ABSTRACT
The activation or inactivation of B-cell lymphoma-2 (Bcl-2) antagonist/killer (Bak) is critical for controlling mitochondrial outer membrane permeabilization-dependent apoptosis. Its pro-apoptotic activity is controlled by intermolecular interactions with the Bcl-2 homology 3 (BH3) domain, which is accommodated in the hydrophobic pocket of Bak. Bcl-2-interacting protein 5 (Bnip5) is a noncanonical BH3 domain-containing protein that interacts with Bak. Bnip5 is characterized by its controversial effects on the regulation of the pro-apoptotic activity of Bak. In the present study, we determined the crystal structure of Bak bound to Bnip5 BH3. The intermolecular association appeared to be typical at first glance, but we found that it is maintained by tight hydrophobic interactions together with hydrogen/ionic bonds, which accounts for their high binding affinity with a dissociation constant of 775 nM. Structural analysis of the complex showed that Bnip5 interacts with Bak in a manner similar to that of the Bak-activating pro-apoptotic factor peroxisomal testis-enriched protein 1, particularly in the destabilization of the intramolecular electrostatic network of Bak. Our structure is considered to reflect the initial point of drastic and consecutive conformational and stoichiometric changes in Bak induced by Bnip5 BH3, which helps in explaining the effects of Bnip5 in regulating Bak-mediated apoptosis.
Subject(s)
Proto-Oncogene Proteins c-bcl-2 , bcl-2 Homologous Antagonist-Killer Protein , Proto-Oncogene Proteins c-bcl-2/chemistry , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Protein Domains , bcl-X Protein/metabolism , BH3 Interacting Domain Death Agonist Protein/metabolism , Apoptosis/physiologyABSTRACT
Bak is a pro-apoptotic protein and a member of the Bcl-2 family that plays a key role in apoptosis, a programmed cell death mechanism of multicellular organisms. Its activation under death stimuli triggers the permeabilization of the mitochondrial outer membrane that represents a point of no return in the apoptotic pathway. This process is deregulated in many tumors where Bak is inactivated, whereas in other cases like in neurodegeneration, it exhibits an excessive response leading to disorders such as the Alzheimer disease. Members of the Bcl-2 family share a common 3D structure, exhibiting an extremely similar orthosteric binding site, a place where both pro and antiapoptotic proteins bind. This similarity raises a selectivity issue that hampers the identification of new drugs, capable of altering Bak activation in a selective manner. An alternative activation site triggered by antibodies has been recently identified, opening the opportunity to undertake new drug discovery studies. Despite this recent identification, an exhaustive study to identify cryptic pockets as prospective allosteric sites has not been yet performed. Thus, the present study aims to characterize novel hotspots in the Bak structure. For this purpose, we have carried out extensive molecular dynamics simulations using three different Bak systems including Bak in its apo form, Bak in complex with its endogen activator Bim and an intermediate form, set up by removing Bim from the previous complex. The results reported in the present work shed some light on future docking studies on Bak through the identification of new prospective allosteric sites, not previously described in this protein.
Subject(s)
Molecular Dynamics Simulation , bcl-2 Homologous Antagonist-Killer Protein , Bcl-2-Like Protein 11/metabolism , Allosteric Site , Prospective Studies , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/metabolism , ApoptosisABSTRACT
Apoptosis is important for development and tissue homeostasis, and its dysregulation can lead to diseases, including cancer. As an apoptotic effector, BAK undergoes conformational changes that promote mitochondrial outer membrane disruption, leading to cell death. This is termed "activation" and can be induced by peptides from the human proteins BID, BIM, and PUMA. To identify additional peptides that can regulate BAK, we used computational protein design, yeast surface display screening, and structure-based energy scoring to identify 10 diverse new binders. We discovered peptides from the human proteins BNIP5 and PXT1 and three non-native peptides that activate BAK in liposome assays and induce cytochrome c release from mitochondria. Crystal structures and binding studies reveal a high degree of similarity among peptide activators and inhibitors, ruling out a simple function-determining property. Our results shed light on the vast peptide sequence space that can regulate BAK function and will guide the design of BAK-modulating tools and therapeutics.
Subject(s)
Apoptosis Regulatory Proteins , Proto-Oncogene Proteins , Humans , Proto-Oncogene Proteins/chemistry , Apoptosis Regulatory Proteins/chemistry , Bcl-2-Like Protein 11 , bcl-X Protein/metabolism , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Apoptosis/physiology , Peptides , bcl-2-Associated X Protein/metabolism , Proto-Oncogene Proteins c-bcl-2/chemistryABSTRACT
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.
Subject(s)
Proto-Oncogene Proteins c-bcl-2 , bcl-2 Homologous Antagonist-Killer Protein , Apoptosis/physiology , Humans , Lipids , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/metabolism , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/metabolismABSTRACT
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.
Subject(s)
Apoptosis , bcl-2 Homologous Antagonist-Killer Protein , Antibodies , Apoptosis/physiology , BH3 Interacting Domain Death Agonist Protein/metabolism , Mitochondrial Membranes/metabolism , Protein Structure, Secondary , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/geneticsABSTRACT
BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric "BH3-in-groove" triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.
Subject(s)
Apoptosis/physiology , Membrane Proteins/metabolism , Mitochondria/metabolism , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , BH3 Interacting Domain Death Agonist Protein/chemistry , BH3 Interacting Domain Death Agonist Protein/genetics , BH3 Interacting Domain Death Agonist Protein/metabolism , Cell Death , Crystallography, X-Ray , Humans , Ligands , Liposomes , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mitochondria/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , bcl-2 Homologous Antagonist-Killer Protein/chemistryABSTRACT
Systematic incorporation of ring-constrained ß- and γ-amino acid residues into α-helix mimetics engenders stable helical secondary structures. In this paper, functional α/ß/γ-helical peptidomimetics were explored for mimicry of BH3 helical domains, Bim as a pioneering study. The Bim-based α/ß/γ-peptides in an αγααßα-hexad repeat with five helical turns inhibited the interaction between Bak and Bcl-xL with excellent resistance towards proteolytic digestion. Further optimization of the α/ß/γ-backbone strategy will considerably expand the utility of functional α/ß/γ-peptidomimetics, in particular due to its prominent stability against proteolysis.
Subject(s)
Peptidomimetics/chemistry , Amino Acid Sequence , Peptidomimetics/metabolism , Protein Conformation, alpha-Helical , Protein Domains , Proteolysis , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-X Protein/chemistry , bcl-X Protein/metabolismABSTRACT
Apoptosis, the intrinsic programmed cell death process, is mediated by the Bcl-2 family members Bak and Bax. Activation via formation of symmetric core dimers and oligomerization on the mitochondrial outer membrane (MOM) leads to permeabilization and cell death. Although this process is linked to the MOM, the role of the membrane in facilitating such pores is poorly understood. We recently described Bak core domain dimers, revealing lipid binding sites and an initial role of lipids in oligomerization. Here we describe simulations that identified localized clustering and interaction of triacylglycerides (TAGs) with a minimized Bak dimer construct. Coalescence of TAGs occurred beneath this Bak dimer, mitigating dimer-induced local membrane thinning and curvature in representative coarse-grain MOM and model membrane systems. Furthermore, the effects observed as a result of coarse-grain TAG cluster formation was concentration dependent, scaling from low physiological MOM concentrations to those found in other organelles. We find that increasing the TAG concentration in liposomes mimicking the MOM decreased the ability of activated Bak to permeabilize these liposomes. These results suggest that the presence of TAGs within a Bak-lipid membrane preserves membrane integrity and is associated with reduced membrane stress, suggesting a possible role of TAGs in Bak-mediated apoptosis.
Subject(s)
Liposomes , bcl-2 Homologous Antagonist-Killer Protein , Apoptosis , Lipids , Liposomes/metabolism , Mitochondrial Membranes/metabolism , bcl-2 Homologous Antagonist-Killer Protein/analysis , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
Apoptosis plays an essential role in maintaining cellular homeostasis and preventing cancer progression. Bcl-xL, an anti-apoptotic protein, is an important modulator of the mitochondrial apoptosis pathway and is a promising target for anticancer therapy. In this study, we identified octenidine as a novel Bcl-xL inhibitor through structural feature-based deep learning and molecular docking from a library of approved drugs. The NMR experiments demonstrated that octenidine binds to the Bcl-2 homology 3 (BH3) domain-binding hydrophobic region that consists of the BH1, BH2, and BH3 domains in Bcl-xL. A structural model of the Bcl-xL/octenidine complex revealed that octenidine binds to Bcl-xL in a similar manner to that of the well-known Bcl-2 family protein antagonist ABT-737. Using the NanoBiT protein-protein interaction system, we confirmed that the interaction between Bcl-xL and Bak-BH3 domains within cells was inhibited by octenidine. Furthermore, octenidine inhibited the proliferation of MCF-7 breast and H1299 lung cancer cells by promoting apoptosis. Taken together, our results shed light on a novel mechanism in which octenidine directly targets anti-apoptotic Bcl-xL to trigger mitochondrial apoptosis in cancer cells.
Subject(s)
Artificial Intelligence , Imines/pharmacology , Pyridines/pharmacology , bcl-X Protein/antagonists & inhibitors , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line , Cell Proliferation/drug effects , Humans , Imines/chemistry , Molecular Docking Simulation , Neoplasms/pathology , Protein Binding/drug effects , Pyridines/chemistry , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-X Protein/chemistryABSTRACT
Permeabilization of the outer mitochondrial membrane by pore-forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro-apoptotic Bak during pore formation, high-resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX-MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high-resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3-only proteins. Furthermore, we determined the first high-resolution structure of the Bak transmembrane helix. Upon activation, α-helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane-bound state. In line with this finding, comparative protein folding experiments with Bak and anti-apoptotic BclxL suggest that α-helix 1 in Bak is a metastable structural element contributing to its pro-apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α-helix 1 yielded Bak variants with delayed pore-forming activity. These insights will contribute to a better mechanistic understanding of Bak-mediated membrane permeabilization.
Subject(s)
Liposomes/chemistry , Membrane Lipids/chemistry , Proto-Oncogene Proteins c-bcl-2/chemistry , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-X Protein/chemistry , Binding Sites , Cloning, Molecular , Deuterium Exchange Measurement , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Liposomes/metabolism , Membrane Lipids/metabolism , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Folding , Protein Interaction Domains and Motifs , Protein Multimerization , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thermodynamics , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-X Protein/genetics , bcl-X Protein/metabolismABSTRACT
BAK and BAX, the effectors of intrinsic apoptosis, each undergo major reconfiguration to an activated conformer that self-associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms of this reconfiguration in the presence of a membrane have yet to be fully elucidated. To explore the metamorphosis of membrane-bound BAK, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS). The HDX-MS profile of BAK on liposomes comprising mitochondrial lipids was consistent with known solution structures of inactive BAK. Following activation, HDX-MS resolved major reconfigurations in BAK. Mutagenesis guided by our HDX-MS profiling revealed that the BCL-2 homology (BH) 4 domain maintains the inactive conformation of BAK, and disrupting this domain is sufficient for constitutive BAK activation. Moreover, the entire N-terminal region preceding the BAK oligomerisation domains became disordered post-activation and remained disordered in the activated oligomer. Removal of the disordered N-terminus did not impair, but rather slightly potentiated, BAK-mediated membrane permeabilisation of liposomes and mitochondria. Together, our HDX-MS analyses reveal new insights into the dynamic nature of BAK activation on a membrane, which may provide new opportunities for therapeutic targeting.
Subject(s)
Liposomes/chemistry , Membrane Lipids/chemistry , Proto-Oncogene Proteins c-bcl-2/chemistry , bcl-2 Homologous Antagonist-Killer Protein/chemistry , Animals , Binding Sites , Cloning, Molecular , Deuterium Exchange Measurement , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Liposomes/metabolism , Membrane Lipids/metabolism , Mice , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Folding , Protein Interaction Domains and Motifs , Protein Multimerization , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thermodynamics , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolismABSTRACT
A body of data supports the existence of core (α2-α5) dimers of BAK and BAX in the oligomeric, membrane-perturbing conformation of these essential apoptotic effector molecules. Molecular structures for these dimers have only been captured for truncated constructs encompassing the core domain alone. Here, we report a crystal structure of BAK α2-α8 dimers (i.e., minus its flexible N-terminal helix and membrane-anchoring C-terminal segment) that has been obtained through the activation of monomeric BAK with the detergent C12E8. Core dimers are evident, linked through the crystal by contacts via latch (α6-α8) domains. This crystal structure shows activated BAK dimers with the extended latch domain present. Our data provide direct evidence for the conformational change converting BAK from inert monomer to the functional dimer that destroys mitochondrial integrity. This dimer is the smallest functional unit for recombinant BAK or BAX described so far.
Subject(s)
Detergents/chemistry , Protein Multimerization , bcl-2 Homologous Antagonist-Killer Protein/chemistry , Amino Acid Sequence , Animals , Liposomes , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Protein Structure, Secondary , bcl-2 Homologous Antagonist-Killer Protein/metabolismABSTRACT
In metazoans, Bcl-2 family proteins are major regulators of mitochondrially mediated apoptosis; however, their evolution remains poorly understood. Here, we describe the molecular characterization of the four members of the Bcl-2 family in the most primitive metazoan, Trichoplax adhaerens All four trBcl-2 homologs are multimotif Bcl-2 group, with trBcl-2L1 and trBcl-2L2 being highly divergent antiapoptotic Bcl-2 members, whereas trBcl-2L3 and trBcl-2L4 are homologs of proapoptotic Bax and Bak, respectively. trBax expression permeabilizes the mitochondrial outer membrane, while trBak operates as a BH3-only sensitizer repressing antiapoptotic activities of trBcl-2L1 and trBcl-2L2. The crystal structure of a trBcl-2L2:trBak BH3 complex reveals that trBcl-2L2 uses the canonical Bcl-2 ligand binding groove to sequester trBak BH3, indicating that the structural basis for apoptosis control is conserved from T. adhaerens to mammals. Finally, we demonstrate that both trBax and trBak BH3 peptides bind selectively to human Bcl-2 homologs to sensitize cancer cells to chemotherapy treatment.
Subject(s)
Apoptosis , bcl-2 Homologous Antagonist-Killer Protein , Animals , Humans , Mammals/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolismSubject(s)
Apoptosis , Mitochondrial Membranes/metabolism , Phospholipids/metabolism , Protein Multimerization , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Animals , Humans , Mitochondrial Membranes/chemistry , Protein Binding , Protein Domains , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/metabolismABSTRACT
BAK and BAX are essential mediators of apoptosis that oligomerize in response to death cues, thereby causing permeabilization of the mitochondrial outer membrane. Their transition from quiescent monomers to pore-forming oligomers involves a well-characterized symmetric dimer intermediate. However, no essential secondary interface that can be disrupted by mutagenesis has been identified. Here we describe crystal structures of human BAK core domain (α2-α5) dimers that reveal preferred binding sites for membrane lipids and detergents. The phospholipid headgroup and one acyl chain (sn2) associate with one core dimer while the other acyl chain (sn1) associates with a neighboring core dimer, suggesting a mechanism by which lipids contribute to the oligomerization of BAK. Our data support a model in which, unlike for other pore-forming proteins whose monomers assemble into oligomers primarily through protein-protein interfaces, the membrane itself plays a role in BAK and BAX oligomerization.
Subject(s)
Membrane Lipids/metabolism , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Binding Sites , Crystallography, X-Ray , Humans , Membrane Lipids/chemistry , Molecular Docking Simulation , Protein Binding , Protein Multimerization , bcl-2 Homologous Antagonist-Killer Protein/chemistryABSTRACT
H1.2 is a key mediator of apoptosis following DNA double-strand breaks. The link between H1.2 and canonical apoptotic pathways is unclear. One study found that H1.2 stimulates cytochrome c (Cyt c) release; in contrast, apoptosis-inducing factor was found to be released in another study. The C-terminal domain (CTD) of H1.2 has been implicated in the latter pathway, but activation of the proapoptotic protein BCL-2 homologous antagonist/killer (BAK) is a common denominator in both pathways. This study aimed to determine whether the CTD of H1.2 is also responsible for mitochondrial Cyt c release and whether a previously identified K/RVVKP motif in the CTD mediates the response. This study investigated if H1.2 mediates apoptosis induction through direct interaction with BAK. We established that the CTD of H1.2 stimulates mitochondrial Cyt c release in vitro in a mitochondrial permeability transition-independent manner and that the substitution of a single valine with threonine in the K/RVVKP motif abolishes Cyt c release. Additionally, we showed that H1.2 directly interacts with BAK with weak affinity and that the CTD of H1.2 mediates this binding. Using two 20-amino acid peptides derived from the CTD of H1.2 and H1.1 (K/RVVKP motif inclusive), we determined the main residues involved in the direct interaction with BAK. We propose that H1.2 operates through the K/RVVKP motif by directly activating BAK through inter- and intramolecular interactions. These findings expand the view of H1.2 as a signal-transducing molecule that can activate apoptosis in a BAK-dependent manner.
Subject(s)
Apoptosis , Cytochromes c/metabolism , Histones/metabolism , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Amino Acid Motifs , Amino Acid Sequence , Histones/chemistry , Humans , Mitochondria/metabolism , Models, Molecular , Molecular Dynamics Simulation , Protein Conformation , Protein Domains , Sequence Homology , bcl-2 Homologous Antagonist-Killer Protein/chemistryABSTRACT
Many cellular stresses are transduced into apoptotic signals through modification or up-regulation of the BH3-only subfamily of BCL2 proteins. Through direct or indirect mechanisms, these proteins activate BAK and BAX to permeabilize the mitochondrial outer membrane. While the BH3-only proteins BIM, PUMA, and tBID have been confirmed to directly activate BAK through its canonical BH3 binding groove, whether the BH3-only proteins BMF, HRK or BIK can directly activate BAK is less clear. Here we show that BMF and HRK bind and directly activate BAK. Through NMR studies, site-directed mutagenesis, and advanced molecular dynamics simulations, we also find that BAK activation by BMF and possibly HRK involves a previously unrecognized binding groove formed by BAK α4, α6, and α7 helices. Alterations in this groove decrease the ability of BMF and HRK to bind BAK, permeabilize membranes and induce apoptosis, suggesting a potential role for this BH3-binding site in BAK activation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/genetics , Binding Sites/genetics , Cells, Cultured , Humans , Jurkat Cells , Magnetic Resonance Spectroscopy , Mice, Knockout , Mitochondrial Membranes/metabolism , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Domains , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Sequence Homology, Amino Acid , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/geneticsABSTRACT
Integrin αvß3 was reported as positive regulators of tumorigenesis and highly expressed in cancer stem cells and kinds of cancers, thus, it is an appealing target for cancer treatment. Nanomedicine with targeting delivery ability has developed rapidly and shown its great therapeutic potential in cancer therapy. Proteins are ideal material for nanomedicine regarding to their excellent biocompatibility, and protein-only self-assembled nanoparticles technology provides a robust method to produce protein nanoparticles. Pro-apoptotic proteins or peptides, such as BAK, have attracted increasing attention in the inhibition of tumor growth. However, the self-assembled nanoparticles of BAK targeting to integrin αvß3 over-expressed tumor cells need to be investigated. In this study, we designed recombinant proteins with BH3 BAK as active domain and RGD peptides as targeting ligands to self-assemble into protein nanoparticles (named as PN2-1 et al.), then experimentally evaluated the nanoparticle size, fluorescence feature, stability, targeting ability and cytotoxicity to tumor cells in vitro. The results showed that the protein nanoparticles containing RGD peptides had a uniform particle size with an diameter of approximately 23â¯nm. PN2-1 had notable inhibition to cell proliferation of C6 cells, C26 cells and MCF-7 cells, with a lower IC50 than the nanoparticles which only had BAK motif without RGD peptide. PN2-1 had higher cellular uptake into C6 cells than MCF-7 cells. Our results demonstrate that the RGD peptide could enhance the cytotoxicity of BAK nanoparticles to tumor cells and increase their tumor targeting ability. This study provides an insight into the design and development of integrin αvß3 targeting protein nanoparticle for cancer treatment.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Drug Carriers , Integrin alphaVbeta3/metabolism , Nanoparticles , Neoplasms/drug therapy , Oligopeptides/metabolism , bcl-2 Homologous Antagonist-Killer Protein/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Dose-Response Relationship, Drug , Drug Compounding , Drug Stability , Female , Humans , Inhibitory Concentration 50 , MCF-7 Cells , Mice , Neoplasms/metabolism , Neoplasms/pathology , Oligopeptides/chemistry , Particle Size , Rats , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolismABSTRACT
Mitochondrial Outer Membrane (MOM) Permeabilization (MOMP) is a critical event in the mitochondrial types of apoptosis. MOMP is controled by the proteins of the Bcl-2 family and its two proapoptotic members Bak and Bax are the key effectors of MOMP. Voltage-dependent anion channel 2 (VDAC2) is an integral membrane protein that plays an important role in the regulation of Bak and Bax apoptotic function, but underlying mechanisms are not fully understood. In the present article, the mechanisms of MOMP regulation mediated by VDAC2 were explored using structure-based modeling. We show that Bak, prior to an apoptotic stimulus, possesses two low-energy conformations of high shape - and polar complementarity in respect to VDAC2, resulting in two high-affinity modes of Bak binding to VDAC2, one with Bak fully residing in the cytosol and the other with Bak α9 helix inserted into the membrane. Even higher binding affinity of VDAC2 for tBid (truncated Bid/p15) was established, suggesting the tBid-mediated displacement of Bak from the VDAC2/Bak complex resulting in the formation of the VDAC2/tBid complex. The structural analysis of the interaction of this complex with Bax revealed a very high binding affinity of this complex for Bax, suggesting the recruitment of Bax to the MOM by this complex under apoptotic conditions. Besides, we revealed one more low-energy structure of Bax of high binding affinity towards the VDAC2/tBid complex and with helix α9 inserted into the membrane.
Subject(s)
Apoptosis , Voltage-Dependent Anion Channel 2/metabolism , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolism , Computational Biology , Humans , Mitochondrial Membranes/metabolism , Models, Molecular , Protein Conformation , Voltage-Dependent Anion Channel 2/chemistry , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2-Associated X Protein/chemistryABSTRACT
BACKGROUND: While tumor suppressor p53 functions primarily as a transcription factor in the nucleus, cellular stress can cause p53 to translocate to the mitochondria and directly trigger a rapid apoptotic response. We have previously shown that fusing p53 (or its DNA binding domain, DBD, alone) to the mitochondrial targeting signal (MTS) from Bak or Bax can target p53 to the mitochondria and induce apoptosis in gynecological cancer cell lines including cervical cancer cells (HeLa; wt p53), ovarian cancer cells (SKOV-3; p53 267del non-expressing), and breast cancer cells (T47D; L194F p53 mutation). However, p53 with Bak or Bax MTSs have not been previously tested in cancers with strong dominant negative (DN) mutant p53 which are capable of inactivating wt p53 by homo-oligomerization. Since p53-Bak or Bax MTS constructs act as monomers, they are not subject to DN inhibition. For this study, the utility of p53-Bak or p53-Bax MTS constructs was tested for ovarian cancers which are known to have varying p53 statuses, including a strong DN contact mutant p53 (Ovcar-3 cells), a p53 DN structural mutant (Kuramochi cells), and a p53 wild type, low expressing cells (ID8). RESULTS: Our mitochondrial p53 constructs were tested for their ability to localize to the mitochondria in both mutant non-expressing p53 (Skov-3) and p53 structural mutant (Kuramochi) cell lines using fluorescence microscopy and a nuclear transcriptional activity assay. The apoptotic activity of these mitochondrial constructs was determined using a mitochondrial outer membrane depolarization assay (TMRE), caspase assay, and a late stage cell death assay (7-AAD). We also tested the possibility of using our constructs with paclitaxel, the current standard of care in ovarian cancer treatment. Our data indicates that our mitochondrial p53 constructs are able to effectively localize to the mitochondria in cancer cells with structural mutant p53 and induce apoptosis in many ovarian cancer cell lines with different p53 statuses. These constructs can also be used in combination with paclitaxel for an increased apoptotic effect. CONCLUSIONS: The results suggest that targeting p53 to mitochondria can be a new strategy for ovarian cancer treatment.