Author:

Abstract:

The anti-apoptotic proteins Bcl-2 and Bcl-Xl are highly similar in sequence and structure, and so far considered as virtually exchangeable for regulating apoptosis. Both proteins conventionally promote cell survival by employing their hydrophobic cleft in the heterodimerizion process with pro-apoptotic Bcl-2 members, like Bax and Bak, and consequently preserving mitochondrial integrity. In addition to this, Bcl-2 safeguards against MOMP by preventing mitochondrial Ca2+ overload. The latter role seems primarily due to Bcl-2’s interaction with ER–resident IP3Rs on their central, regulatory domain, thereby preventing IP3R-dependent Ca2+ release and transfer to the mitochondria. The only Bcl-2 portion sufficient for IP3R inhibition is the N-terminal BH4 domain, which is exclusive for the anti-apoptotic Bcl-2-family members and also reported to be essential for their pro-survival function. Bcl-Xl too has been reported to bind and modulate IP3Rs but the molecular determinants involved in both binding partners are still unknown. In particular, Bcl-Xl may bind to the receptor on a different site, located close to the C-terminal, and stimulate basal/physiological IP3R-mediated Ca2+ oscillations in favor of a proper mitochondrial respiration. In this respect, Bcl-Xl seems to predominantly enhance the pro-survival IP3Rs signaling instead of hampering the anti-apoptotic one.During this Ph.D. research, we first examined side-by-side the molecular features of Bcl-2 and Bcl-Xl in this seemingly conflicting regulation of IP3R-dependent Ca2+ signaling and apoptosis. In more detail, we mainly compared the anti-apoptotic inhibition of IP3R-mediated Ca2+ release by the BH4 domains of Bcl-2 versus Bcl-Xl. In molecular (pull downs; surface plasmon resonance [SPR] analysis) and functional (Ca2+ measurements and apoptotic assays) experiments, the BH4-domain biology of Bcl-2 and Bcl-Xl at the level of IP3Rs appeared very different. BH4-Bcl-2 protected against Ca2+-mediated apoptosis by binding the IP3Rs and inhibiting IP3-induced Ca2+ release, whereas BH4-Bcl-Xl protection proceeded without any binding and regulation of the IP3R channel although acting on the same signaling cascade. Interestingly, the selective molecular liaison between Bcl-2 and the IP3Rs relates to a Lys17 in BH4-Bcl-2 that is not conserved in BH4-Bcl-Xl, in which it corresponds to an Asp residue, having an opposite charge. Next, we examined the structural features of the Bcl-2-BH4 domain in even more detail. Hence, we investigated the role played by the three-dimensional structure of a Bcl-2-BH4 peptide in the aforedescribed IP3R interaction. We thereby found that the isolated Bcl-2-BH4 domain requires an alpha-helical organization for binding the IP3Rs and to inhibit the pro-apoptotic channel activity.Finally, we extended our study towards an initial characterization of the full-length Bcl-2 and Bcl-Xl interactions with their proposed binding sites on IP3R1. In this way, we partially solved a molecular controversy in the Ca2+ field by demonstrating that the full-length Bcl-2 bound with similar efficiency to the two reported interaction sites on IP3R1, while Bcl-Xl bound more effectively to the IP3R1 C-terminal site.Collectively, these data support a selective and unique inhibition of the IP3Rs by Bcl-2. Bcl-2-BH4 domain is therefore in charge of targeting the channel’s central, regulatory domain and of curbing the pro-apoptotic ER Ca2+ release. Accordingly, even though both Bcl-2 and Bcl-Xl interact with the site next to the IP3R1-channel pore, Bcl-Xl is more likely acting as an endogenous IP3R sensitizer and promoter of pro-survival ER Ca2+ release. Indeed, its BH4 domain does not bear any function towards the IP3Rs and seems instead protecting against Ca2+-mediated apoptosis by acting downstream of the IP3Rs probably just prior to the MOMP.