Pore-forming Toxin Research Articles

Role of Gasdermin E in the Biogenesis of Apoptotic Cell-Derived Exosomes.

The gasdermins are a family of pore-forming proteins that has recently been suggested to play a central role in pyroptosis. In this study, we describe the novel roles of gasdermins in the biogenesis of apoptotic cell-derived exosomes. In apoptotic human HeLa and HEK293 cells, GSDMA, GSDMC, GSDMD, and GSDME increased the release of apoptotic exosomes. GSDMB and DFNB59, in contrast, negatively affected the release of apoptotic exosomes. GSDME at its full-length and cleaved forms was localized in the exosomes and exosomal membrane. Full-length and cleaved forms of GSDME are suggested to increase Ca2+ influx to the cytosol through endosomal pores and thus increase the biogenesis of apoptotic exosomes. In addition, the GSDME-mediated biogenesis of apoptotic exosomes depended on the ESCRT-III complex and endosomal recruitment of Ca2+-dependent proteins, that is, annexins A2 and A7, the PEF domain family proteins sorcin and grancalcin, and the Bro1 domain protein HD-PTP. Therefore, we propose that the biogenesis of apoptotic exosomes begins when gasdermin-mediated endosomal pores increase cytosolic Ca2+, continues through the recruitment of annexin-sorcin/grancalcin-HD-PTP, and is completed when the ESCRT-III complex synthesizes intraluminal vesicles in the multivesicular bodies of dying cells. Finally, we found that GSDME-bearing tumors released apoptotic exosomes to induce inflammatory responses in the invivo mouse 4T1 orthotropic model of BALB/c breast cancer. The data indicate that the switch from apoptosis to pyroptosis could drive the transfer of mass signals to nearby or distant living cells and tissues by way of extracellular vesicles, and that gasdermins play critical roles in that process.

Graphene Quantum Dots Nonmonotonically Influence the Horizontal Transfer of Extracellular Antibiotic Resistance Genes via Bacterial Transformation.

Graphene quantum dots (GQDs) coexist with antibiotic resistance genes (ARGs) in the environment. Whether GQDs influence ARG spread needs investigation, since the resulting development of multidrug-resistant pathogens would threaten human health. This study investigates the effect of GQDs on the horizontal transfer of extracellular ARGs (i.e., transformation, a pivotal way that ARGs spread) mediated by plasmids into competent Escherichia coli cells. GQDs enhance ARG transfer at lower concentrations, which are close to their environmental residual concentrations. However, with further increases in concentration (closer to working concentrations needed for wastewater remediation), the effects of enhancement weaken or even become inhibitory. At lower concentrations, GQDs promote the gene expression related to pore-forming outer membrane proteins and the generation of intracellular reactive oxygen species, thus inducing pore formation and enhancing membrane permeability. GQDs may also act as carriers to transport ARGs into cells. These factors result in enhanced ARG transfer. At higher concentrations, GQD aggregation occurs, and aggregates attach to the cell surface, reducing the effective contact area of recipients for external plasmids. GQDs also form large agglomerates with plasmids and thus hindering ARG entrance. This study could promote the understanding of the GQD-caused ecological risks and benefit their safe application.

A bacterial binary toxin system that kills both insects and aquatic crustaceans: Photorhabdus insect-related toxins A and B.

Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed that their homologs from Vibrio parahaemolyticus also play critical roles in the pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimps. Based on the structural features of the PirA/PirB toxins, it was suggested that they might function in the same way as a Bacillus thuringiensis Cry pore-forming toxin. However, unlike Cry toxins, studies on the PirA/PirB toxins are still scarce, and their cytotoxic mechanism remains to be clarified. In this review, based on our studies of V. parahaemolyticus PirAvp/PirBvp, we summarize the current understanding of the gene locations, expression control, activation, and cytotoxic mechanism of this type of toxin. Given the important role these toxins play in aquatic disease and their potential use in pest control applications, we also suggest further topics for research. We hope the information presented here will be helpful for future PirA/PirB studies.

Identification of the pore-forming and binding domains of the Sneathia vaginalis cytopathogenic toxin A.

The association between Sneathia vaginalis and preterm birth is emerging. The Gram-negative anaerobe produces a large exotoxin, the cytopathogenic toxin A (CptA), that forms pores in human epithelial cells and red blood cells. The structure of the toxin has not been determined, but in silico analysis predicts that a large amino-terminal region of the protein is globular and separated from the carboxy-terminal tandem repeats by a disordered region. We found that a recombinant protein consisting of the predicted structured amino-terminal portion of CptA and devoid of the repeat region was sufficient to permeabilize epithelial cells and red blood cells. The repeat region was capable of binding to epithelial cells but did not permeabilize them or lyse red blood cells. CptA is the only S. vaginalis virulence factor that has been examined mechanistically to date, and this analysis sets the foundation for an understanding of how this novel pore-forming toxin exerts its activity.

An ancient defense mechanism: Conservation of gasdermin-mediated pyroptosis.

The gasdermins are a family of pore-forming proteins involved in various cellular processes such as cell death and inflammation. A new study in PLOS Biology explores the evolutionary history of gasdermins across metazoans, highlighting the conservation and divergence of gasdermin E.

Murine Eosinophils Release IL-1β via a Non-Lytic Mechanism Dependent on Gasdermin D (GSDMD) Expression but Independent of GSDMD Proteolytic Processing

Abstract Physiological roles for gasdermin D (GSDMD) in pyroptosis and IL-1β release during inflammasome signaling have been well-characterized in macrophages. This involves GSDMD cleavage by caspase-1 to generate N-GSDMD products that assemble as macropores in the plasma membrane (PM) to mediate direct IL-1β efflux and eventual pyroptosis. Granulocytes, including neutrophils and eosinophils, are also important sources of IL-1β in multiple innate immune responses. In neutrophils, we have previously reported that GSDMD expression and proteolytic processing to N-GSDMD is required for IL-1β release but is independent from accumulation of PM N-GSDMD pores and pyroptosis due to alternative trafficking of N-GSDMD to azurophilic granules and autophagosomes; this atypical GSDMD-dependent IL-1β release is mediated in part by secretory autophagy. We have now tested whether this alternative GSDMD-dependent mechanism for non-lytic IL-1β release is also operative in eosinophils using primary murine bone marrow-derived eosinophils (BMDE) from wildtype (C57Bl/6) or Gsdmd−/−mice and stimulated with activators of NLRP3 inflammasome signaling. NLRP3 inflammasome-activated BMDE rapidly process and release IL-1β via a mechanism dependent on GSDMD expression but independent of GSDMD proteolytic processing, N-GSDMD pore accumulation in the PM, and pyroptosis. This requirement for GSDMD in IL-1β release from BMDE is operative during the initial phases (~60 min) of NLRP3 inflammasome activation but is then complemented by a yet-be-defined GSDMD-independent pathway with prolonged stimulation. These findings indicate a role for GSDMD as a chaperone rather than pore-forming protein and distinct from mechanisms defined in neutrophils. Supported by grants from (NIH P01-AI141350, R01-EY014362).

Study on Cellular Localization of Bin Toxin and its Apoptosis-inducing Effect on Human Nasopharyngeal Carcinoma Cells.

Bacterial pore-forming toxins, BinA and BinB together known as the binary toxin are potent insecticidal proteins, that share structural homology with antitumor bacterial parasporin-2 protein. The underlying molecular mechanism of Bin toxin-induced cancer cell cytotoxicity requires more knowledge to understand whether the toxin induced human cytotoxic effects occur in the same way as that of parasporin-2 or not. In this study, anticancer properties of Lysinibacillus sphaericus derived Bin toxin on HK1 were evaluated through MTT assay, morphological analysis and lactate dehydrogenase efflux assay. Induction of apoptosis was determined from RT-qPCR, caspase activity and cytochrome c release assay. Internalization pattern of Bin toxin in HK1 cells was studied by confocal laser-scanning microscopic analysis. Activated Bin toxin had strong cytocidal activity to HK1 cancer cell line at 24 h postinoculation. Both BinA and BinB treated HK1 cells showed significant inhibition of cell viability at 12 μM. Induction of apoptotic mediators from RT-qPCR and caspase activity analyses indicated the activation of programmed cell death in HK1 cells in response to Bin toxin treatment. Internalization pattern of Bin toxin studied by using confocal microscopy indicated the localization of BinA on cell surface and internalization of BinB in the cytoplasm of cancer cells as well as colocalization of BinA with BinB. Evaluation of cytochrome c release also showed the association of BinB and BinA+BinB with mitochondria. Bin toxin is a cytotoxic protein that induces cytotoxic and apoptotic events in HK1 cells, and may have high therapeutic potential as an anti-cancer agent.

AIM2-mediated Immunity to PlasmodiumInfection in Hepatocytes

Abstract Malaria, caused by Plasmodiumparasites is a severe disease affecting millions of people around the world. Plasmodiumundergoes obligatory development and replication in the hepatocytes, before initiating the life-threatening clinical disease of blood-stage malaria. Although the innate immune responses that control Plasmodiuminfection and development in the liver are known to limit clinical malaria and transmission, the mechanism of such responses remain relatively unknown. Here we demonstrate that the DNA of Plasmodiumis sensed by AIM2 (absent in melanoma 2) receptors in the infected hepatocytes, resulting in Caspase-1 activation. However, we observed that Caspase-1 undergoes an unconventional and incomplete proteolytic processing in such hepatocytes, resulting in the activation of the membrane pore-forming protein, Gasdermin D (GSDMD), without the concurrent activation of the inflammasome-associated pro-inflammatory cytokines such as interleukin (IL)-1 and IL-18. Nevertheless, the AIM2-mediated Caspase-1 activation pathway induced the elimination of Plasmodium-infected hepatocytes and a resultant control of malaria in the liver. Supported by grants from NIH (R01 AI168307)

Lyososome Membrane Permeabilization Induces Plasma Membrane Macropores to Activate K +-Efflux-Dependent NLRP3 Inflammasomes in Murine Dendritic Cells

Abstract NLRP3 is a cytosolic sensor protein in myeloid leukocytes that recruits the adaptor protein ASC and activates caspase-1 in functional NLRP3 inflammasomes. Caspase-1 coordinately cleaves pro-IL-1β to bioactive IL-1β and gasdermin D (GSDMD) to induce N-GSDMD macropores in the plasma membrane (PM). The latter mediate efflux of mature IL-1β and initiate progression to pyroptosis, a lytic cell death. NLRP3 activation is triggered by perturbed cellular homeostasis with decreased cytosolic [K +] being the best-characterized signal. The required K +efflux is typically induced by the ionophore nigericin or extracellular ATP as an agonist for P2X7 receptor channels. K +efflux-dependent activation of NLRP3 is also triggered by soluble or particulate agents which accumulate in lysosomes to induce lysosomal membrane permeabilization (LMP) and release of hydrolytic enzymes into the cytosol; particulates include alum widely used as a vaccine adjuvant. The mechanisms by which LMP rapidly triggers changes in plasma membrane permeability remain undefined. We used a model of murine bone marrow derived dendritic cells (BMDC) treated with the soluble LMP agent Leu-Leu-O-methyl ester (LLME) to elicit K+ efflux and NLRP3 activation. Notably, this K+ efflux was coincident with a rapidly induced influx of propidiumiodide (PI), a 668 Da organic salt routinely used to track assembly of plasma membrane macropores such as GSDMD. Equivalent LLME-induced PI influx was observed in wildtype and Gsdmd−/−BMDC and was unaffected by inhibition of NLRP3 or caspase-1. These observations indicate that LMP triggers rapid activation of an endogenous pore-forming protein distinct from GSDMD. Current studies are evaluating gasdermin E or pannexin-1 as candidates. Supported by grants from the NIH (P01-AI141350 and R01-EY014362)

Structural changes upon membrane insertion of the insecticidal pore-forming toxins produced by Bacillus thuringiensis.

Different Bacillus thuringiensis (Bt) strains produce a broad variety of pore-forming toxins (PFTs) that show toxicity against insects and other invertebrates. Some of these insecticidal PFT proteins have been used successfully worldwide to control diverse insect crop pests. There are several studies focused on describing the mechanism of action of these toxins that have helped to improve their performance and to cope with the resistance evolved by different insects against some of these proteins. However, crucial information that is still missing is the structure of pores formed by some of these PFTs, such as the three-domain crystal (Cry) proteins, which are the most commercially used Bt toxins in the biological control of insect pests. In recent years, progress has been made on the identification of the structural changes that certain Bt insecticidal PFT proteins undergo upon membrane insertion. In this review, we describe the models that have been proposed for the membrane insertion of Cry toxins. We also review the recently published structures of the vegetative insecticidal proteins (Vips; e.g. Vip3) and the insecticidal toxin complex (Tc) in the membrane-inserted state. Although different Bt PFTs show different primary sequences, there are some similarities in the three-dimensional structures of Vips and Cry proteins. In addition, all PFTs described here must undergo major structural rearrangements to pass from a soluble form to a membrane-inserted state. It is proposed that, despite their structural differences, all PFTs undergo major structural rearrangements producing an extended α-helix, which plays a fundamental role in perforating their target membrane, resulting in the formation of the membrane pore required for their insecticidal activity.

The sphingolipids ceramide and inositol phosphorylceramide protect the Leishmania major membrane from sterol-specific toxins

The accessibility of sterols in mammalian cells to exogenous sterol-binding agents has been well-described previously, but sterol accessibility in distantly related protozoa is unclear. The human pathogen Leishmania major uses sterols and sphingolipids distinct from those used in mammals. Sterols in mammalian cells can be sheltered from sterol-binding agents by membrane components, including sphingolipids, but the surface exposure of ergosterol in Leishmania remains unknown. Here, we used flow cytometry to test the ability of the L.major sphingolipids inositol phosphorylceramide (IPC) and ceramide to shelter ergosterol by preventing binding of the sterol-specific toxins streptolysin O and perfringolysin O and subsequent cytotoxicity. In contrast to mammalian systems, we found that Leishmania sphingolipids did not preclude toxin binding to sterols in the membrane. However, we show that IPC reduced cytotoxicity and that ceramide reduced perfringolysin O- but not streptolysin O-mediated cytotoxicity in cells. Furthermore, we demonstrate ceramide sensing was controlled by the toxin L3 loop, and that ceramide was sufficient to protect L.major promastigotes from the anti-leishmaniasis drug amphotericin B. Based on these results, we propose a mechanism whereby pore-forming toxins engage additional lipids like ceramide to determine the optimal environment to sustain pore formation. Thus, L. major could serve as a genetically tractable protozoan model organism for understanding toxin-membrane interactions.

Engineering BinB Pore-Forming Toxin for Selective Killing of Breast Cancer Cells.

Breast cancer is one of the most common cancers in women worldwide. Conventional cancer chemotherapy always has adverse side effects on the patient's healthy tissues. Consequently, combining pore-forming toxins with cell-targeting peptides (CTPs) is a promising anticancer strategy for selectively destroying cancer cells. Here, we aim to improve the target specificity of the BinB toxin produced from Lysinibacillus sphaericus (Ls) by fusing a luteinizing hormone-releasing hormone (LHRH) peptide to its pore-forming domain (BinBC) to target MCF-7 breast cancer cells as opposed to human fibroblast cells (Hs68). The results showed that LHRH-BinBC inhibited MCF-7 cell proliferation in a dose-dependent manner while leaving Hs68 cells unaffected. BinBC, at any concentration tested, did not affect the proliferation of MCF-7 or Hs68 cells. In addition, the LHRH-BinBC toxin caused the efflux of the cytoplasmic enzyme lactate dehydrogenase (LDH), demonstrating the efficacy of the LHRH peptide in directing the BinBC toxin to damage the plasma membranes of MCF-7 cancer cells. LHRH-BinBC also caused MCF-7 cell apoptosis by activating caspase-8. In addition, LHRH-BinBC was predominantly observed on the cell surface of MCF-7 and Hs68 cells, without colocalization with mitochondria. Overall, our findings suggest that LHRH-BinBC could be investigated further as a potential cancer therapeutic agent.

Patch repair protects cells from the small pore-forming toxin aerolysin.

Aerolysin family pore-forming toxins damage the membrane, but membrane repair responses used to resist them, if any, remain controversial. Four proposed membrane repair mechanisms include toxin removal by caveolar endocytosis, clogging by annexins, microvesicle shedding catalyzed by MEK, and patch repair. Which repair mechanism aerolysin triggers is unknown. Membrane repair requires Ca2+, but it is controversial if Ca2+ flux is triggered by aerolysin. Here, we determined Ca2+ influx and repair mechanisms activated by aerolysin. In contrast to what is seen with cholesterol-dependent cytolysins (CDCs), removal of extracellular Ca2+ protected cells from aerolysin. Aerolysin triggered sustained Ca2+ influx. Intracellular Ca2+ chelation increased cell death, indicating that Ca2+-dependent repair pathways were triggered. Caveolar endocytosis failed to protect cells from aerolysin or CDCs. MEK-dependent repair did not protect against aerolysin. Aerolysin triggered slower annexin A6 membrane recruitment compared to CDCs. In contrast to what is seen with CDCs, expression of the patch repair protein dysferlin protected cells from aerolysin. We propose aerolysin triggers a Ca2+-dependent death mechanism that obscures repair, and the primary repair mechanism used to resist aerolysin is patch repair. We conclude that different classes of bacterial toxins trigger distinct repair mechanisms.

IgG Fc-binding protein positively regulates the assembly of pore-forming protein complex βγ-CAT evolved to drive cell vesicular delivery and transport

Cell membranes form barriers for molecule exchange between the cytosol and the extracellular environments. βγ-CAT, a complex of pore-forming protein BmALP1 (two βγ-crystallin domains with an aerolysin pore-forming domain) and the trefoil factor BmTFF3, has been identified in toad Bombina maxima. It plays pivotal roles, via inducing channel formation in various intracellular or extracellular vesicles, as well as in nutrient acquisition, maintaining water balance, and antigen presentation. Thus, such a protein machine should be tightly regulated. Indeed, BmALP3 (a paralog of BmALP1) oxidizes BmALP1 to form a water-soluble polymer, leading to dissociation of the βγ-CAT complex and loss of biological activity. Here, we found that the B.maxima IgG Fc-binding protein (FCGBP), a well-conserved vertebrate mucin-like protein with unknown functions, acted as a positive regulator for βγ-CAT complex assembly. The interactions among FCGBP, BmALP1, and BmTFF3 were revealed by co-immunoprecipitation assays. Interestingly, FCGBP reversed the inhibitory effect of BmALP3 on the βγ-CAT complex. Furthermore, FCGBP reduced BmALP1 polymers and facilitated the assembly of βγ-CAT with the biological pore-forming activity in the presence of BmTFF3. Our findings define the role of FCGBP in mediating the assembly of a pore-forming protein machine evolved to drive cell vesicular delivery and transport.

The natural product, echinatin, protects mice from methicillin-resistant Staphylococcus aureus pneumonia by inhibition of alpha-hemolysin expression.

Antimicrobial resistance (AMR) is a global, multifaceted crisis that poses significant challenges to the successful eradication of devastating pathogens, particularly methicillin-resistant Staphylococcus aureus (MRSA), a persistent superbug that causes devastating infections. The scarcity of new antibacterial drugs is obvious, and antivirulence strategies that reduce the pathogenicity of bacteria by weakening their virulence have become the subject of intense investigation. Alpha-hemolysin (Hla), a cytolytic pore-forming toxin, has a pivotal role in S. aureus pathogenesis. Here, we demonstrated that echinatin, a natural compound isolated from licorice, effectively inhibited the hemolytic activity of MRSA at 32 μg/mL. In addition, echinatin did not interfere with bacterial growth and had no significant cytotoxicity at the inhibitory concentration of S. aureus hemolysis. Heptamer formation tightly correlated with Hla-mediated cell invasion, whereas echinatin did not affect deoxycholic acid-induced oligomerization of Hla. Echinatin affected hemolytic activity through indirect binding to Hla as confirmed by the neutralization assay and cellular thermal shift assay (CETSA). Furthermore, qRT-PCR and western blot analyses revealed that echinatin suppressed Hla expression at both the mRNA and protein levels as well as the transcript levels of Agr quorum-sensing system-related genes. Additionally, when echinatin was added to a coculture system of A549 cells and S. aureus, it significantly reduced cell damage. Importantly, echinatin exhibited a significant therapeutic effect in an MRSA-induced mouse pneumonia model. In conclusion, the present findings demonstrated that echinatin significantly inhibits the hemolysin effect and may be a potential candidate compound for combating drug-resistant MRSA infections.

Gamma-Hemolysin Components: Computational Strategies for LukF-Hlg2 Dimer Reconstruction on a Model Membrane.

The gamma-hemolysin protein is one of the most common pore-forming toxins expressed by the pathogenic bacterium Staphylococcus aureus. The toxin is used by the pathogen to escape the immune system of the host organism, by assembling into octameric transmembrane pores on the surface of the target immune cell and leading to its death by leakage or apoptosis. Despite the high potential risks associated with Staphylococcus aureus infections and the urgent need for new treatments, several aspects of the pore-formation process from gamma-hemolysin are still unclear. These include the identification of the interactions between the individual monomers that lead to the formation of a dimer on the cell membrane, which represents the unit for further oligomerization. Here, we employed a combination of all-atom explicit solvent molecular dynamics simulations and protein-protein docking to determine the stabilizing contacts that guide the formation of a functional dimer. The simulations and the molecular modeling reveal the importance of the flexibility of specific protein domains, in particular the N-terminus, to drive the formation of the correct dimerization interface through functional contacts between the monomers. The results obtained are compared with the experimental data available in the literature.

Amphibian pore-forming protein βγ-CAT drives extracellular nutrient scavenging under cell nutrient deficiency

Nutrient acquisition is essential for animal cells. βγ-CAT is a pore-forming protein (PFP) and trefoil factor complex assembled under tight regulation identified in toad Bombina maxima. Here, we reported that B.maxima cells secreted βγ-CAT under glucose, glutamine, and pyruvate deficiency to scavenge extracellular proteins for their nutrient supply and survival. AMPK signaling positively regulated the expression and secretion of βγ-CAT. The PFP complex selectively bound extracellular proteins and promoted proteins uptake through endolysosomal pathways. Elevated intracellular amino acids, enhanced ATP production, and eventually prolonged cell survival were observed in the presence of βγ-CAT and extracellular proteins. Liposome assays indicated that high concentration of ATP negatively regulated the opening of βγ-CAT channels. Collectively, these results uncovered that βγ-CAT is an essential element in cell nutrient scavenging under cell nutrient deficiency by driving vesicular uptake of extracellular proteins, providing a new paradigm for PFPs in cell nutrient acquisition and metabolic flexibility.

Single-molecule fingerprinting of protein-drug interaction using a funneled biological nanopore

In drug discovery, efficient screening of protein-drug interactions (PDIs) is hampered by the limitations of current biophysical approaches. Here, we develop a biological nanopore sensor for single-molecule detection of proteins and PDIs using the pore-forming toxin YaxAB. Using this YaxAB nanopore, we demonstrate label-free, single-molecule detection of interactions between the anticancer Bcl-xL protein and small-molecule drugs as well as the Bak-BH3 peptide. The long funnel-shaped structure and nanofluidic characteristics of the YaxAB nanopore enable the electro-osmotic trapping of diverse folded proteins and high-resolution monitoring of PDIs. Distinctive nanopore event distributions observed in the two-dimensional (ΔI/Io-versus-IN) plot illustrate the ability of the YaxAB nanopore to discriminate individual small-molecule drugs bound to Bcl-xL from non-binders. Taken together, our results present the YaxAB nanopore as a robust platform for label-free, ultrasensitive, single-molecule detection of PDIs, opening up a possibility for low-cost, highly efficient drug discovery against diverse drug targets.

Abstract 5731: Apoptosis Inducing Agent 1 enhances cancer therapy-induced apoptosis by direct interaction with BAX and BAK

Abstract Many of the most effective anti-cancer therapies induce apoptosis in cancer cells by damaging DNA. Even when effective at eradicating primary cancers, DNA-damaging anti-cancer agents cause mutations that can lead to the development of secondary cancers. Furthermore, DNA damage responses are dependent on wild-type p53, which is mutated in over 50% of cancers and causes resistance to therapies. Fortunately, many p53 mutant cancers continue to express high levels of the pro-apoptotic, pore-forming proteins BAX and BAK and are consequently “primed for apoptosis.” Primed cells are highly sensitive to pro-apoptotic signals and this vulnerability can be exploited using inhibitors of pro-survival BCL-2 family proteins such as the BCL-2 inhibitor ABT-199, which has been successful as therapy for chronic lymphocytic and acute myeloid leukemias (AML). Furthermore, most irreplaceable cells within healthy tissues are apoptosis resistant and express low levels of BAX and BAK. We therefore hypothesized that direct activators of BAX or BAK could be effective single-agent therapies for primed cancers and chemo-/radio-sensitizers for unprimed cancers. Using WT versus BAX -/- BAK -/- HeLa cells, we performed a high-throughput screen of nearly 100,000 compounds to identify small molecules that could directly activate BAX or BAK. 196 compounds were identified as hits and multiple orthogonal validation studies identified Apoptosis Inducing Agent 1 (AIA1) as the most specific and potent putative activator of BAX and BAK. Further validation studies demonstrated that AIA1 directly induces cytochrome c release from mitochondria in a BAX/BAK dependent manner and strongly enhances BIM-mediated activation of BAX and BAK and permeabilization of liposomes. Single-agent AIA treatment triggers apoptotic cell death in a broad panel of cancer cell lines and also sensitizes cancer cells to chemotherapeutic agents and especially BH3 mimetics targeting pro-survival BCL-2 family proteins. Importantly, AIA1 sensitizes ovarian cancer cells to BCL-XL inhibitors and AML cells to BCL-2 inhibitors regardless of p53 status. In vivo, AIA1 suppresses tumor growth and prolongs overall survival in both ovarian cancer and AML xenograft models without causing weight loss, thrombocytopenia or leukopenia. Notably, unprimed cancer cells that don’t immediately undergo apoptosis in response to single-agent AIA1 treatment upregulate expression of BAX and BAK, pro-survival proteins (BCL-XL, BCL-2, MCL-1) and pro-apoptotic proteins (BIM, BID, PUMA, Noxa) to produce a more highly primed apoptosis pathway and setting the stage for increased sensitivity to BH3 mimetics. Based on these findings, AIA1 may exploit and induce apoptotic vulnerabilities in cancers in a p53-independent manner and represent a safer strategy to target primed cancer cells while eliminating the potential for secondary malignancies and p53-mediated resistance. Citation Format: Xingping Qin, Cameron Fraser, Adam Presser, Johan KE Spetz, Stacey J. Yu, Gary A. Bradshaw, Marian Kalocsay, Bo R. Rueda, Tudor Moldoveanu, Kristopher A. Sarosiek. Apoptosis Inducing Agent 1 enhances cancer therapy-induced apoptosis by direct interaction with BAX and BAK. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5731.

Structural basis for GSDMB pore formation and its targeting by IpaH7.8

Gasdermins (GSDMs) are pore-forming proteins that play critical roles in host defence through pyroptosis1,2. Among GSDMs, GSDMB is unique owing to its distinct lipid-binding profile and a lack of consensus on its pyroptotic potential3–7. Recently, GSDMB was shown to exhibit direct bactericidal activity through its pore-forming activity4. Shigella, an intracellular, human-adapted enteropathogen, evades this GSDMB-mediated host defence by secreting IpaH7.8, a virulence effector that triggers ubiquitination-dependent proteasomal degradation of GSDMB4. Here, we report the cryogenic electron microscopy structures of human GSDMB in complex with Shigella IpaH7.8 and the GSDMB pore. The structure of the GSDMB–IpaH7.8 complex identifies a motif of three negatively charged residues in GSDMB as the structural determinant recognized by IpaH7.8. Human, but not mouse, GSDMD contains this conserved motif, explaining the species specificity of IpaH7.8. The GSDMB pore structure shows the alternative splicing-regulated interdomain linker in GSDMB as a regulator of GSDMB pore formation. GSDMB isoforms with a canonical interdomain linker exhibit normal pyroptotic activity whereas other isoforms exhibit attenuated or no pyroptotic activity. Overall, this work sheds light on the molecular mechanisms of Shigella IpaH7.8 recognition and targeting of GSDMs and shows a structural determinant in GSDMB critical for its pyroptotic activity.