Project A04

Roles of lipids in Wnt secretion and signaling

Project leaders:  Britta Brügger and Michael Boutros

Abstract

Wnt signaling is a process intricately linked to lipids. Newly synthesized Wnt proteins undergo a sequence of co- and posttranslational modifications in the ER including disulfide bond formation, N-glycosylation and acylation. The nature of the covalently linked fatty acid is under debate, and conflicting data exist regarding types and sites of acylation. Using the expertise from the Brügger group in lipid biology and the Boutros group in Wnt signaling, we aim to shed light on the roles of lipids in Wnt transport and signaling. In the first funding period we have established cell lines, chemical biology tools and a mass spectrometric workflow to study Wnt acylation. We have found that Wnt11, in contrast to Wnt3 and Wnt5a, can be secreted from cells in the absence of both the palmitoleoyltransferase Porcupine and the Wnt secretory factor Evi/Wls. Our data also suggest that different types and sites of Wnt acylation exist, that might follow different routes of secretion together with distinct signaling activities. Furthermore, we have identified the N-terminus of Wnt11 as the determinant for Evi/Wls-independent secretion and non-canonical signaling activity. Building on our results from the first funding period we will focus on determining the types and sites of Wnt11 acylation. We plan to advance the mass spectrometric workflow towards quantitative monitoring of changes in Wnt11 acylation profiles, which will enable us to compare different cellular states or different Wnt variants. Using a combination of cellular and chemical biology approaches together with site-specific mutagenesis of Wnt proteins we will study the role of Wnt acylation patterns in regulating Wnt functions. We will continue our studies towards investigating a role of zDHHC5 in Wnt function, and will screen for PORCN-independent acyltransferases acting on Wnt11. We will also study how membrane lipids contribute to Wnt trafficking and signaling, including the investigation of a potential role of ether lipid and sphingolipid homeostasis in p24-dependent transport of Wnt proteins. In summary, our project will provide novel insights into the roles lipids and lipid modifications play in regulating Wnt trafficking and signaling.

Project-related publications

  • Kurth, M., F. Lolicato, A. Sandoval-Perez, H. Amaya-Espinosa, A. Teslenko, I. Sinning, R. Beck, B. Brügger*, and C. Aponte-Santamaría*. 2020. Cholesterol Localization around the Metabotropic Glutamate Receptor 2. J Phys Chem B. 124:9061-9078. PMID: 32954729
  • Rafiee, M.R., G. Sigismondo, M. Kalxdorf, L. Förster, B. Brügger, J. Béthune, and J. Krijgsveld. 2020. Protease-resistant streptavidin for interaction proteomics. Mol Syst Biol. 16:39370. PMID: 32400114
  • Wozny, K., W.D. Lehmann, M. Wozny, B.S. Akbulut, and B. Brügger. 2019. A method for the quantitative determination of glycerophospholipid regioisomers by UPLC-ESI-MS/MS. Anal Bioanal Chem. 411:915-924. PMID: 30580388
  • Hammerschmidt, P., D. Ostkotte, H. Nolte, M.J. Gerl, A. Jais, H.L. Brunner, H.G. Sprenger, M. Awazawa, H.T. Nicholls, S.M. Turpin-Nolan, T. Langer, M. Krüger, B. Brügger, and J.C. Brüning. 2019. CerS6-Derived Sphingolipids Interact with Mff and Promote Mitochondrial Fragmentation in Obesity. Cell. 177:1536-1552. PMID: 31150623
  • Voloshanenko, O., U. Schwartz, D. Kranz, B. Rauscher, M. Linnebacher, I. Augustin, and M. Boutros. 2018. beta-catenin-independent regulation of Wnt target genes by RoR2 and ATF2/ATF4 in colon cancer cells. Sci Rep. 8:3178. PMID: 29453334
  • Voloshanenko, O., P. Gmach, J. Winter, D. Kranz, and M. Boutros. 2017. Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families. FASEB J. 31:4832-4844. PMID: 28733458
  • Gerl, M.J., V. Bittl, S. Kirchner, T. Sachsenheimer, H.L. Brunner, C. Lüchtenborg, C. Özbalci, H. Wiedemann, S. Wegehingel, W. Nickel, P. Haberkant, C. Schultz, M. Krüger, and B. Brügger. 2016. Sphingosine-1-phosphate lyase deficient cells as a tool to study protein lipid interactions. Plos One. 11:e0153009. PMID: 27100999
  • Gerl, M.J., T. Sachsenheimer, M. Grzybek, U. Coskun, F.T. Wieland, and B. Brügger. 2014. Analysis of transmembrane domains and lipid modified peptides with matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry. Anal Chem. 86:3722-6. PMID: 24628620