Mechanism of inhibition of the tumor suppressor Patched by Sonic Hedgehog

Abstract

The Hedgehog cell–cell signaling pathway is crucial for animal development, and its misregulation is implicated in numerous birth defects and cancers. In unstimulated cells, pathway activity is inhibited by the tumor suppressor membrane protein, Patched. Hedgehog signaling is triggered by the secreted Hedgehog ligand, which binds and inhibits Patched, thus setting in motion the downstream events in signal transduction. Despite its critical importance, the mechanism by which Hedgehog antagonizes Patched has remained unknown. Here, we show that vertebrate Patched1 inhibition is caused by direct, palmitate-dependent interaction with the Sonic Hedgehog ligand. We find that a short palmitoylated N-terminal fragment of Sonic Hedgehog binds Patched1 and, strikingly, is sufficient to inhibit it and to activate signaling. The rest of Sonic Hedgehog confers high-affinity Patched1 binding and internalization through a distinct binding site, but, surprisingly, it is not absolutely required for signaling. The palmitate-dependent interaction with Patched1 is specifically impaired in a Sonic Hedgehog mutant causing human holoprosencephaly, the most frequent congenital brain malformation, explaining its drastically reduced potency. The palmitate-dependent interaction is also abolished in constitutively inhibited Patched1 point mutants causing the Gorlin cancer syndrome, suggesting that they might adopt a conformation distinct from the wild type. Our data demonstrate that Sonic Hedgehog signals via the palmitate-dependent arm of a two-pronged contact with Patched1. Furthermore, our results suggest that, during Hedgehog signaling, ligand binding inhibits Patched by trapping it in an inactive conformation, a mechanism that explains the dramatically reduced activity of oncogenic Patched1 mutants.

Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm

“…7b). The structure provides a nice explanation for the recent study that a palmitoylated N-terminal 22-residue peptide could partially activate Hh signaling by binding to Ptch1 36 . An unexpected discovery is the insertion of the cholesteryl moiety into ESBS of Ptch1-B, which will naturally block binding of cholesterol to this site, further block the transport of cholesterol along this path.…”

Association of Sonic Hedgehog with the extracellular matrix requires its zinc-coordination fold

“…Our observations that the catalytic domain of ShhN is required for its localization to the ECM provides further evidence that Shh is a peptidase, and that this activity is required for non-cell autonomous signaling. Distinct from the peptidase domain is the extreme N-terminal end of ShhN which binds to Ptch1 (Gong et al, 2018;Qi et al, 2018a;2018b) and suffices to alter Ptch1 activity (Tukachinsky et al, 2016), further demonstrating the dispensability of the peptidase activity to activate the Shh response.…”

“…showing that the N-terminal 22 residues of Shh that are not part of the peptidase domain, mediate binding to Ptch1 (Gong et al, 2018;Qi et al, 2018a;2018b) and suffice to regulate Ptch1 activity (Tukachinsky et al, 2016). Some bacteria have genes coding for peptidases that coordinate zinc and calcium identically to Shh (Rebollido-Rios et al, 2014;Roelink, 2018).…”

Endothelial cell response to Hedgehog ligands depends on their processing

“…Notably, Ptch1 inhibition and internalization were shown to be carried out by separable parts of Shh. 32 explaining why some Hh ligands-induced effects may be induced by any form of Hh ligands while other may be specific for one form or another. Consistently, it is currently not known why 19 N-Shh is a strong activator of Gli1 transcription in fibroblast while 17 N-Dhh is not 22 .…”