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（“聚糖”）复合物来相互交流的能力。实现这一目标的一种方法是使用化学技巧来改变聚合物（如蛋白质）的生物特性，通过添加和去除小的带电化学物质作为调节手段。这些事件，更准确地称为“翻译后修饰”，充当改变信息流的开关，并决定引起的不同生物结果的类型，如细胞运动，生长，存活或死亡。我们的提案旨在开发工具来评估向聚糖或蛋白质添加特定化学基团（称为硫酸盐）的情况。我们已经知道硫酸化是对聚糖聚合物（例如糖胺聚糖）和酪氨酸氨基酸（蛋白质的组分）的修饰，但我们目前无法以所需的精度化学控制硫酸化。羟基（-OH）硫酸化由称为磺基转移酶（ST）的酶家族催化，并且是细胞生物学许多方面的核心但知之甚少的调节剂。事实上，我们已经知道聚糖硫酸化对于细胞-细胞和宿主-微生物相互作用是重要的，支持细胞外和细胞内细胞信号传导途径中的限速事件，包括对细胞老化、细菌感染和神经变性至关重要的过程。以细胞内酪氨酸硫酸化为例的蛋白质硫酸化也导致蛋白质-蛋白质相互作用的研究不足的变化，例如伴随病毒感染和免疫功能的变化。酶促硫酸化被认为发生在高尔基体的内腔中，其中预定用于分泌（即在细胞外起作用）的蛋白质在不同区域用不同数量的硫酸基团修饰。由于两者都发生在酪氨酸上，酪氨酸磷酸化和酪氨酸硫酸化之间的竞争潜力代表了硫酸化在蛋白质-蛋白质相互作用水平上对细胞信号传导的潜在影响的一个例子。然而，硫酸化的分析是没有重点的，它吸引了很少的战略资金，既不是特定的聚糖或蛋白质修饰，使努力研究其全球意义的挑战。我们认为，由于它支撑了这么多的基础生物学，硫酸化研究迫切需要一个协调一致的研究战略，以开发新的化学探针，可用于扰动和分析硫酸化。为了实现这一目标，需要新的高通量测定来测量蛋白质和聚糖硫酸化，以支持可能对硫酸化领域产生重大影响的化学生物学筛选。事实上，以技术为基础的方法来分析不同的化学基团，磷酸盐，已经导致了我们对细胞如何交流的理解的革命，并且对于在结构生物学，细胞信号传导和通信以及药物设计领域工作的生物学家非常重要，对世界各地的生物技术和制药工业产生了显着的连锁反应。我们最近已经表明，小分子与ST的结合可以通过使用差示扫描荧光法的原理的“热稳定性测定”来检测，其中ST在存在和不存在不同化学物质的情况下被加热（导致展开）。化学物质的结合改变了磺基转移酶对解折叠的反应，为发现这些酶的第一个细胞渗透性化学抑制剂的新测定奠定了基础。我们的建议将建立在这些测定，允许硫酸化进行研究，在真实的时间使用一个更高的通量格式，形成新的屏幕使用一个大面板的优化化学品的基础。总之，这些新的技术平台将导致发现用于研究生物硫酸化的新探针。

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.