Biological Role

Biological role

Bcl-2 Protein Interactions

Bcl-2 is critically important within the intrinsic apoptotic pathway, activated as a result of DNA damage or via survival factor withdrawal of molecules such as cytokines and growth factors. Bcl-2 is an anti-apoptotic protein with a C-terminal transmembrane domain that facilitates its localisation to the outer mitochondrial membrane. Bcl-2 inhibits the actions of Bak and Bax, two pro-apoptotic proteins implicated within the formation of the permeability transition pore which increase the permeability of the outer mitochondrial membrane and results in the release of pro-apoptotic proteins from the mitochondria such as SMAC, AIF and cytochrome C. These proteins lead to the apoptotic associated caspase cascade of proteases that dismantle cellular components leading to cell death. In Bcl-2 knockouts, there is spontaneous activation and subsequent homo-oligomerisation of Bak and Bax which constitute the permeability transition pores. Interaction between with Bcl-2 occurs via the BH3 domain of Bax mainly. However, evidence suggests that Bcl-2 inhibits Bak also due to its spontaneous homo-oligomerisation in the absence of Bcl-2. The tight interaction between Bax and Bcl-2 arises due to the BH3 domain interacting with a hydrophobic groove of Bcl-2. BH3 domains insert as an amphipathic alpha-helix and make hydrophobic interactions and hydrogen bonds with the Bcl-2 protein within the region of the hydrophobic groove for all BH3-hydrophobic groove interactions. This is key as the BH3 domain for Bak and Bax is associated with dimer formation and subsequent homo-oligomerisation. Interaction between Bcl-2 and Bax via the BH3 domain of Bax could abrogate dimer formation. This is compounded by an emerging body of evidence suggests upon activation, there is initial BH3-hydrophobic groove association to form homodimers of Bak and Bax. Subsequently, the homodimers associate to form oligomers via interface interactions between alpha-6 helices in formation of the permeability transition pore.

Bcl-2 itself is also subject to regulation through interactions with pro-apoptotic proteins that neutralise Bcl-2. This is typical of pro-apoptotic BH3-only proteins such as Puma, Noxa and Bim. Noxa gene expression is dependent upon p53. DNA damage is believed to upregulate Noxa via the actions of p53. The BH3 domain of Noxa is able to interact with the hydrophobic BH3 binding groove of Bcl-2. This interaction is believed to have a neutralising affect over Bcl-2 to supress its functions of Bax/Bak inhibition of homo-oligomerisation and subsequent permeability transition pore formation. Puma mediates its pro-apoptotic functions through binding anti-apoptotic proteins, including Bcl-2 through its BH3 domain. Experimental evidence suggests the BH3 domain interacts with the hydrophobic groove of Bcl-2 as an amphipathic alpha-helix to neutralise the pro-apoptotic role of Bcl-2 over Bak and Bax.

The intrinsic pathway of apoptosis can also be stimulated by withdrawal of survival factors. Implicated within are proteins such as Bim. Bim is usually in complex with with Beclin but phosphorylation by JNK as a result of survival factor withdrawal alleviates this interaction and Bim instead confers its pro-apoptotic function. It is through its BH3 domain that Bim interacts with the hydrophobic groove of Bcl-2. The indirect model of apoptosis activation suggests that Bim acts to neutralise Bcl-2 and its actions over Bak and Bax that form the permeability transition pore.

The hydrophobic groove of Bcl-2 is formed by alpha helices 2, 3, 4, 5 and 7 which all contribute.

Click the links for further information on Bcl-2's structure, representative proteins, and sequence comparisons.

References:

[1] Leibowitz B, Yu J. Mitochondrial signaling in cell death via the Bcl-2 family. Cancer biology & therapy. 2010;9(6):417-422. [Pubmed]

[2] Vela L, Gonzalo O, Naval J, Marzo I. Direct Interaction of Bax and Bak Proteins with Bcl-2 Homology Domain 3 (BH3)-only Proteins in Living Cells Revealed by Fluorescence Complementation. The Journal of Biological Chemistry. 2013;288(7):4935-4946. doi:10.1074/jbc.M112.422204. [Pubmed]

[3] O’Neill KL, Huang K, Zhang J, Chen Y, Luo X. Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane. Genes & Development. 2016;30(8):973-988. doi:10.1101/gad.276725.115. [Pubmed]

[4] Ku B, Liang C, Jung JU, Oh B-H. Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX. Cell Research. 2011;21(4):627-641. doi:10.1038/cr.2010.149. [Pubmed]

[5] Petros, A. M., Olejniczak, E. T., and Fesik, S. W. (2004). Structural biology of the Bcl-2 family of proteins. Biochim. Biophys. Acta 1644, 83–94. doi: 10.1016/j.bbamcr.2003.08.012 [Pubmed]

[6] Westphal D, Dewson G, Czabotar PE, Kluck RM. Molecular biology of Bax and Bak activation and action. Biochim Biophys Acta. 1813;2011:521–31. [PubMed]

[7] Oda E., Ohki R., Murasawa H., Nemoto J., Shibue T., Yamashita T., Tokino T., Taniguchi T., Tanaka N. Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science. 2000;288:1053–1058. [PubMed]

[8] Smith AJ, Dai H, Correia C, et al. Noxa/Bcl-2 Protein Interactions Contribute to Bortezomib Resistance in Human Lymphoid Cells. The Journal of Biological Chemistry. 2011;286(20):17682-17692. doi:10.1074/jbc.M110.189092. [Pubmed]

[9] Karim CB, Michel Espinoza-Fonseca L, James ZM, et al. Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation. Scientific Reports. 2015;5:14557. doi:10.1038/srep14557. [Pubmed]

[10] Adams J, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007;26(9):1324-1337. doi:10.1038/sj.onc.1210220. [Pubmed]

[11] Shamas-Din A, Brahmbhatt H, Leber B, Andrews DW.. BH3-only proteins: orchestrators of apoptosis. Biochim Biophys Acta (2011) 1813(4):508–20.10.1016/j.bbamcr.2010.11.024 [Pubmed]

[12] Sionov RV, Vlahopoulos SA, Granot Z. Regulation of Bim in Health and Disease. Oncotarget. 2015;6(27):23058-23134. [Pubmed]

[13] O’Connor L, Strasser A, O’Reilly LA, et al. Bim: a novel member of the Bcl-2 family that promotes apoptosis. The EMBO Journal. 1998;17(2):384-395. doi:10.1093/emboj/17.2.384. [Pubmed]

[14] Jiang T, Liu M, Wu J, Shi Y. Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(8):2074-2079. doi:10.1073/pnas.1525616113. [Pubmed]

Protein Interactions

Bcl-2 Family

The interplay between the three subclasses of the BCL-2 family determines the fate of cells in response to developmental or stress signals. The mechanism of how BAX and BAK are activated by the BH3-only proteins in dying cells has been of intense investigation. Two main models have been put forth:

A widely accepted model is the direct activation model, which suggests that the “sensitizer” or “inactivator” BH3-only proteins (BAD, BIK, BMF, Hrk and Noxa) release the “activator” BH3-only proteins (BIM, BID and possibly PUMA) sequestered by the anti-apoptotic BCL-2 subfamily members, and that these “activators” are required for activating inert BAX/BAK via a direct but transient binding interaction.
The other model, called indirect activation model, suggests that the anti-apoptotic BCL-2 proteins inhibit apoptosis by sequestering a small proportion of the activated BAX and BAK in healthy cells, and that a subset of the BH3-only proteins, including the “activators”, engages the anti-apoptotic proteins to release BAX and BAK in dying cells. In this model, the “activator” BH3-only proteins are not directly involved in the activation of BAX/BAK.

The indirect model is opposed in the field mainly by two reasons. First, BAX is mostly cytosolic and monomeric, with a minor fraction bound to the OMM, where most of the anti-apoptotic BCL-2 proteins are found in healthy cells. Second, BAX has been considered to interact with the anti-apoptotic BCL-2 proteins only weakly, if it does, and therefore the significance of these interactions has been elusive. Nevertheless, mutational and other studies strongly support the view that the anti-apoptotic proteins should be able to engage the BH3 domain of Bax to prevent it from mediating apoptosis.

References:

[1] Ku B, Liang C, Jung JU, Oh B-H. Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX. Cell Research. 2011;21(4):627-641. doi:10.1038/cr.2010.149. [Nature]

[1]

The Bcl-2 Family

The Bcl-2 family proteins are central regulators of the mitochondrion-mediated apoptotic cell death. They are characterized by containing up to four conserved stretches of amino acids, known as Bcl-2 homology (BH) domains. They are usually grouped into three distinct subclasses:

One subclass is composed of BAX and BAK that mediate apoptosis by triggering destabilization of the outer mitochondrial membrane (OMM) and consequently releasing the apoptogenic factors, such as cytochrome c, from mitochondria to the cytosol.

Another subclass is composed of the BH3-only proteins (including BIM, BAD, PUMA and Noxa) that sense and convey pro-death signals and ultimately activate downstream BAX and BAK. While BAX and BAK contain the BH1 through BH3 domains and are homologous to each other, the BH3-only proteins are unrelated to each other except that they all contain the BH3 domain.

The activation of BAX/BAK is suppressed by the remaining subclass composed of BCL-2 itself, BCL-xL, BCL-w, MCL-1 and A1 all of which contain the four BH domains.

The figure below shows the Bcl-2 family and its subdivisions. These have been separated into anti-apoptotic, executioners, and BH3-only proteins. The pro-apoptotic proteins have already been divided into their executioner or BH3-only classes.