Download Structure/Function studies on urokinase plasminogen activator

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
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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.
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