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

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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.