Apoptosis: Degradation in Preventing Death

The commitment of a cell to apoptotic programmed cell death is as essential as it is irreversible – too much of which has been linked to neurodegradation and dysfunction; too little, an accumulation of mutant cells, transmission of defective genes, and proliferation of aggressive cancers. Classically mediated by stress, the canonical apoptosis activation pathway in mammalian cells is branched into two major sections: (i) a caspase 8-depedent extrinsic pathway that is triggered through death receptor stress signaling; (ii) a holocytochrome c-dependent intrinsic pathway mediated by apoptosome multimerization1. In the intrinsic apoptosis central dogma, it is believed that pro-apoptotic proteins effectors BAK/BAX, activated by BH3-only initiators and sequestered by multi-domain antiapoptotic protein BCL-2, are exclusively necessary in outer mitochondrial membrane permeability and therefore apoptotic signaling. However, a recent investigation by Llambi et al. in 2016 revealed an alternative non-canonical mitochondria outer membrane permeabilization (MOMP)–mediated apoptotic pathway mediated by BOK protein, one that’s independent of BH3-only protein signaling and BCL-2 inhibition2.
 Upon its initial discovery in 1997, BCL-2 ovarian killer (BOK) shared striking sequence and structural homology to BAK/BAX 2,3. Aside from its ability to slightly heterodimerize with anti-apoptotic BCL-2 and induced death in the ovarian cells, its role in apoptosis was largely ambiguous3. Further in vivo and in vitro studies involving BOK-/- specimen failed to illustrate significant impact on proliferation of lymphomas and development in general4. To clarify the true effect of BOK on apoptosis, Llambi et al. generated BOK-/- mouse embryonic fibroblast (MEF) mutants as well as a Dox-inducible Venus-BOK fluorescent fusion MEF line. Upon failure to find BOK protein despite presence of mRNA in the WT lines and Dox induction treatment, the authors proposed that BOK is under endogenous constitutive degradation2. As predicted, BOK expression upon treatment with proteasome inhibitors was observed through fluorescent imaging and induction of apoptosis, the latter of which was also attenuated by subsequent BOK-targeted siRNA silencing2. In a double knockout BAK-/-/BAX-/- background, Llambi et al. also demonstrated caspase-dependent cell death with analogous characteristics of apoptosis (blebbing) after enforcing BOK expression via proteasome inhibition. The authors thus have expertly shown that BOK-triggered apoptosis acts in a BAK/BAX-independent manner (Fig.1).
Next, Llambi and colleagues focused the mechanism in which BOK degradation was controlled, leading them to explore BOK’s connection to the ER-associated degradation (ERAD) pathway in an apoptotic context. Endogenously, when overloaded with misfolded protein or oligomerized complexes, the cell activates endogenous ubiquitin- and proteasome-dependent mechanisms to degrade the proteins to relieve ER stress. The activated mechanism in mammalian cell utilizes gp78 for ubiquitination on lysine residues and valosin-containing protein (VCP) for elongation of the ubiquitin chain, as well as PERK for sensing and signaling5. Nonconservative lysine-to-arginine substitutions of BOK promoted stable expression by preventing gp78 E3-executed ubiquination2. Further live confocal microscopy illustrated strong colocalization of BOK with the gp78 Ub-ligase complex. Llambi et al. also showed that knockdowns of gp78 and VCP lead to consistent BOK expression and induced stronger death phenotype.

Figure 1. BOK-mediated apoptosis pathway in comparison to canonical intrinsic pathway. The BOK protein is regulated by components in the ERAD system (green box) and is normally degraded by endogenous proteasomes. It is interestingly independent from BH3 activation and anti-apoptotic BCL-2 protein suppression in inducing MOMP. Image adapted from2.

Finally, by compromising ERAD through inhibition of PERK along with TN stimulation, they revealed that the gp78 complex (responsible for BOK degradation) was in turn degraded under these conditions2. Through these perturbations of the components of the ERAD system, the authors beautifully showed the dependency of BOK-mediated apoptosis on the ER stress response (Fig.1).
Further delving into the mechanism in which BOK is controlled, Llambi et al. compared its effects to that of BAK/BAX. Similar to BAK/BAX apoptotic effector, stabilized BOK expression initiated MOMP and therefore release of holocytochrome c2. The authors then dissected the necessity of BOK localization to the mitochondria on its activity by generating three variants of BOK: (i) truncation of the C-terminus transmembrane domain therefore cytosolic sequestering BOK; (ii) anchoring to the plasma membrane; (iii) anchoring BOK mitochondrial outer membrane. It was observed that only the latter BOK form, anchored to the mitochondria, was functional. Differing from BAK/BAX, whose activation is BH3-only protein dependent, BOK in its endogenous multimeric form is constitutively competent and sufficient in permeabilizing the OMM2. Finally, through affinity precipitation and examining enzyme kinetics, BOK was not found to be sequestered by BCL-2 as BAK/BAX is (Fig.1).
In summary, Llambi and colleagues have extrapolated a novel pathway from a previously-known yet unexplored BCL-2 family member protein BOK through which intrinsic apoptosis may be induced. This pathway differs from the BAX/BAK pathway in that being constitutively active it does not require BID activation6. Therefore, it is controlled by constant degradation associated with the integrity of ERAD activity, the disruption of which promotes stable BOK expression and triggering subsequent MOMP and apoptosis, much unlike the underlying mechanism of BAK/BAX6,7. Finally and most remarkably, BOK activity is not attenuated by BCL-2 antiapoptotic protein, giving it great potential in pharmacology. Despite its excellent and meticulous methodology, a limitation of this investigation lies in the in vitro nature of this investigation. Whether a similar pattern observed in MEF’s translates across cell types (aside from ovaries and fibroblasts) as well as microenvironments are left unanswered and should be explored before application to medicine.
Nevertheless, the discovery of the BOK pathway provides great knowledge in expanding the understanding of intrinsic apoptotic signaling, as it provides a molecular basis and further detail for the extraordinary connection between ERAD PERK and apoptotic signaling. Additionally, it provides potential insight into the evolutionary history of apoptosis and its corresponding triggers, since many cases of apoptosis are often implicated (and logically so) to failed control of protein synthesis and folding5. Finally, considering the BOK-mediated apoptosis pathway is not limited by the many components (BH3-only-protein/BID) in play for the canonical intrinsic pathway, it provides an alternative route of inducing apoptosis unaffected by perturbation of such proteins. This is especially significant in the front of cancer development, in which upregulation of BCL-2 anti-apoptotic proteins are often correlated to8. Being unresponsive to BCL-2 anti-apoptotic protein, BOK-mediated apoptosis possesses great medical significance, as it holds great potential serving as a very attractive model for developing novel cancer-targeting therapeutics.

REFERENCES

  1. Miyamoto, Shigeki. “Death Signaling 2.” Biochem 630. Madison. 21 Nov. 2016. Lecture.
  2. Llambi, Fabien, Yue-Ming Wang, Bernadette Victor, Mao Yang, Desiree M. Schneider, Sébastien Gingras, Melissa J. Parsons, Janet H. Zheng, Scott A. Brown, Stéphane Pelletier, Tudor Moldoveanu, Taosheng Chen, and Douglas R. Green. “BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation.” Cell 165.2 (2016): 421-33
  3. Hsu, S. Y., A. Kaipia, E. Mcgee, M. Lomeli, and A. J. W. Hsueh. “Bok Is a Pro-apoptotic Bcl-2 Protein with Restricted Expression in Reproductive Tissues and Heterodimerizes with Selective Anti-apoptotic Bcl-2 Family Members.” Proceedings of the National Academy of Sciences 94.23 (1997): 12401-2406
  4. Ke, F., A. Voss, J. B. Kerr, L. A. O’reilly, L. Tai, N. Echeverry, P. Bouillet, A. Strasser, and T. Kaufmann. “BCL-2 Family Member BOK Is Widely Expressed but Its Loss Has Only Minimal Impact in Mice.” Cell Death and Differentiation 20.1 (2012): 183
  5. Tsai, Y. C., and A. M. Weissman. “The Unfolded Protein Response, Degradation from the Endoplasmic Reticulum, and Cancer.” Genes & Cancer 1.7 (2010): 764-78.
  6. Westphal, Dana, Grant Dewson, Peter E. Czabotar, and Ruth M. Kluck. “Molecular Biology of Bax and Bak Activation and Action.” Biochimica Et Biophysica Acta (BBA) – Molecular Cell Research 1813.4 (2011): 521-31 
  7. Luna-Vargas, Mark P.a., and Jerry Edward Chipuk. “Physiological and Pharmacological Control of BAK, BAX, and Beyond.” Trends in Cell Biology 26.12 (2016): 906-17 
  8. Yip, K. W., and J. C. Reed. “Bcl-2 Family Proteins and Cancer.” Oncogene 27.50 (2008): 6398-40

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2017-12-09T19:38:06+00:00 May 1st, 2017|