biostudies-literatureNakamura TBundesministerium für Bildung und ForschungCourant Forschungszentrum Geobiologie, Georg-August-Universität GöttingenDeutsche ForschungsgemeinschaftEuropean Research CouncilHorizon 2020 Framework ProgrammeHorizon 2020BMBF BerlinJSPSJapan Society for the Promotion of Science100710https://www.ebi.ac.uk/biostudies/studies/S-EPMC10985226biostudies-literatureUnknown4(3)Ferroptosis, a regulated cell death hallmarked by unrestrained lipid peroxidation, plays a pivotal role in the pathophysiology of various diseases, making it a promising therapeutic target. Glutathione peroxidase 4 (GPX4) prevents ferroptosis by reducing (phospho)lipid hydroperoxides, yet evaluation of its actual activity has remained arduous. Here, we present a tangible method using affinity-purified GPX4 to capture a snapshot of its native activity. Next to measuring GPX4 activity, this improved method allows for the investigation of mutational GPX4 activity, exemplified by the GPX4^U46C mutant lacking selenocysteine at its active site, as well as the evaluation of GPX4 inhibitors, such as RSL3, as a showcase. Furthermore, we apply this method to the second ferroptosis guardianCell reports methodsA tangible method to assess native ferroptosis suppressor activity.PMC1098522646150717722H02278471011418GA 88475418K0819822KK025320KK0363CO 291/10-146138541201EJ2205BTRR 353Mishima ENakagawa KIto JWahida ANakamura TMourao ASDConrad MfalseA tangible method to assess native ferroptosis suppressor activity.Ferroptosis, a regulated cell death hallmarked by unrestrained lipid peroxidation, plays a pivotal role in the pathophysiology of various diseases, making it a promising therapeutic target. Glutathione peroxidase 4 (GPX4) prevents ferroptosis by reducing (phospho)lipid hydroperoxides, yet evaluation of its actual activity has remained arduous. Here, we present a tangible method using affinity-purified GPX4 to capture a snapshot of its native activity. Next to measuring GPX4 activity, this improved method allows for the investigation of mutational GPX4 activity, exemplified by the GPX4^U46C mutant lacking selenocysteine at its active site, as well as the evaluation of GPX4 inhibitors, such as RSL3, as a showcase. Furthermore, we apply this method to the second ferroptosis guardian2024-01-01T00:00:00Z2024 Mar2026-06-03T04:40:20.004Z2024-12-04T06:18:56.762ZS-EPMC109852263840154010.1016/j.crmeth.2024.100710