Membrane Pore Formation Research Articles

Attenuates reactive oxygen species: induced pyroptosis via activation of the Nrf2/HO-1 signal pathway in models of trigeminal neuralgia

In this study, we examined the impact of demyelinating and neuroinflammation on trigeminal neuralgia (TN) by utilizing models of chronic constriction injury to the infraorbital nerve (CCI). The CCI rats were treated with either VX-765 (an inhibitor of caspase-1) or a control solution of PBS/DMSO to observe the effects on neurobehavioral and neuropathological outcomes. The histochemical changes, pyroptosis-related proteins were assessed using immunohistochemistry, Elisa, and western blotting. RSC96 cells were pretreated with belnacasan (VX-765, an inhibitor of caspase-1), Gasdermin D(GSDMD)-targeting siRNAs, cobalt protoporphyrin (CoPP) or zinc protoporphyrin (Znpp) before being exposed to H2O2. Following these treatments, the Reactive oxygen species (ROS) level, cell viability, percentage of pyroptosis, pyroptosis-related proteins, nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1 level was measured. The scanning electron microscopy revealed increased ball-like bulge and membrane pore formation in the CCI group. In the CCI and CCI+ Vehicle groups, we found ROS level and expression of pyroptosis-related proteins increased. While, treatment with VX-765resulted in a decreased expression of GSDMD, IL-1, IL-18, and caspase-1 decreased. In the in-vitro study, RSC96 cells showed mild pyroptosis and overall mild edema after being exposed to H2O2. The ROS level, percentage of pyroptosis, pyroptosis-related proteins, Nrf2 and HO-1 level increased significantly in the H2O2 group. While, the percentage of pyroptosis and pyroptosis-related proteins decreased significantly in the H2O2 + VX-765 group, H2O2 + siRNA group, and H2O2 + VX-765 + siRNA group. After treatment with HO-1-inhibitor Znpp and HO-1-activator Copp, the percentage of pyroptosis and pyroptosis-related proteins increased and decreased significantly respectively. In conclusions, the pyroptosis of Schwann cell in the CCI model generated the demyelination of TN nerve. The ROS is an upstream event of NLRP3 inflammasome activation which induced eventual pyroptosis. The Nrf2/HO-1 signaling pathway could protect the H2O2-induced pyroptosis in RSC96 cells.

Control of Osmotic-Engine-Driven Liposomes Using Biological Nanopores

Liposome-based molecular robots that molecular systems are integrated into a giant liposome have been proposed; they are expected to be applied in the fields of medicine, environmental science, food science, and energy science. However, the performance of these molecular robotic components, including intelligence, sensors, and actuators, still hinders their practical use. In particular, the actuators used in the molecular robots, such as molecular motors, do not provide sufficient performance to move the giant liposomes. Hence, we propose an osmotic-engine-driven liposome and demonstrate the migration of liposomes in a microfluidic channel by applying a salt concentration difference between the front and rear of the liposome. Although the migration mechanism is simple and has the potential to provide sufficient mobility performance, control techniques for the movement speed and on/off switching are not established. Herein, we describe a speed control method of osmotic-engine-driven liposomes using pore-forming membrane proteins. In this study, we evaluated the effect of reconstituted α-hemolysin (αHL) nanopores on the water permeability through lipid bilayers. Thereafter, we demonstrated the change in displacement speeds of liposomes with and without nanopores. We expect the speed control method using nanopores to be applied to the liposome-based molecular robots.

2,2',4,4'-Tetrabromodiphenyl ether and cadmium co-exposure activates aryl hydrocarbon receptor pathway to induce ROS and GSDME-dependent pyroptosis in renal tubular epithelial cells.

We have previously found that a mixture exposure of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and cadmium (Cd) causes kidney damage; however, the mechanism was not fully understood. The aryl hydrocarbon receptor (AhR) is a ligand-receptor transcription factor that plays an important role in the adaptive response or metabolic detoxification of environmental toxins. Thus, this study aimed to examine the role of AhR in kidney toxicity. BDE-47 (50 μM) or Cd (5 μM) exposure reduced cell viability in renal tubular epithelial cells (HKC), with a larger effect observed in co-treatment. The cell morphology presented pyroptotic changes, including swollen cells, large bubbles, and plasma membrane pore formation. The gene expressions of AhR, heat shock protein 90 (Hsp90), AhR nuclear translocator (ARNT), and cytochrome P450 1B1 (CYP1B1) were increased, while CYP1A1 was decreased. Reactive oxygen species (ROS) were generated, which was reduced by the AhR antagonist CH223191. The apoptosis, necrosis, and intracellular lactated hydrogenase (LDH) release was elevated, and this was attenuated by N-acetylcysteine (NAC). Furthermore, the pyroptosis pathway was activated with increased protein levels of cleaved-caspase-3 and gasdermin E N-terminal (GSDME-NT), while caspase-8, caspase-3, and GSDME were decreased. These effects were alleviated by NAC and CH223191. Our data demonstrate a combined effect of BDE-47 and Cd on nephrotoxicity by activating AhR to induce ROS contributing to GSDME-dependent pyroptosis, and retardation of the AhR pathway could reduce this toxicity.

Surface Acoustic Wave (SAW)‐Based Sonoporation of Single‐Adherent‐Cell

Sonoporation refers to the formation of tiny transient pores in cell plasma membranes by using ultrasound to increase the permeability to bioactive materials. Recently, the Sonoporation technique has been widely researched in cell treatment applications, such as gene transfection. However, due to the random distribution of microbubbles, it is challenging to perform controllable and precise localized sonoporation of a target cell. In this work, a device based on the surface acoustic wave is developed to achieve a selective manipulation and cavitation of microbubbles. The device consists of a pair of microbubble positioning slant‐finger interdigital transducers (SFITs) for moving a selected microbubble in a two‐dimensional plane. Meanwhile, another narrow‐frequency‐band SFIT is integrated into the device for local cavitation control of the same microbubble. As a result, a microbubble can be transported orthogonal to and along the acoustic transmission path by continuously adjusting the input frequency and relative phase. Upon reaching the target cell, the selected microbubble can be cavitated without exciting other microbubbles, resulting in local sonoporation. The resolution of phase‐based positioning and frequency‐based transportation are 8.3 and 7.0 µm with 45° and 10 kHz settings, respectively.

Led-mediated instantaneous pore formation in membranes driven by structural transformation and deprotonation of phospholipids

The mechanisms by which molecules can be efficiently delivered directly into cells are of interest for fundamental cell biological research. Light-emitting diodes (LED) can be used to deliver substances into cells, but there is limited information on how LEDs alter the structure of the plasma membrane. In this study, we determined the mechanisms by which LED induces the instantaneous pore formation in the plasma membrane. We manufactured three types of artificial membrane (AM) composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), or a mixed form of binary PC/PE and investigated the surfaces and internal structural transformation and chemical changes caused by LED stimulation. LED irradiation (blue and near-infrared wavelengths) decreased the size of the three AMs due to the formation of pores on the surface and internal AM. PE and PC/PE AMs underwent LED-mediated structural transformation. In the PE AMs, chemical changes occurred through deprotonation of the ammonia cation (NH3+) located at the terminal of the head group of the phospholipid to an amine group (NH2). Furthermore, pore formation occurred in plasma membranes from cell lines and was used to deliver cell-impermeable molecules. Our study highlights a fundamental mechanism underlying pore formation at the molecular level.

Amyloid Precursor Protein Changes Arrangement in a Membrane and Its Structure Depending on the Cholesterol Content.

One of the hallmarks of Alzheimer's disease (AD) is the accumulation of amyloid beta (Aβ) peptides in the brain. The processing of amyloid precursor protein (APP) into Aβ is dependent on the location of APP in the membrane, membrane lipid composition and, possibly, presence of lipid rafts. In this study, we used atomic force microscopy (AFM) to investigate the interaction between transmembrane fragment APP672-726 (corresponding to Aβ1-55) and its amyloidogenic mutant L723P with membranes combining liquid-ordered and liquid-disordered lipid phases. Our results demonstrated that most of the APP672-726 is located either in the liquid-disordered phase or at the boundary between ordered and disordered phases, and hardly ever in rafts. We did not notice any major changes in the domain structure induced by APP672-726. In membranes without cholesterol APP672-726, and especially its amyloidogenic mutant L723P formed annular structures and clusters rising above the membrane. Presence of cholesterol led to the appearance of concave membrane regions up to 2 nm in depth that were deeper for wild type APP672-726. Thus, membrane cholesterol regulates changes in membrane structure and permeability induced by APP that might be connected with further formation of membrane pores.

Probing electrophysiological activity of amphiphilic Dynorphin A in planar neutral membranes reveals both ion channel-like activity and neuropeptide translocation

Dynorphin A (DynA) is an endogenous neuropeptide that besides acting as a ligand of the κ-opioid receptor, presents some non-opioid pathophysiological properties associated to its ability to induce cell permeability similarly to cell-penetrating peptides (CPPs). Here, we use electrophysiology experiments to show that amphiphilic DynA generates aqueous pores in neutral membranes similar to those reported previously in charged membranes, but we also find other events thermodynamically incompatible with voltage-driven ion channel activity (i.e. non-zero currents with no applied voltage in symmetric salt conditions, reversal potentials that exceed the theoretical limit for a given salt concentration gradient). By comparison with current traces generated by other amphiphilic molecule known to spontaneously cross membranes, we hypothesize that DynA could directly translocate across neutral bilayers, a feature never observed in charged membranes following the same electrophysiological protocol. Our findings suggest that DynA interaction with the cellular membrane is modulated by the lipid charge distribution, enabling either passive ionic transport via membrane remodeling and pore formation or by peptide direct internalization independent of cellular transduction pathways.

Exploring the mechanism of photosensitizer conjugation on membrane perturbation of antimicrobial peptide: A multiscale molecular simulation study

Antimicrobial peptides (AMPs) exert their biological functions by perturbation with cellular membrane. Conjugation of AMPs with photosensitizer (PS) is a promising strategy for enhancing the efficacy and reducing systemic toxicity of AMPs. However, it is still elusive how the conjugated PS impacts the perturbation of AMPs on cell membrane from molecular level. Here, we addressed this issue by a multiscale computational strategy on pyropheophorbide-a (PPA) conjugated K6L9 (PPA-K6L9), a PS-AMP conjugate developed by us previously. Our atomistic molecular dynamics (MD) simulations revealed that the porphyrin moiety of PPA enhanced the stability of the conjugate in a lipid bilayer membrane model. Moreover, such moiety also maintained the amphipathic structure of K6L9, which is crucial for membrane pore formation. Coarse-grained MD simulations further showed that the conjugates aggregated in membrane environment and formed more stable toroidal pores with respect to K6L9 alone, suggesting the conjugation of PPA may enhance the membrane-disruption activity of K6L9. Consistent with this, our cellular experiments confirmed that PPA-K6L9 was more toxic to 4 T1 tumor cells than K6L9. This study provides insights into the mechanism by which PS-AMP conjugates disrupt cellular membranes and could aid in the design of more potent AMP conjugates.

Leveraging Plasma Membrane Repair Therapeutics for Treating Neurodegenerative Diseases.

Plasma membrane repair is an essential cellular mechanism that reseals membrane disruptions after a variety of insults, and compromised repair capacity can contribute to the progression of many diseases. Neurodegenerative diseases are marked by membrane damage from many sources, reduced membrane integrity, elevated intracellular calcium concentrations, enhanced reactive oxygen species production, mitochondrial dysfunction, and widespread neuronal death. While the toxic intracellular effects of these changes in cellular physiology have been defined, the specific mechanism of neuronal death in certain neurodegenerative diseases remains unclear. An abundance of recent evidence indicates that neuronal membrane damage and pore formation in the membrane are key contributors to neurodegenerative disease pathogenesis. In this review, we have outlined evidence supporting the hypothesis that membrane damage is a contributor to neurodegenerative diseases and that therapeutically enhancing membrane repair can potentially combat neuronal death.

Pyroptosis-related noncoding RNAs and cancer involvement

Abstract Cell pyroptosis, an inflammatory and programmed cell necrosis, is also an important cause of multiple organ damage and immunosuppression. Pyroptosis, a new kind of programmed cell death, leads to cleavage of the pyroptotic protein Gasdermin family and ultimately to membrane pore formation, cell shrinkage, plasma membrane cleavage, chromatin disruption and release of pro-inflammatory components. Various studies found that pyroptosis had a key role in cancer and inflammatory diseases. Gasdermin family is key protein in pyrotopsis. Non-coding RNAs have a potential role in cell pyrotosis which regulate immunity and cell death. This article reviews cell pyroptosis, GSDM family and the regulatory role of non-coding RNAs.

#4359 LOSS OF GASDERMIN D LEADS TO AN EXACERBATION OF KIDNEY DAMAGE IN NEPHROCALCINOSIS-RELATED CHRONIC KIDNEY DISEASE

Abstract Background and Aims Gasdermin D (GSDMD) is a membrane-targeting and pore-forming protein that is primarily expressed in immune cells. In the proinflammatory cell death process known as pyroptosis, cleaved GSDMD plays a crucial role through cell membrane pore formation, leading to the release of proinflammatory cytokines and membrane rapture. Despite this, the majority of GSDMD's other physiological functions remain to be elucidated. Previous reports have implicated GSDMD in the pathogenesis of kidney disease, utilizing some acute kidney injury (AKI) animal models and a few chronic kidney disease (CKD) animal models. However, it remains uncertain if GSDMD contributes to the pathogenesis of nephrocalcinosis-related CKD. Therefore, this study aimed to investigate the role of GSDMD in in this context. Method In our study, 8-week-old GSDMD-knockout (KO) mice and wildtype (WT) mice received an oxalate-rich diet (50 μmol/g sodium oxalate) for three weeks. All mice were sacrificed on day 21 and kidneys were harvested after the sacrifice. Plasma and urine samples were collected at different time points before the sacrifice. The glomerular filtration rate (GFR) was measured on day 0 and day 20 to assess kidney excretion function. Histological analysis was performed using Periodic Acid-Schiff (PAS) staining to assess renal injury, Sirius Red staining to check renal fibrosis, Pizzolato staining to evaluate calcium crystal deposition, F4/80 staining to evaluate the infiltration of macrophages, TUNEL staining to check for cell death, RIPK3 and phosphorylated MLKL (pMLKL) staining to detecting necroptosis. And GSDMD staining was also performed to examine the expression of GSDMD in the kidney. Results Pizzolato staining showed no differences in calcium crystal deposition between WT mice and KO mice. GFR was decreased in both WT mice and KO mice after the oxalate-rich diet (day 0 WT: 199.5±40.1 μl/min vs KO 175.0±44.8 μl/min, day 20 WT: 156.5±37.9 μl/min vs KO: 73.0±48.1 μl/min), and ΔGFR was much greater in KO mice (WT: −22.4±26.5% vs KO: −59.4±19.3%). The level of blood urea nitrogen was significantly higher in KO mice (WT: 41.9±3.90 mg/dl vs KO: 58.7±22.5 mg/dl) in accordance with the result of the GFR. PAS staining revealed more severe kidney injury in KO mice, and Sirius Red staining indicated a higher degree of renal fibrosis in KO mice. Furthermore, F4/80 staining showed greater infiltration of macrophages in KO mice. Although TUNEL staining showed no significant differences in positive cells between WT and KO, KO mice showed greater positive areas implicating more necroptosis in RIPK3 and pMLKL staining. GSDMD staining revealed that injured tubular cells during the chronic phase express GSDMD (Fig. 1). In in vitro experiments, we evaluated the functionality of macrophages such as phagocytic ability and migratory ability using bone marrow-derived macrophages (BMDMs), but we didn't find any significant disparities between BMDMs from WT mice and those from KO mice. Conclusion Genetic deletion of GSDMD resulted in more severe kidney injury, macrophage infiltration and fibrosis in mice with nephrocalcinosis-related CKD. Our results suggest a potential role for pyroptosis, especially GSDMD, in contributing to renal tubular cell necroptosis, a process downstream of calcium-oxalate crystal formation and deposition, in the progression of nephrocalcinosis-related CKD, similar to previous reports in mouse models of AKI.

Molecular mechanisms of gasdermin D pore-forming activity.

The regulated disruption of the plasma membrane, which can promote cell death, cytokine secretion or both is central to organismal health. The protein gasdermin D (GSDMD) is a key player in this process. GSDMD forms membrane pores that can promote cytolysis and the release of interleukin-1 family cytokines into the extracellular space. Recent discoveries have revealed biochemical and cell biological mechanisms that control GSDMD pore-forming activity and its diverse downstream immunological effects. Here, we review these multifaceted regulatory activities, including mechanisms of GSDMD activation by proteolytic cleavage, dynamics of pore assembly, regulation of GSDMD activities by posttranslational modifications, membrane repair and the interplay of GSDMD and mitochondria. We also address recent insights into the evolution of the gasdermin family and their activities in species across the kingdoms of life. In doing so, we hope to condense recent progress and inform future studies in this rapidly moving field in immunology.

Effect of dual-frequency thermosonication, food matrix, and germinants on Alicyclobacillus acidoterrestris spore germination

The off-odors associated with spoilage of acidic beverages are linked to the germination and growth of Alicyclobacillus acidoterrestris (AAT) spores. As a consequence, we determined the influence of nutrients, non-nutrient germinants, dual-frequency thermosonication (DFTS), and food matrix on spore germination. AAT spores in orange juice (OJ), supplemented by L-alanine (L-ala), had the highest germination rate and lowest DPA content at 10 h of incubation. The formation of microscopic pores in cell membranes during DFTS caused irreversible damage in AAT spores in citrate buffer solution (CBS); however, it stimulated AAT spore germination in CBS containing L-ala. Hence, the germination potential was established in the order: L-ala > Calcium dipicolinate > asparagine, glucose, fructose, and potassium ion mixture (AGFK) > L-valine. The conductivity analysis indicated that membrane damage could be a key factor contributing to the artificial germination in CBS. AFM images revealed that after 2 h of adding L-ala, the protein content increased with increased germinated cells. TEM showed that membrane poration and coat detachment were the main pre-germination morphological changes detected after DFTS treatment. This study provides evidence that germination stimulated with DFTS might be an effective strategy for reducing A. acidoterrestris spores in fruit juices.

Gasdermin D-mediated pyroptosis: mechanisms, diseases, and inhibitors.

Gasdermin D (GSDMD)-mediated pyroptosis and downstream inflammation are important self-protection mechanisms against stimuli and infections. Hosts can defend against intracellular bacterial infections by inducing cell pyroptosis, which triggers the clearance of pathogens. However, pyroptosis is a double-edged sword. Numerous studies have revealed the relationship between abnormal GSDMD activation and various inflammatory diseases, including sepsis, coronavirus disease 2019 (COVID-19), neurodegenerative diseases, nonalcoholic steatohepatitis (NASH), inflammatory bowel disease (IBD), and malignant tumors. GSDMD, a key pyroptosis-executing protein, is linked to inflammatory signal transduction, activation of various inflammasomes, and the release of downstream inflammatory cytokines. Thus, inhibiting GSDMD activation is considered an effective strategy for treating related inflammatory diseases. The study of the mechanism of GSDMD activation, the formation of GSDMD membrane pores, and the regulatory strategy of GSDMD-mediated pyroptosis is currently a hot topic. Moreover, studies of the structure of caspase-GSDMD complexes and more in-depth molecular mechanisms provide multiple strategies for the development of GSDMD inhibitors. This review will mainly discuss the structures of GSDMD and GSDMD pores, activation pathways, GSDMD-mediated diseases, and the development of GSDMD inhibitors.

The Bacillus cereus toxin alveolysin disrupts the intestinal epithelial barrier by inducing microtubule disorganization through CFAP100.

Bacillus cereus is a Gram-positive bacterium that mainly causes self-limiting emetic or diarrheal illness but can also cause skin infections and bacteremia. Symptoms of B. cereus ingestion depend on the production of various toxins that target the gastric and intestinal epithelia. From a screen of bacterial isolates from human stool samples that compromised intestinal barrier function in mice, we identified a strain of B. cereus that disrupted tight and adherens junctions in the intestinal epithelium. This activity was mediated by the pore-forming exotoxin alveolysin, which increased the production of the membrane-anchored protein CD59 and of cilia- and flagella-associated protein 100 (CFAP100) in intestinal epithelial cells. In vitro, CFAP100 interacted with microtubules and promoted microtubule polymerization. CFAP100 overexpression stabilized microtubules in intestinal epithelial cells, leading to disorganization of the microtubule network and perturbation of tight and adherens junctions. The disruption of cell junctions by alveolysin depended on the increase in CFAP100, which in turn depended on CD59 and the activation of PI3K-AKT signaling. These findings demonstrate that, in addition to forming membrane pores, B. cereus alveolysin can permeabilize the intestinal epithelium by disrupting epithelial cell junctions in a manner that is consistent with intestinal symptoms and may allow the bacteria to escape the intestine and cause systemic infections. Our results suggest the potential value of targeting alveolysin or CFAP100 to prevent B. cereus-associated intestinal diseases and systemic infections.

Determining the Functional Oligomeric State of Membrane-Associated Protein Oligomers Forming Membrane Pores on Giant Lipid Vesicles.

Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane-associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.

Membranolytic Activity Profile of Nonyl 3,4-Dihydroxybenzoate: A New Anti-Biofilm Compound for the Treatment of Dermatophytosis.

The ability of dermatophytes to live in communities and resist antifungal drugs may explain treatment recurrence, especially in onychomycosis. Therefore, new molecules with reduced toxicity that target dermatophyte biofilms should be investigated. This study evaluated nonyl 3,4-dihydroxybenzoate (nonyl) susceptibility and mechanism of action on planktonic cells and biofilms of T. rubrum and T. mentagrophytes. Metabolic activities, ergosterol, and reactive oxygen species (ROS) were quantified, and the expression of genes encoding ergosterol was determined by real-time PCR. The effects on the biofilm structure were visualized using confocal electron microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). T. rubrum and T. mentagrophytes biofilms were susceptible to nonyl and resistant to fluconazole, griseofulvin (all strains), and terbinafine (two strains). The SEM results revealed that nonyl groups seriously damaged the biofilms, whereas synthetic drugs caused little or no damage and, in some cases, stimulated the development of resistance structures. Confocal microscopy showed a drastic reduction in biofilm thickness, and transmission electron microscopy results indicated that the compound promoted the derangement and formation of pores in the plasma membrane. Biochemical and molecular assays indicated that fungal membrane ergosterol is a nonyl target. These findings show that nonyl 3,4-dihydroxybenzoate is a promising antifungal compound.

DDX3X Deficiency attenuates Pyroptosis Induced by Oxygen-Glucose Deprivation/Reoxygenation in N2a Cells.

NOD‑like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis is strongly related to cerebral ischemia/reperfusion (I/R) injury. DDX3X, the DEAD-box family's ATPase/RNA helicase, promotes NLRP3 inflammasome activation. However, whether DDX3X deficiency attenuates NLRP3 inflammasome-mediated pyroptosis induced by cerebral I/R injury. This study investigated whether DDX3X deficiency attenuates NLRP3 inflammasome-mediated pyroptosis in N2a cells after oxygen-glucose deprivation/ reoxygenation (OGD/R) treatment. In vitro model of cerebral I/R injury, mouse neuro2a (N2a) cells subjected to OGD/R were treated with the knockdown of DDX3X. Cell counting kit-8 (CCK-8) assay and Lactate dehydrogenase (LDH) cytotoxicity assay were conducted to measure cell viability and membrane permeability. Double immunofluorescence was performed to determine the pyroptotic cells. Transmission electron microscopy (TEM) was used to observe morphological changes of pyroptosis. Pyroptosis-associated proteins were analyzed by Western blotting. The OGD/R treatment reduced cell viability, increased pyroptotic cells and released LDH compared to the control group. TEM showed membrane pore formation of pyroptosis. Immunofluorescence showed that GSDMD was translocated from the cytoplasm to the membrane after OGD/R treatment. Western blotting showed that the expression of DDX3X, and pyroptosis-related proteins (NLRP3, cleaved-Caspase1, and GSDMD-N) were increased after OGD/R treatment. Nevertheless, DDX3X knockdown markedly improved cell viability and reduced LDH release, expression of pyroptosis-related proteins, and N2a cells pyroptosis. DDX3X knockdown significantly inhibited membrane pore formation and GSDMD translocation from cytoplasm to membrane. This research demonstrates for the first time that DDX3X knockdown attenuates OGD/R‑induced NLRP3 inflammasome activation and pyroptosis, which implies that DDX3X may become a potential therapeutic target for cerebral I/R injury.

Abstract 655: Necroptosis promotes tumor growth through IL1-alpha release and recruitment of immunosuppressive myeloid cells

Abstract Programmed cell death (PCD) was originally limited to apoptosis, but now includes pyroptosis, ferroptosis, necroptosis, and paraptosis. Necroptosis has attracted particular interest as an approach for immunotherapy as activation of the RIPK1/RIPK3 necrosome phosphorylates MLKL to form membrane pores, leading to loss of integrity and enhanced ability to induce an antigen-specific immune response in models of vaccination. Here we sought to determine if necroptosis within tumor cells was an important regulatory of anti-tumor immunity. Deficiency in Ripk3 or Mlkl did not impact tumor incidence or growth in genetic models of mammary carcinoma or lung adenocarcinoma. Similarly, the growth of syngeneic cell lines was not altered by loss of Ripk3 or Mlkl in vitro or in vivo. We next examined the induction of apoptosis or necroptosis in syngeneic tumor models using genetic constructs with doxycycline-controlled expression. In line with published data, subcutaneous vaccination demonstrated that RIPK3-induced necroptosis promoted an antigen-specific CD8+ T cell response greater than that observed with caspase 8-induced apoptosis. Surprisingly however, when necroptosis was induced within established PyMT-B6 orthotopic mammary tumors, we observed significantly reduced survival, which correlated with higher neutrophil and macrophage recruitment, along with lower CD8+ T cell infiltration and IFN-γ expression. In vitro, necroptosis induced with either RIPK3 or MLKL greatly enhanced expression of Cxcl1 and Cxcl2, a property that could be recapitulated by transferring dead cell supernatant to live cells, suggesting the presence of an alarmin or damage-associated molecular pattern (DAMP). We therefore knocked out MyD88, which serves as a critical adapter protein to a number of surface receptors, including the TLR family, the IL1 receptor, and RAGE. The loss of MyD88 completely eliminated the ability of tumor cells to express Cxcl1 in response to supernatant from necrotic cells. Expression was not driven by release of HMGB1 or signaling through RAGE or TLRs. Instead, we found that blocking the interleukin (IL)1 receptor or IL1α, but not IL1β, prevented Cxcl1 expression. IL1α is a constitutively expressed by some tumors, suggesting a potential role for IL1α in regulating immune responses to therapies that induce necrotic-like forms of cell death. In support of this, Il1a expression was strongly predictive of progression free survival in triple-negative breast cancer. These findings suggest that IL1α has an unexplored role in regulating the tumor microenvironment. By understanding how IL1α regulates the immune response during cytotoxic therapy, our findings may explain why certain therapies are immunogenic (or not) in different cancers. Citation Format: Kay Hänggi, Aysenur Keske, Alycia Gardner, Jie Li, Olabisi Osunmakinde, Daiana Celias, Amer Beg, Brian Ruffell. Necroptosis promotes tumor growth through IL1-alpha release and recruitment of immunosuppressive myeloid cells [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 655.

Gallocin A, an Atypical Two-Peptide Bacteriocin with Intramolecular Disulfide Bonds Required for Activity.

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic gut pathogen associated with colorectal cancer. We previously showed that colonization of the murine colon by SGG in tumoral conditions was strongly enhanced by the production of gallocin A, a two-peptide bacteriocin. Here, we aimed to characterize the mechanisms of its action and resistance. Using a genetic approach, we demonstrated that gallocin A is composed of two peptides, GllA1 and GllA2, which are inactive alone and act together to kill "target" bacteria. We showed that gallocin A can kill phylogenetically close relatives of the pathogen. Importantly, we demonstrated that gallocin A peptides can insert themselves into membranes and permeabilize lipid bilayer vesicles. Next, we showed that the third gene of the gallocin A operon, gip, is necessary and sufficient to confer immunity to gallocin A. Structural modeling of GllA1 and GllA2 mature peptides suggested that both peptides form alpha-helical hairpins stabilized by intramolecular disulfide bridges. The presence of a disulfide bond in GllA1 and GllA2 was confirmed experimentally. Addition of disulfide-reducing agents abrogated gallocin A activity. Likewise, deletion of a gene encoding a surface protein with a thioredoxin-like domain impaired the ability of gallocin A to kill Enterococcus faecalis. Structural modeling of GIP revealed a hairpin-like structure strongly resembling those of the GllA1 and GllA2 mature peptides, suggesting a mechanism of immunity by competition with GllA1/2. Finally, identification of other class IIb bacteriocins exhibiting a similar alpha-helical hairpin fold stabilized with an intramolecular disulfide bridge suggests the existence of a new subclass of class IIb bacteriocins. IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus (SGG), previously named Streptococcus bovis biotype I, is an opportunistic pathogen responsible for invasive infections (septicemia, endocarditis) in elderly people and is often associated with colon tumors. SGG is one of the first bacteria to be associated with the occurrence of colorectal cancer in humans. Previously, we showed that tumor-associated conditions in the colon provide SGG with an ideal environment to proliferate at the expense of phylogenetically and metabolically closely related commensal bacteria such as enterococci (1). SGG takes advantage of CRC-associated conditions to outcompete and substitute commensal members of the gut microbiota using a specific bacteriocin named gallocin, recently renamed gallocin A following the discovery of gallocin D in a peculiar SGG isolate. Here, we showed that gallocin A is a two-peptide bacteriocin and that both GllA1 and GllA2 peptides are required for antimicrobial activity. Gallocin A was shown to permeabilize bacterial membranes and kill phylogenetically closely related bacteria such as most streptococci, lactococci, and enterococci, probably through membrane pore formation. GllA1 and GllA2 secreted peptides are unusually long (42 and 60 amino acids long) and have very few charged amino acids compared to well-known class IIb bacteriocins. In silico modeling revealed that both GllA1 and GllA2 exhibit a similar hairpin-like conformation stabilized by an intramolecular disulfide bond. We also showed that the GIP immunity peptide forms a hairpin-like structure similar to GllA1/GllA2. Thus, we hypothesize that GIP blocks the formation of the GllA1/GllA2 complex by interacting with GllA1 or GllA2. Gallocin A may constitute the first class IIb bacteriocin which displays disulfide bridges important for its structure and activity and might be the founding member of a subtype of class IIb bacteriocins.