New tools and technology to evaluate biological sulphation

生物体的生存取决于不同类型的细胞通过在正确的时间在正确的地点组装正确的蛋白质和碳水化合物(‘多糖’)复合体来相互沟通的能力。实现这一点的一种方法是使用化学的技巧来改变聚合物的生物属性，例如蛋白质，通过添加和移除小电荷化学物质作为一种调节手段。这些事件，更准确地说，被称为“翻译后修饰”，充当改变信息流的开关，并决定所引发的不同生物结果的类型，如细胞运动、生长、存活或死亡。我们的建议旨在开发工具来评估在多糖或蛋白质中添加一种特定的化学基团，称为硫酸盐。我们已经知道，硫化是对葡聚糖聚合物(如糖胺多聚糖)和酪氨酸氨基酸(蛋白质的组成部分)的修饰，但我们目前无法以所需的精度对硫化进行化学控制。羟基(-OH)硫化是由一类称为硫基转移酶(STS)的酶催化的，是细胞生物学许多方面的核心调节因子，但知之甚少。事实上，我们已经知道，糖硫化对于细胞-细胞和宿主-微生物的相互作用是重要的，支持细胞外和细胞内信号通路中的限速事件，包括对细胞衰老、细菌感染和神经退化至关重要的过程。以细胞内酪氨酸硫化为例的蛋白质硫化也导致蛋白质-蛋白质相互作用的变化研究较少，例如伴随病毒感染和免疫功能的变化。酶促硫化作用被认为发生在高尔基体的管腔中，在那里，预定要分泌的蛋白质(即细胞外的功能)在不同的区域被不同数量的硫酸盐基团装饰。由于两者都发生在酪氨酸上，酪氨酸磷酸化和酪氨酸硫化之间的潜在竞争代表了硫化在蛋白质-蛋白质相互作用水平上对细胞信号的潜在影响的一个例子。然而，对硫酸盐的分析没有重点，它吸引的战略资金很少，而且既不是针对多糖修饰，也不是针对蛋白质修饰，这使得努力研究其全球意义具有挑战性。我们认为，由于硫酸盐是基础生物学的基础，硫酸盐研究迫切需要协调一致的研究战略，以开发可用于干扰和分析硫酸盐的新化学探针。为了实现这一点，需要新的高通量分析来测量蛋白质和葡聚糖的硫化，以支持可能对硫酸盐领域有相当大影响的化学生物学筛选。事实上，对另一种化学基团磷酸盐的技术分析方法导致了我们对细胞通信方式的理解的一场革命，对从事结构生物学、细胞信号和通信以及药物设计领域的生物学家来说很重要，对世界各地的生物技术和制药行业产生了显著的连锁反应。我们最近证明，小分子与STS的结合可以通过使用差示扫描荧光原理的“热稳定性分析”来检测，在不同化学物质的存在和不同的情况下，ST被加热(导致展开)。化学物质的结合改变了硫酸盐转移酶对去折叠的反应，形成了一种发现这些酶的第一种细胞通透性化学抑制剂的新方法的基础。我们的建议将建立在这些分析的基础上，允许使用更高吞吐量的格式实时研究硫酸盐，形成使用大量优化化学物质的新屏幕的基础。总而言之，这些新的技术平台将导致发现研究生物硫酸盐的新探测器。

The survival of organisms depends upon the ability of different cell types to communicate with each other by assembling the correct complexes of proteins and carbohydrates ('glycans') at the correct time in the correct place. One way this is achieved is to use the tricks of chemistry to change the biological properties of polymers, such as proteins, by adding and removing small charged chemicals as a means of regulation. These events, more accurately called 'post-translational modifications', act as switches to change information flow and dictate the types of different biological outcomes elicited, such as cell movement, growth, survival or death. Our proposal aims to develop tools to evaluate the addition of a specific chemical group, called sulphate, to glycans or proteins. We already know that sulphation is a modification on glycan polymers (e.g. glycosaminoglycans) and tyrosine amino acids (components of proteins), but we are currently unable to control sulphation chemically with the desired precision. Hydroxyl group (-OH) sulphation is catalysed by a family of enzymes called sulphotransferases (STs), and is a central, yet poorly understood, regulator of many aspects of cell biology. Indeed, we already know that glycan sulphation is important for cell-cell and host-microbe interactions, supporting rate-limiting events in extracellular and intracellular cell signalling pathways, including processes critical for cellular ageing, bacterial infection and neurodegeneration. Protein sulphation, exemplified by intracellular tyrosine sulphation, also leads to poorly-studied changes in protein-protein interactions such as those that accompany viral infection and immune function. Enzymatic sulphation is thought to occur in the lumen of the Golgi apparatus, where proteins destined for secretion (i.e. function outside cells) are decorated with different numbers of sulphate groups in different regions. Since both occur on tyrosine, the potential for competition between tyrosine phosphorylation and tyrosine sulphation represents an example of the potential impact of sulphation on cellular signalling at the level of protein-protein interactions. However, the analysis of sulphation is unfocused, it attracts little strategic funding, and is neither specific for glycan nor protein modifications, making efforts to study its global significance challenging. We are of the opinion that since it underpins so much of basic biology, sulphation research urgently requires a concerted research strategy to develop new chemical probes that can be used to perturb and analyse sulphation. To accomplish this, new high-throughput assays to measure protein and glycan sulphation are required to support chemical biology screens that might have considerable impact on the sulphation field. Indeed, technology-based approaches for the analysis of a different chemical group, phosphate, has led to a revolution in our understanding of how cells communicate, and has been important for biologists working in the areas of structural biology, cell signalling and communication and drug design, with remarkable knock-on effects on biotechnology and pharmaceutical industries across the world. We have recently shown that the binding of small molecules to STs can be detected by a 'thermal stability assay' using the principles of differential scanning fluorimetry, where the ST is heated up (leading to unfolding) in the presence and absence of different chemicals. Binding of chemicals changes the response of sulphotransferase to unfolding, forming the basis for a new assay to discover the first cell permeable chemical inhibitors of these enzymes. Our proposal will build upon these assays to permit sulphation to be studied in real time using an a higher-throughput format, forming the basis for new screens using a large panel of optimised chemicals. Together, these new technological platforms will lead to the discovery of new probes for studying biological sulphation.