Function and regulation of the reductive stress response

项目摘要 线粒体是重要的细胞器，为细胞提供ATP和代谢构件，但也发挥关键作用。 作为协调免疫反应或细胞存活的信号枢纽。线粒体蛋白质突变 阻碍发育并引起疾病，如神经变性，以及线粒体活性下降， 被认为是衰老的标志为了防止这种后果，细胞采用保守的信号通路， 检测和减轻线粒体失调。本提案的一个主要目标是剖析 线粒体信号通路，还原应激反应，保护电子的激活 运输链（ETC）通过感测不变的ETC副产物，活性氧（ROS）。 还原性应激反应建立在Cys氧化还原开关上：在健康细胞中，FNIP1中的Cys残基是 氧化，这稳定了这种蛋白质，并允许它下调ETC活动。当细胞耗尽活性氧， 然而，FNIP1 Cys残基被还原，FNIP1被E3连接酶CUL2FEM1B识别。的 FNIP1随后的泛素化和蛋白酶体降解去除了这个线粒体看门人， 激活ETC并重新给细胞提供ROS因此，FNIP1和CUL2FEM1B是神经元的感觉模块。 还原性应激反应重要的是，过度激活E3连接酶的FEM1B突变引起综合征。 发育延迟，表明组织形成和体内平衡需要严格调节还原性 应激反应 该提案将剖析三种关键的调节模式，以确保准确的还原压力信号。 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， 在这种已经减少的环境中关键的FNIP1 Cys残基尚不清楚。我们发现底物和酶 的还原性应激反应通过TOM复合物锚定在线粒体外膜上， 连接氧化线粒体膜间隙和还原细胞质的通道。在我们的第一 目的，我们将剖析这种定位如何影响还原应激信号。我们希望这项工作能揭示 膜通道作为E3连接酶共接头的新功能。此外，它可能会让我们确定 ROS的来源，介导还原性应激信号，从而揭示了一个受欢迎的生理作用， ROS信号。 接下来我们将重点关注细胞周期对还原性应激信号的调节。长久以来， 提示控制细胞分裂，并且我们确实发现CUL2FEM1B的过度激活抑制增殖。 这一结果表明，ROS通过还原性应激反应控制细胞周期。根据这一概念，我们 鉴定了细胞周期调节因子RNF187，其促进生长因子下游的细胞周期进程 信号传导，作为CUL2FEM1B的抑制剂。我们的初步数据表明，RNF187和CUL2FEM1B合作， 限制另一种E3连接酶AMBRA 1，其驱动细胞周期蛋白D降解，从而阻止DNA的起始 复制的在我们的第二个目标中，我们将剖析这种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。我们希望这项工作能够解释氧化还原应激 在肿瘤发生过程中，它可能对发育或组织稳态产生负面影响。 虽然我们的第一个目标是解决调节的生理模式，但我们最终将开发方法， 药理学控制还原应激信号传导。当还原性应激反应调节ETC时， 激活还原应激E3连接酶CUL2FEM1B为我们提供了一个独特的机会，以增加ETC输出 在由线粒体下降或抑制驱动的病理学中。此外，由于CUL2FEM1B作用于线粒体， 表面，靶向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 用于更有效和更特异性地集中降解病理蛋白质。在我们最后的目标，我们将建立 我们发现了从CUL2FEM1B中取代蛋白质抑制剂的化合物，从而激活FNIP1 降解和ETC功能。这项工作将为进一步研究大肠杆菌相关蛋白的降解奠定基础 作为一种新的模式，在衰老或神经退行性疾病中提供治疗益处。 我们的建议采取综合的遗传学，生物化学和药理学方法来剖析 还原性应激反应的调节作为保守的线粒体应激反应。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治疗方法的发展， 使目前几乎没有治疗选择的神经退行性疾病患者受益。

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.