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The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target., (© 2024. The Author(s).)

The B-cell lymphoma 2 (BCL2) family members, BCL2-associated protein X (BAX) and BCL2 homologous antagonist killer (BAK), are required for programmed cell death via the mitochondrial pathway. When cells are stressed, damaged or redundant, the balance of power between the BCL2 family of proteins shifts towards BAX and BAK, allowing their transition from an inactive, monomeric state to a membrane-active oligomeric form that releases cytochrome c from the mitochondrial intermembrane space. That oligomeric state has an essential intermediate, a symmetric homodimer of BAX or BAK. Here we describe crystal structures of dimers of the core domain of BAX, comprising its helices α2-α5. These structures provide an atomic resolution description of the interactions that drive BAX homo-dimerisation and insights into potential interaction between core domain dimers and membrane lipids. The previously identified BAK lipid-interacting sites are not conserved with BAX and are likely to determine the differences between them in their interactions with lipids. We also describe structures of heterodimers of BAK/BAX core domains, yielding further insight into the differences in lipid binding between BAX and BAK., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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., (© 2023 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

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.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (© 2022. Crown.)

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., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Systematic incorporation of ring-constrained β- and γ-amino acid residues into α-helix mimetics engenders stable helical secondary structures. In this paper, functional α/β/γ-helical peptidomimetics were explored for mimicry of BH3 helical domains, Bim as a pioneering study. The Bim-based α/β/γ-peptides in an αγααβα-hexad repeat with five helical turns inhibited the interaction between Bak and Bcl-x L with excellent resistance towards proteolytic digestion. Further optimization of the α/β/γ-backbone strategy will considerably expand the utility of functional α/β/γ-peptidomimetics, in particular due to its prominent stability against proteolysis.

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

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., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

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., (© 2021 The Authors.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

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.

Integrin α v β 3 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 α v β 3 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 23 nm. PN2-1 had notable inhibition to cell proliferation of C6 cells, C26 cells and MCF-7 cells, with a lower IC 50 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 α v β 3 targeting protein nanoparticle for cancer treatment., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

The E3 ubiquitin ligase Parkin is a key effector of the removal of damaged mitochondria by mitophagy. Parkin determines cell fate in response to mitochondrial damage, with its loss promoting early onset Parkinson's disease and potentially also cancer progression. Controlling a cell's apoptotic response is essential to co-ordinate the removal of damaged mitochondria. We report that following mitochondrial damage-induced mitophagy, Parkin directly ubiquitinates the apoptotic effector protein BAK at a conserved lysine in its hydrophobic groove, a region that is crucial for BAK activation by BH3-only proteins and its homo-dimerisation during apoptosis. Ubiquitination inhibited BAK activity by impairing its activation and the formation of lethal BAK oligomers. Parkin also suppresses BAX-mediated apoptosis, but in the absence of BAX ubiquitination suggesting an indirect mechanism. In addition, we find that BAK-dependent mitochondrial outer membrane permeabilisation during apoptosis promotes PINK1-dependent Parkin activation. Hence, we propose that Parkin directly inhibits BAK to suppress errant apoptosis, thereby allowing the effective clearance of damaged mitochondria, but also promotes clearance of apoptotic mitochondria to limit their potential pro-inflammatory effect., (© 2018 The Authors.)

Progress in drug delivery is hampered by a lack of efficient strategies to target drugs with high specificity and precise spatiotemporal regulation. The remote control of nanoparticles and drugs with light allows regulation of their action site and dosage. Peptide-based drugs are highly specific, non-immunogenic, and can be designed to cross the plasma membrane. In order to combine target specificity and remote control of drug action, here we describe a versatile strategy based on a generalized template to design nanoswitchable peptides that modulate protein-protein interactions upon light activation. This approach is demonstrated to promote photomodulation of two important targets involved in apoptosis (the interactions Bcl-xL-Bak and MDM2-p53), but can be also applied to a large pool of therapeutically relevant protein-protein interactions mediated by α-helical motifs. The template can be adjusted using readily available information about hot spots (residues contributing most to the binding energy) at the protein-protein interface of interest., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The continuous wave (CW) and pulse electron paramagnetic resonance (EPR) methods enable the measurement of distances between spin-labeled residues in biopolymers including proteins, providing structural information. Here we describe the CW EPR deconvolution/convolution method and the four-pulse double electron-electron resonance (DEER) approach for distance determination, which were applied to elucidate the organization of the BAK apoptotic pores formed in the lipid bilayers.

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.

B-cell lymphoma 2 (Bcl-2) family proteins are the central regulators of apoptosis, functioning via mitochondrial outer membrane permeabilization. The family members are involved in several stages of apoptosis regulation. The overexpression of the anti-apoptotic proteins leads to several cancer pathological conditions. This overexpression is modulated or inhibited by heterodimerization of pro-apoptotic BH3 domain or BH3-only peptides to the hydrophobic groove present at the surface of anti-apoptotic proteins. Additionally, the heterodimerization displayed differences in binding affinity profile among the pro-apoptotic peptides binding to anti-apoptotic proteins. In light of discovering the novel peptide/drug molecules that contain the potential to inhibit specific anti-apoptotic protein, it is necessary to understand the molecular basis of recognition between the protein and its binding partner (peptide or ligand) along with its binding energies. Therefore, the present work focused on deciphering the molecular basis of recognition between pro-apoptotic Bak peptide binding to different anti-apoptotic (Bcl-xL, Bfl-1, Bcl-W, Mcl-1, and Bcl-2) proteins using advanced Molecular Dynamics (MD) approach such as Molecular Mechanics-Generalized Born Solvent Accessible. The results from our investigation revealed that the predicted binding free energies showed excellent correlation with the experimental values (r 2  = .95). The electrostatic (ΔG ele ) contributions are the major component that drives the interaction between Bak peptides and different anti-apoptotic peptides. Additionally, van der Waals (ΔG vdw ) energies also play an indispensible role in determining the binding free energy. Furthermore, the decomposition analysis highlighted the comprehensive information about the energy contributions of hotspot residues involved in stabilizing the interaction between Bak peptide and different anti-apoptotic proteins.

Bax and Bak are members of the Bcl-2 family and core regulators of the intrinsic pathway of apoptosis. Upon apoptotic stimuli, they are activated and oligomerize at the mitochondrial outer membrane (MOM) to mediate its permeabilization, which is considered a key step in apoptosis. However, the molecular mechanism underlying Bax and Bak function has remained a key question in the field. Here, we review recent structural and biophysical evidence that has changed our understanding of how Bax and Bak promote MOM permeabilization. We also discuss how the spatial regulation of Bcl-2 family preference for binding partners contributes to regulate Bax and Bak activation. Finally, we consider the contribution of mitochondrial composition, dynamics and interaction with other organelles to apoptosis commitment. A new perspective is emerging, in which the control of apoptosis by Bax and Bak goes beyond them and is highly influenced by additional mitochondrial components., (© 2017 Federation of European Biochemical Societies.)

Global cleavage of cellular proteins by activated caspases is a hallmark of apoptosis, which causes biochemical collapse of the cell. Recent studies suggest that, rather than completely destroying a protein, caspase cleavage can confer novel characteristics or functions. In this respect, the post-caspase role of Bcl-2 family proteins remains uncharacterized. Here, we showed that Mcl-1, a pro-survival member of the Bcl-2 family, was cleaved by caspase-3 in non-small cell lung cancer (NSCLC) cells undergoing chemotherapeutic agent-triggered apoptosis. Caspase cleavage partially impaired the anti-apoptotic activity of Mcl-1 by reducing its mitochondrial localization and impeding its association with the permeability transition pore-forming protein Bak. However, the stability of cleaved Mcl-1 was markedly enhanced because it was more refractory to ubiquitination-dependent proteasomal degradation, thereby improving cell viability to a greater extent than full-length Mcl-1 when transiently expressed in NSCLC cells. These findings shed new light on the role of Mcl-1 in apoptosis and suggest potential novel targets for optimizing the tumoricidal capacity of chemotherapy.

Certain BH3-only proteins transiently bind and activate Bak and Bax, initiating their oligomerization and the permeabilization of the mitochondrial outer membrane, a pivotal step in the mitochondrial pathway to apoptosis. Here we describe the first crystal structures of an activator BH3 peptide bound to Bak and illustrate their use in the design of BH3 derivatives capable of inhibiting human Bak on mitochondria. These BH3 derivatives compete for the activation site at the canonical groove, are the first engineered inhibitors of Bak activation, and support the role of key conformational transitions associated with Bak activation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

BAK and BAX are the essential effectors of apoptosis because without them a cell is resistant to most apoptotic stimuli. BAK and BAX undergo conformation changes to homooligomerize then permeabilize the mitochondrial outer membrane during apoptosis. How BCL-2 homology 3 (BH3)-only proteins bind to activate BAK and BAX is unclear. We report that BH3-only proteins bind inactive full-length BAK at mitochondria and then dissociate following exposure of the BAK BH3 and BH4 domains before BAK homodimerization. Using a functional obstructive labeling approach, we show that activation of BAK involves important interactions of BH3-only proteins with both the canonical hydrophobic binding groove (α2-5) and α6 at the rear of BAK, with interaction at α6 promoting an open groove to receive a BH3-only protein. Once activated, how BAK homodimers multimerize to form the putative apoptotic pore is unknown. Obstructive labeling of BAK beyond the BH3 domain and hydrophobic groove did not inhibit multimerization and mitochondrial damage, indicating that critical protein-protein interfaces in BAK self-association are limited to the α2-5 homodimerization domain., Competing Interests: The authors declare no conflict of interest.

A chemical reaction that is triggered by a specific RNA molecule might provide opportunities for the design of artificial feedback loops. We envision a peptidyl transfer reaction in which mRNA encoding an antiapoptotic protein would instruct the synthesis of apoptosis-inducing peptides. In this study, we used the RNA-programmed synthesis of a 16-mer peptide derived from the BH3 domain of the protein Bak, which inhibits the antiapoptotic protein Bcl-x L . The reaction involves the transfer of a thioester-linked donor peptide fragment from one PNA conjugate to an acceptor peptide-PNA conjugate. We asked two key questions. What are the chemical requirements that allow RNA-templated synthesis of a 16-mer peptide to proceed at lower (nanomolar) concentrations of RNA, that is, the concentration range found in cancer cells? Will such reactions provide sufficient amounts of peptide product and sufficient affinity to interfere with the targeted protein-protein interaction? Perhaps surprisingly, the lengths of the peptides involved in peptidyl transfer chemistry have little effect on the achievable rate enhancements. However, the nature of the thioester C terminus, the distance between the targeted template annealing sites, and template affinity play important roles. The investigation revealed guidelines for the reaction design for peptidyl transfer with low amounts (1-10 nm) of RNA, yet still provide sufficient product to antagonize a protein-protein interaction., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

During apoptosis, Bak and Bax undergo major conformational change and form symmetric dimers that coalesce to perforate the mitochondrial outer membrane via an unknown mechanism. We have employed cysteine labelling and linkage analysis to the full length of Bak in mitochondria. This comprehensive survey showed that in each Bak dimer the N-termini are fully solvent-exposed and mobile, the core is highly structured, and the C-termini are flexible but restrained by their contact with the membrane. Dimer-dimer interactions were more labile than the BH3:groove interaction within dimers, suggesting there is no extensive protein interface between dimers. In addition, linkage in the mobile Bak N-terminus (V61C) specifically quantified association between dimers, allowing mathematical simulations of dimer arrangement. Together, our data show that Bak dimers form disordered clusters to generate lipidic pores. These findings provide a molecular explanation for the observed structural heterogeneity of the apoptotic pore., Competing Interests: The authors declare that no competing interests exist.

Cellular commitment to the mitochondrial pathway of apoptosis is accomplished when proapoptotic B cell chronic lymphocytic leukemia/lymphoma (BCL)-2 proteins compromise mitochondrial integrity through the process of mitochondrial outer membrane permeabilization (MOMP). For nearly three decades, intensive efforts focused on the identification and interactions of two key proapoptotic BCL-2 proteins: BCL-2 antagonist killer (BAK) and BCL-2-associated X (BAX). Indeed, we now have critical insights into which BCL-2 proteins interact with BAK/BAX to either preserve survival or initiate MOMP. In contrast, while mitochondria are targeted by BAK/BAX, a molecular understanding of how these organelles govern BAK/BAX function remains less clear. Here, we integrate recent mechanistic insights of proapoptotic BCL-2 protein function in the context of mitochondrial environment, and discuss current and potential pharmacological opportunities to control MOMP in disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A critical process in apoptosis is the permeabilization of the mitochondrial outer membrane (MOM). This process is known to be regulated by the multi-domain Bcl-2 family proteins. For example, the pro-apoptotic proteins Bax and Bak are responsible for forming pores at MOM. The anti-apoptotic proteins (including Bcl-2, Mcl-1 and Bcl-xL), on the other hand, can inhibit this pore-forming process. Interestingly, although these two subgroups of proteins perform opposite apoptotic functions, their structures are very similar. This raises two highly interesting questions: (1) Why do these structurally similar proteins play opposite roles in apoptosis? (2) What are the roles of different functional domains of a Bcl-2 family protein in determining its apoptotic property? In this study, we generated a series of deletion mutants and substitution chimera, and used a combination of molecular biology, bio-informatics and living cell imaging techniques to answer these questions. Our major findings are: (1) All of the Bcl-2 family proteins appear to possess an intrinsic pro-apoptotic property. (2) The N-termini of these proteins play an active role in suppressing their pro-apoptotic function. (3) The apoptotic potency is positively correlated with membrane affinity of the alpha 5/6 helix domains. (4) Charge distribution flanking the alpha 5/6 helices is also important for the apoptotic potency. These findings explain why different members of Bcl-2 family proteins with similar domain composition can function oppositely in the apoptotic process.

The goal of the present study was to ascertain the differential performance of a long molecular dynamics trajectory versus several shorter ones starting from different points in the phase space and covering the same sampling time. For this purpose, we selected the 16-mer peptide Bak 16 BH3 as a model for study and carried out several samplings in explicit solvent. These samplings included an 8 μs trajectory (sampling S1); two 4 μs trajectories (sampling S2); four 2 μs trajectories (sampling S3); eight 1 μs trajectories (sampling S4); 16 0.5 μs trajectories (sampling S5), and 80 0.1 μs trajectories (sampling S6). Moreover, the 8 μs trajectory was further extended to 16 μs to have reference values of the diverse properties measured. The diverse samplings were compared qualitatively and quantitatively. Among the former, we carried out a comparison of the conformational profiles of the peptide using cluster analysis. Moreover, we also gained insight into the interchange among these structures along the sampling process. Among the latter, we computed the number of new conformational patterns sampled with time using strings defined from the conformations attained by each of the residues in the peptide. We also compared the locations and depths of the obtained minima on the free energy surface using principal component analysis. Finally, we also compared the helical profiles per residue at the end of the sampling process. The results suggest that a few short molecular dynamics trajectories may provide better sampling than one unique trajectory. Moreover, this procedure can also be advantageous to avoid getting trapped in a local minimum. However, caution should be exercised since short trajectories need to be long enough to overcome local barriers surrounding the starting point and the required sampling time depends on the number of degrees of freedom of the system under study. An effective way to gain insight into the minimum MD trajectory length is to monitor the convergence of different structural features, as shown in the present work.

Nuclear magnetic resonance (NMR) spectroscopy has the intrinsic capabilities to investigate proteins in native environments. In general, however, NMR relies on non-natural protein purity and concentration to increase the desired signal over the background. We here report on the efficient and specific hyperpolarization of low amounts of a target protein in a large isotope-labeled background by combining dynamic nuclear polarization (DNP) and the selectivity of protein interactions. Using a biradical-labeled ligand, we were able to direct the hyperpolarization to the protein of interest, maintaining comparable signal enhancement with about 400-fold less radicals than conventionally used. We could selectively filter out our target protein directly from crude cell lysate obtained from only 8 mL of fully isotope-enriched cell culture. Our approach offers effective means to study proteins with atomic resolution in increasingly native concentrations and environments., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In mitochondrial apoptosis, Bak is activated by death signals to form pores of unknown structure on the mitochondrial outer membrane via homooligomerization. Cytochrome c and other apoptotic factors are released from the intermembrane space through these pores, initiating downstream apoptosis events. Using chemical crosslinking and double electron electron resonance (DEER)-derived distance measurements between specific structural elements in Bak, here we clarify how the Bak pore is assembled. We propose that previously described BH3-in-groove homodimers (BGH) are juxtaposed via the 'α3/α5' interface, in which the C-termini of helices α3 and α5 are in close proximity between two neighboring Bak homodimers. This interface is observed concomitantly with the well-known 'α6:α6' interface. We also mapped the contacts between Bak homodimers and the lipid bilayer based on EPR spectroscopy topology studies. Our results suggest a model for the lipidic Bak pore, whereby the mitochondrial targeting C-terminal helix does not change topology to accommodate the lining of the pore lumen by BGH.

During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2-α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1-α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to α1. An antibody (clone 3C10) to the Bax α1-α2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates α1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under α1. Our identification of the α1-α2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins.

The disruption of aberrant protein-protein interactions (PPIs) with synthetic agents remains a challenging goal in contemporary medicinal chemistry but some progress has been made. One such dysregulated PPI is that between the anti-apoptotic Bcl-2 proteins, including myeloid cell leukemia-1 (Mcl-1), and the α-helical Bcl-2 homology-3 (BH3) domains of its pro-apoptotic counterparts, such as Bak. Herein, we describe the discovery of small-molecule inhibitors of the Mcl-1 oncoprotein based on a novel chemotype. Particularly, re-engineering of our α-helix mimetic JY-1-106 into 2,6-di-substituted nicotinates afforded inhibitors of comparable potencies but with significantly decreased molecular weights. The most potent inhibitor 2-(benzyloxy)-6-(4-chloro-3,5-dimethylphenoxy)nicotinic acid (1 r: Ki =2.90 μm) likely binds in the p2 pocket of Mcl-1 and engages R263 in a salt bridge through its carboxylic acid, as supported by 2D (1) H-(15) N HSQC NMR data. Significantly, inhibitors were easily accessed in just four steps, which will facilitate future optimization efforts., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Evidence for association between asthma and the unfolded protein response is emerging. Endoplasmic reticulum resident protein 57 (ERp57) is an endoplasmic reticulum-localized redox chaperone involved in folding and secretion of glycoproteins. We have previously demonstrated that ERp57 is upregulated in allergen-challenged human and murine lung epithelial cells. However, the role of ERp57 in asthma pathophysiology is unknown., Objectives: Here we sought to examine the contribution of airway epithelium-specific ERp57 in the pathogenesis of allergic asthma., Methods: We examined the expression of ERp57 in human asthmatic airway epithelium and used murine models of allergic asthma to evaluate the relevance of epithelium-specific ERp57., Results: Lung biopsy specimens from asthmatic and nonasthmatic patients revealed a predominant increase in ERp57 levels in epithelium of asthmatic patients. Deletion of ERp57 resulted in a significant decrease in inflammatory cell counts and airways resistance in a murine model of allergic asthma. Furthermore, we observed that disulfide bridges in eotaxin, epidermal growth factor, and periostin were also decreased in the lungs of house dust mite-challenged ERp57-deleted mice. Fibrotic markers, such as collagen and α smooth muscle actin, were also significantly decreased in the lungs of ERp57-deleted mice. Furthermore, adaptive immune responses were dispensable for house dust mite-induced endoplasmic reticulum stress and airways fibrosis., Conclusions: Here we show that ERp57 levels are increased in the airway epithelium of asthmatic patients and in mice with allergic airways disease. The ERp57 level increase is associated with redox modification of proinflammatory, apoptotic, and fibrotic mediators and contributes to airways hyperresponsiveness. The strategies to inhibit ERp57 specifically within the airways epithelium might provide an opportunity to alleviate the allergic asthma phenotype., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.

Despite palliative treatments, glioblastoma (GBM) remains a devastating malignancy with a mean survival of about 15 months after diagnosis. Programmed cell-death is de-regulated in almost all GBM and the re-activation of the mitochondrial apoptotic pathway through exogenous bioactive proteins may represent a powerful therapeutic tool to treat multidrug resistant GBM. We have reported that human Bak protein integrated in Liposomes (LB) was able, in vitro, to activate the mitochondrial apoptotic pathway in colon cancer cells. To evaluate the anti-tumor effects of LB on GBM, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and Western blot analysis were performed on GL26 murine cell line. LB treatment shows a dose-dependent inhibition of cell viability, followed by an up-regulation of Bax and a down-modulation of JNK1 proteins. In GL26-bearing mice, two different routes of administration were tested: intra-tumor and intravenous. Biodistribution, tumor growth and animal survival rates were followed. LB show long-lasting tumor accumulation. Moreover, the intra-tumor administration of LB induces tumor growth delay and total tumor regression in about 40% of treated mice, while the intravenous injection leads to a significant increased life span of mice paralleled by an increased tumor cells apoptosis. Our findings are functional to the design of LB with potentiated therapeutic efficacy for GBM.

Proteins belonging to the BCL2 family are key modulators of apoptosis that establish a complex network of interactions among themselves and with other cellular factors to regulate cell fate. It is well established that mitochondrial membranes are the main locus of action of all BCL2 family proteins, but it is difficult to obtain a precise view of how BCL2 family members operate at the native mitochondrial membrane environment during apoptosis. Here, we used minimalist model systems and multiple fluorescence-based techniques to examine selected membrane activities of MCL1 and BAK under apoptotic-like conditions. We show that three distinct apoptosis-related factors (i.e. the BCL2 homology 3 ligand cBID, the mitochondrion-specific lipid cardiolipin, and membrane geometrical curvature) all promote membrane association of BCL2-like structural folds belonging to both MCL1 and BAK. However, at the same time, the two proteins exhibited distinguishing features in their membrane association modes under apoptotic-like conditions. In addition, scanning fluorescence cross-correlation spectroscopy and FRET measurements revealed that the BCL2-like structural fold of MCL1, but not that of BAK, forms stable heterodimeric complexes with cBID in a manner adjustable by membrane cardiolipin content and curvature degree. Our results add significantly to a growing body of evidence indicating that the mitochondrial membrane environment plays a complex and active role in the mode of action of BCL2 family proteins., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Like the commonly used immunoprecipitation (IP) approach, this procedure for the detection of activated Bak or Bax by intracellular flow cytometry is based on the principle that Bak and Bax, during activation, expose occluded amino-terminal epitopes that can be recognized by conformation-specific antibodies. Flow cytometric analysis requires fewer cells and is less time-consuming than IP. Further, in contrast to IP, flow cytometry produces a quantifiable assessment of the percentage of cells containing activated Bak or Bax, which can be correlated with cell death., (© 2015 Cold Spring Harbor Laboratory Press.)

Activation of both Bax and Bak during apoptosis involves significant conformation change. Investigation of this phenomenon by immunoprecipitation (IP) requires a detergent such as CHAPS that does not induce significant conformation change. IP with conformation-specific Bax or Bak antibodies is observed in CHAPS only following an apoptotic stimulus, whereas the same antibodies will immunoprecipitate from both nonapoptotic and apoptotic cells in the presence of Triton X-100. Thus, the latter detergent can serve as a positive control for IP, as described here., (© 2015 Cold Spring Harbor Laboratory Press.)

Blue native PAGE (BN-PAGE) uses Coomassie dye rather than denaturing SDS to provide a negative charge to proteins for electrophoresis. As such, it is a useful assay for investigating native supramolecular membrane complexes without the need for cross-linking. As Bak and Bax oligomers form in the mitochondrial outer membrane, and they can be efficiently monitored by BN-PAGE. Furthermore, BN-PAGE performed in conjunction with gel-shift using conformation-specific antibodies can provide additional information regarding the activation state of Bak or Bax in specific membrane complexes., (© 2015 Cold Spring Harbor Laboratory Press.)

Bax is a pro-apoptotic Bcl-2 family protein. The activated Bax translocates to mitochondria, where it forms pore and permeabilizes the mitochondrial outer membrane. This process requires the BH3-only activator protein (i.e. tBid) and can be inhibited by anti-apoptotic Bcl-2 family proteins such as Bcl-xL. Here by using single molecule fluorescence techniques, we studied the integration and oligomerization of Bax in lipid bilayers. Our study revealed that Bax can bind to lipid membrane spontaneously in the absence of tBid. The Bax pore formation undergoes at least two steps: pre-pore formation and membrane insertion. The activated Bax triggered by tBid or BH3 domain peptide integrates on bilayers and tends to form tetramers, which are termed as pre-pore. Subsequent insertion of the pre-pore into membrane is highly dependent on the composition of cardiolipin in lipid bilayers. Bcl-xL can translocate Bax from membrane to solution and inhibit the pore formation. The study of Bax integration and oligomerization at the single molecule level provides new evidences that may help elucidate the pore formation of Bax and its regulatory mechanism in apoptosis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.

Previous studies have shown that human immunodeficiency virus (HIV) protease cleaves procaspase 8 to a fragment, termed Casp8p41, that lacks caspase activity but nonetheless contributes to T cell apoptosis. Herein, we show that Casp8p41 contains a domain that interacts with the BH3-binding groove of pro-apoptotic Bak to cause Bak oligomerization, Bak-mediated membrane permeabilization, and cell death. Levels of active Bak are higher in HIV-infected T cells that express Casp8p41. Conversely, targeted mutations in the Bak-interacting domain diminish Bak binding and Casp8p41-mediated cell death. Similar mutations in procaspase 8 impair the ability of HIV to kill infected T cells. These observations support a novel paradigm in which HIV converts a normal cellular constituent into a direct activator that functions like a BH3-only protein., (© 2014 Sainski et al.)