BH3 Domains of BH3-Only Proteins Differentially Regulate Bax-Mediated Mitochondrial Membrane Permeabilization Both Directly and Indirectly

Abstract

Using a Bax-dependent membrane-permeabilization assay, we show that peptides corresponding to the BH3 domains of Bcl-2 family "BH3-only" proteins have dual functions. Several BH3 peptides relieved the inhibition of Bax caused by the antiapoptotic Bcl-x(L) and/or Mcl-1 proteins, some displaying a specificity for either Bcl-x(L) or Mcl-1. Besides having this derepression function, the Bid and Bim peptides activated Bax directly and were the only BH3 peptides tested that could potently induce cytochrome c release from mitochondria in cultured cells. Furthermore, Bax activator molecules (cleaved Bid protein and the Bim BH3 peptide) synergistically induced cytochrome c release when introduced into cells along with derepressor BH3 peptides. These observations support a unified model of BH3 domain function, encompassing both positive and negative regulation of other Bcl-2 family members. In this model, the simple inhibition of antiapoptotic functions is insufficient to induce apoptosis unless a direct activator of Bax or Bak is present.

The BCL-2 protein family, BH3-mimetics and cancer therapy

“…Pertinently, the potent BH3-only proteins bind avidly to all prosurvival BCL-2 family members and can also engage BAX/ BAK, whereas the less potent ones have more select binding specificities for the pro-survival BCL-2 family members and reportedly do not bind to BAX or BAK. 31,32 Mechanisms of BH3-only protein activation. Because of their position at the apex of the BCL-2-regulated apoptotic pathway, the BH3-only proteins act as a fulcrum, determining whether the scales tip in favour of cell death or in favour of cell survival.…”

BH3-only proteins and their roles in programmed cell death

“…Using a large array of synthetic BH3 peptides, two groups later showed that some of these peptides (Bid, Bim and Puma) could directly induce oligomerization of Bax and Bak to release cytochrome c, whereas the others could not (Letai et al, 2002;Kuwana et al, 2005;Kim et al, 2006b). The term 'activator' was chosen to describe this particular ability.…”

“…Various groups have reported evidence to support the existence of the two proposed classes of BH3-only proteins. Kuwana et al (2005) used an innovative Bax-dependent membrane permeabilization assay to show that recombinant Bim and truncated Bid BH3 peptides were able to activate Bax directly whereas other BH3-only proteins required the presence of prosurvival proteins to induce permeabilization. To circumvent the shortcomings of using short BH3 peptides, another group used full-length proteins to assess the 'two class' model (Kim et al, 2006b).…”

“…The direct activation model defines Bim and Bid as the sole BH3-only direct activators of Bax and Bak (Letai et al, 2002;Kuwana et al, 2005). However, Willis et al showed that Bax and Bak can mediate apoptosis without discernable association with the putative BH3-only activators, even in cells with no Bim or Bid (Willis et al, 2007).…”

Frequent loss of expression of the pro-apoptotic protein Bim in renal cell carcinoma: evidence for contribution to apoptosis resistance

“…Activators (initially this group was limited to Bid and Bim but was later extended to include Puma) are suggested directly to bind Bax and cause its activation. Sensitizers, on the other hand, lack this capacity, and their function is limited to binding to prosurvival Bcl-2 proteins, thereby liberating activators and contributing to apoptosis (Kuwana et al, 2002(Kuwana et al, , 2005Letai et al, 2002;Kim et al, 2006). However, a central prediction of this model is that the combined loss of activators blocks all Bax/Bak-dependent apoptosis, but this is not the case (Kim et al, 2006).…”