Taking the Occam's Razor Approach to Hedgehog Lipidation and Its Role in Development

Abstract

All Hedgehog (Hh) proteins signal from producing cells to distant receiving cells despite being synthesized as N-and C-terminally lipidated, membrane-tethered molecules. To explain this paradoxical situation, over the past 15 years, several hypotheses have been postulated that tie directly into this property, such as Hh transport on cellular extensions called cytonemes or on secreted vesicles called lipophorins and exosomes. The alternative situation that tight membrane association merely serves to prevent unregulated Hh solubilization has been addressed by biochemical and structural studies suggesting Hh extraction from the membrane or proteolytic Hh release. While some of these models may act in different organisms, tissues or developmental programs, others may act together to specify Hh short- and long-range signaling in the same tissues. To test and rank these possibilities, we here review major models of Hh release and transport and hypothesize that the (bio)chemical and physical properties of firmly established, homologous, and functionally essential biochemical Hh modifications are adapted to specify and determine interdependent steps of Hh release, transport and signaling, while ruling out other steps. This is also described by the term "congruence", meaning that the logical combination of biochemical Hh modifications can reveal their true functional implications. This combined approach reveals potential links between models of Hh release and transport that were previously regarded as unrelated, thereby expanding our view of how Hhs can steer development in a simple, yet extremely versatile, manner.

Keywords: Hedgehog; morphogen transport; posttranslational modification.

Figure 1

Biosynthesis and release models of dual-lipidated Hh, using vertebrate Shh as an example. The 45 kDa ShhNC precursors are secreted into the ER and their signal sequence is processed (A). This is followed by cholesterol esterification of the C-terminal peptide by intein-related autoprocessing/cholesterol esterification (B,C). Subsequent palmitoylation of N-terminal peptides is catalysed by Hhat. This generates bioactive dual-lipidated Shh (D). After export to the cell surface, Shh firmly tethers to the outer membrane leaflet of the plasma membrane (E) and generates large clusters (F), using cell-surface-associated HSPGs as scaffolds (not shown for clarity). Crystal lattice interactions, biochemical assays and in vitro cell culture assays suggested that N-terminal peptides of one molecule in the cluster block Ptc receptor binding sites (red) of its neighbour, rendering the molecule inactive. Several Shh release mechanisms have been proposed: direct, Scube2-mediated cholesterol extraction from the membrane (G), proteolytic processing of both lipidated peptides (H), micelle formation (K) or Hh piggybacking on exosomes or lipoprotein particles and transport on extended, filopodia-like structures called cytonemes (L). Note that one way to explain strongly reduced bioactivity of non-palmitoylated Shh is that proteolytic processing of Shh N-termini is impaired during release, resulting in soluble clusters with their receptor binding sites still blocked (F’,I). Proteolytic processing, in contrast, reverses the Ptc blockade by N-terminal peptides in the cluster (J) and thereby activates the protein. The Scube2-assisted in vitro conversion of inactive unprocessed Shh into truncated fully bioactive Shh supported this mechanism (F–H). I, J: Tetrameric Shh crystal lattice interactions as part of a larger extended structure are shown. P: palmitate, C: cholesterol.