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Biochimica Et Biophysica Acta Mar 2012In response to apoptotic stimuli, the pro-apoptotic protein Bax inserts in the outer mitochondrial membrane, resulting in the formation of pores and the release of...
In response to apoptotic stimuli, the pro-apoptotic protein Bax inserts in the outer mitochondrial membrane, resulting in the formation of pores and the release of several mitochondrial components, and sealing the cell's fate. To study the binding of Bax to membranes, we used an in vitro system consisting of 50nm diameter liposomes prepared with a lipid composition mimicking that of mitochondrial membranes in which recombinant purified full-length Bax was inserted via activation with purified tBid. We detected the association of the protein with the membrane using fluorescence fluctuation methods, and found that it could well be described by an equilibrium between soluble and membrane-bound Bax and that at a high protein-to-liposome ratio the binding seemed to saturate at about 15 Bax proteins per 50nm diameter liposome. We then obtained structural data for samples in this saturated binding regime using small-angle neutron scattering under different contrast matching conditions. Utilizing a simple model to fit the neutron data, we observed that a significant amount of the protein mass protrudes above the membrane, in contrast to the conjecture that all of the membrane-associated Bax states are umbrella-like. Upon protein binding, we also observed a thinning of the lipid bilayer accompanied by an increase in liposome radius, an effect reminiscent of the action of antimicrobial peptides on membranes.
Topics: Humans; Liposomes; Mitochondria; Models, Molecular; Neutron Diffraction; Protein Structure, Tertiary; Scattering, Small Angle; bcl-2-Associated X Protein
PubMed: 22037145
DOI: 10.1016/j.bbamem.2011.10.007 -
Parasites & Vectors Aug 2021The salivary glands of female ticks degenerate rapidly by apoptosis and autophagy after feeding. Bcl-2 family proteins play an important role in the apoptosis pathways,...
BACKGROUND
The salivary glands of female ticks degenerate rapidly by apoptosis and autophagy after feeding. Bcl-2 family proteins play an important role in the apoptosis pathways, but the functions of these proteins in ticks are unclear. We studied Bcl-2 and Bax homologs from Rhipicephalus haemaphysaloides and determined their functions in the degeneration of the salivary glands.
METHODS
Two molecules containing conserved BH (Bcl-2 family homology) domains were identified and named RhBcl-2 and RhBax. After protein purification and mouse immunization, specific polyclonal antibodies (PcAb) were created in response to the recombinant proteins. Reverse transcription quantitative PCR (RT-qPCR) and western blot were used to detect the presence of RhBcl-2 and RhBax in ticks. TUNEL assays were used to determine the level of apoptosis in the salivary glands of female ticks at different feeding times after gene silencing. Co-transfection and GST pull-down assays were used to identify interactions between RhBcl-2 and RhBax.
RESULTS
The RT-qPCR assay revealed that RhBax gene transcription increased significantly during feeding at all tick developmental stages (engorged larvae, nymphs, and adult females). Transcriptional levels of RhBcl-2 and RhBax increased more significantly in the female salivary glands than in other tissues post engorgement. RhBcl-2 silencing significantly inhibited tick feeding. In contrast, RhBax interference had no effect on tick feeding. TUNEL staining showed that apoptosis levels were significantly reduced after interference with RhBcl-2 expression. Co-transfection and GST pull-down assays showed that RhBcl-2 and RhBax could interact but not combine in the absence of the BH3 domain.
CONCLUSIONS
This study identified the roles of RhBcl-2 and RhBax in tick salivary gland degeneration and finds that the BH3 domain is a key factor in their interactions.
Topics: Animals; Apoptosis; Female; In Situ Nick-End Labeling; Mice; Proto-Oncogene Proteins; Rhipicephalus; Salivary Glands; bcl-2-Associated X Protein
PubMed: 34348769
DOI: 10.1186/s13071-021-04879-z -
Birth Defects Research Nov 2022During early development, alcohol exposure causes apoptotic cell death in discrete regions of the embryo which are associated with distinctive patterns of later-life...
BACKGROUND
During early development, alcohol exposure causes apoptotic cell death in discrete regions of the embryo which are associated with distinctive patterns of later-life abnormalities. In gastrulation, which occurs during the third week of human pregnancy, alcohol targets the ectoderm, the precursor of the eyes, face, and brain. This midline tissue loss leads to the craniofacial dysmorphologies, such as microphthalmia and a smooth philtrum, which define fetal alcohol syndrome (FAS). An important regulator of alcohol-induced cell death is the pro-apoptotic protein Bax. The current study determines if mice lacking the Bax gene are less susceptible to the pathogenic effects of gastrulation-stage alcohol exposure.
METHODS
Male and female Bax mice mated to produce embryos with full ( ) or partial ( ) Bax deletions, or Bax wild-type controls. On Gestational Day 7 (GD 7), embryos received two alcohol (2.9 g/kg, 4 hr apart), or control exposures. A subset of embryos was collected 12 hr later and examined for the presence of apoptotic cell death, while others were examined on GD 17 for the presence of FAS-like facial features.
RESULTS
Full Bax deletion reduced embryonic apoptotic cell death and the incidence of fetal eye and face malformations, indicating that Bax normally facilitates the development of alcohol-induced defects. An RNA-seq analysis of GD 7 Bax and Bax embryos revealed 63 differentially expressed genes, some of which may interact with the Bax deletion to further protect against apoptosis.
CONCLUSIONS
Overall, these experiments identify that Bax is a primary teratogenic mechanism of gastrulation-stage alcohol exposure.
Topics: Animals; Female; Humans; Male; Mice; Pregnancy; bcl-2-Associated X Protein; Ethanol; Fetal Alcohol Spectrum Disorders; Gastrulation; Maternal Exposure
PubMed: 35396933
DOI: 10.1002/bdr2.2009 -
Proceedings of the National Academy of... Feb 2013Dysregulation of the "intrinsic" apoptotic pathway is associated with the development of cancer and autoimmune disease. Bak and Bax are two proapoptotic members of the...
Dysregulation of the "intrinsic" apoptotic pathway is associated with the development of cancer and autoimmune disease. Bak and Bax are two proapoptotic members of the Bcl-2 protein family with overlapping, essential roles in the intrinsic apoptotic pathway. Their activity is critical for the control of cell survival during lymphocyte development and homeostasis, best demonstrated by defects in thymic T-cell differentiation and peripheral lymphoid homeostasis caused by their combined loss. Because most bak(-/-)bax(-/-) mice die perinatally, the roles of Bax and Bak in immunological tolerance and prevention of autoimmune disease remain unclear. We show that mice reconstituted with a Bak/Bax doubly deficient hematopoietic compartment develop a fatal systemic lupus erythematosus-like autoimmune disease characterized by hypergammaglobulinemia, autoantibodies, lymphadenopathy, glomerulonephritis, and vasculitis. Importantly, these mice also develop a multiorgan autoimmune disease with autoantibodies against most solid glandular structures and evidence of glandular atrophy and necrotizing vasculitis. Interestingly, similar albeit less severe pathology was observed in mice containing a hematopoietic compartment deficient for only Bak, a phenotype reminiscent of the disease seen in patients with point mutations in BAK. These studies demonstrate a critical role for Bak and an ancillary role for Bax in safeguarding immunological tolerance and prevention of autoimmune disease. This suggests that direct activators of the intrinsic apoptotic pathway, such as BH3 mimetics, may be useful for treatment of diverse autoimmune diseases.
Topics: Animals; Apoptosis; Autoantibodies; Autoimmune Diseases; Blotting, Western; Chemokines; Crosses, Genetic; Cytokines; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Histological Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Fluorescence; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 23349374
DOI: 10.1073/pnas.1215097110 -
Journal of B.U.ON. : Official Journal... 2019To evaluate the effect on breast cancer cell proliferation and apoptosis after silencing the HCCR-1 and to study its mechanism.
PURPOSE
To evaluate the effect on breast cancer cell proliferation and apoptosis after silencing the HCCR-1 and to study its mechanism.
METHODS
HCCR-1 siRNA was transfected into the breast cancer cell line MCF -7, and mRNA and protein level of HCCR-1 and Bax were evaluated by real-time quatitative PCR (qRT-PCR) and Western blotting, respectively. The cell proliferation and apoptosis were studied by MTT assay and flow cytometry.
RESULTS
The apoptosis rate in the experimental, control and blank groups were 32.57±2.35%, 3.53±0.60% and 3.15±0.46% respectively. The apoptosis rate of MCF-7 cells was significantly increased in the experimental group, compared with the other two groups (p<0.05). The A490 value in the experimental, control and blank groups were: 24h: 0.78±0.06, 1.18±0.05, 1.24±0.05; 48h: 1.09±0.05, 1.48±0.02, 1.54±0.04; 72h: 1.29±0.01, 1.81±0.02, 1.84±0.04. The proliferation of MCF-7 cells was significantly decreased in the experimental group, compared with the other two groups (p<0.05). The mRNA levels of HCCR-1 and Bax in the experimental, control and blank groups were: HCCR-1 0.46±0.03, 1.01±0.11, 1.00; and Bax 4.40±0.99, 1.03±0.10, 1.00. The protein levels were: HCCR-1 0.62±0.07, 0.89±0.09, 0.94±0.17; and Bax 0.95±0.22, 0.67±0.19, 0.69±0.11. The expressions of mRNA and protein of HCCR-1 were significantly reduced, however the expressions of mRNA and protein of Bax were significantly increased in the experimental group compared with the other two groups (p<0.05).
CONCLUSIONS
HCCR-1 siRNA transfection causes significant increase in the apoptosis and decreases in the proliferation of MCF-7 cells.These effects are related to the upregulation of the Bax expression in the MCF-7 cells.
Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; MCF-7 Cells; Middle Aged; Neoplasm Staging; Proto-Oncogene Proteins; RNA Interference; RNA, Messenger; RNA, Small Interfering; Transfection; bcl-2-Associated X Protein
PubMed: 31424657
DOI: No ID Found -
Cell Death and Differentiation Feb 2014The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying... (Review)
Review
The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying the multiple members of this family, characterizing their form and function and understanding how their interactions determine whether a cell lives or dies. In this review we focus on the recent progress made in characterizing the proapoptotic Bcl-2 family members, Bax and Bak. This progress has resolved longstanding controversies, but has also challenged established theories in the apoptosis field. We will discuss different models of how these two proteins become activated and different 'modes' by which they are inhibited by other Bcl-2 family members. We will also discuss novel conformation changes leading to Bak and Bax oligomerization and speculate how these oligomers might permeabilize the mitochondrial outer membrane.
Topics: Apoptosis; Humans; Mitochondrial Membranes; Protein Binding; Protein Multimerization; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 24162660
DOI: 10.1038/cdd.2013.139 -
European Review For Medical and... Dec 2017To investigate the expression and correlation of B-cell lymphoma-2 (Bcl-2) and Bax in the parotid gland after leading duct ligation in rat.
OBJECTIVE
To investigate the expression and correlation of B-cell lymphoma-2 (Bcl-2) and Bax in the parotid gland after leading duct ligation in rat.
MATERIALS AND METHODS
Atrophy of the right parotid was induced by ligating the right Stensen's duct of rats. Immunohistochemical labeling was performed to study the changes in number and distribution of Bcl-2 and Bax in each step of glandular atrophy, and every group at 1, 3, 5, 7, 14, 21, 30, 60, 90, 150, 180 days after ligation.
RESULTS
Bcl-2 and Bax showed a low level of expression in normal glandular tissues. At different time points after the ligation of the main duct, Bcl-2 was highly expressed in the duct cells, and the absorbance value reached a peak value at 21-day (3.02+0.10). The 1 D expression of Bax was found in some of the cells in the 3 D, and the expression of Bax reached the peak (1.99+0.10), and the expression of Bcl-2 and Bax were decreased in some cells. Bcl-2/Bax ratio increased at 1 day-21 day, and then decreased and stabilized.
CONCLUSIONS
The expression of Bax and Bcl-2 after ligation of the parotid gland is closely related to the process of the parotid gland atrophy.
Topics: Animals; Atrophy; Ligation; Parotid Gland; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Salivary Ducts; bcl-2-Associated X Protein
PubMed: 29243771
DOI: 10.26355/eurrev_201712_13914 -
Cell Death and Differentiation Nov 2018Bax is a Bcl-2 protein critical for apoptosis induction. In healthy cells, Bax is mostly a monomeric, cytosolic protein, while upon apoptosis initiation it inserts into...
Bax is a Bcl-2 protein critical for apoptosis induction. In healthy cells, Bax is mostly a monomeric, cytosolic protein, while upon apoptosis initiation it inserts into the outer mitochondrial membrane, oligomerizes, and forms pores that release proapoptotic factors like Cytochrome c into the cytosol. The structures of active Bax and its homolog Bak are only partially understood and the topology of the proteins with respect to the membrane bilayer is controversially described in the literature. Here, we systematically review and examine the protein-membrane, protein-water, and protein-protein contacts of the nine helices of active Bax and Bak, and add a new set of topology data obtained by fluorescence and EPR methods. We conclude based on the consistent part of the datasets that the core/dimerization domain of Bax (Bak) is water exposed with only helices 4 and 5 in membrane contact, whereas the piercing/latch domain is in peripheral membrane contact, with helix 9 being transmembrane. Among the available structural models, those considering the dimerization/core domain at the rim of a toroidal pore are the most plausible to describe the active state of the proteins, although the structural flexibility of the piercing/latch domain does not allow unambiguous discrimination between the existing models.
Topics: Dimerization; Humans; Mitochondrial Membranes; Protein Conformation, alpha-Helical; Protein Structure, Tertiary; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 30185826
DOI: 10.1038/s41418-018-0184-6 -
Mechanisms of Ageing and Development Jan 2017Bcl-2 family members form a network of protein-protein interactions that regulate apoptosis through permeabilization of the mitochondrial outer membrane. Deciphering... (Review)
Review
Bcl-2 family members form a network of protein-protein interactions that regulate apoptosis through permeabilization of the mitochondrial outer membrane. Deciphering this intricate network requires streamlined experimental models, including the heterologous expression in yeast. This approach had previously enabled researchers to identify domains and residues that underlie the conformational changes driving the translocation, the insertion and the oligomerization of the pro-apoptotic protein Bax at the level of the mitochondrial outer membrane. Recent studies that combine experiments in yeast and in mammalian cells have shown the unexpected effect of the anti-apoptotic protein Bcl-xL on the priming of Bax. As demonstrated with the BH3-mimetic molecule ABT-737, this property of Bcl-xL, and of Bcl-2, is crucial to elaborate about how apoptosis could be reactivated in tumoral cells.
Topics: Animals; Biphenyl Compounds; Humans; Mitochondrial Membranes; Neoplasms; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Saccharomyces cerevisiae; Sulfonamides; bcl-2-Associated X Protein; bcl-X Protein
PubMed: 27112371
DOI: 10.1016/j.mad.2016.04.007 -
FEBS Letters Jan 2016Bax-dependent mitochondrial permeabilization during apoptosis is controlled by multiple factors, including the phosphorylation by the protein kinase AKT. We used the...
Bax-dependent mitochondrial permeabilization during apoptosis is controlled by multiple factors, including the phosphorylation by the protein kinase AKT. We used the heterologous co-expression of human Bax and AKT1 in yeast to investigate how the kinase modulates the different steps underlying Bax activation. We found that AKT activated Bax and increased its cellular content. Both effects were dependent on Ser184, but a phosphorylation of this residue did not fully explain the effects of AKT. Additional experiments with mutants substituted on Ser184 suggested that the regulation of Bax dynamic equilibrium between the cytosol and mitochondria might be more tightly regulated by Bcl-xL when Bax is phosphorylated.
Topics: Amino Acid Substitution; Apoptosis; Cytosol; Gene Deletion; Haploidy; Humans; Mitochondria; Mutation; Phosphorylation; Phosphoserine; Protein Interaction Domains and Motifs; Protein Processing, Post-Translational; Protein Stability; Protein Transport; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Saccharomyces cerevisiae; Serine; bcl-2-Associated X Protein; bcl-X Protein
PubMed: 26763134
DOI: 10.1002/1873-3468.12030