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

铁凋亡是一种不依赖半胱天冬酶的调节性坏死形式，其特征在于在细胞膜中产生铁依赖性脂质过氧化物。虽然脂质过氧化如何导致铁凋亡细胞溶解和死亡仍然是未知的，但质膜破裂释放促炎性损伤相关分子模式（DAMP），导致坏死性炎症和先天免疫系统的激活。在此背景下，铁凋亡与诸如缺血/再灌注损伤、组织损伤和器官死亡、神经退行性疾病和癌症的疾病有关。因此，阐明在ferroptosis膜破裂的分子机制，不仅是生物学，但也medical relevance.The总体目标是了解脂质过氧化反应如何触发质膜透化导致细胞死亡。我们先前发现，铁凋亡执行的最后一步涉及质膜上纳米孔的打开，这导致细胞死亡前持续的高胞质钙和细胞肿胀。建立在这些结果和我们的专业知识，在规定的细胞死亡膜透化机制的特点，在本提案的第一个目标，我们将确定在ferroptotic细胞的细胞膜的生物物理特性的改变。我们将使用先进的显微镜和生物物理工具来检查其渗透性，流动性和横向组织，跨膜不对称性和机械性能的变化。在第二个目标中，我们将通过脂质组学分析来确定在亚铁代谢进展过程中亚细胞膜中产生的过氧化脂质种类和衍生物。最后，在第三个目标中，我们将验证先前已知和新鉴定的脂质种类对铁中毒死亡的功能相关性，并将它们与第一个目标中鉴定的膜改变联系起来。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凋亡的分子理解。

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.