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

  1. Jazlyn P Borges
  2. Ragnhild SR Sætra
  3. Allen Volchuk
  4. Marit Bugge
  5. Pascal Devant
  6. Bjørnar Sporsheim
  7. Bridget R Kilburn
  8. Charles L Evavold
  9. Jonathan C Kagan
  10. Neil M Goldenberg
  11. Trude Helen Flo
  12. Benjamin Ethan Steinberg  Is a corresponding author
  1. Program in Neuroscience and Mental Health, Hospital for Sick Children, Canada
  2. Centre of Molecular Inflammation Research, Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology, Norway
  3. Program in Cell Biology, Hospital for Sick Children, Canada
  4. Division of Gastroenterology, Boston Children's Hospital and Harvard Medical School, United States
  5. Ragon Institute of MGH, MIT and Harvard, United States
  6. Department of Anesthesia and Pain Medicine, Hospital for Sick Children, Canada
  7. Department of Anesthesiology and Pain Medicine, University of Toronto, Canada

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

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
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.

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
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.

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
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 4 with 2 supplements see all
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.

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
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.

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 membrane rupture. Necroptosis represents another programmed cell death pathway, important to inflammatory responses, pathogen detection, and tissue repair (Bertheloot et al., 2021). Unlike pyroptosis, necrosis, and post-apoptosis lysis, NINJ1 does not appear to be needed for necroptosis-induced plasma membrane rupture (Kayagaki et al., 2021), potentially due to the membrane-disrupting capacity of the necroptosis MLKL pore (Flores-Romero et al., 2020). This is particularly notable as glycine does not prevent TNF-induced necroptosis (Figure 2—figure supplement 2 and Chen et al., 2014), further buttressing the association between NINJ1 and glycine.

Our data demonstrate that glycine treatment and genetic ablation or silencing of NINJ1 yield similar functional and morphological outcomes in both human and mouse macrophages subjected to lytic cell death programs. Mechanistically, our data suggest that glycine cytoprotection results from interference with NINJ1 clustering within the plasma membrane. This finding is further supported by the observations that (1) glycine treatment of NINJ1 knockout cells confers no additional protection against cellular rupture; (2) glycine cytoprotection is independent of upstream programmed lytic cell death signaling; and (3) amino acids structurally similar to glycine inhibit NINJ1 clustering and membrane rupture with the same potency hierarchy as previously reported for glycine cytoprotection in pyroptosis (Figure 1—figure supplement 1B–C and Loomis et al., 2019).

The mechanism by which glycine interferes with NINJ1 clustering can be either direct or indirect. Our in vitro CD studies and liposomal rupture system, respectively, indicate that glycine does not interfere with the secondary structure or lytic function of the NINJ1 α-helix. It remains plausible that glycine still interacts directly with this domain or other components of the NINJ1 N-terminus in cells to interfere with NINJ1 clustering, consistent with our presented data. Alternatively, glycine may act on an unidentified intermediate that modulates NINJ1 clustering within the plasma membrane. Our data in pyroptotic cells suggest that such an intermediate would reside between active GSDMD and NINJ1. In an indirect model, any of a plasma membrane-associated protein, membrane lipid, or cellular metabolic pathway could be the target. In addition, it remains unclear whether glycine’s cytoprotective activity occurs on the intracellular or extracellular side of the plasma membrane. To the best of our knowledge, there is no direct evidence to suggest whether glycine’s cytoprotective effect is specifically mediated from the intracellular or extracellular side. Both extracellular (Weinberg et al., 2016) and intracellular (Rühl and Broz, 2022) sites of action have been proposed. Our in vitro data suggest that glycine is not acting directly on the N-terminal α-helix, which is thought to be extracellular; however, we do not otherwise delineate the topology of glycine’s mechanism of action.

Defining the site of action would provide insight into the molecular mechanism by which glycine inhibits NINJ1 clustering in the plasma membrane and ultimately help determine whether glycine directly or indirectly engages NINJ1. Indeed, how NINJ1 is activated by lytic cell death pathways, its clustering trigger, and method of plasma membrane disruption also remain important and outstanding questions in the field. Due to the ubiquity of lytic cell death pathways in human health and disease and the potency of glycine cytoprotection, answers to these questions will greatly advance the development of therapeutics against pathologic conditions associated with aberrant lytic cell death pathways.

Materials and methods

Cells

Primary BMDMs were harvested from the femurs and tibia of wildtype mixed-sex cohorts of C57Bl/6 mice. The ends of cleaned bones were cut and centrifuged to collect bone marrow into sterile PBS. The cell suspension was washed with PBS and plated in DMEM with 10 ng/mL M-CSF (315-02; Peprotech Inc, Cranbury, NJ, USA). Following 5 days of culture, the BMDM were detached from the dishes with TBS with 5 mM EDTA, resuspended in fresh DMEM, and plated. ASC-expressing RAW264.7 mouse macrophages (RAW-ASC; raw-asc, InvivoGen, San Diego, CA, USA) are engineered to stably express murine ASC, which is absent in the parental RAW 264.7 macrophage cell line (Pelegrin et al., 2008) and therefore are able to undergo pyroptosis. iBMDM expressing Cas9 was obtained from Dr Jonathan Kagan (Boston Children’s Hospital, Boston) and described previously (Evavold et al., 2018). Cell lines were authenticated and negative for mycoplasma contamination.

Isolation and differentiation of human primary macrophages

Buffy coats and pooled serum from healthy donors were provided by the blood bank at St. Olavs Hospital (Trondheim, Norway), after informed consent and with approval by the Regional Committee for Medical and Health Research Ethics (No. 2009/2245). Peripheral blood mononuclear cells were isolated by density gradient using Lymphoprep (Axis-Shield Diagnostics, StemCell Technologies, Cologne, Germany) according to the manufacturer’s instructions. Monocytes were selected by α-CD14 bead isolation (Miltenyi Biotech, Lund, Sweden) and seeded in RPMI 1640 supplemented with 10% serum and 10 recombinant macrophage colony-stimulating factor (M-CSF; R&D Systems, Minneapolis, MN, USA) for 3 days. At day 3, the medium was replaced with RPMI 1640 without M-CSF, and siRNA treatment was initiated.

iPSDMs culture and production

iPSCs were obtained from European Bank for induced pluripotent Stem Cells (EBiSC, https://ebisc.org/about/bank), distributed by the European Cell Culture Collection of Public Health England (Department of Health, United Kingdom). iPSCs were maintained in Vitronectin XF (StemCell Technologies) coated plates with mTeSR Plus medium (StemCell Technologies). Cells were passaged with ReLeSR (StemCell Technologies) and plated in media containing 10 μM Rho-kinase inhibitor Y-27632 (StemCell Technologies). Embryonic body (EB) formation and myeloid differentiation are based on and adapted from a previously reported protocol (van Wilgenburg et al., 2013). Briefly, iPSCs were washed with PBS before Versene (Gibco-Thermo Fisher, Waltham, MA, USA) was added and cells incubated at 37°C for 5 min to generate a single cell suspension of iPSCs. Cells were counted, washed with PBS, and resuspended to a final concentration of 10,000 cells/50 μL in EB media: mTeSR Plus medium (StemCell Technologies), 50 ng/mL BMP-4 (R&D Systems, 315 BP-010), 20 ng/mL SCF (R&D Systems, 255-SC-050), and 50 ng/mL VEGF (R&D Systems, 293-VE-050). Cell suspension, supplemented with 10 μM Y-27632, was added into a 384-well Spheroid Microplate (Corning. #3830), 50 µL/well. The microplate was centrifuged at 1000 RPM for 3 min, and incubated at 37°C, 5% CO2 for 4 days. EBs were fed at day 2 by adding 50 μL of fresh EB medium. After 4 days, EBs were harvested and seeded into a gelatin coated T175 flask in X-VIVO-15 (Lonza) media, supplemented with 100 ng/mL M-CSF (Preprotech, 300–25), 25 ng/mL IL-3 (Preprotech, 200–03), 2 mM glutamax (Gibco-Thermo Fisher), and 0.055 mM β-mercaptoethanol (Gibco-Thermo Fisher). Media was changed every week, and EB-culture was renewed after 4 months. Monocytes produced were routinely checked with regards to phenotype using a defined flow cytometry panel of expected surface markers. Monocytes were harvested once a week and differentiated into macrophages in RPMI 1640 with 10% fetal calf serum and 100 ng/mL M-CSF for 5–7 days.

NINJ1 knock-out cell line generation and siRNA silencing of NINJ1

RAW-ASC and iBMDM cells were transfected with custom CRISPR gRNA plasmid DNA (U6-gRNA:CMV-Cas-9–2 A-tGFP; Sigma-Aldrich, Oakville, Canada) using FuGENE HD transfection reagent (Promega, Madison, WI, USA). The Ninj1 target region sequence was GCCAACAAGAAGAGCGCTG. 24 hr later, the cells were FACS sorted for GFP into 96 well plates. Individual colonies were expanded and tested for NINJ1 expression by western blot. NINJ1 knockdown in hMDMs was performed by siRNA using Lipofectamine RNAiMAX (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Cells were transfected with 20 nM pooled siRNA against NINJ1 (Thermo Scientific, HSS107188, HSS107190, HSS181529) or non-silencing control siRNA (Qiagen, Hilden, Germany, 1027310). hMDMs were siRNA-treated two times (day 3 and day 5) before medium was changed to RPMI with 1% serum (day 6), and cells were allowed to rest overnight prior to stimulation. Knockdown of NINJ1 was confirmed by western blotting.

Generation of N-terminal HA-NINJ1 construct and cellular expression

PCR reactions were performed using ConeAmp HiFi master mix (Takara). Primers for mouse Ninj1 (insert) PCR reaction were: forward primer 5’gat tac gct gag tcg ggc act gag ga3’ and reverse primer 5’ctc ggt acc cta ctg ccg ggg cgc ca3’. Template DNA for the PCR reaction was mouse Ninj1 cDNA clone from Origene (Cat. No. MR225037). The template vector pcDNA3.1-HA was obtained from Addgene. Primers for pcDNA3.1-HA PCR (vector) reaction were: forward primer 5’cgg cag tag ggt acc gag ctc gga3’ and reverse primer 5’gcc cga ctc agc gta atc tgg aac atc3’. PCR reaction products were cleaned up using the NucleoSpin PCR Clean-up kit (Macherey-Nagel). The PCR products contained 18 bp overlapping regions between the pcDNA3.1-HA tag vector and the mouse NINJ1 insert. This allows for fusion of the insert and vector using the In-Fusion Snap Assembly EcoDry Cloning Kit (Takara, 638954) and transformation into Stellar competent bacterial cells (Takara). DNA sequencing with CMV-F primer confirmed Ninj1 insertion into the pcDNA3.1-HA vector and that NINJ1 was in-frame with the N-terminal HA-tag. HeLa cells (ATCC #CCL-2) were transfected with pcDNA 3.1HA-NINJ1 using Fugene HD transfection reagent (Promega) according to the manufacturer’s protocol.

Inducible iBMDM-doxy-moNINJ1 system

iBMDMs with doxycycline-inducible expression of the N-terminal fragment of GSDMD fused to BFP are published (Evavold et al., 2021). We used the same stock of iBMDMs from Cas9-expressing mice in which the TET3G transactivator protein had introduced previously (Evavold et al., 2021) to generate cell lines with dox-inducible expression of NINJ1 carrying an N-terminal FLAG-tag. cDNA encoding full-length murine NINJ1 was ordered as a gBlock from Integrated DNA Techologies. This sequence was amplified by PCR to append an N-terminal FLAG-tag and cloned into pRETROX-TRE3G (TakaraBio) using BamHI and NotI restriction sites and into pMSCV IRES EGFP using NotI and SalI restriction sites. To introduce dox-inducible FLAG-NINJ1, 2.5×106 Platinum-GP retrovirus packaging cells were seeded in a TC-treated 10 cm dish. On the next day, cells were transfected with 3 μg pCMV-VSV-G and 9 μg pRETROX-TRE3G encoding FLAG-NINJ1 using Lipofectamine 2000. After 18 hr, media was exchanged to 6 mL of fresh complete DMEM. The same day, 0.5×106 Cas9- and TET3G-expressing iBMDMs were seeded per well of 6-well plate. The next day, retroviral supernatant was harvested and filtered through 0.45 μm syringe filter, supplemented with polybrene (1:2000; EMD Millipore), and spun onto cells (1250 g, 30°C, 1 hr). 6 mL of fresh cDMEM were added to retrovirus packaging cells, and spinfection was repeated the next day. 3 days after the second transduction, transduced cells were selected by growth in complete DMEM including 1.5 mg/mL G-418 and 10 μg/mL puromycin. To ensure maximal transgene expression, which correlates with the ability of FLAG-NINJ1 to induce cell death and lysis, FLAG-NINJ1 was also stably overexpressed in these cells using retroviral transduction with an MSCV(murine stem cell virus) retrovirus. Retrovirus production and spinfections were performed as described above, only using pMSCV IRES EGFP-FLAG-NINJ1 instead of pRETROX-TRE3G construct. Cells were then sorted on a BD Melody cell sorter for maximal EGFP expression (top 5–10%). Clonal cell lines were derived by limited serial dilution.

1×105 cells per well in a black 96-well plate in DMEM (Gibco)/10% FBS(fetal bovine serum) (R&D, S11550) were induced with 2 µg/mL doxycycline (Sigma-Aldrich) in the presence or absence of increasing concentrations of glycine (Sigma-Aldrich 67126) in a total volume of 200 μL for 8 hr (iBMDM GSDMD-NT) or 12 hr (iBMDM FLAG-NINJ1) before supernatants were harvested for LDH-assay (Invitrogen) and cells detached (PBS/4 mM EDTA) for flow cytometry (Becton Dickinson Fortessa). iBMDM GSDMD-NT express BFP and were analyzed directly; iBMDM NINJ1 were stained with anti-FLAG APC antibody (1:100, Abcam Ab 72569; RRID: AB_1310127) in PBS/2% FBS, 2.5 mM EDTA on ice and washed once before analysis.

Pyroptosis, apoptosis, necrosis, and necroptosis induction

Pyroptosis was activated in BMDM primed with (0.5 µg/mL) LPS from Escherichia coli serotype 055:B5, which was first reconstituted at a stock concentration of 1 mg/mL. In primary BMDM, after 4.5 hr of LPS priming, pyroptosis was induced with 20 µM nigericin (Sigma N7143; stock 10 mM in ethanol) for 30 min. iBMDM were primed with LPS for 3 hr, followed by pyroptosis induction with 10 µM nigericin for 2 hr. Apoptosis was induced in non-primed macrophages using the Bcl-2 inhibitor venetoclax (ABT-199; Tocris, Bristol, United Kingdom; 6960) at 25 µM for 16 hr. Necrosis was induced by treating non-primed macrophages with pneumolysin (0.5 μg/mL) for 15 min in iBMDM or 45 min in primary BMDM. Pneumolysin was obtained from Dr John Brumell (Hospital for Sick Children, Toronto). In hMDMs, pyroptosis was induced by priming with 1 μg/mL LPS from E. coli serotype 0111:B4 for 2 hr, followed by stimulation with 20.7 μM nigericin (Invivogen tlrl-nig; stock 5 mg/mL in ethanol) for 2 hr and 15 min. Necroptosis was induced by treating cells with pan-caspase inhibitor zVAD (25 uM, Invivogen, tlrl-vad) and SMAC mimetic BV6 (10 uM, Invivogen, inh-bv6) for 30 min, before stimulation with recombinant human TNF-α (10 ng/mL, Peprotech, 300–01 A) for 4 hr. iPSDMs were stimulated with 200 ng/mL LPS for 3 hr and 20.7 μM nigericin for 1–1.5 hr.

Where indicated, mouse cells were treated with 5 mM glycine (Sigma, G7126) at the time of cell death induction. Human cells were pretreated with glycine (Millipore, Burlington, MA, USA; 104201) at a concentration of 50 mM for at least 10 min before stimuli was added.

LDH assay

BMDM were seeded at 200,000 cells per well in 12-well plates, treated as indicated, and cytotoxicity assayed by LDH release assay the following day. At the end of the incubations, cell culture supernatants were collected, cleared of debris by centrifugation for 5 min at 500× g. The cells were washed once with PBS then lysed in lysis buffer provided in the LDH assay kit (Invitrogen C20300). Supernatants and lysates were assayed for LDH using an LDH colorimetric assay kit as per the manufacturer’s instructions (Invitrogen C20300). hMDMs were seeded at 500,000 cells per well in 12-well plates and differentiated as described above. iPSDMs were seeded at 180,000–450,000 cells per well in 12-well plates and differentiated as described above. After cells were stimulated as described in figure legends, supernatants were harvested, and LDH release was assayed (Invitrogen C20301). Briefly, equal amounts of supernatant and reaction mixture were mixed and incubated for 30 min, followed by absorbance reads at 490 and 680 nm. To calculate the LDH release, the 680 nm background was subtracted from the 490 nm absorbance reads before further processing.

Western blot cleavage GSDMD, caspase-1, and IL-1β

iBMDMs were seeded at 106 cells per well in 12-well plates overnight, primed with 1 μg/mL E. coli LPS (Enzo; Serotype O111:B4) for 4 hr, or left unprimed before stimulation with 20 μM of nigericin (Invivogen) in 500 μL Opti-MEM (Gibco) with no serum. After 2 hr, 50 μL of supernatant was collected for analysis by ELISA. 125 μL of concentrated 5× SDS loading buffer with reducing agent TCEP(tris(2-carboxyethyl)phosphine) was added to each well to stop the reaction. This treatment ensured the capture of proteins in both the supernatant and the cell lysates for western blot analysis. Proteins were separated by SDS-PAGE and detected by western blot using primary antibodies against GSDMD (Abcam ab209845; RRID: #AB_2783550), Caspase-1 (Adipogen AG-20B-0042-C100; RRID: #AB_2755041), pro-IL-1β (Genetex GTX74034; RRID: #AB_378141), and actin (Sigma A5441; RRID: #AB_476744).

Mitochondrial membrane potential and cellular ATP measurements

hMDMs were differentiated at 30,000 cells per well in glass-bottom 96-well plates (Cellvis, P96-1.5H-N). Parallel plates were prepared for live-cell imaging and LDH + CellTiter Glo analysis. hMDMs were either left untreated or pre-treated with glycine (50 mM) and primed with 1 μg/mL LPS (E. coli serotype 0111:B4) in RPMI complemented with 1% fetal calf serum for 3 hr before stimulation with 20.7 μM of nigericin (Invivogen). Tetramethylrhodamine ethyl ester perchlorate (TMRE, 200 or 10 nM, Sigma-Aldrich) was added to cells before the plates were placed in a heated incubator (37°C, 5% CO2), and images were taken every 30 min over 18 or 22 hr (two independent experiments). In the second experiment, at the end of LPS priming, cells were labeled with CellTracker Deep Red (0.5 μM Invitrogen) for 15 min and washed before addition of fresh medium with TMRE (10 nM) with or without LPS, nigericin, and glycine. In parallel plates treated identically, supernatants were harvested and assayed for LDH, and cellular ATP was measured using CellTiterGlo (Promega) at 0, 2, 6, and 18/22 hr.

Fluorescence microscopy

Mouse macrophages were cultured on 18 mm glass coverslips in 12-well plates at 150,000 cells per well. Cells were treated as indicated in the figure legends, washed with PBS, fixed in 4% paraformaldehyde (PFA) in PBS at room temperature for 15 min, and permeabilized with 0.1% Tween-20. Cells were then blocked in PBS supplemented with 10% donkey serum and 0.1% Tween-20 for 1 hr at room temperature prior to overnight incubation with primary antibody at 4°C. Rabbit monoclonal anti-mouse NINJ1 was used at 10 μg/mL. Next, cells were washed three times with PBS supplemented with 10% donkey serum prior to the addition of secondary antibody at room temperature for 1 hr. In validation studies of the rabbit anti-mouse NINJ1 monoclonal antibody, cells were stained with and without the primary antibody prior to application of the secondary antibody. Nuclei were labeled using DAPI containing mounting medium (ProLong Diamond Antifade Mountant, Invitrogen P36961). For total internal reflection microscopy, cells were co-stained with recombinant Cholera toxin subunit B conjugated to Fluor488 (Invitrogen C34775) at 1 μg/mL for less than 1 min prior to fixing and staining for NINJ1 as described above. The Cholera toxin subunit B served as a membrane marker to optimize plasma membrane visualization. TIRF imaging of mouse macrophages was conducted on a Zeiss Axio Observer Z1 microscope with a 100× objective and Andor iXon3 885 detector. For STED microscopy, primary mouse macrophages were cultured on 1.5 H 18 mm glass coverslips (Marienfeld) in 12-well plates at 150,000 cells per well. After treatments as indicated in the figure legend, cells were washed with PBS, then fixed in 4% PFA in PBS for 10–15 min at room temperature. Following fixation, cells were washed, then permeabilized in 0.1% Tween-20. Cells were blocked in in PBS supplemented with 10% donkey serum and 0.1% Tween-20 for 1 hr at room temperature, followed by primary antibody incubation in blocking solution overnight at 4°C. Rabbit monoclonal anti-mouse NINJ1 was used at 10 µg/mL. The next day, cells were incubated with Cy3-conjugated secondary antibody (diluted 1:100) in 1% serum in PBST for 1 hr at room temperature. After final washes, coverslips were mounted onto glass slides using ProLong Diamond Antifade Mountant (Invitrogen, P36965). STED microscopy was performed using a Leica TCS SP8 STED 3× microscope using HyD detectors and a 100×/1.4 oil objective. Samples labeled with Cy3-conjugated secondary antibody were excited using a white light laser at 554 nm. Emissions were time-gated 1.30–6.00 ns. A 1.5 W depletion laser at 660 nm was used at 70% maximal power. Images were acquired and deconvolved using Leica LAS X software with Lightning Deconvolution with an xy pixel size of 12.62 nm. For all fluorescence microscopy in mouse macrophages, rabbit monoclonal anti-mouse NINJ1 was obtained from Drs Nobuhiko Kayagaki and Vishva Dixit (Genentech Inc, San Francisco, CA, USA). Where indicated, images of live cell morphology were captured using the epifluorescence mode and with FM 4–64 (Invitrogen, T3166) added right before imaging.

Human primary macrophages were differentiated at 30,000 cells per well in glass-bottom 96-well plates (Cellvis, P96-1.5H-N). After stimuli, the cells were washed with PBS and thereafter stained with Cholera toxin subunit B Alexa Fluor 488 conjugate at 1 μg/mL in PBS for less than 1 min. Cells were then fixed in 4% PFA in PBS at room temperature for 15 min, followed by washing three times with PBS. We permeabilized the cells with PBS containing 0.01% saponin for 10 min at room temperature and thereafter blocked with PBS/0.01% saponin/20% pooled human serum for 3 hr. Cells were either left unstained or incubated with anti-human NINJ1 antibody (mouse IgG2b, R&D Systems, MAB5105, RRID: AB_11128852) or a control Ab (mouse IgG2a, Serotec, MCA929F; RRID: AB_322271) at 10 μg/mL in PBS/0,01% saponin/1% pooled human serum at 4°C for 41 hr and then washed three times with PBS/0.01% saponin/1% pooled human serum. Following this, we incubated the cells with far-red fluorescent rabbit anti-mouse antibody (Invitrogen, A27029, RRID: AB_2536092) at 2 μg/mL in PBS/0.01% saponin/1% pooled human serum at room temperature for 1 hr. Cells were washed three times with PBS/0.01% saponin/1% pooled human serum and thereafter three times with PBS before imaging. TIRF imaging of human macrophages was done on a Zeiss TIRF 3 microscope with a 63× objective and a Hamamatsu ORCA Fusion sCMOS detector. Images of morphology in live cells were captured using the epifluorescence mode and with FM 1–43 (Invitrogen, T3163) added at 5 μg/mL right before imaging.

For TMRE experiments, human macrophages were imaged using a Zeiss TIRF III (Carl Zeiss GmbH, Jena) equipped with an alpha Plan-Apochromat 100×/1.46 oil-immersion objective and a Hamamatsu ORCA-Fusion (C14440-20UP) CMOS camera. A Plan-Apochromat 10×/0.45 air objective was used for the time series experiments. TMRE was excited by a Colibri.2 470 nm LED, and the emission was detected between 510–542 nm. CellTracker Deep Red was excited by a 638 nm diode laser, and the emission was detected between 665 and 711 nm. Image processing was done in FIJI (Schindelin et al., 2012). Automatic counting of TMRE-positive cells was done in CellProfiler v.4.2.4 (Stirling et al., 2021) by using the IdentifyPrimaryObjects module with either Maximum Cross-Entropy adaptive thresholding or Otsu thresholding, and settings were optimized for each dataset to enhance accuracy.

Native and SDS-PAGE of NINJ1

Mouse macrophages were lysed with native-PAGE lysis buffer (150 mM NaCl, 1% Digitonin, 50 mM Tris pH7.5, and 1× Complete Protease Inhibitor). Following centrifugation at 20,800× g for 30 min, lysates were mixed with 4× NativePAGE sample buffer and Coomassie G-250 (ThermoFisher) and resolved using NativePAGE 3–12% gels. For SDS-PAGE, cells were washed with 1× PBS and lysed in RIPA lysis buffer containing protease inhibitors (Protease inhibitor tablet, Pierce A32955). Proteins were resolved using NuPAGE 4–12% Bis-Tris gels (Invitrogen), transferred to PVDF(polyvinylidene difluoride) membranes, and immunoblotted. For HA-NINJ1 overexpression experiments, rabbit monoclonal anti-HA (Cell Signaling Technology, Inc, Danvers, MA, USA; #3724; RRID: AB_1549585) was used. GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA; sc-25778; RRID: AB_10167668 1:1000) was used for a loading control.

hMDMs and iPSDMs were lysed with 1× NativePAGE Sample Buffer containing 1% Digitonin. After centrifugation at 21,000× g for 30 min, protein concentration of lysates was measured using Pierce BCA Protein Assay Kit (ThermoFisher) to ensure equal loading of proteins. For Native-PAGE on hMDMs, lysates were mixed with Novex Tris-Glycine Native Sample Buffer (Invitrogen) and resolved using Novex 4–12% Tris-Glycine gels (Invitrogen). For Native-PAGE on human iPSDMs, lysates were mixed with Coomassie G-250 (ThermoFisher) and resolved using NativePAGE 3–12% Bis-Tris gels (Invitrogen). For SDS-PAGE, lysates were mixed with DTT(dithiothreitol) and NuPAGE LDS sample buffer (Invitrogen), denatured for 5 min at 95 °C, and resolved using NuPAGE 4–12% Bis-Tris gels (Invitrogen). Proteins were transferred to PVDF membranes and immunoblotted. NINJ1 (R&D Systems, MAB5105, RRID: AB_11128852) was used to determine NINJ1 expression, while β-actin (Cell Signaling Technology, Danvers, MA, USA; Cat# 8457, RRID: AB_10950489) was used as loading control.

Cytokine measurements

Supernatants from human iPSDMs and mouse iBMDM were assessed for IL-1β (R&D Systems, DY201 or Invitrogen 88–7013, respectively) by ELISA according to the protocol of the manufacturer.

Large unilamellar vesicle rupture assay

To generate large unilamellar vesicles (LUVs), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, Avanti Polar Lipids) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (sodium salt) (DOPS, Avanti Polar Lipids) were first prepared at 80% DOPC and 20% DOPS, freeze dried, and hydrated with a solution containing 25 mM 6-carboxyfluorescein. The suspension was bath sonicated, freeze-thawed, and extruded (Avanti Mini Extruder) using a Nucleopore 0.1 μm membrane (Whatman) to yield LUVs, which were washed three times in PBS to remove excess 6-carboxyfluorescein. At this concentration, the fluorescence of 6-carboxyflurescein self-quenches. With liposomal rupture, the 6-carboxyfluorescein is released into the bathing solution, thereby releasing the self-quenching with fluorescence monitored in a spectrofluorometer. The liposomal rupture assay was setup by mixing equal amounts of the prepared LUVs with 0.4 mg/mL human NINJ1 α-helix region peptide (HYASKKSAAESMLDIALLMANASQLKAVVE; GenScript Biotech Corporation) with either vehicle or glycine (50 mM) in PBS buffer. Where indicated, a sequence-scrambled analog peptide (IAAAAMKMYLANSLEHAKSLKVVLASQDS; GenScript Biotech Corporation) was used as a negative control for the human NINJ1 α-helix region peptide. Following 45 min, all liposomes were then ruptured by adding Triton X-100 and the maximum fluorescence (cargo release) recorded. The results were converted to percentage cargo release and background control subtracted. The lytic yeast peptide candidalysin (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNK; Peptide Protein Research) was used as an additional positive control.

Circular dichroism

The secondary structure of the human NINJ1 α-helix region (amino acids 40–69; HYASKKSAAESMLDIALLMANASQLKAVVE; GenScript Biotech Corporation) was measured using a Jasco J-1500 spectropolarimeter. For this assay, peptide solutions from a 10 mg/mL stock in 5% DMSO in ddH2O were diluted to 0.2 mg/mL in buffer (20 mM sodium phosphate, pH 7.4, 10 mM NaCl, and 1% sodium dodecyl sulfate) with the final peptide concentration determined using a bicinchoninic acid (BCA) protein assay. NINJ1 helix peptide was titrated with glycine between 0 and 20 mM. 250 µL of the glycine titrated human NINJ1 helix peptide solutions were pipetted into 1 mm path length quartz CD cuvettes, capped, and placed in the Peltier temperature-controlled CD sample holder for analysis. A corresponding buffer blank spectrum was subtracted out from the sample datasets. In all conditions, spectra were recorded between 260 and 190 nm using 50 nm/min scanning speed, 0.5 nm data pitch, 1 s DIT, and 1 nm bandwidth. Between 3 and 20 accumulations were averaged. The CD studies were performed at the Structural and Biophysical Core Facility, The Hospital for Sick Children, Toronto, Canada.

Quantification and statistics

TIRF and STED microscopy images of mouse macrophages were analyzed in Imaris (Oxford Instruments, Abingdon, United Kingdom) software. The Spot tool was used to identify NINJ1 puncta with parameters (estimated puncta XY diameter, region type, and quality filter) set using images from the LPS-primed group and applied to all samples across all experiments. Cells were contoured using the surfaces tool to measure cell area. The density of NINJ1 puncta was calculated on a per-cell basis by dividing the total number of puncta within the contoured plasma membrane area. TIRF microscopy images of human primary macrophages were analyzed in FIJI, applying thresholds (triangle algorithm) and using the analyze particles tool to segment cells in the Cholera toxin subunit B channel. The find maxima command was used to identify NINJ1 puncta within these cell outlines, and the measurement tool was used to measure the area of the cell outlines. The density of NINJ1 puncta was calculated on a per-cell basis by dividing the number of puncta within a cell outline with its area.

Statistical testing was calculated using Prism 8.3.1 or 9.0 (GraphPad Software Inc, La Jolla, CA, USA). Experiments with more than two groups were tested using ANOVA with Tukey’s multiple comparison test. Unless otherwise indicated, presented data are representative of at least three independent experiments or donors and are provided as mean ± SEM. All collected data was analyzed. Unless otherwise specified, for non-quantitative data, such as western blots, experiments were replicated three times with representative images shown in the figures.

Animal and human studies

All animal studies were approved by the Hospital for Sick Children Animal Care Committee (AUP #47781). All human studies were conducted according to the principles expressed in the Helsinki Declaration and approved by the Regional Committee for Medical and Health Research Ethics (No. 2009/2245). Informed consent was obtained from all subjects prior to sample collection.

Appendix 1

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Mus musculus)Ninj1GenBankID: 18081
Gene (Homo sapiens)Ninj1GenBankID: 4814
Strain and strain background (Escherichia coli)Stellar Competent CellsTarakaBioStrain HST08
Genetic reagent (Mus musculus)Ninj1 knockoutKayagaki et al., 2021C57Bl/6 strain background; Mixed-sex cohorts
Cell line (Mus musculus)RAW-ascInvivoGenraw-asc
Cell line (Mus musculus)iBMDM TET3GEvavold et al., 2018
Cell line (Mus musculus)iBMDM inducible doxy-moNINJ1-FLAGThis paperJonathan Kagan laboratory; described in the Methods section
Cell line (Mus musculus)iBMDM inducible doxy-NT-GSDMD-BFPEvavold et al., 2021
Cell line (Homo sapiens)HeLaATCCCCL-2
Cell line (Homo sapiens)Platinum GPCell biolabsRV-103
Transfected construct (human)siRNA to NINJ1Thermo ScientificHSS107188, HSS107190, HSS18152920 nM
Transfected construct (mouse)HA-NINJ1This paperSteinberg laboratory; described in the Methods section
Biological sample (Mus musculus)Primary bone marrow-derived macrophages (wildtype BMDM)This paperFreshly prepared from C57Bl/6 J mice; male and female donors; Steinberg laboratory
Biological sample (Mus musculus)Primary bone marrow-derived macrophages (Ninj1 KO BMDM)This paperFreshly prepared from Ninj1 knockout mice; male and female donors; Steinberg laboratory
Biological sample (Homo sapiens)Primary human macrophages (human MDM)This paperFemale and male donors; information not disclosed; Flo laboratory
Biological sample (Homo sapiens)Induced pluripotent stem-cell (iPSC) derived macrophages (iPSDM)This paperiPSCs were obtained from European Bank for induced pluripotent Stem Cells; Male origin; Flo laboratory
AntibodyAnti-human NINJ1 (Mouse monoclonal)R&D SystemsMAB5105, RRID: AB_11128852Immunofluorescence: 10 µg/mL; Western blot: 2 µg/mL
AntibodyAnti-mouse NINJ1 (Rabbit monoclonal)Other; Kayagaki et al., 2021Gift from Dr Vishva Dixit and Dr Nobuhiko Kayagaki (Genentech, Inc, South San Francisco, USA); 10 µg/mL
AntibodyAnti-β-Actin (Rabbit monoclonal)Cell Signaling Technology8457, RRID: AB_10950489Western blot: 1:1000
AntibodyAnti-β-Actin (Mouse monoclonal)SigmaA5441; RRID: AB_476744Western blot 1:5000
AntibodyCaspase-1 (Mouse monoclonal)AdipogenAG-20B-0042-C100; RRID: #AB_2755041Western blot 1:1000
AntibodyGasdermin D (Rabbit monoclonal)Abcamab209845; RRID: #AB_2783550Western blot 1:1000
AntibodyAnti-pro-IL1B (Rabbit polyclonal)GenetexGTX74034; RRID: #AB_378141Western blot 1:1000
AntibodyAnti-HA (Rabbit monoclonal)Cell Signaling Technology3724; RRID: AB_1549585Western blot: 1:1000
AntibodyHRP conjugated anti-mouse IgG (H+L) (Goat polyclonal)Jackson ImmunoResearch115-035-003Western blot: 1:3000
AntibodyHRP conjugated anti-rabbit IgG (H+L) (Goat polyclonal)Jackson ImmunoResearch111–035003Western blot: 1:3000
AntibodyAnti-GAPDH (Rabbit polyclonal)Santa Cruz Biotechnologysc-25778; RRID: AB_10167668Western blot 1:1000
AntibodyAnti-GAPDH (Mouse monoclonal)Cell Signaling Technology97166; RRID: AB_2756824Western blot: 1:1000
AntibodyAnti-FLAG APC (Mouse monoclonal)AbcamAb72569; RRID: AB_1310127Flow cytometry 1:100
AntibodyRabbit anti-Mouse IgG (H+L) Recombinant Secondary Antibody, Alexa Fluor 647 (Rabbit polyclonal)InvitrogenA27029, RRID: AB_25360922 µg/mL
AntibodyCy3-conjugated AffiniPure Donkey Anti-Rabbit IgG (H+L) (Donkey polyclonal)Jackson ImmunoResearch711-165-152STED: 1:100; TIRF 1:1000
AntibodyMouse IgG2a isotype control (Mouse polyclonal)SerotecMCA929F; RRID: AB_32227110 µg/mL
Recombinant DNA reagentpRETROX-TRE3GTakaraBio631188
Recombinant DNA reagentpRETROX-TRE3G-Flag-NINJ1This paper.Jonathan Kagan laboratory; described in the Methods section
Recombinant DNA reagentpMSCV-Flag-NINJ1 IRES EGFPThis paper.Jonathan Kagan laboratory; described in the Methods section
Recombinant DNA reagentpCMV-VSVGAddgeneAddgene #8454
Recombinant DNA reagentNINJ1 (Myc-DDK tagged) cDNA cloneOrigeneMR225037
Recombinant DNA reagentpcDNA3.1-HAAddgeneAddgene #128034
Peptide, recombinant proteinHuman NINJ1 residues 40–69GenScript BioTech CorpHYASKKSAA
ESMLDIALLM
ANASQLKAVVE
Peptide, recombinant proteinSequence-scrambled analog
of human NINJ1 (residues 40–69)
GenScript BioTech CorpIAAAAMKMY
LANSLEHAK
SLKVVLASQDS
Peptide, recombinant proteinCandidalysinPeptide Protein ResearchSIIGIIMGILG
NIPQVIQIIM
SIVKAFKGNK
Peptide, recombinant proteinTNFPeproTech300–01 A10 ng/mL
Peptide, recombinant proteinPneumolysinOtherGift from Dr John Brumell
(The Hospital for Sick Children,
Toronto, Canada)
Peptide, recombinant proteinIL-3PeptroTech200–0325 ng/mL
Peptide, recombinant proteinM-CSFPeproTech300–25MDM: 10 ng/mL iPSDM:
100 ng/mL
Peptide, recombinant proteinVEGFR&D Systems293-VE-05050 ng/mL
Peptide, recombinant proteinSCFR&D Systems255-SC-05020 ng/mL
Peptide, recombinant proteinBMP-4R&D Systems315 BP-01050 ng/mL
Peptide, recombinant proteinCholera toxin subunit B Alexa
Fluor 488 conjugate
InvitrogenC347751 µg/mL
Commercial assay or kitCyQuant LDH cytotoxicity assay kitThermoFisher ScientificC20301
Commercial assay or kitHuman IL-1β ELISAR&D SystemsDY201
Commercial assay or kitMouse IL-1β ELISAInvitrogen88–7013
Commercial assay or kitCellTiterGlo Luminescent
Cell Viability Assay
PromegaG7570
Chemical compound and drugGlycineSigmaG7126 or 67126 (Sigma); 104201 (Millipore)
Chemical compound and drugFM1-43InvitrogenT31635 µg/mL
Chemical compound and drugFM4-64InvitrogenT31665 µg/mL
Chemical compound and drugCellTracker Deep Red DyeInvitrogenC345650.5 µM
Chemical compound and drugTetramethylrhodamine ethyl ester perchlorate (TMRE)Sigma8791710 nM
Chemical compound and drug1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)Avanti Polar Lipids#850375
Chemical compound and drug1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS)Avanti Polar Lipids#840035
Chemical compound and drugzVADInvivoGentlrl-vad25 µM
Chemical compound and drugBV6InvivoGeninh-bv610 µM
Chemical compound and drugNigericinInvivoGen or Sigmatlrl-nig (InvivoGen); N7146 (Sigma)20 µM
Chemical compound and drugABT-199Tocris696025 µM
Chemical compound and drugLipopolysaccharide from E. coli serotype 0111:B4 or serotype O55:B5 (LPS)SigmaLPS25; L65290.5 or 1 µg/mL
Chemical compound and drugY-27632StemCell Technologies7230210 µM
Chemical compound and drugDoxycyclineSigmaD34472 µg/mL
Software and algorithmImarisOxford InstrumentsRRID: SCR_007370
Software and algorithmFIJI v2.0.0imagej.net/ software/fijiRRID: SCR_002285
Software and algorithmCellProfiler v.4.2.4cellprofiler.org/RRID: SCR_007358
Software and algorithmPRISM v8.3.1 or v9.0GraphPad Software IncRRID: SCR_002798
OtherProLong Diamond Antifade
Mountant
InvitrogenP36961Mountant for immunofluorescnece
studies as described in the
Methods section

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files, which includes the source data for the manuscript figures.

References

Decision letter

  1. Carla V Rothlin
    Senior and Reviewing Editor; Yale University, United States

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Glycine targets NINJ1-mediated plasma membrane rupture to provide cytoprotection" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Nancy Carrasco as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

All reviewers appreciate the importance of the topic. However, they all raise significant concerns with regards to the lack of mechanistic understanding provided on how Glycine inhibition of NINJ1-mediated plasma membrane rupture? This is of great significance and should be explored.

1 – Is there a direct interaction between Glycine and NINJ1?

2 – Can the authors provide evidence on the mechanism/s by which Glycine prevents NINJ1 oligomerization?

Please make note of the specific comments from the reviewers below.

Reviewer #1 (Recommendations for the authors):

What is the mechanism of Glycine inhibition of NINJ1-mediated plasma membrane rupture? Is the mechanism direct or indirect? To increase the impact of the work, the authors would have to show the direct binding of Glycine to NINJ1 and provide biochemical/ structural insights into how Glycine inhibits NINJ1. Direct binding can be measured by SPR analysis using full or partial length NINJ1 protein. Other amino acids that were previously shown to be cytoprotective (i.e. Serine) should be included in this SPR study as well. If inhibition by Glycine is actually indirect, further exploration of this mechanism would need to be provided.

Reviewer #2 (Recommendations for the authors):

Although the authors show that glycine inhibits the formation of large NINJ1 oligomers in cells, their study does not show any direct interaction of glycine with NINJ1 or shed light on the mechanism of NINJ1-mediated cell rupture.

(1) The authors initiated this study by assessing the functional and morphological similarities between NINJ1 knockout and glycine treatment during different types of cell death in both human and murine cells. This requires careful kinetics and concentration-dependent experiments, which this paper falls short of. Besides, the staining patterns with plasma membrane dye exhibited obvious differences between NINJ1 knockout and glycine treatment, although the cells in both conditions showed intact plasma membrane (Figure 1E).

(2) Although NINJ1 has been identified as the key mediator of plasma membrane rupture by forming oligomers on the cell membrane, it remains unknown how NINJ1 is activated and executes its membrane-rupturing function. The authors found glycine treatment blocks NINJ1 oligomerization, which indicates glycine inhibits the activation of NINJ1 during cell death. Thus, besides NINJ1 itself, upstream regulators or co-factors of NINJ1 may be targeted and inactivated upon glycine treatment. Dissecting how NINJ1 inhibition occurs in the presence of glycine would further substantiate the authors' hypothesis proposed in this study, and meanwhile may also delineate the mechanism of how NINJ1 is activated in response to cell death signals.

(3) Biophysical methods to demonstrate a direct in vitro interaction of NINJ1 with glycine, modification of NINJ1 by glycine, or effectiveness of glycine in minimizing leakage of liposomes containing NINJ1 would greatly add to the story and determine whether glycine's effect requires other molecules.

(4) The imaging used in the manuscript is not state-of-the-art and does not take advantage of higher resolution methods available that could be used to visualize NINj1 clustering on pyroptotic balloons and examine how it is altered by glycine.

(5) The native gels seem to indicate 2 NINJ1 bands in control cells that are not dying – the ~40 kD band mentioned a higher band (~200 KD) that might correspond to higher order multimers observed in the control cells.

(6) It would be useful to use necroptotic cells throughout as a negative control for glycine effects.

Reviewer #3 (Recommendations for the authors):

While the work in this manuscript may suggest a possible link between the cytoprotective function of glycine and NINJ1, in its current form the manuscript is largely correlative and does not provide definitive evidence to support the conclusion being made. Substantial experimentation is necessary to demonstrate the direct link between glycine treatment and the prevention of Ninj1 oligomerization.

1. Figure 1E – why are cellular contents nearly completely gone in the NINJ1 knockout cells treated with glycine? I would think that the NINJ1-knockout would have no phenotype in response to glycine treatment, as they both presumably go through the same pathway (ie the presence and absence of glycine in knockout cells should not matter for the morphology of the cells). Can the authors clarify this? The authors claim that the morphology of cells treated with glycine and cells genetically deficient in Ninj1 is the same but it does not seem that this is the case.

2. The use of microscopy, particularly TIRF, to investigate NINJ1 surface dynamics and oligomerization is nice. However, the microscopy in figures 1E, and 2B needs further quantification, as it is quite difficult to determine similarities or differences between panels. Quantification of the microscopy images would be important to allow this. In 2B in particular we see one panel and one close-up inset that basically shows a single cell for each condition, and no untreated condition is shown. This is not sufficient data to make a conclusion about the distribution of NINJ1 under these conditions.

3. What is the evidence that the cells remain viable with the morphology seen in Figure 1E in the presence of glycine or NINJ1 deficiency? Are the cells capable of carrying out biological functions in this state, or are the cells no longer metabolically active, but retain some level of membrane integrity and retain their cellular contents (like LDH and HMGB1)?

4. Prior studies demonstrate that NINJ1 does not contribute to cell lysis during necroptosis. Glycine is likewise not reported to play a role in protective responses after necroptosis. It is therefore surprising that this manuscript finds a role for NINJ1 and glycine in cytoprotection in the setting of necroptosis. This should be clarified and further investigated to account for the possible discrepancy if the authors wish to include necroptosis studies here.

5. The authors report that glycine limits NINJ1 oligomerization. What is the effect of glycine on GSDMD cleavage and oligomerization? In the absence of this data, it is not possible to definitively conclude that the effect of glycine is only on NINJ1.

6. The microscopy describing NINJ1 distribution in the presence of glycine is difficult to parse – it is unclear why the reduction in the number of NINJ1 puncta should be indicative of NINJ1 oligomerization. Again, images in Figure 3B need to be quantified, and it should also be made clear how the assay being quantified is linked to NINJ1 function. The authors quantify the density of puncta. It seems to me that the diameter or volume of the individual puncta should be increased as a relative indicator of aggregation. Is this possible to quantify? A control cell surface protein that would not be expected to behave this way under these conditions is critical in these experiments. In the absence of such a control it is not possible to conclude that the effect of LPS+nigericin on NINJ1 is specific, nor is it possible to conclude the effect of glycine on NINJ1 oligomerization is specific. In general negative controls for the specificity of glycine for NINJ1 in these assays is important to include in order to strengthen the conclusions being made here.

7. The authors are using the effect of glycine on NINJ1 to make the conclusion that the cytoprotective effect of glycine is mediated by interference with NINJ1 oligomerization. However, in their current form, the data are largely correlative. Definitive data to support this conclusion require demonstrating that forced oligomerization of NINJ1 in the presence of glycine restores the release of LDH and other cellular contents or a similar type of experiment that can definitively demonstrate that glycine limits cell lysis by directly regulating NINJ1 oligomerization.

8. It would be important to demonstrate in the authors' hands under the same conditions described here, what are the effect of glycine treatment on other parts of the pyroptosis pathway – casp1 cleavage, gsdmd processing and cleavage, pro-IL-1b levels, and IL-1b secretion.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Glycine targets NINJ1-mediated plasma membrane rupture to provide cytoprotection" for further consideration by eLife. Your revised article has been evaluated by Carla Rothlin (Senior Editor) and a Reviewing Editor.

The manuscript has been significantly improved but there are some remaining issues that need to be addressed before publication, as outlined below:

Please take note of the specific recommendations of Reviewers 1 and 2 below to revise your manuscript.

Reviewer #1 (Recommendations for the authors):

The submitted manuscript reveals that the amino acid, glycine, a known inhibitor of pyroptosis-related plasma membrane rupture, can block NINJ1-dependent plasma membrane rupture, either through direct or indirect mechanisms, perhaps at the level of NINJ1 clustering.

This manuscript confirms that glycine, a known weak inhibitor of pyroptosis-related cellular plasma membrane rupture (IC50 1.6-1.8 mM, PMID: 30975978), can inhibit NINJ1 clustering and consequent plasma membrane rupture.

New Supplement 3 data confirms that glycine does not inhibit pyroptosis (GSDMD-dependent cell death), which is consistent with previous observations in Ninj1-deficient cells (PMID: 33472215).

The mechanism of action for glycine inhibition, however, remains unclear from this manuscript; yet it is likely to be an indirect mechanism, as suggested from previous reports showing inhibitory activity of other amino acids (including alanine and serine (PMID: 30975978)).

Specific feedback

The authors made a herculean effort to revise the manuscript and diligently responded to reviewers’ feedback with new data. However, the mechanism of action through which glycine inhibits plasma membrane rupture is still not clear. Glycine inhibition could be mediated directly or indirectly through NINJ1.

I could recommend the publication if the authors revise the following points.

1. In the abstract, add the following or similar sentences.

Glycine prevents NINJ1 clustering either directly or indirectly.

Glycine does not prevent pyroptosis.

2. In the revised manuscript, include the alanine amino acid data (which is currently only used for rebuttal) and discuss the possible indirect mechanisms of action by glycine.

"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."

Does glycine need to be inside cells to block cell lysis? What is the evidence? This should be discussed with a citation. Amino acid transporter or another mechanism may contribute to the glycine intake.

Reviewer #2 (Recommendations for the authors):

The resubmission has addressed the reviewers' concerns and greatly improved the quality of the manuscript. Although the authors still have not identified the molecular basis of glycine inhibition of cell membrane bursting, they have now clearly shown that glycine only acts at the final step of Ninj1 clustering and membrane disruption, independently of the upstream steps of inflammasome activation and gasdermin pore formation.

However, their conclusion that Ninj1 is the target of glycine is not warranted by their data. In particular (1) their failed attempts to show a physical association of glycine with Ninj1 and (2) the lack of inhibition of Ninj1-mediated liposome leakage, but inhibition of Ninj1-mediated cell membrane rupture in cells, strongly argue that something else in the cellular membrane or associated with it (but not in the liposomes) is affected by glycine that then affects Ninj1 clustering.

It seems highly likely that identifying the target and mechanism behind glycine inhibition will be a very risky project with no easy solution. Therefore the experiments in this paper have gone about as far as they can and provide some very useful and well-controlled data for tackling this difficult project. I, therefore, think that the manuscript is suitable for publication with the current data. However, I feel strongly that the paper needs to be revised to

(1) discuss all the experimental methods that didn't show a physical association of glycine with Ninj1 (I don't think the negative data needs to be shown).

(2) remove the statements in the abstract and discussion that "Ninj1 is the target of glycine".

(3) emphasize that glycine does not inhibit lysosomal membrane perturbing properties of Ninj1 which suggests that a cellular Ninj1- and plasma membrane-associated molecule is likely the target of glycine.

(4) discuss any ideas about how glycine could be working (if you have them) and discuss what approaches could be used to identify the glycine target.

A more precise title would be a good idea (such as "Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death").

Reviewer #3 (Recommendations for the authors):

The authors have thoroughly and comprehensively addressed the comments, and the conclusions of the manuscript is significantly strengthened. The revised manuscript provides new insight into how glycine limits cell lysis and extends our understanding of the mechanisms of NINJ1-mediated cellular rupture. I have no further concerns or comments.

https://doi.org/10.7554/eLife.78609.sa1

Author response

Essential revisions:

All reviewers appreciate the importance of the topic. However, they all raise significant concerns with regards to the lack of mechanistic understanding provided on how Glycine inhibition of NINJ1-mediated plasma membrane rupture? This is of great significance and should be explored.

1 – Is there a direct interaction between Glycine and NINJ1?

2 – Can the authors provide evidence on the mechanism/s by which Glycine prevents NINJ1 oligomerization?

We acknowledge that our initial submission was limited as it did not offer a mechanistic understanding of how glycine inhibited NINJ1-mediated plasma membrane rupture. We demonstrated that glycine treatment prevents NINJ1 clustering as shown though biochemical and microscopy-based assays during activation of cell death pathways. As acknowledged in the Discussion section of our initial submission, this effect could be either indirect or direct action of glycine on NINJ1. In our revised manuscript we have undertaken multiple approaches to strengthen our findings and evaluate the mechanism of action of glycine on NINJ1.

We first turned our attention to whether glycine is able to interfere with NINJ1-mediated cellular rupture independent of upstream signaling pathways. To this end, we generated a doxycycline-inducible NINJ1 overexpression system in macrophages. Using this cell-based system, we demonstrate that glycine dosedependently suppresses NINJ1-mediated cell rupture independent of activation of upstream programmed cell death pathway activation (new Figure 5C-D). This new data is consistent with our finding that glycine does not prevent caspase-1 activation, gasdermin D processing or activation in pyroptosis (new Figure 5A-B). To further address glycine’s mode of action in this system, we evaluated its effect on NINJ1 clustering biochemically and found that glycine suppresses NINJ1 clustering in an over-expression system (new Figure 5E).

We next turned our attention to the amphipathic α-helix in the NINJ1 N-terminus (residues 40-69), which Kayagaki et al. demonstrated to be necessary for NINJ1-mediated plasma membrane rupture (e.g. Figure 4 of Kayagaki et al. Nature 2021). We 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 (new Figure 5F).

A more comprehensive description of our approaches and new findings are as follows:

1. Inducible iBMDM-doxy-moNINJ1 system: In a new collaboration with Dr Jonathan Kagan’s group, we generated a doxycycline-inducible NINJ1 overexpression system in immortalized mouse macrophages. In this system, the overexpression of NINJ1 alone is sufficient to yield lytic cell death without the need for upstream activation mechanisms (e.g. LPS priming and nigericin). The cotreatment of these cells with glycine during induction of NINJ1 expression was able to suppress NINJ1-mediated plasma membrane rupture in a dose-dependent fashion. Notably, glycine did not change the frequency of NINJ1 expressing cells and only the highest concentration of glycine (50mM) slightly reduced NINJ1 surface expression. While these data do not answer whether glycine directly binds NINJ1 to prevent plasma membrane rupture, they further corroborate our findings that glycine is able to suppress NINJ1-mediated plasma membrane rupture and indicate that glycine cytoprotection is independent of upstream cell death pathway mediators. A characterization of the inducible iBMDM-doxy-moNINJ1 system is provided as Figure 5 —figure supplement 1.

– Methods details: iBMDMs with doxycycline-inducible expression of the N-terminal fragment of GSDMD fused to BFP are published (Evavold, Hafner-Bratkovic, Cell 2021). We used the same stock of iBMDMs from Cas9-expressing mice in which the TET3G transactivator protein had been introduced previously (Evavold et al., 2021) to generate cell lines with dox-inducible expression of NINJ1 carrying an N-terminal FLAG-tag. CDNA encoding full-length murine NINJ1 was ordered as a gBlock from Integrated DNA Technologies. This sequence was amplified by PCR to append an N-terminal FLAG-tag and cloned into pRETROX-TRE3G (TakaraBio) using BamHI and NotI restriction sites and into pMSCV IRES EGFP using NotI and SalI restriction sites. To introduce dox-inducible FLAG-NINJ1, 2.5 x 106 Platinum-GP retrovirus packaging cells were seeded in a TC-treated 10 cm dish. On the next day, cells were transfected with 3 μg pCMV-VSV-G and 9 μg pRETROX-TRE3G encoding FLAG-NINJ1 using Lipofectamine 2000. After 18h, media was exchanged to 6 ml of fresh complete DMEM. The same day, 0.5 x 106 Cas9- and TET3G-expressing iBMDMs were seeded per well in a 6-well plate. The next day, retroviral supernatant was harvested and filtered through 0.45 μm syringe filter, supplemented with polybrene (1:2000; EMD Millipore) and spun onto cells (1250 g, 30 °C, 1h). 6 ml of fresh cDMEM were added to retrovirus packaging cells and spinfection was repeated the next day. Three days after the second transduction, transduced cells were selected by growth in complete DMEM including 1.5 mg/ml G-418 and 10 μg/ml puromycin. To ensure maximal transgene expression, which correlates with the ability of FLAG-NINJ1 to induce cell death and lysis, FLAG-NINJ1 was also stably overexpressed in these cells using retroviral transduction with an MSCV retrovirus. Retrovirus production and spinfections were performed as described above, only using pMSCV IRES EGFP-FLAG-NINJ1 instead of pRETROX-TRE3G construct. Cells were then sorted on a BD Melody cell sorter for maximal EGFP expression (Top 5-10%). Clonal cell lines were derived by limited serial dilution. 1 x 105 cells per well in a black 96-well plate in DMEM (Gibco)/10% FBS (R and D, S11550) were induced with 2 ug/ml doxycycline (Σ-Aldrich) in the presence or absence of increasing concentrations of glycine (Σ-Aldrich 67126) in a total volume of 200 μl for 8 hours (iBMDM GSDMD-NT) or 12 hours (iBMDM FLAG-NINJ1) before supernatants were harvested for LDH-assay (Invitrogen) and cells detached (PBS/4 mM EDTA) for flow cytometry (Becton Dickinson Fortessa). iBMDM GSDMD-NT express BFP and were analyzed directly; iBMDM NINJ1 were stained with an APC-labeled anti-FLAG antibody (1:100, Abcam ab72569) in PBS/2% FBS, 2.5 mM EDTA on ice and washed once before analysis.

2. Transient HA-mNINJ1 transfection system. In a complementary experiment to our inducible system, we performed transient, over-expression of HA-tagged mNINJ1 in HeLa cells, which have no detectable endogenous NINJ1. Analysis of NINJ1 clustering by native-PAGE revealed HAmNINJ1 aggregate at high molecular weight, which was prevented by 5 mM glycine treatment (new panel Figure 5E of our revised manuscript). This observation recapitulates the behaviour we observed in macrophages during pyroptosis, forms of necrosis, and post-apoptosis secondary necrosis.

– Method details: PCR reactions were performed using ConeAmp HiFi master mix (Takara). Primers for mouse NINJ1 (insert) PCR reaction were: Forward primer 5’gat tac gct gag tcg ggc act gag ga3’ and reverse primer 5’ctc ggt acc cta ctg ccg ggg cgc ca3’. Template DNA for the PCR reaction was mouse cDNA clone from Origene (Cat. No. MR225037). The template vector pcDNA3.1-HA was obtained from Addgene. Primers for pcDNA3.1-HA PCR (vector) reaction were: Forward primer 5’cgg cag tag ggt acc gag ctc gga3’ and reverse primer 5’gcc cga ctc agc gta atc tgg aac atc3’. PCR reaction products were cleaned up using the NucleoSpin PCR Clean-up kit (Macherey-Nagel). The PCR products contained 18bp overlapping regions between the pcDNA3.1-HA tag vector and the mouse NINJ1 insert. This allows for fusion of the insert and vector using the In-Fusion Snap Assembly EcoDry Cloning Kit (Takara, 638954) and transformation into Stellar competent bacterial cells (Takara). DNA sequencing with CMV-F primer confirmed NINJ1 insertion into the pcDNA3.1-HA vector and that NINJ1 was in-frame with the N-terminal HA-tag. HeLa cells were transfected with pcDNA 3.1HA-NINJ1 using Fugene HD transfection reagent (Promega) according to the manufacturer’s protocol.

3. In vitro liposomal rupture system. As reported by Kayagaki et al., the N terminus of NINJ1 contains a purported amphipathic helix between amino acids 40 to 69, which is necessary for NINJ1mediated plasma membrane rupture (Figure 4 of their paper in Nature 2021). Accordingly, we next turned our attention to this amphipathic helix as a potential target of glycine’s mode of action. To evaluate whether glycine functionally and directly interacts with this purported helix, we employed an in vitro large unilamellar vesicle (LUVs) liposomal system where we measure peptide-induced liposomal rupture using a fluorescence reporter. A similar system was used to demonstrate that the NINJ1 amphipathic helix is sufficient to rupture LUVs (Extended Data Figure 7C, Kayagaki et al. Nature 2021). Whereas the NINJ1α-helix leads to liposomal rupture, co-treatment with glycine (50 mM) did not suppress this effect, suggesting that glycine does not directly target the lytic function of this NINJ1 domain (new panel Figure 5F of our revised manuscript).

– Method details: To generate large unilamellar vesicles, 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC, Avanti Polar Lipids) and 1,2-dioleoyl-sn-glycero-3-phospho-lserine (sodium salt) (DOPS, Avanti Polar Lipids) were first prepared at 80% DOPC and

20% DOPS, freeze dried and hydrated with a solution containing 25 mM 6carboxyfluorescein. The suspension was bath sonicated, freeze-thawed and extruded (Avanti Mini Extruder) using a Nucleopore 0.1 μm membrane (Whatman) to yield large unilamellar vesicles. The liposomes were washed three times in PBS to remove remaining 6carboxyfluorescein not contained within the liposomes (spin at 13 000 rpm for 5 min). At this concentration, the fluorescence of 6-carboxyflurescein self-quenches inside the liposomes. With liposomal rupture, the 6-carboxyfluorescein is released into the bathing solution thereby releasing the self-quenching with fluorescence monitored in a spectrofluorometer (excitation 492 nm; emission 512 nm). The liposomal rupture assay was setup by mixing equal amounts of the prepared LUVs with 0.4 mg ml−1 human NINJ1 αhelix region peptide (HYASKKSAAESMLDIALLMANASQLKAVVE; GenScript Biotech Corporation) with either vehicle or glycine (50 mM) in PBS buffer. Where indicated, a sequence-scrambled analogue peptide (IAAAAMKMYLANSLEHAKSLKVVLASQDS; GenScript Biotech Corporation) was used as a negative control for the human NINJ1 α-helix region peptide. Following 45 minutes, all liposomes were then ruptured by adding Triton X100 and the maximum fluorescence (cargo release) recorded. The results were converted to percentage cargo release and background control subtracted. The lytic yeast peptide candidalysin was used as an additional positive control.

Additional biophysical methods to probe for a direct interaction between NINJ1 and glycine: While our liposomal rupture assay suggests that glycine does not interfere with the lytic function of the NINJ1 αhelix in an artificial liposomal system, it remains plausible that glycine still interacts with this domain or other components of the NINJ1 N-terminus to interfere with NINJ1 clustering, which would be consistent with our presented data. As a result, we attempted a variety of biophysical approaches to evaluate a direct interaction between the NINJ1 N-terminus (including the α-helix) and glycine.

4. Circular dichroism spectroscopy: We first interrogated the N-terminal α-helix by circular dichroism (CD) spectroscopy in the presence of glycine. Like the studies by Kayagaki et al., the CD spectrum of this region reveals an α-helical secondary structure with characteristic downward deflections at 208 nm and 222 nm. Glycine alone did not affect the CD spectrum of the helix, indicating that it did not alter the stability of the helical secondary structure.

– Methods details: The secondary structure of the human NINJ1 α-helix region (amino acids 40-69; HYASKKSAAESMLDIALLMANASQLKAVVE; GenScript Biotech Corporation) was measured using a Jasco J-1500 spectropolarimeter. For this assay, peptide solutions from a 10 mg/mL stock in 5% DMSO in ddH2O were diluted to 0.2 mg/mL in buffer (20 mM sodium phosphate, pH 7.4, 10 mM NaCl and 1% sodium dodecyl sulfate) with the final peptide concentration determined using a bicinchoninic acid (BCA) protein assay. NINJ1 helix peptide was titrated with glycine between 0 and 20 mM. 250 µL of the glycine titrated human NINJ1 helix peptide solutions were pipetted into 1 mm path length quartz CD cuvettes, capped, and placed in the Peltier temperature-controlled CD sample holder for analysis. A corresponding buffer blank spectrum was subtracted out from the sample datasets. In all conditions, spectra were recorded between 260-190 nm using 50 nm/min scanning speed, 0.5 nm data pitch, 1 second DIT, and 1 nm bandwidth. Between 3 and 20 accumulations were averaged.

5. Bio-layer interferometry and surface plasmon resonance: Our Reviewers suggested we evaluate a direct interaction between NINJ1 and glycine using surface plasmon resonance (SPR). To this end, we trialed bio-layer interferometry as a similar approach. In these experiments, we probed for an interaction between the NINJ1 α-helix and a histidine-tagged poly-glycine peptide made of a concatenated chain of 10 glycine residues. These experiments were confounded by non-specific binding of the NINJ1 α-helix to both Ni-NAD and anti-His antibody bio-layer interferometry probes. Therefore, the resulting binding data (not shown) were not interpretable. For completeness, we include a detailed description of the protocol we undertook to evaluate the glycine and NINJ1 interaction by bio-layer interferometry.

– Methods details: Either Ni-NTA biosensors (Sartorius Part # 18-5101) or anti-Penta-His biosensors (Sartorius Part # 18-5120) were used for each experiment in a 96-well plate (Greiner Part #655209) as required by the Octet RH16 Biolayer Inferometer (BLI) instrument manufacturer (Sartorius). Polyhistidine-tagged poly-glycine ligand

(HHHHHHGGGGGGGGGG, stock solution of 10 mg/mL, 3.13 mM, in 5% DMSO in ddH2O) was immobilized to the Ni-NTA or anti-Penta-His biosensors at 200 nM and then dipped into a titration of the human NINJ1 α-helix peptide (residues 40-69 as above; (GenScript Biotech Corporation)) analyte in assay buffer (20 mM sodium phosphate, pH 7.4, 10mM NaCl and 1% sodium dodecyl sulfate). Assay volume per well was 50 µL at a temperature of 25°C. The assay baseline drift was subtracted out using the protein-bound biosensor dipped into buffer instead of the respective analyte. A negative control experiment was included where the ligand was not loaded onto the biosensors and a titration of the same concentrations of analyte were tested to determine bare” biosensor binding profiles. A blocking step was added using Superblock (Thermo Fisher cat. #37515). The general BLI step conditions used were as follows: 1. Biosensor check in assay buffer (30 sec). 2. HisPolyG Peptide (ligand) immobilization (10 min). 3. Blocking with Superblock (3 min). 4. Baseline in assay buffer (3 min). 5. NINJ1 Helix Peptide analyte titration association (10 min). 6. Dissociation back into aseline buffer (10 min).

6. NINJ1 N-terminus and dynamic light scattering. As we did not see any evidence of a direct interaction between glycine and the α-helix of NINJ1 by CD or our in vitro liposomal assay, we next posited that glycine may interact with other regions of the NINJ1 N-terminus. This hypothesis would be consistent with our cellular data demonstrating that glycine interferes with NINJ1 clustering as residues purported to be involved in NINJ1 homotypic interactions may fall within the N terminus but outside the helical region (Bae SJ et al. J Cell Biochem 2017). To address this hypothesis, we attempted to purify the full N-terminus of mouse NINJ1 (residues 1 through 71) expressed in bacteria. In brief, His6-SUMO-mNINJ1 (residues 1-71) was made in a pET SUMO vector with TA cloning and expressed in BL21 Rosetta E. coli, prior to purification. Methodological details are provided below. The SUMO tag improved protein solubility. However, following removal of the His6-SUMO tag, the mNINJ1(1-71) aggregated into the insoluble pellet fraction across a variety of protein concentration, pH and salt concentrations. Its insolubility is consistent with NINJ1’s ability to aggregate / cluster within membranes during lytic cell death but precluded the use of the purified N-terminus in biochemical studies. As a mitigating strategy, we employed the His6-SUMOmNINJ1(1-71) protein in a series of dynamic light scattering experiments. In these experiments, we monitored for an effect of glycine on protein aggregation by measuring the extent of dispersion through varying glycine concentrations. While we observed a small effect of 5 mM glycine, the extent of protein dispersion was inconsistent across different glycine doses. We did not pursue these investigations given that they made use of the His6-SUMO-mNINJ1(1-71) protein.

– Methods details: Dynamic light scattering (DLS) experiments were conducted on a DynaPro Plate Reader III (Wyatt Technology). His6-SUMO-mNINJ1(1-71) protein, at the supplied stock concentration (2 mg/mL) and buffer, was titrated with varying concentrations of Glycine (40mM, 20mM, 10mM, 5mM, 1mM) and analyzed. Samples were transferred to a low volume 384-well microplate (Corning #3540) using 20 µL per well. The DLS experiment was conducted at 25°C with 10 acquisitions of 5 seconds, with laser autoattenuation enabled. Both intensity-weighted and number-weighted (right) regularization analyses were conducted.

Please make note of the specific comments from the reviewers below.

Reviewer #1 (Recommendations for the authors):

What is the mechanism of Glycine inhibition of NINJ1-mediated plasma membrane rupture? Is the mechanism direct or indirect? To increase the impact of the work, the authors would have to show the direct binding of Glycine to NINJ1 and provide biochemical/ structural insights into how Glycine inhibits NINJ1. Direct binding can be measured by SPR analysis using full or partial length NINJ1 protein. Other amino acids that were previously shown to be cytoprotective (i.e. Serine) should be included in this SPR study as well. If inhibition by Glycine is actually indirect, further exploration of this mechanism would need to be provided.

We now provide mechanistic studies into how glycine inhibits NINJ1-mediated plasma membrane rupture. A detailed description of these studies and new results are provided above. In brief, we demonstrate that glycine dose-dependently inhibits NINJ1-mediated rupture independent of action of upstream cell death pathways. Biophysical studies using circular dichroism demonstrated no evidence that glycine interacts with NINJ1 α-helix in its N-terminus (residues 40-69) consistent with our observation that glycine does not prevent its lytic function in an in vitro liposomal rupture assay. Together with our biochemical and imaging data, these new findings further support the notion that glycine interferes with NINJ1 clustering potentially through indirect interaction.

As our Reviewer also notes, other amino acids have been shown to be cytoprotective but with lower efficacy than glycine. In the context of pyroptosis, the extent of cytoprotection conferred by different amino acids and small molecules has been best characterized by Loomis WP et al. (Cell Death and Disease 2019). In their study, they demonstrate that glycine > alanine >> serine, valine can protect against membrane rupture during pyroptosis but did not implicate or study NINJ1. We have observed a similar pattern in our studies of NINJ1mediated membrane rupture. Representative data are shown here for LPS-primed primary mouse macrophages stimulated to undergo pyroptosis with nigericin without or with co-treatment with the indicated amino acid. These data are not included in our revised manuscript.

Author response image 1
Glycine exhibits cytoprotection as compared to similar amino acisa through inhibition of NINK1 aggregation.

Primary wildtype bone marrow-derived macrophages where induced to undergo pyroptosis in the presence of glycine, alanine, serine or valine at either 5 or 15 mM. (A) Similar to the work of Loomis et al. (Cell Death and Disease 2019), glycine exhibits a greater extent of cytoprotection compared with alanine >> serine, valine as measured by LDH release. (B) BN-PAGE for NINJ1 shows that amino acid treatment limits NINJ1 aggregation in pyroptossi with a similar hierarchy glycine > alanine > serine, valine.

Reviewer #2 (Recommendations for the authors):

Although the authors show that glycine inhibits the formation of large NINJ1 oligomers in cells, their study does not show any direct interaction of glycine with NINJ1 or shed light on the mechanism of NINJ1-mediated cell rupture.

(1) The authors initiated this study by assessing the functional and morphological similarities between NINJ1 knockout and glycine treatment during different types of cell death in both human and murine cells. This requires careful kinetics and concentration-dependent experiments, which this paper falls short of. Besides, the staining patterns with plasma membrane dye exhibited obvious differences between NINJ1 knockout and glycine treatment, although the cells in both conditions showed intact plasma membrane (Figure 1E).

We agree with the Reviewer that kinetics and dose-response of glycine inhibition of cell lysis would be informative. We have included time course data on morphology and cell viability (mitochondria integrity, ATP) in our response to Reviewer #1 Comment #5 above. As suggested by the Reviewer, we have now tested different concentrations of glycine and show dose-dependent inhibition of LDH release from human and mouse primary macrophages in response to LPS priming followed by nigericin, and from mouse iBMDMs in response to doxycycline-induced overexpression of NINJ1 or GSDMD-NT (see response to Essential Revisions above). These data are now included as supplemental material to Figures 1 and 2 in our revised manuscript.

(2) Although NINJ1 has been identified as the key mediator of plasma membrane rupture by forming oligomers on the cell membrane, it remains unknown how NINJ1 is activated and executes its membrane-rupturing function. The authors found glycine treatment blocks NINJ1 oligomerization, which indicates glycine inhibits the activation of NINJ1 during cell death. Thus, besides NINJ1 itself, upstream regulators or co-factors of NINJ1 may be targeted and inactivated upon glycine treatment. Dissecting how NINJ1 inhibition occurs in the presence of glycine would further substantiate the authors' hypothesis proposed in this study, and meanwhile may also delineate the mechanism of how NINJ1 is activated in response to cell death signals.

We agree that delineating the mechanism of NINJ1-mediated plasma membrane rupture is paramount to our understanding of lytic cell death pathways. As mentioned here, at present, it remains unknown how NINJ1 is activated and executes membrane rupture. Using multiple methods, we have demonstrated that glycine interferes with NINJ1 clustering. In the context of pyroptosis, we have previously shown that glycine does not interfere with IL-1β secretion (Volchuk A et al. Nature Communications 2020) in mouse macrophages, and we showed the same in Figure 2 —figure supplement 1 for human stem-cell derived macrophages (see Reviewer #1, Comment #1 above). In our revised manuscript, we expand on these findings by demonstrating that glycine treatment does not interfere with caspase-1 activation, gasdermin D cleavage, and IL-1β secretion. No specific proteins have been shown to link gasdermin D pore formation to NINJ1 activation. Therefore, we cannot evaluate them as potential glycine targets. However, using a doxycycline-inducible overexpression system of NINJ1 we provide new data showing that glycine targets NINJ1 independent of known upstream cell death pathways (see data in Response to Essential Revisions). Given the role of NINJ1 in mediating plasma membrane rupture in multiple lytic cell death pathways, a

generalized activation mechanism common to each of the implicated pathways is anticipated. As the mechanism of NINJ1 is explored and more fully delineated, we anticipate that additional molecular players and processes can be specifically evaluated as potential glycine targets.

(3) Biophysical methods to demonstrate a direct in vitro interaction of NINJ1 with glycine, modification of NINJ1 by glycine, or effectiveness of glycine in minimizing leakage of liposomes containing NINJ1 would greatly add to the story and determine whether glycine's effect requires other molecules.

We thank the reviewer for these helpful suggestions. In our revised submission, we now include (1) in vitro biophysical characterizations of glycine with the a-helix of NINJ1 using a liposomal rupture assay and circular dichroism spectroscopy, and (2) a doxycycline-inducible cellular system to isolate the effect of glycine on NINJ1 from other upstream molecules. These data and methods are described in full above.

(4) The imaging used in the manuscript is not state-of-the-art and does not take advantage of higher resolution methods available that could be used to visualize NINj1 clustering on pyroptotic balloons and examine how it is altered by glycine.

In our initial submission, we used total internal reflection fluorescence (TIRF) microscopy to limit our observations to the plasma membrane devoid of other intracellular compartments. TIRF microscopy is one of the few imaging modalities that allows for the fluorescence signal to be uniformly restricted to the cell’s plasma membrane.

Nevertheless, we acknowledge that TIRF is diffraction limited. As suggested by the Reviewer, we have advanced our imaging of native NINJ1 in macrophages undergoing pyroptosis without and with glycine treatment using stimulated emission depletion (STED) microscopy, a super-resolution fluorescence microscopy technique. These new data are provided in the updated Figure 3 of our revised manuscript. With our STED setup, we have improved our spatial resolution of 271 ± 98 nm with standard confocal microscopy to 63 ± 8 nm as measured by full-width at half maximum (FWHM) of single NINJ1 puncta. Using this approach in LPS-primed macrophages, we now better delineate the clustering of NINJ1 during pyroptosis, which is inhibited by glycine co-treatment. In brief, these data demonstrate that NINJ1 puncta decrease in

density upon pyroptosis stimulation with a corresponding increase in their mean intensity. Glycine treatment prevents this decrease in puncta density and decreased intensity. These new data, therefore, complement and corroborate the results presented in our initial manuscript using (1) native-PAGE, (2) cytotoxicity as measured by LDH release, and (3) TIRF microscopy. Together, our results support the notion that glycine targets NINJ1-mediated plasma membrane rupture and inhibits its clustering within the plasma membrane.

(5) The native gels seem to indicate 2 NINJ1 bands in control cells that are not dying – the ~40 kD band mentioned a higher band (~200 KD) that might correspond to higher order multimers observed in the control cells.

Thank you for this comment and astute observation. We agree that in the native gels of basal or LPS-primed cells, there is indeed a higher band of approximately 150-200 kDa. Of note, a similar band can be seen in the native gels shown in the paper by Kayagaki et al. (Nature 2021; see Figure 4C for example). As our Reviewer describes, this higher band may correspond to higher order multimers observed in the control cells. In our revised manuscript, we now mention this higher band and state that it may correspond to higher order multimers.

(6) It would be useful to use necroptotic cells throughout as a negative control for glycine effects.

We agree with the Reviewer that a negative control would strengthen our findings, and necroptosis is particularly interesting in this regard since previous studies have shown that glycine does not protect necroptotic cells from rupture, even though MLKL pores are formed (Chen X et al. Cell Research 2014). The study by Kayagaki N et al. further suggested that NINJ1 is not mediating PM rupture during necroptosis in mouse macrophages (although a partial phenotype was seen). We have now included data showing that glycine does not inhibit LDH release in necroptotic human primary macrophages (induced by TNF+zVAD+BV6). The new data are provided in the new Figure 2 —figure supplement 2 of our revised manuscript.

Reviewer #3 (Recommendations for the authors):

While the work in this manuscript may suggest a possible link between the cytoprotective function of glycine and NINJ1, in its current form the manuscript is largely correlative and does not provide definitive evidence to support the conclusion being made. Substantial experimentation is necessary to demonstrate the direct link between glycine treatment and the prevention of Ninj1 oligomerization.

1. Figure 1E – why are cellular contents nearly completely gone in the NINJ1 knockout cells treated with glycine? I would think that the NINJ1-knockout would have no phenotype in response to glycine treatment, as they both presumably go through the same pathway (ie the presence and absence of glycine in knockout cells should not matter for the morphology of the cells). Can the authors clarify this? The authors claim that the morphology of cells treated with glycine and cells genetically deficient in Ninj1 is the same but it does not seem that this is the case.

Thank you for this comment and notable observation. We agree that the presented images appear to depict significant differences between the glycine-treated wildtype and Ninj1 knockout cells. In the experiments of Figure 1E, we employ the FM fluorescent membrane dye to visualize the plasma membrane. In the context of pyroptosis stimulation without NINJ1 (or glycine treatment in wildtype cells), the membrane dye slowly enters the cytoplasm to eventually illuminate the intracellular membranes. We speculate that the dye is entering through the relatively large gasdermin D pores. The extent of intracellular fluorescent signal is a function of the time between pyroptosis induction and image acquisition. To avoid confusion, we have reviewed our images and have selected a representative set that were acquired at a more consistent time post-induction of pyroptosis.

2. The use of microscopy, particularly TIRF, to investigate NINJ1 surface dynamics and oligomerization is nice. However, the microscopy in figures 1E, and 2B needs further quantification, as it is quite difficult to determine similarities or differences between panels. Quantification of the microscopy images would be important to allow this. In 2B in particular we see one panel and one close-up inset that basically shows a single cell for each condition, and no untreated condition is shown. This is not sufficient data to make a conclusion about the distribution of NINJ1 under these conditions.

The figures presented in Figures 1E and 2B of our original submission are representative of the general morphology that we observed across multiple cells and independent experiments using wide-field microscopy (not TIRF). In these experiments, we have included LPS-primed but otherwise untreated cells as controls. We thank the reviewer for their positive acknowledgement of our investigation of NINJ1 surface dynamics and clustering by TIRF. As noted elsewhere, we have expanded the quantification of these data and now include additional corroborating super-resolution imaging by stimulated emission depletion (STED) of NINJ1 clustering in pyroptosis (Figures 3 and 4).

3. What is the evidence that the cells remain viable with the morphology seen in Figure 1E in the presence of glycine or NINJ1 deficiency? Are the cells capable of carrying out biological functions in this state, or are the cells no longer metabolically active, but retain some level of membrane integrity and retain their cellular contents (like LDH and HMGB1)?

We thank you for this is an important point. In the work of Kayagaki et al. (Nature 2021) that first positions NINJ1 in plasma membrane rupture, they show that Ninj1 knockout cells induced to undergo pyroptosis are in fact dead but not ruptured. In their paper, they state

“The cells were dead based on their loss of ATP, mitochondrial membrane potential, and motility (Figure 2g, Extended Data Figure 2h, Supplementary Videos 1, 2). Thus, PMR and related events, including LDH release and bubble disintegration, are genetically separable from GSDMD-driven cell death and IL-1β release. PMR is probably an event that occurs after cell death17,18. Of note, BMDMs ceased moving before bubble formation (Supplementary Videos 1, 2). NINJ1-independent loss of mitochondrial membrane potential also preceded PMR (as assessed by release of DD-150) (Extended Data Figure 2h, i).”

We have addressed these findings by evaluating mitochondrial membrane potential as well as ATP loss in human monocyte-derived macrophages stimulated to undergo pyroptosis with and without glycine. Glycinetreated cells lose both their mitochondria membrane potential (as measured by TMRE loss) and ATP comparably to untreated cells (data presented above in Reviewer #1, Comment #5). We (Volchuk et al. Nature Communications 2020) and others (Kayagaki N et al. Nature 2021) have previously shown that the glycine-treated pyroptotic macrophages retain large cellular contents, such as LDH and HMGB1.

4. Prior studies demonstrate that NINJ1 does not contribute to cell lysis during necroptosis. Glycine is likewise not reported to play a role in protective responses after necroptosis. It is therefore surprising that this manuscript finds a role for NINJ1 and glycine in cytoprotection in the setting of necroptosis. This should be clarified and further investigated to account for the possible discrepancy if the authors wish to include necroptosis studies here.

We agree with the Reviewer that the study by Kayagaki N et al. suggested that NINJ1 is not mediating PM rupture during necroptosis (although a partial phenotype was seen), and that previous studies have shown that glycine is not involved in cytoprotection during necroptosis (Chen X et al. Cell Research 2014). We believe this comment could result from a confusion around "necrosis" and "necroptosis". That said, we have now included data showing that glycine does not inhibit LDH release from necroptotic human primary macrophages (see Response #6 to Reviewer #2 above).

5. The authors report that glycine limits NINJ1 oligomerization. What is the effect of glycine on GSDMD cleavage and oligomerization? In the absence of this data, it is not possible to definitively conclude that the effect of glycine is only on NINJ1.

In our original submission, we did not include data that addressed whether glycine affects GSDMD cleavage or oligomerization, although we did show that glycine did not interfere with IL-1β secretion (Figure 4 Supplement 1). While our data demonstrate that glycine interfere with NINJ1 clustering and function in pyroptosis in addition to other forms of lytic cell death, we had not ruled out a potential effect of glycine on proteins upstream of NINJ1 in pyroptosis. We now include additional data that shows that glycine does not affect caspase-1 cleavage, gasdermin D cleavage, IL-1β processing and secretion. In these data, we use IL-1β secretion as a surrogate marker of gasdermin D oligomerization.

6. The microscopy describing NINJ1 distribution in the presence of glycine is difficult to parse – it is unclear why the reduction in the number of NINJ1 puncta should be indicative of NINJ1 oligomerization. Again, images in Figure 3B need to be quantified, and it should also be made clear how the assay being quantified is linked to NINJ1 function. The authors quantify the density of puncta. It seems to me that the diameter or volume of the individual puncta should be increased as a relative indicator of aggregation. Is this possible to quantify? A control cell surface protein that would not be expected to behave this way under these conditions is critical in these experiments. In the absence of such a control it is not possible to conclude that the effect of LPS+nigericin on NINJ1 is specific, nor is it possible to conclude the effect of glycine on NINJ1 oligomerization is specific. In general negative controls for the specificity of glycine for NINJ1 in these assays is important to include in order to strengthen the conclusions being made here.

We thank the Reviewer for these important points. We acknowledge that the size of the individual NINJ1 puncta could increase as a relative indicator of NINJ1 aggregation. Accordingly, in our revised submission, we now include additional analyses of NINJ1 puncta fluorescence intensity in our TIRF images of mouse and human macrophages (see Reviewer #1, Comment #7). This new analysis is complemented by super-resolution fluorescence microscopy to better resolve the size of the NINJ1 puncta. As noted above, we were able to improve our spatial resolution (mean ± SD) of 271 ± 98 nm with standard confocal microscopy to 63 ± 8 nm as measured by full-width at half maximum (FWHM) of single NINJ1 puncta. At this resolution, we still cannot fully resolve the membrane organization of these individual NINJ1 clusters. In addition, we provide Ab staining controls for the mouse and human TIRF imaging.

7. The authors are using the effect of glycine on NINJ1 to make the conclusion that the cytoprotective effect of glycine is mediated by interference with NINJ1 oligomerization. However, in their current form, the data are largely correlative. Definitive data to support this conclusion require demonstrating that forced oligomerization of NINJ1 in the presence of glycine restores the release of LDH and other cellular contents or a similar type of experiment that can definitively demonstrate that glycine limits cell lysis by directly regulating NINJ1 oligomerization.

In our revised submission we have strengthened our data that show that glycine interferes with NINJ1 oligomerization by both biochemical and imaging approaches in two species of macrophages. As described above, we now include informative biophysical studies evaluating a direct interaction of glycine with NINJ1.

While we do not have an engineered system that allows for the forced oligomerization of NINJ1, we now include data using an inducible NINJ1 system whereby the overexpression of NINJ1 alone leads to membrane rupture. These data – described in full above – show that glycine limits NINJ1-induced plasma membrane rupture independent of stimulation of any of pyroptosis, necrosis, or post-apoptosis lysis.

8. It would be important to demonstrate in the authors' hands under the same conditions described here, what are the effect of glycine treatment on other parts of the pyroptosis pathway – casp1 cleavage, gsdmd processing and cleavage, pro-IL-1b levels, and IL-1b secretion.

Thank you for this question. Please see our response to your Comment #5 above. In brief, glycine does not affect other parts of the pyroptosis pathway including caspase-1 cleavage, gasdermin D cleavage, or IL-1β processing and secretion. These data are included in our new Figure 5A-B of our revised manuscript.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Reviewer #1 (Recommendations for the authors):

The submitted manuscript reveals that the amino acid, glycine, a known inhibitor of pyroptosis-related plasma membrane rupture, can block NINJ1-dependent plasma membrane rupture, either through direct or indirect mechanisms, perhaps at the level of NINJ1 clustering.

This manuscript confirms that glycine, a known weak inhibitor of pyroptosis-related cellular plasma membrane rupture (IC50 1.6-1.8 mM, PMID: 30975978), can inhibit NINJ1 clustering and consequent plasma membrane rupture.

New Supplement 3 data confirms that glycine does not inhibit pyroptosis (GSDMD-dependent cell death), which is consistent with previous observations in Ninj1-deficient cells (PMID: 33472215).

The mechanism of action for glycine inhibition, however, remains unclear from this manuscript; yet it is likely to be an indirect mechanism, as suggested from previous reports showing inhibitory activity of other amino acids (including alanine and serine (PMID: 30975978)).

Specific feedback

The authors made a herculean effort to revise the manuscript and diligently responded to reviewers’ feedback with new data. However, the mechanism of action through which glycine inhibits plasma membrane rupture is still not clear. Glycine inhibition could be mediated directly or indirectly through NINJ1.

Thank you very much for your insightful comments regarding our manuscript throughout the review process. We acknowledge that the mechanism of action through which glycine inhibits NINJ1mediated plasma membrane rupture may be direct or indirect. This has been more fully discussed in our revised manuscript as outlined in the specific changes listed below.

I could recommend the publication if the authors revise the following points.

1. In the abstract, add the following or similar sentences.

Glycine prevents NINJ1 clustering either directly or indirectly.

Glycine does not prevent pyroptosis.

We have added the first sentence to our abstract as suggested. For the second sentence (“Glycine does not prevent pyroptosis”), we have modified the abstract to include: “In pyroptosis, glycine preserves cellular integrity but does not affect upstream inflammasome activities or accompanying energetic cell death.” We feel this statement is consistent with our data that demonstrate that while glycine prevents pyroptotic cell membrane rupture, upstream inflammasome activities (e.g. IL-1β processing, GSDMD cleavage and pore formation; Figure 5) and accompanying energetic cell death (e.g. mitochondrial membrane depolarization, cellular ATP loss; Figure 2 —figure supplement 3) persist.

In addition, as suggested by our Reviewer #2 (Comment #2), we have also removed the statements “NINJ1 is the target of glycine…” from both the abstract and discussion. Together, these changes better emphasize that glycine is working at the level of NINJ1 through either direct or indirect mechanisms, consistent with our data.

2. In the revised manuscript, include the alanine amino acid data (which is currently only used for rebuttal) and discuss the possible indirect mechanisms of action by glycine.

Thank you for this suggestion. We now include our alanine (along with serine and valine) data in our revised manuscript in Figure 1 —figure supplement 1.

We have also expanded our discussion of possible indirect mechanisms of action of glycine in the Discussion section of our manuscript (see also Reviewer #2, Comments #3 and #4 below):

“The mechanism by which glycine interferes with NINJ1 clustering can be either direct or indirect. Our in vitro circular dichroism studies and liposomal rupture system respectively indicate that glycine does not interfere with the secondary structure or lytic function of the NINJ1 α-helix. It remains plausible that glycine still interacts directly with this domain or other components of the NINJ1 N-terminus in cells to interfere with NINJ1 clustering, consistent with our presented data. Alternatively, glycine may act on an unidentified intermediate that modulates NINJ1 clustering within the plasma membrane. Our data in pyroptotic cells suggest that such an intermediate would reside between active GSDMD and NINJ1. In an indirect model, any of a plasma membrane-associated protein, membrane lipid, or cellular metabolic pathway could be the target. In addition, it remains unclear whether glycine’s cytoprotective activity occurs on the intracellular or extracellular side of the plasma membrane. To the best of our knowledge, there is no direct evidence to suggest whether glycine’s cytoprotective effect is specifically mediated from the intracellular or extracellular side. Both extracellular (Weinberg et al., 2016) and intracellular (Rühl and Broz, 2022) sites of action have been proposed. Our in vitro data suggest that glycine is not acting directly on the N-terminal α-helix, which is thought to be extracellular; however, we do not otherwise delineate the topology of glycine’s mechanism of action.

Defining the site of action would provide insight into the molecular mechanism by which glycine inhibits NINJ1 clustering in the plasma membrane and ultimately help determine whether glycine directly or indirectly engages NINJ1. Indeed, how NINJ1 is activated by lytic cell death pathways, its clustering trigger, and method of plasma membrane disruption also remain important and outstanding questions in the field. Due to the ubiquity of lytic cell death pathways in human health and disease and the potency of glycine cytoprotection, answers to these questions will greatly advance the development of therapeutics against pathologic conditions associated with aberrant lytic cell death pathways.”

"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."

Does glycine need to be inside cells to block cell lysis? What is the evidence? This should be discussed with a citation. Amino acid transporter or another mechanism may contribute to the glycine intake.

Thank you for alerting us to this important point for clarification. In our manuscript, we do not demonstrate that glycine is acting extra- or intracellularly. We have posited that given glycine’s small hydrodynamic radius (0.28 nm; doi.org/10.1021/j100799a040), it will freely enter a pyroptotic cell based on the structural studies of the gasdermin D pore size (PMID 33883744). Our intention in discussing the permeability of glycine in our Introduction was to emphasize that glycine’s mode of action is not through an osmoprotectant effect. Nevertheless, as noted, amino acid transporters or other mechanisms may indeed contribute to glycine intake.

The question of whether glycine is acting on the intracellular or extracellular side of the plasma membrane is intriguing. To the best of our knowledge, there is no direct evidence to suggest whether glycine’s cytoprotective effect is specifically mediated from the intracellular or extracellular side. Both extracellular (PMID 27066896) and intracellular (PMID 34537232) sites of action have been proposed. Our in vitro data suggest that glycine is not acting directly on the N-terminal a-helix, which is thought to be extracellular; however, we do not otherwise delineate the topology of glycine’s mechanism of action.

In our updated manuscript, we now discuss this question in our Introduction and as part of our Discussion of potential indirect and direct mechanisms.

Reviewer #2 (Recommendations for the authors):

The resubmission has addressed the reviewers' concerns and greatly improved the quality of the manuscript. Although the authors still have not identified the molecular basis of glycine inhibition of cell membrane bursting, they have now clearly shown that glycine only acts at the final step of Ninj1 clustering and membrane disruption, independently of the upstream steps of inflammasome activation and gasdermin pore formation.

However, their conclusion that Ninj1 is the target of glycine is not warranted by their data. In particular (1) their failed attempts to show a physical association of glycine with Ninj1 and (2) the lack of inhibition of Ninj1-mediated liposome leakage, but inhibition of Ninj1-mediated cell membrane rupture in cells, strongly argue that something else in the cellular membrane or associated with it (but not in the liposomes) is affected by glycine that then affects Ninj1 clustering.

It seems highly likely that identifying the target and mechanism behind glycine inhibition will be a very risky project with no easy solution. Therefore the experiments in this paper have gone about as far as they can and provide some very useful and well-controlled data for tackling this difficult project. I, therefore, think that the manuscript is suitable for publication with the current data. However, I feel strongly that the paper needs to be revised to

(1) Discuss all the experimental methods that didn't show a physical association of glycine with Ninj1 (I don't think the negative data needs to be shown).

Thank you for this suggestion, which buttresses our in vitro liposomal rupture data shown in Figure 5F. In our revised manuscript, we include a description and discussion of the experimental methods that did not show a physical association of glycine with NINJ1. Namely, circular dichroism (CD) of the N-terminal α-helix (residues 40-69) through a glycine titration from 0 to 20 mM did not affect the CD spectrum of the helix, indicating that it did not alter the stability of the helical secondary structure. These data are consistent with our findings that glycine does not prevent liposomal rupture induced by the α-helix (Figure 5F) and have been incorporated into a new Figure 5 —figure supplement 2.

We have not included the biolayer interferometry experiments in our revised manuscript because these experiments were confounded by non-specific binding of the NINJ1 α-helix to both Ni-NAD and anti-His antibody bio-layer interferometry probes. Therefore, the resulting binding data were not interpretable. Similarly, we have not incorporated our work with a His6-SUMO-tagged NINJ1 N terminus (residues 171) peptide. Unfortunately, following removal of the His6-SUMO tag, the mNINJ1(1-71) aggregated into the insoluble pellet fraction across a variety of protein concentrations, pH and salt concentrations. Its insolubility is consistent with NINJ1’s ability to aggregate / cluster within membranes during lytic cell death but precluded the use of the purified N-terminus in biochemical (or liposomal rupture) assays.

(2) Remove the statements in the abstract and discussion that "Ninj1 is the target of glycine".

As suggested, we have removed the statements “NINJ1 is the target of glycine…” from both the abstract and discussion.

(3) Emphasize that glycine does not inhibit lysosomal membrane perturbing properties of Ninj1 which suggests that a cellular Ninj1- and plasma membrane-associated molecule is likely the target of glycine.

Thank you for this important point. We have revised our Discussion section to emphasize that our finding that glycine does not inhibit liposomal membrane rupture by the N-terminal α-helix of NINJ1 is consistent with glycine targeting a plasma membrane-associated molecule or other non-NINJ1 molecular target. Because the α-helix of NINJ1’s N terminus is the minimally sufficient region for liposomal rupture, however, our in vitro findings do not exclude the possibility that other parts of full length NINJ1 are regulatory domains targeted by glycine. Accordingly, we now state that in cells glycine may act indirectly by targeting a plasma membrane-associated protein, the lipid membrane, or a metabolic pathway. A direct mechanism is still plausible through an interaction with a region of NINJ1 outside the N-terminal α-helix.

(4) Discuss any ideas about how glycine could be working (if you have them) and discuss what approaches could be used to identify the glycine target.

As noted above in our response # 3 (and Reviewer #1, Comment #3), we have clarified in our main text that glycine’s specific molecular target remains unknown and that both direct and indirect mechanisms remain possible. As suggested during our review, biophysical methods of testing for direct binding of glycine to either the full-length protein or the full N-terminus would provide important insight into potential direct mechanisms. Similarly, elucidating the mechanism by which NINJ1 is activated will most likely provide answers to how glycine works (and vice versa).

A more precise title would be a good idea (such as "Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death").

Thank you for this suggestion. We agree that “Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death” precisely encompasses our presented work. We have changed the title accordingly.

https://doi.org/10.7554/eLife.78609.sa2

Article and author information

Author details

  1. Jazlyn P Borges

    Program in Neuroscience and Mental Health, Hospital for Sick Children, Toronto, Canada
    Contribution
    Formal analysis, Investigation, Writing – original draft, Writing – review and editing
    Contributed equally with
    Ragnhild SR Sætra
    Competing interests
    No competing interests declared
  2. Ragnhild SR Sætra

    Centre of Molecular Inflammation Research, Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology, Trondheim, Norway
    Contribution
    Formal analysis, Validation, Investigation, Methodology, Writing – review and editing
    Contributed equally with
    Jazlyn P Borges
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8248-0460
  3. Allen Volchuk

    Program in Cell Biology, Hospital for Sick Children, Toronto, Canada
    Contribution
    Formal analysis, Validation, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
  4. Marit Bugge

    Centre of Molecular Inflammation Research, Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology, Trondheim, Norway
    Contribution
    Formal analysis, Validation, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
  5. Pascal Devant

    Division of Gastroenterology, Boston Children's Hospital and Harvard Medical School, Boston, United States
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9743-6764
  6. Bjørnar Sporsheim

    Centre of Molecular Inflammation Research, Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology, Trondheim, Norway
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  7. Bridget R Kilburn

    Program in Neuroscience and Mental Health, Hospital for Sick Children, Toronto, Canada
    Contribution
    Formal analysis, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0171-9370
  8. Charles L Evavold

    Ragon Institute of MGH, MIT and Harvard, Boston, United States
    Contribution
    Supervision, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3913-5626
  9. Jonathan C Kagan

    Division of Gastroenterology, Boston Children's Hospital and Harvard Medical School, Boston, United States
    Contribution
    Supervision, Funding acquisition, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2364-2746
  10. Neil M Goldenberg

    1. Program in Cell Biology, Hospital for Sick Children, Toronto, Canada
    2. Department of Anesthesia and Pain Medicine, Hospital for Sick Children, Toronto, Canada
    3. Department of Anesthesiology and Pain Medicine, University of Toronto, Toronto, Canada
    Contribution
    Formal analysis, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2785-1852
  11. Trude Helen Flo

    1. Centre of Molecular Inflammation Research, Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology, Trondheim, Norway
    2. Division of Gastroenterology, Boston Children's Hospital and Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Methodology, Writing – original draft, Project administration, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2569-0381
  12. Benjamin Ethan Steinberg

    1. Program in Neuroscience and Mental Health, Hospital for Sick Children, Toronto, Canada
    2. Department of Anesthesia and Pain Medicine, Hospital for Sick Children, Toronto, Canada
    3. Department of Anesthesiology and Pain Medicine, University of Toronto, Toronto, Canada
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Methodology, Writing – original draft, Project administration, Writing – review and editing
    For correspondence
    benjamin.steinberg@sickkids.ca
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3070-0548

Funding

International Anesthesia Research Society (Mentored Research Award)

  • Benjamin Ethan Steinberg

Department of Anesthesiology and Pain Medicine, University of Toronto (Early Investigator Award)

  • Benjamin Ethan Steinberg

Research Council of Norway (287696)

  • Trude Helen Flo

Ragon Institute of MGH, MIT and Harvard (Ragon Early Independence Fellowship)

  • Charles L Evavold

National Institutes of Health (AI133524)

  • Jonathan C Kagan

Boehringer Ingelheim Fonds (PhD Fellowship)

  • Pascal Devant

National Institutes of Health (P30DK3485)

  • Jonathan C Kagan

National Institutes of Health (AI116550)

  • Jonathan C Kagan

National Institutes of Health (AI093589)

  • Jonathan C Kagan

Research Council of Norway (223255)

  • Trude Helen Flo

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Dr Nobuhiko Kayagaki and Dr Vishva Dixit for providing the rabbit monoclonal anti-mouse NINJ1 antibody, Dr John Brumell for providing pneumolysin, and Anne Marstad for technical assistance. We thank Dr Greg Wasney at the Structural and Biophysical Core Facility, The Hospital for Sick Children, for his technical assistance with the circular dichroism studies. All imaging of the mouse and human cells was performed at the SickKids Imaging Facility at The Hospital for Sick Children and the Cellular and Molecular Imaging Core Facility at NTNU, respectively. Graphical abstract created with BioRender.com. The authors declare no competing financial interests.

This work was supported by a Mentored Research Award from the International Anesthesia Research Society and an Early Investigator Award from the Department of Anesthesiology and Pain Medicine, University of Toronto to BES; by grants (287696, 223255) from the Research Council of Norway to THF; a Ragon Early Independence Fellowship to CLE; NIH grants AI133524, AI093589, AI116550, and P30DK34854 to JCK; and a fellowship to PD by the Boehringer Ingelheim Fonds.

Ethics

Human subjects: All human studies were conducted according to the principles expressed in the Helsinki Declaration and approved by the Regional Committee for Medical and Health Research Ethics (No. 2009/2245). Informed consent was obtained from all subjects prior to sample collection.

All animal studies were approved by the Hospital for Sick Children Animal Care Committee (AUP #47781).

Senior and Reviewing Editor

  1. Carla V Rothlin, Yale University, United States

Publication history

  1. Preprint posted: December 12, 2021 (view preprint)
  2. Received: March 14, 2022
  3. Accepted: December 5, 2022
  4. Accepted Manuscript published: December 5, 2022 (version 1)
  5. Accepted Manuscript updated: December 8, 2022 (version 2)
  6. Version of Record published: December 15, 2022 (version 3)

Copyright

© 2022, Borges, Sætra et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Jazlyn P Borges
  2. Ragnhild SR Sætra
  3. Allen Volchuk
  4. Marit Bugge
  5. Pascal Devant
  6. Bjørnar Sporsheim
  7. Bridget R Kilburn
  8. Charles L Evavold
  9. Jonathan C Kagan
  10. Neil M Goldenberg
  11. Trude Helen Flo
  12. Benjamin Ethan Steinberg
(2022)
Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death
eLife 11:e78609.
https://doi.org/10.7554/eLife.78609
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