Investigating the Role of Cysteamine Dioxygenase in Oxygen Sensing

本项目属于《化学生物学与生物化学》EPSRC研究领域，旨在研究半胱胺双加氧酶(ADO)的氧气传感作用。氧稳态对人体内的各种细胞过程是必不可少的，因此了解人体对氧水平变化的生化反应是很重要的。ADO最近被确定为一种与蛋白质降解有关的氧敏感酶1 ADO氧化各种蛋白质的N末端半胱氨酸，包括G蛋白信号转导RGS4和RGS5的调节器。一旦被氧化，N端的半胱氨酸被精氨基转移酶识别，精氨基转移酶在蛋白质的N端添加精氨酸残基。精氨酸在这个位置会破坏稳定，因此促进了蛋白质的降解。本项目旨在进一步研究ADO的这种活性，以便更好地了解它在人体氧气感应中的作用。该项目的第一个目标是确定ADO的底物，以了解其在氧依赖蛋白质稳定性中的作用范围。这将包括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。将产生一个共识序列，这将有助于识别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展示选择的改编，可以使用被鉴定为ADO底物的序列进一步适配该共识序列。生成的动力学数据将有助于研究ADO催化的氧敏感性是否依赖于底物，从而研究ADO底物在缺氧中的相对重要性。第二个目标是开发ADO的多肽和小分子抑制剂，作为正交化学探针来研究该酶在细胞中的生化作用。多肽往往具有更有利的结合特性，而小分子探针往往具有更好的物理化学特性，能够更容易地穿透细胞。牛津大学将使用一致的底物序列以及通过与Kawamura小组合作的mRNA展示选择2来开发多肽抑制剂。悉尼大学的M.White博士还可以将多肽抑制剂用于ADO的共结晶研究。最后，还建议进行片段筛选，与GSK合作，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的分析方法，能够使用最近开发的半胱氨酸亚磺酸探针3来检测双氧半胱氨酸。该分析方法可用于大规模筛选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无价的，并将加速实现其他目标的工作。该项目的第四个目标是对中间物种进行ADO探测的机理研究，确定限速步骤并确定氧反应的动力学。硫醇双加氧酶氧化半胱氨酸的机制还不是很清楚，有关ADO催化的信息可能会为更好地理解其他硫醇双加氧酶的机制提供途径。

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.