Auto-activation of the Apoptosis Protein Bax Increases Mitochondrial Membrane Permeability and Is Inhibited by Bcl-2

Abstract

Interactions among Bcl-2 family proteins mediated by Bcl-2 homology (BH) regions transform apoptosis signals into actions.The interactions between BH3 region-only proteins and multi-BH region proteins such as Bax and Bcl-2 have been proposed to be the dominant interactions required for initiating apoptosis. Experimental evidence also suggests that both homo-and hetero-interactions are mediated primarily by the BH3 regions in all Bcl-2 family proteins and contribute to commitment to or inhibition of apoptosis. We found that a peptide containing the BH3 helix of Bax was not sufficient to activate recombinant Bax to permeabilize mitochondria. However, an extended peptide containing the BH3 helix and additional downstream sequences activated Bax to permeabilize mitochondria and liposomes. Bcl-2 inhibited the membrane-permeabilizing activity of peptide-activated Bax. This activity of Bcl-2 was inhibited by the extended but not the BH3-only peptide despite both peptides binding to Bcl-2 with similar affinity. Further, membrane-bound Bax activation intermediates directly activated soluble Bax further permeabilizing the membrane. Bcl-2 inhibited Bax auto-activation. We therefore propose that Bax auto-activation amplifies the initial death signal produced by BH3-only proteins and that Bcl-2 functions as an inhibitor of Bax auto-activation.The dominant form of programmed cell death, apoptosis, plays an essential role in embryogenesis and tissue homeostasis of multicellular organisms by removing unwanted or damaged cells. Impaired regulation of apoptosis is implicated in a variety of diseases including cancer and stroke. Two kinds of signals can trigger apoptosis, death signals received by death receptors on cell surface, and stress signals such as structural and genotypic damage. The latter apoptotic signals mainly route through mitochondria to provoke activation of caspases and nucleases that cleave critical proteins and DNAs thereby dismantling the cell (1, 2).Apoptosis is regulated by a complicated series of interactions between Bcl-2 family proteins. These interactions include hetero-and homo-interactions of proteins containing either multiple Bcl-2 homology (BH) 3 regions (BH1-3 or BH1-4) or a single (BH3) region. After receiving certain apoptotic stimuli, pro-apoptotic BH3-only proteins induce the release of cytochrome c and other pro-apoptotic proteins from mitochondria by triggering permeabilization of the mitochondrial outer membrane (MOM) by the multi-BH domain proteins Bax and Bak (3, 4). One proposed mechanism of MOM permeabilization involves BH3-only protein-induced conformational changes in Bax and/or Bak leading to their oligomerization in the MOM (5-7).BH3-only proteins are found in a variety of different subcellular locations and share very limited sequence similarity, often only in the short BH3 region. There is insufficient sequence similarity in BH3 region sequences to permit unambiguous identification from sequence alone. It appears that BH3 region proteins act as sensors within the cell, reporting on...

Where Killers Meet--Permeabilization of the Outer Mitochondrial Membrane during Apoptosis

“…In that case, the exposed Bax BH3 domain would act just like the BH3 domain of a direct activator BH3-only protein. This would be consistent with the observation that a small number of BH3-only proteins can recruit a molar excess of Bax to the membrane through a Bax-autoactivating mechanism (Tan et al 2006). Indeed, experimental evidence obtained with a stapled Bax BH3 peptide similar to the Bim peptide described above suggests self-propagating autoactivation of Bax (Gavathiotis et al 2010).…”

“…Bax is subsequently recruited by tBid and integrates into the membrane, which can be inhibited by the addition of Bcl-XL. When Bax becomes activated at the membrane, the rate-limiting step is its oligomerization, which involves the recruitment and activation of more Bax molecules (Tan et al 2006;Lovell et al 2008). While Bax oligomerizes, liposome contents are released because of membrane permeabilization at the same time.…”

Direct activation of full-length proapoptotic BAK

“…S7D). Finally, to explore whether the distinct trigger sites for initiating direct activation of BAK/BAX by BID/BIM BH3 helices are invoked during the self-propagation and/or oligomeric self-association steps of BAK/BAX activation (28,40,41), we generated BAK and BAX pSAHBs corresponding to the BAK and BAX BH3 helices for crosslinking analyses. Consistent with the results observed for BID and BIM pSAHBs, BAK and BAX pSAHBs crosslinked exclusively to residues of the canonical BH3-binding pocket of FL-BAK (Fig.…”

Two different HPV-11E6 fusion proteins trap p53 in the cytoplasm and induce apoptosis

“…10,11 However, some recent reports have indicated that p53 can mediate apoptosis when engaged directly from the cytoplasm. [12][13][14] Therefore, changes in the subcellular distribution of p53 partly contribute to its function. 15,16 Little is known about whether the overexpression of low risk E6 protein alters the distribution of p53 protein, and what happens next.…”