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

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.

The long term of this research programme is to generate macromolecular reagents which can recognise phosphorylate histidine. This research project therefore has the potential to have a transformative effect on research in cell and molecular biology. Reagents which can bind to and identify phosphorylated histidine selectively have wide potential applications in research on cell signalling and the biochemistry of multiple disease states. The beneficiaries of this research may be in the private sector both nationally and internationally in addition to those in the academic research environment. Any current uncertainty about the relative importance of histidine phosphorylation in cellular systems will be resolved by the results of this research programme - using the selected reagents it will be possible to profile histidine phosphorylation events in whole cell extracts, this is currently impossible. Histidine phosphorylation has already been identified in multiple disease states, it is unknown how many more it may play a role in. In addition to these questions, the macromolecular reagents will have immediate utility for those researchers studying bacterial two-component signal regulation and how this can control antibiotic resistance. There is also potential for the chemical reagents generated during this programme to be used as lead compounds in antimicrobial compound development. All of these applications could have long term benefits for the nation's health, and potentially their wealth should commercial exploitation occur within the United Kingdom. The impact upon academic and industrial research programmes will be immediate. If suitable reagents exist, the research communities will use adopt their use as soon as they become readily available. Commercial exploitation may be feasible at this point via licensing of these reagents. The long-term impact of the reagents on the nation's health and wealth will be in the longer term, for example the development of potential pharmaceutical agents using these reagents in research or as lead compounds will take 5-15 years to have a commercial impact. The impact upon the career prospects of the post-doctoral worker employed in the project will be significant. The project would ideally suit a recent graduate in synthetic organic chemistry and will provide them with an ideal opportunity to widen their skill set in interdisciplinary science allowing them to gain expertise in protein chemistry and biophysical assays.