# Elucidating the Mechanisms of Lipid Droplet Protein Degradation

> **NIH NIH F31** · UNIVERSITY OF CALIFORNIA BERKELEY · 2020 · $40,987

## Abstract

PROJECT SUMMARY
The growing global epidemic of metabolic disease is a pressing public health issue. As the prevalence of
disorders such as obesity, insulin resistance, and nonalcoholic fatty liver continue to climb, the need for a
thorough understanding of cellular lipid storage mechanisms has become increasingly imperative. Most
metabolic disorders involve the aberrant accumulation of lipid in tissues such as the liver and heart, leading to
devastating systemic health effects. Within cells, lipids are stored in cytosolic organelles called lipid droplets
(LDs), which consist of a neutral lipid core of triacylglycerols and cholesterol esters bounded by a phospholipid
monolayer. Associated with the monolayer are multiple regulatory proteins and enzymes that control the dynamic
sequestration and release of the lipid reserves, allowing LD proteins to influence the metabolism of the entire
cell. Although LD proteins have essential roles in maintaining cellular lipid homeostasis, little is known regarding
the regulation of LD proteins themselves – in particular, the pathways that control LD protein abundance.
Although several studies report a role for the ubiquitin-proteasome system (UPS) in modulating LD protein levels,
the identities of the required ubiquitination machinery (e.g. E3 ubiquitin ligases and E2 ubiquitin conjugating
enzymes) and the pathways by which they exert control remain unclear. To address these fundamental
questions, I propose 1) a genome-wide, fluorescence-based CRISPR/Cas9 screen to identify the degradation
pathway of the LD protein perilipin-2 (PLIN2), and 2) follow-up studies to interrogate how impaired PLIN2
degradation impacts global cellular metabolism. I have characterized a human hepatoma Cas9-expressing
fluorescent reporter cell line in which endogenous PLIN2 is tagged with GFP. Flow cytometry, Western blotting,
and fluorescence microscopy analyses confirmed that PLIN2-GFP is expressed at endogenous levels, localizes
to LDs, and is degraded by the proteasome. The validated PLIN2-GFP cell line was employed in a pilot screen
of a 10-guide-per-gene lentiviral sublibrary of single guide RNAs (sgRNAs) enriched in UPS genes. This screen
identified several candidate UPS factors that I hypothesize are involved in PLIN2 degradation. My proposed
studies include the completion of a comprehensive, genome-wide screen, validation of candidate genes,
characterization of PLIN2 degradation pathways, and examination of the functional consequences of impaired
PLIN2 clearance. These studies will elucidate the mechanisms of LD protein regulation by the UPS, providing
novel insights into how cells maintain lipid homeostasis. Knowledge of LD protein degradation pathways will
allow for an understanding of how alterations in LD protein regulation contribute to the pathogenesis of metabolic
disease.

## Key facts

- **NIH application ID:** 9891849
- **Project number:** 5F31DK121477-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA BERKELEY
- **Principal Investigator:** Melissa Roberts
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $40,987
- **Award type:** 5
- **Project period:** 2019-04-01 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9891849

## Citation

> US National Institutes of Health, RePORTER application 9891849, Elucidating the Mechanisms of Lipid Droplet Protein Degradation (5F31DK121477-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9891849. Licensed CC0.

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