Signaling mechanisms that detect stress and maintain homeostasis

项目摘要 米拉的建议集中在两个重叠的领域：应激反应调节和 体内基于氧化还原的信号转导。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。在这一领域已经学到了很多，但我们仍然有一个非常不完整的理解 一些压力是如何被检测到的，包括活性小分子，如ROS。多年来我的 该小组研究了线虫的应激反应和衰老，重点是Nrf2转录因子同源基因 SKN-1。NRF2介导对反应性小分子的保守解毒反应，但有许多 更多的功能，对健康和疾病都非常重要。在我们定义的线虫中工作 SKN-1/NRF2调节和功能的若干方面，包括其在长寿保障方面的主要作用。 我们最近发现了一种令人兴奋的SKN-1/NRF调节机制，它构成了 这是一个新的研究方向。我们发现SKN-1和人类NRF2在内质网被局部ROS激活 由应激诱导的内质网、氮氧化物酶激活或线粒体产生的信号。这个信号 在ER未折叠蛋白传感器IRE-1的激酶激活环内诱导单个Cys的硫苯化， 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 通过第二次亚磺化反应。P38信号转而激活SKN-1/Nrf2。值得注意的是，其他的激酶 AKT，p70S6K，ROCK1)似乎是通过相同的半胱氨酸的亚磺化来调节的。这个 数据显示，一种意想不到的IRE-1功能由氧化还原开关调节，氧化还原开关是SKN-1的主要应力传感器。 1/Nrf2，以及氧化还原压力如何影响如此多的细胞过程的一个可能的理论基础。他们还建议 基于半胱氨酸的信令的范围和功能的多样性比人们通常所认识的要广泛得多。 在我们拟议的研究中，我们将继续确定SKN-1/Nrf2的调控机制及其 在体内发挥作用，也将使我们在利用线虫的优势方面撒网更广 Cys氧化还原信号在压力、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的调节模型及其在体内的功能。我们也将同样研究SKN- 1/Nrf2由伴侣调节，我们已经确定的另一种可能涉及氧化还原的机制 信号转导，并根据筛选结果开发SKN-1/Nrf2调控的新模型。使用体量 光谱(MS)，我们将合作识别在胁迫下半胱氨酰化的线虫蛋白质 和生长条件。我们将调查这一修饰的调节和体内影响 从我们的MS数据中选择的候选基因，如上所示。线虫将是这项工作的理想选择 由于Cas9/CRISPR基因组编辑和表型分析的相对快速。我们的研究将 揭示关乎根本利益的应激反应调控机制，并采取重大步骤 确定体内氧化还原信号的新机制靶点和功能含义。

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.