Function and regulation of the reductive stress response

项目总结 线粒体是为细胞提供三磷酸腺苷和新陈代谢构件的基本细胞器，但也起着关键作用 发挥信号中枢的作用，协调免疫反应或细胞生存。线粒体蛋白的突变 阻碍发育并导致疾病，如神经退化，线粒体活性下降是 被认为是衰老的标志。为了防止这种后果，细胞使用保守的信号通路， 检测并缓解线粒体失调。这项提案的一个主要目标是剖析对 线粒体信号通路，还原应激反应，保护电子的激活 运输链(ETC)通过感知不变的ETC副产物、活性氧物种(ROS)。 还原应激反应建立在半胱氨酸氧化还原开关上：在健康细胞中，FNIP1中的半胱氨酸残基是 氧化，这稳定了这个蛋白质，并允许它下调ETC活性。当细胞耗尽ROS时， 然而，FNIP1的半胱氨酸残基减少，FNIP1被E3连接酶CUL2FEM1B识别。这个 随后FNIP1的泛素化和蛋白酶体降解移除了这个线粒体守门人，从而重新- 激活ETC并为电池重新供应ROS。因此，FNIP1和CUL2FEM1B是 还原应激反应。重要的是，FEM1B过度激活E3连接酶的突变会导致综合征 发育迟缓，表明组织形成和动态平衡需要严格的还原调节 压力反应。 这项提案将剖析确保准确减少压力信号的三种关键监管模式。 我们将首先研究还原应激信号的空间控制。与所有泛素化反应一样，FNIP1 CUL2FEM1B的修饰发生在细胞质中，但细胞如何调节细胞的氧化状态 在这种已经还原的环境中，关键的FNIP1半胱氨酸残留量尚不清楚。我们发现底物和酶 的还原应激反应通过TOM复合体锚定在线粒体膜外，一个 连接氧化线粒体膜间间隙和还原细胞质的通道。在我们的第一次 目的，我们将剖析这种局部化如何影响还原应激信号。我们期待这项工作将揭示 膜通道作为E3连接酶共适配子的新功能。此外，它可能会让我们准确地定位 ROS的来源，它介导了减少压力的信号，从而揭示了 ROS正在发送信号。 接下来，我们将重点介绍细胞周期对还原应激信号的调节。ROS长期以来一直是 建议控制细胞分裂，我们确实发现CUL2FEM1B的过度激活抑制了细胞的增殖。 这一结果表明，ROS通过还原应激反应控制细胞周期。本着这一理念，我们 发现了细胞周期调节因子RNF187，它促进生长因子下游的细胞周期进程 信号转导，作为CUL2FEM1B的抑制因子。我们的初步数据表明，RNF187和CUL2FEM1B合作 限制另一种E3连接酶，AMBRA1，它驱动细胞周期蛋白D的降解，从而阻止DNA的启动 复制。在我们的第二个目标中，我们将剖析E3连接酶串扰的机制基础，以揭示 ROS信号如何整合到细胞周期程序中。我们希望这项研究能够解释氧化还原压力是如何 它是否会在肿瘤发生过程中对发育或组织动态平衡产生负面影响？ 虽然我们的首要目标是解决生理调节模式，但我们最终将开发出应用于 还原应激信号的药理控制。当还原压力反应调谐ETC时， 激活还原应激E3连接酶CUL2FEM1B为我们提供了增加ETC产量的独特机会 在由线粒体衰退或抑制所驱动的病理中。此外，由于CUL2FEM1B作用于线粒体 表面，靶向该E3连接酶的化合物可以转化为定位的蛋白水解靶向嵌合体 以便更有效和更具体地集中降解病理性蛋白质。在我们的最终目标中，我们将建立 关于我们发现的化合物可以取代CUL2FEM1B，从而激活两个FNIP1 降解性和ETC功能。这项工作将为线粒体相关蛋白的降解奠定基础 作为一种在衰老或神经退行性疾病中提供治疗益处的新方法。 我们的建议采用了遗传学、生化和药理学的综合方法来剖析 作为保守的线粒体应激反应的还原应激反应的调节。这项工作将揭示 氧化还原信号的基本原理，并可能导致一种新的疗法的开发，可以 使目前几乎没有治疗选择的神经退行性疾病的患者受益。

PROJECT SUMMARY Mitochondria are essential organelles that supply cells with ATP and metabolic building blocks, but also play key roles as signaling hubs that orchestrate the immune response or cell survival. Mutations in mitochondrial proteins impede development and cause diseases, such as neurodegeneration, and a decline in mitochondrial activity is considered a hallmark of aging. To prevent such consequences, cells employ conserved signaling pathways that detect and alleviate mitochondrial dysregulation. It is a major goal of this proposal to dissect the regulation of a mitochondrial signaling pathway, the reductive stress response, which safeguards activation of the electron transport chain (ETC) through sensing an invariant ETC byproduct, reactive oxygen species (ROS). The reductive stress response is built on a Cys redox switch: in healthy cells, Cys residues in FNIP1 are oxidized, which stabilizes this protein and allows it to downregulate ETC activity. When cells run out of ROS, however, the FNIP1 Cys residues become reduced and FNIP1 is recognized by the E3 ligase CUL2FEM1B. The subsequent ubiquitylation and proteasomal degradation of FNIP1 removes this mitochondrial gatekeeper to re- activate the ETC and re-supply cells with ROS. FNIP1 and CUL2FEM1B are therefore the sensory module of the reductive stress response. Importantly, mutations in FEM1B that hyperactivate this E3 ligase cause syndromic developmental delay, showing that tissue formation and homeostasis require tight regulation of the reductive stress response. This proposal will dissect three crucial modes of regulation that ensure accurate reductive stress signaling. We will first investigate spatial control of reductive stress signaling. As with all ubiquitylation reactions, FNIP1 modification by CUL2FEM1B takes places in the cytoplasm, yet how cells can modulate the oxidation state of the critical FNIP1 Cys residues in this already reducing environment is unclear. We found that substrate and enzyme of the reductive stress response are anchored on the outer mitochondrial membrane via the TOM complex, a channel that connects the oxidative mitochondrial intermembrane space with the reducing cytoplasm. In our first aim, we will dissect how this localization impacts reductive stress signaling. We expect that this work will reveal a novel function of a membrane channel as an E3 ligase co-adaptor. Moreover, it will likely allow us to pinpoint the source of ROS that mediate reductive stress signaling, thereby revealing a sought-after physiological role for ROS in signaling. We will next focus on the regulation of reductive stress signaling by the cell cycle. ROS have long been suggested to control cell division, and we had indeed found that hyperactivation of CUL2FEM1B inhibits proliferation. This result implied that ROS control the cell cycle via the reductive stress response. In line with this notion, we identified the cell cycle regulator RNF187, which promotes cell cycle progression downstream of growth factor signaling, as an inhibitor of CUL2FEM1B. Our preliminary data suggest that RNF187 and CUL2FEM1B collaborate to restrict another E3 ligase, AMBRA1, which drives cyclin D degradation and thereby prevents initiation of DNA replication. In our second aim, we will dissect the mechanistic underpinnings of this E3 ligase crosstalk to reveal how ROS signaling is integrated into the cell cycle program. We expect this work to explain how redox stress can its exert negative consequences onto development or onto tissue homeostasis during tumorigenesis. While our first aims address physiological modes of regulation, we will finally develop methods to exert pharmacological control over reductive stress signaling. As the reductive stress response tunes the ETC, activating the reductive stress E3 ligase CUL2FEM1B provides us with a unique opportunity to increase ETC output in pathologies driven by mitochondrial decline or inhibition. Moreover, because CUL2FEM1B acts on mitochondrial surfaces, compounds that target this E3 ligase could be converted into localized proteolysis-targeting chimera for more efficient and more specific focused degradation of pathological proteins. In our last aim, we will build on our discovery of compounds that displace protein inhibitors from CUL2FEM1B,, thereby activating both FNIP1 degradation and ETC function. This work will lay the foundation for mitochondria-associated protein degradation as a new modality to provide therapeutic benefit during aging or in neurodegenerative disease. Our proposal takes an integrated genetic, biochemical, and pharmacological approach to dissect the regulation of the reductive stress response as a conserved mitochondrial stress response. This work will reveal fundamental principles of redox signaling and may lead to the development of a new therapeutics that could benefit patients of neurodegenerative diseases that currently have few, if any, treatment options.