Mitochondrial Thiol Regulation and Programmed Cell Death in Yeast

蛋白质是控制大多数生物过程的关键分子。每种蛋白质都是由采用独特三维结构的氨基酸的线性聚合物组成的。人们越来越认识到，半胱氨酸氨基酸在调节许多不同蛋白质的活性方面起着关键作用。这是因为半胱氨酸氨基酸在硫醇基团中含有硫原子，这解释了半胱氨酸在氧化和还原反应中的高反应性。这意味着半胱氨酸残基在许多不同蛋白质中的氧化还原状态，如酶和转录因子，可以深刻地影响它们的活性。因此，不足为奇的是，细胞氧化还原平衡的改变与许多疾病过程有关。这项工作计划将研究细胞如何维持线粒体蛋白质的氧化还原状态。了解线粒体氧化还原调节尤为重要，因为线粒体功能障碍和疾病之间存在许多既定的联系。该项目的重点将是谷胱甘肽/谷氧还蛋白和硫氧还蛋白系统，它们构成了主要的细胞氧化还原系统。我们在申请中描述的初步数据表明，线粒体硫氧还蛋白的氧化还原状态对生长很重要，其氧化诱导一种形式的程序性细胞死亡(PCD)。PCD是健康生物体发育的正常过程，细胞以受控、规范的方式死亡。这是一种细胞自杀的形式，可以在包括压力在内的各种刺激下诱导。这很重要，因为细胞死亡过程与许多疾病过程有关，许多医学治疗和干预都是通过PCD发挥作用的。我们将使用酵母模型系统来系统地检测和定义线粒体氧化还原调节系统的细胞浓度和氧化还原状态。重要的是，我们将在诱导PCD的条件下检查这些系统，以确定线粒体硫氧还蛋白在这一过程中的调节作用。遗传学方法将被用来理解线粒体硫氧还蛋白如何调节细胞死亡。一项关键技术将是突变线粒体硫氧还蛋白中的半胱氨酸残基，以直接检测它们在调节PCD中的作用，这是使用各种作用于细胞死亡途径的已知标记物来测量的。线粒体是大多数生物体中活性氧物种的主要来源，这被认为是激活PCD的关键信号。众所周知，蛋白质中的硫醇基团对氧化特别敏感，因此在项目的最后部分，我们将检验线粒体硫氧还蛋白系统作为激活PCD的活性氧物种的传感器的假设。

Proteins are key molecules that control most biological processes. Each protein is made-up of a linear polymer of amino acids which adopt a unique three-dimensional structure. It is becoming increasingly recognized that the cysteine amino acid can play a key role in regulating the activity of many different proteins. This is because cysteine amino acids contain a sulphur atom in a thiol group which accounts for its high reactivity in oxidation and reduction reactions. This means that the redox state of cysteine residues in many different proteins, such as enzymes and transcription factors, can profoundly influence their activity. Not surprisingly therefore, alterations in the cellular redox balance are implicated in many disease processes. This work programme will investigate how cells maintain the redox state of mitochondrial proteins. Understanding mitochondrial redox regulation is particularly important since there are many established links between mitochondrial dysfunction and disease. The focus of this project will be on the glutathione/glutaredoxin and thioredoxin systems which constitute the main cellular redox systems. Our preliminary data, described in the application, show that the redox state of the mitochondrial thioredoxin is important for growth and its oxidation induces a form of programmed cell death (PCD). PCD is a normal process in the development of healthy organisms where cells die in a controlled, regulated fashion. It is a form of cell suicide which can be induced in response to a variety of stimuli including stress. It is important because cell death processes have been implicated in numerous disease processes and many medical treatments and interventions act through PCD. We will use the yeast model system to systematically examine and define the cellular concentrations and redox state of the mitochondrial redox regulatory systems. Importantly, we will examine these systems under conditions that induce PCD to determine the regulatory role of the mitochondrial thioredoxin in this process. Genetic approaches will be used to understand how the mitochondrial thioredoxin regulates cell death. A key technique will be to mutate the cysteine residues in the mitochondrial thioredoxin to directly examine their role in regulating PCD, measured using various known markers which act in the cell death pathway. Mitochondria are the main source of reactive oxygen species in most organisms and this is thought to represent a key signal that activates PCD. Thiol groups in proteins are known to be particularly sensitive to oxidation and so the final part of the project we will test the hypothesis that the mitochondrial thioredoxin system acts as a sensor of reactive oxygen species to activate PCD.