Protein-protein interactions of the BCL-2 family

The BCL-2 family of proteins are important regulators of mitochondrial
apoptosis, comprising both pro- and anti-apoptotic members that interact
with one another at the mitochondrial outer membrane to determine cellular
fate. Dysregulation of their activities in the cell is implicated in many forms of
cancer; the development of molecules able to mimic and modulate their
interactions is thus highly desirable and has been the subject of a great deal
of research effort. The use of techniques including structure based design
and peptidomimetic approaches has produced some notable successes in
this area, but few have successfully transitioned from laboratory to clinic,
and the search for more and better ways to develop such molecules
continues.
In this thesis, I present a novel approach to identifying binding partners for
BCL-2 proteins, which uses phage display experiments and the production
of non-antibody scaffold proteins called Affimers. Five BCL-2 family proteins
were selected as targets for study, comprising a good cross section of proand
anti-apoptotic members. In the following chapters, I first describe the
work undertaken to purify and characterise these target proteins, then detail
the work done to identify and purify Affimer binding partners for each of
them. Finally, I report on structural studies carried out to explore the
mechanism by which BAX, a pro-apoptotic family member, forms death
inducing oligomers.
Taken together, the results of this project lay the foundations for further
structural studies of BAX oligomerisation, and demonstrate that the use of
phage display to generate selectively binding non-antibody scaffold proteins
can provide useful additions to the existing array of BCL2 family interacting
molecules.