Signaling mechanisms that detect stress and maintain homeostasis

检测压力和维持体内平衡的信号机制

• 批准号: 9276991
• 负责人: T Keith Blackwell
• 金额: $ 43.65万
• 依托单位: JOSLIN DIABETES CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-20 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9276991
• 关键词: 70-kDa Ribosomal Protein S6 Kinases; Acute; Affect; Aging; Area; CRISPR/Cas technology; Caenorhabditis elegans; Cell physiology; Data; Disease; Drug Metabolic Detoxication; Enzyme Activation; Event; Growth; Health; Homeostasis; Human; Human Development; Longevity; MAPK14 gene; Mass Spectrum Analysis; Mediating; Metabolic; Mitochondria; Modeling; Modification; Molecular Chaperones; Nox enzyme; Organism; Orthologous Gene; Oxidation-Reduction; Phenotype; Phosphotransferases; Play; Proteins; Proto-Oncogene Proteins c-akt; ROCK1 gene; Reactive Oxygen Species; Regulation; Research; Role; Screening Result; Signal Transduction; Stress; Work; base; biological adaptation to stress; genome editing; in vivo; interest; response; sensor; small molecule; stress reactivity; transcription factor

Project Summary This MIRA proposal focuses on two overlapping areas: stress response regulation and the functions of redox-based signaling in vivo. It is a fundamentally important problem how organisms detect and respond to different forms of stress. Much has been learned in this area but we still have a very incomplete understanding of how some stresses are detected, including reactive small molecules such as ROS. For many years my group has studied stress responses and aging in C. elegans, focusing on the Nrf2 transcription factor ortholog SKN-1. Nrf2 mediates a conserved detoxification response to reactive small molecules but has many additional functions, and is of great importance in health and disease. Working in C. elegans we have defined a number of aspects of SKN-1/Nrf2 regulation and functions, including its major role in longevity assurance. We have recently uncovered an exciting mechanism of SKN-1/Nrf regulation that forms the basis for this new research direction. We find that SKN-1 and human Nrf2 are activated at the ER by a localized ROS signal that can derive from the ER, NOX enzyme activation induced by stress, or mitochondria. This signal induces sulfenylation of a single Cys within the kinase activation loop of the ER unfolded protein sensor IRE-1, resulting in acute inhibition of the IRE-1 unfolded protein response and activation of p38 signaling at IRE-1 through a second sulfenylation event. p38 signaling in turn activates SKN-1/Nrf2. Remarkably, other kinases of major interest (AKT, p70S6K, ROCK1) seem to be regulated through sulfenylation of the same Cys. The data reveal an unexpected IRE-1 function that is regulated by a redox switch, a major stress sensor for SKN- 1/Nrf2, and a possible rationale for how redox stress can affect so many cellular processes. They also suggest that the scope and functional versatility of Cys-based signaling are much wider than is generally appreciated. In our proposed research we will continue to identify mechanisms of SKN-1/Nrf2 regulation and their functions in vivo, but will also cast our net wider in utilizing the advantages of C. elegans to explore mechanisms and functions of Cys redox signaling in the context of stress, growth, and other conditions. We will refine models for IRE-1 regulation of SKN-1/Nrf2 and its functions in vivo. We will also similarly study SKN- 1/Nrf2 regulation by the chaperone TRIC, another mechanism we have identified that may involve redox signaling, and build upon screening results to develop new models for SKN-1/Nrf2 regulation. Using mass spectrometry (MS), we will collaboratively identify C. elegans proteins that are Cys-sulfenylated under stress and growth conditions. We will investigate regulatory and in vivo implications of this modification for the kinases indicated above, and candidates chosen from our MS data. C. elegans will be ideal for this work because of the relative rapidity of Cas9/CRISPR genome editing, and phenotypic analyses. Our research will reveal stress-responsive regulatory mechanisms of fundamental interest, and take major steps towards identifying new mechanistic targets and functional implications of redox signaling in vivo.

项目概要 MIRA 提案重点关注两个重叠领域：压力反应调节和 基于氧化还原的体内信号传导。生物体如何检测和响应是一个根本性的重要问题。 不同形式的压力。在这方面我们已经学到了很多东西，但我们的理解仍然非常不完整 研究如何检测某些应激，包括活性小分子（例如 ROS）。多年来我的 研究小组研究了秀丽隐杆线虫的应激反应和衰老，重点关注 Nrf2 转录因子直系同源物 SKN-1。 Nrf2 介导对反应性小分子的保守解毒反应，但有许多 附加功能，对健康和疾病非常重要。在秀丽隐杆线虫中我们定义了 SKN-1/Nrf2 调节和功能的许多方面，包括其在长寿保证中的主要作用。 我们最近发现了一种令人兴奋的 SKN-1/Nrf 调节机制，该机制构成了 这个新的研究方向。我们发现 SKN-1 和人类 Nrf2 在 ER 处被局部 ROS 激活 信号可以源自应激诱导的 ER、NOX 酶激活或线粒体。这个信号 诱导 ER 未折叠蛋白传感器 IRE-1 激酶激活环内单个 Cys 的磺酰化， 导致 IRE-1 未折叠蛋白反应的急性抑制和 IRE-1 上 p38 信号的激活 通过第二次磺酰化事件。 p38 信号转而激活 SKN-1/Nrf2。值得注意的是，其他激酶 主要兴趣（AKT、p70S6K、ROCK1）似乎是通过相同半胱氨酸的磺酰化来调节的。这 数据揭示了一个意想不到的 IRE-1 功能，该功能由氧化还原开关调节，氧化还原开关是 SKN- 的主要压力传感器 1/Nrf2，以及氧化还原应激如何影响如此多的细胞过程的可能原理。他们还建议 基于 Cys 的信号传导的范围和功能多样性比通常认为的要广泛得多。 在我们提出的研究中，我们将继续确定 SKN-1/Nrf2 调节机制及其作用 体内功能，但也将扩大我们的网络，利用秀丽隐杆线虫的优势来探索 Cys 氧化还原信号在应激、生长和其他条件下的机制和功能。我们 将完善 IRE-1 对 SKN-1/Nrf2 的调节及其体内功能的模型。我们也会类似地研究SKN- 分子伴侣 TRIC 调节 1/Nrf2，这是我们发现的另一种可能涉及氧化还原的机制 信号传导，并根据筛选结果开发 SKN-1/Nrf2 调控的新模型。使用质量 光谱分析 (MS)，我们将合作鉴定压力下半胱氨酸磺酰化的秀丽隐杆线虫蛋白 和生长条件。我们将研究这种修饰对监管和体内的影响 上述激酶以及从我们的 MS 数据中选择的候选激酶。线虫将是这项工作的理想选择 因为 Cas9/CRISPR 基因组编辑和表型分析的相对速度。我们的研究将 揭示根本利益的压力反应监管机制，并采取重大步骤 确定体内氧化还原信号传导的新机制目标和功能影响。