# Sterol Probes of Hedgehog Protein Processing

> **NIH NIH R15** · COLLEGE OF ENVIRONMENTAL SCI & FORESTRY · 2021 · $445,995

## Abstract

PROJECT SUMMARY/ ABSTRACT
Hedgehog protein is unique by being covalently modified by cholesterol. This is critical to its
signaling role in embryonic development, and also in regulating adult stem cells. Malfunction of
hedgehog signaling leads to severe birth defects and is involved in many cancers.
Cholesterylation is essential to Hedgehog function and is catalyzed by the C-terminal domain of
the Hedgehog precursor protein. Many of the deleterious effects of mutations in cholesterol
biosynthesis are thought to involve Hedgehog signaling. The sterol binding region of the catalytic
domain is poorly understood because it is intrinsically too disordered to be amenable to
crystallography or protein NMR. The overall goal of this proposal to provide information about the
sterol-binding domain through the use of modified sterols. A library of 23 sterols bearing a methyl
group at each position of the sterol nucleus will be assembled by organic synthesis. The methyl
groups will be put in place by methylenation of the corresponding ketones, followed by reduction.
Both stereoisomers will be obtained at each secondary position. The angular methyl groups will
be introduced by literature methods. Where precedence does not exist (positions 8 and 9),
attempts will be made to apply methods for C5-methylation. This library will be tested by a
collaborator in a FRET-based Hedgehog processing assay. The results will establish which sites
of the sterol contact the binding site of the protein. The synthetic strategy allows adding steric bulk
(methyl to ethyl) to better define the binding region. Mutagenesis studies by the same collaborator
which regain function with a blocked sterol will determine which amino acids contact which regions
of the sterol molecule. Another library of 23 sterols, this time bearing fluorine atoms at each
position, will be synthesized using largely the same precursors that were used to make the methyl
sterol library. This library will be used for 19F-NMR based studies to map the binding site of the
protein. Covalent inhibitors of the processing reaction based on this library will also be synthesized
and studied. The NMR studies will be carried by a collaborating structural biologist. Hedgehog
protein modified by the sterol analogs may be useful to study downstream signaling interactions.
The two sterol libraries may be useful in the study of other protein-sterol interactions.

## Key facts

- **NIH application ID:** 10292176
- **Project number:** 1R15GM143714-01
- **Recipient organization:** COLLEGE OF ENVIRONMENTAL SCI & FORESTRY
- **Principal Investigator:** Jose L Giner
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $445,995
- **Award type:** 1
- **Project period:** 2021-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10292176

## Citation

> US National Institutes of Health, RePORTER application 10292176, Sterol Probes of Hedgehog Protein Processing (1R15GM143714-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10292176. Licensed CC0.

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