New insights into the anti-apoptotic mechanism of natural polyphenols in complex with Bax protein.

Excessive apoptosis can kill normal cells and lead to liver damage, heart failure and neurodegenerative diseases. Polyphenols are secondary metabolites of plants that can interact with proteins to inhibit toxins and disease-related apoptosis. Bax is the major pro-apoptotic protein that disrupts the outer mitochondrial membrane to induce apoptosis, but limited studies have focused on the interaction between polyphenols and Bax and the associated anti-apoptotic mechanisms, especially at the atomic level. In this article, we collected 69 common polyphenols for active ingredient screening targeting Bax. Polyphenols with better and worse molecular docking scores were selected, and their anti-apoptosis effects were compared using the H2O2-induced HepG2 cell model. The interactions between the selected polyphenols and Bax protein were analyzed using molecular dynamics simulation to explore the molecular mechanism underlying the anti-apoptosis effect. Secoisolariciresinol diglucoside (SDG) and Epigallocatechin-3-gallate (EGCG) with the best affinity for Bax (-6.76 and -6.52 kcal/mol) reduced the expression of cytochrome c and caspase 3, decreasing the apoptosis rate from 52 to 11% and 12%. Molecular dynamics simulation results showed that Bim unfolded the α1-α2 loop of Bax, and disrupted the non-bond interactions between the loop (Pro-43, Glu-44 and Leu-45) and surface (Ile-133, Arg-134 and Met-137) residues, with binding free energy changed from -15.0 to 0 kJ/mol. The hydrogen bonds and van der Waals interactions formed between polyphenols and Bax prevented the unfolding of the loop. Taken together, our results proved that polyphenols can inhibit apoptosis by maintaining the unactivated conformation of Bax to reduce outer mitochondrial membrane damage.Communicated by Ramaswamy H. Sarma.

Single-molecule scale quantification reveals interactions underlying protein-protein interface: from forces to non-covalent bonds.

Protein-protein interactions (PPIs) between the B-cell lymphoma 2 (Bcl-2) family are considered a major driving force in cell cycle regulation and signaling. However, how this interfacial noncovalent interaction is achieved molecularly remains poorly understood. Herein, anti-apoptotic protein (Bcl-2) and pro-apoptotic protein (BAX) were used as models and their PPIs were explored for the first time using atomic force microscopy-based single-molecule force spectroscopy (SMFS) and in silico approaches. In addition, we used advanced analytical models, including multiple kinetic models, thermodynamic models, Poisson distributions, and contact angle molecular recognition to fully reveal the complexity of the BAX/Bcl-2 interaction interfaces. We propose that the binding kinetics between BAX/Bcl-2 are mainly mediated by specific (hydrogen bonding) and non-specific forces (hydrophobic interactions and electrostatic interactions) and show that the complicated multivalent binding interaction induces stable BAX/Bcl-2 complexes. This study enriches our understanding of the molecular mechanisms by which BAX interacts with Bcl-2. It provides valuable insights into the physical factors that need to be considered when designing PPI inhibitors.

Expanding roles of BCL-2 proteins in apoptosis execution and beyond.

The proteins of the BCL-2 family are known as key regulators of apoptosis, with interactions between family members determining permeabilisation of the mitochondrial outer membrane (MOM) and subsequent cell death. However, the exact mechanism through which they form the apoptotic pore responsible for MOM permeabilisation (MOMP), the structure and specific components of this pore, and what roles BCL-2 proteins play outside of directly regulating MOMP are incompletely understood. Owing to the link between apoptosis dysregulation and disease, the BCL-2 proteins are important targets for drug development. With the development and clinical use of drugs targeting BCL-2 proteins showing success in multiple haematological malignancies, enhancing the efficacy of these drugs, or indeed developing novel drugs targeting BCL-2 proteins is of great interest to treat cancer patients who have developed resistance or who suffer other disease types. Here, we review our current understanding of the molecular mechanism of MOMP, with a particular focus on recently discovered roles of BCL-2 proteins in apoptosis and beyond, and discuss what implications these functions might have in both healthy tissues and disease.

Creation of distinctive Bax-lipid complexes at mitochondrial membrane surfaces drives pore formation to initiate apoptosis.

Apotosis is an essential process tightly regulated by the Bcl-2 protein family where proapoptotic Bax triggers cell death by perforating the mitochondrial outer membrane. Although intensively studied, the molecular mechanism by which these proteins create apoptotic pores remains elusive. Here, we show that Bax creates pores by extracting lipids from outer mitochondrial membrane mimics by formation of Bax/lipid clusters that are deposited on the membrane surface. Time-resolved neutron reflectometry and Fourier transform infrared spectroscopy revealed two kinetically distinct phases in the pore formation process, both of which were critically dependent on cardiolipin levels. The initially fast adsorption of Bax on the mitochondrial membrane surface is followed by a slower formation of pores and Bax-lipid clusters on the membrane surface. Our findings provide a robust molecular understanding of mitochondrial membrane perforation by cell-killing Bax protein and illuminate the initial phases of programmed cellular death.

Anti-apoptotic Bcl-2 protein in apo and holo conformation anchored to the membrane: comparative molecular dynamics simulations.

The interaction between the anti-apoptotic Bcl-2 protein and its antagonist Bax is essential to the regulation of the mitochondrial pathway of apoptosis. For this work, we built models by homology of Bcl-2 full-sequence length in monomeric form (apo-Bcl-2) and in complex with the BH3 domain of Bax (holo-Bcl-2). The Bcl-2 protein was analyzed with its transmembrane domain anchored to a lipidic bilayer of DPPC, imitating physiological conditions. We performed molecular dynamics (MD) simulations using the GROMACS program. Conformational changes showed that the flexible loop domain (FLD) tends to fold on itself and move towards the main core. Furthermore, the BH3 peptide of pro-apoptotic protein Bax, showed an allosteric stabilizing effect on FLD upon being bound to the hydrophobic cleft of the anti-apoptotic protein Bcl-2, causing a reduction in its structural flexibility. However, FLD is distal from the main core of Bcl-2. Principal component analysis (PCA) showed a weak correlation between FLD residues and BH3 peptide from Bax. Upon MD simulations, several new contacts appeared between FLD and some α-helices of the core of Bcl-2, which contribute to maintaining the stability of Bcl-2. This knowledge sheds light on the behavior of Bcl-2 in the cell's native environment.Communicated by Ramaswamy H. Sarma.

Expanding the Biological Importance of Protein Structures: Insight into Dynamic Biological Function from Protein Folding Theory Analyses.

While recent developments in the determination of the three-dimensional structure of proteins have rapidly progressed, there remains a difficult challenge of studying proteins that exhibit dynamic behavior as part of their biological functions in environments considerably different than how their three-dimensional structure was determined. This study investigates the dynamic behavior of Bax, a member of the Bcl-2 family of proteins, during the regulation of apoptosis in the context of its published three-dimensional structure. The location of Bax in live cells is an equilibrium between the cytosol and outer-mitochondrial membrane. However, the regions of Bax that have been determined to be responsible for this equilibrium are shown to be inaccessible to engage in these interactions, namely, the C-terminal helix, according to the solved three-dimensional structure. Therefore, the analyses that have been applied to identify chain folding initiation sites (CFIS) and propose unfolding pathways have also been applied to the three-dimensional structure of Bax to provide a rationale for how Bax can engage in the dynamic behavior that is part of its biological function. The analyses identified regions in Bax that contribute to its stability and regions that could be susceptible to conformational changes, including the C-terminal helix, and, consequently, dynamic behavior. Experimental observations confirmed the classification of these regions. Consequently, the utilization of methods to identify CFIS on three-dimensional structures can be an effective tool to help expand our knowledge about the biological function of proteins that exhibit dynamic behavior.

Protein-protein and protein-lipid interactions of pore-forming BCL-2 family proteins in apoptosis initiation.

Apoptosis is a common cell death program that is important in human health and disease. Signaling in apoptosis is largely driven through protein-protein interactions. The BCL-2 family proteins function in protein-protein interactions as key regulators of mitochondrial poration, the process that initiates apoptosis through the release of cytochrome c, which activates the apoptotic caspase cascade leading to cellular demolition. The BCL-2 pore-forming proteins BAK and BAX are the key executors of mitochondrial poration. We review the state of knowledge of protein-protein and protein-lipid interactions governing the apoptotic function of BAK and BAX, as determined through X-ray crystallography and NMR spectroscopy studies. BAK and BAX are dormant, globular α-helical proteins that participate in protein-protein interactions with other pro-death BCL-2 family proteins, transforming them into active, partially unfolded proteins that dimerize and associate with and permeabilize mitochondrial membranes. We compare the protein-protein interactions observed in high-resolution structures with those derived in silico by AlphaFold, making predictions based on combining experimental and in silico approaches to delineate the structural basis for novel protein-protein interaction complexes of BCL-2 family proteins.

Mutation in MCL1 predicted loop to helix structural transition stabilizes MCL1-Bax binding interaction favoring cancer cell survival.

Myeloid cell leukemia-1 (MCL1), an anti-apoptotic BCL-2 family protein plays a major role in the control of apoptosis as the regulator of mitochondrial permeability which is deregulated in various solid and hematological malignancies. Interaction of the executioner proteins Bak/Bax with anti-apoptotic MCL1 and its cellular composition determines the apoptotic or survival pathway. Mutations act at various levels in the apoptotic process and can contribute to disease. Single nucleotide polymorphism (SNP) in MCL1 gene was focused as they result in changes in the amino acid sequence and have been associated with tumorigenesis. This study highlighted the deleterious MCL1-Bax stabilizing effect of the mutation V220F on MCL1 structure through computational protein-protein interaction predictions and molecular dynamics simulations. The single point mutation at V220F was selected as it is residing at the hydrophobic core region of BH3 conserved domain, the site of Bax binding. The molecular dynamics simulation studies showed increase in stability of the mutated MCL1 before and after Bax binding comparable with the native MCL1. The clusters from free energy landscape found out structural variation in folding pattern with additional helix near the BH3 domain in the mutated structure. This loop to helix structural change in the mutated complex favored stable interaction of the complex and also induced Bax conformational change. Moreover, molecular mechanics-based binding free energy calculations confirmed increased affinity of Bax toward mutated MCL1. Residue-wise interaction network analysis showed the individual residues in Bax binding responsible for the change in stability and interaction due to the protein mutation. In conclusion, the overall findings from the study reveal that the presence of V220F mutation on MCL1 is responsible for the structural confirmational change leading to disruption of its biological functions which might be responsible for tumorigenesis. The mutation could possibly be used as future diagnostic markers in treating cancers.

A kinetic fluorescence polarization ligand assay for monitoring BAX early activation.

Developmental, homeostatic, and pharmacological pro-apoptotic signals converge by activating the BCL-2 family member BAX. Studies investigating molecular regulation of BAX are commonly limited to methodologies measuring endpoint phenotypes and do not assess activation of monomeric BAX. Here, we present FLAMBE, a fluorescence polarization ligand assay for monitoring BAX early activation, that measures activation-induced release of a peptide probe in real time. Using complementary parallel and tandem biochemical techniques, we validate, corroborate, and apply FLAMBE to a contemporary repertoire of BAX modulators, characterizing their contributions within the early steps of BAX activation. Additionally, we use FLAMBE to reveal that historically "dead" BAX mutants remain responsive to activation as quasi-functional monomers. We also identify data metrics for comparative analyses and demonstrate that FLAMBE data align with downstream functional observations. Collectively, FLAMBE advances our understanding of BAX activation and fills a methodological void for studying BAX with broad applications in cell biology and therapeutic development. MOTIVATION In vitro BAX activation studies are invaluable platforms for studying cellular and pharmacological modulators of apoptosis. The gold standard for studying BAX function relies on membrane permeabilization assays, which assess the pore-forming activity of oligomeric BAX. However, there are currently no rapid or kinetic assays to interrogate real-time activation of monomeric BAX in solution, thereby limiting any molecular insights that occur upstream of mitochondrial permeabilization. Furthermore, available methods to observe the activation of monomeric BAX suffer from low throughput and static observations. To address this methodological gap, we developed FLAMBE, a kinetic fluorescence polarization-based assay to measure monomeric BAX activation in solution via concomitant displacement of a labeled peptide. This approach maintains the benefits of rapid kinetic data generation in a low-cost microplate format without requiring specialized equipment or large quantities of protein. FLAMBE compliments available experimental strategies and expands the accessibility of investigators to monitor early steps within the BAX activation continuum.

Structure of the BAK-activating antibody 7D10 bound to BAK reveals an unexpected role for the α1-α2 loop in BAK activation.

Pro-apoptotic BAK and BAX are activated by BH3-only proteins to permeabilise the outer mitochondrial membrane. The antibody 7D10 also activates BAK on mitochondria and its epitope has previously been mapped to BAK residues in the loop connecting helices α1 and α2 of BAK. A crystal structure of the complex between the Fv fragment of 7D10 and the BAK mutant L100A suggests a possible mechanism of activation involving the α1-α2 loop residue M60. M60 mutants of BAK have reduced stability and elevated sensitivity to activation by BID, illustrating that M60, through its contacts with residues in helices α1, α5 and α6, is a linchpin stabilising the inert, monomeric structure of BAK. Our data demonstrate that BAK's α1-α2 loop is not a passive covalent connector between secondary structure elements, but a direct restraint on BAK's activation.

Recombinant TSG-6 protein inhibits the growth of capsule fibroblasts in frozen shoulder via suppressing the TGF-ß/Smad2 signal pathway.

BACKGROUND: The tumor necrosis factor-stimulated gene-6 (TSG-6) has been confirmed to inhibit inflammation. It is now generally accepted that local inflammatory stimulation around shoulder capsule causes proliferative fibrosis. This study aims to investigate the mechanism of recombinant TSG-6 protein inhibiting the growth of capsule fibroblasts in frozen shoulder via the TGF-ß/Smad2 signal pathway. METHODS: Human frozen shoulder capsule tissue was taken for primary and passage culture, and the 3rd generation fibroblasts from pathological frozen shoulder capsule were treated with different concentrations of recombinant TSG-6 protein, or with TGF-ß1 agonist SRI-011381. Immunoconfocal analysis was used to identify the isolated fibroblasts, and MTT assay, colony formation assay, and flow cytometry were used to detect the viability, proliferation, and apoptosis rate of fibroblast. The contents of fibrosis and inflammation indexes COL1A1, TNF-α, IL-6, and IL-1ß in the cell supernatant were detected using ELISA and then further examined by qRT-PCR. The expression of Bax, Bcl-2, and proteins related to TGF-ß/Smad2 pathway were detected by Western Blot. RESULTS: Compared with the blank control group, fibroblasts intervened with TSG-6 (2 µg and 5 µg) showed significantly decreased viability and proliferation ability and enhanced cell apoptosis, concurrent with the reductions in Bcl-2 expression; COL1A1, TNF-α, IL-6, and IL-1ß levels; and the expression of TGF-ß1 and phosphorylated Smad22, and an increase in Bax expression, while SRI-011381 treatment would reverse the effect of recombinant TSG-6 protein. CONCLUSION: Recombinant TSG-6 protein inhibited the growth of primary fibroblasts from human frozen shoulder capsule by suppressing the TGF-ß/Smad2 signaling pathway.

The conformational stability of pro-apoptotic BAX is dictated by discrete residues of the protein core.

BAX is a pro-apoptotic member of the BCL-2 family, which regulates the balance between cellular life and death. During homeostasis, BAX predominantly resides in the cytosol as a latent monomer but, in response to stress, transforms into an oligomeric protein that permeabilizes the mitochondria, leading to apoptosis. Because renegade BAX activation poses a grave risk to the cell, the architecture of BAX must ensure monomeric stability yet enable conformational change upon stress signaling. The specific structural features that afford both stability and dynamic flexibility remain ill-defined and represent a critical control point of BAX regulation. We identify a nexus of interactions involving four residues of the BAX core α5 helix that are individually essential to maintaining the structure and latency of monomeric BAX and are collectively required for dimeric assembly. The dual yet distinct roles of these residues reveals the intricacy of BAX conformational regulation and opportunities for therapeutic modulation.

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane.

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.

Membrane-dependent amyloid aggregation of human BAX α9 (173-192).

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C-terminal α-helix (α9) of the proapoptotic Bcl-2 family protein BAX. The isolated α9 fragment (residues 173-192) is also competent to disrupt model membranes, and the structures of its membrane-associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two-dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the ß-sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.

Eltrombopag directly inhibits BAX and prevents cell death.

The BCL-2 family protein BAX has essential activity in mitochondrial regulation of cell death. While BAX activity ensures tissue homeostasis, when dysregulated it contributes to aberrant cell death in several diseases. During cellular stress BAX is transformed from an inactive cytosolic conformation to a toxic mitochondrial oligomer. Although the BAX transformation process is not well understood, drugs that interfere with this process are useful research tools and potential therapeutics. Here, we show that Eltrombopag,  an FDA-approved drug,  is a direct inhibitor of BAX. Eltrombopag binds the BAX trigger site distinctly from BAX activators, preventing them from triggering BAX conformational transformation and simultaneously promoting stabilization of the inactive BAX structure. Accordingly, Eltrombopag is capable of inhibiting BAX-mediated apoptosis induced by cytotoxic stimuli. Our data demonstrate structure-function insights into a mechanism of BAX inhibition and reveal a mechanism for Eltrombopag that may expand its use in diseases of uncontrolled cell death.

Plant Mitophagy in Comparison to Mammals: What Is Still Missing?

Mitochondrial homeostasis refers to the balance of mitochondrial number and quality in a cell. It is maintained by mitochondrial biogenesis, mitochondrial fusion/fission, and the clearance of unwanted/damaged mitochondria. Mitophagy represents a selective form of autophagy by sequestration of the potentially harmful mitochondrial materials into a double-membrane autophagosome, thus preventing the release of death inducers, which can trigger programmed cell death (PCD). Recent advances have also unveiled a close interconnection between mitophagy and mitochondrial dynamics, as well as PCD in both mammalian and plant cells. In this review, we will summarize and discuss recent findings on the interplay between mitophagy and mitochondrial dynamics, with a focus on the molecular evidence for mitophagy crosstalk with mitochondrial dynamics and PCD.

Computational analysis of binding free energies, hotspots and the binding mechanism of Bcl-xL/Bcl-2 binding to Bad/Bax.

The anti-apoptotic proteins B-cell lymphoma-extra large (Bcl-xL) and B-cell lymphoma/leukemia-2 (Bcl-2) are members of the Bcl-2 protein family, and they play important roles in regulating apoptosis and cell cycle retardation. However, the binding mechanisms of Bcl-xL/Bcl-2 with their associated agonists, including Bcl-2-associated death promoter (Bad) and Bcl-2-associated X protein (Bax), are not well understood. In the present study, the recently developed interaction entropy approach was employed for the calculation of entropic contribution, and the computational alanine scanning method was used to identify the hot spot in the protein-protein interactions between Bcl-xL/Bcl-2 and Bad/Bax. The calculated binding free energies and their ranks for the four systems were in good agreement with the experimental results. Computational analysis shows that there are more hot-spot residues in the Bcl-xL/Bad complex than that in the Bcl-xL/Bax complex, leading to a stronger binding affinity in the former. It is interesting to find that the reason for the stronger binding affinity of Bcl-2 to Bad than to Bax is different for the Bcl-xL system. Although there are more hot-spot residues in the Bcl-2/Bax system than in the Bcl-2/Bad complex, there are also more negatively contributing residues in the Bcl-2/Bax. Our study identified Arg104, Tyr105, Leu116, and Leu134 to be the common key residues in the Bcl-xL complexes, and Arg107, Tyr108, Phe112, Gln118, Leu137, Arg146, and Tyr202 are common key residues in the Bcl-2 complexes. These results would provide valuable information for the design of potent inhibitors of Bcl-xL/Bcl-2.

Expression and regulation of ccBAX by miR-124 in the caudal fin cell of C. auratus gibelio upon cyprinid herpesvirus 2 infection.

Bcl2 family proteins play a critical role in cell death or survival. BAX, the death-promoting protein of bcl2 family, mediated mitochondrial pathway inducing cells' apoptosis in mammal. MiRNAs have been implicated as negative regulators down-regulating genes' expression after post-transcriptional level. At present, little is known about the regulatory mechanism of miRNA on the Bcl2 family proteins during CyHV-2 infection in silver crucian carp (Carassius auratus gibelio). In this study, the ccBAX (silver crucian carp BAX) gene was cloned and expressed, and polyclonal antibodies were raised in mouse against the purified ccBAX-GST fusion protein. The structure analysis indicated that ccBAX protein included four conserve domains (BH1, BH2, BH3 and transmembrane domains) and the expression of ccBAX protein occurred throughout the cells. Furthermore, two miRNAs (miR-124 and miRNA-29b) were identified to negatively regulate ccBAX gene expression in GiCF cell. miR-124 was found to suppress the expression of WT-ccBAX (wild type), but not the MT-ccBAX (mutant). Overall, the results demonstrated that the expression of the ccBAX gene was significantly down-regulated by miR-124 in silver crucian carp (Carassius auratus gibelio) during CyHV-2 infection.

Humanin selectively prevents the activation of pro-apoptotic protein BID by sequestering it into fibers.

Members of the B-cell lymphoma (BCL-2) protein family regulate mitochondrial outer membrane permeabilization (MOMP), a phenomenon in which mitochondria become porous and release death-propagating complexes during the early stages of apoptosis. Pro-apoptotic BCL-2 proteins oligomerize at the mitochondrial outer membrane during MOMP, inducing pore formation. Of current interest are endogenous factors that can inhibit pro-apoptotic BCL-2 mitochondrial outer membrane translocation and oligomerization. A mitochondrial-derived peptide, Humanin (HN), was reported being expressed from an alternate ORF in the mitochondrial genome and inhibiting apoptosis through interactions with the pro-apoptotic BCL-2 proteins. Specifically, it is known to complex with BAX and BID. We recently reported the fibrillation of HN and BAX into ß-sheets. Here, we detail the fibrillation between HN and BID. These fibers were characterized using several spectroscopic techniques, protease fragmentation with mass analysis, and EM. Enhanced fibrillation rates were detected with rising temperatures or pH values and the presence of a detergent. BID fibers are similar to those produced using BAX; however, the structures differ in final conformations of the BCL-2 proteins. BID fibers display both types of secondary structure in the fiber, whereas BAX was converted entirely to ß-sheets. The data show that two distinct segments of BID are incorporated into the fiber structure, whereas other portions of BID remain solvent-exposed and retain helical structure. Similar analyses show that anti-apoptotic BCL-xL does not form fibers with humanin. These results support a general mechanism of sequestration of pro-apoptotic BCL-2 proteins into fibers by HN to inhibit MOMP.