School of Biosciences

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Current PhD opportunities:

Graduate Teaching Assistantships, deadline for applications 31 January 2017

Dissecting the structure, dynamics and mechanism of drug efflux pumps associated with antimicrobial resistance

Supervisor: Dr Chris Mulligan

Antimicrobial resistance is a major global health concern. In the EU alone there are ~25,000 deaths per year directly associated with drug resistant bacteria, costing an estimated €1.5 billion in extra healthcare costs and loss of productivity. Unchecked, the global death toll is predicted to exceed 10 million per year, costing over 100 trillion dollars in loss of productivity by 2050. As well as the need for new drug discovery, developing the means to combat resistance itself, by inhibiting known resistance mechanisms, is a promising approach, as it could breathe new life into drugs currently rendered ineffective and prolong the effectiveness of newly developed drugs.

One of the most effective mechanisms bacteria have developed to resist the effects of antimicrobial agents is to pump them out of the cell before they can do any damage. Multidrug resistance transporters are transmembrane molecular machines responsible for the majority of drug efflux in bacteria. Single multidrug resistance transporters can expel a huge range of structurally diverse substrates, thus undermining the efficacy of a wide variety of drugs in one fell swoop. Understanding the mechanisms of drug efflux transporters, and ways of inhibiting them, will increase both the efficacy and longevity of current and future antimicrobial agents.

This project will focus on understanding the molecular mechanisms of secondary active drug efflux pumps; in particular those from the multidrug and toxic compound extrusion (MATE) family of transporters. MATEs confer resistance to several pathogenic bacteria, including Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile. Much is known about the structure of the MATE transporters from crystallographic studies; yet the mechanism by which these transporters pump drugs across the membrane remains elusive. The goal of this project is to probe the structure, function and dynamics of several members of the MATE family to understand how they work. The project will focus on three major questions; how do these proteins recognise such a diverse range of drugs; how do they harness an energy source and couple it to drug pumping, and what conformational changes are required for this process? To do this, the structure and activity of the MATE transporters will be probed using assays in bacterial cells as well as with purified protein in detergent solution and reconstituted into proteoliposomes (purified protein inserted into artificial membranes). A suite of molecular biology methods, as well as biochemical and biophysical tools, including spectrofluorometry, chemical crosslinking, and advanced in vitro transport assays, will be used to elucidate the mechanism of drug efflux.

A thorough mechanistic understanding how these transporters work will give us a better understanding of this important antimicrobial resistance mechanism and potentially provide a basis for future drug design.Antimicrobial resistance is a major global health concern. In the EU alone there are ~25,000 deaths per year directly associated with drug resistant bacteria, costing an estimated €1.5 billion in extra healthcare costs and loss of productivity. Unchecked, the global death toll is predicted to exceed 10 million per year, costing over 100 trillion dollars in loss of productivity by 2050. As well as the need for new drug discovery, developing the means to combat resistance itself, by inhibiting known resistance mechanisms, is a promising approach, as it could breathe new life into drugs currently rendered ineffective and prolong the effectiveness of newly developed drugs.

One of the most effective mechanisms bacteria have developed to resist the effects of antimicrobial agents is to pump them out of the cell before they can do any damage. Multidrug resistance transporters are transmembrane molecular machines responsible for the majority of drug efflux in bacteria. Single multidrug resistance transporters can expel a huge range of structurally diverse substrates, thus undermining the efficacy of a wide variety of drugs in one fell swoop. Understanding the mechanisms of drug efflux transporters, and ways of inhibiting them, will increase both the efficacy and longevity of current and future antimicrobial agents.

This project will focus on understanding the molecular mechanisms of secondary active drug efflux pumps; in particular those from the multidrug and toxic compound extrusion (MATE) family of transporters. MATEs confer resistance to several pathogenic bacteria, including Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile. Much is known about the structure of the MATE transporters from crystallographic studies; yet the mechanism by which these transporters pump drugs across the membrane remains elusive. The goal of this project is to probe the structure, function and dynamics of several members of the MATE family to understand how they work. The project will focus on three major questions; how do these proteins recognise such a diverse range of drugs; how do they harness an energy source and couple it to drug pumping, and what conformational changes are required for this process? To do this, the structure and activity of the MATE transporters will be probed using assays in bacterial cells as well as with purified protein in detergent solution and reconstituted into proteoliposomes (purified protein inserted into artificial membranes). A suite of molecular biology methods, as well as biochemical and biophysical tools, including spectrofluorometry, chemical crosslinking, and advanced in vitro transport assays, will be used to elucidate the mechanism of drug efflux.

A thorough mechanistic understanding how these transporters work will give us a better understanding of this important antimicrobial resistance mechanism and potentially provide a basis for future drug design.

The Graduate Teaching Assistantship (GTA) provides a postgraduate research student with financial support in return for 96 hours per year of teaching. The stipend paid equals the full UK Research Council rate of £14,296 (rate for 2016/17) plus tuition fees at the home/EU rate. International applicants should make provision to meet the difference between Home /EU and International fees.
For further information on the Graduate Teaching Assistantship scheme go to: https://www.kent.ac.uk/scholarships/search/FNADGTA00001

Applications for Postgraduate study should be made via the University's online application page:: https://www.kent.ac.uk/courses/postgraduate/apply/index.html

 

 

Investigating APOBEC3 cytosine deaminase regulation in a model of HPV-driven neoplasia

Supervisor: Dr Tim Fenton

Recent cancer genome sequence analyses suggest that somatic mutagenesis catalysed by the antiviral APOBEC3 (A3) cytosine deaminase enzymes is a major carcinogenic mechanism1. This field has exploded in the last three years, attracting great interest throughout the cancer research community and already a number of A3 inhibitor programmes have been initiated, both in academia and industry, with the aim of limiting acquired resistance by suppressing de novo mutagenesis during treatment2, 3. However, at present we know very little about A3-mediated genomic mutagenesis, which A3 enzymes are responsible in given cancers, how they are activated and whether A3 targeting by small molecule drugs or vaccination are feasible therapeutic approaches for cancer.

In our previous work, analysing gene expression and genomic alterations in human papillomavirus (HPV)-driven cancers, we uncovered a key role for one or more A3 enzymes and demonstrated A3-mediated mutagenesis of the PIK3CA proto-oncogene across multiple cancer types4. Transcript levels of two A3 family genes (APOBEC3A (A3A) and APOBEC3B (A3B)) are increased in HPV-infected keratinocytes and APOBECs target HPV DNA, which is localized in the nucleus5-9. This project will involve using a model of HPV-driven keratinocyte transformation to understand how A3 proteins become deregulated, leading to off-target activity against the host cell genomic DNA.

Hypothesis:

In normal cells, A3 enzymes are tightly regulated to avoid damage to genomic DNA. We hypothesize that in addition to mRNA changes, regulation of A3 proteins is also altered in HPV-infected cells, resulting in nuclear A3 activity necessary for editing of viral DNA but also increasing the risk of genomic mutagenesis.

Experimental Approach:

The aim of this project is to identify mechanisms by which A3 enzymes are regulated at the protein level in normal cells and to understand how this regulation is perturbed in HPV-infected cells. The high sequence homology between the seven human A3 genes has hampered development of specific antibodies against the individual proteins, thus post-translational A3 regulation has remained largely unexplored. To overcome this, we have used CRISPR/Cas9 gene editing to add epitope tags to the endogenous A3A and A3B genes in keratinocytes, allowing detection of the proteins by western blotting and immunofluorescence microscopy. This project is based on using these novel tools to investigate A3 protein regulation in detail. A3A and A3B expression and localization will be visualized under different conditions, including interferon stimulation and expression of HPV proteins. A3 binding proteins will be identified using mass spectrometry. The roles of selected binding proteins in regulating A3 enzymes and the functional importance of these interactions for preserving genomic integrity will be investigated.

The project will employ a variety of cell biology, biochemical and molecular biology techniques in addition to those listed above, including affinity purification, subcellular fractionation, cytosine deamination and DNA damage assays, retroviral gene transduction and CRISPR/Cas9 gene editing. There will also be opportunities to visit and learn techniques from our collaborators on this project, at the University of Cambridge and at the Francis Crick Institute in London.

References:

1. Henderson, S. & Fenton, T. APOBEC3 genes: retroviral restriction factors to cancer drivers. Trends in molecular medicine 21, 274-284 (2015).
2. Harris, R.S. Molecular mechanism and clinical impact of APOBEC3B-catalyzed mutagenesis in breast cancer. Breast Cancer Res 17, 8 (2015).
3. Swanton, C., McGranahan, N., Starrett, G.J. & Harris, R.S. APOBEC Enzymes: Mutagenic Fuel for Cancer Evolution and Heterogeneity. Cancer Discov 5, 704-712 (2015).
4. Henderson, S., Chakravarthy, A., Su, X., Boshoff, C. & Fenton, T.R. APOBEC-Mediated Cytosine Deamination Links PIK3CA Helical Domain Mutations to Human Papillomavirus-Driven Tumor Development. Cell Reports 7, 1833-1841 (2014).
5. Kukimoto, I. et al. Hypermutation in the E2 gene of human papillomavirus type 16 in cervical intraepithelial neoplasia. Journal of Medical Virology 87, 1754-1760 (2015).
6. Vartanian, J.-P., Guetard, D., Henry, M. & Wain-Hobson, S. Evidence for Editing of Human Papillomavirus DNA by APOBEC3 in Benign and Precancerous Lesions. Science 320, 230-233 (2008).
7. Vieira, V.C. et al. Human papillomavirus E6 triggers upregulation of the antiviral and cancer genomic DNA deaminase APOBEC3B. mBio 5 (2014).
8. Wang, Z. et al. APOBEC3 Deaminases Induce Hypermutation in Human Papillomavirus 16 DNA upon Beta Interferon Stimulation. Journal of Virology 88, 1308-1317 (2014).
9. Warren, C.J. et al. APOBEC3A Functions as a Restriction Factor of Human Papillomavirus. Journal of Virology 89, 688-702 (2015).

The Graduate Teaching Assistantship (GTA) provides a postgraduate research student with financial support in return for 96 hours per year of teaching. The stipend paid equals the full UK Research Council rate of £14,296 (rate for 2016/17) plus tuition fees at the home/EU rate. International applicants should make provision to meet the difference between Home /EU and International fees.
For further information on the Graduate Teaching Assistantship scheme go to: https://www.kent.ac.uk/scholarships/search/FNADGTA00001

Applications for Postgraduate study should be made via the University's online application page:: https://www.kent.ac.uk/courses/postgraduate/apply/index.html

 

 

How does CLIC-1 inserts into lipid membranes in healthy and tumour cells?

Supervisor: Dr Jose Ortega-Roldan

Cancer is a leading cause of disease worldwide. In 2012 approximately 14 million new cases were diagnosed, and there were 8.2 million cancer related deaths, with the number of new cases expected to rise about 70% over the next two 20 years.
The main challenge in cancer treatment is how to design selective drugs targeting exclusively tumour cells. Ion channels, and chloride channels in particular, are key target for potential drugs, but specificity towards tumour cells still remains a problem.

CLIC-1 has emerged as a promising candidate because it localises to the plasma membrane only in tumour cells. In healthy cells it can exist as both cytoplasmic soluble protein or integral membrane embedded intracellular ion channel. This unusual equilibrium is regulated by reactive oxidative species (ROS) and pH changes, common features of tumour cells. Two members of this family, CLIC1 and CLIC4, have been directly implicated in tumour development and identified as novel therapeutic targets.

The goal of this project is to understand the mechanism of CLIC1 insertion into lipid membranes and membrane mimetic systems using solution Nuclear Magnetic Resonance (NMR) in combination with Circular Dichroism and other biophysical tools. Initially, different protein constructs will be screened for favourable in vitro lipid insertion using biochemical assays. State-of-the-art NMR methods will be applied to characterize with atomic detail the soluble and membrane bound states of CLIC1, as well as the mechanism of insertion.

A second part of the project aims to understand the chloride transport function of CLIC-1 and its modulation by drugs. We intend to do use a multidisciplinary approach that integrates X-ray crystallography, solution NMR, electron microscopy and in-cell fluorescence microscopy.

Any mechanistic information obtained for CLIC1 will be of great interest for the development of conformation-specific pharmacological inhibitors and regulators of CLIC1 that could lead to new avenues for cancer treatment.

The Graduate Teaching Assistantship (GTA) provides a postgraduate research student with financial support in return for 96 hours per year of teaching. The stipend paid equals the full UK Research Council rate of £14,296 (rate for 2016/17) plus tuition fees at the home/EU rate. International applicants should make provision to meet the difference between Home /EU and International fees.
For further information on the Graduate Teaching Assistantship scheme go to: https://www.kent.ac.uk/scholarships/search/FNADGTA00001

Applications for Postgraduate study should be made via the University's online application page:: https://www.kent.ac.uk/courses/postgraduate/apply/index.html

 

 

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School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ

Last Updated: 02/12/2016