Enhanced Potency of Human Sonic Hedgehog by Hydrophobic Modification

Abstract

Post-translational modifications of the developmental signaling protein Sonic hedgehog (Shh) by a long-chain fatty acid at the N-terminus and cholesterol at the C-terminus greatly activate the protein in a cell-based signaling assay. To investigate the structural determinants of this activation phenomenon, hydrophobic and hydrophilic moieties have been introduced by chemical and mutagenic methods to the soluble N-terminal signaling domain of Shh and tested in both in vitro and in vivo assays. A wide variety of hydrophobic modifications increased the potency of Shh when added at the N-terminus of the protein, ranging from long-chain fatty acids to hydrophobic amino acids, with EC(50) values from 99 nM for the unmodified protein to 0.6 nM for the myristoylated form. The N-myristoylated Shh was as active as the natural form having both N- and C-terminal modifications. The degree of activation appears to correlate with the hydrophobicity of the modification rather than any specific chemical feature of the adduct; moreover, substitution with hydrophilic moieties decreased activity. Hydrophobic modifications at the C-terminus of Shh resulted in only a 2-3-fold increase in activity, and no activation was found with hydrophobic modification at other surface positions. The N-terminal modifications did not appear to alter the binding affinity of the Shh protein for the transfected receptor protein, Patched, and had no apparent effect on structure as measured by circular dichroism, thermal denaturation, and size determination. Activation of Desert Hh through modification of its N-terminus was also observed, suggesting that this is a common feature of Hh proteins.

Sonic Hedgehog and Other Soluble Factors from Differentiating Embryoid Bodies Inhibit Pancreas Development

“…The discrepancy in Shh concentrations (EB-CM vs. recombinant Shh-N) and levels of target gene induction is concordant with the 164-fold difference in potency between the natural lipid-modified Shh-N and its recombinant counterpart, though it can also be attributed to the presence of Indian Hedgehog which is expressed but not detectable by our assay [26,45,46]. Such differences in potency were shown to be dependent on lipid modifications that normally occur during Hedgehog processing by eukaryotic cells, a phenomenon that is bypassed in the course of recombinant protein production, resulting in a much less active peptide [45,[47][48][49][50]. Despite its low level, this concentration remains in the range with potential inhibitory effect on pancreas.…”

Identification of a small molecule inhibitor of the hedgehog signaling pathway: Effects on basal cell carcinoma-like lesions

“…The Hh-responsive (Gli 8x -luciferase) reporter construct (a gift from Brigid Hogan, Vanderbilt University, Nashville, TN) was transfected into the 10T1͞2 cells, and stable clones were selected in 1 g͞ml Geneticin (GIBCO͞BRL, Grand Island, NY). In these studies, a modified form of Shh that has an octyl moiety coupled to its N terminus (Shh shown to be much more potent in cell-based assays (21). The clone (s12) showing the strongest induction of luciferase activity (8-to 10-fold) on addition of Shh OCT was selected for use.…”

Mechanism of inhibition of the tumor suppressor Patched by Sonic Hedgehog

“…ShhΔ9 binds the high-affinity site but does not inhibit Ptch1 because it cannot engage the low-affinity site; this also explains why ShhΔ9 acts as dominant-negative toward wild-type Shh (20). Interestingly, N-terminal modification with other fatty acids in addition to palmitate also increases Shh potency (29); furthermore, Shh in which the N-terminal cysteine is replaced by two isoleucines (ShhC24II) is significantly more potent than the unmodified ligand (29). We speculate that the palmitate-binding site in Ptch1 can interact with a wide range of hydrophobic moieties, explaining the increased potency of Shh thus modified.…”