Author response: Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death

Abstract

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract First recognized more than 30 years ago, glycine protects cells against rupture from diverse types of injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target of glycine that mediates cytoprotection, however, remains elusive. Here, we show that glycine works at the level of NINJ1, a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptosis lysis. NINJ1 is thought to cluster within the plasma membrane to cause cell rupture. We demonstrate that the execution of pyroptotic cell rupture is similar for human and mouse NINJ1 and that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in macrophages undergoing lytic cell death. Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. In pyroptosis, glycine preserves cellular integrity but does not affect upstream inflammasome activities or accompanying energetic cell death. By positioning NINJ1 clustering as a glycine target, our data resolve a long-standing mechanism for glycine-mediated cytoprotection. This new understanding will inform the development of cell preservation strategies to counter pathologic lytic cell death. Editor's evaluation It's been widely known that the amino acid Glycine can work as a cytoprotectant and inhibit cell death-associated plasma membrane rupture. However, a long-standing question has been: how does Glycine cytoprotection work? In this manuscript, the authors demonstrate that Glycine treatment inhibits the clustering of NINJ1 to preserve membrane integrity, which provides a significant advance in the cell death field. https://doi.org/10.7554/eLife.78609.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The amino acid glycine has long been known to protect cells against plasma membrane rupture induced by a diverse set of injurious stimuli. This cytoprotective effect was first described in 1987, where glycine was found to protect renal tubular cells against hypoxic injury (Weinberg et al., 1987). Since, similar observations have been replicated across immune, parenchymal, endothelial, and neuronal cell types in multiple injury models (reviewed in Weinberg et al., 2016). These include the lytic cell death pathways pyroptosis (Loomis et al., 2019; Volchuk et al., 2020) and necrosis (Estacion et al., 2003). While the robust cytoprotective effects of glycine are widespread, the molecular target and mechanism by which glycine protects cells and tissues remain elusive. Many cellular processes have been investigated but ultimately ruled out as underlying this phenomenon (Weinberg et al., 2016). These include glycine or ATP metabolic pathways (Weinberg et al., 1991), intracellular calcium or pH regulation (Weinberg et al., 1994), cytoskeleton stabilization (Chen et al., 1997), and chloride conductance (Loomis et al., 2019). It has been speculated that glycine targets a membrane receptor but is unlikely to involve canonical glycine receptors (Loomis et al., 2019; Weinberg et al., 2016). Lastly, as a cytoprotective agent, glycine has oftentimes been described as an osmoprotectant (Fink and Cookson, 2006). However, its small size should generally allow free passage through the large membrane conduits involved in lytic cell death pathways, such as the gasdermin D pores formed during pyroptosis that are known to allow transport of small proteins and ions across lipid bilayers (Evavold et al., 2018; Heilig et al., 2018; Xia et al., 2021). The free permeability of the plasma membrane to glycine through large conduits or amino acid transporters would negate a protective osmotic effect. Moreover, it remains unknown whether glycine’s cytoprotective effect is through an extracellular or intracellular mode of action and, therefore, whether glycine’s entry into the cell is required for cytoprotection. A mechanistic knowledge of how glycine preserves cellular integrity would inform the development of cell and tissue preservation strategies. This mechanistic understanding of glycine cytoprotection has clear clinical implications, such as for limiting inflammation in ischemia-reperfusion injuries where multiple lytic cell death pathways are activated to cause tissue damage and morbidity (Del Re et al., 2019). Given how widely observed the phenomenon is, it stands to reason that glycine targets a late downstream process common to multiple tissue injury models. Notably, the transmembrane protein ninjurin-1 (NINJ1) was recently identified as the common executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptosis lysis (Kayagaki et al., 2021). Post-apoptosis lysis refers to the cell membrane rupture that occurs when phagocytes are unable to scavenge apoptotic cells that have otherwise completed the apoptotic program (Silva, 2010). NINJ1 is a 16 kilodalton protein with two transmembrane regions that reside in the plasma membrane with prior reports, suggesting a function in cell adhesion (Araki et al., 1997; Araki and Milbrandt, 1996). NINJ1 has been implicated in multiple inflammatory conditions, such as nerve injury, atherosclerosis, ischemic brain injury, and tumorigenesis (Araki and Milbrandt, 1996; Jeon et al., 2020; Kim et al., 2020; Yang et al., 2017). The mechanism by which NINJ1 mediates lytic cell death coincides with its clustering within the plasma membrane, and this multimerization is required for membrane disruption by an unknown mechanism (Kayagaki et al., 2021). Here, we hypothesized that glycine targets NINJ1 to mediate its cytoprotective effect. We first demonstrate that NINJ1 knockout or silencing functionally and morphologically phenocopies glycine cytoprotection in mouse and human primary macrophages stimulated to undergo various forms of lytic cell death. Next, we show that glycine treatment prevents NINJ1 clustering within the plasma membrane, thereby preserving its integrity without affecting upstream programmed cell death signaling. By identifying NINJ1-dependent plasma membrane rupture as a glycine target, our data help resolve a long-standing mechanism of glycine cytoprotection. Results NINJ1 deficiency functionally and morphologically phenocopies glycine cytoprotection The published literature suggests a potential association between NINJ1 and glycine given that NINJ1 knockout and glycine treatment protect cells against a common set of lytic cell death pathways. Moreover, in pyroptosis, both allow for IL-1β secretion through the gasdermin D pore while preventing final membrane lysis (Kayagaki et al., 2021; Volchuk et al., 2020). We first sought to extend these associations by further characterizing functional and morphological similarities between NINJ1 knockout and glycine cytoprotection in pyroptosis, necrosis, and post-apoptosis lysis. Using a CRISPR-Cas9 system, we generated a NINJ1 knockout cell line in mouse immortalized bone marrow-derived macrophages (iBMDMs; Figure 1A). Pyroptosis was induced in LPS(lipopolysaccharide)-primed wildtype and NINJ1 knockout iBMDM with nigericin (20 µM, 2 hr) in the absence or presence of 5 mM glycine. Glycine treatment at 5 mM was based on the reported literature (Loomis et al., 2019; Volchuk et al., 2020) and a dose-finding study (Figure 1—figure supplement 1). We performed a colorimetric assay to test for LDH release, a common marker of cell rupture. Both NINJ1 knockout and glycine treatment in wildtype iBMDM protected against cytotoxicity (Figure 1B). Similarly, cell membrane integrity was preserved following apoptosis (Figure 1C) and necrosis (Figure 1D) induced with venetoclax (25 µM, 16 hr) and pneumolysin (0.5 µg/mL, 15 min), respectively. Importantly, in NINJ1 knockout macrophages, treatment with glycine during lytic cell death did not confer any additional protection against cell lysis, suggesting that these distinct perturbations affect the same process (Figure 1B–D). Figure 1 with 2 supplements see all Download asset Open asset NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection. (A) Immunoblot analysis demonstrating NINJ1 knockout in immortalized bone marrow-derived macrophage (iBMDM). GAPDH(glyceraldehyde-3-phosphate dehydrogenase) is presented as a loading control. (B–D) Wildtype and NINJ1 knockout iBMDM were induced to undergo pyroptosis (LPS + nigericin), post-apoptosis lysis (secondary necrosis; venetoclax, ABT-199), or necrosis (pneumolysin) with or without 5 mM glycine treatment. Cytotoxicity was evaluated by measuring the levels of LDH in the supernatant. Cytotoxicity decreased in glycine-treated wildtype cells comparably to NINJ1 knockout across each of (B) pyroptosis, (C) post-apoptosis lysis, and (D) necrosis. Glycine treatment of NINJ1 KO cells provided no additional protection to knockout cells treated without glycine. Data are expressed as supernatant LDH as a % of total LDH from lysates and supernatants, from n = 3 independent experiments for each cell death type. Individual data points are shown along with their mean. * p<0.05 by ANOVA with Tukey’s multiple comparison correction. (E) LPS-primed wildtype iBMDM induced to undergo pyroptosis in the presence of glycine demonstrate similar plasma membrane ballooning to NINJ1 knockout iBMDM induced to undergo pyroptosis. Membrane ballooning is shown in live cells labeled with the plasma membrane dye FM4-64. The corresponding brightfield image is shown in the inset. Scale bar 15 µm. Figure 1—source data 1 Numerical values for experimental data plotted in Figure 1. https://cdn.elifesciences.org/articles/78609/elife-78609-fig1-data1-v3.xlsx Download elife-78609-fig1-data1-v3.xlsx When visualizing glycine-treated pyroptotic macrophages, we observed prominent plasma membrane ballooning (Figure 1E). This impressive morphology was comparable to that of LPS-primed NINJ1 knockout macrophages stimulated to undergo pyroptosis with nigericin, which we (Figure 1E) and others (Kayagaki et al., 2021) have observed. Together, these functional and morphologic similarities buttress the association between glycine cytoprotection and NINJ1-mediated plasma membrane rupture. To further corroborate the above findings, we conducted pyroptosis assays in RAW mouse macrophages reconstituted with the inflammasome adaptor ASC (hereafter referred to as RAW-ASC). NINJ1 protein expression was ablated through CRISPR-Cas9 targeting as compared with the parental line (Figure 1—figure supplement 2A). Pyroptosis stimulation in this orthogonal murine macrophage model demonstrates NINJ1 deficiency functionally (Figure 1—figure supplement 2B) and morphologically (Figure 1—figure supplement 2C) phenocopies glycine treatment as was seen in the iBMDM system. Human NINJ1 is 90% homologous to the mouse protein and was shown to be toxic when overexpressed in HEK293T cells (Kayagaki et al., 2021), but it is not known if endogenous NINJ1 is activated and mediates plasma membrane rupture in human cells undergoing programmed cell death. We knocked down NINJ1 in primary human monocyte-derived macrophages (hMDMs) using siRNA specific to NINJ1 (siNINJ1) and compared to non-targeting control siRNA (siCtrl) with an efficiency approaching 70% (Figure 4—figure supplement 2A). Both NINJ1 knockdown and glycine (50 mM; Figure 2—figure supplement 1) independently and completely prevented pyroptosis (LDH release) induced by nigericin (20 µM, 2 hr) in LPS-primed hMDMs. Notably, again there was no additional effect with combined NINJ1 deficiency and glycine treatment arguing that NINJ1 and glycine are involved in the same process in primary human cell death pathways (Figure 2A). In contrast, human macrophages induced to undergo necroptosis (zVAD 20 µM + SMAC mimetic BV6 10 µM followed by TNF 10 ng/mL) were unprotected by glycine treatment (Figure 2—figure supplement 2), consistent with the observation that Ninj1 knockout does not confer protection in necroptosis likely due to sufficient cytolytic activity of MLKL pores (Kayagaki et al., 2021). Like mouse macrophages, both siNINJ1 and glycine sustained the ballooned morphology of pyroptotic hMDMs (Figure 2B). While pyroptotic human macrophages maintained their plasma membrane integrity in the presence of glycine as demonstrated by suppressed LDH release, we next confirmed that they had nevertheless lost viability as measured by the loss of mitochondrial membrane potential and cellular ATP content (Figure 2—figure supplement 3). Figure 2 with 3 supplements see all Download asset Open asset NINJ1 silencing in primary human monocyte-derived macrophages (hMDMs) functionally and morphologically phenocopies glycine cytoprotection. Primary hMDMs were treated with siRNA targeting NINJ1 (siNINJ1) or non-targeted control (siCtrl). (A) Wildtype and NINJ1-silenced hMDMs were LPS-primed and induced to undergo pyroptosis (nigericin) with or without 50 mM glycine. LDH release in the supernatants was measured to assess cytotoxicity. Cytotoxicity was reduced in glycine-treated wildtype cells, as well as in NINJ1-silenced cells. Glycine treatment did not yield additional protection from pyroptotic cell death in NINJ1-silenced cells. Data are expressed as % of LDH in supernatant from siCtrl-treated cells stimulated to undergo pyroptosis from n=4 independent donors. Data points from each donor are shown along with their mean and SD. **** p<0.0001 by two-way ANOVA with Tukey’s multiple comparison correction. (B) Nigericin- and glycine-treated LPS-primed wildtype hMDMs show similar ballooning morphology as nigericin-treated LPS-primed NINJ1-silenced hMDMs. Membrane ballooning is shown in live cells labeled with the plasma membrane dye FM1-43. The corresponding brightfield image is shown in the inset. Scale bar 20 μm. Figure 2—source data 1 Numerical values for experimental data plotted in Figure 2. https://cdn.elifesciences.org/articles/78609/elife-78609-fig2-data1-v3.xlsx Download elife-78609-fig2-data1-v3.xlsx Together, these data using macrophage models from mouse and human demonstrate that genetic deletion or silencing of NINJ1 functionally and morphologically parallels glycine-mediated cytoprotection. Glycine targets NINJ1 clustering to prevent membrane rupture We therefore next posited that glycine targets NINJ1 as part of its cytoprotective effect. The working model of NINJ1-mediated membrane rupture involves its clustering within the plasma membrane (Kayagaki et al., 2021). Point mutants (e.g. K45Q and A59P) that prevent membrane rupture also fail to cluster upon induction of pyroptosis (Kayagaki et al., 2021), further reinforcing a connection between the clustering process and the capacity of NINJ1 to rupture membranes. To establish whether glycine directly interferes with NINJ1-mediated cell death, we next evaluated the effect of glycine treatment on NINJ1 clustering during cell lysis in pyroptosis, post-apoptosis lysis, and necrosis. Using a biochemical native-PAGE approach that maintains native protein interactions, endogenous NINJ1 of otherwise untreated primary mouse bone marrow-derived macrophages (BMDM) migrates at approximately 40 kDa, potentially indicative of NINJ1 dimers or trimers in unstimulated cells (Figure 3A) and consistent with its known ability to form homotypic interactions (Bae et al., 2017). By native-PAGE, a band of approximately 160–200 kDa is also observed in the basal state, suggesting that higher-order multimers of NINJ1 consistent with decamers or higher-order multimers may also be present in the plasma membrane of unstimulated macrophages (Figure 3A). In response to pyroptosis (nigericin in LPS-primed BMDM), necrosis (pneumolysin), and apoptosis (venetoclax) induction in primary BMDM, the endogenous NINJ1 signal shifts to a high molecular weight aggregate, suggestive of a clustering process. Importantly, this shift is completely abrogated by glycine treatment (Figure 3A). Using this same native-PAGE approach, glycine treatment of primary hMDMs (Figure 4A) and mouse RAW-ASC cells (Figure 1—figure supplement 1D) undergoing pyroptosis prevented the shift of NINJ1 to a high molecular weight band. Finally, glycine inhibited NINJ1 oligomerization and pyroptotic lysis (as measured by LDH release) in LPS-primed and nigericin-treated human macrophages derived from induced pluripotent stem cells (iPSCs) derived macrophages (iPSDMs) without impairing secretion of IL-1β, confirming previous findings that glycine acts downstream of GSDMD pore formation (Figure 4—figure supplement 2B–D). Figure 3 with 1 supplement see all Download asset Open asset Glycine targets NINJ1 oligomerization to prevent membrane rupture in primary mouse macrophages. (A) Primary bone marrow-derived macrophage (BMDM) was induced to undergo pyroptosis (nigericin), apoptosis (venetoclax, ABT-199), or necrosis (pneumolysin) with or without glycine treatment. Native-PAGE analysis of endogenous NINJ1 demonstrates a shift to high molecular weight aggregate upon cell death stimulation, which is abrogated by glycine treatment. (B) Total internal reflection microscopy of NINJ1 in LPS-primed primary BMDM reveals that endogenous NINJ1 resides in discrete puncta within the plasma membrane. Cell membrane outline (dotted white line) was determined using fluorescently labeled cholera toxin subunit B (not shown) as a plasma membrane marker. Scale bar 20 µm. Inset shows magnified area demarcated by the white box. Anti-mouse NINJ1 antibody validation for immunofluorescence is provided in Figure 3—figure supplement 1. Quantification of the density (C) and mean fluorescence intensity (D) of NINJ1 puncta in LPS-primed primary macrophages at baseline or stimulated to undergo pyroptosis (nigericin 20 µM for 30 min) without or with glycine (5 mM). NINJ1 puncta become less dense and brighter upon pyroptosis induction, consistent with NINJ1 plasma membrane clustering. Glycine limits this redistribution. Violin plot of NINJ1 puncta density from three pooled independent experiments. Data points superimposed on the violin plots are the mean NINJ1 puncta densities for the three independent experiments (10–23 cells measured per replicate with >675 NINJ1 puncta identified per replicate). Bars represent mean ± SEM of the NINJ1 densities for the n=3 independent experiments. * p<0.05, ** p<0.01, and **** p<0.0001 by Kruskal–Wallis test with Dunn’s multiple comparison correction. (E) Representative confocal (top) and stimulated emission depletion (STED; bottom) images of LPS-primed primary mouse bone marrow-derived macrophage (BMDM) at baseline or stimulated to undergo pyroptosis (nigericin 20 µM for 30 min) without or with glycine (5 mM). Full-width at half maximum of identified NINJ1 puncta shows a resolution of 271±98 nm and 63±8 nm (mean ± SD) for standard confocal and STED microscopy, respectively. Scale bar 0.5 µm; inset scale bar 20 µm. (F–G) Quantification of the normalized NINJ1 puncta density (F) and mean fluorescence intensity (G) from the STED images. The NINJ1 puncta become less dense with a corresponding increase in fluorescence intensity upon pyroptosis induction, consistent with NINJ1 plasma membrane clustering. Glycine limits these effects. Violin plot of the individual NINJ1 puncta from three pooled independent experiments (8–14 cells per replicate with >4964 NINJ1 puncta identified per replicate). Bars represent mean ± SEM of the NINJ1 densities for the n=3 independent experiments. * p<0.05 and **** p<0.0001 by Kruskal–Wallis test with Dunn’s multiple comparison correction. Figure 3—source data 1 Numerical values for experimental data plotted in Figure 3. https://cdn.elifesciences.org/articles/78609/elife-78609-fig3-data1-v3.xlsx Download elife-78609-fig3-data1-v3.xlsx Figure 4 with 2 supplements see all Download asset Open asset Glycine targets NINJ1 oligomerization to prevent membrane rupture in primary human macrophages. (A) Native-PAGE analysis of endogenous NINJ1 from human monocyte-derived macrophages (hMDMs) stimulated to undergo pyroptosis displays a shift to higher molecular weight, which is abrogated by glycine treatment. NINJ1 levels are almost absent in siNINJ1-treated cells (quantification in Figure 4—figure supplement 2A), and no shift is seen after LPS and nigericin treatment. (B) Total internal reflection fluorescence (TIRF) microscopy of LPS-primed primary hMDMs shows endogenous NINJ1 in discrete plasma membrane puncta. Cell membrane outline (dotted white line) was determined using fluorescently labeled cholera toxin subunit B (not shown) as a plasma membrane marker. Scale bar 20 μm. Anti-human NINJ1 antibody validation for immunofluorescence is provided in Figure 4—figure supplement 1. Quantification of the NINJ1 puncta density (C) and fluorescence intensity (D) in LPS-primed hMDMs at baseline or stimulated to undergo pyroptosis (nigericin 20.7 μM for 2 hr) without or with glycine (50 mM). Violin plot of NINJ1 puncta density and intensity quantified from images of cells from four independent donors. Mean of NINJ1 puncta densities from three cells per donor per condition shown as superimposed datapoints (different symbols are used for individual donors) along with median and quartiles for each condition. Figure 4—source data 1 Numerical values for experimental data plotted in Figure 4. https://cdn.elifesciences.org/articles/78609/elife-78609-fig4-data1-v3.xlsx Download elife-78609-fig4-data1-v3.xlsx To corroborate these biochemical findings, we proceeded to evaluate native NINJ1 oligomerization within the plasma membrane by total internal reflection fluorescence (TIRF) microscopy in primary BMDM and hMDMs induced to undergo pyroptosis. TIRF microscopy allows for the preferential excitation of a thin (~150 nm) layer that includes the ventral plasma membrane of adherent cells, thereby eliminating background fluorescence from structures outside this focal plane (e.g. endomembrane compartments). In LPS-primed BMDMs and hMDMs, NINJ1 appears in discrete, small puncta that are diffusely distributed across the plasma membrane in both cell types (Figure 3B, Figure 4B). Upon induction of pyroptosis, the density of these puncta decreases, and their intensity increases (Figure 3C–D, Figure 4C–D), consistent with the clustering or aggregation of the low-order multimers into high-order oligomers as seen on the native PAGE (Figure 3A, Figure 4A). Co-treatment with glycine abrogates the formation of these clusters (Figure 3B–D, Figure 4B–D). While the use of TIRF microscopy allowed us to localize our evaluation of NINJ1 clustering to the plasma membrane, it inadequately resolved the spatial clustering due to its diffraction limitation. To partially overcome this limitation, we next probed NINJ1 membrane clustering using stimulated emission depletion (STED), a super-resolution fluorescence microscopy approach, in primary BMDM. With STED, we improved upon the resolution from a full-width half-maximum by confocal microscopy of 271±98 nm to 63±8 nm (Figure 3E). In the basal state, NINJ1 was observed in small discrete puncta, which decreased in density (Figure 3F) and increased in intensity (Figure 3G) upon pyroptosis induction with nigericin. Co-treatment with glycine (5 mM) limited clustering (Figure 3F–G). Together, our biochemical and microscopy-based data are consistent with the notion that during pyroptosis, low-order NINJ1 oligomers cluster within the plasma membrane, a phenomenon suppressed by glycine. Glycine targets NINJ1 clustering to confer cytoprotection independently of upstream programmed lytic cell death signaling Thus far, our data do not rule out the possibility that glycine is able to target proteins upstream of NINJ1 in pyroptosis. To test whether this is indeed the case, we evaluated caspase-1 activation, GSDMD processing, and IL-1β processing and secretion in iBMDM induced with nigericin with and without 5 mM glycine. Co-treatment with glycine had no effect on these upstream processes (Figure 5A–B), supporting the hypothesis that the target of glycine is downstream and independent of GSDMD activation and oligomerization. To confirm that glycine interferes with NINJ1-mediated cellular rupture independent of upstream signaling pathways, we generated a doxycycline-inducible NINJ1 overexpression system in mouse macrophages (Figure 5—figure supplement 1) based on the rationale that sufficient overexpression of NINJ1 can drive spontaneous lysis as seen in 293T systems (Kayagaki et al., 2021). Using this cell-based system, we demonstrate that glycine dose-dependently suppresses NINJ1-mediated cell rupture independent of activation of upstream programmed cell death pathway activation (Figure 5C). Expression of N-terminal GSDMD using a similar inducible system was able to trigger a glycine-inhibitable cellular rupture (Figure 5D). To further address glycine’s mode of action, we evaluated glycine’s effect on NINJ1 clustering biochemically and found that glycine suppresses NINJ1 clustering when NINJ1 is overexpressed (Figure 5E). Figure 5 with 2 supplements see all Download asset Open asset Glycine inhibits NINJ1 clustering but not upstream pyroptosis signaling to confer cytoprotection. (A–B) Wildtype immortalized bone marrow-derived macrophage (iBMDM) was primed with 1 μg/mL LPS for 4 hr or left unprimed before stimulation of primed cells with 20 μM nigericin for 2 hr in the presence or absence of 5 mM glycine. (A) Processing of GSDMD, caspase-1, and IL-1β was assessed by western blot. (B) Release of IL-1β into the cell culture supernatant was quantified by ELISA. ELISA results show mean ± SEM of three independent experiments. Western blots are representative of three independent experiments. ***p<0.001 and ****p<0.0001 by one-way ANOVA with Tukey’s multiple comparison correction. (C–D) Glycine dose-dependently inhibits plasma membrane rupture in mouse macrophages overexpressing NINJ1 or the N-terminal fragment of GSDMD. iBMDMs with doxycycline-inducible expression of FLAG-tagged NINJ1 (C) or the N-terminal fragment of GSDMD fused to BFP(blue fluorescent protein) (D) were incubated with 2 µg/mL doxycycline and increasing concentrations of glycine for 12 hr or 8 hr, respectively, before analyses of cell rupture (LDH release) and protein expression. Left: LDH release in supernatants relative to full lysis controls. Middle: frequencies of NINJ1-FLAG-positive or GSDMD-NT-BFP-positive cells. Right: surface expression of NINJ1-FLAG or GSDMD-NT-BFP by mean fluorescence intensity. * p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 by two-sided ANOVA with Tukey’s multiple comparison correction. (E) HeLa cells were transiently transfected with HA-tagged mouse NINJ1 in the presence or absence of glycine (5 mM). Native-PAGE analysis of ectopically expressed NINJ1 demonstrates a shift to high molecular weight aggregate, which is abrogated by glycine treatment. (F) Large unilamellar vesicles (LUVs; gray circles) were made containing 25 mM carboxyfluorescein (CF) at which the CF fluorescence self-quenches. NINJ1 α-helix peptide (corresponding to amino acids 40–69 of human NINJ1) is added to the LUV suspension. Ruptured LUV release CF, which no longer self-quenches. The resulting increase in fluorescence is monitored using a spectrofluorometer. Detergent is added to rupture all remaining liposomes to capture the maximum attainable fluorescence. LUV rupture by N-terminus NINJ1 α-helix without and with glycine (50 mM), scrambled NINJ1 α-helix peptide, or the cytolytic yeast peptide candidalysin compared to vehicle. Glycine does not prevent LUV rupture by the N-terminal NINJ1 α-helix. * p<0.05 by ANOVA with Tukey’s multiple comparison correction. Figure 5—source data 1 Numerical values for experimental data plotted in Figure 5. https://cdn.elifesciences.org/articles/78609/elife-78609-fig5-data1-v3.xlsx Download elife-78609-fig5-data1-v3.xlsx We next turned our attention to the purported amphipathic α-helix in the NINJ1 N-terminus (residues 40–69 of human NINJ1), which others have shown to be necessary for NINJ1-mediated plasma membrane rupture (Kayagaki et al., 2021). Circular dichroism (CD) of the α-helix at varying glycine concentrations between 0 and 20 mM did not affect the CD spectrum, indicating that at the trialed concentrations, glycine did not alter the stability of the helical secondary structure of this region of the NINJ1 N-terminus (Figure 5—figure supplement 2). We then functionally evaluated whether glycine interferes with the membrane lytic function of the helix using a reconstituted liposomal rupture assay. In this system, we demonstrate that while the α-helix was able to rupture liposomes, glycine did not interfere with this effect (Figure 5F). These data suggest that glycine can block full-length NINJ1 clustering and membrane rupture in cellulo but that additional unknown players may be affected within cells, or the mode of action is separate from the minimally sufficient lytic α-helix of the NINJ1 N-terminus as shown by intact liposome rupture. Discussion The protective effect of glycine treatment is a pervasive feature of many cell death pathways, yet the mechanism of action was previously unknown. Here, we position the plasma membrane clustering of the pan-death protein NINJ1 as an elusive glycine target that mediates cytoprotection. In our studies, we focused on fundamental cell death pathways in both mouse and human macrophages and show that glycine prevents NINJ1 clustering, a process required for plasma mem