Institute of Metabolism and Cell Death, Molecular Targets & Therapeutics Center, Helmholtz Zentrum München, 85764 Neuherberg, Bavaria, Germany.
Institute of Metabolism and Cell Death, Molecular Targets & Therapeutics Center, Helmholtz Zentrum München, 85764 Neuherberg, Bavaria, Germany; Laboratory of Food Function Analysis, Tohoku University Graduate School of Agricultural Science, Sendai, Miyagi 980-8572, Japan.
Cell Rep Methods. 2024 Mar 25;4(3):100710. doi: 10.1016/j.crmeth.2024.100710. Epub 2024 Feb 24.
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 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 guardian, ferroptosis suppressor protein 1, to validate the newly identified ferroptosis inhibitor WIN62577. Together, these methods open up opportunities for evaluating alternative ferroptosis suppression mechanisms.
铁死亡是一种受脂质过氧化作用不受控制调节的细胞死亡方式，在多种疾病的病理生理学中起着关键作用，使其成为一种有前途的治疗靶点。谷胱甘肽过氧化物酶 4（GPX4）通过减少（磷酸）脂质氢过氧化物来防止铁死亡，但对其实际活性的评估仍然很困难。在这里，我们提出了一种使用亲和纯化的 GPX4 来捕捉其天然活性的具体方法。除了测量 GPX4 活性外，这种改进的方法还可以研究突变 GPX4 的活性，例如缺乏活性位点硒半胱氨酸的 GPX4 突变体，以及评估 GPX4 抑制剂，如 RSL3，作为一个范例。此外，我们将该方法应用于第二种铁死亡保护蛋白铁死亡抑制蛋白 1，以验证新鉴定的铁死亡抑制剂 WIN62577。总之，这些方法为评估替代的铁死亡抑制机制提供了机会。
