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The transmembrane BAX inhibitor-1-containing motif 6 (TMBIM6) is suggested to modulate apoptosis by regulating calcium homeostasis in the endoplasmic reticulum (ER). However, the precise molecular mechanism underlying this calcium regulation remains poorly understood. To shed light on this issue, we investigated all negatively charged residues in BsYetJ, a bacterial homolog of TMBIM6, using mutagenesis and fluorescence-based functional assays. We reconstituted BsYetJ in membrane vesicles with a lipid composition similar to that of the ER. Our results show that the charged residues E49 and R205 work together as a major gate, regulating calcium conductance in these ER-like lipid vesicles. However, these residues become largely inactive when reconstituted in other lipid environments. In addition, we found that D195 acts as a minor filter compared to the E49-R205 dyad. Our study uncovers a previously unknown function of BsYetJ/TMBIM6 in the calcium-dependent inactivation of BsYetJ, providing a framework for the development of a lipid-dependent mechanistic model of BsYetJ that will facilitate our understanding of calcium-dependent apoptosis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yun-Wei Chiang reports financial support was provided by National Science and Technology Council of Taiwan. Yun-Wei Chiang reports financial support was provided by Ministry of Education of Taiwan., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: BAX activation is a crucial step for commitment to apoptosis. Several activators, such as BimBH3-based therapeutic peptides and cleaved Bid (cBid) protein, can trigger BAX-mediated apoptosis, but it is unclear whether they proceed through the same pathway., Methods: Here we utilize PEGylation-based approach, which is shown to efficiently shield individual binding grooves in BAX from activators, to investigate and reveal that the activators take different routes to induce BAX-mediated apoptosis. Various spectroscopic/biochemical tools, including electron spin resonance, circular dichroism, fluorescence recovery after photobleaching, and label-transfer assay, were employed to reveal details in the processes., Results: We observe a key mutant BAX 164-PEG that acts differently in response to cBid and BimBH3 stimuli. While BimBH3 directly interacts with the trigger groove (TG) to induce the conformational changes in BAX that includes the release of α9 from the canonical groove (CG) and oligomerization, cBid engages with CG and works with mitochondrial lipids to fully activate BAX., Conclusion: PEGylation-based approach is proven useful to shield individual binding grooves of BAX from apoptotic stimuli. Groove engagement in CG of BAX is required for a full cBid-induced BAX activation. This study has identified differences in the pathways involved during the initiation of BAX activation by full-length cBid protein versus synthetic BimBH3-based peptides., General Significance: Our finding is potentially valuable for therapeutic application as the pore-forming activity of 164-PEG is independent from the cBid-mediated apoptotic pathways, but can be administrated by the synthetic short peptides., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

BCL-2-associated X (BAX) protein acts as a gatekeeper in regulating mitochondria-dependent apoptosis. Under cellular stress, BAX becomes activated and transforms into a lethal oligomer that causes mitochondrial outer membrane permeabilization (MOMP). Previous studies have identified several structural features of the membrane-associated BAX oligomer; they include the formation of the BH3-in-groove dimer, the collapse of the helical hairpin α5-α6, and the membrane insertion of α9 helix. However, it remains unclear as to the role of lipid environment in determining the conformation and the pore-forming activity of the BAX oligomers. Here we study molecular details of the membrane-associated BAX in various lipid environments using fluorescence and ESR techniques. We identify the inactive versus active forms of membrane-associated BAX, only the latter of which can induce stable and large membrane pores that are sufficient in size to pass apoptogenic factors. We reveal that the presence of CL is crucial to promoting the association between BAX dimers, hence the active oligomers. Without the presence of CL, BAX dimers assemble into an inactive oligomer that lacks the ability to form stable pores in the membrane. This study suggests an important role of CL in determining the formation of active BAX oligomers., (Copyright © 2018 Elsevier B.V. All rights reserved.)