Visualization of BOK pores independent of BAX and BAK reveals a similar mechanism with differing regulation

Abstract

BOK is a poorly understood member of the BCL-2 family of proteins that has been proposed to function as a pro-apoptotic, BAX-like effector. However, the molecular mechanism and structural properties of BOK pores remain enigmatic. Here, we show that the thermal stability and pore activity of BOK depends on the presence of its C-terminus as well as on the mitochondrial lipid cardiolipin. We directly visualized BOK pores in liposomes by electron microscopy, which appeared similar to those induced by BAX, in line with comparable oligomerization properties quantified by single molecule imaging. In addition, super-resolution STED imaging revealed that BOK organized into dots and ring-shaped assemblies in apoptotic mitochondria, also reminiscent of those found for BAX and BAK. Yet, unlike BAX and BAK, the apoptotic activity of BOK was limited by partial mitochondrial localization and was independent of and unaffected by other BCL-2 proteins. These results suggest that, while BOK activity is kept in check by subcellular localization instead of interaction with BCL-2 family members, the resulting pores are structurally similar to those of BAX and BAK.

Apoptotic mitochondrial poration by a growing list of pore‐forming BCL‐2 family proteins

“…The lipid composition determines the efficiency of membrane permeabilization by BOK with maximal activity observed in vesicles rich in cardiolipin (phosphatidylcholine (PC):CL 8:2) compared to intermediate and low activity supported in vesicles with lipid composition resembling mitochondria or 100% PC, respectively. [ 51 ] At the mitochondria, BOK was visualized by STED microscopy and underwent oligomerization into ring‐like structures, as described for BAK and BAX (Figure 3C). Like BAK and BAX, BOK exhibited concentration‐dependent oligomerization in supported lipid bilayers (SLBs) prepared from mixtures of PC:CL 8:2 liposomes and fluorescently labeled BOK‐∆TM.…”

“…Supporting the essential role of BOK's TM in mitochondrial targeting, when transiently expressed in AKO cells BOK‐∆TM was largely cytosolic exhibiting no apoptotic activity whereas FL‐BOK exhibited significant albeit half of the activity of BAX achieved on transient expression in these cells. [ 51 ] FL‐BOK localized at the mitochondria and ER, and their contact sites. In contrast, the chimeric BOK protein with the BCL‐xL TM replacing that of BOK, BOK‐TM xL , was more potent in triggering apoptosis presumably by targeting the mitochondria better than FL‐BOK.…”

Bok: real killer or bystander with non-apoptotic roles?

“…Subsequent studies revealed that Bok is actually expressed in all cells and tissues ( Ke et al, 2012 ; Naim and Kaufmann, 2020 ; Bonzerato et al, 2022 ) and confirmed that it can induce apoptosis when over-expressed in a variety of cell types ( Echeverry et al, 2013 ; Einsele-Scholz et al, 2016 ; Llambi et al, 2016 ; Schulman et al, 2016 ; Szczesniak et al, 2021b ). Also, cell-free experiments showed that Bok can permeabilize liposomes or artificial MOMs, like its pro-apoptotic counterparts Bak and Bax ( Llambi et al, 2016 ; Fernandez-Marrero et al, 2017 ; Zheng et al, 2018 ; Shalaby et al, 2022 ). So, there is no doubt that over-expressed Bok in an exogenous context, or Bok in a cell-free system, can induce MOMP and apoptosis.…”

“…During intrinsic apoptosis signaling, Bak and Bax oligomerize to form pores that permeabilize the mitochondrial outer membrane (MOM), releasing proteins like cytochrome c into the cytosol, which in turn activate a downstream signaling cascade and ultimately cause cell death ( Westphal et al, 2014 ). This irreversible process is known as mitochondrial outer membrane permeabilization (MOMP) and various studies have indicated that Bcl-2-related ovarian killer, Bok, might also be a MOMP mediator ( Llambi et al, 2016 ; Fernandez-Marrero et al, 2017 ; Zheng et al, 2018 ; Shalaby et al, 2022 ). Indeed, in many reviews ( Moldoveanu and Czabotar, 2020 ; Shalaby et al, 2020 ; Vandenabeele et al, 2022 ; Moldoveanu, 2023 ), Bok is grouped together with Bak and Bax as a bona fide MOMP mediator.…”

Endogenous Bok is stable at the endoplasmic reticulum membrane and does not mediate proteasome inhibitor-induced apoptosis

“…Bcl-2-related ovarian killer (Bok) is a member of the Bcl-2 protein family network that helps control cell viability ( Moldoveanu et al, 2014 ; Kale et al, 2018 ; Kalkavan and Green, 2018 ), but its place in that network and its role within the cell remains unclear and controversial ( D'Orsi et al, 2017 ; Joshi et al, 2020 ; Naim and Kaufmann, 2020 ; Shalaby et al, 2020 ; Means and Katz, 2021 ). On one hand, it has been shown that Bok over-expression in mammalian cells can trigger apoptosis ( Echeverry et al, 2013 ; Einsele-Scholz et al, 2016 ; Llambi et al, 2016 ; Stehle et al, 2018 ) and that purified recombinant Bok can permeabilize liposomes ( Llambi et al, 2016 ; Fernandez-Marrero et al, 2017 ; Zheng et al, 2018 ; Shalaby et al, 2022 ), indicating that Bok may trigger mitochondrial outer membrane permeabilization (MOMP), similarly to the better-characterized pro-apoptotic proteins Bak and Bax ( Moldoveanu et al, 2014 ; Kale et al, 2018 ; Kalkavan and Green, 2018 ; Pena-Blanco and Garcia-Saez, 2018 ). Further, it has been proposed that Bok is constitutively pro-apoptotic, with Bok expression maintained at very low and “safe” levels by activity of the ubiquitin-proteasome pathway (UPP) ( Llambi et al, 2016 ), and that Bok accumulates and causes cell death only if its UPP-dependent processing is blocked with proteasome inhibitors ( Llambi et al, 2016 ; Naim and Kaufmann, 2020 ; Shalaby et al, 2020 ).…”