Enhanced potency of human Sonic hedgehog by hydrophobic modification

Area of Science:

• Biochemistry
• Molecular Biology
• Developmental Biology

Background:

• Sonic hedgehog (Shh) is a crucial developmental signaling protein.
• Post-translational modifications, including N-terminal lipidation and C-terminal cholesterolylation, are known to activate Shh.
• The structural basis for this activation remains incompletely understood.

Purpose of the Study:

• To investigate the structural determinants of Shh activation by examining the effects of N-terminal modifications.
• To determine the relationship between hydrophobicity and Shh signaling potency.
• To explore if N-terminal modification is a conserved activation mechanism in Hedgehog proteins.

Main Methods:

• Chemical and mutagenic methods were used to introduce hydrophobic and hydrophilic moieties to the N-terminal signaling domain of Shh.
• In vitro and in vivo assays were employed to test the activity of modified Shh proteins.
• Binding affinity, structural integrity (circular dichroism, thermal denaturation, size determination), and signaling potency (EC50 values) were assessed.

Main Results:

• Hydrophobic modifications at the N-terminus significantly increased Shh potency, with N-myristoylated Shh showing activity comparable to the naturally modified form.
• Shh activation correlated with the hydrophobicity of the N-terminal modification, while hydrophilic substitutions decreased activity.
• N-terminal modifications did not affect Shh binding to its receptor Patched or its overall structure.
• Similar activation was observed for Desert Hh upon N-terminal modification.

Conclusions:

• N-terminal hydrophobicity is a key determinant of Sonic hedgehog signaling potency.
• Lipidation at the N-terminus is sufficient for significant Shh activation.
• The findings suggest a conserved mechanism of N-terminal modification for activating Hedgehog family proteins.