Computational design of potent and selective BAK and BAX binders
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ABSTRACT:
PROVIDER: PRJNA1107244 | ENA |
REPOSITORIES: ENA
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Project description:There are numerous binders of the pro-survival BCL2 family proteins such as BCL2, MCL1, and BCL-XL, but development of potent and selective binders of their pro-apoptotic counterparts BAK and BAX has remained a major unsolved challenge. We use computational protein design to generate 13 kDa binders of BAK and BAX with 400 pM and 3 nM affinity, orders of magnitude higher than any existing native or designed binder, and with greater than 100-fold specificity against pro-survival BCL2 family members. The crystal structure of the BAKᐧɑBAK2 complex is very close to the computational design model, with the binder making specific interactions extending out from the canonical BH3-binding groove. Liposome- and cell-based analyses reveal that ɑBAK2 inhibits membrane permeabilization when in excess of BAK, but activates BAK when BAK is in excess. Structural analyses indicate that binding of ɑBAK2 results in partial unfolding and exposure of BAK’s BH3 domain. Similar to ɑBAK2, ɑBAX2 activates BAX at low concentrations and does not activate BAX at high concentrations. This work provides valuable insight into design of small molecule or protein inhibitors of BAK and BAX; inhibition requires high affinity binding as well as a saturating concentration of binder at the site of action. Our designs are the first binders with the high specificity required for efficient modulation of apoptosis via direct interaction with BAK and BAX and they provide highly selective molecular probes for addressing outstanding cell biological questions about cell death.
2025-05-01 | GSE266473 | GEO
Project description:MEF WT, MEF DKO(Bax/Bak), MEF DKO(Bax/Bak) expressing rabbit Serca2a, MEF DKO(Bax/Bak) expressing Bak at the endoplasmic reticulum both control and treated with Noco 100 nM for 48h
2023-06-12 | E-MTAB-10997 | biostudies-arrayexpress
Project description:Intrinsic apoptosis is critical for normal physiology including the prevention of tumor formation. BAX and BAK, which are essential for mediating this process and for the cytotoxic action of many anti-cancer drugs, are thought to be regulated through similar mechanisms and act redundantly to drive apoptosis. Here we have established the various mitochondrial complexes that contain VDAC1, VDAC2, VDAC3 and BAX or BAK.
2018-10-29 | PXD011195 | Pride
Project description:Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples.
2022-10-01 | GSE186088 | GEO
Project description:The BCL-2 family proteins are central regulators of apoptosis. However, cells doubly deficient for BAX and BAK or overexpressing BCL-2 still succumb to oxidative stress upon DNA damage or loss of matrix attachment. Our studies indicate a central role for the transcription factor deltaNp63alpha (referred to as p63 from this point forward) in this form of non-apoptotic cell death. We used microarrays to investigate gene expression in apoptosis-incompetent cells expressing a control vector or p63, at baseline and following exposure to genotoxic stress, to further elucidate molecular mechanisms regulating this type of non-apoptotic death and the role of p63 in this context.
2020-10-25 | GSE106214 | GEO
Project description:Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death
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Project description:Investigating the role of Bax/Bak as a driver of inflammation in aging
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Project description:Senescent cells drive age-related tissue dysfunction partially via the induction of a chronic senescence-associated secretory phenotype (SASP). Mitochondria are major regulators of the SASP; however, the underlying mechanisms have not been elucidated. Mitochondria are often essential for apoptosis, a cell fate distinct from cellular senescence. During apoptosis, widespread mitochondrial outer membrane permeabilization (MOMP) commits a cell to die. Here, we find that MOMP occurring in a subset of mitochondria is a feature of cellular senescence. This process, called minority MOMP (miMOMP), depends on the formation of BAX and BAK macropores leading to the release of mitochondrial DNA (mtDNA) into the cytosol, which in turn activates the cGAS-STING pathway, a major regulator of the SASP. Importantly, we found that inhibition of MOMP in vivo decreases inflammatory markers and improves healthspan in aged mice. Our results reveal, unexpectedly, that apoptosis and senescence are regulated by similar mitochondrial-dependent mechanisms, and that sub-lethal mitochondrial apoptotic stress is a major driver of the SASP. We also provide proof-of-concept that inhibition of miMOMP-induced inflammation may be a new therapeutic avenue to improve healthspan.
2023-08-16 | GSE235225 | GEO
Project description:BAK and BAX, the effectors of intrinsic apoptosis, undergo major reconfiguration to an activated conformer that self-associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms that describe 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) on liposomes comprising mitochondrial lipids. The HDX-MS profile of BAK on a membrane was broadly consistent with the known solution structures of inactive BAK. Following activation, HDX-MS resolved major reconfigurations in BAK. Mutagenesis led by our HDX-MS profiling revealed that the BCL-2 homology (BH) 4 domain maintains BAK in its inactive conformation and disrupting this was sufficient for constitutive BAK activation. Moreover, the entire BAK N-terminus that precedes the BAK oligomerisation domains became disordered post-activation and remained disordered in the activated oligomer. Cleavage of the N-terminus potentiated BAK-mediated membrane permeabilisation on liposomes and mitochondria. Together, HDX-MS reveals new insights into the dynamic nature of BAK activating conformation change in a membrane that will reveal new opportunities for therapeutic targeting.
2021-08-09 | PXD027288 | Pride
Project description:Expression data from BAX/BAK double knockout mouse embryonic fibroblast at baseline and following exposure to genotoxic stress.
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