Wagner, A. G. | Search bioRxiv

bioRxiv2026

In vitro reconstitution of vertebrate Sonic Hedgehog protein cholesterolysis

Extracellular secretion of the oncogenic sonic hedgehog signaling ligand is contingent on its release from a precursor protein through peptide bond cholesterolysis, mediated by the hedgehog C-terminal domain, SHhC. In this work, we describe the in vitro reconstitution of cholesterolysis activity for SHhC domains from vertebrate model organisms, Xenopus laevis (Xla) and Danio rerio (Dre). Cholesterolysis is assayed continuously in multi-well plates by monitoring changes in fluorescence resonance energy transfer (FRET) from an engineered precursor construct, expressed in E. coli and purified in soluble form. Using this FRET assay, we found that Xla and Dre SHhC exhibit high substrate stereospecificity, accepting cholesterol, (KM, 1-2 {micro}M, cholesterolysis t1/2 of [~]11 min) while rejecting the 3-alpha epimer, epi-cholesterol (KM > 100 {micro}M, t1/2 > 10 hr). By screening a 96-member detergent/surfactant library for compatibility with SHhC activity, we identify cationic detergents that inhibit cholesterolysis and find a shared preference for the zwitterionic n-dodecyl-phosphocholine (DPC, Fos-choline-12), which supported the fastest reaction kinetics. Lastly, we report that alanine point mutation at a conserved aspartate residue (D46A) in Xla SHhC and Dre SHhC blocks cholesterolysis; however, activity could be chemically rescued with rationally designed hyper-nucleophilic sterols. Of those sterols, 2-beta carboxy cholestanol was active as a substrate with D46A variants only; the remaining sterols were accepted by both D46A and wild-type SHhC. In summary, we have established the first in vitro kinetic assay to continuously monitor enzymatic activity of wild-type and mutant vertebrate SHhC domains in multi-well plates, a key step toward pharmacological manipulation of Sonic hedgehog protein biosynthesis in vivo.

biochemistry↗

bioRxiv2021

Nanomolar, noncovalent antagonism of hedgehog cholesterolysis: exception to the irreversibility rule for protein autoprocessing inhibition.

Hedgehog (Hh) signaling ligands undergo carboxy terminal sterylation through specialized autoprocessing, called cholesterolysis. Sterylation is brought about intramolecularly in a single turn-over by an enzymatic domain, called HhC. HhC is found in precursor Hh proteins only. Through cholesterolysis, HhC is cleaved from the precursor. Attempts to identify molecules that inhibit intramolecular cleavage/sterylation activity of HhC have resulted in antagonists that bind HhC irreversibly through covalent mechanisms, as is commonplace for protein autoprocessing inhibitors. Here we report an exception to the "irreversibility rule" for protein autoprocessing inhibition. Using a FRET-based activity assay for HhC, we screened a focused library of sterol-like analogs for HhC cholesterolysis inhibitors. We identified and validated four structurally related noncovalent inhibitors, which were then used for SAR studies. The most effective derivative, tBT-HBT, binds HhC reversibly with an IC50 of 300 nM. An allosteric binding site for tBT-HBT, encompassing interactions from the two subdomains of HhC, is suggested by kinetic analysis, mutagenesis studies, and photoaffinity labeling. A striking resemblance is found between the inhibitors described here and a family of noncovalent, allosteric activators of HhC, which we described previously. The inhibitor/activator duality appears to be mediated by the same allosteric site, which displays sensitivity to subtle differences in the structure of a heterocycle substituent on the effector molecule.

biochemistry↗