# Structural and biophysical investigation of Scap regulation by cholesterol

> **NIH NIH F31** · UT SOUTHWESTERN MEDICAL CENTER · 2024 · $40,127

## Abstract

Cholesterol plays a multifaceted role in the regulation of animal cell membranes and an inability to control
excessive cholesterol levels has been implicated in many human diseases including atherosclerosis, fatty liver
disease and cardiovascular disease. Due to its dual nature (essential but toxic in excess), cellular cholesterol
content is tightly regulated. Cellular cholesterol levels are controlled through the feedback regulation of the Scap-
SREBP pathway, which is comprised of a collection of membrane proteins in the endoplasmic reticulum (ER).
The primary proteins involved in the cholesterol sensing pathway include several integral membrane proteins:
Scap, transcription factors named Sterol Regulatory-Element Binding Proteins (SREBPs), and Insigs. When ER
cholesterol levels drop below a threshold, Scap transports SREBPs to the Golgi, where proteolytic activation of
SREBPs results in cholesterol synthesis and uptake. When ER cholesterol levels rise above a threshold, Insig
binds Scap and prevents Scap-SREBP from traveling to the Golgi, terminating further cholesterol synthesis and
uptake. The central question of this research proposal is: how does Scap sense changes in ER membrane
cholesterol? Understanding the structural and biophysical role of cholesterol’s influence on Scap will uncover
the molecular mechanism of feedback regulation of the Scap-SREBP pathway. Preliminary work has yielded a
Scap-Insig complex cryo-EM reconstruction enabling the modeling of cholesterol at the Scap-Insig interface.
While investigating Scap residues involved in the cholesterol interaction, multiple residues were also identified
to play a role in SREBP-transport. Therefore aim 1 proposes to determine the cryo-EM structures of Scap in a
SREBP-transporting conformation and in the presence of cholesterol-saturated detergent micelles or
reconstituted into cholesterol-rich lipid bilayer nanodiscs. A complete set of structures will enable deciphering of
the mechanism of how cholesterol influences Scap to stop transporting SREBPs and instead allow for Insig-
binding. Recent moderate-resolution (~4 angstrom) structures of Scap and the Scap-Insig complex suggest a
large conformational change in the luminal extramembranous globular domain (L1L7) of Scap between the Insig-
free and Insig-bound states. Therefore aim 2 seeks to study how cholesterol influences the movement of the
L1L7 domain by developing and implementing a structure guided single-molecule FRET (Fluorescence
Resonance Energy Transfer, smFRET) assay to assess the conformations of purified Scap in the presence of
cholesterol. Preliminary studies have generated and confirmed the cellular regulation of Scap constructs meant
for labeling experiments. To understand the progression of the conformational changes seen in the current
structures, movement of the L1L7 domain will be measured at high-speed resolution using smFRET. Together
these studies will provide a better structural and biophysical understanding of...

## Key facts

- **NIH application ID:** 10996426
- **Project number:** 1F31HL176187-01
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Blake Christopher Williams
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $40,127
- **Award type:** 1
- **Project period:** 2025-02-01 → 2028-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10996426

## Citation

> US National Institutes of Health, RePORTER application 10996426, Structural and biophysical investigation of Scap regulation by cholesterol (1F31HL176187-01). Retrieved via MORVS 2026-10-24 from https://api.morvs.ai/grant/nih/10996426. Licensed CC0.

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