Chemical probes to decode the subcellular redox-regulated proteome

这项提议旨在创造新的小分子工具来绘制细胞中的分子过程。细胞内的蛋白质执行生命的基本功能，影响我们等生物体的健康和疾病的方方面面。然而，细胞不是一袋分子，而是错综复杂地划分成具有不同化学环境的隔间。相同的蛋白质可能存在于不同的隔间，但以不同的方式修饰(修饰)，与不同的蛋白质组相互作用，或执行不同的催化功能。我们能够绘制出这些“兼职”蛋白质的地图--它们在哪里，它们的修饰根据它们在哪个隔间有什么不同，以及它们在不同的隔间里有什么功能--这些工具和方法有助于我们理解基本的细胞生物学。细胞隔间的不同之处之一是它们的活性氧物种(ROS)和氧化还原(还原-氧化)状态的水平不同。ROS是细胞新陈代谢的结果，但也是关键的信号分子，使生物系统能够对不断变化的环境做出快速反应。ROS是通过氧化还原敏感蛋白半胱氨酸残基的翻译后修饰来检测的。这种化学信号机制被认为在许多疾病中被解除调控，如癌症、糖尿病和炎症，细胞维持氧化还原稳定状态的能力缺乏可塑性可能是衰老的一个组成部分。植物通过感知氧化还原变化来调节它们的生长，并决定细胞的命运，例如在干旱和逆境时期。然而，目前的方法不能捕捉到活细胞特定隔室中蛋白质氧化还原相关修饰的全局和分子图像，因为当细胞被打开进行分析时，这些信息会丢失或扭曲。在这个项目中，我们将开发新的化学工具，可以针对不同的细胞隔室。在这里，他们将释放一枚反应性弹头，以捕获处于不同氧化还原状态的半胱氨酸残留物。我们将设计、综合和测试工具，探索两种新的方法来屏蔽反应性弹头，以便按需释放。然后，我们将展示我们的新方法，设计工具来靶向一种名为过氧化物酶体的间隔，该间隔通过尚未完全了解的机制参与氧化还原信号传递。我们将使用我们的工具来研究植物细胞中过氧化体中被扰乱的氧化还原状态如何影响这一区域中的半胱氨酸修饰。基于增强细胞抗氧化剂应该有有益效果的前提的治疗方法在很大程度上未能实现。这可能是因为缺乏关于氧化还原过程如何调节细胞生物学以及它们如何在有机体的生命周期中发生变化的基础知识。在单个细胞隔间水平上研究氧化还原信号的工具将使我们能够理解这种生物学。

This proposal aims to create new small molecule tools to map molecular processes in cells. Proteins inside cells perform the fundamental functions of life, impacting on all aspects of health and disease in organisms such as ourselves. A cell is not a bag of molecules, however, but intricately divided into compartments possessing different chemical environments. The same protein may be present in different compartments but modified (decorated) in distinct ways, interacting with different groups of proteins, or performing different catalytic functions. Tools and methods that enable us to map these 'moonlighting' proteins - where they are, how their modifications differ depending on which compartment they are in, and what their function is in different compartments - contribute to our understanding of fundamental cell biology.One way in which cellular compartments differ is in their level of reactive oxygen species (ROS) and redox (reduction-oxidation) state. ROS are produced as a consequence of cell metabolism, but are also crucial signalling molecules that enable biological systems to respond rapidly to changing environments. ROS are detected via post-translational modifications on the cysteine residues of redox-sensitive proteins. This chemical signalling mechanism is thought to be deregulated in many diseases, such as cancer, diabetes and inflammation, and a lack of plasticity in the ability of a cell to maintain redox steady state may be an integral part of ageing. Plants sense redox changes to regulate their growth and determine cell fate, for example in times of drought and stress. However, current methods cannot capture a global and molecular picture of redox-related modifications on proteins in specific compartments of live cells, as this information is lost or distorted when cells are broken open for analysis.In this project we will develop new chemical tools that can be directed to different cellular compartments. Here they will release a reactive 'warhead' to capture cysteine residues in different redox states. We will design, synthesise and test tools, exploring two new approaches to mask the reactive warhead so that it can be released on demand. We will then showcase our new approach by designing tools to target a compartment called the peroxisome, which is involved in redox signalling via mechanisms that are not yet fully understood. We will use our tools to study how perturbed redox status in peroxisomes in plant cells influences cysteine modifications in this compartment.Therapeutic approaches based on the premise that boosting cellular antioxidants should have beneficial effects have largely failed to materialise. This is likely due to a lack of fundamental knowledge on how redox processes regulate cell biology and how they change over the lifetime of an organism. Tools to study redox signalling at the level of individual cellular compartments will enable our understanding of this biology.