Function and regulation of the reductive stress response

还原应激反应的功能和调节

基本信息

批准号：
10717394
负责人：
Michael P Rape
金额：
$ 30.23万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10717394
关键词：
26S proteasome Address Affect Aging Biochemical Cardiomyopathies Cell Cycle Cell Cycle Progression Cell Cycle Regulation Cell Death Cell Survival Cell division Cells Collaborations Complement Cyclin D1 Cytoplasm Data Development Developmental Delay Disorders Diabetes Mellitus Disease Drops Electron Transport Ensure Environment Enzymes Excision Foundations Gatekeeping Genetic Goals Growth Factor Heart Homeostasis Immune response Immune signaling Inflammatory Ion Channel Malignant Neoplasms Mediating Metabolic Metabolic Control Metabolic Diseases Metabolism Methods Mitochondria Mitochondrial Proteins Modality Modeling Modification Mutation Nerve Degeneration Neurodegenerative Disorders Organelles Outer Mitochondrial Membrane Output Oxidation-Reduction Oxidative Phosphorylation Pathologic Pathology Pathway interactions Patients Physiological Play Population Production Proliferating Protac Proteins Reaction Reactive Oxygen Species Regulation Replication Initiation Role Running Sensory Signal Pathway Signal Transduction Source Stress Surface TOM translocase Testing Therapeutic Tissues Viral Work biological adaptation to stress cellular targeting combat gain of function mutation genetic analysis human disease inhibitor innovation neuron development novel novel therapeutics oxidation pharmacologic preservation prevent programs protein degradation protein misfolding recruit repaired response sensor transmission process tumorigenesis ubiquitin-protein ligase

项目摘要

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.

项目概要线粒体是为细胞提供 ATP 和代谢构件的重要细胞器，同时也发挥着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导致疾病，例如神经退行性变，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传导途径检测并缓解线粒体失调。该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，保护电子的激活通过感测不变的 ETC 副产物活性氧 (ROS) 来控制传输链 (ETC)。还原应激反应建立在 Cys 氧化还原开关的基础上：在健康细胞中，FNIP1 中的 Cys 残基氧化，从而稳定该蛋白质并使其能够下调 ETC 活性。当细胞耗尽ROS时，然而，FNIP1 Cys 残基减少，并且 FNIP1 被 E3 连接酶 CUL2FEM1B 识别。这随后 FNIP1 的泛素化和蛋白酶体降解去除了这个线粒体看门人以重新激活 ETC 并为细胞重新供应 ROS。因此，FNIP1 和 CUL2FEM1B 是传感器的传感模块还原应激反应。重要的是，FEM1B 的突变使这种 E3 连接酶过度激活，从而导致综合征发育迟缓，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，以确保准确的还原应激信号。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 CUL2FEM1B 的氧化态在这种已经还原的环境中，关键的 FNIP1 Cys 残留物尚不清楚。我们发现底物和酶还原应激反应的一部分通过 TOM 复合物锚定在线粒体外膜上，连接氧化线粒体膜间空间与还原细胞质的通道。在我们的第一个目标是，我们将剖析这种定位如何影响还原应激信号。我们期望这项工作能够揭示膜通道作为 E3 连接酶协同适配器的新功能。此外，它可能会让我们查明介导还原应激信号传导的 ROS 来源，从而揭示了其广受追捧的生理作用 ROS在信号传导中的作用。接下来我们将重点关注细胞周期对还原应激信号的调节。 ROS 长期以来建议控制细胞分裂，我们确实发现 CUL2FEM1B 的过度激活会抑制增殖。这一结果表明ROS通过还原应激反应控制细胞周期。本着这个理念，我们确定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 AMBRA1，该酶驱动细胞周期蛋白 D 降解，从而阻止 DNA 的起始复制。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如何它会对肿瘤发生过程中的发育或组织稳态产生负面影响。虽然我们的首要目标是解决生理调节模式，但我们最终将开发方法来发挥作用对还原应激信号的药理学控制。当还原应激反应调整 ETC 时，激活还原应力 E3 连接酶 CUL2FEM1B 为我们提供了增加 ETC 输出的独特机会由线粒体衰退或抑制引起的病理。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局部蛋白水解靶向嵌合体更有效、更具体地降解病理蛋白。在我们的最后一个目标中，我们将建立我们发现了可以取代 CUL2FEM1B 蛋白抑制剂的化合物，从而激活 FNIP1 降解和ETC功能。这项工作将为线粒体相关蛋白降解奠定基础作为一种在衰老或神经退行性疾病期间提供治疗益处的新方式。我们的建议采用综合遗传、生化和药理学方法来剖析还原应激反应作为保守的线粒体应激反应的调节。这部作品将揭示氧化还原信号传导的基本原理，可能会导致新疗法的开发使目前几乎没有治疗选择（如果有的话）的神经退行性疾病患者受益。