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Uncovering the molecular basis of Epstein-Barr virus-induced cancers

Primary Supervisor: Professor Tim Dafforn, School of Biosciences

Secondary supervisor: Professor Lawrence Young / Dr Corinne Smith

PhD project title: Uncovering the molecular basis of Epstein-Barr virus-induced cancers

University of Registration: University of Birmingham

Project outline:

The association of Epstein-Barr virus (EBV) infection with the development of certain lymphomas and carcinomas is well established. Despite being the most common virus infection in humans, under certain circumstances EBV directly contributes to the oncogenic process resulting in tumours carrying multiple copies of the circular virus genome in every cancer cell and expressing a limited set of virus-encoded genes. The association of EBV with these cancers provides opportunities to exploit the virus as both a diagnostic and prognostic biomarker as well as for the development of therapeutic approaches that specifically target EBV-encoded proteins.

While EBV expresses several proteins in virus-associated tumour cells, LMP1 is considered to be the major oncoprotein on account of its ability to transform B cells and rat fibroblast in vitro rendering them tumourigenic in nude mice. Although showing no appreciable homology to known cellular proteins, LMP1 displays functional similarities to the tumour necrosis factor receptor (TNFR) family members: TNFR1 and CD40.These functional similarities are attributed to the recruitment of a number of TNFR-associated signalling proteins, which include TNFR-associated factors (TRAFs) and the TNFR-associated death domain protein (TRADD), amongst others. However, unlike TNFR1 and CD40, LMP1 signals in a constitutive, ligand independent manner.

LMP1 is a 66kDa integral membrane protein comprising a short amino cytoplasmic N-terminus, six hydrophobic alpha-helical transmembrane (TM) spanning regions and a large 200 amino acid cytoplasmic tail. The TM domains self-aggregate and participate in intermolecular oligomerisation while the large cytosolic C-terminus possesses most of the signalling capacity of the protein. It is through this self-aggregation that LMP1 behaves as a constitutively active TNF receptor, signalling via numerous pathways commonly deregulated in cancer including NF-kB, ERK-MAPK, PI3K/Akt and JNK/SAPK. The precise nature of LMP1 structure and these intermolecular interactions remain unknown.

In 2009 we developed a revolutionary new polymer-based method (SMALP) for the production of the membrane proteins. The method negates the problematic use of detergents in making membrane proteins, instead encapsulating them, complete with a portion of phospholipid membrane in a 10 nm diameter nanodisc. Since this time we have shown that the SMALP methods in generically applicable to a wide range of membranes proteins including G-protein coupled receptors, ion channels, pumps and enzymes. The resulting preparations show a high degree of activity and a stability that is substantially enhanced over detergent purified samples.

In the past 3 years it has also become apparent that SMALP particles containing membrane proteins provide a perfect basis for structural studies using Cryo-electron microscopy. The structure of the proteins within the particles being solved using single particle analysis methods. This makes the system perfectly suited to providing the first high resolution structural data on the LMP1 complex in situ in the membrane. This can then form the basis for unravelling the complexes of LMP1 with its partner protein in turn providing data that will aid the development of anti EB treatments.

Objectives:

  • Developing the SMALP method for LMP1 extraction
  • Determination of the secondary structure, oligomerisation state and affinity for partners SMALP-LMP1
  • Determination of the high-resolution structure of LMP1 using Cryo-electron microscopy

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology

Techniques that will be undertaken during the project:

  • Protein expression
  • SMA polymer production and modification
  • SMALP extraction
  • Protein purification including chromatography
  • Structural determination using Circular Dichoism, Analytical Ultracentrifugation, Cryo-Electron Microscopy
  • Ligand binding determination using SPR, MST and Fluorescence

Contact: Professor Tim Dafforn, University of Birmingham