A novel inhibitory BAK antibody enables assessment of non-activated BAK in cancer cells.

BAX and BAK are pro-apoptotic members of the BCL2 family that are required to permeabilize the mitochondrial outer membrane. The proteins can adopt a non-activated monomeric conformation, or an activated conformation in which the exposed BH3 domain facilitates binding either to a prosurvival protein or to another activated BAK or BAX protein to promote pore formation. Certain cancer cells are proposed to have high levels of activated BAK sequestered by MCL1 or BCLXL, thus priming these cells to undergo apoptosis in response to BH3 mimetic compounds that target MCL1 or BCLXL. Here we report the first antibody, 14G6, that is specific for the non-activated BAK conformer. A crystal structure of 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites involved in the unfolding of BAK during its activation. In mitochondrial experiments, 14G6 inhibited BAK unfolding triggered by three diverse BAK activators, supporting crucial roles for both α1 dissociation and separation of the core (α2-α5) and latch (α6-α9) regions in BAK activation. 14G6 bound the majority of BAK in several leukaemia cell lines, and binding decreased following treatment with BH3 mimetics, indicating only minor levels of constitutively activated BAK in those cells. In summary, 14G6 provides a new means of assessing BAK status in response to anti-cancer treatments.

A case report of coexisting multinodular goiter with carotid body tumor.

INTRODUCTION: Carotid body tumor (CBT), a neuroendocrine neoplasm, and benign multinodular goiter (BMNG) are distinct pathologies affecting the neck region. Although rare, they can occur concurrently. This case contributes to the limited evidence regarding the association between these distinct pathologies and their operative management. CASE PRESENTATION: The patient was a 45-year-old female with a palpable mass on the right side of her neck. She was diagnosed with Shamblin type III non-secretory CBT alongside BMNG. The surgical intervention included resection of the CBT, carotid artery bypass, and Dunhill thyroidectomy. DISCUSSION: This case is the third reported instance of coexisting CBT and BMNG. Their causative relationship is evident in the literature without a clear explanation of the underlying mechanism. Both conditions are treated surgically. Dunhill thyroidectomy for BMNG is a safer option, offering more flexibility and advantages over other thyroidectomies. CONCLUSION: This case highlights the complexity of managing such dual pathologies and may provide further evidence of their association.

Sequence differences between BAX and BAK core domains manifest as differences in their interactions with lipids.

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.

Performance comparison of machine learning driven approaches for classification of complex noises in quick response code images.

Quick response codes (QRCs) are found on many consumer products and often encode security information. However, information retrieval at receiving end may become challenging due to the degraded clarity of QRC images. This degradation may occur because of the transmission of digital images over noise channels or limited printing technology. Although the ability to reduce noises is critical, it is just as important to define the type and quantity of noises present in QRC images. Therefore, this study proposed a simple deep learning-based architecture to segregate the image as either an original (normal) QRC or a noisy QRC and identifies the noise type present in the image. For this, the study is divided into two stages. Firstly, it generated a QRC image dataset of 80,000 images by introducing seven different noises (speckle, salt & pepper, Poisson, pepper, localvar, salt, and Gaussian) to the original QRC images. Secondly, the generated dataset is fed to train the proposed convolutional neural network (CNN)-based model, seventeen pre-trained deep learning models, and two classical machine learning algorithms (Naïve Bayes (NB) and Decision Tree (DT)). XceptionNet attained the highest accuracy (87.48%) and kappa (85.7%). However, it is worth noting that the proposed CNN network with few layers competes with the state-of-the-art models and attained near to best accuracy (86.75%). Furthermore, detailed analysis shows that all models failed to classify images having Gaussian and Localvar noises correctly.

RhopH2 and RhopH3 export enables assembly of the RhopH complex on P. falciparum-infected erythrocyte membranes.

RhopH complexes consists of Clag3, RhopH2 and RhopH3 and are essential for growth of Plasmodium falciparum inside infected erythrocytes. Proteins are released from rhoptry organelles during merozoite invasion and trafficked to the surface of infected erythrocytes and enable uptake of nutrients. RhopH3, unlike other RhopH proteins, is required for parasite invasion, suggesting some cellular processes RhopH proteins function as single players rather than a complex. We show the RhopH complex has not formed during merozoite invasion. Clag3 is directly released into the host cell cytoplasm, whilst RhopH2 and RhopH3 are released into the nascent parasitophorous vacuole. Export of RhopH2 and RhopH3 from the parasitophorous vacuole into the infected erythrocyte cytoplasm enables assembly of Clag3/RhopH2/RhopH3 complexes and incorporation into the host cell membrane concomitant with activation of nutrient uptake. This suggests compartmentalisation prevents premature channel assembly before intact complex is assembled at the host cell membrane.

Concomitant trans-sternal repair of Morgagni hernia and ventricular septal defect in a patient with Down syndrome: A case report.

INTRODUCTION: Morgagni hernia is a rare type of hernia occurring secondary to potential anterior-medial defects in the diaphragm. The association of the defect with congenital cardiac pathologies and Down syndrome are well known. The defect is repaired usually by trans-abdominal or transthoracic approaches. Trans-sternal repair of the hernia is preferred in patients undergoing concomitant open heart surgery. CASE PRESENTATION: A 2-year-old child with Down syndrome underwent concomitant repair of Morgagni hernia and closure of his ventricular septal defect under cardiopulmonary bypass. The hernia was corrected by the sternotomy approach, without opening the hernia content, before the correction of the cardiac pathology. The patient made an uneventful recovery and was discharged on the 4th postoperative day. DISCUSSION: Preoperative diagnosis of diaphragmatic hernia in congenital heart disease is important to decrease mortality rate. However, trans-sternal exposure of the defect is also possible, as in this case, in patients undergoing open heart surgery for congenital cardiac defects. The defect can be repaired by this approach, concomitantly with the cardiac anomaly, no need for an additional incision and without opening the hernia sac. CONCLUSION: Our experience, although very limited, in patients who are suffering from Morgagni hernia and concomitant congenital heart defects shows that simultaneous repair of Morgagni hernia through midline sternotomy prior to cardiac procedure is effective. As Morgagni hernia can be accompanied with many congenital cardiac anomalies, cardiac surgeons should be familiar with the trans-sternal approach to the defect.

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

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

Membrane permeabilization is mediated by distinct epitopes in mouse and human orthologs of the necroptosis effector, MLKL.

Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL's N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.

The Bak core dimer focuses triacylglycerides in the membrane.

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.

Ion currents through Kir potassium channels are gated by anionic lipids.

Ion currents through potassium channels are gated. Constriction of the ion conduction pathway at the inner helix bundle, the textbook gate of Kir potassium channels, has been shown to be an ineffective permeation control, creating a rift in our understanding of how these channels are gated. Here we present evidence that anionic lipids act as interactive response elements sufficient to gate potassium conduction. We demonstrate the limiting barrier to K+ permeation lies within the ion conduction pathway and show that this gate is operated by the fatty acyl tails of lipids that infiltrate the conduction pathway via fenestrations in the walls of the pore. Acyl tails occupying a surface groove extending from the cytosolic interface to the conduction pathway provide a potential means of relaying cellular signals, mediated by anionic lipid head groups bound at the canonical lipid binding site, to the internal gate.

Dynamic reconfiguration of pro-apoptotic BAK on membranes.

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.

BCL-XL antagonism selectively reduces neutrophil life span within inflamed tissues without causing neutropenia.

Neutrophils help to clear pathogens and cellular debris, but can also cause collateral damage within inflamed tissues. Prolonged neutrophil residency within an inflammatory niche can exacerbate tissue pathology. Using both genetic and pharmacological approaches, we show that BCL-XL is required for the persistence of neutrophils within inflammatory sites in mice. We demonstrate that a selective BCL-XL inhibitor (A-1331852) has therapeutic potential by causing apoptosis in inflammatory human neutrophils ex vivo. Moreover, in murine models of acute and chronic inflammatory disease, it reduced inflammatory neutrophil numbers and ameliorated tissue pathology. In contrast, there was minimal effect on circulating neutrophils. Thus, we show a differential survival requirement in activated neutrophils for BCL-XL and reveal a new therapeutic approach to neutrophil-mediated diseases.

BAK core dimers bind lipids and can be bridged by them.

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.

A small molecule interacts with VDAC2 to block mouse BAK-driven apoptosis.

Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.

Parkin inhibits BAK and BAX apoptotic function by distinct mechanisms during mitophagy.

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.

Ensemble Properties of Bax Determine Its Function.

BAX and BAK are essential mediators of intrinsic apoptosis that permeabilize the mitochondrial outer membrane. BAX activation requires its translocation from cytosol to mitochondria where conformational changes cause its oligomerization. To better understand the critical step of translocation, we examined its blockade by mutation near the C terminus (P168G) or by antibody binding near the N terminus. Similarities in the crystal structures of wild-type and BAX P168G but significant other differences suggest that cytosolic BAX exists as an ensemble of conformers, and that the distribution of conformers within the ensemble determines the different functions of wild-type and mutant proteins. We also describe the structure of BAX in complex with an antibody, 3C10, that inhibits cytosolic BAX by limiting exposure of the membrane-associating helix α9, as does the P168G mutation. Our data for both means of BAX inhibition argue for an allosteric model of BAX regulation that derives from properties of the ensemble of conformers.

CD52 glycan binds the proinflammatory B box of HMGB1 to engage the Siglec-10 receptor and suppress human T cell function.

CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein, is released in a soluble form following T cell activation and binds to the Siglec (sialic acid-binding Ig-like lectin)-10 receptor on T cells to suppress their function. We show that binding of CD52-Fc to Siglec-10 and T cell suppression requires the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). CD52-Fc bound specifically to the proinflammatory Box B domain of HMGB1, and this in turn promoted binding of the CD52 N-linked glycan, in α-2,3 sialic acid linkage with galactose, to Siglec-10. Suppression of T cell function was blocked by anti-HMGB1 antibody or the antiinflammatory Box A domain of HMGB1. CD52-Fc induced tyrosine phosphorylation of Siglec-10 and was recovered from T cells complexed with HMGB1 and Siglec-10 in association with SHP1 phosphatase and the T cell receptor (TCR). Thus, soluble CD52 exerts a concerted immunosuppressive effect by first sequestering HMGB1 to nullify its proinflammatory Box B, followed by binding to the inhibitory Siglec-10 receptor, triggering recruitment of SHP1 to the intracellular immunoreceptor tyrosine-based inhibitory motif of Siglec-10 and its interaction with the TCR. This mechanism may contribute to immune-inflammatory homeostasis in pathophysiologic states and underscores the potential of soluble CD52 as a therapeutic agent.

Conformational switching of the pseudokinase domain promotes human MLKL tetramerization and cell death by necroptosis.

Necroptotic cell death is mediated by the most terminal known effector of the pathway, MLKL. Precisely how phosphorylation of the MLKL pseudokinase domain activation loop by the upstream kinase, RIPK3, induces unmasking of the N-terminal executioner four-helix bundle (4HB) domain of MLKL, higher-order assemblies, and permeabilization of plasma membranes remains poorly understood. Here, we reveal the existence of a basal monomeric MLKL conformer present in human cells prior to exposure to a necroptotic stimulus. Following activation, toggling within the MLKL pseudokinase domain promotes 4HB domain disengagement from the pseudokinase domain αC helix and pseudocatalytic loop, to enable formation of a necroptosis-inducing tetramer. In contrast to mouse MLKL, substitution of RIPK3 substrate sites in the human MLKL pseudokinase domain completely abrogated necroptotic signaling. Therefore, while the pseudokinase domains of mouse and human MLKL function as molecular switches to control MLKL activation, the underlying mechanism differs between species.

Conversion of Bim-BH3 from Activator to Inhibitor of Bak through Structure-Based Design.

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.

Mutation to Bax beyond the BH3 domain disrupts interactions with pro-survival proteins and promotes apoptosis.

Pro-survival members of the Bcl-2 family of proteins restrain the pro-apoptotic activity of Bax, either directly through interactions with Bax or indirectly by sequestration of activator BH3-only proteins, or both. Mutations in Bax that promote apoptosis can provide insight into how Bax is regulated. Here, we describe crystal structures of the pro-survival proteins Mcl-1 and Bcl-x(L) in complex with a 34-mer peptide from Bax that encompasses its BH3 domain. These structures reveal canonical interactions between four signature hydrophobic amino acids from the BaxBH3 domain and the BH3-binding groove of the pro-survival proteins. In both structures, Met-74 from the Bax peptide engages with the BH3-binding groove in a fifth hydrophobic interaction. Various Bax Met-74 mutants disrupt interactions between Bax and all pro-survival proteins, but these Bax mutants retain pro-apoptotic activity. Bax/Bak-deficient mouse embryonic fibroblast cells reconstituted with several Bax Met-74 mutants are more sensitive to the BH3 mimetic compound ABT-737 as compared with cells expressing wild-type Bax. Furthermore, the cells expressing Bax Met-74 mutants are less viable in colony assays even in the absence of an external apoptotic stimulus. These results support a model in which direct restraint of Bax by pro-survival Bcl-2 proteins is a barrier to apoptosis.