Chemical probes to decode the subcellular redox-regulated proteome

该提案旨在创建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，影响生物体（如我们自己）的健康和疾病的各个方面。然而，细胞并不是一袋分子，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区室中，但以不同的方式修饰（装饰），与不同的蛋白质组相互作用，或执行不同的催化功能。这些工具和方法使我们能够绘制这些“兼职”蛋白质-它们在哪里，它们的修饰如何根据它们所在的区室而有所不同，以及它们在不同区室中的功能-有助于我们理解基本的细胞生物学。细胞区室不同的一种方式是它们的活性氧（ROS）水平和氧化还原（还原-氧化）状态。ROS是细胞代谢的结果，但也是使生物系统能够对变化的环境做出快速反应的关键信号分子。ROS通过氧化还原敏感蛋白的半胱氨酸残基上的翻译后修饰来检测。这种化学信号传导机制被认为在许多疾病中失调，例如癌症，糖尿病和炎症，并且细胞维持氧化还原稳态的能力缺乏可塑性可能是衰老的一个组成部分。植物感知氧化还原变化来调节其生长并决定细胞命运，例如在干旱和胁迫时期。然而，目前的方法无法捕捉到活细胞特定区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区室。在这里，它们将释放一个反应性的“弹头”，以捕获不同氧化还原状态的半胱氨酸残基。我们将设计，合成和测试工具，探索两种新的方法来掩盖反应弹头，以便它可以按需释放。然后，我们将展示我们的新方法，通过设计工具来靶向一个称为过氧化物酶体的隔室，该隔室通过尚未完全理解的机制参与氧化还原信号传导。我们将使用我们的工具来研究植物细胞中过氧化物酶体的氧化还原状态如何影响半胱氨酸修饰。基于提高细胞抗氧化剂应该具有有益效果的前提的治疗方法在很大程度上未能实现。这可能是由于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生物体的一生中发生变化的基础知识。在单个细胞区室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.