Lipid droplets (LDs) are ubiquitous lipid storage organelles composed of a neutral lipid core surrounded by a phospholipid monolayer that is decorated with integral and peripheral proteins. Accurate identification of LD proteins using biochemical fractionation methods has been challenging due to the presence of contaminant proteins from co-fractionating organelles. Here, we describe a method to identify high-confidence LD proteomes that employs an engineered ascorbate peroxidase (APEX2) to induce spatially and temporally restricted biotinylation of LD proteins. This proximity labeling method can be broadly applied to define the composition of the LD proteome in any cultured cell line and can be utilized to examine LD proteome dynamics.

Lipid droplets (LDs) are conserved, endoplasmic reticulum (ER)-derived organelles that act as a dynamic cellular repository for neutral lipids. Numerous studies have examined the composition of LD proteomes by using mass spectrometry to identify proteins present in biochemically isolated buoyant fractions that are enriched in LDs. Although many bona fide LD proteins were identified, high levels of non-LD proteins that contaminate buoyant fractions complicate the detection of true LD proteins. To overcome this problem, we recently developed a proximity-labeling proteomic method to define high-confidence LD proteomes. Moreover, employing this approach, we discovered that ER-associated degradation impacts the composition of LD proteomes by targeting select LD proteins for clearance by the 26S proteasome as they transit between the ER and LDs. These findings implicate the ER as a site of LD protein degradation and underscore the high degree of crosstalk between ER and LDs.

Lipid droplets (LDs) are ubiquitous, endoplasmic reticulum (ER)-derived organelles that mediate the sequestration of neutral lipids (e.g. triacylglycerol and sterol esters), providing a dynamic cellular storage depot for rapid lipid mobilization in response to increased cellular demands. LDs have a unique ultrastructure, consisting of a core of neutral lipids encircled by a phospholipid monolayer that is decorated with integral and peripheral proteins. The LD proteome contains numerous lipid metabolic enzymes, regulatory scaffold proteins, proteins involved in LD clustering and fusion, and other proteins of unknown functions. The cellular role of LDs is inherently determined by the composition of its proteome and alteration of the LD protein coat provides a powerful mechanism to adapt LDs to fluctuating metabolic states. Here, we review the current understanding of the molecular mechanisms that govern LD protein targeting and degradation. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.

Lipid droplet (LD) functions are regulated by a complement of integral and peripheral proteins that associate with the bounding LD phospholipid monolayer. Defining the composition of the LD proteome has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions. To overcome this limitation, we developed a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. Application of this approach to two different cell types identified the vast majority of previously validated LD proteins, excluded common contaminating proteins, and revealed new LD proteins. Moreover, quantitative analysis of LD proteome dynamics uncovered a role for endoplasmic reticulum-associated degradation in controlling the composition of the LD proteome. These data provide an important resource for future LD studies and demonstrate the utility of proximity labeling to study the regulation of LD proteomes.

Ferroptosis is a regulated form of cell death associated with the iron-dependent accumulation of phospholipid hydroperoxides. Inducing ferroptosis is a promising approach to treat therapy-resistant cancer. Ferroptosis suppressor protein 1 (FSP1) promotes ferroptosis resistance in cancer by generating the antioxidant form of coenzyme Q10 (CoQ). Despite the important role of FSP1, few molecular tools exist that target the CoQ-FSP1 pathway. Through a series of chemical screens, we identify several structurally diverse FSP1 inhibitors. The most potent of these compounds, ferroptosis sensitizer 1 (FSEN1), is an uncompetitive inhibitor that acts selectively through on-target inhibition of FSP1 to sensitize cancer cells to ferroptosis. Furthermore, a synthetic lethality screen reveals that FSEN1 synergizes with endoperoxide-containing ferroptosis inducers, including dihydroartemisinin, to trigger ferroptosis. These results provide new tools that catalyze the exploration of FSP1 as a therapeutic target and highlight the value of combinatorial therapeutic regimes targeting FSP1 and additional ferroptosis defense pathways.

Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids^1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols^3,4. Ferroptosis has previously been implicated in the cell death that underlies several degenerative conditions², and induction of ferroptosis by the inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death⁵. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines⁶, which suggests that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR-Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis-resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q₁₀ (CoQ) (also known as ubiquinone-10), which acts as a lipophilic radical-trapping antioxidant that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumour xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutic agents.