Investigating the Role of Cysteamine Dioxygenase in Oxygen Sensing

本项目福尔斯属于EPSRC“化学生物学与生物化学”研究领域，旨在研究半胱胺双加氧酶（ADO）的氧传感作用。氧稳态对人体内的各种细胞过程至关重要，因此了解人体对氧水平变化的生化反应非常重要。ADO最近被鉴定为参与蛋白质降解的氧敏感酶。1 ADO氧化多种蛋白质的N-末端半胱氨酸，包括G蛋白信号转导调节剂RGS 4和RGS 5。一旦被氧化，N-末端半胱氨酸就被N-乙酰基转移酶识别，其将精氨酸残基添加到蛋白质的N-末端。精氨酸在这个位置是不稳定的，因此促进了蛋白质的降解。该项目旨在进一步研究ADO的这种活性，以便更好地了解其在人类氧感测中的作用。该项目的第一个目标是确定ADO的底物，以了解其在氧依赖性蛋白质稳定性中的作用范围。这将涉及在体外筛选各种含有N-末端半胱氨酸的多肽，并分析最具活性的底物的氧敏感性和其他动力学性质。将生成一个共有序列，该序列应有助于鉴定人类蛋白质组中其他可能的ADO底物。通过与纽卡斯尔的Kawamura小组合作进行的mRNA展示选择的适应，可以使用鉴定为ADO底物的序列进一步适应该共有序列。所产生的动力学数据将是有用的调查是否氧敏感性的ADO催化是底物依赖性，因此相对重要性的ADO底物在hypoxia.The第二个目标是开发肽和小分子抑制剂的ADO，这可能是用来作为正交化学探针，以调查细胞中的酶的生化作用。肽通常具有更有利的结合特性，而小分子探针往往具有更好的理化特性，并且能够更容易地穿透细胞。肽抑制剂将在牛津大学使用的共识底物序列，以及通过mRNA展示选择2与Kawamura组合作开发。肽抑制剂也可用于与ADO的共结晶研究。悉尼大学的白色。最后，还建议与GSK合作进行片段筛选，以寻找基于小分子代谢物的ADO抑制剂。第三个目标是开发一种高通量检测方法，能够使用最近开发的半胱氨酸亚磺酸探针3检测双氧半胱氨酸。该测定可用于大规模筛选，以选择ADO的底物和抑制剂，对于化学探针开发的片段筛选部分将是非常宝贵的，并将加速实现其他目标的工作。该项目的第四个目标是进行ADO探测中间物种的机理研究，确定限速步骤和确定氧反应动力学。巯基双加氧酶半胱氨酸氧化的机制还没有很好地理解，ADO催化的信息可以提供一个更好地理解其他巯基双加氧酶机制的途径。

This project falls within the "Chemical Biology and Biological Chemistry" EPSRC research area and aims to investigate the oxygen sensing role of cysteamine dioxygenase (ADO). Oxygen homeostasis is essential to various cellular processes within the human body and it is therefore important to understand the body's biochemical response to changes in oxygen levels. ADO has recently been identified as an oxygen-sensitive enzyme involved in protein degradation.1 ADO oxidises the N-terminal cysteine of a variety of proteins, including regulators of G-protein signalling RGS4 and RGS5. Once oxidised the N-terminal cysteine is recognised by arginyl transferases, which add an arginine residue to the N-terminus of the protein. Arginine is destabilising at this position, so degradation of the protein is promoted. This project aims to further investigate this activity of ADO in order to better understand its role in oxygen sensing in humans. The first objective of this project is to identify substrates of ADO in order to understand the scope of its role in oxygen-dependent protein stability. This will involve screening of a variety of polypeptides containing N-terminal cysteines in vitro and analysis of the most active substrates' oxygen sensitivity and other kinetic properties. A consensus sequence will be generated that should aid identification of other possible ADO substrates in the human proteome. This consensus sequence may be further adapted using sequences identified as ADO substrates through an adaptation of an mRNA display selection carried out in collaboration with the Kawamura group in Newcastle. The kinetic data generated will be useful for investigating whether the oxygen sensitivity of ADO catalysis is substrate dependent and therefore the relative importance of the ADO substrates in hypoxia.The second objective is to develop peptide and small molecule inhibitors of ADO, which could be used as orthogonal chemical probes to investigate the biochemical role of the enzyme in cells. Peptides often have more favourable binding properties, while small molecule probes tend to have better physiochemical properties and are able to penetrate cells more easily. Peptide inhibitors will be developed in Oxford using the consensus substrate sequence as well as through an mRNA display selection2 in collaboration with the Kawamura group. Peptide inhibitors may also be used for cocrystallisation studies with ADO by Dr. M. White at the University of Sydney. Finally, it is also proposed to carry out a fragment screen, working with GSK, to find a small molecule metabolite-based inhibitor of ADO. The third objective will be to develop a high-throughput assay capable of detecting the dioxygenated cysteine using recently developed cysteine sulfinic acid probes3. This assay could be used in large-scale screens to select for substrates and inhibitors of ADO, will be invaluable for the fragment screen portion of chemical probe development and will accelerate work towards other objectives. The fourth objective for this project is to carry out mechanistic studies on ADO probing for intermediate species, identifying the rate limiting step and determining the kinetics of oxygen reactivity. The mechanism of cysteine oxidation by thiol dioxygenases is not well understood; information on ADO catalysis may provide a route to better understanding of other thiol dioxygenase mechanisms.