Paracatalytic induction: Subverting specificity in hedgehog protein autoprocessing with small molecules

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Hedgehog (Hh) proteins undergo autoprocessing, a self-cleavage reaction essential for their biological activity.
• This native process involves the Hh C-terminal domain (HhC) utilizing cholesterol as a nucleophile to cleave the precursor protein.
• Dysregulation of Hh signaling is implicated in various diseases, making Hh pathway modulation a therapeutic target.

Purpose of the Study:

• To describe methods for discovering paracatalytic inducers of Hedgehog (Hh) protein autoprocessing.
• To characterize these novel antagonists that inhibit Hh autoprocessing via a cholesterol-independent mechanism.
• To provide protocols for identifying and validating these inducers in both in vitro and cellular systems.

Main Methods:

• Development of in vitro Förster Resonance Energy Transfer (FRET)-based assays.
• Establishment of in-cell bioluminescence assays.
• Characterization of small molecules that alter HhC substrate specificity.

Main Results:

• Identification of paracatalytic inducers as a novel class of Hh autoprocessing antagonists.
• Demonstration that these inducers shift HhC specificity from cholesterol to solvent water.
• Observation that cholesterol-independent autoproteolysis yields a non-native Hh product with diminished signaling capacity.

Conclusions:

• Paracatalytic inducers represent a new strategy for inhibiting Hh protein processing.
• These antagonists offer a mechanism to generate non-native Hh forms with reduced biological activity.
• The described assays facilitate the discovery and characterization of potential therapeutics targeting the Hh pathway.