi/Wls with candidates from the Wnt secretory pathway. The exact mechanism of the interactions between Evi/Wls and several proteins in the ER, associated with loading palmitoylated Wnt onto Evi/Wls and shuttling this complex into COPII vesicles, is still missing to date. In this project, we aim to fill this gap by characterizing these interactions. We will investigate the biochemical and structural properties of Evi/Wls and its interactions with ERAD factors in the absence of Wnt ligands. In addition to biochemical interaction assays, we aim to determine 3D structures of Evi/Wls complexes using an integrated structural biology approach, including crystallography and cryogenic-electron microscopy (cryo-EM). We will characterize Evi/Wls interacting proteins, determine complex stoichiometries, and define interactions by using advanced tools such as cross-linking mass spectrometry (XL-MS), hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) spectroscopy. These tools will also be applied to other Wnt secretion candidates and novel candidates identified in our in vivo screen. Taking together, combining biochemical, biophysical and genetic approaches, we aim to arrive at a mechanistic understanding of Wnt protein secretion.
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