Identifying binding partners and molecular functions of the Bcl-2 family member Bok

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Despite its discovery 15 years ago, Bok remains a poorly characterised member of the Bcl-2 family. Initially reported to be expressed selectively in reproductive tissues it has meanwhile become clear that Bok is much more widely expressed and detectable in most tissue. Based on its amino acid sequence, Bok resembles the pro-apoptotic multi BH domain members Bax and Bak and, accordingly, enforced expression of Bok leads to apoptosis in many cell types. Intriguingly, a recent report identified the Bok gene to be deleted in human cancers with high frequency. We could recently show that Bok-induced apoptosis relies on a mechanism largely dependent on Bax or Bak. Furthermore, we could demonstrate that major portions of Bok are localised at non-mitochondrial sites, in particular at the membranes of the Golgi apparatus and the endoplasmic reticulum (ER). Consistent with its subcellular localisation we found that Bok-deficient cells display with an aberrant unfolded protein response upon treatment with ER stressors. With the here-proposed project, which involves multiple collaborations with principal investigators within our Research Group, we aim to better understand the molecular functions of Bok. We have recently generated a Bok-deficient mouse model and acquired a large arsenal of molecular tools facilitating analysis of Bok. We first want to analyse Bokcontaining multiprotein complexes isolated from various intracellular membranes of healthy and dying cells. We will then isolate and identify Bok binding proteins by non-biased approaches using mass spectrometry and subsequently test the biological relevance of newly identified protein interactions. We will characterise the oligomerisation and poreforming potential of recombinant Bok in cell free systems and assist in the quantitative measurement of Bok interactions to other Bcl-2 family members. Another major aim consists in the elucidation of the molecular mechanisms that regulate Bok-induced apoptosis and the potential of Bok to sensitise cancer cells towards classical chemotherapeutics. Finally, we would like to clarify the underlying mechanisms that lead to a disturbed ER stress response in the absence of Bok, using both in vitro and in vivo approaches in the mouse.