# A Residual N-Terminal Peptide Enhances Signaling of Depalmitoylated Hedgehog to the Patched Receptor

## TL;DR

This study shows that a specific N-terminal peptide is needed for the signaling of a modified form of the Sonic hedgehog protein to its receptor.

## Contribution

The study reveals that a residual N-terminal peptide compensates for the loss of N-palmitate in Hedgehog signaling.

## Key findings

- ShhC, a cholesterol-modified variant of Sonic hedgehog, remains bioactive without N-palmitate.
- Removing N-terminal peptides longer than eight amino acids inactivates depalmitoylated ShhC.
- The findings suggest a new mechanism for Hedgehog signaling involving the N-terminal peptide.

## Abstract

During their biosynthesis, Sonic hedgehog (Shh) morphogens are covalently modified by cholesterol at the C-terminus and palmitate at the N-terminus. Although both lipids initially anchor Shh to the plasma membrane of producing cells, it later translocates to the extracellular compartment to direct developmental fates in cells expressing the Patched (Ptch) receptor. Possible release mechanisms for dually lipidated Hh/Shh into the extracellular compartment are currently under intense debate. In this paper, we describe the serum-dependent conversion of the dually lipidated cellular precursor into a soluble cholesteroylated variant (ShhC) during its release. Although ShhC is formed in a Dispatched- and Scube2-dependent manner, suggesting the physiological relevance of the protein, the depalmitoylation of ShhC during release is inconsistent with the previously postulated function of N-palmitate in Ptch receptor binding and signaling. Therefore, we analyzed the potency of ShhC to induce Ptch-controlled target cell transcription and differentiation in Hh-sensitive reporter cells and in the Drosophila eye. In both experimental systems, we found that ShhC was highly bioactive despite the absence of the N-palmitate. We also found that the artificial removal of N-terminal peptides longer than eight amino acids inactivated the depalmitoylated soluble proteins in vitro and in the developing Drosophila eye. These results demonstrate that N-depalmitoylated ShhC requires an N-peptide of a defined minimum length for its signaling function to Ptch.