Abstract 3704: The C-terminal transmembrane domain of BAX is essential for BAX auto-inhibition

Abstract

Abstract Apoptosis is a complex and highly regulated cellular suicide program culminating in a caspase-dependent self-destruction. As a critical step of intrinsic apoptosis, pro-apoptotic effector Bcl-2 proteins BAX, BAK and BOK form pores in the mitochondrial outer membrane (MOM) which causes Cytochrome C release, followed by caspase-activation and ultimately cell death. In contrast to BAK and BOK, BAX mainly resides in the cytosol and translocates to mitochondria when activated by pro-apoptotic signals. During activation and translocation, BAX undergoes conformational changes including dislodging of the c-terminal α-9 helix from the hydrophobic groove and insertion in the MOM. Most studies concerning BAX activation focus on the interaction of the BH3 domain and the hydrophobic groove, which is essential for BAX inhibition by members of the Bcl-2 family. In contrast, how modifications of the α-9 transmembrane domain (TMD) can impair BAX inhibition and membrane insertion remains understudied so far, so that functional roles of the TMD in BAX activation and translocation are yet to be defined. To determine the role of the BAX-TMD in activation and translocation, we generated several plasmids encoding for BAX chimeras in which the original BAX-TMD sequence was swapped for TMDs of other Bcl-2 proteins. The chimeric BAX proteins were expressed in human cell lines and cell death as well as protein localisation was investigated. Surprisingly, we found that swapping the BAX-TMD for another effector TMD increases BAX pro-apoptotic activity assessed via Annexin-V staining and flow cytometry. In addition, confocal microscopy revealed that BAX shows an increased tendency to mitochondrial localisation and oligomerisation when bearing TMDs of BAK or BOK. Interestingly, co-expressed anti-apoptotic BCL-2 an antagonistic binding-partner of BAX co-localises with BAX chimeras but poorly prevents cell death induced by the BAX chimera with the BAK-TMD. These findings strongly support a crucial regulatory role of the BAX-TMD in activation and translocation. In fact, the increased apoptotic potential of the BAX-TMD chimeras as well as augmented mitochondrial localisation suggest an auto-inhibitory function of the BAX-TMD ensured by its affinity to the BAX hydrophobic groove. Moreover, the decreased inhibitory capacity of Bcl-2 for the BAK-TMD-containing BAX chimera could point to an involvement of the BAX-TMD in interaction with BCL-2. In contrast to the archetypical understanding of the TMD as a mere membrane anchor, these results also indicate that the TMD can regulate the activity of BAX and other Bcl-2 family proteins. Citation Format: Tobias Benjamin Beigl, Sandra Weller, Benjamin Schäfer, Walter Erich Aulitzky, Hans-Georg Kopp, Markus Rehm, Frank Essmann. The C-terminal transmembrane domain of BAX is essential for BAX auto-inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3704.