Membrane alterations in ferroptosis: from lipid oxidation to pore-formation

铁下垂是一种不依赖半胱氨酸天冬氨酸氨基转移酶的调节性坏死，其特征是在细胞膜上产生铁依赖的过氧化脂质。虽然目前尚不清楚脂质过氧化是如何导致铁链细胞溶解和死亡的，但质膜破裂会释放促炎损伤相关的分子模式(DAMP)，导致坏死性炎症和天然免疫系统的激活。在这种背景下，铁性下垂与诸如缺血/再灌注损伤、组织损伤和器官死亡、神经退行性疾病和癌症等疾病有关。因此，阐明铁下垂时膜破裂的分子机制不仅具有生物学意义，而且具有医学意义。本项目的首要目标是了解脂质过氧化如何触发质膜通透性，从而导致细胞死亡。我们之前发现，铁下垂执行的最后一步是在质膜上打开纳米孔，导致持续的高胞浆钙和细胞死亡前的细胞肿胀。基于这些结果和我们在表征受控细胞死亡中的膜通透性机制方面的专业知识，在这项建议的第一个目标中，我们将确定铁链细胞中细胞膜的生物物理性质的变化。我们将使用先进的显微镜和生物物理工具来检测其渗透性、流动性和侧向组织、跨膜不对称性和力学性能的变化。在第二个目标中，我们将通过脂体学分析来确定在铁下垂进展过程中，亚细胞膜中产生了哪些过氧化脂质及其衍生物。最后，在第三个目标中，我们将验证先前已知和新发现的脂类与铁眼症死亡的功能相关性，并将它们与第一个目标中确定的膜变化联系起来。这项研究的预期结果将通过建立脂质过氧化和细胞死亡之间的机制联系来促进我们对铁性下垂的分子理解。

Ferroptosis is a caspase-independent form of regulated necrosis characterized by the generation of iron-dependent lipid peroxides in cellular membranes. While it is still unknown how lipid peroxidation leads to ferroptotic cell lysis and death, plasma membrane rupture releases pro-inflammatory damage-associated molecular patterns (DAMPs) leading to necroinflammation and activation of the innate immune system. In this context, ferroptosis has been linked to diseases such as ischemia/reperfusion injury, tissue damage and organ demise, neurodegenerative diseases and cancer. Elucidation of the molecular mechanisms governing membrane rupture during ferroptosis is therefore not only of biological, but also of medical relevance.The overarching goal of this project is to understand how lipid peroxidation triggers plasma membrane permeabilization leading to cell death. We previously found that the final step of ferroptosis execution involves the opening of nanopores at the plasma membrane that cause sustained high cytosolic calcium and cell swelling prior to cell death. Building on these results and our expertise on characterizing membrane permeabilization mechanisms in regulated cell death, in the first aim of this proposal we will determine the alterations in the biophysical properties of cellular membranes in ferroptotic cells. We will use advanced microscopy and biophysical tools to examine the changes in their permeability, fluidity and lateral organization, transmembrane asymmetry and mechanical properties. In the second aim, we will identify by lipidomic analysis which peroxidized lipid species and derivatives are generated in subcellular membranes during ferroptotic progression. Finally, in the third aim, we will validate the functional relevance of previously known and newly identified lipid species for ferroptotic death and relate them to the membrane alterations identified in the first aim. The expected outcome of this research will advance our molecular understanding of ferroptosis by establishing a mechanistic link between lipid peroxidation and execution of cell death.