Processed Sonic hedgehog - an active signaling protease?

• Applicants: Professor Dr. Kay Grobe; Professor Dr. Oliver Schilling
• Subject Area: Developmental Biology ; Biochemistry ; Evolutionary Cell and Developmental Biology (Zoology)
• Term: from 2013 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 246417003

• Final Report Year: 2020

Final Report Abstract

Although our original attempts to prove the putative roles of Shh proteolytic activity in Hh gradient formation in vivo were unsuccessful, observations made in this project initiated or contributed to other projects. One of them currently addresses the newly discovered possibility that catalytic Shh processing off its lipidated tags is required for its release and signaling during early development, as outlined above. Two other now published projects are of particular importance because they address the current dogma that lipidated Hh is released from the plasma membrane of producing cells by the extracting activity of the RND efflux pump Dispatched (Disp), is transported by Scube2 and signals to Ptc receptors on receiving cells via both lipids. Yet, despite its popularity, this model not only lacks true in vivo support, it is even refuted by the available data. For example, overexpressed mouse Disp can functionally replace Drosophila Dispatched in vivo, despite the fact that flies do not express Scube2 orthologs, and sterol-sensing domains (SSD) in Disp-related SSD proteins NPC1 and Ptc are involved in different aspects of homeostasis of free cellular cholesterol, but not in the extraction of lipidated proteins. Finally, Scube2 is not strictly required for Hh signaling in vertebrates, because the zebrafish you (=Scube2 KO) phenotype is the weakest in its class of mutants that disrupt the Hh signal transduction pathway, and because Shh overexpression can bypass Hh patterning defects as a consequence of Scube1-3 depletion in vivo. Consistent with this, overexpressed Hh can also be released independent of Disp function. Therefore, the question of Hh release and transport is all but settled, and the progress made in our in vitro and in vivo studies supports Disp- and Scube2-regulated shedding as the underlying Hh release mode. In addition, methodological work has contributed to progressing the field of proteomics with regard to protease specificity determination and quantitative feature determination.

Publications

• Automated peptide mapping and protein-topographical annotation of proteomics data. BMC Bioinformatics. 2014 Jun 19;15:207
  Videm P, Gunasekaran D, Schröder B, Mayer B, Biniossek ML, Schilling O
  
• Signaling domain of Sonic hedghog as cannibalistic calcium-regulated zinc peptidase. PLoS Comput Biol. 2014, 10(7): e1003707
  Rebollido-Rios R., Bandari, S., Wilms, C., Jakuschev, S., Vortkamp, A., Grobe, K. and Hoffmann, D.
  
• (2017). Molecular determinants of Hedgehog shedding and activation. Dissertation
  Kastl, Philipp (geb. Schulz)
  
• Enrichment of protein N-termini by charge reversal of internal peptides. Proteomics. 2015 Jul;15(14):2470-8
  Lai ZW, Gomez-Auli A, Keller EJ, Mayer B, Biniossek ML, Schilling O
  
• Lysosomal protein turnover contributes to the acquisition of TGFβ-1 induced invasive properties of mammary cancer cells. Mol Cancer. 2015 Feb 15;14:39
  Kern U, Wischnewski V, Biniossek ML, Schilling O, Reinheckel T
  
• Bridging the gap: Heparan sulfate and Scube2 assemble Sonic hedgehog release complexes at the surface of producing cells. Sci Rep. 2016, 6: 26435
  Jakobs, P., Schulz, P., Ortmann, C., Schürmann, S., Exner, S., Rebollido-Rios, R., Dreier, R., Seidler, D.G. and Grobe, K.
  
• Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini. Mol Cell Proteomics. 2016 Jun;15(6):2203-13
  Lai ZW, Weisser J, Nilse L, Costa F, Keller E, Tholen M, Kizhakkedathu JN, Biniossek M, Bronsert P, Schilling O
  
• Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics. Mol Cell Proteomics. 2016 Jul;15(7):2515-24
  Biniossek ML, Niemer M, Maksimchuk K, Mayer B, Fuchs J, Huesgen PF, McCafferty DG, Turk B, Fritz G, Mayer J, Haecker G, Mach L, Schilling O
  
• OpenMS: a flexible open-source software platform for mass spectrometry data analysis. Nat Methods. 2016 Aug 30;13(9):741-8
  Röst HL, Sachsenberg T, Aiche S, Bielow C, Weisser H, Aicheler F, Andreotti S, Ehrlich HC, Gutenbrunner P, Kenar E, Liang X, Nahnsen S, Nilse L, Pfeuffer J, Rosenberger G, Rurik M, Schmitt U, Veit J, Walzer M, Wojnar D, Wolski WE, Schilling O, Choudhary JS, Malmström L, Aebersold R, Reinert K, Kohlbacher O
  
• Yeast membrane proteomics using leucine metabolic labelling: Bioinformatic data processing and exemplary application to the ER-intramembrane protease Ypf1. Biochim Biophys Acta. 2016 Oct;1864(10):1363-71
  Nilse L, Avci D, Heisterkamp P, Serang O, Lemberg MK, Schilling O
  
• Ca2+ coordination controls Sonic hedgehog structure and its Scube2-regulated release. J Cell Sci. 2017. 130(19), 3261-3271
  Jakobs, P., Schulz, P., Schürmann, S., Niland, S., Exner, S., Rebollido-Rios, R., Manikowski, D., Hoffmann, D., Seidler, D.G. and Grobe, K.
  
• Disrupting Hedgehog Cardin-Weintraub sequence and positioning changes cellular differentiation and compartmentalization in vivo. Development 2018 (145)
  Kastl, P., Manikowski, D., Steffes, G., Schürmann, S., Bandari, S., Klämbt, C. and Grobe K.
  
• Proteolytic processing of palmitoylated Hedgehog peptides specifies the 3-4 intervein region of the Drosophila wing. Elife. 2018;7. pii: e33033
  Schürmann, S., Steffes, G., Manikowski, D., Kastl, P., Malkus, U., Bandari, S., Ohlig, S., Ortmann, C., Rebollido-Rios, R., Otto, M., Nüsse, H., Hoffmann, D., Klämbt, C., Galic, M., Klingauf, J. and Grobe, K.
  
• Soluble Heparin and Heparan Sulfate Glycosaminoglycans Interfere with Sonic Hedgehog Solubilization and Receptor Binding. Molecules. 2019 (8). pii: E1607
  Manikowski, D., Jakobs, P., Jboor, H. and Grobe K