Bid chimeras indicate that most BH3-only proteins can directly activate Bak and Bax,and show no preference for Bak versus Bax

The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-x_L according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.The Bcl-2 family of proteins controls the mitochondrial pathway of apoptosis, a process often dysregulated in cancer and other diseases.^{1, 2, 3} Apoptotic triggers including DNA damage and oncogene activation cause the synthesis or activation of one or more pro-apoptotic Bcl-2 homology region 3 (BH3)-only proteins,^{1, 2, 3, 4} a subfamily that includes Bid, Bim, Puma, Noxa, Bad, Bik, Bmf and Hrk. These proteins then engage via their BH3 domain with other Bcl-2 family members. BH3-only proteins that can directly bind and activate the Bcl-2 effector proteins Bak or Bax are called ‘activators''.⁵ When Bak or Bax become activated and oligomerize in the mitochondrial outer membrane (MOM), the apoptotic ‘switch'' has flipped and the cell is committed to cell death. The prosurvival members (Bcl-2, Bcl-x_L, Mcl-1, Bcl-w, Bfl-1/A1 and Bcl-B) inhibit apoptosis by specifically binding both the BH3-only proteins and activated Bak and Bax.^{6, 7, 8, 9, 10, 11} Thus, the cell''s complement of prosurvival proteins, Bak, and Bax, determines the sensitivity of that cell to each BH3-only protein, and by extension to each type of pro-apoptotic stimulus.A thorough understanding of BH3-only proteins is crucial for the development of cancer therapeutics such as the new class of anti-cancer molecules called BH3 mimetics that are showing significant promise in clinical trials.^{12, 13} The binding of BH3-only proteins to prosurvival proteins has been well-characterized and revealed significant preferences for engaging different members.^{6, 8, 9} How BH3-only proteins bind and activate Bak and Bax remains less understood for several reasons. First, generating stable recombinant BH3-only proteins is difficult because, except for Bid, they are intrinsically disordered^{14, 15, 16} and because most contain hydrophobic C-terminal membrane anchors.¹⁷ Thus, most in vitro studies of BH3-only proteins have used synthetic peptides corresponding to the BH3 domains, C-terminally truncated recombinant proteins or in vitro translated (IVT) proteins. Second, BH3-only reagents bind poorly to recombinant Bak and Bax in the absence of membranes, although detergents and liposomes may substitute for the MOM.^{18, 19, 20} Third, activation of Bak and Bax on mitochondria can be complicated by the presence of other proteins such as prosurvival proteins. Indeed, genetically altering BH3-only protein levels in mice resulted in complex phenotypes due to multiple interactions between family members, precluding firm conclusions as to which BH3-only proteins are direct activators.^{18, 21, 22}Bid and Bim are direct activators according to a variety of approaches,^{5, 8, 9, 23, 24} and were recently proposed to be specific for Bak and Bax, respectively.²⁵ Early studies using Noxa BH3 peptides^{5, 8} and IVT Noxa⁹ concluded that Noxa was not an activator. However, in more recent studies a Noxa BH3 peptide²³ and purified recombinant NoxaΔC²⁰ were found to be activators of both Bak and Bax. Puma has also been described as both an activator^{26, 27} and not an activator.^{8, 28} Du et al.²³ analyzed the full panel of BH3 peptides and classified Bim as a strong activator, Bid, Noxa and Bmf as moderate activators, and Puma, Bik and Hrk as weak activators. The only BH3-only member that has never been described as an activator is Bad.While BH3 peptides and recombinant truncated BH3-only proteins have been useful for in vitro studies, new reagents that target mitochondria may better reflect the behavior of the parent proteins. As Bid is stable as a recombinant protein, we generated chimeras of Bid in which the BH3 domain of Bid was replaced with that of seven other BH3-only proteins. This is a similar approach to the Bim chimeras used for expression in cells¹⁸ and in mice.²⁹ More recently, truncated Bid (tBid) chimeras containing the BH3 domains of Bim, Bak and Bax as well as those of the prosurvival proteins, have been generated as IVT proteins.¹¹To compare the ability of BH3-only proteins to activate Bak and Bax in vitro, we incubated Bid chimeras and BH3 peptides with mitochondria containing either Bak or Bax. We found that the membrane-targeted Bid chimeras were much more potent activators than their related BH3 peptides, and that all BH3 domains except for Bad and Noxa were activators to some extent. We conclude that activation of Bak and Bax may be underestimated by studies using BH3 peptides, and that even BH3-only proteins such as Bik, Bmf and Hrk that are often considered unable to activate Bak or Bax, may act as activators under certain conditions.