Processed Sonic hedgehog - an active signaling protease?

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A major challenge in developmental biology is to understand how cells coordinate developmental behaviors with that of their neighbours. Cells often employ secreted signaling molecules such as the Hedgehog (Hh) morphogens to control developmental growth and patterning. Hh is an unusual signaling molecule, however. All vertebrate Hh family members are metalloproteins (containing a tetrahedrally coordinated zinc ion) that are synthesized in dually lipid-modified form (N-terminally palmitoylated, C-terminally cholesterol-linked). This results in metalloprotein multimerization and firm tethering to the surface of producing cells. Yet, Hhs do get released from producing cells and form lipidation-dependent concentration gradients. This raises the question of how Hh release is achieved, and specifically, how Hh release is linked to morphogen gradient formation. Previously, we showed that N-terminal lipidation is the prerequisite for the processing of an associated N-terminal peptide during sheddase-mediated Sonic Hh (Shh) release from producing cells. In addition to Shh solubilization, removal of N-terminal peptides results in the exposure of the Shh tetrahedral zinc-coordination site, which serves as the receptor (Patched, Ptc) binding site. This site was previously blocked by the N-terminal peptide. Importantly, the Shh zinc-coordination site and lysostaphin-type protease active sites are strikingly similar, indicating that Shh bears a functional protease active site. In this grant proposal, we thus suggest that N-terminal peptide processing during release is coupled to the conversion of an inactive, membrane-tethered Shh zymogen into an active zinc-metalloprotease. By processing extracellular matrix constituents, and possibly via autodegradation, Shh proteolytic activation may then help shape its extracellular gradient. To test this idea, we aim to characterize in vitro and in vivo Shh targets and specificity via modern, system-wide protease substrate profiling technology. We also plan to test putative roles of functional Hh zinc-coordination sites in the well-characterized Drosophila system in vivo. From these experiments, we expect fascinating insights into proteolysis-dependent Hh gradient formation in vivo and in vitro, with important implications for development and disease.