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Dr Joanna Fox

Supervisor Details

J Fox

Contact Details

Dr Joanna Fox

Department of Molecular and Cell Biology, University of Leicester

Research Interests

Research group activity

Regulation of the cellular life–death switch is essential in healthy cells for normal foetal development and for the clearance of damaged cells. The process of programmed cell death or apoptosis therefore has to be tightly controlled and regulated. Aberrant regulation of cell death has been implicated in numerous human diseases, including AIDS, degenerative diseases, autoimmune diseases and cancer. It is becoming increasing clear that a detailed understanding of the apoptotic signaling pathways, and the key proteins that regulate them is required if they are to be successfully modulated for therapeutic benefit.

There are two main apoptosis pathways; the extrinsic pathway is triggered by signals external to the cell binding to death receptors at the cell surface and initiating formation of the Death-Inducing Signaling Complex (DISC). The intrinsic pathway, however, is triggered by signals within the cell such as DNA damage. Intrinsic or Mitochondrial-driven apoptosis involves mitochondrial outer membrane permeabilization (MOMP) and depends on the activation of BCL-2 effector proteins, BAK and BAX. The pre-dominance of death over survival signals determines whether MOMP takes place, a process involving oligomerization and pore formation by activated BAK/BAX that releases apoptogenic factors from the mitochondria and assists the dissipation of the mitochondrial transmembrane potential, a process that is the point of no return and ultimately consigns a cell to death. BAK and BAX therefore exert their effects at a nodal commitment point in the apoptotic cascade by integrating apoptotic versus survival signals from diverse stimuli to determine cell fate.

My research focuses on regulation of the effector protein BAK. Detailed structural analysis has been undertaken to determine the conformational changes of BAK which occur to facilitate dimer and multimer formation committing the cell to undergo MOMP and apoptotic cell death. Initiation of this process is tightly controlled. I have characterised a novel phospho-regulatory mechanism involved in regulating BAK availability to become activated, and identified key proteins complexes involved in determining both phosphorylation and activation status of BAK.

I aim to establish the molecular architecture of the key protein complexes that regulate the ability of BAK to commit a cell to intrinsic apoptosis. This detailed analysis will provide mechanistic insight into the phospho-regulation of BAK and how the apoptotic threshold of cells is determined. In addition, structural studies of the protein-protein interactions may guide future drug development.

Research Groups

Regulation and molecular architecture of apoptotic protein complexes


Project Details

Dr Fox is the supervisor on the below project:

Structural-guided PROTAC targeting of BMX to modulate apoptotic sensitivity in disease

Secondary Supervisor(s): Dr James Hodgkinson

University of Registration:University of Leicester

BBSRC Research Themes:

Apply here!

Deadline: 23 May, 2024


Project Outline

What determines at the molecular level whether a cell lives or dies? Regulation of the cellular life–death switch is essential in healthy cells for normal foetal development and for the clearance of damaged cells. Aberrant regulation of cell death has been implicated in numerous human diseases, including AIDS, degenerative diseases, autoimmune diseases and particularly cancer, resulting in an unmet need to understand the fundamental mechanisms which exist in cells to regulate apoptosis. Commitment to apoptosis and regulation of the cell life-death switch is dependent largely on protein-protein interactions and the formation of multi-protein complexes, yet the regulation of these complexes is still not fully elucidated.

One crucial member of the apoptosis machinery is BAK. Once activated, BAK permeabilises the mitochondrial membrane, releasing factors which commit a cell to death. BH3 mimetics have been developed, which inhibit anti-apoptotic BCL-2 proteins allowing activation of BAK and BAX. Some of these agents now have FDA approval, but they have on-target toxicity resulting in severe limitations to their use in clinical settings (1).

The unmet need is still for a drug that promotes apoptosis, without significant side effects.

In ground-breaking work, we identified the first and obligatory step in BAK activation (2), which is regulated by tyrosine kinase BMX. BMX phosphorylates BAK on residue Y108, and locks BAK in an inactive conformation making it insensitive to activators, blocking apoptosis. Removal of BMX protein via RNA interference has been shown to sensitize cells to apoptotic cell death (2).

Our therapeutic hypothesis therefore is that removal of BMX will reverse the apoptotic block observed in apoptosis resistant cells and result in increased levels of de-phosphorylated BAK, re-sensitising cells to a wide range of chemotherapeutic drugs, radiation therapies and other existing cancer treatments. This innovative approach will enable a therapeutic window to be created, which can be exploited to increase cell killing with reduced doses of current therapies. Not only making these existing agents more efficacious, but has the potential to reduce side effects and increase quality of life during therapy.

PROteolysis Targeting Chimeras (PROTACs) regulate protein function by degrading target proteins instead of inhibiting them. The removal of the protein via proteolysis can have many advantages over small molecule inhibition, such as enhanced selectivity for the target protein. This project will use a structure-guided approach to design, synthesise and test PROTACs against BMX to potentiate apoptotic cell death in cancer cells.

Aim 1

Utilise structural biology techniques including X-ray crystallography and NMR to determine the structure of tyrosine kinase BMX and use this and previously determined structures of individual domains of BMX to design and synthesise PROTAC molecules

Aim 2

Characterise binding of the PROTAC molecules to BMX both in vitro using structural techniques (x-ray crystallography and NMR) and biophysical methods to detect and analyse binding to recombinant proteins

Aim 3

Characterise the PROTAC molecules in cell-based models. Initially the molecules will be characterised in paired cell line model +/- BMX overexpression to determine the effect on cell growth, sensitivity to induction of apoptotic cell death and cellular morphology. These studies would then be extended to a panel of normal and cancer prostate cell lines to determine if a therapeutic window can be developed in these cell lines with the combination of PROTAC molecules in combination with chemotherapeutics routinely used to treat prostate cancer

Techniques

  • Synthetic organic chemistry/ Medicinal chemistry
  • Recombinant protein expression and purification
  • Structural Biology Methods – NMR and X-ray Crystallography
  • Biophysical techniques to study the impact of the ProTAC molecules on protein structure, and function, including circular dichonism and isothermal calorimetry.
  • Cell culture
  • Flow cytometry (FACS) analysis
  • Cell survival and proliferation assays

Previous Projects

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