About the Project
Phagosomes are intracellular vesicles that are formed by phagocytosing particles such as bacteria or apoptotic cells by specialised immune cells. Consequently, they are an essential component of the innate immunity defence mechanism. Phagosomes mature by fusing with other organelles such as early and late endosomes and finally lysosomes where the particles are degraded. This process is essential for innate and adaptive immunity, as antigens from the phagocytosed particle are presented on the surface of antigen presenting cells. In recent years, the phagosome has been recognised as a hub for innate immune signalling [1] in which protein phosphorylation plays a number of essential roles [2].
To date, phosphorylation-mediated signalling in vertebrates has focussed on characterisation of serine, threonine and tyrosine residues, yet growing evidence has implicated roles for non-canonical phosphorylation of e.g. histidine, in critical cellular processes. Efforts at characterising phosphohistidine (pHis) are hampered by the fact that this modification is extremely prone to hydrolysis. Unlike the relatively stable canonical phosphoesters (Ser, Thr, Tyr), the phosphoramidate bond of pHis is both acid and heat labile. Traditional biochemical, cellular and proteomics techniques for analysing protein phosphorylation (e.g. boiling, low pH phosphopeptide isolation) are therefore unsuitable for pHis characterisation [3]. Very recently, pan-pHis antibodies have been reported. Excitingly, these revealed a concentration of pHis around the outer membrane of phagosomes in macrophages [4]. Moreover, one of the few known His kinases, NME2, is associated with the outer membrane, suggesting that amongst other functions, histidine phosphorylation may play a role in phagocytosis, phagosome maturation and/or innate immune signalling from the phagosome. We have recently developed novel methodology (termed UPAX) that permits enrichment of peptides carrying these ‘unstable’ non-canonical phosphorylation sites, for subsequent characterisation using mass spectrometry [5]. Consequently, we are in a unique position to exploit our methodology and define and explore the role of histidine phosphorylation-mediated signalling in phagosome biology upon infection.
[1] Pauwels et al., Trends in Immunology, 2017
[2] Trost et al., Immunity, 2009
[3] Gonzalez-Sanchez et al.,Biochem Soc Trans. 2013
[4] Fuhs et al., Cell, 2015
[5] Hardman et al., bioRxiv 202820
This project is a collaboration between the active research groups of Prof. C Eyers (University of Liverpool) who has extensive experience in phosphorylation-mediated cell signalling and has developed the mass spectrometry-based strategy for identification of non-canonical sites of phosphorylation (including, but not limited to, pHis), and Prof. M Trost (Newcastle University) whose expertise lies in understanding macrophage innate immune responses and phagosome biology using a combination of biochemical, cell biology and proteomics approaches. By combining their unique areas of expertise and interests, we are in an ideal position to better understand the innate immune response and regulation of phagosomes by both canonical and non-canonical phosphorylation events, and will provide training in numerous key and transferable skills.
Our main objectives are:
1. Quantify the regulated canonical and non-canonical phosphorylation networks during phagocytosis and on phagosomes in macrophages using our novel UPAX methodology in combination with LC/MS/MS
2. Characterise the role of NME2 in phagosome biology by generating a NME2 knock-out macrophage cell line by CRISPR/Cas9 and compare canonical and non-canonical phosphoproteomes.
3. Interrogate the roles of key pHis-sites in phagosome maturation and innate immune signalling by pHis site mutation and functional characterisation
Key outcomes for the student include:
1. A portfolio of training in vulnerable skill sets, including i) in-depth knowledge of state-of-the-art instrumentation for proteomics, ii) computational/bioinformatics analysis of MS data to elucidate biological signalling mechanisms; iii) cell and chemical biology strategies to interrogate macrophage and phagosome biology, including CRISPR/Cas9 knock-out cell line generation
2. Immersion in state-of-the-art research programme aimed at elucidating roles of non-canonical phosphorylation in human biology
https://www.liverpool.ac.uk/integrative-biology/staff/claire-eyers/
https://www.liverpool.ac.uk/pfg/
http://www.ncl.ac.uk/camb/staff/profile/matthiastrost.html#background
Funding Notes
This studentship is part of the MRC Discovery Medicine North (DiMeN) partnership and is funded for 3.5 years. Including the following financial support:
Tax-free maintenance grant at the national UK Research Council rate
Full payment of tuition fees at the standard UK/EU rate
Research training support grant (RTSG)
Travel allowance for attendance at UK and international meetings
Opportunity to apply for Flexible Funds for further training and development
Please carefully read eligibility requirements and how to apply on our website, then use the link on this page to submit an application: https://goo.gl/jvPe1N