VDAC2-dependent regulation of the mitochondrial apoptotic mediators, Bak and Bax

Bak and Bax are the central mediators of the intrinsic programmed cell death. During apoptosis, structural re-organization of Bax and Bak induced by activated BH3-only proteins allows them to form oligomers to permeabilise the mitochondrial outer membrane (MOM). Although structural studies have provided crucial evidence for conformational changes of Bak and Bax in response to apoptotic signals, the regulation of Bak and Bax in unstimulated cells remains to be fully deciphered. Voltage-dependent anion channel isoform 2 (VDAC2) has been reported to both negatively and positively regulate proapoptotic activity of Bak. This thesis further clarifies and characterises the BakVDAC2 interaction and possible involvement of VDAC2 in Bax regulation at the MOM. The absence of VDAC2 in mouse embryonic fibroblasts (MEFs) prevented efficient mitochondrial import of Bak leading to less Bak present at the MOM and a significant population of cytosolic Bak. It was also found that the mislocalised cytosolic Bak in Vdac2-/- MEFs is rapidly degraded via an unknown mechanism, potentially contributing to the low Bak total level in Vdac2-/- MEFs. The direct interaction between Bak and VDAC2 was successfully confirmed and VDAC2 was shown to be a crucial component of the inactive Bak complex. Sitedirected mutagenesis study also revealed that both cytosolic and transmembrane domains of Bak are essential for modulating the formation of the Bak-VDAC2 complex. Potential components of the Bak-VDAC2 complex were identified using mass spectrometry. The ability of VDAC2 to interfere with pro-apoptotic activity of Bax was reexamined and VDAC2 appeared to facilitate the efficient association and translocation of Bax to mitochondria. Bax associated with VDAC2 within a 440 kDa complex that was independent of Bak. Whether VDAC2 has an inhibitory effect on Bax after its integration into the MOM requires further investigation. To further evaluate the obtained results from Vdac2-/- MEFs, VDAC2-deficient HCT116 cells were successfully generated using TALEN technology. Initial characterisation supported the important role of VDAC2 in the biogenesis of the 440 kDa Bak and Bax complexes and in regulating Bak expression level within human cells. In conclusion, VDAC2 specifically regulates mitochondrial apoptosis through modulating pro-apoptotic functions of Bak and Bax. The work in this thesis provides important insights into a critical step of Bak and Bax regulation by nonBcl-2 proteins.

Submission note: A thesis submitted in the total fulfilment of the requirement for the degree of Doctor of Philosophy to the Department of Biochemistry, La Trobe Institute for Molecular Science, School of Molecular Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Melbourne.