In Vitro Reconstitution of Vertebrate Sonic Hedgehog Protein Cholesterolysis; Native and Engineered Sterol Specificity
- 1Department of Chemistry, Binghamton University, State University of New York, Binghamton, New York 13902, United States
- 2Department of Chemistry, State University of New York College of Environmental Science and Forestry, Syracuse, New York 13210, United States
Abstract
Extracellular secretion of the oncogenic sonic hedgehog signaling ligand is contingent on release from a precursor protein through peptide-bond cholesterolysis, mediated by the hedgehog C-terminal domain, SHhC. In this work, we first describe the in vitro reconstitution of SHhC domains from vertebrate model organisms, Xenopus laevis (Xla) and Danio rerio (Dre). Cholesterolysis is assayed continuously in multiwell plates by monitoring changes in fluorescence resonance energy transfer (FRET) from a bacterially expressed, engineered precursor construct. Using this FRET assay, we found that Xla and Dre SHhC exhibit high substrate stereospecificity, accepting cholesterol (K M, 1–2 μM, cholesterolysis t 1/2 of ∼11 min) while rejecting the 3-alpha epimer, epi-cholesterol (K M > 100 μM, t 1/2 > 10 h). Next, screening a 96-member detergent/surfactant library for compatibility with SHhC activity, we observed a shared preference for the zwitterionic n-dodecylphosphocholine (DPC, Fos-choline-12), while finding that cationic detergents appeared incompatible with cholesterolysis. Lastly, we report that an alanine point mutation at a conserved aspartate residue (D46A) in Xla SHhC and Dre SHhC enables engineering of substrate specificity away from cholesterol and toward rationally designed hypernucleophilic sterols via chemical rescue. Of those sterols, 2-β-carboxycholestanol 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 a FRET-based kinetic assay to continuously monitor enzymatic activity of vertebrate SHhC domains, a key step toward pharmacological manipulation of Sonic hedgehog protein biosynthesis, while also identifying the first SHhC mutant/sterol pairing for orthogonal sterolysis.
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BibTeX
@article{seidel2026vitro,
title = {{In Vitro Reconstitution of Vertebrate Sonic Hedgehog Protein Cholesterolysis; Native and Engineered Sterol Specificity}},
author = {Dayton C. Seidel and Andrew G. Wagner and John L. Pezzullo and Katherine A. Thayer and Seth Beadle and Margot L. Olejarczyk and Logan R. Bartlett and José-Luis Giner and Brian P. Callahan},
journal = {Biochemistry},
year = {2026},
month = oct,
doi = {10.1021/acs.biochem.6c00321},
url = {https://doi.org/10.1021/acs.biochem.6c00321},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - In Vitro Reconstitution of Vertebrate Sonic Hedgehog Protein Cholesterolysis; Native and Engineered Sterol Specificity
AU - Dayton C. Seidel
AU - Andrew G. Wagner
AU - John L. Pezzullo
AU - Katherine A. Thayer
AU - Seth Beadle
AU - Margot L. Olejarczyk
AU - Logan R. Bartlett
AU - José-Luis Giner
AU - Brian P. Callahan
PY - 2026
DA - 2026/10/13
JO - Biochemistry
DO - 10.1021/acs.biochem.6c00321
UR - https://doi.org/10.1021/acs.biochem.6c00321
AB - Extracellular secretion of the oncogenic sonic hedgehog signaling ligand is contingent on release from a precursor protein through peptide-bond cholesterolysis, mediated by the hedgehog C-terminal domain, SHhC. In this work, we first describe the in vitro reconstitution of SHhC domains from vertebrate model organisms, Xenopus laevis (Xla) and Danio rerio (Dre). Cholesterolysis is assayed continuously in multiwell plates by monitoring changes in fluorescence resonance energy transfer (FRET) from a bacterially expressed, engineered precursor construct. Using this FRET assay, we found that Xla and Dre SHhC exhibit high substrate stereospecificity, accepting cholesterol (K M, 1–2 μM, cholesterolysis t 1/2 of ∼11 min) while rejecting the 3-alpha epimer, epi-cholesterol (K M > 100 μM, t 1/2 > 10 h). Next, screening a 96-member detergent/surfactant library for compatibility with SHhC activity, we observed a shared preference for the zwitterionic n-dodecylphosphocholine (DPC, Fos-choline-12), while finding that cationic detergents appeared incompatible with cholesterolysis. Lastly, we report that an alanine point mutation at a conserved aspartate residue (D46A) in Xla SHhC and Dre SHhC enables engineering of substrate specificity away from cholesterol and toward rationally designed hypernucleophilic sterols via chemical rescue. Of those sterols, 2-β-carboxycholestanol 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 a FRET-based kinetic assay to continuously monitor enzymatic activity of vertebrate SHhC domains, a key step toward pharmacological manipulation of Sonic hedgehog protein biosynthesis, while also identifying the first SHhC mutant/sterol pairing for orthogonal sterolysis.
SN - 0006-2960
SN - 1520-4995
ER -