Mechanism of palytoxin-induced ferroptosis in HaCaT cells via targeting TrxR1

PLTX (Palytoxin) is a fatty-chain polyether marine biotoxin originally produced by benthic dinoflagellates, particularly Ostreopsis ovata and Ostreopsis siamensis. Via the marine food web, PLTX bioaccumulates in numerous organisms such as corals and shellfish, and ranks among the most lethal non-proteinaceous natural compounds described to date. Thus far, its toxicological mechanism remains unclear, and no specific clinical antidote is available. In this study, we employed a limited proteolysis‑based chemical proteomics approach (Lip‑SMap) to analyze the protection of protease cleavage sites conferred by PLTX binding. Combined with DIA (data‑independent acquisition) mass spectrometry in HaCaT (human immortalized keratinocyte) cells, we identified TrxR1 (thioredoxin reductase 1) as an intracellular direct target of PLTX. Molecular docking simulations revealed that PLTX forms stable hydrogen bonds with the active‑site residues Q230 and N234 of TrxR1. BLI (Biolayer interferometry) further confirmed a moderate‑to‑high affinity interaction between them (KD = 5.415 × 10^-1 μM). Confocal fluorescence imaging demonstrated significant spatial colocalization of PLTX with TrxR1 in the cytoplasm, providing visual evidence for their direct intracellular interaction. Functional assays showed that PLTX specifically inhibits TrxR1 activity in a concentration‑dependent manner, thereby compromising the cystine/GSH/GPx4 antioxidant axis. The ensuing cascade-glutathione (GSH) depletion, loss of GPx4 (glutathione peroxidase 4) activity, and build-up of reactive oxygen species (ROS) and malondialdehyde (MDA)-ultimately drives the cell toward ferroptosis. Importantly, overexpression of TrxR1 significantly reversed PLTX‑induced GSH depletion, the decrease in GPx4 activity, and MDA accumulation, confirming the central role of TrxR1 in PLTX toxicity. Collectively, our findings uncover a previously unrecognized mechanism by which PLTX induces ferroptosis through direct inhibition of TrxR1 in HaCaT cells. These findings not only offer a molecular basis for understanding the toxicity of PLTX, but also identify TrxR1 as a potential therapeutic target. As such, they provide a theoretical foundation for the prevention and treatment of marine toxin poisoning, as well as for the development of related antidotes.