GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death
2016; Springer Nature; Volume: 35; Issue: 16 Linguagem: Inglês
10.15252/embj.201694696
ISSN1460-2075
AutoresLorenzo Sborgi, Sebastian Rühl, Estefania Mulvihill, Joka Pipercevic, Rosalie Heilig, Henning Stahlberg, Christopher J. Farady, Daniel J. Müller, Petr Brož, Sebastian Hiller,
Tópico(s)Streptococcal Infections and Treatments
ResumoArticle14 July 2016Open Access Source DataTransparent process GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death Lorenzo Sborgi Lorenzo Sborgi Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Sebastian Rühl Sebastian Rühl Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Estefania Mulvihill Estefania Mulvihill orcid.org/0000-0002-7074-2371 Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland Search for more papers by this author Joka Pipercevic Joka Pipercevic Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Rosalie Heilig Rosalie Heilig Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Henning Stahlberg Henning Stahlberg orcid.org/0000-0002-1185-4592 Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Christopher J Farady Christopher J Farady Novartis Institutes for BioMedical Research, Forum 1, Basel, Switzerland Search for more papers by this author Daniel J Müller Daniel J Müller Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland Search for more papers by this author Petr Broz Corresponding Author Petr Broz [email protected] Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Sebastian Hiller Corresponding Author Sebastian Hiller [email protected] orcid.org/0000-0002-6709-4684 Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Lorenzo Sborgi Lorenzo Sborgi Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Sebastian Rühl Sebastian Rühl Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Estefania Mulvihill Estefania Mulvihill orcid.org/0000-0002-7074-2371 Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland Search for more papers by this author Joka Pipercevic Joka Pipercevic Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Rosalie Heilig Rosalie Heilig Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Henning Stahlberg Henning Stahlberg orcid.org/0000-0002-1185-4592 Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Christopher J Farady Christopher J Farady Novartis Institutes for BioMedical Research, Forum 1, Basel, Switzerland Search for more papers by this author Daniel J Müller Daniel J Müller Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland Search for more papers by this author Petr Broz Corresponding Author Petr Broz [email protected] Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Sebastian Hiller Corresponding Author Sebastian Hiller [email protected] orcid.org/0000-0002-6709-4684 Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Author Information Lorenzo Sborgi1,‡, Sebastian Rühl1,‡, Estefania Mulvihill2, Joka Pipercevic1, Rosalie Heilig1, Henning Stahlberg1, Christopher J Farady3, Daniel J Müller2, Petr Broz *,1 and Sebastian Hiller *,1 1Biozentrum, University of Basel, Basel, Switzerland 2Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland 3Novartis Institutes for BioMedical Research, Forum 1, Basel, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: +41 6126 72342; E-mail: [email protected] *Corresponding author. Tel: +41 6126 72082; E-mail: [email protected] The EMBO Journal (2016)35:1766-1778https://doi.org/10.15252/embj.201694696 See also: MM Gaidt & V Hornung (October 2016) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi-protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill-characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro. We show that the N-terminal fragment of caspase-1-cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N-terminal fragment of caspase-1-cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome-inserted GSDMD at nanometer resolution by cryo-electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death. Synopsis Inflammatory caspases induce a lytic type of cell death known as pyroptosis by cleaving the protein gasdermin D. New findings show that cleaved gasdermin D targets the plasma membrane, where it forms a large permeability pore. Pyroptosis induction involves the cleavage of gasdermin D by inflammatory caspases. The N-terminal fragment of gasdermin D targets cellular membranes and leads to plasma membrane permeabilization. Gasdermin D targets and permeabilizes liposome membranes after in vitro cleavage by caspase-1. Introduction Inflammatory caspases (caspase-1, human caspase-4, human caspase-5, and murine caspase-11) are a group of cysteine-dependent aspartate-directed proteases that is essential for host innate immune defense. Caspase-1 is activated within large multi-protein complexes termed inflammasomes, which are assembled by the protein pyrin or members of the NOD-like receptor (NLR) and PYHIN protein families (Latz et al, 2013; von Moltke et al, 2013). These proteins act as cytosolic pattern-recognition receptors (PRRs) and detect a variety of pathogen-associated molecular patterns (PAMPs) or endogenous danger signals (DAMPs). In contrast, the bacterial cell wall component lipopolysaccharide (LPS), one of the strongest immune-system activators, leads to the assembly of "non-canonical" inflammasomes through activation of caspase-4, caspase-5 or caspase-11 (Kayagaki et al, 2011, 2013; Hagar et al, 2013; Shi et al, 2014). The downstream signaling pathways that follow the activation of inflammatory caspases and how the active caspases initiate these events are still poorly understood (Lamkanfi, 2011). Initial work has identified the pro-inflammatory cytokine interleukin (IL)-1β as a key substrate of caspase-1 (Thornberry et al, 1992). Subsequently, it was found that caspase-1, as well as caspase-11 and its human orthologs caspase-4 and caspase-5, induces a novel programmed cell death pathway that is characterized by cell swelling, lysis, and the release of cytoplasmic content (Fink & Cookson, 2007; Kayagaki et al, 2011; Shi et al, 2014), presumably as a result of the formation of membrane pores (Fink et al, 2008). Since this type of cell death is morphologically distinct from apoptosis and intrinsically pro-inflammatory, it was named pyroptosis, from the Greek pyro (fire or fever) and ptosis (to fall) (Bergsbaken et al, 2009). The physiological function of pyroptosis is thought to be the prevention of intracellular pathogen replication and to re-expose pathogens to extracellular killing mechanisms (Miao et al, 2010). Several landmark studies have recently identified GSDMD (gasdermin D), a member of the gasdermin protein family, as an essential mediator of pyroptosis in human and murine cells (He et al, 2015; Kayagaki et al, 2015; Shi et al, 2015). GSDMD is required for pyroptosis induction after canonical and non-canonical inflammasome activation and is processed by caspase-1, caspase-11, caspase-4, and caspase-5, but not by apoptotic caspases. The N-terminal fragment of GSDMD (GSDMDNterm) was found sufficient to induce cell death with the morphological features of pyroptosis (Kayagaki et al, 2015; Shi et al, 2015), and overexpression of the C-terminal domain GSDMDCterm was found to block GSDMDNterm-dependent cell death (Shi et al, 2015). These results gave rise to the hypothesis that caspase-dependent cleavage releases the N-terminal domain from an inhibitory interaction with the C-terminus, allowing GSDMDNterm to induce cell death by a yet undefined mechanism. Since pyroptosis had long been speculated to involve the formation of a plasma membrane pore, immediate destruction of the electrochemical gradient, and subsequent osmotic lysis of the host cell (Lamkanfi, 2011), it is likely that GSDMDNterm either promotes the formation of this pore or itself has pore-forming activity (Broz, 2015). Here, we investigate the functional role of GSDMDNterm in live cells and in vitro. We demonstrate that after cleavage by caspase-1, GSDMDNterm targets cellular membranes and that it induces the formation of a large permeability pore in the plasma membrane. In vitro experiments with purified recombinant GSDMD show that GSDMDNterm forms large pores in liposomes. We visualize these with cryo-electron and atomic force microscopy. Overall, these results close the gaps in the pyroptotic signaling pathway by providing the proof that GSDMD is the final and direct executor of pyroptotic cell death. Results The N-terminal GSDMD fragment induces the formation of a large plasma membrane pore Pyroptotic cell death involves the formation of a plasma membrane pore, cell swelling, and rupture of the plasma membrane. To investigate whether GSDMD, a recently identified mediator of pyroptosis, mediates pore formation directly, we developed a doxycycline-inducible system to express the N-terminal fragment (GSDMDNterm) of mouse GSDMD in HEK293T cells. Doxycycline treatment caused cell death in HEK293T cells harboring the GSDMDNterm-expressing plasmid, but not in cells harboring a vector control, in a concentration-dependent manner (Fig 1A). To characterize whether GSDMDNterm expression induced a lytic type of cell death, characteristic for pyroptosis, we next measured the amount of LDH (lactate dehydrogenase) release after doxycycline-induced expression of the GSDMDNterm (Fig 1B). Increasing levels of GSDMDNterm expression resulted in increased levels of LDH release, indicating that ectopic expression of the GSDMDNterm induced death through cell lysis. Microscopy analysis showed that GSDMDNterm-induced death had the morphological features of pyroptosis, that is, cell swelling and nuclear condensation. To estimate the size of the GSDMDNterm-induced plasma membrane pore, we next employed an osmoprotection assay based on the addition of polyethylene glycols (PEGs) of increasing molecular weight. Addition of these high-molecular polymers can prevent water influx through pores and the resulting swelling and osmotic lysis, if the molecular diameter of the agent is larger than the diameter of the pore (Appendix Fig S1A). We induced GSDMDNterm expression by doxycycline addition in HEK293T cells in the presence of PEGs and measured LDH release as a readout for osmotic lysis and propidium iodide (PI) staining as a measure of plasma membrane pore formation (Fig 1C and D). Only the largest sized agent, PEG3000, was able to reduce LDH release partially, while smaller PEGs did not reduce cell lysis. Importantly, PEG3000 did not prevent PI influx, indicating that it does not block the pore directly, but functions as an osmoprotectant. PEGs in the range of 600–3,000 Da did not induce significant levels of cell death when added to cells (Appendix Fig S1B), but larger PEGs could not be used, since they proved to be cytotoxic. Figure 1. GSDMDNterm induces the formation of a large plasma membrane pore A. Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TetOne3G-eGFP plasmid only (vector) or pRetroX TetOne3G-eGFP harboring the N-terminal fragment of GSDMD. Cells were treated with the indicated concentrations of doxycycline 24 h post-transfection, and the percentage of GFP-positive cells was determined 16 h later by flow cytometry. B. LDH release from HEK293T cells transfected with the pRetroX TetOne3G-eGFP plasmid only (vector) or pRetroX TetOne3G-eGFP harboring the N-terminal fragment of GSDMD. At 24 h post-transfection, cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined. Graphs show mean and s.d. of quadruplicate wells. C, D. PI staining of and LDH release from HEK293T cells transfected with pRetroX TetOne3G-eGFP harboring the N-terminal fragment of GSDMD in the presence of osmoprotectants. At 24 h post-transfection, PEGs of the indicated molecular weights were added to a final concentration of 30 mM, cells were treated with 250 ng ml−1 doxycycline for 8 h, and the level of PI staining (C) or LDH release (D) was determined. E, F. PI staining of and LDH release from LPS-primed primary BMDMs infected with log-phase S. typhimurium for the indicated time points in the presence of PEGs of the indicated molecular weight (numbers on the x-axis, 30 mM final concentration). Data information: Graphs show mean and s.d. of quadruplicate wells (B–F) or the mean and s.d. of duplicate wells (A). *P < 0.05 as determined by Student's t-test. Data are representative of at least three independent experiments. Download figure Download PowerPoint Infection of primary murine bone marrow-derived macrophages (BMDMs) with Salmonella enterica serovar Typhimurium (S. typhimurium) activates the NLRC4 inflammasome (Mariathasan et al, 2004) and results in caspase-1- and GSDMD-dependent pyroptosis and cytokine release (Appendix Fig S1C and D). To estimate the size of the GSDMD-dependent plasma membrane pore in BMDMs, we measured cell lysis as a function of time in the presence of PEGs of increasing size (Fig 1E and F). Consistent with the osmoprotection experiment done in HEK293T cells, we observed that only PEG3000 had a small protective effect, while all smaller PEGs did not prevent pyroptosis. PI influx was not affected by any of the osmoprotectants. IL-1β release was also partially affected by PEG treatment (Appendix Fig S1E); consistent with the observation that pyroptosis is required for efficient release of the mature cytokine in BMDMs (Shi et al, 2015). Overall, these experiments suggest that GSDMD-dependent pyroptosis involves the formation of a plasma membrane pore with an inner diameter of over 3.5 nm, the estimated molecular size of PEG3000 (Scherrer & Gerhardt, 1971). The N-terminal GSDMD fragment targets cellular membranes GSDMDNterm might itself form a pore in the plasma membrane or alternatively initiate other events that result in pore formation (Broz, 2015). To define the fate of GSDMDNterm after caspase-1-dependent cleavage of GSDMD, we followed caspase-1 activation, GSDMD processing, and cell death over time in immortalized wild-type macrophages infected with S. typhimurium (Fig 2A, Appendix Fig S2A). The processed caspase-1 p20 fragment, an indicator of caspase-1 activation, appeared within 20 min after infection in the supernatant of macrophages. GSDMD processing correlated with caspase-1 activation and was detectable in the cell lysate as well as in the cell supernatant. LDH release was also detectable at the same time points (Fig 1F and Appendix Fig S1C). Based on these data, we decided to determine the subcellular localization of full-length GSDMD and GSDMDNterm in either uninfected cells or in cells infected with S. typhimurium for 10 and 20 min. Cells were harvested at each of these time points and subjected to subcellular fractionation as outlined (Fig 2B). GSDMD full length was exclusively found in the cytosolic fraction (S150) in uninfected cells (Fig 2C and Appendix Fig S2B), in line with the notion that it is a soluble, cytosolic protein. After infection, full-length GSDMD, but very little GSDMDNterm was detected in the S150 fraction. Instead, the majority of GSDMDNterm was found in the P150 fraction and in the P10 fraction, correlating with the presence of the plasma membrane marker Na+K+ ATPase. The Na+K+ ATPase was also strongly present in the P10, presumably since it is secreted via the ER/Golgi pathway. The mitochondrial marker VDAC, a porin of the outer mitochondrial membrane, did not correlate with the GSDMDNterm and was mainly found in the P0.7 and P10 fractions, but not in the P150 fraction. Overall, these results suggest that the GSDMDNterm targets membranes after caspase-1-mediated cleavage. To characterize the interaction, we isolated plasma membrane fractions of BMDMs after S. typhimurium infection and subjected them to different treatments (Fig 2D). Conditions known to release membrane-associated proteins or destabilize protein–protein interactions (Gatfield & Pieters, 2000), such as high salt (1 M NaCl) and sodium carbonate (pH 11) did not solubilize GSDMDNterm. Extraction of the plasma membrane with 0.02% digitonin, a cholesterol-sequestering detergent, did not solubilize GSDMDNterm either. Only the disruption of the membrane with low concentrations of the detergent SDS (0.1%) was able to fully solubilize GSDMDNterm. Consistently, extraction of BMDMs membranes with 1% Triton was also able to partly solubilize GSDMDNterm (Appendix Fig S2C). Thus, GSDMDNterm integrates into cellular membranes in a cholesterol-independent manner, and this integration is associated with formation of a pore and cell lysis. Figure 2. GSDMDNterm localizes to cellular membranes after inflammasome activation Immunoblot analysis of cleaved GSDMD in culture supernatants and full-length GSDMD, cleaved GSDMD and α-tubulin in the cell lysates of immortalized LPS-primed WT macrophages left uninfected (NS) or infected for 10–40 min with log-phase S. typhimurium (MOI = 50). Schematic representation of the subcellular fractionation shown in (C). Fractionation and immunoblot analysis for GSDMD, Na+K+ ATPase, VDAC (voltage-dependent anion channel), HDAC1 (histone deacetylase 1), and GAPDH (glycerinaldehyd-3-phosphate dehydrogenase) of WT macrophages infected for 10 and 20 min with log-phase S. typhimurium (MOI = 50). Fractionation was carried out as described in the 4 section, and equivalent amount of protein was loaded per lane. Extraction of cleaved GSDMD from isolated membranes of WT macrophages infected for 10 min with log-phase S. typhimurium (MOI = 50). Extraction was carried in variable conditions as described in the 4 section. Source data are available online for this figure. Source Data for Figure 2 [embj201694696-sup-0002-SDataFig2.zip] Download figure Download PowerPoint The N-terminal fragment of GSDMD associates to liposomes in vitro These findings encouraged us to attempt the reconstitution of a possible pore-forming function of GSDMDNterm in vitro. We established recombinant expression for full-length human GSDMD in E. coli BL21(DE3) expression cells. The protein expressed well with yields of 1.5 mg l−1 cell culture. Notably, following the same protocol, GSDMDNterm did not express in E. coli BL21(DE3) cells to detectable levels by SDS–PAGE, in agreement with the hypothesis that the protein might have a toxic effect on the host cells. Full-length GSDMD was isolated and purified to homogeneity (Appendix Fig S3A). It elutes as a monodisperse, homogeneous elution peak from size exclusion chromatography, at the position of the expected monomeric species. Thermal denaturation showed a melting point of 43°C, indicating that the protein is folded and can be thermally denatured. Upon incubation with different human caspases, we confirmed that recombinant GSDMD is cleaved by caspase-1, but not by the apoptotic caspase-3 or caspase-8 (Shi et al, 2015). Recombinant GSDMD is thus a functional substrate of its native enzyme. Then, we characterized the time dependence of GSDMD cleavage (Fig 3A). About 5 nM of caspase-1 cleaves more than 50% of 2 μM of GSDMD in 40 min. GSDMD cleavage by caspase-1 results in a 30-kDa N-terminal (GSDMDNterm) and a 22-kDa C-terminal (GSDMDCterm) fragment. In aqueous solution in the absence of a lipidic phase, GSDMDNterm is not soluble and forms aggregates, as demonstrated in a cross-linking experiment (Fig 3B). After cleavage, the N-terminus is highly cross-linked by DSS (disuccinimidyl suberate), while the 22-kDa GSDMDCterm remains soluble. To determine whether the poorly soluble GSDMDNterm associates with lipids, GSDMD was incubated in the presence or absence of active caspase-1 with unilamellar liposomes made of either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or from E. coli polar lipid extract. Ultracentrifugation allowed for separation of the liposomes from the soluble fraction. Whereas full-length GSDMD did not associate with either the DMPC or the E. coli polar extract liposomes, the GSDMDNterm fully associated with either of the two membrane mimetics. GSDMDCterm did not associate with the liposomes (Fig 3C). Therefore, the soluble GSDMDCterm domain is acting in full-length GSDMD not only as an inhibitor of GSDMDNterm but also as a solubility tag for the intrinsically insoluble and lipophilic GSDMDNterm, preventing it from aggregation and membrane association. Once cleaved, GSDMDNterm associates strongly to available membranes. DMPC liposomes are made from a chemically pure compound, showing that membrane association by GSDMDNterm does not require any specific receptors in the membrane. Figure 3. GSDMDNterm targets liposomes after caspase-1 cleavage Human GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. The protein was cleaved in a time-dependent manner into two bands of 31 kDa (GSDMDNterm) and 22 kDa (GSDMDCterm). Cross-linking experiment of full-length and cleaved GSDMD. GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. After enzymatic cleavage, GSDMDNterm is highly cross-linked by DSS, resulting in the gel-impenetrating species highlighted by the arrow. GSDMDCterm is not cross-linked. GSDMD at a concentration of 1 μM was incubated at room temperature with 5 nM caspase-1 and liposomes composed of 4 mM DMPC or polar lipid extract derived from E. coli. After 2 h, the lipid fraction (L) was separated from the supernatant (S) by ultracentrifugation at 4°C for 1 h at 120,000 g. Source data are available online for this figure. Source Data for Figure 3 [embj201694696-sup-0003-SDataFig3.zip] Download figure Download PowerPoint The N-terminal fragment of GSDMD forms pores in liposomes Next, we asked whether liposome-associated GSDMDNterm forms transmembrane pores. Liposomes filled with a self-quenching concentration of the fluorophore 6-carboxyfluorescein were incubated with recombinant full-length GSDMD in the presence or absence of active caspase-1 (Fig 4). Release of the fluorophore from the liposome interior results in a strong reduction in the concentration-dependent self-quenching effect and consequently in an increase of the overall fluorescence signal, as demonstrated by chemical rupture of the liposomes with the detergent Triton X in a control experiment (Appendix Fig S5). Neither caspase-1 nor full-length GSDMD alone was able to release dye from the liposomes, but in the presence of both GSDMD and caspase-1, dye release was observed, indicating the formation of permeability pores with open diameters of at least the molecular size of 6-carboxyfluorescein (≈1 nm). The dye release reaction from liposomes includes at least three kinetic steps: the first step is the proteolytic cleavage of GSDMD by caspase-1, the second step is the membrane association, and the third step is pore formation of GSDMDNterm. Whereas the first step follows classical Michaelis–Menten kinetics, the reaction mechanisms of the second and third steps may include additional oligomerization steps with non-trivial concentration dependence. In an attempt to visualize the concentration dependence of the overall reaction, we measured the kinetics of dye release as a function of GSDMD concentration at a constant caspase-1 concentration of 5 nM (Fig 4A). At a GSDMD concentration of 520 nM, the dye release is very efficient so that in 20 min, already more than 90% of the total fluorescence signal is observed. With decreasing GSDMD concentration, the overall reaction rate decreases, but the overall dye release nonetheless reaches 100%, showing that sufficient GSDMD is available to permeate all liposomes. This conclusion breaks down at a GSDMD concentration of 65 nM, where only about 50% of the liposomes are permeated at late time points. Figure 4. GSDMDNterm causes liposome permeability by pore formation A–I. Dye release time courses from liposomes as a percentage of maximal release. (A) Five different reactions, where 5 nM caspase-1 and 400 μM 6-carboxyfluorescein-loaded liposomes prepared with E. coli polar lipids were incubated with GSDMD concentrations of (nM): 520, 260, 130, 100, 65 (colored dark to light orange). The time point of 20 min is highlighted by a vertical dashed line. (B) Five different reactions, where 130 nM of GSDMD and 400 μM 6-carboxyfluorescein-loaded liposomes prepared with E. coli extract polar lipid, were incubated with caspase-1 concentration of (nM): 15, 8, 5, 2.5, 1.2 (colored dark to light blue). The time point of 20 min is highlighted by a vertical dashed line. (C) Three different reactions, where 5 nM caspase-1 and 400 μM 6-carboxyfluorescein-loaded liposomes prepared with porcine brain total lipid extract, were incubated with GSDMD concentrations of (nM): 520, 260, 100 (colored dark to light green). (D) Dye release at 20-min reaction as a function of GSDMD (dark orange) and caspase-1 (dark blue) concentrations. Error bars for three independent experiments are shown. (E) Two different sets of reactions, where wild-type GSDMD (dark to light orange) and the mutant GSDMDI104N (dark to light blue) were independently incubated at the concentrations of 260, 130, and 65 nM with 5 nM caspase-1 and 400 μM 6-carboxyfluorescein-loaded liposomes. The time point of 60 min is highlighted by a vertical dashed line. (F) Dye release at 60 min of reaction as a function of GSDMD wild-type (dark orange) and GSDMDI104N (dark blue) concentration. Error bars for three independent experiments are shown. (G–I) Dye release from 400 μM liposomes loaded with the 6-carboxyfluorescein derivates FD-20, FD-40, and FD-150, with variable Stokes diameters, as indicated. 130 nM of GSDMD and 5 nM caspase-1 were incubated with the liposomes. For each experiment, a representative from three independent experiments is shown. The corresponding raw data are shown in Appendix Figure S5. Download figure Download PowerPoint We then measured the kinetics of dye release as a function of the caspase-1 concentration in the range 1.2–15 nM, while keeping the GSDMD concentration constant at 130 nM (Fig 4B). In this experiment, by decreasing the concentration of caspase-1, we expect a reduction of the availability of cleaved GSDMDNterm by the initial protease cleavage step, and consequently, we observe a deceleration of the dye release reaction. The total amount of GSDMD is always sufficient to permeate all liposomes in the setup, and consequently, we observe 100% dye release levels in all measurements. Importantly, GSDMD did not only permeate liposomes made of bacterial lipid extract (Fig 4A and B), but similarly also liposomes from a eukaryotic source (Fig 4C). In a next experiment, we examined the functionality of the I105N mutant of GSDMD. This mutant had played a key role in the discovery of GSDMD, since it had previously been identified as a loss-of-function mutant in mouse models (Kayagaki et al, 2015). We generated the analogous mutation I104N in GSDMD and expressed and purified the mutant protein with the same biochemical protocols as the wild-type protein. GSDMDI104N is cleaved by caspase-1 with kinetics indistinguishable from wild-type GSDMD (Appendix Fig S4A and B). In the dye release assay, at high protein concentrations, GSDMDI104N is able to form functional pores with only minor differences to the wild-type protein. At reduced proteins concentrations, however, it showed reduced activity compared to the wild-type protein (Fig 4E and F). Consistent with these data and previously published work (Kayagaki et al, 2015), we found that GSDMDI104N could also induce cell death of HEK293T cells when expressed by a doxycycline-inducible promoter, although significantly less than the WT protein (Appendix Fig S4D). Similarly, expression of the I104N mutant of human GSDMD in immortalized Gsdmd-deficient mouse macrophages partially restored pyroptosis after Salmonella infection when compared to wild-type human GSDMD (Appendix Fig S4C). The quantitative differences observed here may well translate into an effective loss-of-function effect in whole animals, and our experiments thus confirm the functional deficiency o
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