The cell biology of ferroptosis
铁死亡的细胞生物学

Ferroptosis is a non-apoptotic cell death mechanism that requires the redox-active metal iron^1,2. Free intracellular iron or iron-containing enzymes react with oxygen and polyunsaturated fatty acid (PUFA)-containing lipids to generate high levels of membrane lipid peroxides — the defining feature of ferroptosis in relation to other forms of cell death³ (Box 1). These membrane lipid peroxides can be lethal to the cell when they accumulate at high levels. Any cell in the biosphere containing iron, oxygen and PUFA-containing lipids may therefore be at risk of undergoing ferroptosis. For this reason, cells have evolved powerful enzyme-catalysed mechanisms to defend against oxidative membrane damage and the onset of ferroptosis^4,5 (Fig. 1a). Ferroptosis is a physiological process with a role in homeostasis, namely tumour suppression^6,7. Ferroptosis may also be activated in acute and chronic disease conditions⁸. Accordingly, there is considerable interest in understanding the nature and regulation of this mechanism.
铁死亡是一种非凋亡性细胞死亡机制，需要氧化还原活性金属铁^{1 , 2} 。细胞内游离铁或含铁酶与氧和含多不饱和脂肪酸(PUFA) 的脂质发生反应，产生高水平的膜脂过氧化物——铁死亡与其他形式的细胞死亡相关的决定性特征³ （框1 ）。这些膜脂过氧化物在高水平积累时可能对细胞致命。因此，生物圈中任何含有铁、氧和含有 PUFA 的脂质的细胞都可能面临铁死亡的风险。因此，细胞进化出强大的酶催化机制来防御氧化膜损伤和铁死亡的发生^{4 , 5} （图1a ）。铁死亡是一种具有稳态作用的生理过程，即肿瘤抑制^{6 , 7} 。铁死亡也可能在急性和慢性疾病中被激活⁸ 。因此，人们对了解这种机制的性质和调节非常感兴趣。

a, Ferroptosis execution can be distinguished from the regulation of ferroptosis sensitivity. Ferroptosis execution involves membrane lipid peroxidation, leading to plasma membrane rupture and cell death. The glutathione (GSH)–glutathione peroxidase 4 (GPX4) and NADPH–ferroptosis suppressor protein 1 (FSP1) systems limit membrane lipid peroxidation and inhibit ferroptosis. Ferroptosis sensitivity is dictated by the propensity of the cell to accumulate high levels of membrane lipid peroxides. Whether a cell will accumulate high levels of lipid peroxides relates to the amount of iron and oxidizable lipids in the cell, and to the status of the cellular oxidant-producing and antioxidant systems. The regulation of ferroptosis sensitivity likely encompasses hundreds of distinct metabolites, enzymes and other biomolecules that impinge upon the membrane lipid peroxidation and membrane-rupturing mechanisms. b, Plasma membrane rupture is the key event that results in cell death during ferroptosis. It involves the accumulation of phospholipid hydroperoxides, which may be generated enzymatically (for example, via lipoxygenases) and non-enzymatically through radical-mediated reactions. Phospholipid peroxidation has several consequences for the cell, including altered ion fluxes (for example, increased Piezo-mediated Ca²⁺ uptake, increased transient receptor potential (TRP)-mediated Ca²⁺ and Na⁺ uptake, and reduced Na⁺/K⁺ ATPase-mediated Na⁺ export and K⁺ uptake), water ingress and biophysical effects on the membrane. Cell swelling and increased membrane stiffness can further activate ion channels (for example, Piezo and TRP) in a feedforward manner, further enhancing ion fluxes and accelerating plasma membrane rupture. c, Important ferroptosis defence mechanisms localize to the plasma membrane. The system x_c^– antiporter imports cystine in exchange for glutamate. In the cytosol, cystine is rapidly reduced to cysteine. Cysteine can be used to synthesize (reduced) GSH. GSH is the cofactor for GPX4, an enzyme that can convert toxic phospholipid peroxides (L-OOH) into benign lipid alcohols (L-OH). Parallel to GPX4, the oxidoreductase FSP1 uses NAD(P)H to regenerate the reduced form of radical-trapping antioxidants (for example, coenzyme Q10 (CoQ) or vitamin K), which in turn terminate the lipid peroxidation process by donating electrons to phospholipid peroxyl radicals (LOO^•). Receptor-mediated endocytosis influences ferroptosis sensitivity. For example, the uptake of iron in complex with transferrin by the transferrin receptor enhances ferroptosis sensitivity while uptake of the selenium-rich protein SEPP1 by its cognate receptor LDL receptor-related protein 8 (LRP8) suppresses ferroptosis sensitivity. Iron, and presumably selenium in the form of selenocysteine, are subsequently released from the lysosome. Iron can react with soluble and lipid peroxides to generate hydroxyl and lipid alkoxyl radicals that promote lipid peroxidation. Cysteine and selenium released from the lysosome can be used to synthesize GSH and the selenoprotein GPX4, respectively. ACSL4, acyl-CoA synthetase long-chain family member 4; CHMP, charged multivesicular body protein; CoA, coenzyme A; GR, GSH reductase; GSSG, oxidized GSH; PKCβII, protein kinase Cβ; PUFA, polyunsaturated fatty acid.
a ，铁死亡的执行可以与铁死亡敏感性的调节区分开来。铁死亡执行涉及膜脂过氧化，导致质膜破裂和细胞死亡。谷胱甘肽 (GSH)-谷胱甘肽过氧化物酶 4 (GPX4) 和 NADPH-铁死亡抑制蛋白 1 (FSP1) 系统限制膜脂过氧化并抑制铁死亡。铁死亡敏感性由细胞积累高水平膜脂过氧化物的倾向决定。细胞是否会积累高水平的脂质过氧化物与细胞中铁和可氧化脂质的量以及细胞产生氧化剂和抗氧化系统的状态有关。铁死亡敏感性的调节可能涉及数百种不同的代谢物、酶和其他影响膜脂质过氧化和膜破裂机制的生物分子。 b ，质膜破裂是铁死亡过程中导致细胞死亡的关键事件。它涉及磷脂氢过氧化物的积累，其可以通过酶促（例如，通过脂氧合酶）和非酶促通过自由基介导的反应产生。磷脂过氧化对细胞有多种影响，包括改变离子通量（例如，增加压电介导的 Ca ²⁺吸收、增加瞬时受体电位 (TRP) 介导的 Ca ²⁺和 Na ⁺吸收以及减少 Na ⁺ /K ⁺ ATP 酶介导的 Na ⁺输出和 K ⁺吸收）、水进入和对膜的生物物理影响。 细胞肿胀和膜硬度增加可以前馈方式进一步激活离子通道（例如，Piezo 和 TRP），进一步增强离子通量并加速质膜破裂。 c ，重要的铁死亡防御机制定位于质膜。系统 x _c ^–逆向转运蛋白导入胱氨酸以换取谷氨酸。在细胞质中，胱氨酸迅速还原为半胱氨酸。半胱氨酸可用于合成（还原型）GSH。 GSH 是 GPX4 的辅助因子，GPX4 是一种可以将有毒的磷脂过氧化物 (L-OOH) 转化为良性脂质醇 (L-OH) 的酶。与 GPX4 类似，氧化还原酶 FSP1 使用 NAD(P)H 再生还原形式的自由基捕获抗氧化剂（例如辅酶 Q10 (CoQ) 或维生素 K），从而通过向磷脂提供电子来终止脂质过氧化过程过氧自由基(LOO ^• )。受体介导的内吞作用影响铁死亡敏感性。例如，转铁蛋白受体对与转铁蛋白复合物的铁的摄取增强了铁死亡的敏感性，而富硒蛋白SEPP1通过其同源受体LDL受体相关蛋白8（LRP8）的摄取则抑制了铁死亡的敏感性。随后，铁以及可能以硒代半胱氨酸形式存在的硒从溶酶体中释放出来。铁可以与可溶性过氧化物和脂质过氧化物反应，生成羟基和脂质烷氧基自由基，促进脂质过氧化。从溶酶体释放的半胱氨酸和硒可分别用于合成谷胱甘肽和硒蛋白GPX4。 ACSL4，酰基辅酶A合成酶长链家族成员4； CHMP，带电多泡体蛋白； CoA、辅酶A； GR、谷胱甘肽还原酶； GSSG，氧化谷胱甘肽； PKCβII，蛋白激酶Cβ； PUFA，多不饱和脂肪酸。

Full size image 全尺寸图像

We can distinguish the execution of ferroptosis from mechanisms that regulate ferroptosis sensitivity. The execution of ferroptosis involves membrane lipid peroxidation, the aberrant movement of ions across the plasma membrane, cell swelling and plasma membrane rupture⁹. Cellular sensitivity to ferroptosis — the likelihood that a given stimulus will cause plasma membrane rupture through the ferroptosis mechanism — is regulated positively and negatively by molecules and pathways that control lipid metabolism, iron homeostasis, redox regulation and related processes. Accordingly, sensitivity to ferroptosis is governed by hundreds of enzymes, reactions and molecules in the cell¹⁰. These species are distributed throughout the plasma membrane and cytosol, as well as in different organelles. Depending on the cell type and the ferroptosis-inducing condition, different metabolites, enzymes and organelles appear more important than others for ferroptosis execution and the regulation of ferroptosis sensitivity¹⁰. Understanding this context-specific regulation of ferroptosis sensitivity presents an interesting challenge.
我们可以将铁死亡的执行与调节铁死亡敏感性的机制区分开来。铁死亡的发生涉及膜脂过氧化、离子穿过质膜的异常运动、细胞肿胀和质膜破裂⁹ 。细胞对铁死亡的敏感性（特定刺激通过铁死亡机制导致质膜破裂的可能性）受到控制脂质代谢、铁稳态、氧化还原调节和相关过程的分子和途径的正向和负向调节。因此，对铁死亡的敏感性由细胞¹⁰中的数百种酶、反应和分子控制。这些物种分布在整个质膜和细胞质以及不同的细胞器中。根据细胞类型和铁死亡诱导条件，不同的代谢物、酶和细胞器对于铁死亡的执行和铁死亡敏感性的调节似乎比其他代谢物更重要¹⁰ 。了解铁死亡敏感性的这种特定环境调节提出了一个有趣的挑战。

In this Review, we examine ferroptosis mechanisms at the cellular level. We first describe the events that lead to lethal plasma membrane rupture. We then discuss how different enzymes and molecules regulate ferroptosis sensitivity from different locations in the cell. Finally, we point out examples of how these processes may be integrated between different regions of the cell to execute or prevent ferroptosis. Throughout this Review, we highlight unanswered questions that exist in connection with the cell biology of ferroptosis.
在这篇综述中，我们研究了细胞水平的铁死亡机制。我们首先描述导致致命质膜破裂的事件。然后我们讨论不同的酶和分子如何从细胞的不同位置调节铁死亡敏感性。最后，我们指出了如何在细胞的不同区域之间整合这些过程以执行或预防铁死亡的示例。在这篇综述中，我们强调了与铁死亡的细胞生物学有关的未解答的问题。

Plasma membrane rupture is the terminal event in many forms of cell death. For some forms of non-apoptotic cell death, such as necroptosis and pyroptosis, this terminal process involves permeabilization of the plasma membrane by pore-forming proteins such as mixed lineage kinase domain-like pseudokinase (MLKL), gasdermin D and ninjurin 1 (refs. ^11,12). Ninjurin 1 may facilitate the execution of ferroptosis in some but not all cells^9,13. Otherwise, there is little evidence that the execution of ferroptosis requires a specific pore-forming protein. Rather, ferroptosis execution and regulation involve a distinct set of lipid-centric mechanisms that govern plasma membrane integrity.

Ferroptosis execution at the plasma membrane

The execution of ferroptosis involves the peroxidation of PUFA-containing phospholipids and the formation of lipid hydroperoxides^14,15. Once formed, lipid hydroperoxides can react with iron to generate highly reactive lipid radicals and become further modified or truncated in ways that likely contribute to the execution of ferroptosis¹⁶. Lipid hydroperoxides may be synthesized by lipoxygenase enzymes¹⁷ or formed through chemical reactions between PUFAs, soluble reactive oxygen species (ROS) and iron¹⁰. It is controversial which mechanism is most important for ferroptosis and it seems likely that both mechanisms are relevant in one cell type or another^17,18,19,20. Within the membrane, dozens of different PUFA-containing phospholipid species can be peroxidized to contribute to the execution of ferroptosis, with PUFA-phosphatidylethanolamines being especially important in many cells^{14,21,22,23,24,25,26}. When cells are exposed to ferroptosis-inducing conditions, lipid peroxides seem to initially accumulate within the endoplasmic reticulum (ER) and other organelles and subsequently at the plasma membrane prior to membrane rupture^27,28,29,30 (Box 2).

Events downstream of plasma membrane lipid peroxidation that promote ferroptosis execution have recently been clarified (Fig. 1b). Increasing lipid peroxidation raises membrane tension, which in turn activates Piezo1 and transient receptor potential (TRP) mechanosensitive ion channels⁹. Channel opening leads to Ca²⁺ and Na⁺ influx and K⁺ efflux. These ion fluxes are enhanced by the simultaneous inactivation of the plasma membrane Na⁺/K⁺ ATPase, possibly due to altered interactions between this protein and peroxidized plasma membrane phospholipids⁹. Collectively, these changes lead to a loss of ionic homeostasis and osmotic cell swelling that eventually result in plasma membrane rupture. Plasma membrane rupture can be transiently delayed by polyethylene glycol molecules that block membrane nanopores to prevent the flow of ions and water across the plasma membrane^31,32. However, polyethylene glycol molecules only delay the onset of ferroptosis and do not completely prevent plasma membrane rupture over longer periods of time³¹. It seems likely that, as more lipids become peroxidized, this disrupts the biophysical properties of the plasma membrane³³ so severely that plasma membrane rupture becomes inevitable.
最近已经阐明了质膜脂质过氧化下游促进铁死亡执行的事件（图1b ）。脂质过氧化的增加会提高膜张力，进而激活 Piezo1 和瞬时受体电位 (TRP) 机械敏感离子通道⁹ 。通道开放导致Ca ²⁺和Na ⁺流入以及K ⁺流出。这些离子通量通过质膜 Na ⁺ /K ⁺ ATP 酶的同时失活而增强，这可能是由于该蛋白质与过氧化质膜磷脂之间的相互作用发生改变⁹ 。总的来说，这些变化导致离子稳态丧失和渗透细胞肿胀，最终导致质膜破裂。质膜破裂可以通过聚乙二醇分子暂时延迟，聚乙二醇分子阻塞膜纳米孔以防止离子和水流过质膜^{31、32 。}然而，聚乙二醇分子只能延迟铁死亡的发生，并不能在较长时间内完全防止质膜破裂³¹ 。似乎有可能，随着更多的脂质被过氧化，这会严重破坏质膜³³的生物物理性质，以致质膜破裂变得不可避免。

Suppression of membrane lipid peroxidation
抑制膜脂过氧化

Ferroptosis is normally inhibited by the continuous activity of coupled enzyme-metabolite systems that prevent the accumulation of membrane lipid peroxides to toxic levels. These systems are, in fact, the targets of small molecules that helped elucidate the ferroptosis mechanism in the first place (see Box 2 for a description of common ferroptosis inducers and inhibitors). The enzyme glutathione peroxidase 4 (GPX4) reduces potentially toxic lipid hydroperoxides to less dangerous lipid alcohols^34,35. GPX4 appears to be the most critical enzyme for preventing lipid hydroperoxide accumulation in most cells^35,36 (Fig. 1c). Several isoforms of GPX4 exist, including those designated as cytosolic, mitochondrial and nuclear. Cytosolic GPX4 seems the most essential for preventing ferroptosis, although mitochondrial GPX4 may also help inhibit ferroptosis in some cases^37,38,39. Cytosolic GPX4 does not contain an obvious plasma membrane-targeting element. However, modelling studies indicate that a patch of positively charged lysine and arginine residues may facilitate the electrostatic interactions with phospholipids that help keep GPX4 in the vicinity of the plasma membrane⁴⁰.
铁死亡通常受到偶联酶代谢系统的持续活性的抑制，该系统可防止膜脂过氧化物累积至有毒水平。事实上，这些系统是小分子的靶标，这些小分子首先帮助阐明了铁死亡机制（参见方框2 ，了解常见铁死亡诱导剂和抑制剂的描述）。谷胱甘肽过氧化物酶 4 (GPX4) 将潜在有毒的脂质氢过氧化物还原为危险性较低的脂质醇^{34 、 35} 。 GPX4 似乎是防止大多数细胞中脂质氢过氧化物积累的最关键酶^{35 、 36} （图1c ）。 GPX4 存在多种亚型，包括细胞质亚型、线粒体亚型和核亚型。胞质 GPX4 似乎对于预防铁死亡最重要，尽管线粒体 GPX4 在某些情况下也可能有助于抑制铁死亡^{37 , 38 , 39} 。胞质 GPX4 不包含明显的质膜靶向元件。然而，模型研究表明，一片带正电的赖氨酸和精氨酸残基可能促进与磷脂的静电相互作用，从而有助于将 GPX4 保持在质膜附近⁴⁰ 。

Ferroptosis suppressor protein 1 (FSP1; also known as AIFM2) inhibits plasma membrane lipid peroxidation in parallel to GPX4 (Fig. 1c). FSP1 is an NAD(P)H- and FAD-dependent oxidoreductase that reduces coenzyme Q10 (CoQ)^41,42 and vitamin K⁴³. The reduced forms of CoQ and vitamin K act as lipophilic radical-trapping antioxidants (RTAs) that can terminate lipid peroxidation chain reactions. FSP1 localizes specifically to the plasma membrane and to lipid droplets^41,42. During translation, the N-terminal initiator methionine of FSP1 is removed and the 14-carbon fatty acid myristate is covalently attached to the subsequent glycine residue^41,42. The irreversibly conjugated myristate anchors FSP1 to the plasma membrane and is necessary for its anti-ferroptotic activity^41,42. Targeting FSP1 selectively to the plasma membrane is sufficient to suppress ferroptosis, implying that this is the key site where endogenous RTAs function to suppress ferroptosis⁴¹. Co-immunoprecipitation and biochemistry data support the possibility that human FSP1 exists as a dimer⁴⁴, but whether this is the functional state of FSP1 at the membrane is unknown. In addition to CoQ and vitamin K, other endogenous metabolites can serve as RTA inhibitors of ferroptosis, including tetrahydrobiopterin^23,45, vitamin E and vitamin A⁴⁶. The relative contribution of these different metabolic ferroptosis suppressors appears to vary between cell types.
铁死亡抑制蛋白1（FSP1；也称为AIFM2）与GPX4同时抑制质膜脂质过氧化（图1c ）。 FSP1 是一种 NAD(P)H 和 FAD 依赖性氧化还原酶，可还原辅酶 Q10 (CoQ) ^{41 、 42}和维生素 K ⁴³ 。还原形式的 CoQ 和维生素 K 作为亲脂性自由基捕获抗氧化剂 (RTA)，可以终止脂质过氧化链反应。 FSP1特异性定位于质膜和脂^滴41、42 。在翻译过程中，FSP1的N端起始子甲硫氨酸被去除，并且14碳脂肪酸肉豆蔻酸酯共价连接至随后的甘氨酸残基^{41、42 。}不可逆结合的肉豆蔻酸酯将 FSP1 锚定在质膜上，并且是其抗铁死亡活性所必需的^{41 , 42} 。将 FSP1 选择性地靶向质膜足以抑制铁死亡，这意味着这是内源性 RTA 发挥抑制铁死亡作用的关键位点⁴¹ 。免疫共沉淀和生物化学数据支持人类 FSP1 以二聚体形式存在的可能性⁴⁴ ，但这是否是 FSP1 在膜上的功能状态尚不清楚。除了 CoQ 和维生素 K 之外，其他内源性代谢物也可以作为铁死亡的 RTA 抑制剂，包括四氢生物蝶呤^{23 、 45} 、维生素 E 和维生素 A ⁴⁶ 。这些不同的代谢性铁死亡抑制剂的相对贡献似乎因细胞类型而异。

Plasma membrane repair as an anti-ferroptotic mechanism
质膜修复作为抗铁死亡机制

Plasma membrane repair processes oppose the terminal execution of ferroptosis. As noted above, lipid peroxidation can trigger an increase in intracellular Ca²⁺ levels³¹. Increased intracellular Ca²⁺ acts as a signal to recruit charged multivesicular body protein 5 (CHMP5) and CHMP6 — the components of the endosomal sorting complexes required for transport (ESCRT)-III — to the plasma membrane⁴⁷, where they participate in local membrane repair. Indeed, genetic silencing of CHMP5, CHMP6 or CHMP4B can enhance ferroptosis sensitivity to some degree^31,47 (Fig. 1b). However, membrane repair mediated by CHMP4B, CHMP5 and CHMP6 can be overwhelmed by high levels of lipid peroxidation^31,47. Of note, the ESCRT-III complex can also limit protein pore-dependent membrane damage during necroptosis and pyroptosis^48,49 and is therefore a negative regulator of several forms of non-apoptotic cell death.
质膜修复过程反对铁死亡的最终执行。如上所述，脂质过氧化可以引发细胞内Ca ²⁺水平的增加³¹ 。细胞内 Ca ²⁺增加充当信号，将带电多泡体蛋白 5 (CHMP5) 和 CHMP6（运输 (ESCRT)-III 所需的内体分选复合物的成分）招募到质膜⁴⁷ ，在那里它们参与局部膜维修。事实上， CHMP5 、 CHMP6或CHMP4B的基因沉默可以在一定程度上增强铁死亡^{敏感性31、47} （图1b ）。然而，CHMP4B、CHMP5 和 CHMP6 介导的膜修复可能会被高水平的脂质过氧化作用所淹没^{31 、 47} 。值得注意的是，ESCRT-III 复合物还可以限制坏死性凋亡和焦亡期间蛋白质孔依赖性膜损伤^{48 、 49} ，因此是几种形式的非凋亡细胞死亡的负调节因子。

Ferroptosis regulation by plasma membrane transporters and enzymes
质膜转运蛋白和酶对铁死亡的调节

Integral plasma membrane proteins can contribute to the execution and regulation of ferroptosis in many ways, including through the generation of ROS^1,50,51, and to the mediation of iron or metabolite trafficking^{52,53,54,55,56,57}. Here, we focus on proteins that are involved in the import of cysteine and selenium, two metabolites that contribute essentially to the negative regulation of ferroptosis (see also next section) (Fig. 1c).
完整的质膜蛋白可以通过多种方式促进铁死亡的执行和调节，包括通过ROS^{的产生1,50,51} ，以及介导铁^{或代谢物运输52,53,54,55,56,57 。}在这里，我们重点关注参与半胱氨酸和硒输入的蛋白质，这两种代谢物基本上有助于铁死亡的负调节（另见下一节）（图1c ）。

Cysteine is required to synthesize sulfur-containing anti-ferroptotic molecules, including the GPX4 cofactor (reduced) glutathione (GSH)⁵⁸. The cysteine disulfide, cystine, can be imported by the cell surface system x_c^– cystine–glutamate antiporter. Cysteine can also be acquired indirectly via endocytosis of extracellular cysteine-rich proteins, such as albumin, and their subsequent lysosomal catabolism⁵⁹. Extracellular GSH can also directly fuel de novo intracellular GSH synthesis via the γ-glutamyl cycle. Plasma membrane-localized γ-glutamyltransferase 1 (GGT1) cleaves the usual γ-glutamyl bond that links glutamate to cysteine in the GSH tripeptide. The resulting cysteinylglycine dipeptide can then be imported and further catabolized by dipeptidases, including carnosine dipeptidase 2 (CNDP2), to release cysteine for use in GSH synthesis⁶⁰. Some brain cancer cells overcome the need for system x_c^–-mediated cystine import via GGT1-mediated catabolism of extracellular GSH⁶¹. Moreover, in a rare kidney cancer (chromophobe renal cell carcinoma), the absence of GGT1 function renders these cells exquisitely dependent upon extracellular cystine for survival relative to normal tissues⁶². This helps illustrate how differences in protein expression between cells can result in unique sensitivities to pro-ferroptotic stimuli such as cystine deprivation.
合成含硫抗铁死亡分子需要半胱氨酸，包括 GPX4 辅因子（还原型）谷胱甘肽 (GSH) ⁵⁸ 。半胱氨酸二硫化物胱氨酸可以通过细胞表面系统 x _c ^–胱氨酸 – 谷氨酸逆向转运蛋白输入。半胱氨酸也可以通过细胞外富含半胱氨酸的蛋白质（例如白蛋白）的内吞作用及其随后的溶酶体分解代谢来间接获得⁵⁹ 。细胞外谷胱甘肽还可以通过γ-谷氨酰循环直接促进细胞内谷胱甘肽的从头合成。质膜定位的 γ-谷氨酰转移酶 1 (GGT1) 可裂解 GSH 三肽中连接谷氨酸和半胱氨酸的常见 γ-谷氨酰键。然后，所得半胱氨酰甘氨酸二肽可被输入并进一步被二肽酶（包括肌肽二肽酶 2 (CNDP2)）分解代谢，以释放半胱氨酸用于 GSH 合成⁶⁰ 。一些脑癌细胞通过 GGT1 介导的细胞外 GSH ⁶¹分解代谢克服了系统 x _c^介导的胱氨酸输入的需要。此外，在一种罕见的肾癌（嫌色肾细胞癌）中，GGT1 功能的缺失使得这些细胞相对于正常组织而言完全依赖于细胞外胱氨酸的生存⁶² 。这有助于说明细胞之间蛋白质表达的差异如何导致对促铁死亡刺激（例如胱氨酸剥夺）的独特敏感性。

Selenium is found within the active-site selenocysteine of GPX4 and a small number of other enzymes. In cancer cells, high levels of GPX4 translation promote ferroptosis resistance but, under conditions of low selenium, this GPX4 synthesis is inhibited by ribosome stalling and collisions, which causes early GPX4 translation termination (see also next section)⁶³. Selenium can enter the cell by endocytosis of the liver-secreted, selenium-containing carrier protein selenoprotein P, which is bound and endocytosed by cell surface receptors such as the LDL receptor-related protein 8 (LRP8)^63,64,65. In triple-negative breast cancer cells, LRP8 knockout sensitizes cells to ferroptosis inducers⁶³. In MYCN-amplified neuroblastoma cells, loss of LRP8 alone is sufficient to trigger ferroptosis, likely due to coincident low expression of system x_c^– subunits⁶⁵. Selenium can also enter the cell via the conversion of inorganic selenium (that is, selenite) to volatile selenide through a process that involves system x_c^–-dependent cystine uptake, which provides thiols that can reduce extracellular inorganic selenium to selenide, which is then presumed to diffuse across the plasma membrane⁶⁶. How cystine and selenium uptake and metabolism are coordinated to ensure proper GPX4 function is an interesting open question.
硒存在于 GPX4 和少数其他酶的活性位点硒代半胱氨酸中。在癌细胞中，高水平的 GPX4 翻译会促进铁死亡抵抗，但在低硒条件下，这种 GPX4 合成会受到核糖体停滞和碰撞的抑制，从而导致 GPX4 翻译早期终止（另见下一节） ⁶³ 。硒可以通过肝脏分泌的含硒载体蛋白硒蛋白P 的内吞作用进入细胞，该蛋白与细胞表面受体如 LDL 受体相关蛋白 8 (LRP8) ^{63 , 64 , 65}结合并被内吞。在三阴性乳腺癌细胞中，LRP8 敲除使细胞对铁死亡诱导剂敏感⁶³ 。在 MYCN 扩增的神经母细胞瘤细胞中，仅 LRP8 缺失就足以引发铁死亡，这可能是由于系统 x _c ^–亚基的同时低表达所致⁶⁵ 。硒还可以通过无机硒（即亚硒酸盐）转化为挥发性硒化物进入细胞，该过程涉及系统 x _c ^-依赖的胱氨酸摄取，该过程提供硫醇，可将细胞外无机硒还原为硒化物，然后将其转化为硒化物。推测扩散穿过质膜⁶⁶ 。胱氨酸和硒的摄取和代谢如何协调以确保 GPX4 正常发挥功能是一个有趣的悬而未决的问题。

Ferroptosis execution centres on the plasma membrane. However, key ferroptosis regulatory activities occur in the cytosol. The translation of selenoproteins such as GPX4 occurs in the cytosol. Selenocysteine is synthesized directly on a dedicated tRNA (tRNA^[Ser]Sec) in a multistep process. tRNA^[Ser]Sec is first conjugated with serine, which is then phosphorylated, allowing for the subsequent incorporation of selenium from selenophosphate and yielding the selenocysteine-conjugated tRNA. Selenoprotein transcripts are unique for two reasons. First, they contain a UGA codon within the open reading frame. UGA codons are typically stop codons that bind release factors to terminate translation. However, in selenoprotein transcripts, these UGA codons are recoded for selenocysteine insertion. Second, they contain a stem-loop-like structure called the selenocysteine insertion sequence (SECIS) element in the 3′ untranslated region that recruits machinery such as SECIS binding protein 2 to recode the ribosome for insertion of selenocysteine at the UGA codon, rather than the binding of a ribosome release factor. This unique mode of translation makes selenoprotein translation less efficient and prone to early termination, especially under conditions of limiting selenium^63,67.
铁死亡的执行中心是质膜。然而，关键的铁死亡调节活动发生在细胞质中。硒蛋白（例如 GPX4）的翻译发生在细胞质中。硒代半胱氨酸是通过多步骤过程直接在专用 tRNA (tRNA ^[Ser]Sec ) 上合成的。 tRNA ^[Ser]Sec首先与丝氨酸缀合，然后磷酸化，随后从硒磷酸盐中掺入硒并产生硒代半胱氨酸缀合的 tRNA。硒蛋白转录物之所以独特有两个原因。首先，它们在开放阅读框中包含UGA密码子。 UGA密码子通常是结合释放因子以终止翻译的终止密码子。然而，在硒蛋白转录物中，这些 UGA 密码子被重新编码以插入硒代半胱氨酸。其次，它们在 3' 非翻译区包含一个称为硒代半胱氨酸插入序列 (SECIS) 元件的茎环样结构，该元件招募 SECIS 结合蛋白 2 等机制来重新编码核糖体，以便在 UGA 密码子处插入硒代半胱氨酸，而不是核糖体释放因子的结合。这种独特的翻译模式使得硒蛋白翻译效率较低，并且容易提前终止，特别是在限制硒的条件下^{63 , 67} 。

Important anti-ferroptotic metabolites are also synthesized in the cytosol. The GPX4 cofactor GSH is synthesized in a two-step reaction by glutamate cysteine ligase (GCL; comprising a catalytic and a modifier subunit, GCLC and GCLM, respectively) and GSH synthetase (GSS). Cysteine, obtained from imported cystine or other sources, is rate-limiting for GSH synthesis. It is often assumed that GSH alone is sufficient to inhibit ferroptosis. However, this might not be the case. Genetic disruption of GCLC does not seem to induce ferroptosis despite eliminating de novo GSH synthesis⁶⁸. It seems possible that one or more additional cysteine-derived, sulfur-containing metabolites can suppress ferroptosis, in parallel to GSH. For example, co-depletion of GSH and the metabolic intermediate coenzyme A (CoA) is necessary and sufficient to trigger ferroptosis in some cancer cells^69,70. How CoA impacts ferroptosis is unclear but could be related to the requirement for CoA in lipid metabolism or to a direct role for CoA in modulating protein function via post-translational modification (that is, CoAlation). Additionally, hydropersulfides can suppress lipid peroxidation^71,72, and the synthesis of these metabolites requires cysteine. A further possibility is that GSH tripeptide (γ-glutamylcysteinylglycine) serves as a sink for glutamate, which otherwise can act in a pro-ferroptotic manner. Under conditions of intracellular cysteine starvation, GCL remains active and synthesizes an array of alternative dipeptides and tripeptides that contain glutamate and different amino acids in place of cysteine (for example, γ-glutamyl-threonine)⁷³. The synthesis of these unusual metabolites removes free glutamate from the cell, which otherwise enhances pro-ferroptotic oxidative stress through a downstream mechanism that requires further elicidation⁷³.
重要的抗铁死亡代谢物也在细胞质中合成。 GPX4 辅因子 GSH 通过谷氨酸半胱氨酸连接酶（GCL；分别包含催化亚基和修饰亚基，GCLC 和 GCLM）和 GSH 合成酶 (GSS) 进行两步反应合成。从进口胱氨酸或其他来源获得的半胱氨酸是谷胱甘肽合成的限速剂。人们通常认为单独使用 GSH 就足以抑制铁死亡。然而，情况可能并非如此。尽管消除了谷胱甘肽从头合成^{68 ，}但 GCLC的遗传破坏似乎不会诱导铁死亡。一种或多种额外的半胱氨酸衍生的含硫代谢物似乎可能与 GSH 一样抑制铁死亡。例如，GSH 和代谢中间体辅酶 A (CoA) 的共同消耗对于触发某些癌细胞中的铁死亡是必要且充分^的69、70 。 CoA 如何影响铁死亡尚不清楚，但可能与脂质代谢中 CoA 的需求或 CoA 通过翻译后修饰（即 CoAlation）调节蛋白质功能的直接作用有关。此外，氢过硫化物可以抑制脂质过^{氧化71、72} ，并且这些代谢物的合成需要半胱氨酸。另一种可能性是 GSH 三肽（γ-谷氨酰半胱氨酰甘氨酸）充当谷氨酸的接收器，否则谷氨酸可以以促铁死亡的方式发挥作用。在细胞内半胱氨酸饥饿的条件下，GCL 保持活性并合成一系列替代二肽和三肽，其中含有谷氨酸和不同氨基酸代替半胱氨酸（例如，γ-谷氨酰-苏氨酸） ⁷³ 。 这些不寻常代谢物的合成会从细胞中去除游离谷氨酸，否则会通过需要进一步阐明的下游机制增强促铁死亡氧化应激⁷³ 。

NADPH is a key electron carrier in the cell. NADP can be synthesized in the cytosol from nicotinamide adenine dinucleotide (NAD⁺) by NAD kinase (NADK) and then reduced to NADPH by malic enzyme 1 (ME1), isocitrate dehydrogenase (IDH1), or enzymes in the oxidative pentose phosphate pathway⁷⁴. Higher NADPH levels generally correlate with greater ferroptosis resistance⁷⁵; this is sensible as NADPH is used to synthesize or regenerate endogenous antioxidant metabolites such as GSH from oxidized glutathione (GSSG) by glutathione-disulfide reductase (GSR), reduced CoQ from oxidized CoQ and reduced vitamin K from oxidized vitamin K by FSP1 (refs. ^41,42), and reduced tetrahydrobiopterin (BH4) from BH2 by dihydrofolate reductase (DHFR)^23,45. Notably, however, NADPH is also an electron donor for the synthesis of ROS by NOX enzymes at the plasma membrane and by the oxidoreductases cytochrome P450 oxidoreductase (POR) and cytochrome b5 reductase 1 (CYB5R1) at the ER membrane^76,77. Thus, NADPH likely has both pro-ferroptotic and anti-ferroptotic functions whose relative importance will vary between cells.
NADPH 是细胞中关键的电子载体。 NADP 可以在细胞质中通过 NAD 激酶 (NADK) 从烟酰胺腺嘌呤二核苷酸 (NAD ⁺ ) 合成，然后通过苹果酸酶 1 (ME1)、异柠檬酸脱氢酶 (IDH1) 或氧化戊糖磷酸途径中的酶还原为 NADPH ⁷⁴ 。较高的 NADPH 水平通常与较高的铁死亡抗性相关⁷⁵ ；这是合理的，因为 NADPH 用于合成或再生内源性抗氧化代谢物，例如通过谷胱甘肽二硫化物还原酶 (GSR) 从氧化型谷胱甘肽 (GSSG) 中合成 GSH，通过 FSP1 从氧化 CoQ 中还原 CoQ，以及从氧化维生素 K 中还原维生素 K（参考文献 1）。 ^{41 , 42} )，以及从 BH2 还原的四氢生物蝶呤 (BH4)通过二氢叶酸还原酶 (DHFR) ^{23 , 45} 。然而，值得注意的是，NADPH还是质膜上的NOX酶以及ER膜上的氧化还原酶细胞色素P450氧化还原酶(POR)和细胞色素b5还原酶1(CYB5R1)合成ROS的电子供体^{76、77 。}因此，NADPH 可能同时具有促铁死亡和抗铁死亡的功能，其相对重要性因细胞而异。

Recent studies connect NADPH to ferroptosis in unexpected ways. HD domain-containing 3 (HDDC3; also known as MESH1) is proposed to function as an NADPH phosphatase whose activity enhances ferroptosis sensitivity by decreasing the levels of NADPH⁷⁸. Experiments manipulating cytosolic NADK and mitochondrial NADK2 establish that cytosolic NADPH is most critical for governing ferroptosis sensitivity⁷⁸. NADPH may also be directly sensed by an ER-localized enzyme. The ER-localized E3 ubiquitin ligase membrane-associated ring-CH-type finger 6 (MARCHF6) may directly sense cytosolic NADPH levels via a unique C-terminal binding region, increasing the activity of this enzyme and resulting in changes in the levels of key regulators of ferroptosis, including acyl-CoA synthetase long-chain family member 4 (ACSL4) and p53 (ref. ⁷⁹). Remarkably, embryonic development is impaired in Marchf6^–/– animals and this can be partially reverted by a maternal diet enriched in the natural RTA vitamin E. Thus, MARCHF6 may play a role in preventing ferroptosis during development.
最近的研究以意想不到的方式将 NADPH 与铁死亡联系起来。含 HD 结构域 3（HDDC3；也称为 MESH1）被认为具有 NADPH 磷酸酶的功能，其活性通过降低 NADPH 水平来增强铁死亡敏感性⁷⁸ 。操纵细胞质 NADK 和线粒体 NADK2 的实验表明，细胞质 NADPH 对于控制铁死亡敏感性最为关键⁷⁸ 。 NADPH 也可以被内质网定位的酶直接感知。 ER 定位的 E3 泛素连接酶膜相关环 CH 型指 6 (MARCHF6) 可以通过独特的 C 端结合区域直接感测胞质 NADPH 水平，从而增加该酶的活性并导致关键酶水平的变化。铁死亡的调节因子，包括酰基辅酶A合成酶长链家族成员4（ACSL4）和p53（参考文献⁷⁹ ）。值得注意的是， Marchf6 ^–/–动物的胚胎发育受到损害，而这种情况可以通过富含天然 RTA 维生素 E 的母体饮食部分恢复。因此，MARCHF6 可能在发育过程中预防铁死亡中发挥作用。

Most organelles have been linked to the regulation of ferroptosis sensitivity in one way or another, and often in several potentially contradictory ways at once. How these different and sometimes opposing activities are coordinated to generate a unified effect on ferroptosis in a given context is an important open question. Key aspects of ferroptosis regulation by different organelles are described below.
大多数细胞器都以一种或另一种方式与铁死亡敏感性的调节相关，并且常常同时以几种潜在矛盾的方式相关。如何协调这些不同的、有时是相反的活动，以在给定的背景下对铁死亡产生统一的影响是一个重要的悬而未决的问题。不同细胞器铁死亡调节的关键方面如下所述。

Mitochondria 线粒体

Mitochondria are multifunctional organelles that can regulate ferroptosis sensitivity in several different ways (Fig. 2). In cells treated with the system x_c^– inhibitor erastin, mitochondria are smaller, with disorganized cristae^1,80. This represents a potential morphological marker of ferroptosis. However, it is not clear whether changes in mitochondrial morphology contribute to the execution of ferroptosis or are merely a correlate of this process. For example, mitochondria can be eliminated from some cells with little effect on ferroptosis sensitivity⁸¹. In other cells, mitochondria seem to be essential for ferroptosis⁸². Below, we describe examples of the pro-ferroptotic and anti-ferroptotic effects of mitochondria, touching on specific examples of context-dependent effects and areas of disagreement in the literature.
线粒体是多功能细胞器，可以通过几种不同的方式调节铁死亡敏感性（图2 ）。在用系统 x _c ^–抑制剂erastin 处理的细胞中，线粒体较小，嵴混乱^{1 , 80} 。这代表了铁死亡的潜在形态学标记。然而，尚不清楚线粒体形态的变化是否有助于铁死亡的执行，或者仅仅是该过程的相关因素。例如，可以从一些细胞中消除线粒体，而对铁死亡敏感性影响很小⁸¹ 。在其他细胞中，线粒体似乎对于铁死亡至关重要⁸² 。下面，我们描述线粒体的促铁死亡和抗铁死亡作用的例子，涉及背景依赖性效应的具体例子和文献中存在分歧的领域。

Mitochondria play several context-dependent roles in the regulation of ferroptosis sensitivity. a, Metabolic reactions — the breakdown of sugar and amino acids via glycolysis and the tricarboxylic acid (TCA) cycle yields NADH and FADH₂, which pass electrons to the mitochondrial electron transport chain (ETC). The ETC can partially reduce oxygen to generate reactive oxygen species (ROS) such as superoxide (O₂^•−) at complexes I and III. Dismutation of O₂^•− yields hydrogen peroxide, which can result in the formation of hydroxyl radicals (HO•) via reactions with Fe²⁺ in the Fenton reaction. Glutamine can be catabolized to glutamate (a substrate for the system x_c^– antiporter) and then to the TCA cycle intermediate α-ketoglutarate (αKG). αKG synthesis promotes ferroptosis, possibly by enhancing mitochondrial ROS production. b, Iron handling — mitoferrin 1 (MFRN1) and MFRN2 mediate iron transport across the inner mitochondrial membrane. The synthesis of Fe–S clusters and haem prosthetic groups in the mitochondria consumes labile iron that otherwise may accumulate to promote ROS accumulation. c, Mitochondrial integrated stress response — OMA1-dependent proteolytic processing of DAP3 binding cell death enhancer 1 (DELE1) releases a protein fragment that initiates the integrated stress response, culminating in activating transcription factor 4 (ATF4)-dependent transcription of a programme that enhances glutathione metabolism and protects against ferroptosis. d, Coenzyme Q10 (CoQ) synthesis — the final steps in de novo CoQ synthesis occur in the mitochondria. CoQ is employed as a key electron carrier in the ETC. CoQ can be reduced by dihydroorotate dehydrogenase (DHODH) to CoQH₂, which can either transfer electrons to complex III in the ETC or function as a local radical-trapping antioxidant to prevent peroxidation of mitochondrial lipids and limit ferroptosis. In addition, CoQ is trafficked by a processed form of StAR-related lipid transfer protein 7 (STARD7) from the mitochondria to the plasma membrane, where it can contribute to ferroptosis protection mediated by ferroptosis suppressor protein 1. CoA, coenzyme A.
线粒体在铁死亡敏感性的调节中发挥着多种背景依赖性作用。 a ，代谢反应 — 糖和氨基酸通过糖酵解和三羧酸 (TCA) 循环分解，产生 NADH 和 FADH ₂ ，将电子传递到线粒体电子传递链 (ETC)。 ETC可以部分还原氧以在配合物I和III处产生活性氧(ROS)，例如超氧化物(O ₂ ^•− )。 O ₂ ^•−的歧化产生过氧化氢，在芬顿反应中与 Fe ²⁺反应可导致羟基自由基 (HO•) 的形成。谷氨酰胺可分解代谢为谷氨酸（x _c系统的底物^-逆向转运蛋白），然后分解为 TCA 循环中间体 α-酮戊二酸 (αKG)。 αKG 合成可能通过增强线粒体 ROS 产生来促进铁死亡。 b ，铁处理——线粒体铁蛋白 1 (MFRN1) 和 MFRN2 介导铁穿过线粒体内膜的转运。线粒体中 Fe-S 簇和血红素辅基的合成会消耗不稳定的铁，否则这些铁可能会积累以促进 ROS 积累。 c ，线粒体整合应激反应 — DAP3 结合细胞死亡增强子 1 (DELE1) 的 OMA1 依赖性蛋白水解过程释放启动整合应激反应的蛋白片段，最终激活转录因子 4 (ATF4) 依赖的程序转录，从而增强细胞死亡谷胱甘肽代谢并防止铁死亡。 d ，辅酶 Q10 (CoQ) 合成 — CoQ 从头合成的最后步骤发生在线粒体中。 CoQ 被用作 ETC 中的关键电子载体。 CoQ 可以被二氢乳清酸脱氢酶 (DHODH) 还原为 CoQH ₂ ，CoQH 2 可以将电子转移到 ETC 中的复合物 III，或者充当局部自由基捕获抗氧化剂，以防止线粒体脂质过氧化并限制铁死亡。此外，CoQ 通过加工形式的 StAR 相关脂质转移蛋白 7 (STARD7) 从线粒体运输到质膜，在质膜中它可以促进铁死亡抑制蛋白 1 介导的铁死亡保护。CoA，辅酶 A。

Full size image 全尺寸图像

Several mitochondrial activities reduce ferroptosis sensitivity. Mitofusin 1 (MFN1)-mediated mitochondrial fusion may reduce sensitivity to RSL3-induced ferroptosis, at least in cultured cancer cells⁸³. Likewise, in cells with dysfunctional mitochondria, activation of the mitochondrial stress response pathway mediated by metalloendopeptidase OMA1 and DAP3 binding cell death enhancer 1 (DELE1) can inhibit ferroptosis and delay cardiomyopathy⁸⁴. Here, DELE1 activates cytosolic activating transcription factor 4 (ATF4), which in turn promotes GSH synthesis and GPX4 protein stability. Mitochondria are also the site of CoQ metabolite synthesis⁸⁵. CoQ is an electron carrier in the mitochondrial electron transport chain (ETC) but also functions as an important anti-ferroptotic RTA^41,42. Key enzymatic machinery necessary for CoQ synthesis (for example, the COQ proteins) localize to the mitochondria. A specific proteolytically processed version of StAR-related lipid transfer protein 7 (STARD7) transports CoQ from the mitochondria to the plasma membrane, where it can be reduced by FSP1 to inhibit lipid peroxidation and ferroptosis⁸⁶. Whether STARD7 might act at membrane contact sites between the mitochondria and plasma membrane to promote efficient and directional CoQ transfer is unknown. Another important protective role for mitochondria is in the breakdown of PUFAs that could otherwise be incorporated into membrane phospholipids, peroxidized and thereby promote ferroptosis. This catabolic process requires the mitochondrial enzyme 2,4-dienoyl-CoA reductase 1 (DECR1) and, for reasons that remain unclear, appears especially relevant in prostate cancer^87,88. Collectively, these mitochondrial functions would tend to reduce ferroptosis sensitivity.
几种线粒体活性可降低铁死亡敏感性。线粒体融合蛋白 1 (MFN1) 介导的线粒体融合可能会降低对 RSL3 诱导的铁死亡的敏感性，至少在培养的癌细胞中是这样⁸³ 。同样，在线粒体功能障碍的细胞中，金属内肽酶 OMA1 和 DAP3 结合细胞死亡增强子 1 (DELE1) 介导的线粒体应激反应途径的激活可以抑制铁死亡并延缓心肌病⁸⁴ 。在这里，DELE1 激活胞质激活转录因子 4 (ATF4)，进而促进 GSH 合成和 GPX4 蛋白稳定性。线粒体也是 CoQ 代谢物合成的场所⁸⁵ 。 CoQ 是线粒体电子传递链 (ETC) 中的电子载体，同时也是重要的抗铁死亡 RTA ^{41 、 42} 。 CoQ 合成所需的关键酶机制（例如 COQ 蛋白）位于线粒体。 StAR 相关脂质转移蛋白 7 (STARD7) 的特定蛋白水解加工版本将 CoQ 从线粒体转运到质膜，在质膜中它可以被 FSP1 还原，从而抑制脂质过氧化和铁死亡⁸⁶ 。 STARD7 是否可能作用于线粒体和质膜之间的膜接触位点以促进有效和定向的 CoQ 转移尚不清楚。线粒体的另一个重要保护作用是分解多不饱和脂肪酸，否则多不饱和脂肪酸可能会掺入膜磷脂中，发生过氧化，从而促进铁死亡。 这种分解代谢过程需要线粒体酶 2,4-二烯酰辅酶 A 还原酶 1 (DECR1)，并且由于尚不清楚的原因，它似乎与前列腺癌特别相关^{87 , 88} 。总的来说，这些线粒体功能往往会降低铁死亡的敏感性。

Other mitochondrial mechanisms increase ferroptosis sensitivity. For example, the mitochondrial tricarboxylic acid (TCA) cycle promotes ferroptosis sensitivity, at least in response to cystine deprivation⁸⁹. During glutaminolysis, glutamine is converted to glutamate and the TCA cycle intermediate α-ketoglutarate within mitochondria; both glutamate and α-ketoglutarate can promote ferroptosis^53,73,90. In these cases, the precise effector mechanism linking TCA cycle activity with the induction of ferroptosis remains obscure, although enhanced oxidative stress seems to be involved. Mitochondria are also central hubs for the use and metabolism of iron². Increased mitochondrial iron uptake mediated by mitoferrin 1 (MFRN1; encoded by SLC25A37) can enhance ferroptosis sensitivity, although the exact mechanism is unclear⁹¹. Disruption of mitochondrial biosynthesis of Fe–S clusters can also enhance ferroptosis sensitivity. Here, depletion of NFS1 cysteine desulfurase lowers the synthesis of Fe–S clusters, triggering a feedback iron starvation response that increases iron import and heightens ferroptosis sensitivity⁹². It might be the case that cystine deprivation caused by system x_c^– inhibition has unique effects on mitochondrial Fe–S biosynthesis given the need for cysteine-derived sulfur in this process. One model to explain observed differences in the ferroptosis phenotypes caused by cystine deprivation versus direct GPX4 inhibition¹⁰ is that cystine deprivation uniquely perturbs mitochondrial Fe–S cluster biosynthesis⁴⁵. Finally, mitochondrial outer membrane permeabilization, when it occurs in a partial and non-lethal manner throughout the cell, results in both activation of the ATF4 pathway and enhanced sensitivity to ferroptosis induced by GPX4 inhibitors⁹³. This may suggest that the loss of one or more mitochondrial anti-ferroptotic functions can, on balance, overcome the protective effect of ATF4 pathway activation.
其他线粒体机制增加了铁死亡的敏感性。例如，线粒体三羧酸 (TCA) 循环会促进铁死亡敏感性，至少是对胱氨酸剥夺的反应⁸⁹ 。在谷氨酰胺分解过程中，谷氨酰胺在线粒体内转化为谷氨酸和 TCA 循环中间体 α-酮戊二酸；谷氨酸和α-酮戊二酸均可促进铁死亡^{53 , 73 , 90} 。在这些情况下，尽管似乎涉及增强的氧化应激，但将 TCA 循环活性与铁死亡诱导联系起来的精确效应机制仍然不清楚。线粒体也是铁的利用和代谢的中心枢纽² 。线粒体铁蛋白 1（MFRN1；由SLC25A37编码）介导的线粒体铁摄取增加可以增强铁死亡敏感性，但确切机制尚不清楚⁹¹ 。线粒体Fe-S 簇生物合成的破坏也可以增强铁死亡的敏感性。在这里，NFS1 半胱氨酸脱硫酶的消耗降低了 Fe-S 簇的合成，触发反馈铁饥饿反应，增加铁输入并提高铁死亡敏感性⁹² 。考虑到在此过程中需要半胱氨酸衍生的硫，系统 x _c^抑制引起的胱氨酸剥夺可能对线粒体 Fe-S 生物合成具有独特的影响。 一种解释胱氨酸剥夺与直接 GPX4 抑制引起的铁死亡表型差异的模型¹⁰是胱氨酸剥夺独特地扰乱线粒体 Fe-S 簇生物合成⁴⁵ 。最后，当线粒体外膜透化在整个细胞中以部分和非致命的方式发生时，会导致 ATF4 途径的激活并增强对 GPX4 抑制剂诱导的铁死亡的敏感性⁹³ 。这可能表明，一种或多种线粒体抗铁死亡功能的丧失总体上可以克服 ATF4 途径激活的保护作用。

In some cancer cells, mitochondrial ETC activity promotes ferroptosis, at least in response to cystine deprivation, as shown using specific ETC poisons⁸⁹. How the ETC promotes ferroptosis is not clear. One obvious candidate is the production of ROS that might be expected to initiate lipid peroxidation. Indeed, ROS scavengers that target mitochondria can attenuate ferroptosis in some models, implying that mitochondria-derived oxidants may contribute to cell death (Box 2). However, in other cancer cells, depleting the mitochondrial DNA, resulting in complete loss of ETC function, has no effect on ferroptosis sensitivity^1,94. Moreover, ETC function is necessary for the activity of two mitochondrially localized enzymes that may be able to generate reduced CoQ: dihydroorotate dehydrogenase (DHODH)³⁷ and glycerol-3-phosphate dehydrogenase 2 (GPD2)⁹⁵. Accordingly, loss of ETC function might be expected to enhance ferroptosis sensitivity by disrupting the synthesis of reduced mitochondrial CoQ. Overall, it seems likely that the role of the mitochondrial ETC varies by cell type and by the nature of the lethal stimulus. An emerging (and controversial)³⁸ concept is that the mitochondria might assume a greater importance in overall ferroptosis regulation when cytosolic anti-ferroptotic systems are disrupted^37,39,96. This model of a conditional role for mitochondria depending on circumstances could provide a reconciliation for some conflicting observations.
在一些癌细胞中，线粒体 ETC 活性会促进铁死亡，至少是对胱氨酸剥夺的反应，如使用特定 ETC 毒物所示⁸⁹ 。 ETC 如何促进铁死亡尚不清楚。一个明显的候选者是ROS的产生，预计它可能会引发脂质过氧化。事实上，在某些模型中，针对线粒体的 ROS 清除剂可以减弱铁死亡，这意味着线粒体来源的氧化剂可能会导致细胞死亡（框2 ）。然而，在其他癌细胞中，耗尽线粒体 DNA 导致 ETC 功能完全丧失，对铁死亡敏感性没有影响^{1 , 94} 。此外，ETC 功能对于两种可能产生还原 CoQ 的线粒体定位酶的活性是必需的：二氢乳清酸脱氢酶 (DHODH) ³⁷和甘油-3-磷酸脱氢酶 2 (GPD2) ⁹⁵ 。因此，ETC 功能的丧失可能会通过破坏还原线粒体 CoQ 的合成来增强铁死亡敏感性。总体而言，线粒体 ETC 的作用可能因细胞类型和致死刺激的性质而异。一个新兴的（且有争议的） ³⁸概念是，当胞质抗铁死亡系统被破坏时，线粒体可能在整体铁死亡调节中发挥更大的重要性^{37 , 39 , 96} 。这种线粒体根据环境发挥条件作用的模型可以为一些相互矛盾的观察结果提供调和。

Endoplasmic reticulum 内质网

The ER plays an important role in ferroptosis regulation. POR and CYB5R1 are enzymes that reside in the ER membrane and use NADPH to produce ROS that can promote membrane lipid peroxidation^76,77 (Fig. 3a). Indeed, there is good evidence that the ER is one site where membrane lipid peroxidation is initiated during ferroptosis (Box 2). As noted above, whether lipid peroxidation spreads from the ER to the plasma membrane to execute ferroptosis, for example, through vesicular trafficking or phospholipid transfer at membrane contact sites, is an open question.
ER 在铁死亡调节中发挥重要作用。 POR 和 CYB5R1 是驻留在 ER 膜上的酶，它们利用 NADPH 产生 ROS，从而促进膜脂过氧化^{76 、 77} （图3a ）。事实上，有充分的证据表明内质网是铁死亡过程中启动膜脂过氧化的位点之一（框2 ）。如上所述，脂质过氧化是否从内质网扩散到质膜以执行铁死亡，例如通过膜接触位点的囊泡运输或磷脂转移，是一个悬而未决的问题。

a, Cytochrome P450 oxidoreductase (POR) and cytochrome b5 reductase 1 (CYB5R1) are endoplasmic reticulum (ER) enzymes that promote ferroptosis by generating H₂O₂, which can further react with iron to produce reactive oxygen species species that initiate lipid peroxidation, leading to ferroptosis. Lipid peroxidation in the ER is an early event in ferroptosis. Whether damage propagates from the ER or accumulates at a slower rate in other membranes is an open question (see also Box 2). b, The ER is the primary site of lipid synthesis, including the synthesis of glycerolipids, namely phospholipids (PLs) and triacylglycerols (TAGs). The incorporation of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) into PLs or TAGs helps establish the overall sensitivity of the cell to lipid peroxidation and ferroptosis. TAGs are stored within lipid droplets that emerge from the outer leaflet of the ER. PLs are trafficked to the plasma membrane via the secretory pathway or transferred at ER–plasma membrane contact sites. Phosphatidylethanolamine (PE) is often oxidized during ferroptosis. The synthesis of PE involves the transfer of phosphatidylserine from the ER to mitochondria, where it is enzymatically converted to PE prior to transport back to the ER. The relative contributions of these PL trafficking pathways to ferroptosis remain to be defined. c, A complex series of enzymatic steps in the ER mediate the synthesis of cholesterol via the mevalonate pathway. Several intermediates in this pathway protect against ferroptosis. Farnesyl pyrophosphate (FPP) is needed to synthesize the endogenous radical-trapping antioxidants coenzyme Q10 (CoQ) and vitamin K in the mitochondria and Golgi, respectively. Isopentenyl pyrophosphate (IPP) is attached to the selenocysteine tRNA as an isopentenyl moiety, a key modification for its function, promoting the synthesis of selenoproteins, including the anti-ferroptotic enzyme glutathione peroxidase 4 (GPX4). 7-Dehydrocholesterol (7DHC) is highly prone to oxidation and can suppress ferroptosis by competing with PL for oxidation. Squalene also suppresses ferroptosis, but the mechanism is not understood. d, Proteolytic processing of the membrane-tethered transcription factors sterol regulatory element-binding protein 1 (SREBP1) and nuclear factor erythroid 2-related factor 1 (NFE2L1) releases soluble, active transcription factors that traffic to the nucleus and initiate transcriptional programmes to control lipid metabolism and the cellular oxidative stress response. The phosphoinositide 3-kinase (PI3K)–mechanistic target of rapamycin complex 1 (mTORC1) pathway promotes ferroptosis resistance by increasing SREBP1 activation and expression of stearoyl-CoA desaturase 1 (SCD1), which generates ferroptosis-suppressive MUFAs. NFE2L1 is dislocated from the ER into the cytoplasm, where it is deglycosylated by N-glycanase 1 (NGLY1) and proteolytically processed by DNA-damage inducible 1 homologue 2 (DDI2). The amount of NFE2L1 that escapes proteasomal clearance is a determinant of NFE2L1 transcriptional signalling. HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; MVA, mevalonate; GGPP, geranylgeranyl pyrophosphate; UBIAD1, UBIA prenyltransferase domain-containing protein 1.
a , 细胞色素 P450 氧化还原酶 (POR) 和细胞色素 b5 还原酶 1 (CYB5R1) 是内质网 (ER) 酶，通过产生 H ₂ O ₂促进铁死亡，H 2 O 2 可以进一步与铁反应产生活性氧物种，引发脂质过氧化，导致铁死亡。内质网中的脂质过氧化是铁死亡的早期事件。损伤是从内质网传播还是以较慢的速度在其他膜中累积是一个悬而未决的问题（另见框2 ）。 b ，内质网是脂质合成的主要场所，包括甘油脂的合成，即磷脂（PL）和三酰甘油（TAG）。将单不饱和脂肪酸 (MUFA) 和多不饱和脂肪酸 (PUFA) 纳入 PL 或 TAG 有助于建立细胞对脂质过氧化和铁死亡的整体敏感性。 TAG 储存在从 ER 外层出现的脂滴中。 PL 通过分泌途径运输至质膜或在内质网-质膜接触位点转移。磷脂酰乙醇胺 (PE) 在铁死亡过程中经常被氧化。 PE 的合成涉及将磷脂酰丝氨酸从 ER 转移到线粒体，在线粒体中酶促转化为 PE，然后运回 ER。这些 PL 运输途径对铁死亡的相对贡献仍有待确定。 c ，内质网中一系列复杂的酶促步骤通过甲羟戊酸途径介导胆固醇的合成。该途径中的几种中间体可以防止铁死亡。 法尼基焦磷酸 (FPP) 是线粒体和高尔基体中分别合成内源性自由基捕获抗氧化剂辅酶 Q10 (CoQ) 和维生素 K 所必需的。异戊烯焦磷酸 (IPP) 作为异戊烯基部分附着在硒代半胱氨酸 tRNA 上，这是其功能的关键修饰，可促进硒蛋白的合成，包括抗铁死亡酶谷胱甘肽过氧化物酶 4 (GPX4)。 7-脱氢胆固醇 (7DHC) 极易氧化，可通过与 PL 竞争氧化来抑制铁死亡。角鲨烯也能抑制铁死亡，但其机制尚不清楚。 d ，膜束缚转录因子甾醇调节元件结合蛋白 1 (SREBP1) 和核因子红细胞 2 相关因子 1 (NFE2L1) 的蛋白水解过程释放可溶性活性转录因子，这些转录因子运输到细胞核并启动转录程序来控制脂质代谢和细胞氧化应激反应。磷酸肌醇 3 激酶 (PI3K)——雷帕霉素复合物 1 (mTORC1) 通路的机制靶点通过增加 SREBP1 的激活和硬脂酰辅酶 A 去饱和酶 1 (SCD1) 的表达来促进铁死亡抵抗，从而产生铁死亡抑制性 MUFA。 NFE2L1 从 ER 移位到细胞质中，在细胞质中被 N-聚糖酶 1 (NGLY1) 去糖基化，并被 DNA 损伤诱导型 1 同源物 2 (DDI2) 进行蛋白水解加工。逃避蛋白酶体清除的 NFE2L1 量是 NFE2L1 转录信号传导的决定因素。 HMG-CoA，3-羟基-3-甲基戊二酰辅酶A； MVA，甲羟戊酸； GGPP，香叶基香叶基焦磷酸； UBIAD1，UBIA 异戊烯基转移酶结构域含蛋白 1。

Full size image 全尺寸图像

Ferroptosis is a lipid-dependent process. The ER is a major hub for lipid metabolism in the cell. Lipid desaturation, phospholipid synthesis, phospholipid remodelling and biogenesis of lipid droplets are all ER-localized activities (Fig. 3b). Thus, it is no surprise that ER-resident lipid metabolic enzymes contribute importantly to the regulation of ferroptosis sensitivity. Overall, the ferroptotic threshold of the cell can be determined by lipid composition and, more specifically, by the relative levels of different lipid species⁹⁷. Decreasing the ratio of more oxidizable PUFAs to less oxidizable monounsaturated fatty acids (MUFAs) is sufficient to convert cells from ferroptosis sensitive to ferroptosis resistant^{28,98,99,100,101} whereas increasing the PUFA to MUFA ratio can have the opposite effect^22,77. ER-resident enzymes shape the PUFA and MUFA content of cellular lipid pools. For example, fatty acid desaturase 1 (FADS1) and FADS2, which introduce carbon–carbon double bonds into fatty acids, can enhance or reduce ferroptosis sensitivity, respectively, by synthesizing specific PUFAs or MUFAs^102,103. The overall activity of these desaturation processes can be governed within the ER by calcium levels, which are regulated by the tetraspanin membrane-spanning 4-domains subfamily A member 15 (MS4A15)¹⁰⁴.
铁死亡是一个脂质依赖性过程。 ER 是细胞内脂质代谢的主要枢纽。脂质去饱和、磷脂合成、磷脂重塑和脂滴的生物发生都是内质网局部的活动（图3b ）。因此，内质网驻留的脂质代谢酶对铁死亡敏感性的调节发挥重要作用也就不足为奇了。总体而言，细胞的铁死亡阈值可以通过脂质组成来确定，更具体地说，可以通过不同脂质种类的相对水平来确定⁹⁷ 。降低更多可氧化的 PUFA 与更少氧化的单不饱和脂肪酸(MUFA) 的比例足以将细胞从铁死亡敏感转化为铁死亡抗性^{28 , 98 , 99 , 100 , 101}而增加 PUFA 与 MUFA 的比例可能会产生相反的效果^{22 , 77} .内质网驻留酶决定细胞脂质库中 PUFA 和 MUFA 的含量。例如，将碳碳双键引入脂肪酸的脂肪酸去饱和酶1 (FADS1)和FADS2可以通过合成特定的PUFA或MUFA ^{102 、 103}分别增强或降低铁死亡敏感性。这些去饱和过程的总体活性可以在 ER 内由钙水平控制，钙水平由四跨膜蛋白 4 结构域亚家族 A 成员 15 (MS4A15) ¹⁰⁴调节。

Acyl-CoA synthetase long-chain (ACSL) enzymes ‘activate’ both PUFAs and MUFAs to PUFA-CoA and MUFA-CoA species, which can then be incorporated into membrane phospholipids, triacylglycerols (TAGs) or other lipids. ACSL4 and ACSL3 appear generally to function in opposition, with ACSL4-driven PUFA metabolism increasing ferroptosis sensitivity and ACSL3-dependent MUFA metabolism promoting ferroptosis resistance^{24,28,30,99,105,}