# Biophysical study of the recognition of ER proteins for degradation and lipid homeostasis

> **NIH NIH K99** · UT SOUTHWESTERN MEDICAL CENTER · 2021 · $100,000

## Abstract

Protein homeostasis ensures the proper levels of proteins to accomplish their tasks. Targeted protein degradation is
emerging as a critical mechanism for the regulation of membrane cholesterol and sphingolipids. How the proteins in these
pathways, which exist in the ER, are recognized for degradation is poorly understood. Misfolded ER proteins are recognized
by conserved ERAD machinery. However, members of multi-protein complexes are not always misfolded apart from their
cognate partners and require targeted degradation pathways. The degradation of many sterol and lipid biosynthesis proteins
is regulated by their protein-protein interactions or is induced by their metabolic products in negative feedback loops. This
proposal will elucidate the molecular and biophysical basis for selective degradation in sterol and sphingolipid metabolism.
 Cholesterol levels are regulated by the Scap-SREBP system. SREBP2 begins as an integral ER membrane protein. In
conditions of low cholesterol, SREBP2 is transported by the cholesterol-sensor Scap to the Golgi. There, SREBP2 is cleaved
to release its soluble N-terminal transcription factor domain, which traffics to the nucleus and upregulates genes for
cholesterol synthesis and uptake. In my postdoctoral work, I identified a novel degron in the C-terminal regulatory domain
of SREBP2. This motif is necessary for the degradation of the SREBP2 precursor in the absence of Scap and for the
degradation of the C-terminal SREBP2 product created in the Golgi by the cleavage of SREBP2. This C-terminal SREBP2
product must be cleared to allow Scap recycle and interact with additional SREBP2 precursors. The degradation of SREBP2
is mediated by TRC8, an ER-resident E3 ligase. I developed systems to express and purify Scap-SREBP2 complexes for
structural studies. In the K99 period, I will determine the structure of SREBP2-Scap using cutting edge cryo-EM methods
and will use cell-based and biophysical methods to characterize the interaction between SREBP2 and TRC8. These studies
will reveal SREBP2 is recognized by TRC8 and how this is antagonized by the interaction between SREBP2 and Scap.
 In the R00 period, I will establish my independent career by determining the mechanisms by which targeted degradation
accomplishes the regulation of membrane levels sphingolipids. The ER-resident serine palmitoyltransferase (SPT) complex
conducts the rate-limiting step in sphingolipid synthesis. In mammals, SPT’s enzymatic activity is negatively regulated by
three highly conserved proteins (ORMDL1-3), which form a direct complex with the SPT. While there is very little
biochemical or biophysical insight into how SPT functions, recent studies show that ORMDL activity is regulated through
degradation in response to excess sphingolipid metabolites. Moreover, this degradation may be carried out by non-canonical
ERAD pathways. I will use functional assays to determine the E3 ligase recognition motifs in the ORMDLs and conduct a
biophysical study o...

## Key facts

- **NIH application ID:** 10189043
- **Project number:** 1K99GM141261-01
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Daniel Luke Kober
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $100,000
- **Award type:** 1
- **Project period:** 2021-04-01 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10189043

## Citation

> US National Institutes of Health, RePORTER application 10189043, Biophysical study of the recognition of ER proteins for degradation and lipid homeostasis (1K99GM141261-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10189043. Licensed CC0.

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