Re-Writing HIStory: Identification and characterisation of the histidine phosphoproteome

The addition of phosphate (phosphorylation) is a form of code that regulates protein function within cells. Bacteria and yeast contain proteins that are phosphorylated on the amino acid histidine in response to defined environmental changes. This phosphorylation event is critical to allow these organisms to adapt to environmental changes and survive. We currently know very little about histidine phosphorylation in humans and other mammals because the techniques to study them are still in their infancy. However there is evidence that histidine phosphorylation of mammalian proteins may be involved in transferring information about the extracellular environment to the inside of a cell, promoting a cellular response. Phosphorylation of histidine in humans thus appears to have roles in the immune system and in controlling the rate of cell growth. These functional effects could be particularly important medically, because abnormal phosphorylation is already known to be important in diseases such as cancer and diabetes. Dr Claire Eyers at the University of Manchester is developing strategies to identify sites of histidine phosphorylation on a global scale, using state-of-the-art mass spectrometry instrumentation. Deciphering the histidine phosphorylation code using these large-scale 'proteomic' studies will be crucial for understanding what this protein modification actually does in humans. During the course of these studies histidine phosphate containing proteins will be identified and then characterized in detail to assign specific roles for this modification in regulating protein function. Importantly, the development of this methodology and the generation of data from these studies will open the field significantly for further detailed analysis. Dr Eyers predicts that histidine phosphorylation will change the biological function of many proteins, and if histidine phosphorylation is shown to be correlated with one or more disease states, her group will be in a strong position to develop and validate novel disease targets.

All bacteria, fungi and some plants rely on 'two-component' signalling systems, comprising a receptor histidine kinase and a response regulator, to respond rapidly to changes in their environment. Evidence has been accumulating to suggest that mammalian systems rely on histidine phosphorylation of intracellular proteins to regulate certain signalling events including neuronal and T-cell receptor signalling. However, the extent of this modification in mammals is unclear, as the instability of phosphohistidine at low pH makes it difficult to study using classical biochemical techniques. Precise roles for phosphohistidine in mammalian cell have thus been hard to define. I therefore propose to characterise phosphohistidine containing peptides using an analytical strategy based on peptide chromatography and state-of-the art techniques in tandem mass spectrometry. Protein extracts from stable HeLa cell lines inducibly expressing shRNAs against the two known human histidine phosphatases, PHPT1 and LHPP, will be subjected to proteolysis and beta-elimination (to remove phosphate on serine/threonine residues), and the phosphopeptides enriched using strong-cation exchange chromatography at neutral pH. Peptides will then separated by reverse-phase chromatography, also at neutral pH, and phosphate specific neutral loss induced during collision-induced dissociation (CID) employed to permit phosphopeptide identification using electron transfer dissociation (ETD). Follow-up studies will then ascertain the role of phosphohistidine in mammalian cell signalling, firstly using in vitro assays and cell based studies to characterise a select number of modified proteins in detail, and secondly using a bioinformatics approach to class the phosphohistidine regulated proteins. Assessment of the modified proteins and their known cellular functions will thus enable the role(s) of this modification in mammals to be defined in more detail, opening the field significantly for further study.

The research program outlined here has fundamental objectives to improve the scope of phosphorylation site identification in cell extracts by developing methodology specifically for the characterisation of acid-labile sites of modification. When applied to the analysis of human proteins, these techniques will play a key role in helping elucidate the role of phosphohistidine in mammalian cell signalling. It is anticipated, therefore, that the developments made during this programme and directly related follow-on studies will attract wide interest. If further evidence is gathered that supports the current hypothesis of a role for phosphohistidine in regulating cell signalling and proliferation, with an implication in cancer cell biology, these studies could ultimately lead to the identification of novel biomarkers for tumourigenesis, speculatively leading to new approaches to cancer chemotherapy. This could potentially be beneficial to a variety of pharmaceutical and biotechnology companies, generating new lead targets against which small molecule inhibitors can be designed. In addition to providing targets for disease treatment, the characterization of mammalian histidine kinases (and the elucidation of a histidine kinase catalytic domain) may promote the synthesis (or discovery) of a new array of small molecule inhibitors which will prove invaluable to the signalling community and help further define the physiological roles of this histidine phosphorylation. The identification of novel sites of post-translational modification on human proteins, together with the associated bioinformatics studies will open up a new area of study, both for academia and industry. How this is exploited to its full potential will depend greatly on the elucidated regulatory roles that this modification plays. Improved understanding of the mechanisms of protein regulation aids in understanding how cells respond under different conditions. In the long-term, it is therefore conceivable that this study could provide a missing link to help elucidate the mechanisms of onset or development of certain disease states, thus potentially impacting on human and animal health both here and abroad. As expected, the primary output of this research will include publication in peer-reviewed journals and presentations at conferences. The Media Relations Office within the University of Manchester will also be informed of noteworthy research findings. These can then be imparted to the general public using targeted press releases written by the Media Relations Office in collaboration with myself and the PDRA. Should the outcome of the research stimulate general public interest, formal media training will be arranged to enable both the PDRA and myself to conduct personal interviews both with the media and the public. The PDRA employed on this project will be trained in a uniquely desirable set of skills that can be applied to a variety of biochemistry/protein chemistry, signal transduction, mass spectrometry and bioinformatics-related projects. This will make them eminently employable in a wide range of sectors from public through to private. As dissemination of the output from these studies will be coordinated between the PI and the PDRA, the PDRA will also become proficient in data presentation at conferences and in manuscript writing. The PDRA will also be encouraged to participate in a range of community dissemination activities including (but not necessarily limited to) the 'Researchers in Residence' program as part of the Beacon project and Science week, promoting skills in communication of science to a lay audience. Should the output from these studies generate an avenue for commercialization as a result of reagents generated (stable cell lines, antibodies), the University of Manchester Intellectual property (UMIP) office will be contacted to appropriately manage the project.