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. 2024 May 13;15(1):4025.
doi: 10.1038/s41467-024-47634-5.

Oxidative photocatalysis on membranes triggers non-canonical pyroptosis

Chaiheon Lee #  1   2   3 , Mingyu Park #  1   2 , W C Bhashini Wijesinghe #  1 , Seungjin Na  4 , Chae Gyu Lee  1   2 , Eunhye Hwang  1   2   3 , Gwangsu Yoon  1   2 , Jeong Kyeong Lee  1   2 , Deok-Ho Roh  1   2 , Yoon Hee Kwon  3 , Jihyeon Yang  3 , Sebastian A Hughes  5   6 , James E Vince  5   6 , Jeong Kon Seo  7   8 , Duyoung Min  9   10 , Tae-Hyuk Kwon  11   12   13   14   15
Affiliations

Oxidative photocatalysis on membranes triggers non-canonical pyroptosis

Chaiheon Lee et al. Nat Commun. .

Abstract

Intracellular membranes composing organelles of eukaryotes include membrane proteins playing crucial roles in physiological functions. However, a comprehensive understanding of the cellular responses triggered by intracellular membrane-focused oxidative stress remains elusive. Herein, we report an amphiphilic photocatalyst localised in intracellular membranes to damage membrane proteins oxidatively, resulting in non-canonical pyroptosis. Our developed photocatalysis generates hydroxyl radicals and hydrogen peroxides via water oxidation, which is accelerated under hypoxia. Single-molecule magnetic tweezers reveal that photocatalysis-induced oxidation markedly destabilised membrane protein folding. In cell environment, label-free quantification reveals that oxidative damage occurs primarily in membrane proteins related to protein quality control, thereby aggravating mitochondrial and endoplasmic reticulum stress and inducing lytic cell death. Notably, the photocatalysis activates non-canonical inflammasome caspases, resulting in gasdermin D cleavage to its pore-forming fragment and subsequent pyroptosis. These findings suggest that the oxidation of intracellular membrane proteins triggers non-canonical pyroptosis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Photocatalytic cycles of BTP for inducing pyroptosis via membrane oxidation.
a Amphiphilic molecular structure of BTP. b Photocatalytic cycles of BTP. The right circle represents the reductive quenching cycle that produces H2O2 and ∙OH, and the left circle represents oxidative quenching cycle that induces O2•− generation and amino acid oxidation. This oxidative photocatalysis triggers non-canonical pyroptosis. The inserted cell image shows the pyroptotic morphology of HeLa cells with BTP (5 μM) photocatalysis. c Quenching of the fluorescence of BTP by photoinduced electron transfer from H2O. The spectra represent the variation in BTP fluorescence with %H2O (0 − 12%) in acetonitrile. d H2O2 generation assay with DPD and horseradish peroxidase. BTP (50 μM) in normoxic PBS, Ar-bubbled PBS (hypoxic PBS), and DMSO were irradiated by the blue LED (λmax = 450 nm, 66.7 mW·cm−2) for 150 min. At 30 min intervals, the change in absorbance at 551 nm was measured to indicate H2O2 generation. e ∙OH generation assay with HPF. The results represent HPF fluorescence measured under various conditions (See method section) with/without light exposure (blue LED, λmax = 450 nm, 2 J·cm−2) (n = 3 independent experiments). f Electron paramagnetic resonance (EPR) spectroscopy with 10 mM BMPO. A spectrum of ∙OH spin adduct, BMPO-OH, was observed after BTP photocatalysis with H2O2 ([BTP] = 1 mM, [H2O2] = 10 mM) and Fenton reaction as positive control ([Fe2SO4] = 1 mM, [H2O2] = 10 mM). Data are presented as mean ± s.d. **P = 0.00049. Student’s two-tailed t test. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Membrane protein stability assay and single-molecule forced-unfolding assay.
a 15% SDS-PAGE confirming the destabilisation and aggregation of GlpG by BTP photocatalysis. The GlpG sample with 100 μM BTP added was irradiated by blue LED (λpeak = 450 nm, 30 J·cm−2). b Quantification of the GlpG destabilisation with normalised band intensity. Data are presented as mean ± s.d. ***P = 0.001. One-way ANOVA with post-hoc Turkey HSD test (n = 6 independent samples for BTP– conditions, n = 3 independent samples for BTP+ conditions). c Thermal denaturation assay of GlpGs with or without BTP photocatalysis (λpeak = 450 nm, 30 J·cm−2). Each GlpG sample was incubated at various temperatures for 10 min and then analysed by 15% SDS-PAGE (see Methods for details). d Normalised GlpG band intensity of the thermal denaturation assay at each temperature (n = 6 independent samples for BTP– conditions, n = 3 independent samples for BTP+ conditions). e Schematic diagram of single-molecule forced-unfolding assay. The lipid bilayer environment was reconstituted by the bicelle, a lipid bilayer disc composed of lipids and detergents. f Representative force-extension curves of GlpG with or without BTP photocatalysis. The repetitive force scanning of 1–50 pN allows for the observation of repetitive GlpG unfolding. Normal unfolding of GlpG was maintained more than hundred pulling cycles, whereas upon BTP addition, the unfolding forces were drastically reduced. The pulling-cycle number at which the abnormal unfolding ( < 15 pN) appears for the first time is marked as C0. g Scatter plot of unfolding forces for various conditions. The light (+) and light (–) indicates the exposure of blue light (λpeak = 450 nm, 9.16 mW·cm−2) and infrared light (λpeak = 850 nm, 39.51 mW·cm−2), respectively. The lower and upper limits of the box indicate the lower quartile (25%) and the upper quartile (75%), respectively. The central line of the box represents the median value, and each whisker extends from the box limits to the furthest data point within 1.5 times the interquartile range (IQR). The number of unfolding cycles for each condition is 118 (n = 11 molecules), 20 (n = 3 molecules), 48 (n = 3 molecules), 16 (n = 4 molecules), 36 (n = 4 molecules), 52 (n = 4 molecules), and 39 (n = 4 molecules), respectively. h Time span and number of unfolding cycles after BTP addition before the C0 point (n = 4 molecules; mean ± s.d.). Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Comprehensive proteomic profiling of membrane protein oxidation induced by BTP photocatalysis.
a Schematic illustration of the proteomic analysis workflow used to investigate the extent of oxidative modifications induced by BTP photocatalysis. The process involves a multistage search strategy for identifying O-Met and FPOP (fast photochemical oxidation of proteins) modifications. In the first search, 2,120,870 peptide-spectrum matches (PSMs) were found, and in the second search, 1,959,844 PSMs were found. Samples subjected to BTP photocatalysis and control samples were analysed and compared based on the fold change in the average precursor intensity of oxidative modifications (inset). b Proteins categorised by GO subcellular annotations into ‘membrane-specific’, located exclusively on membranes, and ‘membrane-cytosol’, found on both membranes and cytosol. The remaining cytosolic proteins were labelled ‘cytosolic’ proteins. c Volcano plot of O-Met ( + 16 Da) proteome showcasing oxidation focused on membrane-specific proteins versus cytosolic proteins. Proteins with a p-value < 0.05 and Fold Change > 2 were defined as potential oxidation targets of BTP photocatalysis. p values were calculated for Student’s one-tailed t test. d The proportions of oxidised membrane proteins across different organelles based on the O-Met proteome. ‘Others’ include plasma membranes and unidentified locations. e Overview of the 2nd search based on FPOP modifications, showing average oxidation intensities for different amino acids. ‘All AAs’ represents the aggregated intensities of oxidative modifications of these 17 amino acids. The averaged oxidation intensities of ‘membrane-specific’, ‘membrane-cytosolic’, and ‘cytosolic’ proteins for the corresponding amino acids were presented to compare the degree of oxidation of membrane proteins and soluble proteins for each type of amino acid. The averaged oxidation intensities of three control conditions were normalised to 1. f Volcano plots of the proteome other than O-Met, contrasting membrane-specific proteins with cytosolic proteins. Stricter criteria than sole O-Met analysis (p-value < 0.01 and Fold Change > 4) were applied for robust identification of oxidised proteins. p values were calculated for Student’s one-tailed t test. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Functional implication of oxidised membrane proteins by BTP photocatalysis.
a Protein quality control (PQC) related proteins was highlighted on the volcano plot of membrane proteins by their functional categories. Oxidised proteins were defined by the strict criteria (P-value < 0.01, Fold Change > 4), based on the intensity of oxidative modifications of all amino acids excluding Gly, Ser, Thr, and O-Met ( + 16 Da). b The count and percentage of proteins satisfying the oxidation criteria. c Distribution of oxidised membrane proteins across cellular organelles. ‘Others’ include plasma membranes and unidentified locations. d Heatmap comparison of highlighted protein oxidation between experimental and control conditions. e String network of the strictly defined oxidised proteins (P < 0.01, FC > 4), filtered for high interaction confidence (0.9) illustrated with GO biological processes and GO enrichment scores. Node size reflects log2FC values, and disconnected nodes were excluded from the network. Key PQC-related processes were marked in blue. All P values were calculated for Student’s one-tailed t test. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Ca2+ and K+ mobilisation by BTP photocatalysis.
a Mitochondrial Ca2+ assay performed using Rhod-2. HeLa cells were incubated with BTP (5 µM), MitoTrackerTM Deep Red FM (0.5 µM), and Rhod-2 (3 µM). The fluorescence of MitoTracker (cyan) and Rhod-2 (red) was measured using time-series confocal microscopy (t = 0–50 s, 10 s interval) during light exposure (λ = 445 nm, 0.3 mW). The fluorescence of Rhod-2 was enhanced dramatically between 20 and 30 s, implying that Ca2+ mobilisation occurred at this time. Mitochondrial matrix swelling following Ca2+ uptake was also observed after BTP photocatalysis. b Merged images of MitoTracker and Rhod-2 signals at t = 0 and 30 s. c Line-cut analysis of white arrows in (b). d Flowcytometry for Ca2+ mobilisation. HeLa cells were treated with BTP and Rhod-2, and the Rhod-2 fluorescence of each cell was measured before (hv−) and 2 h after (hv+) light exposure (λmax = 450 nm, 10 J·cm−2). e Live-SIM images of ER (red) and mitochondria (cyan) after BTP photocatalysis (λ = 488 nm, 10 mW). Mitochondrial swelling (top), fission, and fusion (bottom) were observed in HeLa cells. Arrows indicate mitochondrial fission (yellow) and fusion (white). f Mitochondrial membrane potential assay using tetramethylrhodamine, ethyl ester (TMRE). HeLa cells were incubated with BTP (10 μM) and TMRE (0.5 μM) and irradiated with blue LED light (λmax = 450 nm, 10 J·cm−2). Box plot analysis of TMRE signals from randomly selected cells (BTP+/hv+ and BTP−/hv−) (n = 18 and 21). The whiskers represent the standard deviations (s.d.), and the box represents to 25% and 75% of the s.d. g Flowcytometry of K+ efflux in HeLa cells. Intracellular K+ was measured with ION K+ Green-2, an intracellular K+ sensor, before (hv−) and 2 h after (hv+) BTP photosensitisation (λmax = 450 nm, 10 J·cm−2). Data are presented as mean ± s.d. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. BTP photocatalysis induced cytotoxicity.
a Live or dead assay with Calcein AM (green) and propidium iodide (PI, red). HeLa cells with BTP photocatalysis were stained by Calcein AM and PI 24 h after light exposure (λmax = 450 nm, 3.7 J·cm−2). The experiment was repeated three times independently, and each experiment showed similar results. b MTT assay of HeLa cells with BTP photocatalysis (λmax = 450 nm, 10 J·cm−2) (n = 4 biologically independent samples). c MTT assays for normoxic/hypoxic pancreatic cancer cells (Panc-1 and MiaPaca-2). All data are presented as mean ± s.d (n = 4 biologically independent samples). *P < 0.05. Student’s two-tailed t test. Source data are provided as a Source Data file.
Fig. 7
Fig. 7. Caspase-4/5-mediated pyroptosis by oxidative photocatalysis on membranes.
a Pyroptotic morphology changes in response to photocatalytic membrane oxidation. Yellow arrows indicate pyroptotic blebbing of dyeing HeLa cells. b Lactate dehydrogenase (LDH) release assay. Ce6 (photodynamic therapy agent) and lipopolysaccharide (LPS) + nigericin were used as positive controls for photooxidation-induced cell death and LPS-induced pyroptosis. (n = 3 biologically independent samples). c Western blots of HeLa cells with BTP photocatalysis for investigating gasdermin D (GSDMD) cleavage. BTP-treated cells were exposed to 3 or 10 J·cm−2 of light energy, and the pyroptotic media (Lane: Media) and cell lysate (Lane: Cell) were obtained individually 2 h after BTP photocatalysis. d Changes in the morphology of wild-type immortalised bone marrow-derived macrophages (WT-iBMDMs) and GSDMD knock-out iBMDM (GSDMD−/− iBMDM) in response to BTP photocatalysis. e LDH release assay for WT- and GSDMD−/− iBMDM exposed to BTP photocatalysis. (n = 4 biologically independent samples). f Western blot analysis of HeLa cells with BTP photocatalysis for investigating caspase-1/3/4/5 cleavage. g Secretion assay for ATP evaluation. (n = 3 biologically independent samples). h Western blot analysis of HeLa cells for examination of interleukin cleavages (IL-18 and IL-1β). All data are presented as mean ± s.d. **P = 0.0018. Student’s two-tailed t test. Source data are provided as a Source Data file.
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