S-palmitoylation is a reversible and widespread post-translational modification, but its role in the regulation of ferroptosis has been poorly understood. Here, we elucidate that GPX4, an essential regulator of ferroptosis, is reversibly palmitoylated on cysteine 66. The acyltransferase ZDHHC20 palmitoylates GPX4 and increases its protein stability. ZDHHC20 depletion or inhibition of protein palmitoylation by 2-BP sensitizes cancer cells to ferroptosis. Moreover, we identify APT2 as the depalmitoylase of GPX4. Genetic silencing or pharmacological inhibition of APT2 with ML349 increases GPX4 palmitoylation, thereby stabilizing the protein and conferring resistance to ferroptosis. Notably, disrupting GPX4 palmitoylation markedly potentiates ferroptosis in xenografted and orthotopically implanted tumor models, and inhibits tumor metastasis through blood vessels. In the chemically induced colorectal cancer model, knockout of APT2 significantly aggravates cancer progression. Furthermore, pharmacologically modulating GPX4 palmitoylation impacts liver ischemia-reperfusion injury. Overall, our findings uncover the intricate network regulating GPX4 palmitoylation, highlighting its pivotal role in modulating ferroptosis sensitivity.
© 2025. The Author(s).

Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.
© 2023, Cabukusta et al.

Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. LTPs localize at membrane contact sites (MCS) where donor and acceptor organelles form a synapse to exchange information and material. The human genome encodes 90 intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. We created a gene knockout library targeting intracellular LTPs and performed whole-cell lipidomics analysis. This analysis identified major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at ER- trans Golgi membrane contact sites. Here, ORP9-ORP11 dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles, as a critical step in sphingomyelin synthesis in trans Golgi membranes. Overall, our LTP knockout library toolbox identifies various proteins controlling cellular lipid levels including the ORP9-ORP11 heterodimer linking phospholipid and sphingolipid metabolisms at ER-Golgi MCS interphase.

Production and release of natriuretic peptides by the stressed heart reduce cardiac workload by promoting vasodilation, natriuresis, and diuresis, which has been leveraged in the recent development of novel heart-failure pharmacotherapies, yet the mechanisms regulating cardiomyocyte exocytosis and natriuretic peptide release remain ill defined. We found that the Golgi S-acyltransferase zDHHC9 palmitoylates Rab3gap1 resulting in its spatial segregation from Rab3a, elevation of Rab3a-GTP levels, formation of Rab3a-positive peripheral vesicles, and impairment of exocytosis that limits atrial natriuretic peptide release. This novel pathway potentially can be exploited for targeting natriuretic peptide signaling in the treatment of heart failure.
© 2023 The Authors.

Mycobacterium tuberculosis (Mtb) hijacks host-derived fatty acids (FAs) to sustain its intracellular growth inside host cells. Here, we present a click-chemistry-based protocol to assess FA import by Mtb in axenic culture or inside mouse macrophages. We describe the use of alkyne analogs of natural FAs as an alternative to structurally altered fluorescent derivatives or hazardous radiolabeled FAs. We also detail quantitative analyses of FA uptake at single bacterial or host cell level by flow cytometry and confocal fluorescence microscopy. For complete details on the use and execution of this protocol, please refer to Laval et al. (2021).1.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.