Ca2+ coordination controls sonic hedgehog structure and its Scube2-regulated release

Abstract

Proteolytic processing of cell-surface-bound ligands, called shedding, is a fundamental system to control cell-cell signaling. Yet, our understanding of how shedding is regulated is still incomplete. One way to increase the processing of dual-lipidated membrane-associated Sonic hedgehog (Shh) is to increase the density of substrate and sheddase. This releases and also activates Shh by the removal of lipidated inhibitory N-terminal peptides from Shh receptor binding sites. Shh release and activation is enhanced by Scube2 [signal sequence, cubulin (CUB) domain, epidermal growth factor (EGF)-like protein 2], raising the question of how this is achieved. Here, we show that Scube2 EGF domains are responsible for specific proteolysis of the inhibitory Shh N-terminus, and that CUB domains complete the process by reversing steric masking of this peptide. Steric masking, in turn, depends on Ca occupancy of Shh ectodomains, unveiling a new mode of shedding regulation at the substrate level. Importantly, Scube2 uncouples processing of Shh peptides from their lipid-mediated juxtamembrane positioning, and thereby explains the long-standing conundrum that N-terminally unlipidated Shh shows patterning activity in Scube2-expressing vertebrates, but not in invertebrates that lack Scube orthologs.

Proteolytic processing of palmitoylated Hedgehog peptides specifies the 3-4 intervein region of the Drosophila wing

“…Our future aim is to distinguish between these possibilities. We also aim to characterize the Hh release factor Shifted ( Glise et al, 2005 ), a soluble protein with structural similarities to vertebrate Scube2 sheddase enhancers ( Jakobs et al, 2014 ; Jakobs et al, 2016 ; Jakobs et al, 2017 ), to identify the elusive ‘Hh sheddase’. Finally, we are currently investigating the important question of whether C-terminal Hh processing contributes to its in vivo biofunction in the wing disc and in other developing tissues requiring Hh signaling over shorter ranges, such as in the developing eye ( Ma et al, 1993 ).…”

Taking the Occam’s Razor Approach to Hedgehog Lipidation and Its Role in Development

“…Indeed, both proteolytic Shh release from the surface of producing cells and Scube2-mediated enhanced Shh processing have been reported [ 71 , 89 , 90 , 91 ]. These reports generate several predictions that can be assessed for their congruence with established functions of Hh PTMs.…”

“…Therefore, the second prediction is that proteolytically processed Hhs are also bioactive and lack palmitoylated N-terminal peptides, as shown for Spitz. Indeed, N-terminally truncated soluble and bioactive vertebrate Shh and invertebrate Hh have been described [ 91 , 92 , 98 ]. This finding, however, seems to be at odds with the established essential role of N-palmitate for Hh biofunction, as demonstrated by genetic Hhat inactivation [ 78 , 79 ] and site-directed mutagenesis of the conserved palmitate acceptor cysteine [ 33 , 43 ].…”

“…This finding, however, seems to be at odds with the established essential role of N-palmitate for Hh biofunction, as demonstrated by genetic Hhat inactivation [ 78 , 79 ] and site-directed mutagenesis of the conserved palmitate acceptor cysteine [ 33 , 43 ]. A solution to this paradox came from the observation that unprocessed N-terminal peptides block Ptc receptor binding sites of adjacent molecules in the multimeric cluster ( Figure 1 F) and that their proteolytic removal exposes these sites to activate the truncated protein [ 90 , 91 ] ( Figure 1 H). Because of its continued membrane association, the essential yet indirect role of N-palmitate is to ensure that only fully processed Hh clusters are released from the cell surface and are simultaneously primed for Ptc binding at the surface of target cells.…”

“…Here, unprocessed N-terminal extended peptides locate to the Hh binding site for its receptor Ptc and the Hh inhibitory 5E1 antibody that binds to the same site [ 100 , 101 , 102 ] ( Figure 1 J). Finally, and notably, Scube2-assisted removal of non-palmitoylated Shh C25A peptides restored Shh biofunction in vitro [ 91 ]. This finding supports the suggestion that N-palmitate is not required for direct Ptc binding and Hh signalling at the receiving cell and explains how non-palmitoylated Shh C25S can retain significant inductive and patterning activity in vertebrate tissues in vivo, as described previously [ 46 , 49 , 50 , 91 ] ( Table 1 ).…”