Quantitative analysis of the operation and control of oxidative protein folding in the yeast endoplasmic reticulum

所有活细胞都具有将蛋白质从内部分泌到外部环境的能力。这种能力有多种用途，包括将营养物质转化为细胞可以吸收的形式，与其他细胞交流，以及形成细胞可以生长的支架结构。细胞分泌蛋白质的能力也被生物加工行业利用，生物加工行业对细胞进行重新编程，以制造和分泌基于蛋白质的一线药物，以对抗癌症、多发性硬化症和关节炎等衰弱疾病。高等(真核)细胞的部分分泌过程是为了确保分泌的蛋白质采用具有最佳活性的结构。蛋白质是氨基酸的聚合序列，它们的折叠结构依赖于其中单个氨基酸之间的相互作用。当两个半胱氨酸氨基酸在它们所含的硫原子之间形成键时，就会发生一种对许多蛋白质的活性至关重要的特定类型的相互作用：这被称为二硫键。二硫键的形成是分泌过程中不可或缺的一部分，涉及一系列特定的酶。这些酶从相互作用的半胱氨酸中移除一个电子，使它们之间形成一个键，从而决定受影响的蛋白质的形状。然后，电子在不同的酶之间传递，最终到达氧原子，氧原子与水反应形成过氧化氢。由于后者在大量存在时是有毒的，必须在进一步的一系列反应中将其去除。这些反应的整个过程被称为氧化蛋白质折叠(OPF)途径，也是本项目的重点。不同类型的高等细胞的OPF之间存在根本差异。我们将探索两种特定细胞类型(简单酵母细胞和复杂人类细胞)的OPF之间的差异，以提高我们对参与氧化折叠的分子机制的理解。这些知识还将提高我们通过基因工程操纵这一途径的能力，以便为生物加工工业产生更好的生产细胞。然而，细胞只分泌相对少量的蛋白质，其OPF机制因此进化为在最小的酶集合上运行。相比之下，许多人类细胞是多产的分泌者，因为它们需要与身体内的其他细胞进行广泛的交流，产生消化食物的酶，或产生免疫系统的分子。因此，人类细胞的OPF要复杂得多，具有不同形式的OPF酶，这些酶只作用于特定类型的靶蛋白。有趣的是，人类细胞也能够使用有毒的过氧化氢来驱动OPF反应，而酵母不能这样做。我们将使用三管齐下的策略来利用这些差异：1)我们将从酵母和人类细胞中分离OPF机制的酶，并将在试管中研究它们的详细性质；2)我们将使用这些实验获得的信息来生成一个计算模型，该模型可以预测细胞内OPF途径的性质；以及3)我们将使用计算模型所做的预测来改变酵母OPF酶的性质，并将它们与人类酶在活的酵母细胞中混合。总体而言，这一策略将使我们能够更好地了解OPF机械是如何运作的，从长远来看，将使我们能够设计出更适合用于生物处理应用的酵母细胞。

All living cells possess the ability to secrete proteins from their interior to their exterior environment. This ability serves a variety of purposes, including the transformation of nutrients into a form where they can be taken up by the cell, communication with other cells, and the formation of scaffold structures on which cells can grow. The ability of cells to secrete proteins is also exploited by the bioprocessing industry, which re-programs cells to make and secrete protein-based frontline drugs against debilitating diseases like cancer, multiple sclerosis and arthritis.Part of the secretion processes in higher (eukaryotic) cells is to ensure that secreted proteins adopt a structure in which they have optimal activity. Proteins are polymeric strings of amino acids, and their folded structure depends on interactions between individual amino acids within them. A specific type of interaction that is critical to the activity of many proteins occurs when two cysteine amino acids form bonds between the sulphur atoms they contain: this is known as a disulphide bond. Disulphide bond formation occurs as an integral part of the secretion process, and involves a cascade of specific enzymes. These enzymes remove an electron from the interacting cysteines, allowing them to form a bond between them that determines the affected protein's shape. The electron is then passed between different enzymes and ultimately onto an oxygen atom, which reacts with water to form hydrogen peroxide. Since the latter is toxic if present in large amounts, it has to be removed in a further series of reactions. The entirety of these reactions is called the oxidative protein folding (OPF) pathway and is the focus of this project.There are fundamental differences between the OPFs of different types of higher cells. We will explore differences between the OPFs of two specific cell types (simple yeast cells and complex human cells) to improve our understanding of the molecular machinery involved in oxidative folding. Such knowledge will also improve our ability to manipulate the pathway by genetic engineering in order to generate better producing cells for the bioprocessing industry.Yeast cells only secrete relatively small amounts of proteins, and their OPF machinery therefore evolved to operate on a minimal enzyme set. In contrast, many human cells are prolific secretors, due to their need to communicate extensively with other cells in the body, to produce enzymes for the digestion of food, or to produce molecules of the immune system. Human cells therefore have a much more complex OPF, with different forms of the OPF enzymes that are only act on specific types of target proteins. Interestingly, human cells are also able to use the toxic hydrogen peroxide to drive the OPF reactions, whereas yeast cannot do this. We will use a three-pronged strategy to exploit these differences: 1), we will isolate the enzymes of the OPF machinery from yeast and human cells and will study their detailed properties in test tubes; 2) we will use the information from these experiments to generate a computational model that can predict properties of the OPF pathways inside cells; and 3) we will use predictions made with the computational model to change properties of the yeast OPF enzymes, and to mix them with human enzymes, in living yeast cells. Overall, this strategy will enable us to better understand how the OPF machinery functions, and in the longer term will enable us to engineer yeast cells that are better suited for use in bioprocessing applications.