C-Terminal Peptide Modifications Reveal Direct and Indirect Roles of Hedgehog Morphogen Cholesteroylation

Abstract

Hedgehog (Hh) morphogens are involved in embryonic development and stem cell biology and, if misregulated, can contribute to cancer. One important post-translational modification with profound impact on Hh biofunction is its C-terminal cholesteroylation during biosynthesis. The current hypothesis is that the cholesterol moiety is a decisive factor in Hh association with the outer plasma membrane leaflet of producing cells, cell-surface Hh multimerization, and its transport and signaling. Yet, it is not decided whether the cholesterol moiety is directly involved in all of these processes, because their functional interdependency raises the alternative possibility that the cholesterol initiates early processes directly and that these processes can then steer later stages of Hh signaling independent of the lipid. We generated variants of the C-terminal Hh peptide and observed that these cholesteroylated peptides variably impaired several post-translational processes in producing cells and Hh biofunction in Drosophila melanogaster eye and wing development. We also found that substantial Hh amounts separated from cholesteroylated peptide tags in vitro and in vivo and that tagged and untagged Hh variants lacking their C-cholesterol moieties remained bioactive. Our approach thus confirms that Hh cholesteroylation is essential during the early steps of Hh production and maturation but also suggests that it is dispensable for Hh signal reception at receiving cells.

Hedgehog lipids: Promotors of alternative morphogen release and signaling?

“…Non-essential roles of the N-palmitate for Hh signaling activation on receiving cells can be further supported by in vitro assays that compare dually lipidated Shh (detergent-extracted from the cell membrane of Shh expressing cells, R&D 8908-SH), as used to generate cryo-EM structures that show lipid involvement, [33,34] and Shh released This reveals that dually lipidated R&D 8908-SH is unlike Shh after its release from Disp-and Scube2-expressing cells. This is also directly confirmed by reverse-phase high-performance liquid chromatography (RP-HPLC) analyses of their different hydrophobicities [17,18] : While R&D 8908-SH and cellular Shh elute in late fractions from the C4 column, consistent with their dual lipidation, Disp/Scube2-solubilized Shh elutes earlier and similar to an unlipidated N-truncated Δ25-38 ShhN control [17] (Figure 2E-F). As observed earlier, immunoblotting revealed that palmitoylation-deficient but cholesteroylated C25A Shh was also released as partially processed (Figure 2D, upper band) and Ntruncated proteins (Figure 2D, lower band).…”

“…This reveals that dually lipidated R&D 8908‐SH is unlike Shh after its release from Disp‐ and Scube2‐expressing cells. This is also directly confirmed by reverse‐phase high‐performance liquid chromatography (RP‐HPLC) analyses of their different hydrophobicities [ 17,18 ] : While R&D 8908‐SH and cellular Shh elute in late fractions from the C4 column, consistent with their dual lipidation, Disp/Scube2‐solubilized Shh elutes earlier and similar to an unlipidated N‐truncated Δ25‐38 ShhN control [ 17 ] (Figure 2E‐F). As observed earlier, immunoblotting revealed that palmitoylation‐deficient but cholesteroylated C25A Shh was also released as partially processed (Figure 2D, upper band) and N‐truncated proteins (Figure 2D, lower band).…”

“…[33][34][35][36] Other recent experiments, however, have indicated that the pathway-activating effect of N-palmitoylation might rather be indirect. Here, both lipid anchors initially tether the Hh molecules to the cell membrane until ectodomain sheddases release them in a regulated manner, leaving the lipidated terminal peptides associated with the cell membrane [18,[45][46][47] (Figure 1, step 4).…”

“…As a proof of concept, the artificial, gradual truncation of the N-terminus progressively exposes the 5E1 binding pseudo-active groove of non-palmitoylated C25S Shh (Figure 2C, bottom), which is in line with increased 5E1 reactivity of truncated Shh found by others. [21] The functional blockade of Hh biofunction by unprocessed N-termini of uncleaved molecules can explain why overexpressed palmitoylation-deficient C85S Hh (the fly equivalent of murine C25S Shh) suppresses Drosophila wing and eye development in a dominant-negative manner [18,45,49] and why gradual artificial truncation of the N-terminal unpalmitoylated peptide attenuates this effect. [45] Moreover, site-directed functional inactivation of the N-terminal Hh processing site strongly suppresses, and sometimes even abolishes Drosophila wing and eye development despite the presence of the N-palmitate, and its additional targeted removal restores their formation.…”

“…[ 33–36 ] Other recent experiments, however, have indicated that the pathway‐activating effect of N‐palmitoylation might rather be indirect. Here, both lipid anchors initially tether the Hh molecules to the cell membrane until ectodomain sheddases release them in a regulated manner, leaving the lipidated terminal peptides associated with the cell membrane [ 18,45–47 ] (Figure 1, step 4). Consistent with this model, if Shh is co‐expressed with Hhat under serum‐depleted conditions (0%–1%, the accepted and generally used condition to study Shh solubilization and activity [ 17,20,21,35 ] ), most solubilized proteins are truncated when compared with their cellular precursors (Fig.…”