Ironing out the mitochondria
消除线粒体

Mitochondria are important inducers of cell death but also respond to multiple cell death triggers from other organelles or from the outside of the cell. For instance, calcium (Ca²⁺) influx from the ER triggers mitochondrial swelling, the opening of the permeability transition pore and the release of mitochondrial proapoptotic factors, including cytochrome c. Ca²⁺ release from the ER is dependent on structures where the ER touches mitochondria. These are called ER–mitochondria contact sites (ERMCS), and their biochemical isolate is referred to as mitochondria-associated membranes (MAMs). Mitochondria are also involved in the regulation of another form of cell death called ferroptosis, but whether MAMs are critically important in this mechanism was unclear. In this issue of Nature Chemical Biology, Zhang et al.¹ reveal a connection between ferroptosis and MAMs through the identification of a small-molecule inhibitor that blocks ferroptosis and MAM formation (Fig. 1).
线粒体是细胞死亡的重要诱导剂，但也对来自其他细胞器或细胞外部的多种细胞死亡触发因素做出反应。例如，钙 (Ca ²⁺ ) 从内质网流入会引发线粒体肿胀、通透性转换孔打开以及线粒体促凋亡因子（包括细胞色素c ）的释放。 ER 中 Ca ^{2+ 的}释放取决于 ER 接触线粒体的结构。这些被称为内质网-线粒体接触位点（ERMCS），它们的生化分离物被称为线粒体相关膜（MAM）。线粒体还参与另一种细胞死亡形式（称为铁死亡）的调节，但 MAM 在此机制中是否至关重要尚不清楚。在本期《自然化学生物学》中，Zhang 等人。^图1通过鉴定阻止铁死亡和MAM形成的小分子抑制剂揭示了铁死亡和MAM之间的联系（图1 ）。

Ferroptosis coincides with the activation of IP₃R Ca²⁺ signaling from the ER toward mitochondria, mediated by its link to VDACs on mitochondria through the GRP75 linker protein and the Sig1R stabilizing protein (σ1R) on MAM domains with peroxidized lipids (red lipid heads, left). By contrast, CGI1746, a chemical inhibitor of ferroptosis, interferes with Sig1R. This then disrupts normal ER–mitochondrion protein interactions on MAMs and reduces lipid peroxidation, a key feature of ferroptosis (right).
铁死亡与 IP ₃ R Ca ²⁺信号从 ER 向线粒体的激活同时发生，该信号通过 GRP75 连接蛋白和 MAM 结构域上的 Sig1R 稳定蛋白 (σ1R) 与线粒体上的 VDAC 连接介导，具有过氧化脂质（红色脂质头） ， 左边）。相比之下，CGI1746（一种铁死亡的化学抑制剂）会干扰 Sig1R。然后，这会破坏 MAM 上正常的 ER-线粒体蛋白相互作用，并减少脂质过氧化，这是铁死亡的一个关键特征（右）。

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GPx4 is a selenoperoxidase found in the cytoplasm, mitochondria and nucleus that uses reduced glutathione (GSH) to transform phospholipid hydroperoxide (PLOOH) into its nontoxic, reduced form². Ferroptosis is triggered through the accumulation of oxidized polyunsaturated fatty acids (PUFAs) either with the GPx4 inhibitor RSL3 or with erastin, which inhibits cystine import by the plasma membrane system x_c^–. Mitochondria are involved in ferroptosis in multiple ways. Aside from their production of mitochondrial reactive oxygen species (ROS) that generate PLOOH, their ER-apposed membranes house acyl-coenzyme A (CoA) synthetase long-chain family member 4 (ACSL4), also known as FACL4. This enzyme, an established marker of MAMs, promotes the production of PUFAs through the incorporation of arachidonic acid into phospholipids³. Given that excessive PUFA levels promote ferroptosis in the presence of a stress trigger, it is therefore not surprising that ACSL4 is a ferroptosis accelerator⁴. Moreover, mitochondrial ROS production increases when the MAM structure undergoes functional changes, such as the activation of ER–mitochondrion Ca²⁺ flux⁵. Therefore, it was expected that ferroptosis depends on the MAM structure, given its functional links to mitochondrial ROS production, which increases oxidized PUFA, as well as to ACSL4, which localizes to MAMs. However, this connection had not been explored in great mechanistic detail.
GPx4 是一种存在于细胞质、线粒体和细胞核中的硒过氧化物酶，它利用还原型谷胱甘肽 (GSH) 将磷脂氢过氧化物 (PLOOH) 转化为其无毒的还原型² 。铁死亡是通过 GPx4 抑制剂 RSL3 或erastin 氧化多不饱和脂肪酸 (PUFA) 的积累而引发的，erastin 会抑制质膜系统 x _c ^–胱氨酸的输入。线粒体以多种方式参与铁死亡。除了产生产生 PLOOH 的线粒体活性氧 (ROS) 之外，它们的内质网膜上还含有酰基辅酶 A (CoA) 合成酶长链家族成员 4 (ACSL4)，也称为 FACL4。这种酶是 MAM 的既定标记，通过将花生四烯酸掺入磷脂中，促进 PUFA 的产生³ 。鉴于过量的 PUFA 水平在存在应激触发的情况下会促进铁死亡，因此 ACSL4 是铁死亡加速器也就不足为奇了⁴ 。此外，当 MAM 结构发生功能变化（例如 ER-线粒体 Ca ²⁺通量⁵的激活）时，线粒体 ROS 的产生会增加。因此，考虑到 MAM 结构与线粒体 ROS 产生（增加氧化的 PUFA）以及定位于 MAM 的 ACSL4 的功能联系，预计铁死亡取决于 MAM 结构。然而，这种联系尚未得到详细的机械细节探讨。

Zhang et al.¹ now firmly establish this long-suspected link between MAMs and ferroptosis. They screened small molecules that inhibit ferroptosis triggered by RSL3 and identified 26 chemicals, many of which are known kinase inhibitors. The authors focused on CGI1746, an inhibitor of Bruton’s tyrosine kinase (BTK). Although they did not detect an involvement of BTK in ferroptosis, they found that CGI1746, surprisingly, acts on the ER-derived Ca²⁺ and the ER transmembrane sigma-1 receptor (Sig1R). Sig1R promotes MAM formation, albeit through poorly defined mechanisms involving oxidative stress and MAM Ca²⁺ signaling (Fig. 1). Zhang et al.¹ provide data showing that CGI1746 can prevent MAM formation, and this is accompanied by a reduction of Ca²⁺ accumulation in the MAM interorganellar cleft during RSL3-mediated ferroptosis. Subsequently, CGI1746 also impairs ER–mitochondrion Ca²⁺ transfer (Fig. 1), normally mediated by inositol 1,4,5-trisphosphate receptors (IP₃Rs), the main ER Ca²⁺ release channels, along with voltage-dependent anion channels (VDACs), their mitochondrial partners that direct Ca²⁺ toward the mitochondrial Ca²⁺ uniporter. In an in vivo model, CGI1746 inhibited ferroptosis-mediated kidney injury.
张等人。 ¹现在牢固地确立了 MAM 与铁死亡之间这种长期怀疑的联系。他们筛选了抑制 RSL3 引发的铁死亡的小分子，并鉴定了 26 种化学物质，其中许多是已知的激酶抑制剂。作者重点关注 CGI1746，一种布鲁顿酪氨酸激酶 (BTK) 抑制剂。尽管他们没有检测到 BTK 参与铁死亡，但令人惊讶的是，他们发现 CGI1746 作用于内质网衍生的 Ca ²⁺和内质网跨膜 sigma-1 受体 (Sig1R)。 Sig1R 促进 MAM 形成，尽管其机制不明确，涉及氧化应激和 MAM Ca ²⁺信号传导（图1 ）。张等人。^图1提供的数据显示CGI1746可以阻止MAM形成，并且这伴随着RSL3介导的铁死亡过程中MAM细胞间裂中Ca ²⁺积累的减少。随后，CGI1746 还会损害 ER-线粒体 Ca ²⁺转移（图1 ），该转移通常由肌醇 1,4,5-三磷酸受体 (IP ₃ Rs)（主要 ER Ca ²⁺释放通道）介导，并且具有电压依赖性阴离子通道 (VDAC)，它们的线粒体伙伴，将 Ca ²⁺引导至线粒体 Ca ²⁺单向转运蛋白。在体内模型中，CGI1746 抑制铁死亡介导的肾损伤。

This work therefore extends the known MAM functions from lipid synthesis and homeostasis, Ca²⁺ signaling, mitochondria metabolism, inflammation and apoptosis to yet another cell death mechanism. At the moment, the mechanistic basis for this observation is not entirely clear. The present study opens up the possibility that oxidized PUFA could form a nucleus for the known enrichment of cholesterol at MAMs, thought to be a requirement for the formation of this signaling platform⁶. Moreover, the authors provide several avenues to pursue in the future. These include potential roles of MAMs in the balance of PUFA synthesis and accumulation, as well as in the control of cellular ROS homeostasis. The former hypothesis is backed up by a known increase of PUFA-sequestering triacylglycerol (TAG), as well as lipid droplet accumulation upon disruption of MAMs⁷. The latter is backed up by the accumulation of ER and mitochondrial ROS upon MAM disruption⁸. Given the plasticity of MAMs, the present findings suggest that the amount of MAM PUFA peroxidation could provide a lipid-based mechanism to control MAM formation upon stress, and a specific elimination of these lipid species could subsequently reduce MAMs upon stress resolution. It remains to be tested whether the control of these lipid species depends on MAM tethering factors such as PACS-2, mitofusin-2 or VAPB, all of which were found to influence ferroptosis. Alternatively, the results presented here suggest that the ER, and not just mitochondria, plays a key role in ferroptosis, suggesting that ER chaperones and redox enzymes could also be involved, in addition to their known roles in protein folding and MAM formation. Overall, Zhang et al.¹ solidify the long-suspected functional link between the formation of ER–mitochondrion contacts known as MAMs and ferroptosis.
因此，这项工作将已知的 MAM 功能从脂质合成和稳态、Ca ²⁺信号传导、线粒体代谢、炎症和细胞凋亡扩展到另一种细胞死亡机制。目前，这一观察结果的机制基础尚不完全清楚。本研究提出了一种可能性，即氧化的 PUFA 可以形成已知的 MAM 胆固醇富集核，这被认为是形成该信号平台的必要条件⁶ 。此外，作者还提供了几种未来追求的途径。其中包括 MAM 在 PUFA 合成和积累平衡以及细胞 ROS 稳态控制中的潜在作用。前一种假设得到了已知的 PUFA 螯合三酰甘油 (TAG) 的增加以及 MAM 破坏后脂滴积累的支持⁷ 。后者得到 MAM 破坏后 ER 和线粒体 ROS 积累的支持⁸ 。鉴于 MAM 的可塑性，目前的研究结果表明，MAM PUFA 过氧化的量可以提供基于脂质的机制来控制应激时 MAM 的形成，并且特定消除这些脂质种类可以随后在应激缓解后减少 MAM。这些脂质种类的控制是否依赖于 MAM 束缚因子（例如 PACS-2、mitofusin-2 或 VAPB）仍有待测试，所有这些因子都被发现影响铁死亡。或者，这里提出的结果表明，内质网（而不仅仅是线粒体）在铁死亡中发挥着关键作用，这表明除了已知的在蛋白质折叠和 MAM 形成中的作用外，内质网伴侣和氧化还原酶也可能参与其中。 总的来说，张等人。^{图 1}证实了人们长期以来怀疑的 ER-线粒体接触（MAM）的形成与铁死亡之间的功能联系。