Bax Molecules Research Articles

Reactive oxygen species involved in apoptosis induction of human respiratory epithelial (A549) cells by Streptococcus agalactiae.

Streptococcus agalactiae (Group B Streptococcus; GBS) is an important pathogen and is associated with pneumonia, sepsis and meningitis in neonates and adults. GBS infections induce cytotoxicity of respiratory epithelial cells (A549) with generation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential (ψm). The apoptosis of A549 cells by GBS was dependent on the activation of caspase-3 and caspase-9 with increased pro-apoptotic Bim and Bax molecules and decreased Bcl-2 pro-survival protein. Treatment of infected A549 cells with ROS inhibitors (diphenyleniodonium chloride or apocynin) prevented intracellular ROS production and apoptosis. Consequently, oxidative stress is included among the cellular events leading to apoptosis during GBS human invasive infections.

Neuroprotective effects of (Val8)GLP-1-Glu-PAL in the MPTP Parkinson’s disease mouse model

Glucagon-like peptide 1 (GLP-1) is a hormone and a growth factor. GLP-1 mimetics are currently on the market as treatments for type 2 diabetes. They also have shown neuroprotective properties in animal models of neurodegenerative disorders. In addition, the GLP-1 mimetic exendin-4 has shown protective effects in animal models of Parkinson’s disease (PD), and a first clinical trial in PD patients showed promising results. (Val8)GLP-1-glu-PAL is a new GLP-1 analogue which has a longer biological half-life than exendin-4. We previously showed that (Val8)GLP-1-glu-PAL has neuroprotective properties. Here we tested the drug in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. MPTP was injected (30mg/kg i.p.) along with (Val8)GLP-1-glu-PAL (25nmol/kg i.p.) once-daily for 8 days. (Val8)GLP-1-glu-PAL showed good effects in preventing the MPTP-induced motor impairment (Rotarod, open field locomotion, swim test), reduction in tyrosine hydroxylase levels (dopamine synthesis) in the substantia nigra, a reduction of activated caspase 3 levels, of TUNEL positive cell numbers, of the pro-apoptotic signaling molecule BAX and an increase in the growth signaling molecule Bcl-2. The results demonstrate that (Val8)GLP-1-glu-PAL shows promise as a novel treatment of PD.

Neuroprotective effects of lixisenatide and liraglutide in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease

Glucagon-like peptide 1 (GLP-1) is a growth factor. GLP-1 mimetics are on the market as treatments for type 2 diabetes and are well tolerated. These drugs have shown neuroprotective properties in animal models of neurodegenerative disorders. In addition, the GLP-1 mimetic exendin-4 has shown protective effects in animal models of Parkinson’s disease (PD), and a clinical trial in PD patients showed promising first results. Liraglutide and lixisenatide are two newer GLP-1 mimetics which have a longer biological half-life than exendin-4. We previously showed that these drugs have neuroprotective properties in an animal model of Alzheimer’s disease. Here we demonstrate the neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. MPTP was injected once-daily (20mg/kg i.p.) for 7days, and drugs were injected once-daily for 14days i.p. When comparing exendin-4 (10nmol/kg), liraglutide (25nmol/kg) and lixisenatide (10nmol/kg), it was found that exendin-4 showed no protective effects at the dose chosen. Both liraglutide and lixisenatide showed effects in preventing the MPTP-induced motor impairment (Rotarod, open-field locomotion, catalepsy test), reduction in tyrosine hydroxylase (TH) levels (dopamine synthesis) in the substantia nigra and basal ganglia, a reduction of the pro-apoptotic signaling molecule BAX and an increase in the anti-apoptotic signaling molecule B-cell lymphoma-2. The results demonstrate that in this study, both liraglutide and lixisenatide are superior to exendin-4, and both drugs show promise as a novel treatment of PD.

Live-Cell Measurements of the Conformational Rearrangements in Bax at the Initiation of Apoptosis

The Bcl-2 family of proteins regulates the activation of apoptosis through the mitochondria pathway. Pro- and anti-apoptotic members of this family keep each other in check until the correct time to commit to apoptosis. The point of no return for this commitment is the permeabilization of the outer-mitochondrial membrane (OMM). Translocation of the pro apoptotic member, Bax, from the cytosol to the mitochondria is the molecular signature of this event. We employed a novel method to reliably detect Forster Resonance Energy Transfer (FRET) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in Bax as this translocation occurs in live cells. In the cytosol, our FRET measurements indicated that the C-terminal helix is exposed instead of tucked away in the core of the protein. This coincided with measurements using fluorescence correlation spectroscopy (FCS) that showed that cytosolic Bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. We propose that this exposed helix allows for this contact to occur. Cross-linking the C-terminal helix (α9) to helix α4 reduced the instances of these interactions while at the same time yielded FRET measurements that are consistent with the α9 helix tucked into the core of the protein. After translocation, our FRET measurements showed that Bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the BH3 domain (helix α2) and the C-terminal helix. These findings have implications for possible contacts with other Bcl-2 proteins to create pores to permeabilize the OMM, which would also be necessary for the regulation of apoptosis.

Atomization Energies of LnX Molecules (Ln = Sm, Eu, and Yb; X = Cl, Br, and I)

The gas phase equilibria Ba + LnX = BaX + Ln (Ln = Sm, Eu, Yb; X = Cl, Br, I) were investigated by Knudsen effusion mass spectrometry using a low energy of ionizing electrons to avoid fragmentation processes. The BaX molecules were used as references with well-established bond energies. The atomization enthalpies ΔatH0° of the LnX molecules were determined to be 427 ± 9 (SmCl), 409 ± 9 (EuCl), 366 ± 9 (YbCl), 360 ± 10 (SmBr), 356 ± 13 (EuBr), 316 ± 9 (YbBr), 317 ± 10 (SmI), 293 ± 10 (EuI), and 283 ± 10 (YbI) kJ·mol–1.

Conformational Rearrangements in the Pro-apoptotic Protein, Bax, as It Inserts into Mitochondria: A CELLULAR DEATH SWITCH

The B-cell lymphoma 2 (Bcl-2) family of proteins regulates the activation of apoptosis through the mitochondria pathway. Pro- and anti-apoptotic members of this family keep each other in check until the correct time to commit to apoptosis. The point of no return for this commitment is the permeabilization of the outer mitochondrial membrane. Translocation of the pro-apoptotic member, Bax, from the cytosol to the mitochondria is the molecular signature of this event. We employed a novel method to reliably detect Förster resonance energy transfer (FRET) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in Bax as this translocation occurs in live cells. In the cytosol, our FRET measurement indicated that the C-terminal helix is exposed instead of tucked away in the core of the protein. In addition fluorescence correlation spectroscopy (FCS) showed that cytosolic Bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. Cross-linking the C-terminal helix (α9) to helix α4 reduced the potential of those interactions to occur. After translocation, our FRET measurements showed that Bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the BH3 domain (helix α2) and the C-terminal helix. These findings have implications for possible contacts with other Bcl-2 proteins necessary for the regulation of apoptosis.

Is resection of the tendon edge necessary to enhance the healing process? An evaluation of the homeostasis of apoptotic and inflammatory processes in the distal 1 cm of a torn supraspinatus tendon: part I

We hypothesize that the expression of proapoptotic and antiapoptotic molecules and cytokines is dependent on the distance from the torn supraspinatus tendon edge and this expression may influence its potential for healing. The aim of this work is to evaluate the expression of proapoptotic Bax molecule and caspases 3, 8, and 9; antiapoptotic Bcl-2 molecule; and proinflammatory tumor necrosis factor (TNF) α and anti-inflammatory interleukin 10 (IL-10) in 3 sections taken from a 1-cm section of the edge of a torn supraspinatus tendon: 3mm distal and 3mm proximal, as well as the remaining 4-mm middle section between them. Nine patients, with a mean age of 58years, were included in the study. All fulfilled strict inclusion criteria regarding the morphology of the tear and reconstruction technique. Samples were taken from the ruptured supraspinatus tendon at the time of arthroscopic repair. Quantitative real-time polymerase chain reaction assay was used for analysis. The expression of caspases 9, 8 and 3; Bax; and TNF-α significantly decreased from the distal to the proximal parts of the tendon edge (P<.05). However, a significant increase in Bcl-2 and IL-10 expression was also found in the same direction (P<.05). Tenocytes can reduce the expression of proapoptotic caspases 3, 8, and 9 and Bax, as well as proinflammatory TNF-α, by increasing the expression of Bcl-2 and IL-10 within 1cm of the supraspinatus edge in a distal to proximal direction. Resection 4 to 7mm from the edge of the torn supraspinatus tendon may enhance the healing process by reaching a reasonable compromise between molecular homeostasis of apoptotic and inflammatory processes and mechanical aspects of rotator cuff reconstruction.

The Point of No Return; 3D Structure of Bax-Mediated Pores in Membrane Bilayers

Bax is a protein that is responsible for the decisive point-of-no-return event in cell death. Bax is normally monomeric, but it transforms into its lethal oligomeric form in response to cell stress. To map this transformation on the molecular level, we used nanometer-scale phospholipid bilayer islands (nanodiscs), each able to accommodate only a single BAX molecule. Using this minimal reconstituted system, we captured structural correlates that precede Bax homo-oligomerization elucidating previously inaccessible steps of the core molecular mechanism by which Bcl-2 family proteins regulate membrane permeabilization. The three-dimensional reconstructions of these membrane assemblies demonstrate that in the presence of a BID BH3 peptide, an individual BAX molecule can insert into the nanodisc membrane, distort the lipid bilayer, and form a pore. This implies that monomeric membrane-inserted BAX is the key functional unit responsible for initiating mitochondrial outer membrane pore formation and destabilization, leading to apoptosis and cell death. This study provides a new set of tools and previously not accessible data to gain new understanding of the molecular basis of mitochrondrial permeabilization.

PUMA binding induces partial unfolding within BCL-xL to disrupt p53 binding and promote apoptosis

Following DNA damage, nuclear p53 induces the expression of PUMA, a BH3-only protein that binds and inhibits the anti-apoptotic BCL-2 repertoire, including BCL-xL. PUMA, unique amongst BH3-only proteins, disrupts the interaction between cytosolic p53 and BCL-xL, allowing p53 to promote apoptosis via direct activation of the BCL-2 effector molecules, BAX and BAK. Structural investigations using nuclear magnetic resonance spectroscopy and X-ray crystallography revealed that PUMA binding induced partial unfolding of two α-helices within BCL-xL. Wild-type PUMA or a PUMA mutant incapable of causing binding-induced unfolding of BCL-xL equivalently inhibited the anti-apoptotic BCL-2 repertoire to sensitize for death receptor (DR)-activated apoptosis, but only wild-type PUMA promoted p53-dependent, DNA damage-induced apoptosis. Our data suggest that PUMA-induced partial unfolding of BCL-xL disrupts interactions between cytosolic p53 and BCL-xL, releasing the bound p53 to initiate apoptosis. We propose that regulated unfolding of BCL-xL provides a mechanism to promote PUMA-dependent signaling within the apoptotic pathways.

Unlatched BAX Pairs for Death

Self-interacting BAX proteins permeabilize outer mitochondrial membranes to trigger apoptotic cell death. Czabotar et al. present two revealing structures of BAX dimers: one dimer has an activator BH3 helix bound into its canonical cleft, and the other dimer exposes a planar hydrophobic face potentially critical for membrane interactions.

Bax Crystal Structures Reveal How BH3 Domains Activate Bax and Nucleate Its Oligomerization to Induce Apoptosis

In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other's surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.

The Dynamics of Bax Channel Formation: Influence of Ionic Strength

Mitochondrial outer membrane permeabilization (MOMP) is a complex multistep process. Studies of MOMP in vivo are limited by the stochastic variability of MOMP between cells and rapid completion of IMS protein release within single cells. In vitro models have provided useful insights into MOMP. We have investigated the dynamics of Bax-mediated MOMP in isolated mitochondria using ionic strength as a tool to control the rate of MOMP. We find that Bax can induce both transient permeabilization, detected by protein release, and more substantial long-lasting permeabilization, measured by the rate of oxidation of added cytochrome c. We found that higher ionic strength causes Bax to form small channels quickly but the expansion of these early channels is impeded. This inhibitory effect of ionic strength is independent of tBid. Channels formed under low ionic strength are not destabilized by raising the ionic strength. Increase in ionic strength also increases the ability of Bcl-xL to inhibit Bax-mediated MOMP. Ionic strength does not affect Bax insertion into mitochondria. Thus, ionic strength influences the assembly of Bax molecules already in membrane into channels. Ionic strength can be used as an effective biophysical tool to study Bax-mediated channel formation.

BH3-only proteins in apoptosis at a glance

Apoptosis is a genetically programmed process for the elimination of damaged or redundant cells by activation of caspases (aspartate-specific cysteine proteases). Caspases cleave vital proteins, leading the cell to fragment into vesicles that are rapidly engulfed by phagocytes (for reviews, see [

Enlightening the impact of immunogenic cell death in photodynamic cancer therapy

EMBO J 31 5, 1062–1079 (2012); published online January172012 In this issue of The EMBO Journal, Garg et al (2012) delineate a signalling pathway that leads to calreticulin (CRT) exposure and ATP release by cancer cells that succumb to photodynamic therapy (PTD), thereby providing fresh insights into the molecular regulation of immunogenic cell death (ICD).

Abstract 5476: Small molecule activation of BAX-mediated apoptosis through direct engagement of the BAX trigger site

Abstract BCL-2 family proteins are key regulators of the mitochondrial apoptotic pathway in health and disease. Anti-apoptotic members such as BCL-2 and BCL-XL have been implicated in the initiation, progression, and chemoresistance of human cancer. The anti-apoptotic BCL-2/BCL-XL groove that binds and sequesters pro-apoptotic BH3 death helices has been successfully targeted by small molecules and peptides. Such compounds induce tumor cell apoptosis and are being advanced in clinical trials as promising next-generation cancer therapeutics. We recently localized a distinct BH3 binding groove on pro-apoptotic BAX and demonstrated that engagement of the novel site by a triggering BIM BH3 helix directly activates BAX (Gavathiotis et al. Nature, 2008), unleashing a succession of key conformational changes that culminates in the formation of a toxic mitochondrial pore (Gavathiotis et al. Mol Cell, 2010). Here we applied these new structural insights in a computational screen to identify small molecules capable of directly engaging the BAX trigger site for therapeutic induction of BAX-mediated apoptosis in human cancer. We confirmed by NMR analysis that a series of identified small molecules indeed bound to the BAX trigger site and in fluorescence polarization assays were strikingly selective for the BH3-binding groove of BAX as compared to that of anti-apoptotic targets. We demonstrate that molecular binding triggers dose-responsive BAX activation, as assessed by in vitro oligomerization, membrane permeabilization, and cell death assays. Importantly, the small molecule BAX activators induce apoptosis of cancer cells in a BAX-dependent manner, highlighting both the selectivity and pharmacologic potential of direct BAX activation as a novel strategy for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5476. doi:10.1158/1538-7445.AM2011-5476

Effect of Ionic Strength on Bax:TBID Mediated Mitochondrial Outer Membrane Permeabilization (MOMP)

Fluctuations in ionic strength have been documented in apoptosis. While the exact molecular identity of the channels through which proteins permeate across the outer mitochondrial membrane is unclear, it is fairly certain that the channel is large in size and sustained over time. We sought to determine the effect of salt concentration on Bax and tBid in inducing MOMP. While high salt buffer (60mM) facilitated more release of inter-membrane space (IMS) proteins (Adenylate Kinase and Sulfite Oxidase) with Bax and tBid than low salt (5mM HEPES buffer), low salt conditions induced more sustained, real-time permeabilization, as measured by accessibility of exogenous cytochrome c to complex IV. There was a dichotomy between the kinetics of IMS protein release and real-time permeabilization at high salt conditions, while under low salt conditions, they were congruent. The effect of ionic strength on sustained permeabilization is biphasic, with maxima at 10mM. The sustained permeabilization is inducible by diluting the salt concentration to that of low salt, but irreversible by incorporating high salt conditions. This observation is inconsistent with a dynamic channel model sensitive to ionic strength. It seems likely that under low salt conditions, there is a co-operative interaction between Bax molecules in the membrane and those in solution, but the growth of the channel by membrane-bound Bax molecules itself is independent of ionic strength. Supported by a grant from NSF (MCB-0641208).

Bax Activation by Engagement with, Then Release from, the BH3 Binding Site of Bcl-xL

Bcl-2 homologues (such as Bcl-x(L)) promote survival in part through sequestration of "activator" BH3-only proteins (such as Puma), preventing them from directly activating Bax. It is thus assumed that inhibition of interactions between activators and Bcl-x(L) is a prerequisite for small molecules to antagonize Bcl-x(L) and induce cell death. The biological properties, described here of a terphenyl-based alpha-helical peptidomimetic inhibitor of Bcl-x(L) attest that displacement of Bax from Bcl-x(L) is also critical. Terphenyl 14 triggers Bax-dependent but Puma-independent cell death, disrupting Bax/Bcl-x(L) interactions without affecting Puma/Bcl-x(L) interactions. In cell-free assays, binding of inactive Bax to Bcl-x(L), followed by its displacement from Bcl-x(L) by terphenyl 14, produces mitochondrially permeabilizing Bax molecules. Moreover, the peptidomimetic kills yeast cells that express Bax and Bcl-x(L), and it uses Bax-binding Bcl-x(L) to induce mammalian cell death. Likewise, ectopic expression of Bax in yeast and mammalian cells enhances sensitivity to another Bcl-x(L) inhibitor, ABT-737, when Bcl-x(L) is present. Thus, the interaction of Bcl-x(L) with Bax paradoxically primes Bax at the same time it keeps Bax activity in check, and displacement of Bax from Bcl-x(L) triggers an apoptotic signal by itself. This mechanism might contribute to the clinical efficiency of Bcl-x(L) inhibitors.

Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion

Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices alpha5 and alpha6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.

FCS Studies of the Pore Formation by Protein BAX in Lipid Membranes

BAX is a pro-apoptotic member of the BCL-2 protein family. At the onset of apoptosis, monomeric, cytoplasmic BAX is activated and translocates to the outer mitochondrial membrane, where it forms oligomeric pores. The biophysical mechanism of BAX pore formation and the structure of the BAX pore are not clear. To study the mechanism of BAX pore formation in lipid membranes we designed an in vitro system employing giant unilamellar vesicles (GUVs) and fluorescently labeled BAX combined with the single-molecule sensitivity technique, dual-focus scanning FCS. Use of scanning FCS in experiments, where two spectrally different populations of BAX molecules interact with a GUV membrane, allowed correlation of the pore formation by BAX with the ability of BAX to oligomerize in lipid membranes. As a result, we show that BAX binds lipid membranes as a monomer and then undergoes oligomerization to form BAX pore protein-lipid complexes. FCS analysis of the populations of GUVs over a period of time showed that BAX pore complexes grow in size and increase in number with time. Analysis of the diffusion coefficients of these BAX complexes using Saffman-Delbruck theory estimates that the in-membrane hydrodynamic radius of a BAX pore complex ranges from 1 to 31 nm. Formation of BAX pore complexes in a lipid membrane is inhibited in the presence of BCL-XL (in-membrane BAX is 100% monomeric) and can be rescued by the addition of cut BID. We also show confocal 3D reconstructions of a giant BAX pore with fluorescent BAX accumulating in the edge of the pore. Lifetime of the giant BAX pore (5-10 min) together with the accumulation of BAX at the edge of the pore and the loss of surface tension in a GUV support the toroidal BAX pore model.

Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax

A central issue in the control of apoptosis is whether its essential mediators Bax and Bak must be restrained by Bcl-2-like prosurvival relatives to prevent their damaging mitochondria and unleashing apoptosis. The issue is particularly vexed for Bax, which is largely a cytosolic monomer in unstressed cells. To determine whether Bax regulation requires its binding by prosurvival relatives, we replaced a conserved aspartate in its BH3 interaction domain with arginine. Bax D68R functioned and behaved like wild-type Bax in localization and activation but had greatly impaired binding to the prosurvival family members. Nevertheless, Bcl-x L remained able to block apoptosis induced by Bax D68R. Whereas cells with sufficient Bcl-x L tolerated expression of Bax D68R, it provoked apoptosis when Bcl-x L was absent, downregulated, or inactivated. Moreover, Bax D68R rendered membrane bound by a C-terminal anchor mutation overwhelmed endogenous Bcl-x L and killed cells. These unexpected results suggest that engagement of Bax by its prosurvival relatives is a major barrier to its full activation. We propose that the Bcl-2-like proteins must capture the small proportion of Bax molecules with an exposed BH3 domain, probably on the mitochondrial membrane, to prevent Bax-imposed cell death, but that Bcl-x L also controls Bax by other mechanisms.