High-Throughput Luminescence Assay for Sulfotransferase Activity

生物发育和生长，适应环境和能源的不断变化，繁殖和抵抗疾病。它们通过有机体之间和有机体内细胞之间的通信实现这些事情，通常是通过改变分子的化学组成。一种常见的方法是添加或移除一种带电的小化学物质，如磷酸盐或硫酸盐，它们充当开关，改变交流的信息。这决定了生物学结果，这可能与细胞运动、细胞死亡和成功(或失败)感染一样多种多样。负硫酸盐基团的加成是由称为磺基转移酶的特定酶来完成的，但我们缺乏轻松测量这些酶活性的技术。磺基转移酶催化蛋白质、大糖(多糖)和许多小分子(包括激素)上的硫酸盐转移到醇(-OH)。目前的研究主要集中在这些酶的小的选择性亚群及其一些硫化产物上。动物体内的葡聚糖硫酸盐化在发育、伤口修复中起关键作用，并与衰老、神经退行性疾病和病毒感染有关。蛋白质在氨基酸酪氨酸上的硫酸化导致与免疫功能和病毒感染性相关的蛋白质-蛋白质相互作用的改变。在植物中，酪氨酸硫化会产生激素，并可被病原体用作其感染策略的一部分。细菌硫酸盐转移酶可以比动物硫化更广泛的小分子靶标，其中一些酶有可能取代目前不可持续的个人护理和食品化学生产。要了解硫酸盐作用的全球意义并利用它，面临着一个不可逾越的障碍：没有简单和容易获得的方法来测量硫酸盐转移酶对目标的硫酸盐作用。结果是零敲碎打的研究，很大程度上无法提供基础知识的深度和广度，以及在动植物健康和产品合成中利用硫酸盐所必需的进展。我们的建议旨在开发一种简单、廉价和通用的分析技术，使所有研究人员能够测量任何生物或人造分子通过磺基转移酶添加硫酸盐的情况。这类分析已被证明是理解和利用对生物分子的相关化学修饰的关键，如磷酸化。硫转移酶使用通用的硫酸盐供体PPS，当其硫酸盐基团转移时，它就成为PAP。测定PAP提供了硫酸盐化的直接测量方法。我们将开发基于稀土元素Eu的分子探针，它在与PAP可逆结合时发出红光。对发射光的测量非常灵敏，并提供了磺基转移酶活性的实时读数。这将与磺基转移酶连接硫酸盐的分子无关。通过这种方式，化验将是快速、实时的，并且不需要化学修饰的试剂、放射性、抗体和通常无法获得的昂贵设备。我们建议的分子探针具有巨大的潜力，可以使磺基转移酶活性的测定成为常规工作，并减少进行高通量筛选分析所需的成本和时间。这些探针将为快速、准确地测定磺基转移酶的动力学和机理提供重要的一步。这将使更好地选择和验证磺基转移酶抑制剂(药物先导)和用于工业用途的突变酶。这项新技术将为理解生物硫酸盐化并在健康和疾病以及产品制造方面利用它铺平道路。

Living things develop and grow, adapt to constant changes in their environment and energy sources, reproduce and resist disease. These things they achieve through communication between organisms and between cells within an organism, often by changing the chemical make-up of molecules. A common route is to add or remove a small charged chemical such as phosphate or sulfate, which act as switches, altering the information that is communicated. This dictates the biological outcome, which may be as varied as cell movement, cell death, and successful (or not) infection. The addition of a negative sulfate group is performed by specific enzymes called sulfotransferases, but we lack the technology to easily measure the activity of these enzymes.Sulfotransferases catalyse the transfer of sulfate to alcohols (-OH) on proteins, large sugars (glycans) and many small molecules, including hormones. There has been focussed research on small selective subsets of these enzymes and on some of their sulfated products. Glycan sulfation in animals plays key roles in development, wound repair and is associated with ageing, neurodegenerative diseases and infection by viruses. Sulfation of proteins on the amino acid tyrosine results in changes in protein-protein interactions related to immune function and virus infectivity. In plants, tyrosine sulfation produces hormones, and can be used by pathogens as part of their infection strategy. Bacterial sulfotransferases can sulfate a wider range of small molecule targets than animals, and some of these enzymes have potential as replacements of currently unsustainable chemical production of personal care and food products.Efforts to understand the global significance of sulfation and to harness it have faced an unsurmountable obstacle: there is no simple and accessible means to measure the sulfation of a target by a sulfotransferase. The consequence is piecemeal research, which is largely unable to deliver the depth and breadth of fundamental knowledge and advances necessary to harness sulfation for our benefit in animal and plant health, and in product synthesis.Our proposal aims to develop a simple, cheap and versatile assay technology that will allow all researchers to measure the addition of a sulfate to any biological or indeed man-made molecule by a sulfotransferase. Such assays have proven to be the key to understanding and exploiting related chemical modifications to biological molecules, such as phosphorylation.Sulfotransferases use a universal sulfate donor, PAPS, which upon transfer of its sulfate group becomes PAP. Measuring PAP provides a direct measure of sulfation. We will develop molecular probes based on a rare earth element, Europium, which emit red light upon bind reversibly to PAP. Measurement of the emitted light is very sensitive and provides a real-time readout of the activity of the sulfotransferase. This will be independent of the molecule the sulfotransferase is attaching the sulfate to. In this way the assay will be fast, real-time and eliminate the need for chemically modified reagents, radioactivity, antibodies and expensive equipment not generally available. Our proposed molecular probes have enormous potential to make the measurement of sulfotransferase activity routine and reduce the cost and time required to conduct high-throughput screening assays. These probes will provide a vital step towards the rapid, accurate determination of sulfotransferase kinetics and mechanism. This will enable better selection and validation of sulfotransferase inhibitors (drug leads), and of mutant enzymes for industrial use. The new technology will pave the way for understanding biological sulfation and exploiting it in the contexts of health and disease, and of product manufacture.