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

Paracatalytic inducers are antagonists that shift the specificity of biological catalysts, resulting in non-native transformations. In this Chapter we describe methods to discover paracatalytic inducers of Hedgehog (Hh) protein autoprocessing. Native autoprocessing uses cholesterol as a substrate nucleophile to assist in cleaving an internal peptide bond within a precursor form of Hh.

A cell-based bioluminescence reporter assay of human Sonic Hedgehog protein autoprocessing to identify inhibitors and activators.

The Sonic Hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and covalently attach cholesterol to the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor's C-terminal enzymatic domain. A method to screen for small molecule regulators of this process may be of therapeutic value.

Specificity Distorted: Chemical Induction of Biological Paracatalysis.

We define paracatalysis as the acceleration of a reaction that appears abnormal or nonphysiological. With the high specificity of enzymes, side reactivity of this kind is typically negligible. However, enzyme paracatalysis can be amplified to levels that are biologically significant through interactions with a special class of small molecule "antagonist", here termed a paracatalytic inducer.

General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing.

Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation.

Protein-Nucleic Acid Conjugation with Sterol Linkers Using Hedgehog Autoprocessing.

Hedgehog (Hh) precursor proteins contain an autoprocessing domain called HhC whose native function is protein cleavage and C-terminal glycine sterylation. The transformation catalyzed by HhC occurs in from a precursor protein and exhibits wide tolerance toward both sterol and protein substrates. Here, we repurpose HhC as a 1:1 protein-nucleic acid ligase, with the sterol serving as a molecular linker.

Sterol A-ring plasticity in hedgehog protein cholesterolysis supports a primitive substrate selectivity mechanism.

Cholesterolysis of Hedgehog family proteins couples endoproteolysis to protein C-terminal sterylation. The transformation is self-catalyzed by HhC, a partially characterized enzymatic domain found in precursor forms of Hedgehog. Here we explore spatial ambiguity in sterol recognition by HhC, using a trio of derivatives where the sterol A-ring is contracted, fused, or distorted.

Chemical Bypass of General Base Catalysis in Hedgehog Protein Cholesterolysis Using a Hyper-Nucleophilic Substrate.

Proteins in the hedgehog family undergo self-catalyzed endoproteolysis involving nucleophilic attack by a molecule of cholesterol. Recently, a conserved aspartate residue (D303, or D46) of hedgehog was identified as the general base that activates cholesterol during this unusual autoprocessing event; mutation of the catalyzing functional group (D303A) reduces activity by >10-fold. Here we report near total rescue of this ostensibly dead general base mutant by a synthetic substrate, 3β-hydroperoxycholestane (3HPC) in which the sterol -OH group is replaced by the hyper nucleophilic -OOH group.

Hedgehog Proteins Consume Steroidal CYP17A1 Antagonists: Potential Therapeutic Significance in Advanced Prostate Cancer.

Abiraterone, a potent inhibitor of the human enzyme CYP17A1 (cytochrome P450c17), provides a last line of defense against ectopic androgenesis in advanced prostate cancer. Herein we report an unprecedented off-target interaction between abiraterone and oncogenic hedgehog proteins. Our experiments indicate that abiraterone and its structural congener, galeterone, can replace cholesterol as a substrate in a specialized biosynthetic event of hedgehog proteins, known as cholesterolysis.