The EMBO Journal, Journal Year: 2016, Volume and Issue: 35(16), P. 1766 - 1778

Published: July 14, 2016

Article14 July 2016Open Access Source DataTransparent process GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death Lorenzo Sborgi Biozentrum, University Basel, Switzerland Search for more papers by this author Sebastian Rühl Estefania Mulvihill orcid.org/0000-0002-7074-2371 Department Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Joka Pipercevic Rosalie Heilig Henning Stahlberg orcid.org/0000-0002-1185-4592 Christopher J Farady Novartis Institutes BioMedical Research, Forum 1, Daniel Müller Petr Broz Corresponding Author [email protected] Hiller orcid.org/0000-0002-6709-4684 Information Sborgi1,‡, Rühl1,‡, Mulvihill2, Pipercevic1, Heilig1, Stahlberg1, Farady3, Müller2, *,1 1Biozentrum, 2Department 3Novartis ‡These authors contributed equally to work *Corresponding author. Tel: +41 6126 72342; E-mail: 72082; The EMBO Journal (2016)35:1766-1778https://doi.org/10.15252/embj.201694696 See also: MM Gaidt & V Hornung (October 2016) PDFDownload PDF article text main figures. Peer ReviewDownload a summary editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures Info Abstract Pyroptosis is lytic type that initiated inflammatory caspases. These caspases are activated within multi-protein inflammasome complexes assemble in response pathogens endogenous danger signals. Pyroptotic has been proposed proceed via plasma pore, but underlying molecular remained unclear. Recently, gasdermin D (GSDMD), member ill-characterized protein family, was identified as caspase substrate an essential mediator pyroptosis. thus candidate formation. Here, we characterize function live cells vitro. We show N-terminal fragment caspase-1-cleaved rapidly targets fraction macrophages it induces pore. In vitro, recombinant tightly binds liposomes forms large permeability pores. Visualization liposome-inserted at nanometer resolution cryo-electron atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate direct final executor death. Synopsis Inflammatory induce known pyroptosis cleaving D. New findings cleaved membrane, where induction involves cleavage cellular membranes leads permeabilization. Gasdermin permeabilizes liposome after vitro caspase-1. Introduction (caspase-1, human caspase-4, caspase-5, murine caspase-11) group cysteine-dependent aspartate-directed proteases host innate immune defense. Caspase-1 termed inflammasomes, which assembled pyrin or members NOD-like receptor (NLR) PYHIN families (Latz et al, 2013; von Moltke 2013). proteins act cytosolic pattern-recognition receptors (PRRs) detect variety pathogen-associated patterns (PAMPs) signals (DAMPs). contrast, bacterial wall component lipopolysaccharide (LPS), one strongest immune-system activators, assembly "non-canonical" inflammasomes through activation caspase-5 caspase-11 (Kayagaki 2011, Hagar Shi 2014). downstream signaling pathways follow how active initiate events still poorly understood (Lamkanfi, 2011). Initial pro-inflammatory cytokine interleukin (IL)-1β key caspase-1 (Thornberry 1992). Subsequently, found caspase-1, well its orthologs caspase-4 novel programmed pathway characterized swelling, lysis, release cytoplasmic content (Fink Cookson, 2007; Kayagaki 2011; 2014), presumably result 2008). Since morphologically distinct from apoptosis intrinsically pro-inflammatory, named pyroptosis, Greek pyro (fire fever) ptosis (to fall) (Bergsbaken 2009). physiological thought be prevention intracellular pathogen replication re-expose extracellular killing mechanisms (Miao 2010). Several landmark studies have recently (gasdermin D), (He 2015; 2015). required canonical non-canonical processed caspase-11, not apoptotic (GSDMDNterm) sufficient morphological features 2015), overexpression C-terminal domain GSDMDCterm block GSDMDNterm-dependent (Shi results gave rise hypothesis caspase-dependent releases inhibitory interaction C-terminus, allowing GSDMDNterm yet undefined mechanism. had long speculated involve immediate destruction electrochemical gradient, subsequent osmotic lysis 2011), likely either promotes itself pore-forming activity (Broz, investigate functional role membrane. experiments purified liposomes. visualize microscopy. close gaps providing proof Results rupture To whether GSDMD, mediates directly, developed doxycycline-inducible system express mouse HEK293T cells. Doxycycline treatment caused harboring GSDMDNterm-expressing plasmid, vector control, concentration-dependent manner (Fig 1A). expression induced death, characteristic next measured amount LDH (lactate dehydrogenase) doxycycline-induced 1B). Increasing levels resulted increased release, indicating ectopic lysis. Microscopy analysis showed GSDMDNterm-induced is, swelling nuclear condensation. estimate size employed osmoprotection assay based on addition polyethylene glycols (PEGs) increasing weight. Addition high-molecular polymers can prevent water influx resulting if diameter agent larger than (Appendix Fig S1A). doxycycline presence PEGs readout propidium iodide (PI) staining measure 1C D). Only largest sized agent, PEG3000, able reduce partially, while smaller did Importantly, PEG3000 PI influx, does functions osmoprotectant. range 600–3,000 Da significant when added S1B), could used, since they proved cytotoxic. Figure 1. A. Cell viability assessed GFP transfected pRetroX TetOne3G-eGFP plasmid only (vector) GSDMD. Cells were treated indicated concentrations 24 h post-transfection, percentage GFP-positive determined 16 later flow cytometry. B. At 8 determined. Graphs mean s.d. quadruplicate wells. C, osmoprotectants. weights concentration 30 mM, 250 ng ml−1 h, level (C) (D) E, F. LPS-primed primary BMDMs infected log-phase S. typhimurium time points weight (numbers x-axis, mM concentration). Data information: wells (B–F) duplicate (A). *P < 0.05 Student's t-test. representative least three independent experiments. Download figure PowerPoint Infection bone marrow-derived (BMDMs) Salmonella enterica serovar Typhimurium (S. typhimurium) activates NLRC4 (Mariathasan 2004) caspase-1- GSDMD-dependent S1C BMDMs, 1E F). Consistent experiment done cells, observed small protective effect, all affected any IL-1β also partially PEG S1E); consistent observation efficient mature suggest inner over 3.5 nm, estimated (Scherrer Gerhardt, 1971). might form alternatively other define fate caspase-1-dependent followed activation, processing, immortalized wild-type 2A, Appendix S2A). p20 fragment, indicator appeared min infection supernatant macrophages. processing correlated detectable lysate supernatant. same 1F S1C). Based data, decided determine subcellular localization full-length uninfected 10 min. harvested each subjected fractionation outlined 2B). full length exclusively (S150) 2C S2B), line notion soluble, protein. After infection, very little detected S150 fraction. Instead, majority P150 P10 fraction, correlating marker Na+K+ ATPase. ATPase strongly present P10, secreted ER/Golgi pathway. mitochondrial VDAC, porin outer correlate mainly P0.7 fractions, caspase-1-mediated cleavage. interaction, isolated fractions them different treatments 2D). Conditions membrane-associated destabilize protein–protein interactions (Gatfield Pieters, 2000), such high salt (1 M NaCl) sodium carbonate (pH 11) solubilize GSDMDNterm. Extraction 0.02% digitonin, cholesterol-sequestering detergent, either. disruption low detergent SDS (0.1%) fully Consistently, extraction 1% Triton partly S2C). Thus, integrates into cholesterol-independent manner, integration associated 2. localizes Immunoblot culture supernatants α-tubulin lysates WT left (NS) 10–40 (MOI = 50). Schematic representation shown (C). Fractionation immunoblot ATPase, VDAC (voltage-dependent anion channel), HDAC1 (histone deacetylase 1), GAPDH (glycerinaldehyd-3-phosphate carried out described 4 section, equivalent loaded per lane. conditions section. available online figure. 2 [embj201694696-sup-0002-SDataFig2.zip] associates encouraged us attempt reconstitution possible established E. coli BL21(DE3) expressed yields 1.5 mg l−1 culture. Notably, following protocol, SDS–PAGE, agreement toxic effect Full-length homogeneity S3A). It elutes monodisperse, homogeneous elution peak exclusion chromatography, position expected monomeric species. Thermal denaturation melting point 43°C, folded thermally denatured. Upon incubation caspases, confirmed caspase-3 caspase-8 Recombinant native enzyme. Then, dependence 3A). About 5 nM cleaves 50% μM 40 30-kDa 22-kDa (GSDMDCterm) fragment. aqueous solution absence lipidic phase, soluble aggregates, demonstrated cross-linking 3B). cleavage, N-terminus highly cross-linked DSS (disuccinimidyl suberate), remains soluble. lipids, incubated unilamellar made 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) polar lipid extract. Ultracentrifugation allowed separation Whereas associate DMPC extract liposomes, two mimetics. 3C). Therefore, acting inhibitor solubility tag insoluble lipophilic GSDMDNterm, preventing aggregation association. Once cleaved, membranes. chemically pure compound, showing association require specific 3. Human room temperature time-dependent bands 31 kDa 22 (GSDMDCterm). Cross-linking enzymatic DSS, gel-impenetrating species highlighted arrow. cross-linked. 1 composed derived coli. (L) separated (S) ultracentrifugation 4°C 120,000 g. 3 [embj201694696-sup-0003-SDataFig3.zip] Next, asked liposome-associated transmembrane Liposomes filled self-quenching fluorophore 6-carboxyfluorescein 4). Release interior strong reduction consequently increase overall fluorescence signal, chemical X control S5). Neither nor alone dye both observed, open (≈1 nm). reaction includes kinetic steps: first step proteolytic second association, third follows classical Michaelis–Menten kinetics, steps may include additional oligomerization non-trivial dependence. reaction, kinetics constant 4A). 520 nM, so min, already 90% total signal observed. With decreasing concentration, rate decreases, nonetheless reaches 100%, permeate This conclusion breaks down 65 about permeated late points. 4. causes A–I. Dye courses maximal release. (A) Five reactions, 400 6-carboxyfluorescein-loaded prepared lipids (nM): 520, 260, 130, 100, (colored dark light orange). vertical dashed line. (B) 130 lipid, 15, 8, 5, 2.5, 1.2 blue). Three porcine brain extract, 100 green). 20-min (dark orange) blue) concentrations. Error bars shown. (E) Two sets mutant GSDMDI104N independently 60 (F) concentration. (G–I) derivates FD-20, FD-40, FD-150, Stokes diameters, indicated. For experiment, corresponding raw S5. then 1.2–15 keeping 4B). expect availability initial protease step, consequently, observe deceleration reaction. always setup, 100% measurements. 4A B), similarly eukaryotic source 4C). examined functionality I105N played discovery previously loss-of-function models generated analogous mutation I104N biochemical protocols indistinguishable S4A B). assay, concentrations, minor differences reduced however, compared 4E published promoter, although significantly less S4D). Similarly, Gsdmd-deficient restored S4C). quantitative here translate effective whole animals, our confirm deficiency o

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