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.
Highlights
Cell fate determination during development is controlled by morphogen gradients
We found that insertional mutagenesis attenuated dominant-negative suppression of Hh-dependent wing and restored eye developmental defects caused by N-terminal peptide modifications in vivo, probably by affecting physical interactions between both proteins at the cell surface
To determine cholesteroylated Hh biofunctions, we analyzed the following C-terminally tagged Hh variants: (i) Hemagglutinin tagged Hh (HhHA, protein sequence YPYDVPDYA inserted between residues H256 and G257), (ii) a streptavidin tagged form (HhStrep, protein sequence WSHPQFEK inserted between residues S250 and I251), and (iii) a variant having amino acids S250-H256 replaced with an HA tag (Hh HA, Figure 1)
Summary
Cell fate determination during development is controlled by morphogen gradients. In vertebrates, three closely related Hedgehog morphogens [Sonic hedgehog (Shh), Indian hedgehog, and Desert hedgehog] directly and concentration dependently regulate cell differentiation, cell proliferation, and tissue polarity during embryogenesis. C-Terminal Hh Processing molecule during endoplasmic reticulum (ER) export, followed by coupled autocatalytic cleavage and cholesteroylation of the 19 kDa N-terminal signaling domain by the autocatalytic cholesterol transferase domain (Porter et al, 1996b). In contrast to the N-palmitate, which directly (Tukachinsky et al, 2016; Qi et al, 2018) or indirectly (Ohlig et al, 2011; Kastl et al, 2018; Schürmann et al, 2018) controls Hh biofunction, artificially truncated Hh cDNA lacking the C-terminal domain is translated into non-cholesteroylated yet bioactive 19 kDa HhN in vitro (Zeng et al, 2001; Dawber et al, 2005) and in vivo (Porter et al, 1996a; Lewis et al, 2001; Li et al, 2006)
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