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International Journal of Molecular... Jul 2021The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the...
The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D-KD decreasing from ≃1.6μm-2 to ≃0.1μm-2) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid.
Topics: Animals; BH3 Interacting Domain Death Agonist Protein; Humans; Lipid Bilayers; Mice; Protein Binding; Protein Conformation; bcl-2-Associated X Protein
PubMed: 34361006
DOI: 10.3390/ijms22158240 -
Neuroscience Research Sep 2022BAX is a Bcl-2 family protein acting on apoptosis. It also promotes mitochondrial fusion by interacting with the mitochondrial fusion protein Mitofusin (Mfn1 and Mfn2)....
BAX is a Bcl-2 family protein acting on apoptosis. It also promotes mitochondrial fusion by interacting with the mitochondrial fusion protein Mitofusin (Mfn1 and Mfn2). Neuronal mitochondria are important for the development and modification of dendritic spines, which are subcellular compartments accommodating excitatory synapses in postsynaptic neurons. The abundance of dendritic mitochondria influences dendritic spine development. Mitochondrial fusion is essential for mitochondrial homeostasis. Here, we show that in the hippocampal neuron of BAX knockout mice, mitochondrial fusion is impaired, leading to decreases in mitochondrial length and total mitochondrial mass in dendrites. Notably, BAX knockout mice also have fewer dendritic spines and less cellular Adenosine 5'triphosphate (ATP) in dendrites. The spine and ATP changes are abolished by restoring mitochondria fusion via overexpressing Mfn1 and Mfn2. These findings indicate that BAX-mediated mitochondrial fusion in neurons is crucial for the development of dendritic spines and the maintenance of cellular ATP levels.
Topics: Adenosine Triphosphate; Animals; Dendritic Spines; GTP Phosphohydrolases; Mice; Mitochondrial Dynamics; Mitochondrial Proteins; bcl-2-Associated X Protein
PubMed: 35688289
DOI: 10.1016/j.neures.2022.06.002 -
Scientific Reports Feb 2022Apoptosis, programmed cell death, plays a central role in haematopoiesis. Mature erythrocytes of non-mammalian vertebrates maintain a permanent nucleus; these cells can...
Apoptosis, programmed cell death, plays a central role in haematopoiesis. Mature erythrocytes of non-mammalian vertebrates maintain a permanent nucleus; these cells can undergo apoptosis (eryptosis), as do other somatic cells of a given non-mammalian vertebrate. In this study, we have investigated the expression and subcellular distribution of Bcl-2, Bcl-X and Bax proteins in the maturation phases and after X-ray irradiation of nucleated erythrocytes of Torpedo marmorata and Caretta caretta and the effect of X-ray irradiation on nucleated circulating erythrocytes of Torpedo marmorata. The cellular distribution of proteins was detected in erythrocytes by using immunocytochemistry at light microscopy and immunoelectron microscopy. The electrophoretic separation and immunoblotting of pro- and anti-apoptotic proteins of immature and mature erythroid cells was performed too, after X-ray irradiation of torpedoes. The results of the immunocytochemical analyses show an increase, in the expression level of Bax in mature as compared to young erythrocytes and a corresponding decrease of Bcl-2 and Bcl-X. This maturation pattern of Bax, Bcl-2 and Bcl-X was abrogated in X-ray irradiated torpedo erythrocytes. On the basis of these observations, Bax, Bcl-2 and Bcl-X seems to play a role in the erythropoiesis of Torpedo marmorata Risso and in Caretta caretta. In conclusion, the same apoptotic proteins of somatic cells appear to be conserved in circulating nucleated erythrocytes thus suggesting to play a role in the maturation of these cells.
Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Erythropoiesis; Proto-Oncogene Proteins c-bcl-2; Vertebrates; bcl-2-Associated X Protein; bcl-X Protein
PubMed: 35228540
DOI: 10.1038/s41598-022-06617-6 -
The Journal of Biological Chemistry Nov 2013The Bcl-2 proapoptotic proteins Bax and Bak mediate the permeabilization of the mitochondrial outer membrane during apoptosis. Current models consider that Bax and Bak...
The Bcl-2 proapoptotic proteins Bax and Bak mediate the permeabilization of the mitochondrial outer membrane during apoptosis. Current models consider that Bax and Bak form pores at the mitochondrial outer membrane that are responsible for the release of cytochrome c and other larger mitochondrial apoptotic factors (i.e. Smac/DIABLO, AIF, and endoglycosidase G). However, the properties and nature of Bax/Bak apoptotic pores remain enigmatic. Here, we performed a detailed analysis of the membrane permeabilizing activity of Bax and Bak at the single vesicle level. We directly visualized that cBid-activated Bax and BakΔC21 can form membrane pores large enough to release not only cytochrome c, but also allophycocyanine, a protein of 104 kDa. Interestingly, the size of Bax and BakΔC21 pores is not constant, as typically observed in purely proteinaceous channels, but evolves with time and depends on protein concentration. We found that Bax and BakΔC21 formed long-lived pores, whose areas changed with the amount of Bax/BakΔC21 but not with cardiolipin concentration. Altogether, our results demonstrate that Bax and BakΔC21 follow similar mechanisms of membrane permeabilization characterized by the formation of protein-permeable pores of dynamic size, in agreement with the proteolipidic nature of these apoptotic pores.
Topics: Animals; Cardiolipins; Humans; Membranes, Artificial; Mice; Multiprotein Complexes; Permeability; Recombinant Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 24100034
DOI: 10.1074/jbc.M113.512087 -
Experimental Biology and Medicine... Jun 2016Cells with DNA damage undergo apoptosis or cellular senescence if the damage cannot be repaired. Recent studies highlight that cellular senescence plays a major role in... (Review)
Review
Cells with DNA damage undergo apoptosis or cellular senescence if the damage cannot be repaired. Recent studies highlight that cellular senescence plays a major role in aging. However, age-associated diseases, including emphysema and neurodegenerative disorders, are caused by apoptosis of lung alveolar epithelial cells and neurons, respectively. Therefore, enhanced apoptosis also promotes aging and shortens the life span depending on the cell type. Recently, we reported that ku70(-) (/) (-)bax(-) (/) (-) and ku70(-) (/) (-)bax(+/) (-) mice showed significantly extended life span in comparison with ku70(-) (/) (-)bax(+/+) mice. Ku70 is essential for non-homologous end joining pathway for DNA double strand break repair, and Bax plays an important role in apoptosis. Our study suggests that Bax-induced apoptosis has a significant impact on shortening the life span of ku70(-) (/) (-) mice, which are defective in one of DNA repair pathways. The lung alveolar space gradually enlarges during aging, both in mouse and human, and this age-dependent change results in the decrease of respiration capacity during aging that can lead to emphysema in more severe cases. We found that emphysema occurred in ku70(-) (/) (-) mice at the age of three-months old, and that Bax deficiency was able to suppress it. These results suggest that Bax-mediated apoptosis induces emphysema in ku70(-) (/) (-) mice. We also found that the number of cells, including bronchiolar epithelial cells and type 2 alveolar epithelial cells, shows a higher DNA double strand break damage response in ku70 KO mouse lung than in wild type. Recent studies suggest that non-homologous end joining activity decreases with increased age in mouse and rat model. Together, we hypothesize that the decline of Ku70-dependent DNA repair activity in lung alveolar epithelial cells is one of the causes of age-dependent decline of lung function resulting from excess Bax-mediated apoptosis of lung alveolar epithelial cells (and their progenitor cells).
Topics: Animals; Apoptosis; DNA Repair; Emphysema; Ku Autoantigen; Longevity; Mice; Mice, Knockout; Survival Analysis; bcl-2-Associated X Protein
PubMed: 27302174
DOI: 10.1177/1535370216654587 -
Molecular Neurobiology Mar 2022The BCL-2 (B-cell lymphoma-2) family of proteins contributes to mitochondrial-based apoptosis in models of neurodegeneration, including glaucomatous optic neuropathy...
The BCL-2 (B-cell lymphoma-2) family of proteins contributes to mitochondrial-based apoptosis in models of neurodegeneration, including glaucomatous optic neuropathy (glaucoma), which degrades the retinal ganglion cell (RGC) axonal projection to the visual brain. Glaucoma is commonly associated with increased sensitivity to intraocular pressure (IOP) and involves a proximal program that leads to RGC dendritic pruning and a distal program that underlies axonopathy in the optic projection. While genetic deletion of the Bcl2-associated X protein (Bax) prolongs RGC body survival in models of glaucoma and optic nerve trauma, axonopathy persists, thus raising the question of whether dendrites and the RGC light response are protected. Here, we used an inducible model of glaucoma in Bax mice to determine if Bax contributes to RGC dendritic degeneration. We performed whole-cell recordings and dye filling in RGCs signaling light onset (αON-Sustained) and offset (αOFF-Sustained). We recovered RGC dendritic morphologies by confocal microscopy and analyzed dendritic arbor complexity and size. Additionally, we assessed RGC axon function by measuring anterograde axon transport of cholera toxin subunit B to the superior colliculus and behavioral spatial frequency threshold (i.e., spatial acuity). We found 1 month of IOP elevation did not cause significant RGC death in either WT or Bax retinas. However, IOP elevation reduced dendritic arbor complexity of WT αON-Sustained and αOFF-Sustained RGCs. In the absence of Bax, αON- and αOFF-Sustained RGC dendritic arbors remained intact following IOP elevation. In addition to dendrites, neuroprotection by Bax generalized to αON-and αOFF-Sustained RGC light- and current-evoked responses. Both anterograde axon transport and spatial acuity declined during IOP elevation in WT and Bax mice. Collectively, our results indicate Bax contributes to RGC dendritic degeneration and distinguishes the proximal and distal neurodegenerative programs involved during the progression of glaucoma.
Topics: Animals; Disease Models, Animal; Glaucoma; Intraocular Pressure; Mice; Retinal Ganglion Cells; bcl-2-Associated X Protein
PubMed: 34984584
DOI: 10.1007/s12035-021-02675-5 -
Cell Cycle (Georgetown, Tex.) 2017It has become more and more evident that the BCL-2 family proteins mediate a wide range of non-apoptotic functions. The pro-apoptotic BAX protein has been reported in...
It has become more and more evident that the BCL-2 family proteins mediate a wide range of non-apoptotic functions. The pro-apoptotic BAX protein has been reported in interphasic nuclei. Whether the nuclear form of BAX could be involved in non-apoptotic function is still unknown. Our study showed for the first time that BAX was associated with chromatin in vitro. Next, we used gain and loss of function approaches to decipher the potential role of nuclear BAX in non-apoptotic cells. In vitro, nuclear BAX promoted cell proliferation in lung epithelial cells and primary human lung fibroblasts by modulating CDKN1A expression. Interestingly, BAX occupancy of CDKN1A promoter was specifically enriched close to the transcription-starting site. Nuclear BAX also modulated the basal myofibroblastic differentiation and migration of primary human lung fibroblasts. Finally, BAX nuclear localization was associated in vivo with the remodelling of lung parenchyma during development, tumorigenesis as well as fibrosis compared to control adult human lungs. Hence, our study established for the first time, a strong link between the nuclear localization of the pro-apoptotic BAX protein and key basic cellular functions in the non-apoptotic setting.
Topics: Apoptosis; Cell Differentiation; Cell Nucleus; Cell Proliferation; Fibroblasts; Humans; Interphase; Mitochondria; Proto-Oncogene Proteins c-bcl-2; bcl-2-Associated X Protein
PubMed: 28933587
DOI: 10.1080/15384101.2017.1371882 -
Sovremennye Tekhnologii V Meditsine 2021was to assess the levels of Всl-2 and Bax proteins in the vascular wall and their correlation with serum cholesterol in patients with stage III-IV atherosclerosis...
UNLABELLED
was to assess the levels of Всl-2 and Bax proteins in the vascular wall and their correlation with serum cholesterol in patients with stage III-IV atherosclerosis obliterans of lower limb arteries.
MATERIALS AND METHODS
The study included 32 patients with stage III-IV atherosclerosis obliterans of the lower limb. Samples of intraoperative material (all three layers of the vascular wall) including an atherosclerotic plaque (AP) were taken during primary open surgery on major leg arteries. As a control, we used samples of the arterial wall without visible signs of atherosclerosis. Based on AP ultrasonography, the patients were divided into two groups: with APs of mixed echogenicity and with hyperechoic (calcified) AP. The vascular samples were crushed and homogenized for further measurements of Всl-2 and Bax proteins; in a separate setup, cholesterol in blood serum was measured.
RESULTS
In patients without atherosclerotic changes, the level of the anti-apoptotic protein Bcl-2 in the arterial wall was 1.25 ng/mg, and that of the pro-apoptotic protein Bax - 4.7 ng/mg. In the case of APs of mixed echogenicity, the expression of Bcl-2 was 1.8 ng/mg (p=0.143) and that of Bax - 5.1 ng/mg (p=0.834), with no significant differences from AP-free vascular wall samples. In the arterial wall containing a heterogeneous calcified AP, the expression of Bcl-2 was 0.9 ng/mg (p=0.143), In contrast, the level of Bax was 6.8 ng/mg, which showed its significant increase as compared with the non-AP controls (p=0.02). In the cases with predominantly hyperechoic AP, the expression of Bcl-2 was significantly lower (p=0.036), and that of Bax - significantly higher (p=0.036) in comparison with AP of mixed echogenicity. In patients with hyperechoic AP, we found a negative correlation between the Bax and Bcl-2 values ( r=-0.315) and a positive correlation between the Bax expression and serum cholesterol (r=0.617).
CONCLUSION
In arterial walls with hyperechoic (calcified) APs, the expression of anti-apoptotic protein Bcl-2 is reduced, and that of pro-apoptotic protein Bax is increased, which indicates the apoptosis activation in advanced atherosclerotic lesions. In patients with such APs, elevated cholesterol levels directly correlate with the increased expression of pro-apoptotic Bax protein (r=0.617).
Topics: Apoptosis; Arteries; Atherosclerosis; Humans; Proto-Oncogene Proteins c-bcl-2; bcl-2-Associated X Protein
PubMed: 34513076
DOI: 10.17691/stm2021.13.2.05 -
Nature Communications Nov 2023Since adult stem cells are responsible for replenishing tissues throughout life, it is vital to understand how failure to undergo apoptosis can dictate stem cell...
Since adult stem cells are responsible for replenishing tissues throughout life, it is vital to understand how failure to undergo apoptosis can dictate stem cell behavior both intrinsically and non-autonomously. Here, we report that depletion of pro-apoptotic Bax protein bestows hair follicle stem cells with the capacity to eliminate viable neighboring cells by sequestration of TNFα in their membrane. This in turn induces apoptosis in "loser" cells in a contact-dependent manner. Examining the underlying mechanism, we find that Bax loss-of-function competitive phenotype is mediated by the intrinsic activation of NFκB. Notably, winner stem cells differentially respond to TNFα, owing to their elevated expression of TNFR2. Finally, we report that in vivo depletion of Bax results in an increased stem cell pool, accelerating wound-repair and de novo hair follicle regeneration. Collectively, we establish a mechanism of mammalian cell competition, which can have broad therapeutic implications for tissue regeneration and tumorigenesis.
Topics: Animals; Tumor Necrosis Factor-alpha; Cell Competition; bcl-2-Associated X Protein; Wound Healing; Hair Follicle; Stem Cells; Mammals
PubMed: 37985759
DOI: 10.1038/s41467-023-41684-x -
Proceedings of the National Academy of... Aug 2021Death receptor-mediated apoptosis requires the mitochondrial apoptosis pathway in many mammalian cells. In response to death receptor signaling, the truncated BH3-only...
Death receptor-mediated apoptosis requires the mitochondrial apoptosis pathway in many mammalian cells. In response to death receptor signaling, the truncated BH3-only protein BID can activate the proapoptotic BCL-2 proteins BAX and BAK and trigger the permeabilization of the mitochondria. BAX and BAK are inhibited by prosurvival BCL-2 proteins through retrotranslocation from the mitochondria into the cytosol, but a specific resistance mechanism to truncated BID-dependent apoptosis is unknown. Here, we report that hexokinase 1 and hexokinase 2 inhibit the apoptosis activator truncated BID as well as the effectors BAX and BAK by retrotranslocation from the mitochondria into the cytosol. BCL-2 protein shuttling and protection from TRAIL- and FasL-induced cell death requires mitochondrial hexokinase localization and interactions with the BH3 motifs of BCL-2 proteins but not glucose phosphorylation. Together, our work establishes hexokinase-dependent retrotranslocation of truncated BID as a selective protective mechanism against death receptor-induced apoptosis on the mitochondria.
Topics: Anti-Bacterial Agents; Antibiotics, Antineoplastic; Apoptosis; Cell Line; Cyclosporine; Dactinomycin; Doxorubicin; Enzyme Inhibitors; Fas Ligand Protein; Gene Deletion; Gene Expression Regulation, Enzymologic; Hexokinase; Humans; Mitochondria; TNF-Related Apoptosis-Inducing Ligand; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 34385311
DOI: 10.1073/pnas.2021175118