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Download Structure/Function studies on urokinase plasminogen activator
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School of Medicine PhD Project Application 2017 Structure/Function studies on urokinase plasminogen activator receptor (uPAR) coding region variants associated with asthma Section 1 – Project Details: Structure/Function studies on urokinase plasminogen activator receptor (uPAR) coding region variants associated with asthma Maximum 800 words, using the following headings Rationale: Asthma is a common chronic disease with an estimated 300 million affected individuals. Worldwide, deaths from asthma have reached more than 180,000 annually. Asthma is considered a complex genetic disorder (1). We have previously identified that genetic polymorphisms spanning the urokinase plasminogen receptor (uPAR) gene are associated with the risk of developing asthma. uPAR is a serine protease receptor that interacts with several ligands and has proteolytic (via plasmin activation) functions including ECM degradation as well as non-proteolytic functions, e.g. cell migration and proliferation. In particular, a polymorphism that results in a Lys220Arg substitution in Domain 3 of uPAR was associated with asthma risk, lung function decline and airway structural changes in multiple asthma/respiratory cohorts (2, 3, 4). Similarly, through sequencing we have identified novel coding region variants only found in the asthma population. The molecular mechanisms underlying these observations remains to be resolved and may be important when targeting this receptor for therapeutic benefit. Figure 1: The Lys220Arg mutation associated with risk of developing asthma is located in Domain 3 of uPAR and is surface exposed and close to the uPA binding site. Aims and methodology: This studentship has three main questions: 1. What is the effect of the Lys220Arg substitution on uPAR expression and function? We will be use both recombinant cell lines expressing the uPAR-Lys220 and uPAR-Arg220 receptors and genotyped primary human bronchial epithelial cells from asthma patients to examine uPAR expression and activity. Key experiments will determine the effect of multiple ligands; e.g. uPA, FXII, VN on uPAR signalling (Erk1/2, Akt and p38MAPK) and plasmin formation in specific allele carriers. Cell proliferation, migration and adhesion will be determined to define functional consequences (5). To provide greater depth to the study we will also use RNA-seq to identify a transcriptomic signature downstream of variant receptors following ligand activation. We will also investigate novel coding region variants identified in our sequencing analyses. 2. What is the effect of the Lys220Arg substitution on uPAR structure? To complement functional analyses we will determine the crystal structure of uPAR1 School of Medicine PhD Project Application 2017 Lys220 and uPAR-Arg220 proteins to 1.25-Å resolution. This will be achieved by recombinant expression and crystallography essentially as described (6). We will also determine the structure for novel coding region variants identified in our sequencing analyses that show functional effects in 1. 3. Is the presence/absence of the Lys220Arg substitution related to specific clinical features of asthma? To complement the functional genetics and protein structure analyses we will further define the clinical implications for asthma patients that carry these uPAR protein variants. This will use the Genetics of Asthma Severity & Phenotypes (GASP) cohort which is an ongoing collection of asthma patients with extensive clinical, immunological and genetic data (currently n=5,000). Key analyses will include stratification of clinical e.g. lung function, comorbidities, severity and immunological e.g. IgE levels, blood eosinophil levels by genotype. Benefits and suitability as a PhD project: This project is ideally placed for a PhD student as it provides unprecedented training in functional genomics (Sayers), protein structural chemistry (Emsley) and population genetics (Sayers). Split between School of Medicine and Pharmacy the student will experience both research environments with additional benefits. Importantly, the studentship builds on published findings and it is anticipated will provide a high impact publication. The project is designed to have several streams and many of the tools are already in place, e.g. expression vectors i.e. we have considered risk mitigation. Key References: 1. 2. 3. 4. 5. 6. Portelli MA, et. al. Clin Exp Allergy. 2015 Jan;45(1):21-31. Ierodiakonou D, et. al. Eur Respir J. 2016 May;47(5):1568-71. Stewart CE, et. al. BMC Med Genet. 2009 Oct 31;10:112. Barton SJ, et. al. J Allergy Clin Immunol. 2009 Jun;123(6):1391-400.e17. Stewart CE, et. al. Thorax. 2012 Jun;67(6):477-87. McEwan PA, et. al. Blood. 2011 Nov 10;118(19):5292-301. Section 2 – Training Provision: Maximum of 250 words. Please detail the training provision that will be made available to the student. The project will be mainly based in the Division of Respiratory Medicine which includes 10 senior academic staff, research fellows, research technicians, nurses and support. The Division has research programmes focussed on the genetics, pharmacogenetics, molecular pathophysiology, translational research (including biomarkers and imaging) and clinical trials of multiple lung disease. The applicants’ group is internationally recognized for research in this area and the student will benefit from exposure to visiting researchers. Specifically, comprehensive training will be provided in; gene characterisation approaches including, bioinformatics, next generation sequencing, primary cell culture: in vitro models e.g. airway epithelium, over expression and protein engineering approaches using lentivirus vectors, i.e. a wide range of core techniques including cloning, site-directed mutagenesis, RT-PCR, transfection, manufacture of stable cell lines. Similarly; in vitro cell biology studies, signalling assays, protein analyses: Western, immunofluorescence, flow cytometry and cell biology: proliferation, migration, apoptosis assays will be learnt. Specialist external training is provided by recognised leaders, e.g. European Bioinformatics Institute and there is also scope to spend time in collaborating laboratories in the UK and overseas. In the School of Pharmacy the Emsley group the student will be trained in insect cell protein expression and purification. Characterisation of the stability and ligand binding of recombinant proteins using a variety of techniques including thermodynamics (ITC, DSC) and surface plasmon 2 School of Medicine PhD Project Application 2017 resonance measurements (SPR). Molecular graphics programs to analyse protein structure and molecular modelling of mutants and variants to examine effects on protein structure. Protein crystallisation and protein crystal structure determination by x-ray crystallography. 3