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.
Synopsis
The ubiquitin ligase Parkin plays a protective role in neurodegenerative disease by removing damaged mitochondria and preventing apoptosis, and by limiting the functions of pro‐apoptotic effector proteins BAK and BAX. Defective control of apoptosis may contribute to the pathogenesis of early onset Parkinson's Disease caused by Parkin mutations.
Parkin activity is induced by mitochondrial damage during apoptosis.
Parkin mono‐ and di‐ubiquitinates BAK at a conserved lysine in its hydrophobic groove.
Ubiquitination reduces BAK oligomerisation and apoptotic activity on mitochondria.
Parkin prevents BAX mitochondrial localisation and apoptotic activity independent of ubiquitination.
Certain Parkinson's Disease‐associated Parkin mutants cannot ubiquitinate BAK and restrain it on mitochondria.
Autophagic clearance of damaged mitochondria and suppression of apoptotic effector proteins are coordinated functions of the Parkin ubiquitin ligase that are lost upon Parkinson's Disease‐associated mutations.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
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.
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.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Granting smart device consumers with information, simply and quickly, is what drives quick response (QR) codes and mobile marketing to go hand in hand. It boosts marketing campaigns and objectives ...and allows one to approach, engage, influence, and transform a wider target audience by connecting from offline to online platforms. However, restricted printing technology and flexibility in surfaces introduce noise while printing QR code images. Moreover, noise is often unavoidable during the gathering and transmission of digital images. Therefore, this paper proposed an automatic and accurate noise detector to identify the type of noise present in QR code images. For this, the paper first generates a new dataset comprising 10,000 original QR code images of varying sizes and later introduces several noises, including salt and pepper, pepper, speckle, Poisson, salt, local var, and Gaussian to form a dataset of 80,000 images. We perform extensive experiments by reshaping the generated images to uniform size for exploiting Convolutional Neural Network (CNN), Support Vector Machine (SVM), and Logistic Regression (LG) to classify the original and noisy images. Later, the analysis is further widened by incorporating histogram density analysis to trace and target highly important features by transforming images of varying sizes to obtain 256 features, followed by SVM, LG, and Artificial Neural Network (ANN) to identify the noise type. Moreover, to understand the impact of symmetry of noises in QR code images, we trained the models with combinations of 3-, 5-, and 7-noise types and analyzed the classification performance. From comparative analyses, it is noted that the Gaussian and Localvar noises possess symmetrical characteristics, as all the classifiers did not perform well to segregate these two noises. The results prove that histogram analysis significantly improves classification accuracy with all exploited models, especially when combined with SVM, it achieved maximum accuracy for 4- and 6-class classification problems.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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 BCLX
, thus priming these cells to undergo apoptosis in response to BH3 mimetic compounds that target MCL1 or BCLX
. 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.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
SYNOPSIS
While BAK is a known executioner protein in apoptotic cell death, how it changes conformation to form oligomeric mitochondrial pores to kill cells remains unclear. Hydrogen‐deuterium exchange mass spectrometry and site‐directed mutagenesis now resolve key events in BAK activation as it occurs on membranes and mitochondria.
Hydrogen‐deuterium exchange mass spectrometry provides new insight into conformation changes of BAK on a mitochondria‐like membrane.
The BAK BH4 domain constrains BAK activity in the absence of a death stimulus.
During BAK activation and oligomerisation, amino acids preceding the BH3 domain become exposed and disordered.
The disordered N‐terminus is not required for BAK to permeabilise membranes and mediate cytochrome c release and may rather inhibit it.
Hydrogen‐deuterium exchange mass spectrometry of BAK on mitochondria‐like membranes identifies domains and conformations critically involved in activation.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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, BAX and BAK transition from an inactive, monomeric state to a membrane‐active oligomer. Oligomeric BAX and BAK are responsible for rupturing the mitochondrial outer membrane. Here, Miller et al. present the crystal structures of the core domain dimers of BAX and of BAX/BAK core domain heterodimers. These provide atomic resolution details of interactions that drive BAX homo‐dimerization and insight into their potential interactions with mitochondrial lipids.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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-xL 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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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