Two-way Dispatched function in Sonic hedgehog shedding and transfer to high-density lipoproteins

Abstract

The Sonic hedgehog (Shh) signaling pathway controls embryonic development and tissue homeostasis after birth. This requires regulated solubilization of dual-lipidated, firmly plasma membrane-associated Shh precursors from producing cells. Although it is firmly established that the resistance-nodulation-division transporter Dispatched (Disp) drives this process, it is less clear how lipidated Shh solubilization from the plasma membrane is achieved. We previously showed that Disp enhances proteolytic Shh solubilization from its lipidated terminal peptide anchors. This process, called shedding, converts tightly membrane-associated hydrophobic Shh precursors into delipidated soluble proteins. We show here that Disp-mediated Shh shedding is modulated by a serum factor that we identify as high-density lipoprotein (HDL). In addition to serving as soluble sinks for free membrane cholesterol, HDLs also accept the cholesterol-modified Shh peptide from Disp. The cholesteroylated Shh peptide is required and sufficient for Disp-mediated transfer because mCherry linked to cholesteroylated peptides associates with HDL in a Disp-dependent manner, but an N-palmitoylated Shh variant that lacks C-cholesterol does not. Disp-mediated Shh transfer to HDL is finalized by proteolytic processing of the palmitoylated N-terminal membrane anchor. The resulting mono-lipidated Shh variant may help meet the demands for Hh activity regulation in different cell types and developing tissues.

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

“…Support for proteolytic processing during release is provided by the increased electrophoretic mobility of the solubilized protein, as determined by SDS-PAGE/immunoblotting, and its decreased hydrophobicity, as determined by RP-HPLC. The proteolytic conversion of Shh during release is physiologically important because it is strictly dependent on the co-expression of the Shh release proteins Disp and Scube2 [ 25 ] (supporting the release mechanism shown in Figure 1 B). The physiological importance of the proteolytic conversion of Shh is further supported by the functionality of the solubilized protein product in vitro and in vivo, although we note that the bioactivity of unpalmitoylated proteins was somewhat reduced in NIH3T3 cells and in the Drosophila eye disc.…”

“…Notably, the replacement of serum with 600 μg/mL of the pharmacological cholesterol chelator methyl-β-cyclodextrin (CD, an oligosaccharide that complexes and shields cholesterol, thereby rendering it soluble) solubilized Shh C and C25A Shh C with their C-terminal cholesteroylated peptides intact, yet again with the N-terminal palmitate removed ( Figure 2 H,I, fractions #32–34 represent N-processed Shh C and C25A Shh C (white arrowhead), Shh C and C25A Shh C fractions #27–28 represent small amounts of dually delipidated soluble Shh [ 32 , 33 , 36 ]). Taken together, these results suggest that physiological or pharmacological sterol acceptors likely interact with the C-terminal cholesteroylated Shh peptide to render it soluble, which in turn may protect it during release and limit the proteolytic processing of Shh to the plasma membrane-anchored N-peptide [ 25 ].…”

“…We first tested the multipotency of our C3H10T1/2 cell line to differentiate into osteoblasts [ 28 ], chondrocytes [ 41 ], or adipocytes [ 42 ]. To this end, C3H10T1/2 cells were cultured in the presence of adipogenic, chondrogenic, and osteogenic supplements for different time periods and their responsiveness was confirmed based on the phenotype and the expression of cell surface markers (as shown in [ 25 ]). We then expressed Shh in serum-containing media in the presence of the physiological release regulators Scube2 [ 15 , 43 ] and Disp [ 12 , 13 ], normalized the proteins by Western blotting (inset), and added similar protein amounts to the C3H10T1/2 cells.…”

“…This proposed mode of Ptch control may represent one point in a conceivable spectrum of Ptch activity. However, the regulation of Ptch1 activity by monolipidated Shh variants carrying only N-palmitate [ 58 ] or only C-cholesterol [ 25 ] has also been demonstrated. Support for these alternative signaling modes comes from the in vivo finding that the unpalmitoylated protein is active in developing tissues, such as the mouse limb bud, because it leads to a spectrum of phenotypes similar to that induced by Shh [ 17 ].…”

“…To this end, we expressed dually lipidated Shh in the presence of 10% serum. Using reverse-phase (RP) high-performance liquid chromatography (HPLC) to compare proteins based on their hydrophobicity, we identified high levels of soluble Shh variants that retained their C-cholesteroylated peptide but lacked their N-terminal palmitoylated peptides as a consequence of proteolytic processing at the plasma membrane [ 25 ]. Throughout this study, we refer to this monolipidated serum-released Hh variant as Hh C /Shh C .…”