Deciphering the Mechanism of Action of the Proapoptotic Protein BOK

Abstract

BCL-2 family members are the main regulators of the mitochondrial pathway of apoptosis. The Bcl-2 family includes both pro-apoptotic and pro-survival proteins. The proapoptotic effectors BAX and BAK oligomerize upon activation to form pores in the mitochondrial outer membrane which is regarded as the point-of-no-return in intrinsic apoptosis. BOK is a poorly understood member of the BCL-2 family of proteins which is classified as a pro-apoptotic protein based on structural homology and the ability to induce apoptosis upon overexpression. BOK is currently proposed to function as a pro-apoptotic BAX-like effector, even if the molecular mechanism and structural properties of BOK pores are still unclear. In contrast to most BCL-2 proteins which are localized to the mitochondria, BOK is mainly localized to the membranes of the endoplasmic reticulum (ER) and the Golgi apparatus. BOK is also known to be overexpressed in several types of cancer, including ovarian cancer and cervical cancer, and is being studied as a potential cancer prognostic marker. In the present work, we directly visualized BOK-induced pores in liposomes using negative staining electron microscopy. The pores were similar to the ones induced by BAX, which suggests that both proteins have related oligomerization properties. This was also backed by single-molecule imaging, which demonstrated that BOK can oligomerize in membranes containing cardiolipin. In addition, we found that the thermal stability of BOK can explain its pore-forming activity, in agreement with previous reports. We also found that BOK apoptotic activity is analogous to and independent of BAX and BAK, and unaffected by other BCL-2 proteins. Interestingly, we show that apoptosis induction by BOK is restricted by its limited mitochondrial localization. Finally, Super resolution STED imaging was used to visualize BOK organization in apoptotic mitochondria, which appeared to form dots and ring-shaped assemblies, similar to what have been reported for BAX and BAK.