Skip to main content
. 2022 Dec 5;11:e78609. doi: 10.7554/eLife.78609

Figure 5. Glycine inhibits NINJ1 clustering but not upstream pyroptosis signaling to confer cytoprotection.

(A–B) Wildtype immortalized bone marrow-derived macrophage (iBMDM) was primed with 1 μg/mL LPS for 4 hr or left unprimed before stimulation of primed cells with 20 μM nigericin for 2 hr in the presence or absence of 5 mM glycine. (A) Processing of GSDMD, caspase-1, and IL-1β was assessed by western blot. (B) Release of IL-1β into the cell culture supernatant was quantified by ELISA. ELISA results show mean ± SEM of three independent experiments. Western blots are representative of three independent experiments. ***p<0.001 and ****p<0.0001 by one-way ANOVA with Tukey’s multiple comparison correction. (C–D) Glycine dose-dependently inhibits plasma membrane rupture in mouse macrophages overexpressing NINJ1 or the N-terminal fragment of GSDMD. iBMDMs with doxycycline-inducible expression of FLAG-tagged NINJ1 (C) or the N-terminal fragment of GSDMD fused to BFP(blue fluorescent protein) (D) were incubated with 2 µg/mL doxycycline and increasing concentrations of glycine for 12 hr or 8 hr, respectively, before analyses of cell rupture (LDH release) and protein expression. Left: LDH release in supernatants relative to full lysis controls. Middle: frequencies of NINJ1-FLAG-positive or GSDMD-NT-BFP-positive cells. Right: surface expression of NINJ1-FLAG or GSDMD-NT-BFP by mean fluorescence intensity. * p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 by two-sided ANOVA with Tukey’s multiple comparison correction. (E) HeLa cells were transiently transfected with HA-tagged mouse NINJ1 in the presence or absence of glycine (5 mM). Native-PAGE analysis of ectopically expressed NINJ1 demonstrates a shift to high molecular weight aggregate, which is abrogated by glycine treatment. (F) Large unilamellar vesicles (LUVs; gray circles) were made containing 25 mM carboxyfluorescein (CF) at which the CF fluorescence self-quenches. NINJ1 α-helix peptide (corresponding to amino acids 40–69 of human NINJ1) is added to the LUV suspension. Ruptured LUV release CF, which no longer self-quenches. The resulting increase in fluorescence is monitored using a spectrofluorometer. Detergent is added to rupture all remaining liposomes to capture the maximum attainable fluorescence. LUV rupture by N-terminus NINJ1 α-helix without and with glycine (50 mM), scrambled NINJ1 α-helix peptide, or the cytolytic yeast peptide candidalysin compared to vehicle. Glycine does not prevent LUV rupture by the N-terminal NINJ1 α-helix. * p<0.05 by ANOVA with Tukey’s multiple comparison correction.

Figure 5—source data 1. Numerical values for experimental data plotted in Figure 5.

Figure 5.

Figure 5—figure supplement 1. Characterization of the inducible immortalized bone marrow-derived macrophage (iBMDM)-doxy-moNINJ1 system.

Figure 5—figure supplement 1.

Two clonal cell lines expressing FLAG-NINJ1 under a doxy-inducible promoter or the parental Cas9 + iBMDM cell line were treated with 2 µg/mL of doxycycline for the indicated times. (A) Expression of FLAG-NINJ1 in whole cell lysates was analyzed by western blot. (B) Cell membrane rupture was assessed by LDH assay. (C) Membrane permeabilization over time was analyzed by PI(propidium iodide) staining. (D–F) Cell surface expression of FLAG-NINJ1 and plasma membrane permeabilization (Sytox Blue positive) was analyzed by flow cytometry.
Figure 5—figure supplement 1—source data 1. Numerical values for experimental data plotted in Figure 5—figure supplement 1.
Figure 5—figure supplement 2. Circular dichroism (CD) spectroscopy of human NINJ1 peptide reveals a stable α-helical secondary structure insensitive to glycine.

Figure 5—figure supplement 2.

(A) CD spectrum of NINJ1 amino acids 40–69 (HYASKKSAAESMLDIALLMANASQLKAVVE) shows the characteristic pattern of an α-helix with two downward deflections at 207 nm and 222 nm. (B) CD spectra of the human NINJ1 α-helix co-incubated with glycine titrated between 0 and 20 mM indicate that the helical secondary structure is generally insensitive to glycine.
Figure 5—figure supplement 2—source data 1. Numerical values for experimental data plotted in Figure 5—figure supplement 2.