New tools and technology to evaluate biological sulphation

点击翻译按钮获取中文摘要

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.