Membrane‐dependent amyloid aggregation of human BAX α9 (173–192)

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C‐terminal α‐helix (α9) of the proapoptotic Bcl‐2 family protein BAX. The isolated α9 fragment (residues 173–192) is also competent to disrupt model membranes, and the structures of its membrane‐associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two‐dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the β‐sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.     

High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation

Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS₄₀₀. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS₁₀₀ and Fab-PAS₂₀₀, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS₁₀₀ and Fab-PAS₂₀₀. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS₁₀₀ vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.     

Emerging roles of lipids in BCL-2 family-regulated apoptosis

Apoptosis is an intricately regulated process required for the health and homeostasis of living systems. The mitochondrial apoptotic pathway depends on the BCL-2 family of pro- and anti-apoptotic members whose interactions form a complex network of checks and balances in regulating cell fate. A diverse set of signals recruits distinct BH3-domain only BCL-2 proteins to trigger activation of the executioner proteins BAX and BAK. In addition to protein components of the apoptotic machinery, literature of the past several decades supports crucial functions for lipids in apoptosis and cooperation between lipid metabolism and BCL-2 proteins. In this review we present the two key examples of ceramide and cardiolipin in apoptosis, focusing particularly on BCL-2 family-regulated pathways at the mitochondrial level. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.     

Platinum resistant cancer cells conserve sensitivity to BH3 domains and obatoclax induced mitochondrial apoptosis

Resistance to cisplatin chemotherapy remains a major hurdle preventing effective treatment of many solid cancers. BAX and BAK are pivotal regulators of the mitochondrial apoptosis pathway, however little is known regarding their regulation in cisplatin resistant cells. Cisplatin induces DNA damage in both sensitive and resistant cells, however the latter exhibits a failure to initiate N-terminal exposure of mitochondrial BAK or mitochondrial SMAC release. Both phenotypes are highly sensitive to mitochondrial permeabilisation induced by exogenous BH3 domain peptides derived from BID, BIM, NOXA (which targets MCL-1 and A1), and there is no significant change in their prosurvival BCL2 protein expression profiles. Obatoclax, a small molecule inhibitor of pro-survival BCL-2 family proteins including MCL-1, decreases cell viability irrespective of platinum resistance status across a panel of cell lines selected for oxaliplatin resistance. In summary, selection for platinum resistance is associated with a block of mitochondrial death signalling upstream of BAX/BAK activation. Conservation of sensitivity to BH3 domain induced apoptosis can be exploited by agents such as obatoclax, which directly target the mitochondria and BCL-2 family.     

FBXL20 promotes breast cancer malignancy by inhibiting apoptosis through degradation of PUMA and BAX

Apoptosis is a programmed cell death that efficiently removes damaged cells to maintain tissue homeostasis. Defect in apoptotic machinery can lead to tumor development, progression, and resistance to chemotherapy. PUMA (p53 upregulated modulator of apoptosis) and BAX (BCL2-associated X protein) are among the most well-known inducers of apoptosis. It has been reported that expression levels of BAX and PUMA are controlled at the posttranslational level by phosphorylation. However, the posttranslational regulation of these proapoptotic proteins remains largely unexplored. In this study, using biochemical, molecular biology, flow cytometric, and immunohistochemistry techniques, we show that PUMA and BAX are the direct target of the F-box protein FBXL20, which restricts their cellular levels. FBXL20 directs the proteasomal degradation of PUMA and BAX in a protein kinase AKT1-dependent manner to promote cancer cell proliferation and tumor growth. Interestingly, inactivation of AKT1 results in activation of another protein kinase GSK3α/β, which facilitates the proteasomal degradation of FBXL20 by another F-box protein, FBXO31. Thus, a switch between two signaling kinases AKT1 and GSK3α/β modulates the functional activity of these proapoptotic regulators, thereby determining cell survival or death. RNAi-mediated ablation of FBXL20 results in increased levels of PUMA as well as BAX, which further enhances the sensitivity of cancer cells to chemotherapeutic drugs. We showed that high level expression of FBXL20 in cancer cells reduces therapeutic drug-induced apoptosis and promotes chemoresistance. Overall, this study highlights the importance of targeting FBXL20 in cancers in conjunction with chemotherapy and may represent a promising anticancer strategy to overcome chemoresistance.     

Wip1 suppresses apoptotic cell death through direct dephosphorylation of BAX in response to γ-radiation

Wild-type p53-induced phosphatase 1 (Wip1) is a p53-inducible serine/threonine phosphatase that switches off DNA damage checkpoint responses by the dephosphorylation of certain proteins (i.e. p38 mitogen-activated protein kinase, p53, checkpoint kinase 1, checkpoint kinase 2, and uracil DNA glycosylase) involved in DNA repair and the cell cycle checkpoint. Emerging data indicate that Wip1 is amplified or overexpressed in various human tumors, and its detection implies a poor prognosis. In this study, we show that Wip1 interacts with and dephosphorylates BAX to suppress BAX-mediated apoptosis in response to γ-irradiation in prostate cancer cells. Radiation-resistant LNCaP cells showed dramatic increases in Wip1 levels and impaired BAX movement to the mitochondria after γ-irradiation, and these effects were reverted by a Wip1 inhibitor. These results show that Wip1 directly interacts with and dephosphorylates BAX. Dephosphorylation occurs at threonines 172, 174 and 186, and BAX proteins with mutations at these sites fail to translocate efficiently to the mitochondria following cellular γ-irradiation. Overexpression of Wip1 and BAX, but not phosphatase-dead Wip1, in BAX-deficient cells strongly reduces apoptosis. Our results suggest that BAX dephosphorylation of Wip1 phosphatase is an important regulator of resistance to anticancer therapy. This study is the first to report the downregulation of BAX activity by a protein phosphatase.     

Organization of the Mitochondrial Apoptotic BAK Pore: OLIGOMERIZATION OF THE BAK HOMODIMERS*

The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX “BH3-in-groove homodimer.” Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a “worm hole.”     

MCL-1 inhibits BAX in the absence of MCL-1/BAX Interaction

The BCL-2 family of proteins plays a major role in the control of apoptosis as the primary regulator of mitochondrial permeability. The pro-apoptotic BCL-2 homologues BAX and BAK are activated following the induction of apoptosis and induce cytochrome c release from mitochondria. A second class of BCL-2 homologues, the BH3-only proteins, is required for the activation of BAX and BAK. The activity of both BAX/BAK and BH3-only proteins is opposed by anti-apoptotic BCL-2 homologues such as BCL-2 and MCL-1. Here we show that anti-apoptotic MCL-1 inhibits the function of BAX downstream of its initial activation and translocation to mitochondria. Although MCL-1 interacted with BAK and inhibited its activation, the activity of MCL-1 against BAX was independent of an interaction between the two proteins. However, the anti-apoptotic function of MCL-1 required the presence of BAX. These results suggest that the pro-survival activity of MCL-1 proceeds via inhibition of BAX function at mitochondria, downstream of its activation and translocation to this organelle.     

Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies

Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the α/β TCR. Replacing C_H1 with Cα and C_L with Cβ, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of Cβ with a canonical β-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the C_H1/C_L and Cα/Cβ domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases.     

Alpha-Melanocyte Stimulating Hormone (α-MSH) Is a Post-Caspase Suppressor of Apoptosis in RAW 264.7 Macrophages

The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) is an important regulator of immune cell activity within the immunosuppressive ocular microenvironment. Its constitutive presence not only suppresses macrophage inflammatory activity, it also participates in retinal pigment epithelial cell (RPE) mediated activation of macrophages to function similar to myeloid suppressor cells. In addition, α-MSH promotes survival of the alternatively activated macrophages where without α-MSH RPE induce apoptosis in the macrophages, which is seen as increased TUNEL stained cells. Since there is little know about α-MSH as an anti-apoptotic factor, the effects of α-MSH on caspase activity, mitochondrial membrane potential, Bcl2 to BAX expression, along with TUNEL staining, and Annexin V binding were examined in RAW 264.7 macrophages under serum-starved conditions that trigger apoptosis. There was no effect of α-MSH on activated Caspase 9 and Caspase 3 while there was suppression of Caspase 8 activity. In addition, α-MSH did not improve mitochondrial membrane potential, change the ratio between Bcl-2 and BAX, nor reduce Annexin V binding. These results demonstrate that the diminution in TUNEL staining by α-MSH is through α-MSH mediating suppression of the apoptotic pathway that is post-Caspase 3, but before DNA fragmentation. Therefore, as α-MSH promotes the alternative activation of macrophages it also provides a survival signal, and the potential for the caspases to participate in non-apoptotic activities that can contribute to an immunosuppressive microenvironment.     

Pro-apoptotic complexes of BAX and BAK on the outer mitochondrial membrane

In multicellular organisms the regulated cell death apoptosis is critically important for both ontogeny and homeostasis. Mitochondria are indispensable for stress-induced apoptosis. The BCL-2 protein family controls mitochondrial apoptosis and initiates cell death through the pro-apoptotic activities of BAX and BAK at the outer mitochondrial membrane (OMM). Cellular survival is ensured by the retrotranslocation of mitochondrial BAX and BAK into the cytosol by anti-apoptotic BCL-2 proteins. BAX/BAK-dependent OMM permeabilization releases the mitochondrial cytochrome c (cyt c), which initiates activation of caspase-9. The caspase cascade leads to cell shrinkage, plasma membrane blebbing, chromatin condensation, and apoptotic body formation. Although it is clear that ultimately complexes of active BAX and BAK commit the cell to apoptosis, the nature of these complexes is still enigmatic. Excessive research has described a range of complexes, varying from a few molecules to several 10,000, in different systems. BAX/BAK complexes potentially form ring-like structures that could expose the inner mitochondrial membrane. It has been suggested that these pores allow the efflux of small proteins and even mitochondrial DNA. Here we summarize the current state of knowledge for mitochondrial BAX/BAK complexes and the interactions between these proteins and the membrane.     

Mutagenesis of the BH3 Domain of BAX Identifies Residues Critical for Dimerization and Killing

The BCL-2 family of proteins is comprised of proapoptotic as well as antiapoptotic members (S. N. Farrow and R. Brown, Curr. Opin. Genet. Dev. 6:45–49, 1996). A prominent death agonist, BAX, forms homodimers and heterodimerizes with multiple antiapoptotic members. Death agonists have an amphipathic α helix, called BH3; however, the initial assessment of BH3 in BAX has yielded conflicting results. Our BAX deletion constructs and minimal domain constructs indicated that the BH3 domain was required for BAX homodimerization and heterodimerization with BCL-2, BCL-X_L, and MCL-1. An extensive site-directed mutagenesis of BH3 revealed that substitutions along the hydrophobic face of BH3, especially charged substitutions, had the greatest affects on dimerization patterns and death agonist activity. Particularly instructive was the BAX mutant mIII-1 (L63A, G67A, L70A, and M74A), which replaced the hydrophobic face of BH3 with alanines, preserving its amphipathic nature. BAXmIII-1 failed to form heterodimers or homodimers by yeast two-hybrid or immunoprecipitation analysis yet retained proapoptotic activity. This suggests that BAX’s killing function reflects mechanisms beyond its binding to BCL-2 or BCL-X_L to inhibit them or simply displace other protein partners. Notably, BAXmIII-1 was found predominantly in mitochondrial membranes, where it was homodimerized as assessed by homobifunctional cross-linkers. This characteristic of BAXmIII-1 correlates with its capacity to induce mitochondrial dysfunction, caspase activation, and apoptosis. These data are consistent with a model in which BAX death agonist activity may require an intramembranous conformation of this molecule that is not assessed accurately by classic binding assays.

Programmed cell death and its morphologic equivalent, apoptosis, are orchestrated by a distinct genetic pathway that is apparently possessed by all multicellular organisms (22). Moreover, the biochemical details of how encoded proteins function are beginning to emerge. The BCL-2 family of proteins constitutes a central decisional point within the common portion of the apoptotic pathway. This family possesses both proapoptotic (BAX, BAK, BCL-X_S, BAD, BIK, BID, HRK, and BIM) and antiapoptotic (BCL-2, BCL-X_L, MCL-1, and A1) molecules (5, 11). The ratio of antiapoptotic to proapoptotic molecules such as BCL-2/BAX determines the response to a proximal apoptotic signal (14). A striking characteristic of many family members is their propensity to form homo- and heterodimers (16, 19). The BCL-2 family has homology clustered principally within four conserved domains called BH1, BH2, BH3, and BH4 (5, 11). The multidimensional nuclear magnetic resonance (NMR) and X-ray crystallographic structure of a BCL-X_L monomer indicates that the BH1-4 domains correspond to α helices 1 to 7. Notably, the BH1, -2, and -3 domains are in close proximity and create a hydrophobic pocket presumably involved in interactions with other BCL-2 family members (13). The NMR analysis of a BCL-X_L-BAK BH3 peptide complex revealed both hydrophobic and electrostatic interactions between the BCL-X_L pocket and a BH3 amphipathic α-helical peptide from BAK (17).Prior mutagenesis studies of BCL-2 and BCL-X_L revealed the importance of BH1 and BH2 domains for both their antiapoptotic function and the capacity to heterodimerize with proapoptotic molecules like BAX or BAK (2, 19, 26). In general, most mutations that disrupt heterodimerization with BAX also lose their death repressor function. However, exceptions do exist; some mutants of BCL-X_L fail to bind BAX or BAK but still repress cell death, suggesting that these functions can be separated for antiapoptotic molecules (2). Moreover, a genetic approach with Bcl-2-deficient and Bax-deficient mice also suggested that BCL-2 and BAX could function independently of one another (10).Deletion studies of the death agonist BAK first implicated the BH3 domain as having the capacity to bind BCL-X_L and promote apoptosis (3). However, the functional significance of BH3 in BAX is uncertain as indicated in the literature. Three deletion analyses indicated the necessity of the BH3 domain in BAX to promote cell death as well as to heterodimerize with BCL-2 (3, 9, 28). Yet, two recent studies reported that BAX functions as a death activator independent of its heterodimerization (21, 27). Moreover, substitution mutants within the BH3 domain showed conflicting specificities of heterodimerization (20, 21, 27).Our initial screen of yeast two-hybrid libraries with BCL-2 as bait yielded multiple clones that possess only the NH₂ terminus of BAX, bearing the BH3 but not the BH1 or the BH2 domains. A similar set of isolates was obtained when BCL-2 (G145A) was used as bait (15). We also noted by deletion analysis and assessment of minimal domains of BAX that the BH3 domain was required for both homodimerization and heterodimerization. Consequently, we undertook an extensive site-directed mutagenesis of the BH3 domain of BAX. These studies demonstrate the importance of the hydrophobic face of the amphipathic α helix of BH3 for the dimerization and cell death activities of BAX. Furthermore, analysis of a BAX mutant indicates that its retained conformation as a cross-linkable dimer at mitochondrial membranes correlates with its intact apoptotic function.     

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis¹. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)¹. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues².In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)^3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation^7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members^7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane. Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization¹⁰. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)¹⁰. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)¹¹. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.     

Direct and selective small-molecule activation of proapoptotic BAX

BCL-2 family proteins are key regulators of the apoptotic pathway. Antiapoptotic members sequester the BCL-2 homology 3 (BH3) death domains of proapoptotic members such as BAX to maintain cell survival. The antiapoptotic BH3-binding groove has been successfully targeted to reactivate apoptosis in cancer. We recently identified a geographically distinct BH3-binding groove that mediates direct BAX activation, suggesting a new strategy for inducing apoptosis by flipping BAX's 'on switch'. Here we applied computational screening to identify a BAX activator molecule that directly and selectively activates BAX. We demonstrate by NMR and biochemical analyses that the molecule engages the BAX trigger site and promotes the functional oligomerization of BAX. The molecule does not interact with the BH3-binding pocket of antiapoptotic proteins or proapoptotic BAK and induces cell death in a BAX-dependent fashion. To our knowledge, we report the first gain-of-function molecular modulator of a BCL-2 family protein and demonstrate a new paradigm for pharmacologic induction of apoptosis.     

Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion

Inflammasome is an intracellular signaling complex of the innate immune system. Activation of inflammasomes promotes the secretion of interleukin 1β (IL-1β) and IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the canonical and non-canonical inflammasome pathways, respectively, are crucial for inflammasome-mediated inflammatory responses. Here we report that gasdermin D (GSDMD) is another crucial component of inflammasomes. We discovered the presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a quantitative mass spectrometry-based analysis. Gene deletion of GSDMD demonstrated that GSDMD is required for pyroptosis and for the secretion but not proteolytic maturation of IL-1β in both canonical and non-canonical inflammasome responses. It was known that GSDMD is a substrate of caspase-1 and we showed its cleavage at the predicted site during inflammasome activation and that this cleavage was required for pyroptosis and IL-1β secretion. Expression of the N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal modification such as tagging with Flag sequence disrupted the function of GSDMD. We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not essential for GSDMD to function. Further analyses of LPS plus nigericin- or Salmonella typhimurium-treated macrophage cell lines and primary cells showed that apoptosis became apparent in Gsdmd^−/− cells, indicating a suppression of apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other inflammasome receptors and ASC, and caspase-1 may partially contribute to the activation of apoptotic caspases in Gsdmd^−/− cells. These data provide new insights into the molecular mechanisms of pyroptosis and reveal an unexpected interplay between apoptosis and pyroptosis.     

A unified model of mammalian BCL-2 protein family interactions at the mitochondria

During apoptosis, the BCL-2 protein family controls mitochondrial outer membrane permeabilization (MOMP), but the dynamics of this regulation remain controversial. We employed chimeric proteins composed of exogenous BH3 domains inserted into a tBID backbone that can activate the proapoptotic effectors BAX and BAK to permeabilize membranes without being universally sequestered by all antiapoptotic BCL-2 proteins. We thus identified two "modes" whereby prosurvival BCL-2 proteins can block MOMP, by sequestering direct-activator BH3-only proteins ("MODE 1") or by binding active BAX?and BAK ("MODE 2"). Notably, we found that MODE 1 sequestration is less efficient and more easily derepressed to promote MOMP than MODE 2. Further, MODE 2 sequestration prevents mitochondrial fusion. We provide a unified model of BCL-2 family function that helps to explain otherwise paradoxical observations relating to MOMP, apoptosis, and mitochondrial dynamics.     

Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX

Interactions between the BCL-2 family proteins determine the cell's fate to live or die. How they interact with each other to regulate apoptosis remains as an unsettled central issue. So far, the antiapoptotic BCL-2 proteins are thought to interact with BAX weakly, but the physiological significance of this interaction has been vague. Herein, we show that recombinant BCL-2 and BCL-w interact potently with a BCL-2 homology (BH) 3 domain-containing peptide derived from BAX, exhibiting the dissociation constants of 15 and 23 nM, respectively. To clarify the basis for this strong interaction, we determined the three-dimensional structure of a complex of BCL-2 with a BAX peptide spanning its BH3 domain. It revealed that their interactions extended beyond the canonical BH3 domain and involved three nonconserved charged residues of BAX. A novel BAX variant, containing the alanine substitution of these three residues, had greatly impaired affinity for BCL-2 and BCL-w, but was otherwise indistinguishable from wild-type BAX. Critically, the apoptotic activity of the BAX variant could not be restrained by BCL-2 and BCL-w, pointing that the observed tight interactions are critical for regulating BAX activation. We also comprehensively quantified the binding affinities between the three BCL-2 subfamily proteins. Collectively, the data show that due to the high affinity of BAX for BCL-2, BCL-w and A1, and of BAK for BCL-X(L), MCL-1 and A1, only a subset of BH3-only proteins, commonly including BIM, BID and PUMA, could be expected to free BAX or BAK from the antiapoptotic BCL-2 proteins to elicit apoptosis.