Abstract: S-palmitoylation is a reversible post-translational modification of proteins. This dynamic process involves the attachment of a palmitoyl group to cysteine residues on proteins. It is catalyzed by palmitoyltransferase. There are 23 members of palmitoyltransferase family in mammalian cells, all of which contain the zinc finger DHHC domain. This conserved domain is crucial for the catalytic activity of these enzymes. Palmitoyltransferase catalyze substrates undergoing S-palmitoylation via the ping-pong mechanism. The enzymes undergo auto-S-acylation, covalently attaching palmitate to themselves, and then transfer the palmitate to the interacting proteins. Thus, interacting proteins of palmitoyltransferase are all potential S-palmitoylation substrates. For instance, S-palmitoylation plays a vital role in regulating cell fate determination, such as cellular proliferation, metabolic regulation, and programmed cell death. Ferroptosis is a novel form of programmed cell death caused by the accumulation of peroxidized lipids. Glutathione peroxidase 4 (GPX4) utilizes glutathione (GSH) to reduce lipid peroxides, preventing the accumulation of peroxidized lipids and playing a crucial role in ferroptosis resistance. The cystine/glutamate transporter (SLC7A11) transports extracellular cystine into cells, maintaining intracellular GSH levels to regulate GPX4 activity. S-palmitoylation of GPX4 and SLC7A11 enhances protein stability and the ferroptosis resistance of tumor cells. Ferroptosis is intricately regulated by numerous signaling pathways. This study explores the role of S-palmitoylation in ferroptosis on the proteomics level. To achieve this, we selected the MDA-MB-231 breast cancer cell line as our research model. MDA-MB-231 cells lack the expression of estrogen receptor, making it sensitive to ferroptosis. This characteristic provides an ideal platform to study the relationship between S-palmitoylation and ferroptosis. S-palmitoylated proteins were enriched by the acyl-resin-assisted capture (Acyl-RAC) method after short-term stimulation with ferroptosis inducer RSL3. 41 kinds of differentially S-palmitoylated proteins were identified through nano LC—MS/MS, and 7 of them were not included in the SwissPalm database. Among them, 32 proteins were upregulated and 5 proteins were downregulated after treatment with RSL3. 10 kinds of differentially S-palmitoylated proteins associated with ferroptosis were screened by bioinformatics analysis. Notably, these contain transferrin receptor protein 1 (TFRC), lactotransferrin (LTF), solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1), and albumin (ALB). TFRC and SLC2A1, which show increased expression in the estrogen receptor-negative breast cancer tissues, suggest their potential as therapeutic targets. Due to the low natural abundance of palmitoyltransferase, the impact of RSL3 stimulation on its S-palmitoylation level wasn't directly detected in the proteomics data. ZDHHC20 is the only plasma membrane-localized palmitoyltransferase that exhibits significant expression differences in the estrogen receptor-negative breast cancer tissues. This unique characteristic suggests that ZDHHC20 may play a role in the ferroptosis process of breast cancer. Ascorbate peroxidase 2 (APEX2) is an engineered protein. APEX2 can biotinylate the interacting proteins within a 20 nm range of the protein of interest in a short time. The biotinylated proteins could be enriched by the streptavidin. 928 kinds of ZDHHC20 interacting proteins were identified by the APEX2-based proximity labeling method, and 610 of them were found to be S-palmitoylated candidates in the SwissPalm database. 11 kinds of ferroptosis markers were enriched among the interacting proteins, including long chain fatty acid-CoA ligase 4 (ACSL4), TFRC, and SLC7A11. This study provides proteomics insights into the regulatory role of protein S-palmitoylation in ferroptosis.

Key words: S-palmitoylation, ferroptosis, acyl-resin-assisted capture, proximity labeling, proteomics