# The mechanistic basis for targeted protein degradation in lipid metabolism

> **NIH NIH R00** · UT SOUTHWESTERN MEDICAL CENTER · 2024 · $249,000

## Abstract

PROJECT SUMMARY (See instructions):
Mammalian cells tightly control the membrane lipid composition of their organelles, and the key
homeostatic machinery for many lipids resides within the endoplasmic reticulum (ER). Proteins in the ER
must respond to changes in lipid levels, but how proteins respond to changes in lipid composition remains
largely unknown. Protein degradation is a key part of feedback loops that regulate cholesterol and
sphingolipid biosynthetic pathways, yet how protein homeostasis is influence by the membrane itself is
unknown. Resolving this basic knowledge gap will provide insights into membrane protein biology and
potentially uncover new directions for treating metabolic diseases. To better understand how the ER
membrane influences protein function and how this feeds back into lipid metabolism, we focus on the
activity ER resident ubiquitin E3 ligases and their substrates. E3 ligases label substrate proteins with
ubiquitin, which causes the substrate proteins to be degraded by the proteasome system. We will study
how these enzymes and their substrate proteins are regulated in the ER membrane and how their activity,
in turn, regulates lipid levels.
In the first aim, we will study how membrane cholesterol levels influence the activity and substrate
recognition of two ER-localized E3 ligases called MARCH6 and TRC8. We will use cellular and biochemical
assays to determine the lipid binding specificity and activity relationships for these two enzymes. We will
use cryo-electron microscopy to elucidate how these enzymes recognize sterols and how membrane lipids
influence the ability of these enzymes to form active complexes with substrate proteins. Through these
efforts we will discover biophysical principles that may apply to a broad range of membrane bound
mammalian E3 ligases. In the second aim, we will begin to uncover how targeted protein degradation
regulates sphingolipid metabolism. We will use molecular dissection techniques along with candidate and
discovery-based approaches to elucidate the molecular determinants of the regulated degradation of
regulatory proteins called ORMDLs that control sphingolipid biosynthesis. Together, these aims will provide
the first insights into substrate recognition of mammalian membrane E3 ligases and a new understanding
of how these membrane sensors function in cells.

## Key facts

- **NIH application ID:** 10878879
- **Project number:** 5R00GM141261-03
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Daniel Luke Kober
- **Activity code:** R00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $249,000
- **Award type:** 5
- **Project period:** 2021-04-01 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10878879

## Citation

> US National Institutes of Health, RePORTER application 10878879, The mechanistic basis for targeted protein degradation in lipid metabolism (5R00GM141261-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10878879. Licensed CC0.

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