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

Lead Research Organisation: University of Leeds
Department Name: Sch of Chemistry

Abstract

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.

Planned Impact

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.

Publications

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Horner KA (2015) Strain-promoted reaction of 1,2,4-triazines with bicyclononynes. in Chemistry (Weinheim an der Bergstrasse, Germany)

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McAllister TE (2013) Prospects for stable analogues of phosphohistidine. in Biochemical Society transactions

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McAllister TE (2012) Triazole phosphohistidine analogues compatible with the Fmoc-strategy. in Organic & biomolecular chemistry

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McAllister TE (2014) Evaluation of the interaction between phosphohistidine analogues and phosphotyrosine binding domains. in Chembiochem : a European journal of chemical biology

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McAllister TE (2011) Fmoc-chemistry of a stable phosphohistidine analogue. in Chemical communications (Cambridge, England)

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Williamson DJ (2012) Efficient N-terminal labeling of proteins by use of sortase. in Angewandte Chemie (International ed. in English)

 
Description Phosphorylation of proteins is a key mechanism for signalling in organisms. We investigated the role of one such modification, histidine phosphorylation, by generating chemical reagents that mimicked the structure of the modified amino acid.

We optimised chemical methods to generate building blocks for use in peptide synthesis and evaluated them using multiple biochemical systems. We also used the analogues in two different immunochemical screening systems.
Exploitation Route Commercialisation of building blocks generated during the project.
Use of building blocks for immunochemical production.
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

 
Description The chemical methods used in this project have now been applied by several different groups of cell biologists. In particular, our methodology was critical to the development of the first monoclonal antibodies with broad spectrum recognition of phosphohistidine by Hunter and co-workers (Cell, 2015). These tools are now being used in a much wider range of cell biological studies with potential applications to studies of disease processes.
First Year Of Impact 2015
Sector Pharmaceuticals and Medical Biotechnology
 
Title Stable phosphohistidine analogues 
Description Stable analogues of phosphohistidine contained in peptides suitable for monoclonal antibody production. Specific substrate peptides for antibody selection. 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact Analogues were used for monoclonal antibody selection by Eyers group (Manchester). Work was superceded by concurrent work by Muir group (Princeton). 
 
Description Antibody selection (Manchester) 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided reagents for antibody selection and screening
Collaborator Contribution Provided funds expertise for antibody selection and MS analysis of panning using antibodies
Impact Monoclonal antibodies produced but did not have selectivity for natural target.
Start Year 2011
 
Description Binding protein evolution 
Organisation University of Leicester
Department Department of Cardiovascular Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of materials for directed evolution of protein-binding domains to phosphohistidine
Collaborator Contribution Carrying out experimentation using resources
Impact No outputs yet
Start Year 2015
 
Description Salters festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact Increased interest in chemistry for school students

Increased application to science courses
Year(s) Of Engagement Activity 2012,2013
 
Description Sixth form open labs 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Increased interest and understanding of research chemistry

Increased applications to STEM courses
Year(s) Of Engagement Activity 2008,2010,2012