Synthetic probes of histidine phosphorylation: new reagents for systems biology and proteomics

DNA测序显示，人类基因组只包含3万种不同的蛋白质。如此数量相对较少的蛋白质能够结合形成一个人，是因为有大量的方法可以对它们进行修改，以控制它们的功能和行为。发生这种情况的主要方法是蛋白质磷酸化，在过去的50年里，大量的研究（现在每年有几千篇论文发表）已经证明了丝氨酸、苏氨酸和酪氨酸的修饰如何控制细胞和整个生物体的生理机能。这些控制和信号通路中的错误与包括癌症在内的许多人类疾病有关。总共有20种天然氨基酸，其他许多氨基酸也可以被磷酸化。我们感兴趣的是探索磷酸化组氨酸的生物化学，这是迄今为止很少研究的。n -连接的磷酸组氨酸不如o -连接的磷酸组氨酸稳定，因此不可能获得能够识别这种修饰的抗体。反过来，这意味着不可能评估这种修饰在细胞中有多普遍，以及它如何参与调节复杂的生化过程。在这个项目中，我们将产生第一类这样的试剂，从而使这一领域的研究迈出了一步。我们将合成一系列稳定的磷酸组氨酸类似物，并将其纳入短肽中。然后，我们将通过测量它们与已知识别和修饰含磷组氨酸蛋白质的蛋白质的结合程度来评估类似物。在此之后，我们将筛选肽适配体库的适配体特异性识别类似物，从而磷酸组氨酸。然后，这种工具将应用于其他生物系统的测定。

DNA sequencing has revealed that the human genome comprises only 30000 distinct proteins. That such a relatively small number of proteins can combine to form a human being is due to the vast number of ways in which they can be modified to control their function and behaviour. The dominant method by which this occurs is protein phosphorylation, a vast body of research in the last fifty years (now comprising several thousand publications/year) has demonstrated how modification of the amino acids serine, threonine and tyrosine control the physiology of the cell and whole organisms. Errors in these control and signalling pathways are implicated in many human diseases including cancer.There are twenty natural amino acids in total and many others can also be phosphorylated. We are interested in exploring the biochemistry of phosphorylated histidine which has been little studied to date. N-linked Phosphohistidine is less stable than its O-linked counterparts and it has not therefore been possible to acquire antibodies capable of identifying this modification. This, in turn, means that it has been impossible to assess how prevalent the modification is in cells and how it is involved in regulating complex biochemical processes. In this project we will generate the first class of such reagents thereby enabling a step change in research in this area.We will synthesize a range of stable analogues of phosphohistidine and incorporate these into short peptides. We will then evaluate the analogues by measuring how well they bind to a protein known to recognise and modify phosphohistidine-containing proteins. Following this we will screen libraries of peptide aptamers for aptamers which specifically recognise the analogue and thereby phosphohistidine. Such a tool will then be applied to the assay of other biological systems.