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

酵母内质网氧化蛋白折叠的运行和控制的定量分析

• 批准号: BB/M009815/1
• 负责人: Mick Tuite
• 金额: $ 74.38万
• 依托单位: University of Kent
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM009815%2F1
• 关键词: Quantitative; analysis; operation; control; oxidative

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.

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、与其他细胞通讯以及形成细胞可以生长的支架结构。生物加工工业也利用细胞分泌蛋白质的能力，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性疾病。高等（真核）细胞中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串，其折叠结构取决于其中各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，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：这称为二硫键。二硫键的形成是分泌过程的一个组成部分，涉及一系列特定的酶。这些酶从相互作用的半胱氨酸中去除电子，使它们在它们之间形成决定受影响蛋白质形状的键。然后电子在不同的酶之间传递，最终传递到氧原子上，氧原子与水反应形成过氧化氢。由于后者如果大量存在是有毒的，因此必须在进一步的一系列反应中将其除去。这些反应的整体被称为氧化蛋白折叠（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）OPF 之间的差异，以增进我们对氧化折叠分子机制的理解。这些知识还将提高我们通过基因工程操纵该途径的能力，以便为生物加工工业产生更好的生产细胞。酵母细胞只分泌相对少量的蛋白质，因此它们的 OPF 机器进化为在最小的酶组上运行。相比之下，许多人类细胞是多产的分泌者，因为它们需要与体内的其他细胞广泛交流，产生消化食物的酶，或产生免疫系统的分子。因此，人类细胞具有更加复杂的 OPF，具有不同形式的 OPF 酶，仅作用于特定类型的靶蛋白。有趣的是，人类细胞也能够利用有毒的过氧化氢来驱动 OPF 反应，而酵母却不能这样做。我们将采用三管齐下的策略来利用这些差异：1）我们将从酵母和人体细胞中分离OPF机器的酶，并在试管中研究它们的详细特性； 2) 我们将使用这些实验的信息生成一个计算模型，可以预测细胞内 OPF 通路的特性； 3) 我们将利用计算模型的预测来改变酵母 OPF 酶的特性，并将它们与活酵母细胞中的人类酶混合。总体而言，这一策略将使我们能够更好地了解 OPF 机械的功能，并且从长远来看将使我们能够设计出更适合在生物加工应用中使用的酵母细胞。

项目成果

Remembering the Past: A New Form of Protein-Based Inheritance

记住过去：基于蛋白质的遗传的新形式

• DOI: 10.1016/j.cell.2016.09.036
• 发表时间: 2016
• 期刊: Cell
• 影响因子: 64.5
• 作者: Tuite M