Infection of Rift Valley fever virus (RVFV), a highly pathogenic mosquito-borne zoonotic virus, triggers severe inflammatory pathogenesis but the underlying mechanism of inflammation activation is currently unclear. Here, we report that the non-structural protein NSs of RVFV triggers mitochondrial damage to activate the NLRP3 inflammasome leading to viral pathogenesis in vivo. It is found that the host transcription inhibition effect of NSs causes rapid down-regulation of myeloid cell leukemia-1(MCL-1), a pro-survival member of the Bcl-2 (B-cell lymphoma protein 2) protein family. MCL-1 down-regulation led to BAK activation in the mitochondria, which triggered mtROS production and release of oxidized mitochondrial DNA (ox-mtDNA) into the cytosol. Cytosolic ox-mtDNA binds and activates the NLRP3 inflammasome triggering NLRP3-GSDMD pyroptosis in RVFV infected cells. A NSs mutant virus (RVFV-NSsRM) that is compromised in inducing transcription inhibition did not trigger MCL-1 down-regulation nor NLRP3-GSDMD pyroptosis. RVFV infection of the Nlrp3-/- mouse model demonstrated that the RVFV-triggered NLRP3 pyroptosis contributed to RVFV inflammatory pathogenesis and fatal infection in vivo. Infection with the RVFV-NSsRM mutant virus similarly showed alleviated inflammatory pathogenesis and reduced fatality rate. Taken together, these results revealed a mechanism by which a virulence factor activates the mitochondrial MCL-1-BAK axis through inducing host transcription inhibition to trigger NLRP3-dependent inflammatory pathogenesis.

Mitochondrial stress, resulting from dysfunction and proteostasis disturbances, triggers the mitochondrial unfolded protein response (UPRMT), which activates gene encoding chaperones and proteases to restore mitochondrial function. Although ATFS-1 mediates mitochondrial stress UPRMT induction in C. elegans, the mechanisms relaying mitochondrial stress signals to the nucleus in mammals remain poorly defined. Here, we explored the role of protein kinase R (PKR), an eIF2α kinase activated by double-stranded RNAs (dsRNAs), in mitochondrial stress signaling. We found that UPRMT does not occur in cells lacking PKR, indicating its crucial role in this process. Mechanistically, we observed that dsRNAs accumulate within mitochondria under stress conditions, along with unprocessed mitochondrial transcripts. Furthermore, we demonstrated that accumulated mitochondrial dsRNAs in mouse embryonic fibroblasts (MEFs) deficient in the Bax/Bak channels are not released into the cytosol and do not induce the UPRMT upon mitochondrial stress, suggesting a potential role of the Bax/Bak channels in mediating the mitochondrial stress response. These discoveries enhance our understanding of how cells maintain mitochondrial integrity, respond to mitochondrial dysfunction, and communicate stress signals to the nucleus through retrograde signaling. This knowledge provides valuable insights into prospective therapeutic targets for diseases associated with mitochondrial stress.

The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target.

Bak is a pore-forming Bcl2 protein that induces apoptosis at the outer mitochondrial membrane, which can either proceed via Bak oligomerization or be inhibited by anti-apoptotic Bcl2 proteins, such as BclxL. BclxL is very efficient in inhibiting Bak pore formation, but the mechanistic basis of this preferred interaction has remained enigmatic. Here, we identify Bakα1 as a second binding site for BclxL and show that it specifically interacts with the Bcl2-homology (BH)3 binding groove of BclxL. The affinity between BclxL and Bakα1 is weaker than with Bak-BH3, suggesting that Bakα1, being exposed early in the pore-forming trajectory, transiently captures BclxL, which subsequently transitions to the proximal BH3 site. Bak variants where the initial transient interaction with BclxL is modulated show a markedly altered response to BclxL inhibition. This work contributes to a better mechanistic understanding of the fine-tuned interactions between different players of the Bcl2 protein family.

BAX and BAK are proapoptotic members of the BCL2 family that directly mediate mitochondrial outer membrane permeabilition (MOMP), a central step in apoptosis execution. However, the molecular architecture of the mitochondrial apoptotic pore remains a key open question and especially little is known about the contribution of lipids to MOMP. By performing a comparative lipidomics analysis of the proximal membrane environment of BAK isolated in lipid nanodiscs, we find a significant enrichment of unsaturated species nearby BAK and BAX in apoptotic conditions. We then demonstrate that unsaturated lipids promote BAX pore activity in model membranes, isolated mitochondria and cellular systems, which is further supported by molecular dynamics simulations. Accordingly, the fatty acid desaturase FADS2 not only enhances apoptosis sensitivity, but also the activation of the cGAS/STING pathway downstream mtDNA release. The correlation of FADS2 levels with the sensitization to apoptosis of different lung and kidney cancer cell lines by co-treatment with unsaturated fatty acids supports the relevance of our findings. Altogether, our work provides an insight on how local lipid environment affects BAX and BAK function during apoptosis.

The voltage-dependent anion channel 2 (VDAC2) is the abundant protein in the outer mitochondrial membrane. Opening VDAC2 pores leads to the induction of mitochondrial energy and material transport, facilitating interaction with various mitochondrial proteins implicated in essential processes such as cell apoptosis and proliferation. To investigate the VDAC2 in lower vertebrates, we identified Lr-VDAC2, a homologue of VDAC2 found in lamprey (Lethenteron reissneri), sharing a sequence identity of greater than 50 % with its counterparts. Phylogenetic analysis revealed that the position of Lr-VDAC2 aligns with the lamprey phylogeny, indicating its evolutionary relationship within the species. The Lr-VDAC2 protein was primarily located in the mitochondria of lamprey cells. The expression of the Lr-VDAC2 protein was elevated in high energy-demanding tissues, such as the gills, muscles, and myocardial tissue in normal lampreys. Lr-VDAC2 suppressed H2O2 (hydrogen peroxide)-induced 293 T cell apoptosis by reducing the expression levels of Caspase 3, Caspase 9, and Cyt C (cytochrome c). Further research into the mechanism indicated that the Lr-VDAC2 protein inhibited the pro-apoptotic activity of BAK (Bcl-2 antagonist/killer) protein by downregulating its expression at the protein translational level, thus exerting an anti-apoptotic function similar to the role of VDAC2 in humans.

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial channel protein VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, while interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease or to promote BAK-mediated apoptosis for cancer therapy.

BH3-mimetics represent promising anti-cancer agents in tumors that rely on the anti-apoptotic function of B-Cell Lymphoma 2 (BCL2) proteins, particularly in leukemia and lymphoma cells primed for apoptosis. Mechanistically, BH3-mimetics may displace pro-apoptotic binding partners thus inducing BAX/BAK-mediated mitochondrial permeabilization followed by cytochrome c release, activation of the caspase cascade and apoptosis. Here, we describe a novel mode of caspase-independent cell death (CICD) induced by BH3-mimetics in a subset of diffuse large B-cell lymphoma (DLBCL) cells. Of note, rather than occurring via necroptosis, CICD induced immediately after mitochondrial permeabilization was associated with transcriptional reprogramming mediated by activation of c-Jun N-terminal Kinase (JNK) signaling and Activator Protein 1 (AP1). Thereby, CICD resulted in the JNK/AP1-mediated upregulation of inflammatory chemokines and increased migration of cytotoxic Natural Killer (NK) cells. Taken together, our study describes a novel mode of CICD triggered by BH3-mimetics that may alter the immune response towards dying cells.

Tamoxifen (TAM) resistance presents a major clinical obstacle in the management of estrogen-sensitive breast cancer, highlighting the need to understand the underlying mechanisms and potential therapeutic approaches. We showed that dysregulated mitochondrial dynamics were involved in TAM resistance by protecting against mitochondrial apoptosis. The dysregulated mitochondrial dynamics were associated with increased mitochondrial fusion and decreased fission, thus preventing the release of mitochondrial cytochrome c to the cytoplasm following TAM treatment. Dynamin-related GTPase protein mitofusin 1 (MFN1), which promotes fusion, was upregulated in TAM-resistant cells, and high MFN1 expression indicated a poor prognosis in TAM-treated patients. Mitochondrial translocation of MFN1 and interaction between MFN1 and mitofusin 2 (MFN2) were enhanced to promote mitochondrial outer membrane fusion. The interaction of MFN1 and cristae-shaping protein optic atrophy 1 (OPA1) and OPA1 oligomerization were reduced due to augmented OPA1 proteolytic cleavage, and their apoptosis-promoting function was reduced due to cristae remodeling. Furthermore, the interaction of MFN1 and BAK were increased, which restrained BAK activation following TAM treatment. Knockdown or pharmacological inhibition of MFN1 blocked mitochondrial fusion, restored BAK oligomerization and cytochrome c release, and amplified activation of caspase-3/9, thus sensitizing resistant cells to apoptosis and facilitating the therapeutic effects of TAM both in vivo and in vitro. Conversely, overexpression of MFN1 alleviated TAM-induced mitochondrial apoptosis and promoted TAM resistance in sensitive cells. These results revealed that dysregulated mitochondrial dynamics contributes to the development of TAM resistance, suggesting that targeting MFN1-mediated mitochondrial fusion is a promising strategy to circumvent TAM resistance.

BAX and BAK are pro-apoptotic members of the BCL2 family that are required to permeabilize the mitochondrial outer membrane. The proteins can adopt a non-activated monomeric conformation, or an activated conformation in which the exposed BH3 domain facilitates binding either to a prosurvival protein or to another activated BAK or BAX protein to promote pore formation. Certain cancer cells are proposed to have high levels of activated BAK sequestered by MCL1 or BCLXL, thus priming these cells to undergo apoptosis in response to BH3 mimetic compounds that target MCL1 or BCLXL. Here we report the first antibody, 14G6, that is specific for the non-activated BAK conformer. A crystal structure of 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites involved in the unfolding of BAK during its activation. In mitochondrial experiments, 14G6 inhibited BAK unfolding triggered by three diverse BAK activators, supporting crucial roles for both α1 dissociation and separation of the core (α2-α5) and latch (α6-α9) regions in BAK activation. 14G6 bound the majority of BAK in several leukaemia cell lines, and binding decreased following treatment with BH3 mimetics, indicating only minor levels of constitutively activated BAK in those cells. In summary, 14G6 provides a new means of assessing BAK status in response to anti-cancer treatments.