Immune responses and leukocyte function - Role of tyrosine and histidine phosphorylation

Leukocyte Inflammation contributes to chronic inflammatory diseases and is difficult to treat with no drugs that specifically inhibit cell function. For example, leukocytes that have migrated into the tissues are more activated and live longer and the mechanisms regulating this change in their phenotype are complex. However, identifying key activation and survival signals and inhibiting their actions could give insights into potential therapeutic approaches.

Reversible protein phosphorylation regulates most cellular processes with mis-regulation linked to many diseases. Protein phosphorylation of specific tyrosine residues has been shown to direct inflammasome activation. We have recently observed that dephosphorylation of several tyrosine phosphoproteins, including ASC and other members of the inflammasome multi-protein complex, is essential for NLRP3/AIM2 inflammasome activation and IL-1β/IL-18 release. This suggests that tyrosine dephosphorylation is permissive for inflammasome activation and IL-1β release. Improved understanding of the mechanisms that underpin inflammasome activation would shed more light on the effects of manipulating inflammatory pathways and provide a basis to identify potential therapeutic interventions against autoinflammatory diseases and many other diseases associated with detrimental inflammation (e.g. cancer, diabetes, etc.). However, not all the pathways and processes involving phosphotyrosine have been delineated and certainly the role of tyrosine dephosphorylation during inflammasome activation is not well understood and requires further investigation. Increasing evidence suggests phosphohistidine, an acid-labile, high-energy post-translational protein modification may play a significant a role in regulation of key cellular processes in mammalian cells and may even be much more abundant than phosphotyrosine.  Our analysis by ³¹P NMR shows high levels of phosphohistidine exist in human cells. Despite its discovery decades prior to phosphotyrosine, the ready availability of antibodies only towards O-phosphorylated species has delayed research on phosphohistidine. Accordingly, the role of histidine phosphorylation during inflammation and inflammasome activation is not characterised and requires investigation. Using our new selective anti-phosphohistidine antibodies, we wish to test the hypothesis that ligand-induced signaling involving phosphohistidine regulates the immune response and leukocyte function.

The aim of the project is to elucidate the role of phosphohistidine innate immune cell function. Using peripheral blood mononuclear cells (PBMCs), we will address the following:

1)     Characterise histidine phosphorylation profile with and without various cell stimuli.

2)     Establish the identity of the histidine phosphorylated proteins by mass spec analysis

3)     Determine the histidine phosphorylation status of the identified proteins in PBMCs from smokers/COPD/bronchiectasis and CF patients. 

The study will seek to contribute to the establishment of new tools to study cells of the innate immunity system, and identify new disease-associated mechanisms and pathways that can be further explored to develop new immunomodulatory treatments for acute and chronic inflammatory diseases.

Experiments will utilize cell lines, freshly isolated leukocytes from human peripheral blood (PBMCs). Phosphohistidine antibodies will be used to characterize the phosphohistidine profile of innate immune cells in response to various stimuli by western blots and flow cytometry. Target proteins will be identified by mass spectrometry.

Entry Requirements:

Candidates must have a first or upper second class honors degree or significant research experience. The interdisciplinary nature of this programme means that we welcome applications from students with backgrounds in any biological, chemical, and/or physical science who are interested in using their skills in addressing biological questions.

How to apply:

Please complete a University Postgraduate Research Application form available here: https://www.sheffield.ac.uk/postgraduate/phd/apply/applying