BCL2 was first identified as a candidate oncogene at the t(14;18) chromosomal breakpoint in human follicular lymphomas, and was later shown to function through promotion of cell survival; thereby providing the first example of an oncogene involved in the regulation of cell death, as opposed to cell growth and proliferation. Evasion of cell death is now considered one of the six hallmarks of cancer, and, in line with the role ascribed to the founding member, the BCL2 family of proteins is now known to play an essential role in cell death.

The BCL2 family consists of both pro- and anti-apoptotic factions, the opposing activity of which regulates the mitochondrial apoptosis pathway. BCL2 family members are characterized by the presence of one or more BCL2 homology (BH) domains, and can be subdivided into three functional groups: the proapoptotic multi-BH (or effector) group (with BH domains 1 to 4), the proapoptotic BH3-only group (with only the BH3 domain), and the prosurvival (or antiapoptotic) group (with BH domains 1 to 4).

The BH1-3 domains of the multi-BH proteins form a canonical BH3 binding groove, and most interactions between BCL2 family proteins involve accommodation of the BH3 domain of a proapoptotic family member within this groove. In addition to the BH domains, most BCL2 family members also contain a C-T membrane targeting sequence, and are localized at intracellular membranes either constitutively or following an apoptotic stimulus.

The proapoptotic multi-BH proteins, Bax and Bak, are directly involved in MOMP, via pore formation within the MOM. Both Bax and Bak are constitutively present as inactive monomers, with Bax found in a soluble, cytosolic form, and Bak associated with the MOM. Upon apoptotic stimuli Bak/Bax undergo activating conformational changes, leading to stable association with the MOM (in the case of Bax), membrane integration, oligomerization, and, ultimately, pore formation. The prosurvival BCL2 family members (including BCL2, BCLXL, Mcl1, A1 and Bclw), which are constitutively present in either soluble or membrane bound form, function as antagonists of Bax/Bak activation and/or pore formation. The proapoptotic BH3- only proteins (including Bik, Noxa, Puma, Bim, Bid, Bad, Bmf, Hrk, BNip1 and BNip3) couple upstream death stimuli to downstream regulation of the multi-BH family members. The BH3-only proteins, as proximal transducers of cell death signaling, are tightly regulated at the transcriptional and/or post-translational level. Bik, Noxa and Puma, for example, are transcriptionally induced by the tumor suppressor p53 in response to DNA damage, while Bid is activated by proteolytic cleavage at the C-terminus, and Bad by dephosphorylation following growth factor withdrawal.

Beyond their canonical role in apoptosis, BCL2 family members regulate not only additional forms of cell death, but also a wide range of physiological processes in healthy cells. As described in the preceding text, BCL2 proteins are involved in the regulation of ER Ca²⁺ stores/signaling, autophagy (as opposed to autophagic cell death), and adaptation to ER stress. BCL2 family members have also been associated with remodeling/ morphogenesis of the ER and mitochondrial network, as well as with cell cycle progression and regulation of glucose metabolism. How BCL2 family members function in these additional pathways, is not, in most cases, fully understood. Furthermore, although it is clear that BCL2 proteins play active roles in both healthy and dying cells, determining how these roles are coordinately regulated presents a challenging task.

Although a vast body of research has focused on the role of the BCL2 family in regulating the permeabilization of the mitochondrial outer membrane that results in intrinsic apoptosis, recent findings suggest that this group of proteins can also regulate other cellular processes, specifically calcium regulation and mitochondrial dynamics, that may ultimately lead to various forms of cell death independent of the mitochondrial outer membrane permeabilization.

Calcium Regulation by the BCL2 Family

The concept that BCL2 regulates intracellular calcium homeostasis was first formed when BCL2 over-expression prevented the reduction in the Ca²⁺concentration of the endoplasmic reticulum (ER) upon the withdrawal of growth factors in hematopoietic cell lines. Since then, both pro- and anti-apoptotic proteins have been found at the ER, regulating Ca²⁺ signaling between the ER and the mitochondria.

High concentration of free Ca²⁺ in the cytosol is toxic to cells. To maintain the necessary Ca²⁺ gradients required for normal cellular processes, the majority of cellular Ca²⁺ is compartmentalized in the ER. The movement of Ca²⁺ across the ER membrane is regulated by two transporters: (1) the sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA) that actively imports Ca²⁺ from the cytosol into the ER lumen and (2) the inositol 1,4,5-trisphosphate (InsP3) receptor, which mediates the transient release of Ca2+ into the cytosol. A significant portion of Ca²⁺ released by the ER is taken up by the mitochondria juxtaposed to ER Ca²⁺ release channels. Cell death ensues when the mitochondria absorb too much of the released Ca²⁺.

The BCL2 family regulates the Ca²⁺ cross-talk between the ER and the mitochondria. BCL2 and BCL-XL physically interact with the InsP3 receptor at the ER to prevent Ca²⁺release from the ER and subsequent apoptosis. BAX and BAK interfere with this process via the inhibition of both BCL2 and BCL-XL. Also, the unphosphorylated form of the BH3-only protein BAD binds to BCL-XL at the mitochondria and displaces VDAC from BCL-XL, leading to Ca²⁺-mediated apoptosis that is independent of BAX/BAK. Furthermore, although the BH3 domain of NOXA is critical for its ability to induce BAX/BAK-dependent apoptosis, its mitochondrial-targeting domain (MTD) can open the mitochondrial permeability transition pore to release Ca²⁺ into the cytosol, triggering necrosis.

Mitochondrial Dynamics and the BCL2 Family

Mitochondrial networks are highly dynamic and undergo remodeling through continuous cycles of fission and fusion. Mitochondrial fission is often associated with cell injury and has recently been shown to be regulated by BCL2 family members.

Activation of BAX/BAK leads rapidly to mitochondrial fission and can occur in concert with the permeabilization of the mitochondrial outer membrane and the release of cytochrome c. Several studies show that BAX/BAK dependent mitochondrial fission can be uncoupled from cytochrome c release. Peptidic BH3 domains of BH3-only proteins and the NOXA MTD induce mitochondrial fission without the release of cytochrome c and cause cell death in a BAX/BAK independent manner. These studies suggest the BCL2 family may regulate mitochondrial morphogenesis apart from their role in intrinsic apoptosis.

Reference

1. Baffy G, Miyashita T, Williamson J R, et al. Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production[J]. Journal of Biological Chemistry, 1993, 268(9): 6511-6519.

2. Hajnóczky G, Davies E, Madesh M. Calcium signaling and apoptosis[J]. Biochemical and biophysical research communications, 2003, 304(3): 445-454.

3. Rolland S G, Conradt B. New role of the BCL2 family of proteins in the regulation of mitochondrial dynamics[J]. Current opinion in cell biology, 2010, 22(6): 852-858.

4. Karbowski M, Norris K L, Cleland M M, et al. Role of Bax and Bak in mitochondrial morphogenesis[J]. Nature, 2006, 443(7112): 658-662.

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