The Spatiotemporal Regulation of the Keap1/Nrf2 pathway

所有活着的有机体都有保护系统，使它们能够在应激条件下成功适应和生存。一种主要的保护途径是Keap1/Nrf2途径。这一途径通常不会以其全部潜力运行，可以被小分子(诱导剂)激活，其中一些小分子存在于富含蔬菜的饮食中。诱导剂与传感蛋白Keap1的半胱氨酸残基发生化学反应，导致转录因子Nrf2的稳定，从而增加具有保护功能的基因网络的表达。先前的工作表明，激活Keap1/Nrf2通路是预防慢性疾病的一种非常有效的策略，但在衰老模型生物中，激活Nrf2的能力逐渐丧失。因此，人们对发现和开发Nrf2活性的小分子调节剂以改善人类健康和延缓衰老的兴趣越来越大。我们最近开发了一种系统，使我们能够在单个活细胞中研究Nrf2和Keap1之间的相互作用，并发现Keap1使用独特的循环机制来靶向Nrf2进行降解。本项目旨在利用我们新建立的实验系统来：(I)确定Keap1的传感器半胱氨酸(最常被亲电分子修饰)对Keap1：Nrf2复合体形成的影响，以及随后的诱导剂处理；(Ii)寻找直接破坏蛋白质的有效非亲电诱导剂：Keap1和Nrf2之间的蛋白质相互作用，并评估这些诱导剂抵御氧化应激的能力，氧化应激是基本上所有慢性病和衰老的关键因素；(Iii)确定Keap1和Nrf2相互作用对p21和p62水平的调节作用，这两个蛋白是Nrf2活性的两个关键蛋白质调节因子，分别参与两个基本的生物学过程，即细胞凋亡和自噬。总之，这些实验将提供对Keap1和Nrf2之间相互作用的控制机制的详细了解。这些知识对于设计和实施旨在调节NRF2活动以促进终身健康和福祉的新战略至关重要。

All living organisms have protective systems that allow their successful adaptation and survival under conditions of stress. One major way of protection is the Keap1/Nrf2 pathway. This pathway does not normally operate at its full potential, and can be activated by small molecules (inducers), some of which are present in vegetable-rich diets. Inducers react chemically with cysteine residues of the sensor protein Keap1, leading to stabilisation of transcription factor Nrf2, and consequently to increased expression of a network of genes with protective functions. Previous work has shown that activation of the Keap1/Nrf2 pathway is a very effective strategy for protection against chronic disease, but that in ageing model organisms, the ability to activate Nrf2 is progressively lost. Thus, there is a growing interest in the discovery and development of small-molecule modulators of Nrf2 activity to improve human health and delay ageing. We have recently developed a system which has allowed us to study the interactions between Nrf2 and Keap1 in single live cells, and discovered that Keap1 uses a unique cyclic mechanism to target Nrf2 for degradation. This project aims to employ our newly-established experimental system in order to: (i) determine the impact of the sensor cysteines of Keap1 which are most commonly modified by electrophiles on the Keap1: Nrf2 complex formation at basal state as well as following inducer treatment; (ii) identify potent non-electrophilic inducers which directly disrupt the protein: protein interaction between Keap1 and Nrf2, and evaluate the ability of such inducers to protect against oxidative stress, a critical contributor to the pathogenesis of essentially all chronic diseases and ageing; (iii) establish the effect on the interaction of Keap1 and Nrf2 of modulating the levels of p21 and p62, two critical protein regulators of the Nrf2 activity which are involved in two fundamental biological processes, apoptosis and autophagy, respectively. Together, these experiments will provide detailed understanding of the mechanisms controlling the interaction between Keap1 and Nrf2. This knowledge is essential for the design and implementation of new strategies aiming the modulation of Nrf2 activity for life-long health and wellbeing.