NINJ1 mediates plasma membrane rupture by cutting and releasing membrane disks.

The membrane protein NINJ1 mediates plasma membrane rupture in pyroptosis and other lytic cell death pathways. Here, we report the cryo-EM structure of a NINJ1 oligomer segmented from NINJ1 rings. Each NINJ1 subunit comprises amphipathic (⍺1, ⍺2) and transmembrane (TM) helices (⍺3, ⍺4) and forms a chain of subunits, mainly by the TM helices and ⍺1. ⍺3 and ⍺4 are kinked, and the Gly residues are important for function. The NINJ1 oligomer possesses a concave hydrophobic side that should face the membrane and a convex hydrophilic side formed by ⍺1 and ⍺2, presumably upon activation. This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. Live-cell and super-resolution imaging uncover ring-like structures on the plasma membrane that are released into the culture supernatant. Released NINJ1 encircles a membrane inside, as shown by lipid staining. Therefore, NINJ1-mediated membrane disk formation is different from gasdermin-mediated pore formation, resulting in membrane loss and plasma membrane rupture.

NINJ1 induces plasma membrane rupture and release of damage-associated molecular pattern molecules during ferroptosis.

Ferroptosis is a regulated form of necrotic cell death caused by iron-dependent accumulation of oxidized phospholipids in cellular membranes, culminating in plasma membrane rupture (PMR) and cell lysis. PMR is also a hallmark of other types of programmed necrosis, such as pyroptosis and necroptosis, where it is initiated by dedicated pore-forming cell death-executing factors. However, whether ferroptosis-associated PMR is also actively executed by proteins or driven by osmotic pressure remains unknown. Here, we investigate a potential ferroptosis role of ninjurin-1 (NINJ1), a recently identified executor of pyroptosis-associated PMR. We report that NINJ1 oligomerizes during ferroptosis, and that Ninj1-deficiency protects macrophages and fibroblasts from ferroptosis-associated PMR. Mechanistically, we find that NINJ1 is dispensable for the initial steps of ferroptosis, such as lipid peroxidation, channel-mediated calcium influx, and cell swelling. In contrast, NINJ1 is required for early loss of plasma membrane integrity, which precedes complete PMR. Furthermore, NINJ1 mediates the release of cytosolic proteins and danger-associated molecular pattern (DAMP) molecules from ferroptotic cells, suggesting that targeting NINJ1 could be a therapeutic option to reduce ferroptosis-associated inflammation.

Necroptosis and Its Involvement in Various Diseases.

Necroptosis is a regulated form of cell death involved in the development of various pathological conditions. In contrast to apoptosis, plasma membrane rupture (PMR) occurs in cells in the relatively early stage of necroptosis; therefore, necroptosis induces a strong inflammatory response. Stimuli, including tumor necrosis factor (TNF), interferon (IFN)α/ß, lipopolysaccharide, polyI:C, and viral infection, induce the formation of necrosomes that lead to membrane rupture and the release of intracellular contents, termed danger-associated molecular patterns (DAMPs). DAMPs are the collective term for molecules that normally reside in the cytoplasm or nucleus in living cells without inducing inflammation but induce strong inflammatory responses when released outside cells. Recent studies have provided a better understanding of the mechanisms underlying PMR and the release of DAMPs. Moreover, necroptosis is involved in various pathological conditions, and mutations in necroptosis-related genes can cause hereditary autoinflammatory syndromes. Thus, manipulating necroptosis signaling pathways may be useful for treating diseases involving necroptosis.

NINJ1 Regulates Platelet Activation and PANoptosis in Septic Disseminated Intravascular Coagulation.

Disseminated intravascular coagulation (DIC), which is closely related to platelet activation, is a key factor leading to high mortality in sepsis. The release of contents from plasma membrane rupture after platelet death further aggravates thrombosis. Nerve injury-induced protein 1 (NINJ1) is a cell membrane protein that mediates membrane disruption, a typical marker of cell death, through oligomerization. Nevertheless, whether NINJ1 is expressed in platelets and regulates the platelet function remains unclear. The aim of this study was to evaluate the expression of NINJ1 in human and murine platelets and elucidate the role of NINJ1 in platelets and septic DIC. In this study, NINJ1 blocking peptide (NINJ126-37) was used to verify the effect of NINJ1 on platelets in vitro and in vivo. Platelet αIIbß3 and P-selectin were detected by flow cytometry. Platelet aggregation was measured by turbidimetry. Platelet adhesion, spreading and NINJ1 oligomerization were examined by immunofluorescence. Cecal perforation-induced sepsis and FeCl3-induced thrombosis models were used to evaluate the role of NINJ1 in platelet, thrombus and DIC in vivo. We found that inhibition of NINJ1 alleviates platelet activation in vitro. The oligomerization of NINJ1 is verified in membrane-broken platelets, which is regulated by the PANoptosis pathway. In vivo studies demonstrate that inhibition of NINJ1 effectively reduces platelet activation and membrane disruption, thus suppressing platelet-cascade reaction and leading to anti-thrombosis and anti-DIC in sepsis. These data demonstrate that NINJ1 is critical in platelet activation and plasma membrane disruption, and inhibition of NINJ1 effectively reduces platelet-dependent thrombosis and DIC in sepsis. This is the first study to reveal the key role of NINJ1 in platelet and its related disorders.

Calcium/P53/Ninjurin 1 Signaling Mediates Plasma Membrane Rupture of Acinar Cells in Severe Acute Pancreatitis.

Ninjurin 1 (NINJ1) is a double-transmembrane cell-surface protein that might mediate plasma membrane rupture (PMR) and the diffusion of inflammatory factors. PMR is a characteristic of acinar cell injury in severe acute pancreatitis (SAP). However, the involvement of NINJ1 in mediating the PMR of acinar cells in SAP is currently unclear. Our study has shown that NINJ1 is expressed in acinar cells, and the expression is significantly upregulated in sodium-taurocholate-induced SAP. The knockout of NINJ1 delays PMR in acinar cells and alleviates SAP. Moreover, we observed that NINJ1 expression is mediated by Ca2+ concentration in acinar cells. Importantly, we found that Ca2+ overload drives mitochondrial stress to upregulate the P53/NINJ1 pathway, inducing PMR in acinar cells, and amlodipine, a Ca2+ channel inhibitor, can reduce the occurrence of PMR by decreasing the concentration of Ca2+. Our results demonstrate the mechanism by which NINJ1 induces PMR in SAP acinar cells and provide a potential new target for treatment of SAP.

A Bioinspired Immunostimulatory System for Inducing Powerful Antitumor Immune Function by Directly Causing Plasma Membrane Rupture.

The Gasdermin protein is a membrane disruptor that can mediate immunogenic pyroptosis and elicit anti-tumor immune function. However, cancer cells downregulate Gasdermin and develop membrane repair mechanisms to resist pyroptosis. Therefore, an artificial membrane disruptor (AMD) that can directly mediate membrane rupture in pyroptosis-deficient cells and induce antitumor immune responses in a controllable manner will be valuable in preclinical and clinical research. A micron-scale Ce6-based AMD that can directly induce plasma membrane rupture (PMR) in gasdermin-deficient tumor cells is established. Micron-scale AMDs localize Ce6 specifically to the plasma membrane without labeling other organelles. Compared to free Ce6 molecules, the use of AMDs results in a higher degree of specificity for the plasma membrane. Due to this specificity, AMDs mediate fast and irreversible PMR under 660 nm red light. Furthermore, the AMDs are capable of inducing programmed cell death and lytic cell death in a catalytic manner, demonstrating that the amount of Ce6 used by AMDs is only one-fifth of that used by Ce6 alone when inducing 80% of cancer cell death. In vivo, the AMDs show specificity for tumor targeting and penetration, suggesting that light-driven programmed cell death is specific to tumors. AMDs are applied to antitumor therapy in gasdermin-deficient tumors, resulting in efficient tumor elimination with minimal damage to major organs when combined with anti-PD-1 therapy. Tumor regression is correlated with PMR-mediated inflammation and T-cell-based immune responses. This study provides new insights for designing bioinspired membrane disruptors for PMR and mediating anti-tumor immunotherapy. Additionally, AMD is a dependable tool for examining the immunogenicity of PMR both in vitro and in vivo.

Membrane Integrity Assay in Ferroptosis.

Ferroptosis is a form of regulated cell death that relies on the accumulation of intracellular iron and subsequent oxidative stress. Ferroptotic cell death is characterized by uncontrolled lipid peroxidation, which leads to plasma membrane damage and rupture. The loss of plasma membrane integrity results in the release of intracellular components, including damage-associated molecular patterns, and can propagate death between cells in a synchronized manner. Understanding the mechanisms of ferroptotic membrane damage is crucial to comprehending this form of cell death. This chapter provides a summary of techniques for detecting plasma membrane integrity in ferroptosis, including transmission electron microscopy analysis, flow cytometry analysis, and assessments of oxidoreductase-mediated membrane damage.

A novel NINJ1-mediated regulatory step is essential for active membrane rupture and common to different cell death pathways.

Plasma membrane rupture (PMR), the final event in lytic cell death that is in part responsible for the release of pro-inflammatory signals, was believed to be a passive event that followed osmotic swelling. Kayagaki et al. 1 have discovered that PMR is, in fact, mediated by ninjurin-1 (NINJ1), adding a novel regulatory step that is conserved across different types of lytic cell death, such as pyroptosis, necroptosis, and apoptosis. PMR is dependent on NINJ1 oligomerization, which is mediated by its highly conserved putative N-terminal α-helix. In vivo data suggest that the NINJ1-dependent secretome that is released upon PMR is likely to modulate antimicrobial host defense, suggesting this additional regulatory step also has physiological relevance.

Recent progress in the mechanistic understanding of NET formation in neutrophils.

Neutrophils are the most abundant circulating white blood cells and one of the major cell types of the innate immune system. Neutrophil extracellular traps (NETs) are a result of the extracellular release of nuclear chromatin from the ruptured nuclear envelope and plasma membrane. The externalized chromatin is an ancient defense weapon for animals to entrap and kill microorganisms in the extracellular milieu, thus protecting animals ranging from lower invertebrates to higher vertebrates. Although the externalized chromatin has the advantage of acting as anti-infective to protect against infections, extracellular chromatin might be problematic in higher vertebrate animals as they have an adaptive immune system that can trigger further immune or autoimmune responses. NETs and their associated nuclear and/or cytoplasmic components may induce sterile inflammation, immune, and autoimmune responses, leading to various human diseases. Though important in human pathophysiology, the cellular and molecular mechanisms of NET formation (also called NETosis) are not well understood. Given that nuclear chromatin forms the backbone of NETs, the nucleus is the root of the nuclear DNA extracellular traps. Thus, nuclear chromatin decondensation, along with the rupture of nuclear envelope and plasma membrane, is required for nuclear chromatin extracellular release and NET formation. So far, most of the literature focuses on certain signaling pathways, which are involved in NET formation but without explanation of cellular events and morphological changes described above. Here, we have summarized emerging evidence and discuss new mechanistic understanding, with our perspectives, in NET formation in neutrophils.

Role of NINJ1 in Gout Flare and Potential as a Drug Target.

Objective: To determine the role of nerve injury-induced protein 1 (NINJ1) introduced plasma membrane rupture (PMR) and damage-associated molecular patterns (DAMPs) release in the pathogenesis and progression of gout and to explore the potential of NINJ1 as a therapeutic target in gout. Methods: Both peripheral blood mononuclear cells (PBMCs) and serum sample from gout patients (n = 58) and healthy controls (n = 16) were collected and processed to NINJ1 expression, lactate dehydrogenase (LDH) detection, NINJ1 inhibition, and NINJ1 expression experiments, respectively. NINJ1 knockdown was carried out by lentivirus in a monosodium urate (MSU) induced rat model, and NINJ1 neutralizing antibody was applied in a MSU induced mouse model. Results: Our results found that NINJ1 was upregulated during a gout flare, and the resulting induction of PMR correlated with gout progression. NINJ1 knockdown significantly reduced the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and joint swelling in the rat model, and NINJ1 neutralizing antibody also significantly reduced gout flare in the mouse model and PBMCs. Moreover, NINJ1 expression is under NLRP3 inflammasome produced interleukin (IL)-1ß control. Conclusion: These results support the notion of a pathogenic role of NINJ1 introduced PMR in gout and provide a detailed mechanism for gout pathogenesis involving inflammatory cell death and DAMPs release introduced by IL-1ß. In addition, targeting NINJ1 might be a potential therapeutic approach for gout.

Uncovering human mixed lineage kinase domain-like activation in necroptosis.

MLKL and its obligate upstream receptor interacting protein kinase 3 are essential components of necroptosis. It is well established that MLKL is the executioner of plasma membrane rupture in necroptosis. In healthy cells MLKL is dormant. Several dormant configurations have emerged from high-resolution structural studies revealing distinct mechanisms of MLKL autoinhibition in mammals. MLKL is activated through the concerted actions of receptor interacting protein kinase 3, which phosphorylates MLKL, and, in the case of the human pathway, inositol phosphate (IP) metabolites synthesized by the IP kinases of the IP metabolic pathway. Here, we highlight recent progress toward understanding the mechanisms of regulation of human MLKL, and survey the latest opportunities for targeting MLKL in pathophysiology.

Direct Activation of Human MLKL by a Select Repertoire of Inositol Phosphate Metabolites.

Necroptosis is an inflammatory form of programmed cell death executed through plasma membrane rupture by the pseudokinase mixed lineage kinase domain-like (MLKL). We previously showed that MLKL activation requires metabolites of the inositol phosphate (IP) pathway. Here we reveal that I(1,3,4,6)P4, I(1,3,4,5,6)P5, and IP6 promote membrane permeabilization by MLKL through directly binding the N-terminal executioner domain (NED) and dissociating its auto-inhibitory region. We show that IP6 and inositol pentakisphosphate 2-kinase (IPPK) are required for necroptosis as IPPK deletion ablated IP6 production and inhibited necroptosis. The NED auto-inhibitory region is more extensive than originally described and single amino acid substitutions along this region induce spontaneous necroptosis by MLKL. Activating IPs bind three sites with affinity of 100-600 µM to destabilize contacts between the auto-inhibitory region and NED, thereby promoting MLKL activation. We therefore uncover MLKL's activating switch in NED triggered by a select repertoire of IP metabolites.