Ubiquitination of gasdermin D N-terminal domain directs its membrane translocation and pore formation during pyroptosis

Ubiquitination of gasdermin D N-terminal domain directs its membrane translocation and pore formation during pyroptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ubiquitination of gasdermin D N-terminal domain directs its membrane translocation and pore formation during pyroptosis Xiufeng Chu, Pengyuan Zheng, Bukhari Ihtisham This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4907061/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2025 Read the published version in Cell Death & Disease → Version 1 posted You are reading this latest preprint version Abstract Gasdermin D (GSDMD) is a critical mediator of pyroptosis, which consists of a N-terminal pore-forming domain and a C-terminal autoinhibitory domain. The free N-terminal domain (GD-NT), which is released through caspase-1/11 cleavage, exhibits distinct features from the full-length GSDMD (GD-FL), including oligomerization, membrane translocation and pore-formation. However, the underlying mechanisms are not well clarified. Here, we found that GD-NT, but GD-FL, was massively ubiquitinated in cells. The K63-linked polyubiquitination of GD-NT at Lys236/237 (human/mouse), catalyzed by TRAF1, directly its membrane translocation and pore-formation during pyroptosis. Inhibition of GD-NT ubiquitination via site mutation or the UBA1 inhibitor PYR-41 suppressed cell death in several pyroptosis cell models. Additionally, the application of PYR-41 in septic mice efficiently suppressed the release of IL-18 and TNF-⍺. Thus, GD-NT ubiquitination is a key regulatory mechanism controlling its membrane localization and activation, which may provide a novel target for modulating immune activity in pyroptosis-related diseases. Biological sciences/Immunology/Cell death and immune response Health sciences/Diseases/Infectious diseases Biological sciences/Molecular biology/Epigenetics Biological sciences/Cell biology/Proteolysis/Ubiquitylation Gasdermin D pyroptosis Ubiquitination PYR-41 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pyroptosis is a unique cell death featured by pore formation in the plasma membrane and subsequent cell swelling 1 . The release of cellular contents during pyroptosis makes it an inflammatory and immunogenic cell death (ICD). With these attributes, pyroptosis plays a pivotal role in the defense against pathogenic organism as well as cancer 2 , 3 . In some cases, however, it participates in the development of pathological conditions 4 . Pyroptosis are strongly associated with sepsis 5 , Cryopyrin-Associated Periodic Syndromes (CAPS) 6 , Macrophage Activation Syndrome (MAS) 7 , and moderately related with type 2 diabetes 8 , 9 , obesity 10 , myocarditis 11 , 12 , atherosclerotic diseases 13 , gouty arthritis 14 – 16 and neurological diseases 17 . GSDMD is the first gasdermin that is identified as a pyroptosis mediator 18 – 20 . In response to canonical or noncanonical inflammasome activators, the activated inflammatory caspases (specifically, caspase-1/4/5 in human, caspase-1/11 in mouse) cleave GD-FL to release the bioactive pore-forming GD-NT fragment (the liberation of GD-NT) 21 – 25 . Not like the GD-FL that exists in monomer state and localizes in cytoplasm, the liberated GD-NT undergoes self-oligomerization, integrating into plasma membrane and creating pores 24 , 26 , 27 . However, the mechanisms underlying the distinct biological activities between GD-FL and GD-NT are not fully understood. Ubiquitination is a critical PTMs in the regulation of inflammatory cell death by regulating the key components of NF-κB and inflammasome signaling pathways, such as NEMO, RIPK1, ASC, NLRP3, caspase-11 and IL-1beta 28 – 34 (Extended Fig. 1 ). Recent studies suggest the role of ubiquitination in the functionality of gasdermin family. Shigella produces pathogenic ubiquitination ligase to degrade GSDMB/GSDMD and deactivate the host defensive system 35 , 36 . Besides, both SYVN1-mediated ubiquitination of GSDMD and OTUD4-mediated deubiquitination of GSDME promote the happening of pyroptosis 37 , 38 , indicating that the host ubiquitination system also contributes to the regulation of gasdermin function. Here, we report that the ubiquitination at Lys237 of GD-NT (but not GD-FL) is a prerequisite mechanism of GSDMD activation. This process enables GD-NT to translocate and form pores in plasma membrane, thereby causing pyroptotic cell death. Our current study identified a novel molecular mechanism of the liberated GD-NT acquiring pyroptotic ability. Results GD-NT, but not GD-FL, possesses K63-linked polyubiquitination In order to investigate the ubiquitination of GSDMD, the vectors expressing GD-FL, GD-NT, GD-CT or caspase-11 were constructed (Fig. 1 A, B). We transfected Flag-GD-FL/caspase-11 into HEK293 cells and performed IP & immunoblot assay. Interestingly, the result showed that GSDMD was slightly ubiquitinated when it was introduced into cell alone. But the ubiquitination level of GSDMD was significantly increased when it was co-transfected with caspase-11 (Fig. 1 C). To further ensure it, we adopted a more rigorous ubiquitination detection method, in which we denatured the cellular proteins using guanidine and subjected to NI/NTA pulldown (Fig. 1 D). Immunoblot (IB) analysis of the precipitants revealed the same ubiquitination pattern as shown in Fig. 1 C. We supposed that the alteration of ubiquitination may take place during GSDMD processing by caspase-11. To test this hypothesis, we independently transfected Flag-GD-FL, Flag-GD-NT or Flag-GD-CT into HEK293 cells. 18 hours later, the cells were subjected to IP assay. IB analysis of the precipitants revealed that GD-NT had a distinct ubiquitination status from GD-FL (Fig. 1 E). Later on, we performed GST-pulldown and NI/NTA pulldown assay under denatured conditions. Consistently, these assays also indicated that GD-NT, but not GD-FL or GD-CT, was massively ubiquitinated (Fig. 1 F, G). To further identify the conjugation form of ubiquitin on GD-NT, we transfected Flag-GD-NT along with either Ub-WT or Ub-K63 only into HEK293 cells and then performed NI/NTA pulldown assay. The result showed that GD-NT was conjugated with K63-linked polyubiquitin chains (Fig. 1 H). Thus, our data suggest that GD-NT, but not GD-FL, is massively modified with K63-ubiquitination. The K63-linked polyubiquitination of GD-NT is regulated by TRAF1-OTUB1 axis Ubiquitination is a dynamic and reversible process, where E3 ligases and deubiquitinases (DUB) catalyze the conjugation and removal of ubiquitin on substrate proteins, respectively 39 , 40 . Additionally, E3 ligase-DUB axis exhibits its specificity by determining the target proteins and their ubiquitination forms. To identify the E3 ligase responsible for the K63-linked ubiquitination of GD-NT, we transfected Flag-GD-NT along with various E3 ligases into HEK293 cells and conducted IP assay. IB analysis of the precipitants indicated that Flag-GD-NT interacted with TRAF1 (Fig. 2 A, B). To assess the influence of TRAF1 on GD-NT ubiquitination, we transfected either TRAF1 or shTRAF1 together with Ub-K63 only and Flag-GD-NT into HEK293 cells, followed by NI/NTA pulldown assay. Our results revealed that TRAF1 significantly enhanced the K63-linked ubiquitination of GD-NT, whereas the ubiquitination level was downregulated by shTRAF1 (Fig. 2 C, D). Interestingly, although the interaction of GD-NT with TRAF2 was not observed in our experiments (Fig. 2 A, B), TRAF2 still enhanced the K63-linked ubiquitination of GD-NT (the last two lanes in Fig. 2 C). This phenomenon, we believe, may be attributed to the formation of a heterodimer between TRAF1 and TRAF2 41 , or possibly involving another factor that we have yet to identify. Next, to identify the DUB responsible for the K63-linked ubiquitination of GD-NT, we transfected Flag-GD-NT along with different DUBs into HEK293 cells and performed IP assay. The result showed that Flag-GD-NT interacted with both OTUB1 and OTUB2 (Fig. 2 E). Subsequently, either Ub-WT or Ub-K63 only were co-transfected with HA-GD-NT and/or OTUB1/2, followed by NI/NTA pulldown. IB analysis of the precipitants showed that, although both OTUB1 and OTUB2 affected the ubiquitination of GD-NT, only OTUB1 significantly reduced the K63-linked ubiquitination of GD-NT (Fig. 2 F, G). In consistent with this finding, shRNA-mediated silencing of OTUB1 significantly increased the K63-linked ubiquitination of GD-NT (Fig. 2 H). Therefore, our data suggest that the K63-linked ubiquitination of GD-NT is regulated by the TRAF1-OTUB1 axis. GD-NT possesses the K63-linked polyubiquitination through its Lys237 To identify the ubiquitin conjugation sites on GD-NT, we searched the online PhosphoSitePlus® database (PSP, https://www.phosphosite.org ), which provides comprehensive information for the study of ubiquitination as well as other mammalian post-translational modifications (PTMs) 42 . Based on Low ThroughPut and High ThroughPut datasets obtained from PSP database 38 , 43 , several amino acids were considered as the potential ubiquitinated sites, including Lys204, Lys205, and Lys237 in mouse GSDMD (refer to Lys203, Lys204, and Lys236 in human). Sequence alignment of GSDMD among different species revealed an evolutionary conservation of these lysine residues (Fig. 3 A). To assess the ubiquitination on Lys204, Lys205, and Lys237 of GD-NT (but not GD-FL), we created the non-ubiquitinatable mutants GD-NT-K237R and GD-NT-K204/205R. Firstly, we performed Missense3D analysis and found no evidence of artificial structural damage resulting from these “K to R” substitutions, indicating that these constructs could be used for subsequent ubiquitination studies (Fig. 3 B). Then, we transfected HEK293 cells with GD-NT-WT, GD-NT-K237R or GD-NT-K204/205R and conducted IP assay. The results showed that K237R, but not K204/205R, significantly impaired the K63-linked polyubiquitination of GD-NT (Fig. 3 C-E). This difference was even much obvious in the subsequent NI-NTA pulldown assay (Fig. 3 F, G). Intriguingly, we observed that GD-NT-K237 also possessed K48-linked polyubiquitination, which is primarily associated with proteasomal degradation (Fig. H). However, this modification did not affect GD-NT stability in our experimental conditions (Fig. 3 I), suggesting that the K48-linked polyubiquitination on GD-NT-K237 represents a non-functional modification. The K63-linked polyubiquitination controls GD-NT pyroptotic activity As a specific pattern of PTMs, the K63-linked polyubiquitination generally controls various properties of protein including protein-protein interaction, translocation and activation. To assess whether the K63-linked polyubiquitination of Lys237 influences GD-NT pore-forming and cytolytic activity, we transfected HEK293 cells with GD-NT-WT, GD-NT-K237R or GD-NT-K204/205R. 24 hours later, culture medium and whole cell lysate were harvested for Immunoblot analysis, respectively. Interestingly, GD-NT-K237R was not detected in culture medium, suggesting that this GD-NT mutant can not permeabilize the plasma membrane and translocate into the culture medium (Fig. 4 A). To understand how the K63-linked polyubiquitination of Lys237 affects GD-NT cytolytic activity, we further investigated the subcellular localization and oligomerization of GD-NT-K237R using various strategies 26 . Firstly, we transfected HeLa cells with GD-FL, GD-NT-WT, GD-NT-K237R or GD-NT-K204/205R, and then performed confocal immunofluorescent microscopy to visualize the localization of these constructs. Our result showed that GD-NT-K237R clustered near the plasma membrane, which was different from GD-NT`s even distribution along the plasma membrane instead (Fig. 4 B). To precisely define GD-NT-K237R subcellular localization, HEK293 cells were transfected with these constructs and then fractionated into five compartments (soluble cytoplasmic, membrane, soluble nuclear, chromatin-bound nuclear and insoluble cytoskeletal content) for immunoblot analysis. These results showed that GD-NT-K237R were less abundant in membrane and insoluble cytoskeletal fractions in comparison with GD-NT-WT (Fig. 4 C, D). Next, to assess the oligomer formation of GD-NT-K237R, HEK293 cells were transfected with these constructs and subsequently subjected to immunoblot under non-reducing conditions. We found that GD-NT-K237R failed to form the oligomers (Fig. 4 E). Additionally, these transfected cells were fractionated through size exclusion chromatography (SEC)/gel filtration under native conditions and then subjected to immunoblot under reducing conditions. The result revealed that GD-NT-K237R exhibited a diffuse distribution pattern, whereas GD-NT-WT formed a single huge oligomer (≥ 440 kDa) (Fig. 4 F). Collectively, these findings suggest that GD-NT losses pyroptotic activity when it is modified with K63-linked polyubiquitin chains at Lys237. GD-NT-K237R fails to mediated pyroptosis in vitro and in vivo To directly evaluate the influence of ubiquitination on GD-NT-mediated pyroptosis, HEK293 cells were transfected with GD-NT-WT, GD-NT-K237R or GD-NT-K204/205R, and then subjected to cell death/survival assessment. Phase-contrast imaging indicated that, unlike GD-NT-WT and GD-NT-K204/205R that exhibited cellular toxicity, the mutant GD-NT-K237R caused no obvious morphological changes (Fig. 5 A). Consistently, subsequent LDH-based cell death assay and ATP-based cell viability assay suggested that GD-NT-K237R was incapable of mediating pyroptosis. To further investigate the pyroptotic activity of GD-NT-K237R, we employed a xenograft model in NSG mice using HeLa cells that has Dox-inducible expression of GD-NT-WT, GD-NT-K237R or GD-NT-K204/205R. Dox was administrated via intraperitoneal injection to induce the expression of GD-NT. In line with the in vitro findings, the xenografts expressing GD-NT-WT had a slower growth rate than those expressing the empty vector. In contrast, GD-NT-K237R did not efficiently suppress xenograft growth (Fig. 5 D-F). These data suggest that the K63-linked polyubiquitination on Lys237 prevents GD-NT from mediating pyroptosis. UBA1 inhibitor PYR-41 suppresses GD-NT-mediated pyroptosis in vitro and in vivo As K63-linked ubiquitination plays a positive role in GD-NT pyroptotic activity, we sought to evaluate the effects of ubiquitination-targeting small molecules on GD-NT-mediated pyroptosis. Due to the absence of selective molecules targeting TRAF1 or OTUB1, we instead evaluated the universal ubiquitination modulators, including UBA1 inhibitor PYR-41 and MLN7243, NAE inhibitor MLN4924, SAE inhibitor TAK-981 and the proteasome inhibitor Bortezomib. First, to test the influence of PYR-41 on GD-NT ubiquitination status, HEK293 cells were transfected with His-Ub and Flag-GD-NT, and then treated with PYR-41. Ni/NTA pulldown assay indicated that PYR-41 significantly reduce the ubiquitination of GD-NT (Fig. 6 A). Next, to evaluate the effects of PYR-41 on GD-NT-mediated pyroptosis, we established a pyroptosis model cell line, "293-tetO-GD-NT", in which the transgene GD-NT is expressed in response to Dox. Immunoblot assay revealed a peak expression of GD-NT at 4th hour post-Dox. Fluorescent imaging indicated that more than 95% cells underwent pyroptosis at 24th hour post-Dox (Extended Fig. 6 ). We then used 293-tetO-GD-NT cells to assess the effects of PYR-41 on GD-NT-mediated pyroptosis via different strategies, including Phase-contrast imaging, Flow cytometry, LDH-based cell death assay and ATP-based cell viability assay. Notably, the treatment of PYR-41 significantly reduced pyroptosis in 293-tetO-GD-NT cells (Fig. 6 B-F). Sepsis is the leading cause of death in intensive care units (ICUs) throughout the globe 44 . It is characterized by sustained excessive inflammation and immune suppression as well as organ dysfunction. Failure of more than a hundred clinical trials in search of a possible cure is attributed to the complexity of mediators and pathways involved in sepsis. Accumulating evidences suggest that GD-NT-mediated pyroptosis of macrophage is the main pathological basis of sepsis 5 , 45 , and the potential therapeutic targets in pyroptosis may provide future direction for sepsis treatment. In this study, we investigated the effects of PYR-41 in LPS/Nigericin-induced pyroptosis of immortalized bone marrow-derived macrophages (iBMDM) and LPS-induced septic mice. Surprisingly, we found that PYR-41 reduced iBMDM pyroptosis as well as the release of IL-18 and TNF-⍺ of septic mice (Fig. 6 G-J). Thus, we concluded that Inhibition of GD-NT ubiquitination suppresses its mediated pyroptosis and reduces the severity of septic mice (Fig. 6 K). On the other hand, in the context of cancer, GD-NT-mediated pyroptosis of tumor cells releases numerous immunogenic products and may enhance anti-tumor immunity 1 , 4 , 46 , 47 . Pharmaceutical modulation of GD-NT ubiquitination in cancer requires further investigations. Discussion Although coming from the same original amino acid sequence, GD-FL and the liberated GD-NT are two proteins that exhibit significant differences. Firstly, GD-FL solely stays in cytoplasm while GD-NT spreads in both cytoplasm and plasma membrane. Secondly, GD-FL is generally kept in monomer state while GD-NT is kept in oligomer state. Finally, GD-FL and GD-NT, as we previously reported, had different interactomes 26 . An increasing number of studies suggest that the distinct biological features are attributed to Post-Translational Modifications (PTMs). For example, the intermediate of the tricarboxylic acid cycle, fumarate, and its dimethyl form DMF react with the cysteine residue of GD-FL to form succination, thus preventing GD-FL from interacting with caspase-11 and the subsequent cleavage 48 . With the help of palmitoyl acyltransferases ZDHHC5 and ZDHHC9, GD-FL undergoes palmitoylation at Cys191/192 (human/mouse). This modification does not affect the cleavage of GD-FL by inflammatory caspases, but it makes GD-NT translocate into plasma membrane and form pores in a much higher efficient way 49 . More than this, we previously found that AMPK interacts with GD-NT, but not GD-FL. It phosphorylates the Ser46 residue of GD-NT to suppress its translocation and pore-forming activity 26 (Extended Fig. 2 ). These modifications affect the functions of GD-FL and GD-NT through regulating their structure, localization, stability and trafficking. Ubiquitination of gasdermin got our attention due to the works of Vishva and Alto in 2021 35,36 . They found that the ubiquitin ligase IpaH7.8 secreted by intracellular Shigella causes the ubiquitination and subsequent degradation of GSDMD and GSDMB, thereby disabling the host defensive system of pyroptosis (Extended Fig. 3 ). Given that mammalian cells hold a more complex ubiquitination system, we suppose the processing and functioning of gasdermin proteins are tightly regulated by the precise conjugation of ubiquitin in host cells. In the current study, we found that only GD-NT, but not GD-FL, underwent K63-type polyubiquitin chains on Lys237, which directed its translocation and oligomerization. TRAF1-OTUB1 axis was responsible for the ubiquitination and de-ubiquitination on GD-NT-K237. What`s more, we noticed that the ubiquitination on Lys237 did not affect the cleavage of GD-FL by caspase-11 and the production of GD-NT. Thus, we concluded that GD-NT possesses a unique ubiquitination status that differs significantly from GD-FL, and the ubiquitination on Lys237 is a post-cleavage event that help GD-NT translocate, form pore in plasma membrane and therefore promote pyroptosis. Consistent with this, the UBA1 (E1) inhibitor PYR-41 significantly suppressed GD-NT-mediated pyroptosis in 293-tetO-GD-NT cells, iBMDM and septic mice. Thus, our study elucidates the unique role of ubiquitylation in the regulation of GD-NT pyroptotic activity. In addition, we believe that manipulation of PTMs of GD-FL or GD-NT by small molecules, such as PYR-41, DMF, palmostatin B (PMB), metformin, and disulfiram 50 , is potentially paving a therapeutic path in pyroptosis-related diseases. Intriguingly, some of these chemicals have exhibited promising outcomes in the animal models of septic shock 51 (Extended Fig. 10). Materials and Methods Data reporting No statistical methods were used to predetermine the sample size. The samples were not randomized. The investigators were not blinded to allocation during the experiments and the evaluation of the results. Cell lines and cell culture conditions HEK293, HeLa, and iBMDM cells were purchased from ATCC, and were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ mL penicillin and 100 µg/mL streptomycin. All cells were tested for mycoplasma by PCR and were authenticated by morphology only. Plasmids pDB-His-MBP-mGSDMD (Addgene, 123365) was a gift from Hao Wu. pTRIPZ-shNS (Addgene, 127696) was a gift from Sandra Demaria. The vectors expressing GD-NT, human TRAFs, human DUBs were generated by the standard PCR cloning strategy. Truncation mutation or point mutation plasmids were generated using QuickChange Primer Design Program and mutagenesis kit (Agilent Technologies). All plasmids were verified by DNA sequencing and IB analysis. Reagent and antibodies LPS O111:B4 (L2630), Doxycycline (D3447) were obtained from Sigma-Aldrich. Subcellular protein fractionation kit (78840) was obtained from Thermo Fisher. ATP (tlrl-atpl) was obtained from InvivoGen. StrataClean resin (400714) and chemical competent cells (200315) were obtained from Agilent. Bortezomib (B125789) and Carfilzomib (C127870) were obtained from Aladdin. PYR-41 (P798006) and Pevonedistat (MLN4924, P872287) were obtained from MACKLIN. For FCAS analysis, Annexin V-FITC/PI apoptosis kit (KGA1102) were obtained from KeyGEN BioTECH. For ELISA, mouse IL-1beta kit (EM0109), mouse IL-18 kit (EM1158), mouse TNF-alpha kit (EM0183), mouse HMGB1 kit (EM0382) were obtained from Finetest. For immunoblot, Human Reactive Cell Death and Autophagy Antibody Sampler Kit (#42867), anti-LSD1 (#2184) and anti-caspase-3 (#9662) were purchased from Cell Signaling Technology. Anti-Flag (F1804) and anti-HA (H6908) were obtained from Sigma-Aldrich. Anti-β-Actin (66009-1-Ig) and anti-GST (10000-0-AP) were purchased from Proteintech. Anti-Na/K-ATPase α1 (PTM-5533) were purchased from PTO BIO. Stable cell lines Lentivirus was produced in HEK293 cells by transfection of the lentiviral vector with psPAX2 (Addgene) and pMD2.G (Addgene). Lentiviral super- natants were collected, filtered through 0.45-µm filters, and used to transduce HEK293 cells. Polybrene infection/transfection reagent (Millipore, 10µg/ml) was added to increase the efficiency of lentiviral infection. After 2 days of transduction, puromycin (Sigma, 2 µg/ml) was added to select the transduced cells. Empty lentiviral vectors were used to generate control cells. The expression of relevant genes in stable cell lines was verified by immunoblot. Immunoblots and immunoprecipitation Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP- 40) supplemented with protease inhibitors (A32953, Thermo Fisher) and phosphatase Inhibitors (B15002, Bimake). The protein concentrations of lysates were measured using the Beckman Coulter DU-800 spectro- photometer and the Bio-Rad protein assay reagent. Same amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, cell lysates containing 1mg of total proteins were incubated with anti-Flag agarose (A2220, Sigma) or anti-HA Agarose (A2095, Sigma) for 4 hours at 4°C. Precipitants were washed three times with EBC buffer and resolved by SDS-PAGE followed by immunoblot analysis with indicated antibodies. Protein enrichment from the culture medium or peritoneal fluid To enrich the proteins in the culture medium, 1 ml of culture medium was centrifuged at 14,000 x g for 10 min at 4°C to remove cellular debris. 10 µl StrataClean resin (400714, Agilent) was then added for 1-hour incubation on a rotator at 4°C. The supernatant was removed by centrifugation. The resin was harvested and suspended in 50 µl 2 x loading buffer for immunoblot. In septic mice model, the proteins in flushed peritoneal fluid were also enriched in the same way. Size exclusion chromatography (SEC) SEC was performed using an AKTA Purifier system (GE Healthcare, Buckinghamshire, England). A HiLoad 16/600 Superdex 200 column (GE Healthcare) was equilibrated with cell lysis buffer. The column was calibrated using a gel filtration calibration kit (GE Healthcare). Each standard protein was dissolved in cell lysis buffer and chromatographed on the column separately. The filtered protein samples were then fractionated on the column (1.0ml/min; 2ml/fraction). For immunoblot analysis, proteins in fractionated eluent were enriched with 10µl StrataClean resin and harvested in 30 µl of 2 x loading buffer. Cytotoxicity assay and cell viability assay Cell death and cell viability were performed using Non-Radioactive Cytotoxicity Assay kit (G1780, Promega) and CellTiter-Glo Luminescent Cell Viability Assay kit (G7571, Promega), respectively. Briefly, 5 ×10 3 cells were cultured in 96-well plates with Opaque wall. At the desired time points, cell death was determined by titrating the amount of lactate dehydrogenase released into the culture medium, and cell viability was determined by the ATP levels within cells, according to the manufacturer’s instructions. Mouse studies All procedures were conducted in compliance with American guidelines for the care and use of laboratory animals and were approved by Zhengzhou University Animal Care Committee in accordance with institutional animal care and use guidelines. Female wild-type C57BL/6 mice, and NSG mice (6–8 weeks old) were purchased from Vital River Laboratories. All mice were housed in the Zhengzhou University Animal Facility. For xenograft experiment, before inoculation, cell viability was determined using trypan blue exclusion test (minimum of 98% cell viability). 1.0 ×10 6 of were subcutaneously injected into the right flank of mice. Tumor growth was monitored every other day. Tumor volume (mm3) is calculated via the “(W x W x L) / 2” formula, where L is the longest diameter and W is the shortest diameter. Necropsy and tumor collection were performed at the end of tumor size recording. For the septic model, mice were pretreated with PYR-41 (10 mg/ml, i.p.) on day-14 and the following every other day. on day0, LPS (5mg/ml, i.p.) were injected to induce sepsis. Serums were harvested for ELISA assay at 6 hours post-LPS. Flow cytometry FCAS analysis were performed according to the manufacture`s guideline. Briefly, cells were digested using trypsin without EDTA to get cell suspension. Then, cells were washed with PBS twice, suspended with 500µL of Binding Buffer to form a single-cell suspension. After adding 5µL of Annexin V-FITC + 5µL of Propidium Iodide, cells were incubated at room temperature for 5 minutes before conducting flow cytometry observation. Immunofluorescence staining fluorescent microscopy For Immunofluorescence staining, dish with special glass bottom were used. Cells were fixed with 100% methanol (chilled at -20℃) at RT for 5 min. To block unspecific binding of the antibodies, cells were incubated with 1% BSA, 22.52 mg/ml glycine in PBST (PBS + 0.1% Tween 20) for 30 min (alternative is 10% serum from the species that the secondary antibody are raised in). For immunostaining, cells were incubated with the diluted antibody in 1% BSA in PBT in a humidified chamber for 1 hour at RT or overnight at 4 ℃, and then incubated with the secondary antibody in 1% BSA for 1 hour at RT in the dark. At last, cells were counter stained with 0.1-1 µg/ml Hoechst or DAPI for 1 min, mounted and subjected to microscopic analysis. Notes: Between steps, cells were washed with PBS for 3 times, 5 min each wash. For fluorescent microscopy of living cells, PI (10 µg/ml) or DAPI (1 µg/ml) was added into culture medium. 10 minutes later, cells were imaged under fluorescent microscope. Statistical analysis Student`s t-test was used to determine the differences between the two groups. Differences between tumor growth curves were compared by calculating the area-under-curve (AUC) values for each sample and then comparing different groups using Student’s t-test. The results are presented as the mean and standard error of the mean (SEM). Statistical significance was assigned to P < 0.5%. Tumor-free survival and Kaplan- Meier analysis were performed using GraphPad Prism Version 5.04 for Windows. Declarations Reporting summary Further information on research design is available in the Research Reporting Summary linked to this document. Data availability All relevant data are available in the Source Data (for Figs. 1–6) or supplementary information associated with this study. Acknowledgements This project was supported by Research fund for the Doctoral program of the Fifth Affiliated Hospital of Zhengzhou University (pb2024kyqdj03), Supporting Program for Young Talent Innovation Teams of Zhengzhou University (32320688), Henan Medical Key Technologies R & D Program (LHGJ20220570), Henan Natural Science Foundation (242300421283) and Tianjian Advanced Biomedical Laboratory Key Research and Development Project. Author contributions XC and IB conceived thec study and designed the experiments. XC performed the experiments and written first draft. IB and XC reviewed and revised the manuscript. All the authors have discussed the results and approved the final version of the manuscript for publication. Competing interests The authors declare no competing interests. Ethics approval and consent to participate We did not include human samples. References Yu, P. et al. Pyroptosis: mechanisms and diseases. 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Additional Declarations There is no duality of interest Supplementary Files GDUbSM20240808.docx Uncroppedoriginalwesternblot20240808.pdf Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2025 Read the published version in Cell Death & Disease → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4907061","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":340720385,"identity":"6ad4f631-0bf0-4e29-8966-112a0b6af97d","order_by":0,"name":"Xiufeng Chu","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-4878-5834","institution":"Houston Methodist Research Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiufeng","middleName":"","lastName":"Chu","suffix":""},{"id":340720386,"identity":"acc7e4ec-8226-4e1f-a713-bd88c559b7bb","order_by":1,"name":"Pengyuan Zheng","email":"","orcid":"","institution":"Houston Methodist Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pengyuan","middleName":"","lastName":"Zheng","suffix":""},{"id":340720387,"identity":"7f9249fa-b1a7-4426-a980-421553f3eb57","order_by":2,"name":"Bukhari Ihtisham","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bukhari","middleName":"","lastName":"Ihtisham","suffix":""}],"badges":[],"createdAt":"2024-08-13 12:30:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4907061/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4907061/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-07475-6","type":"published","date":"2025-03-17T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66059754,"identity":"3ff60e8d-9cee-4935-92ef-82636da2fee0","added_by":"auto","created_at":"2024-10-07 09:56:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":279317,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGD-NT, but not GD-FL, possesses massive K63-linked polyubiquitin.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Schematic illustration of the cleavage of mouse GSDMD, the proteases responsible for the specific cleavage and the constructs created for the investigation of GSDMD ubiquitination.\u003c/p\u003e\n\u003cp\u003eB. Schematic illustration depicts the structure of mouse GSDMD. Structure of mouse GD-NT was downloaded from RCSB PDB (PDB code: 6N9N) and PyMOL was used to generate structure model of GD-FL, GD-NT.\u003c/p\u003e\n\u003cp\u003eC. 293T cells were transfected with Flag-GD-FL and/or caspase-11. 24 hours later, the cells were harvested and subjected to Flag-IP assay. The precipitants and WCL (whole cell lysate) were immunoblotted with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eD. His-Ub was transfected with Flag-GD-FL and/or caspase-11 in 293T cells. 24 hours later, the cells were harvested and subjected to Ni-NTA Pulldown assay. The precipitants and WCL were immunoblotted with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eE. Similar with (B), except that 293T cells were transfected with Flag-GD-FL, NT or CT, respectively. The mark “*” indicated that the band of GD-CT overlapped with the unspecific band from the anti-Flag primary antibody.\u003c/p\u003e\n\u003cp\u003eF. 293T cells were transfected with GST-GD-FL or NT and treated with or without MG132 (10 μM, 6h). 24 hours later, the cells were harvested and subjected to GST Pulldown assay. The precipitants and WCL were immunoblotted with indicated antibodies. \u003cbr\u003e\nG. similar with (C), except that His-Ub was transfected with Flag-GD-FL, NT or CT, respectively.\u003c/p\u003e\n\u003cp\u003eH. His-Ub-WT or K63 only was transfected with Flag-GD-NT in 293T cells. 24 hours later, the cells were harvested and subjected to Ni-NTA Pulldown assay. The precipitants and WCL were immunoblotted with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eIn C, E and F, to keep the integrity of ubiquitin-chain attached to substrate proteins, NEM (10 mM) was added to inhibit the activity of cysteine peptidases. To prevent the influence of GSDMD interactome, protein samples for IP were denatured via heating at 100℃ for 5 minutes to break protein-protein interaction.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4907061/v1/a31bc41395dc0aa736385204.jpg"},{"id":66058769,"identity":"9b3b7ea1-76ed-4683-a760-e9cef2bdcc3b","added_by":"auto","created_at":"2024-10-07 09:47:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":230129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe K63-linked polyubiquitin of GD-NT is regulated by TRAF1-OTUB1 axis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-B. Interaction of GD-NT with E3 ligase. HEK293T cells were transfected with Flag-GD-NT and different HA-E3. 24 hours later, the cells were harvested for Flag-IP assay (A) and HA-IP assay (B). The preciptants were subjected to immunoblot with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eC-D. Deubiquitylation of GD-NT by DUBs. HEK293T cells were transfected with Flag-GD-NT, His-Ub-K63 only and HA-TRAF/2 (C) or shTRAF1 (D). 24 hours later, the cells were harvested for Ni-NTA pull down assays. The precipitants and WCL were immunoblotted with with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eE. Interaction of GD-NT with DUBs. HEK293T cells were transfected with HA-GD-NT and different Flag-DUB. 24 hours later, the cells were harvested for IP assay. The preciptants were subjected to immunoblot with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eF. Deubiquitylation of GD-NT by DUBs. HEK293T cells were transfected with HA-GD-NT, His-Ub-WT and Flag-OTUB1 or Flag-OTUB2. 24 hours later, the cells were harvested for Ni-NTA pull down assays. The precipitants and WCL were immunoblotted with with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eG. HEK293T cells were transfected with HA-GD-NT, His-Ub-K63 only and Flag-OTUB1 or Flag-OTUB2. 24 hours later, the cells were harvested for Ni-NTA pull down assays. The precipitants and WCL were immunoblotted with with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eH. Similar to (G), ecxpet that shOTUB1 was used to knockdown the expression of instead of OTUB1.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4907061/v1/092b221d2a8c9d01dcf92cdd.jpg"},{"id":66061243,"identity":"e6999788-8e6a-4a9f-bafd-fb4124b1134c","added_by":"auto","created_at":"2024-10-07 10:12:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":289656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe amino acid Lys237 of GD-NT possess K63-type ubiquitination.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Lys237 of GD-NT is an evolutionarily conserved site. Shown is the sequence homology analysis of GD-NT among various organisims including H. sapiens, P. troglodytes, M. mulatta, C. lupus, B. taurus, M. musculus, and R. norvergicus.\u003c/p\u003e\n\u003cp\u003eB. Structural damage analysis of GSDMD site mutation. Structural damage was assessed with the online tool Missense3D (http://www.sbg.bio.ic.ac.uk/missense3d). No structural damage was detected during the creation of GD-NT mutants via site-directed mutagenesis.\u003c/p\u003e\n\u003cp\u003eC. HEK293 cells were transfected with Flag-GD-NT, K237R, K204/205R. 24 hours later, the cells were harvested for Flag-IP assay. The precipitants and WCL were immunoblotted with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eD. His-Ub-WT was transfected with Flag-GD-NT, K237R, K204/205R into HEK293 cells. 24 hours later, the cells were harvested for Flag-IP assay. The precipitants and WCL were immunoblotted with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eE. Similar to (D), except that His-Ub-K63 only was used instead of His-Ub-WT.\u003c/p\u003e\n\u003cp\u003eF. His-Ub-WT was transfected with Flag-GD-NT, K237R, K204/205R into HEK293 cells. 24 hours later, the cells were harvested for Ni/NTA pulldown assay. The precipitants and WCL were immunoblotted with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eG. Similar to (F), except that His-Ub-K63 only was used instead of His-Ub-WT.\u003c/p\u003e\n\u003cp\u003eH. Similar to (F), except that His-Ub-K63 only was used instead of His-Ub-WT.\u003c/p\u003e\n\u003cp\u003eI. HEK293 cells were transfected with Flag-GD-NT-WT or K237R. 12 hours later, the cells were treated with CHX (50 μg/mL) and then harvested at different time points. for Flag-IP assay. The WCL were immunoblotted with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eIn C, D and E, to keep the integrity of ubiquitin-chain attached to substrate proteins, NEM (10 mM) was added to inhibit the activity of cysteine peptidases. To prevent the influence of GSDMD interactome, protein samples for IP were denatured via heating at 100℃ for 5 minutes to break protein-protein interaction.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4907061/v1/b5f2729fb7b8e24fd8ec43af.jpg"},{"id":66059752,"identity":"cd711bb8-16ef-4a0b-9038-555740562d04","added_by":"auto","created_at":"2024-10-07 09:56:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":234275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUbiquitination on Lys237 of GD-NT affects its localization and oligomerization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Detection of GD-NT in culture medium. HEK293 cells were transfected with the indicated constructs. 16 hours later, the culture medium and WCL were harvested and immunoblotted with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eB. Distribution pattern of GD-NT-WT or its mutants. HeLa cells were transfected with the indicated constructs. Shown are representative confocal microscopy images of the distribution of ectopic Flag-GD-FL, GD-NT-WT, GD-NT-K237R or GD-NT-K204/205R (green) co-stained with DAPI (blue).\u003c/p\u003e\n\u003cp\u003eC-D. Subcellular localization of GD-NT-WT or its mutants. HEK293 cells were transfected with the indicated constructs. 16 hours later, cells were harvested and subjected to sample preparation using Thermo Fisher Subcellular Fractionation Kit. Membrane fractions (C) as well as other fractions (D) were immunoblotted with indicated antibodies. WCL was included as an input control.\u003c/p\u003e\n\u003cp\u003eE, Oligomerization of GD-NT. HEK293 cells were transfected with the indicated constructs. 16 hours later, cells were harvested and subjected to IB analysis with indicated antibodies under reducing or non-reducing conditions.\u003c/p\u003e\n\u003cp\u003eF, Fractionation via SEC/gel filtration. HEK293 cells were transfected with the indicated constructs. 16 hours later, cells were harvested for fractionation with SEC/gel filtration. Eluent protein fractions were immunoblotted with indicated antibodies. Input represents 10% of proteins used for SEC/gel filtration. Data are representative of at least two independent experiments.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4907061/v1/278c41c342f6cd904f3427fd.jpg"},{"id":66060109,"identity":"8d03b6fd-a468-425d-9987-97d2226a5303","added_by":"auto","created_at":"2024-10-07 10:04:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":215612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUbiquitinated GD-NT loses the ability to mediate pyroptosis in vivo and in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. HEK293 cells were transfected with Flag-GD-NT-WT or its mutants. Phase-contrast images were taken 24 hours after transfection (left panel). Additionally, the cells were harvested for IB analysis with indicated antibodies 16 hours after transfection (right panel).\u003c/p\u003e\n\u003cp\u003eB. Similar to (A), except that the cells were harvested for LDH-based Cytotoxicity Assay 24 hours after transfection.\u003c/p\u003e\n\u003cp\u003eC. Similar to (A), except that the cells were harvested for ATP-based Cell Viability Assay 24 hours after transfection.\u003c/p\u003e\n\u003cp\u003eD-F, HeLa tumor xenograft in NSG mice. HeLa cells expressing Dox-inducible GD-NT-WT or its mutants (0.5 x 10\u003csup\u003e6\u003c/sup\u003e cells per mouse) were subcutaneously implanted into the right flank of NSG mice (n = 6 mice per group). Dox (50 mg/kg, i.p.) was administrated on d6, d8, d10, d12, d14 and d16. Tumor growth was recorded every other day. Shown are tumor growth (D), tumor image (E) and tumor weight (F).\u003c/p\u003e\n\u003cp\u003eIn B, C, D and F, error bars represent variation range of duplicated experiments. In B, C and F, differences among groups were analyzed by two-tailed Student`s \u003cem\u003et\u003c/em\u003e-test (means ± s.e.m). In D, the areas under the growth curves were compared by two-tailed Student`s \u003cem\u003et\u003c/em\u003e-test (means ± s.e.m). Data are representative of at least two independent experiments.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4907061/v1/df69ad99929a85c459ac11bc.jpg"},{"id":66058772,"identity":"4a02392b-6c4c-4b27-bc9b-051661f54f5a","added_by":"auto","created_at":"2024-10-07 09:48:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":324969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePYR-41 suppressed GD-NT ubiquitination and reduced GD-NT-mediated pyroptosis in vivo and in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. 293-tetO-His-Ub cells were used to ease the ubiquitination detection. Dox (2 µg/ml) were added to induce the expression of His-Ub. 24 hours after Dox, the cells were transfected with Flag-GD-NT and simultaneously treated with PYR-41 (50 µM). 24 hours after transfection, the cells were harvested for Ni-NTA Pulldown. The precipitants and WCL were immunoblotted with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eB. 293-tetO-GD-NT were treated Dox (2 µg/ml) to induce the expression of GD-NT. Different concentrations of PYR-41 were added at the same time as Dox. 6 hours after Dox/PYR41, the cells were harvested for immunoblot with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eC. Similar to (B), except that phase-contrast images were taken 16 hours after Dox/PYR41. The cells floating in culture medium were removed, and the adherent cells (considered as living cells) were subjected to phase-contrast imaging.\u003c/p\u003e\n\u003cp\u003eD. 293-tetO-GD-NT were treated Dox (2 µg/ml) to induce the expression of GD-NT. PYR-41 (50 µM) were added at the same time as Dox. 12 hours after Dox/PYR41, the cells were harvested for flow cytometry and Annexin V/PI staining kit.\u003c/p\u003e\n\u003cp\u003eE. Similar to (D), except that the cells were harvested 12 hours post-Dox and subjected to LDH-based Cytotoxicity Assay.\u003c/p\u003e\n\u003cp\u003eF. Similar to (D), except that the cells were harvested 12 hours post-Dox and subjected to ATP-based Cell Viability Assay.\u003c/p\u003e\n\u003cp\u003eG. Phase-contrast images of iBMDM cells. The cells were treated with LPS (1μg/ml) and (PYR41 50 μM)) for 10 hours before stimulation with nigericin for 30 minutes. The cells floating in culture medium were removed, and the adherent cells (considered as living cells) were subjected to phase-contrast imaging.\u003c/p\u003e\n\u003cp\u003eH. Murine septic model. C57BL/6 naïve mice were intraperitoneally injected with LPS (5mg/ml for cytokine analysis, 20 mg/ml for organ damage and survival analysis) and/or PYR-41 (10 mg/ml) according to the formula.\u003c/p\u003e\n\u003cp\u003eI-J. Cytokine release in septic mice. C57BL/6 naïve mice were intraperitoneally injected with LPS (5mg/ml) and/or PYR-41 (10 mg/ml) according to the formula. 8 hours after LPS, the serums were harvested for ELISA assay to detect the level of IL-18 (I), TNF-⍺ (J).\u003c/p\u003e\n\u003cp\u003eK. Scientific hypothesis: The ubiquitination modification of GD-NT mediated by TRAF1/2 and OTUB2 enables GD-NT to obtain the ability to form polymers and drill holes on the cell membrane. The UBA1 inhibitor PYR-41 can significantly block GD-NT mediated cell apoptosis.\u003c/p\u003e\n\u003cp\u003eIn E, F, I, and J, differences among groups were analyzed by two-tailed Student`s t-test (means ± s.e.m). Error bars represent the variation range of duplicated experiments. Data are representative of at least two independent experiments.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4907061/v1/8930eeddeff48d89e3153f65.jpg"},{"id":78729605,"identity":"884afebf-9489-48df-8c39-61e69ada1624","added_by":"auto","created_at":"2025-03-18 07:11:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2630883,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4907061/v1/dc98d896-c22d-4155-b062-02702bdde811.pdf"},{"id":66058773,"identity":"6a59a6af-abde-42ac-b369-ab524c114b03","added_by":"auto","created_at":"2024-10-07 09:48:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1409325,"visible":true,"origin":"","legend":"","description":"","filename":"GDUbSM20240808.docx","url":"https://assets-eu.researchsquare.com/files/rs-4907061/v1/f9eb19a75f418c9e36218523.docx"},{"id":66058777,"identity":"64fc7644-9171-4463-a3bc-9aff48acfd03","added_by":"auto","created_at":"2024-10-07 09:48:00","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2053824,"visible":true,"origin":"","legend":"","description":"","filename":"Uncroppedoriginalwesternblot20240808.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4907061/v1/8e25e71b6dd856d3fb4cf94d.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"Ubiquitination of gasdermin D N-terminal domain directs its membrane translocation and pore formation during pyroptosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePyroptosis is a unique cell death featured by pore formation in the plasma membrane and subsequent cell swelling\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The release of cellular contents during pyroptosis makes it an inflammatory and immunogenic cell death (ICD). With these attributes, pyroptosis plays a pivotal role in the defense against pathogenic organism as well as cancer\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In some cases, however, it participates in the development of pathological conditions\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Pyroptosis are strongly associated with sepsis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, Cryopyrin-Associated Periodic Syndromes (CAPS)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, Macrophage Activation Syndrome (MAS)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, and moderately related with type 2 diabetes\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, obesity\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, myocarditis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, atherosclerotic diseases\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, gouty arthritis\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and neurological diseases\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGSDMD is the first gasdermin that is identified as a pyroptosis mediator\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In response to canonical or noncanonical inflammasome activators, the activated inflammatory caspases (specifically, caspase-1/4/5 in human, caspase-1/11 in mouse) cleave GD-FL to release the bioactive pore-forming GD-NT fragment (the liberation of GD-NT)\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Not like the GD-FL that exists in monomer state and localizes in cytoplasm, the liberated GD-NT undergoes self-oligomerization, integrating into plasma membrane and creating pores\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. However, the mechanisms underlying the distinct biological activities between GD-FL and GD-NT are not fully understood.\u003c/p\u003e \u003cp\u003eUbiquitination is a critical PTMs in the regulation of inflammatory cell death by regulating the key components of NF-κB and inflammasome signaling pathways, such as NEMO, RIPK1, ASC, NLRP3, caspase-11 and IL-1beta\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32 CR33\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (Extended Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Recent studies suggest the role of ubiquitination in the functionality of gasdermin family. Shigella produces pathogenic ubiquitination ligase to degrade GSDMB/GSDMD and deactivate the host defensive system\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Besides, both SYVN1-mediated ubiquitination of GSDMD and OTUD4-mediated deubiquitination of GSDME promote the happening of pyroptosis\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, indicating that the host ubiquitination system also contributes to the regulation of gasdermin function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHere, we report that the ubiquitination at Lys237 of GD-NT (but not GD-FL) is a prerequisite mechanism of GSDMD activation. This process enables GD-NT to translocate and form pores in plasma membrane, thereby causing pyroptotic cell death. Our current study identified a novel molecular mechanism of the liberated GD-NT acquiring pyroptotic ability.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGD-NT, but not GD-FL, possesses K63-linked polyubiquitination\u003c/h2\u003e \u003cp\u003eIn order to investigate the ubiquitination of GSDMD, the vectors expressing GD-FL, GD-NT, GD-CT or caspase-11 were constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). We transfected Flag-GD-FL/caspase-11 into HEK293 cells and performed IP \u0026amp; immunoblot assay. Interestingly, the result showed that GSDMD was slightly ubiquitinated when it was introduced into cell alone. But the ubiquitination level of GSDMD was significantly increased when it was co-transfected with caspase-11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To further ensure it, we adopted a more rigorous ubiquitination detection method, in which we denatured the cellular proteins using guanidine and subjected to NI/NTA pulldown (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Immunoblot (IB) analysis of the precipitants revealed the same ubiquitination pattern as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. We supposed that the alteration of ubiquitination may take place during GSDMD processing by caspase-11. To test this hypothesis, we independently transfected Flag-GD-FL, Flag-GD-NT or Flag-GD-CT into HEK293 cells. 18 hours later, the cells were subjected to IP assay. IB analysis of the precipitants revealed that GD-NT had a distinct ubiquitination status from GD-FL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Later on, we performed GST-pulldown and NI/NTA pulldown assay under denatured conditions. Consistently, these assays also indicated that GD-NT, but not GD-FL or GD-CT, was massively ubiquitinated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G).\u003c/p\u003e \u003cp\u003eTo further identify the conjugation form of ubiquitin on GD-NT, we transfected Flag-GD-NT along with either Ub-WT or Ub-K63 only into HEK293 cells and then performed NI/NTA pulldown assay. The result showed that GD-NT was conjugated with K63-linked polyubiquitin chains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Thus, our data suggest that GD-NT, but not GD-FL, is massively modified with K63-ubiquitination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eThe K63-linked polyubiquitination of GD-NT is regulated by TRAF1-OTUB1 axis\u003c/h2\u003e \u003cp\u003eUbiquitination is a dynamic and reversible process, where E3 ligases and deubiquitinases (DUB) catalyze the conjugation and removal of ubiquitin on substrate proteins, respectively\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Additionally, E3 ligase-DUB axis exhibits its specificity by determining the target proteins and their ubiquitination forms. To identify the E3 ligase responsible for the K63-linked ubiquitination of GD-NT, we transfected Flag-GD-NT along with various E3 ligases into HEK293 cells and conducted IP assay. IB analysis of the precipitants indicated that Flag-GD-NT interacted with TRAF1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). To assess the influence of TRAF1 on GD-NT ubiquitination, we transfected either TRAF1 or shTRAF1 together with Ub-K63 only and Flag-GD-NT into HEK293 cells, followed by NI/NTA pulldown assay. Our results revealed that TRAF1 significantly enhanced the K63-linked ubiquitination of GD-NT, whereas the ubiquitination level was downregulated by shTRAF1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Interestingly, although the interaction of GD-NT with TRAF2 was not observed in our experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B), TRAF2 still enhanced the K63-linked ubiquitination of GD-NT (the last two lanes in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This phenomenon, we believe, may be attributed to the formation of a heterodimer between TRAF1 and TRAF2\u003csup\u003e41\u003c/sup\u003e, or possibly involving another factor that we have yet to identify.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, to identify the DUB responsible for the K63-linked ubiquitination of GD-NT, we transfected Flag-GD-NT along with different DUBs into HEK293 cells and performed IP assay. The result showed that Flag-GD-NT interacted with both OTUB1 and OTUB2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Subsequently, either Ub-WT or Ub-K63 only were co-transfected with HA-GD-NT and/or OTUB1/2, followed by NI/NTA pulldown. IB analysis of the precipitants showed that, although both OTUB1 and OTUB2 affected the ubiquitination of GD-NT, only OTUB1 significantly reduced the K63-linked ubiquitination of GD-NT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G). In consistent with this finding, shRNA-mediated silencing of OTUB1 significantly increased the K63-linked ubiquitination of GD-NT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Therefore, our data suggest that the K63-linked ubiquitination of GD-NT is regulated by the TRAF1-OTUB1 axis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGD-NT possesses the K63-linked polyubiquitination through its Lys237\u003c/h2\u003e \u003cp\u003eTo identify the ubiquitin conjugation sites on GD-NT, we searched the online PhosphoSitePlus\u0026reg; database (PSP, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.phosphosite.org\u003c/span\u003e\u003cspan address=\"https://www.phosphosite.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which provides comprehensive information for the study of ubiquitination as well as other mammalian post-translational modifications (PTMs)\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Based on Low ThroughPut and High ThroughPut datasets obtained from PSP database\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, several amino acids were considered as the potential ubiquitinated sites, including Lys204, Lys205, and Lys237 in mouse GSDMD (refer to Lys203, Lys204, and Lys236 in human). Sequence alignment of GSDMD among different species revealed an evolutionary conservation of these lysine residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the ubiquitination on Lys204, Lys205, and Lys237 of GD-NT (but not GD-FL), we created the non-ubiquitinatable mutants GD-NT-K237R and GD-NT-K204/205R. Firstly, we performed Missense3D analysis and found no evidence of artificial structural damage resulting from these \u0026ldquo;K to R\u0026rdquo; substitutions, indicating that these constructs could be used for subsequent ubiquitination studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Then, we transfected HEK293 cells with GD-NT-WT, GD-NT-K237R or GD-NT-K204/205R and conducted IP assay. The results showed that K237R, but not K204/205R, significantly impaired the K63-linked polyubiquitination of GD-NT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-E). This difference was even much obvious in the subsequent NI-NTA pulldown assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G). Intriguingly, we observed that GD-NT-K237 also possessed K48-linked polyubiquitination, which is primarily associated with proteasomal degradation (Fig. H). However, this modification did not affect GD-NT stability in our experimental conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), suggesting that the K48-linked polyubiquitination on GD-NT-K237 represents a non-functional modification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eThe K63-linked polyubiquitination controls GD-NT pyroptotic activity\u003c/h2\u003e \u003cp\u003eAs a specific pattern of PTMs, the K63-linked polyubiquitination generally controls various properties of protein including protein-protein interaction, translocation and activation. To assess whether the K63-linked polyubiquitination of Lys237 influences GD-NT pore-forming and cytolytic activity, we transfected HEK293 cells with GD-NT-WT, GD-NT-K237R or GD-NT-K204/205R. 24 hours later, culture medium and whole cell lysate were harvested for Immunoblot analysis, respectively. Interestingly, GD-NT-K237R was not detected in culture medium, suggesting that this GD-NT mutant can not permeabilize the plasma membrane and translocate into the culture medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo understand how the K63-linked polyubiquitination of Lys237 affects GD-NT cytolytic activity, we further investigated the subcellular localization and oligomerization of GD-NT-K237R using various strategies\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Firstly, we transfected HeLa cells with GD-FL, GD-NT-WT, GD-NT-K237R or GD-NT-K204/205R, and then performed confocal immunofluorescent microscopy to visualize the localization of these constructs. Our result showed that GD-NT-K237R clustered near the plasma membrane, which was different from GD-NT`s even distribution along the plasma membrane instead (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo precisely define GD-NT-K237R subcellular localization, HEK293 cells were transfected with these constructs and then fractionated into five compartments (soluble cytoplasmic, membrane, soluble nuclear, chromatin-bound nuclear and insoluble cytoskeletal content) for immunoblot analysis. These results showed that GD-NT-K237R were less abundant in membrane and insoluble cytoskeletal fractions in comparison with GD-NT-WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). Next, to assess the oligomer formation of GD-NT-K237R, HEK293 cells were transfected with these constructs and subsequently subjected to immunoblot under non-reducing conditions. We found that GD-NT-K237R failed to form the oligomers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Additionally, these transfected cells were fractionated through size exclusion chromatography (SEC)/gel filtration under native conditions and then subjected to immunoblot under reducing conditions. The result revealed that GD-NT-K237R exhibited a diffuse distribution pattern, whereas GD-NT-WT formed a single huge oligomer (\u0026ge;\u0026thinsp;440 kDa) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Collectively, these findings suggest that GD-NT losses pyroptotic activity when it is modified with K63-linked polyubiquitin chains at Lys237.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGD-NT-K237R fails to mediated pyroptosis in vitro and in vivo\u003c/h2\u003e \u003cp\u003eTo directly evaluate the influence of ubiquitination on GD-NT-mediated pyroptosis, HEK293 cells were transfected with GD-NT-WT, GD-NT-K237R or GD-NT-K204/205R, and then subjected to cell death/survival assessment. Phase-contrast imaging indicated that, unlike GD-NT-WT and GD-NT-K204/205R that exhibited cellular toxicity, the mutant GD-NT-K237R caused no obvious morphological changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Consistently, subsequent LDH-based cell death assay and ATP-based cell viability assay suggested that GD-NT-K237R was incapable of mediating pyroptosis. To further investigate the pyroptotic activity of GD-NT-K237R, we employed a xenograft model in NSG mice using HeLa cells that has Dox-inducible expression of GD-NT-WT, GD-NT-K237R or GD-NT-K204/205R. Dox was administrated via intraperitoneal injection to induce the expression of GD-NT. In line with the in vitro findings, the xenografts expressing GD-NT-WT had a slower growth rate than those expressing the empty vector. In contrast, GD-NT-K237R did not efficiently suppress xenograft growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F). These data suggest that the K63-linked polyubiquitination on Lys237 prevents GD-NT from mediating pyroptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eUBA1 inhibitor PYR-41 suppresses GD-NT-mediated pyroptosis in vitro and in vivo\u003c/h2\u003e \u003cp\u003eAs K63-linked ubiquitination plays a positive role in GD-NT pyroptotic activity, we sought to evaluate the effects of ubiquitination-targeting small molecules on GD-NT-mediated pyroptosis. Due to the absence of selective molecules targeting TRAF1 or OTUB1, we instead evaluated the universal ubiquitination modulators, including UBA1 inhibitor PYR-41 and MLN7243, NAE inhibitor MLN4924, SAE inhibitor TAK-981 and the proteasome inhibitor Bortezomib.\u003c/p\u003e \u003cp\u003eFirst, to test the influence of PYR-41 on GD-NT ubiquitination status, HEK293 cells were transfected with His-Ub and Flag-GD-NT, and then treated with PYR-41. Ni/NTA pulldown assay indicated that PYR-41 significantly reduce the ubiquitination of GD-NT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, to evaluate the effects of PYR-41 on GD-NT-mediated pyroptosis, we established a pyroptosis model cell line, \"293-tetO-GD-NT\", in which the transgene GD-NT is expressed in response to Dox. Immunoblot assay revealed a peak expression of GD-NT at 4th hour post-Dox. Fluorescent imaging indicated that more than 95% cells underwent pyroptosis at 24th hour post-Dox (Extended Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). We then used 293-tetO-GD-NT cells to assess the effects of PYR-41 on GD-NT-mediated pyroptosis via different strategies, including Phase-contrast imaging, Flow cytometry, LDH-based cell death assay and ATP-based cell viability assay. Notably, the treatment of PYR-41 significantly reduced pyroptosis in 293-tetO-GD-NT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-F).\u003c/p\u003e \u003cp\u003eSepsis is the leading cause of death in intensive care units (ICUs) throughout the globe\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. It is characterized by sustained excessive inflammation and immune suppression as well as organ dysfunction. Failure of more than a hundred clinical trials in search of a possible cure is attributed to the complexity of mediators and pathways involved in sepsis. Accumulating evidences suggest that GD-NT-mediated pyroptosis of macrophage is the main pathological basis of sepsis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, and the potential therapeutic targets in pyroptosis may provide future direction for sepsis treatment. In this study, we investigated the effects of PYR-41 in LPS/Nigericin-induced pyroptosis of immortalized bone marrow-derived macrophages (iBMDM) and LPS-induced septic mice. Surprisingly, we found that PYR-41 reduced iBMDM pyroptosis as well as the release of IL-18 and TNF-⍺ of septic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-J). Thus, we concluded that Inhibition of GD-NT ubiquitination suppresses its mediated pyroptosis and reduces the severity of septic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eOn the other hand, in the context of cancer, GD-NT-mediated pyroptosis of tumor cells releases numerous immunogenic products and may enhance anti-tumor immunity\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Pharmaceutical modulation of GD-NT ubiquitination in cancer requires further investigations.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough coming from the same original amino acid sequence, GD-FL and the liberated GD-NT are two proteins that exhibit significant differences. Firstly, GD-FL solely stays in cytoplasm while GD-NT spreads in both cytoplasm and plasma membrane. Secondly, GD-FL is generally kept in monomer state while GD-NT is kept in oligomer state. Finally, GD-FL and GD-NT, as we previously reported, had different interactomes\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. An increasing number of studies suggest that the distinct biological features are attributed to Post-Translational Modifications (PTMs). For example, the intermediate of the tricarboxylic acid cycle, fumarate, and its dimethyl form DMF react with the cysteine residue of GD-FL to form succination, thus preventing GD-FL from interacting with caspase-11 and the subsequent cleavage\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. With the help of palmitoyl acyltransferases ZDHHC5 and ZDHHC9, GD-FL undergoes palmitoylation at Cys191/192 (human/mouse). This modification does not affect the cleavage of GD-FL by inflammatory caspases, but it makes GD-NT translocate into plasma membrane and form pores in a much higher efficient way\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. More than this, we previously found that AMPK interacts with GD-NT, but not GD-FL. It phosphorylates the Ser46 residue of GD-NT to suppress its translocation and pore-forming activity\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e (Extended Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These modifications affect the functions of GD-FL and GD-NT through regulating their structure, localization, stability and trafficking.\u003c/p\u003e \u003cp\u003eUbiquitination of gasdermin got our attention due to the works of Vishva and Alto in 2021\u003csup\u003e35,36\u003c/sup\u003e. They found that the ubiquitin ligase IpaH7.8 secreted by intracellular Shigella causes the ubiquitination and subsequent degradation of GSDMD and GSDMB, thereby disabling the host defensive system of pyroptosis (Extended Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Given that mammalian cells hold a more complex ubiquitination system, we suppose the processing and functioning of gasdermin proteins are tightly regulated by the precise conjugation of ubiquitin in host cells. In the current study, we found that only GD-NT, but not GD-FL, underwent K63-type polyubiquitin chains on Lys237, which directed its translocation and oligomerization. TRAF1-OTUB1 axis was responsible for the ubiquitination and de-ubiquitination on GD-NT-K237. What`s more, we noticed that the ubiquitination on Lys237 did not affect the cleavage of GD-FL by caspase-11 and the production of GD-NT. Thus, we concluded that GD-NT possesses a unique ubiquitination status that differs significantly from GD-FL, and the ubiquitination on Lys237 is a post-cleavage event that help GD-NT translocate, form pore in plasma membrane and therefore promote pyroptosis. Consistent with this, the UBA1 (E1) inhibitor PYR-41 significantly suppressed GD-NT-mediated pyroptosis in 293-tetO-GD-NT cells, iBMDM and septic mice.\u003c/p\u003e \u003cp\u003eThus, our study elucidates the unique role of ubiquitylation in the regulation of GD-NT pyroptotic activity. In addition, we believe that manipulation of PTMs of GD-FL or GD-NT by small molecules, such as PYR-41, DMF, palmostatin B (PMB), metformin, and disulfiram\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, is potentially paving a therapeutic path in pyroptosis-related diseases. Intriguingly, some of these chemicals have exhibited promising outcomes in the animal models of septic shock\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e (Extended Fig.\u0026nbsp;10).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData reporting\u003c/h2\u003e \u003cp\u003eNo statistical methods were used to predetermine the sample size. The samples were not randomized. The investigators were not blinded to allocation during the experiments and the evaluation of the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and cell culture conditions\u003c/h2\u003e \u003cp\u003eHEK293, HeLa, and iBMDM cells were purchased from ATCC, and were maintained in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ mL penicillin and 100 \u0026micro;g/mL streptomycin. All cells were tested for mycoplasma by PCR and were authenticated by morphology only.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids\u003c/h2\u003e \u003cp\u003epDB-His-MBP-mGSDMD (Addgene, 123365) was a gift from Hao Wu. pTRIPZ-shNS (Addgene, 127696) was a gift from Sandra Demaria. The vectors expressing GD-NT, human TRAFs, human DUBs were generated by the standard PCR cloning strategy. Truncation mutation or point mutation plasmids were generated using QuickChange Primer Design Program and mutagenesis kit (Agilent Technologies). All plasmids were verified by DNA sequencing and IB analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eReagent and antibodies\u003c/h2\u003e \u003cp\u003eLPS O111:B4 (L2630), Doxycycline (D3447) were obtained from Sigma-Aldrich. Subcellular protein fractionation kit (78840) was obtained from Thermo Fisher. ATP (tlrl-atpl) was obtained from InvivoGen. StrataClean resin (400714) and chemical competent cells (200315) were obtained from Agilent. Bortezomib (B125789) and Carfilzomib (C127870) were obtained from Aladdin. PYR-41 (P798006) and Pevonedistat (MLN4924, P872287) were obtained from MACKLIN.\u003c/p\u003e \u003cp\u003eFor FCAS analysis, Annexin V-FITC/PI apoptosis kit (KGA1102) were obtained from KeyGEN BioTECH. For ELISA, mouse IL-1beta kit (EM0109), mouse IL-18 kit (EM1158), mouse TNF-alpha kit (EM0183), mouse HMGB1 kit (EM0382) were obtained from Finetest. For immunoblot, Human Reactive Cell Death and Autophagy Antibody Sampler Kit (#42867), anti-LSD1 (#2184) and anti-caspase-3 (#9662) were purchased from Cell Signaling Technology. Anti-Flag (F1804) and anti-HA (H6908) were obtained from Sigma-Aldrich. Anti-β-Actin (66009-1-Ig) and anti-GST (10000-0-AP) were purchased from Proteintech. Anti-Na/K-ATPase α1 (PTM-5533) were purchased from PTO BIO.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStable cell lines\u003c/h2\u003e \u003cp\u003eLentivirus was produced in HEK293 cells by transfection of the lentiviral vector with psPAX2 (Addgene) and pMD2.G (Addgene). Lentiviral super- natants were collected, filtered through 0.45-\u0026micro;m filters, and used to transduce HEK293 cells. Polybrene infection/transfection reagent (Millipore, 10\u0026micro;g/ml) was added to increase the efficiency of lentiviral infection. After 2 days of transduction, puromycin (Sigma, 2 \u0026micro;g/ml) was added to select the transduced cells. Empty lentiviral vectors were used to generate control cells. The expression of relevant genes in stable cell lines was verified by immunoblot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblots and immunoprecipitation\u003c/h2\u003e \u003cp\u003eCells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP- 40) supplemented with protease inhibitors (A32953, Thermo Fisher) and phosphatase Inhibitors (B15002, Bimake). The protein concentrations of lysates were measured using the Beckman Coulter DU-800 spectro- photometer and the Bio-Rad protein assay reagent. Same amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, cell lysates containing 1mg of total proteins were incubated with anti-Flag agarose (A2220, Sigma) or anti-HA Agarose (A2095, Sigma) for 4 hours at 4\u0026deg;C. Precipitants were washed three times with EBC buffer and resolved by SDS-PAGE followed by immunoblot analysis with indicated antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eProtein enrichment from the culture medium or peritoneal fluid\u003c/h2\u003e \u003cp\u003eTo enrich the proteins in the culture medium, 1 ml of culture medium was centrifuged at 14,000 x g for 10 min at 4\u0026deg;C to remove cellular debris. 10 \u0026micro;l StrataClean resin (400714, Agilent) was then added for 1-hour incubation on a rotator at 4\u0026deg;C. The supernatant was removed by centrifugation. The resin was harvested and suspended in 50 \u0026micro;l 2 x loading buffer for immunoblot. In septic mice model, the proteins in flushed peritoneal fluid were also enriched in the same way.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSize exclusion chromatography (SEC)\u003c/h2\u003e \u003cp\u003eSEC was performed using an AKTA Purifier system (GE Healthcare, Buckinghamshire, England). A HiLoad 16/600 Superdex 200 column (GE Healthcare) was equilibrated with cell lysis buffer. The column was calibrated using a gel filtration calibration kit (GE Healthcare). Each standard protein was dissolved in cell lysis buffer and chromatographed on the column separately. The filtered protein samples were then fractionated on the column (1.0ml/min; 2ml/fraction). For immunoblot analysis, proteins in fractionated eluent were enriched with 10\u0026micro;l StrataClean resin and harvested in 30 \u0026micro;l of 2 x loading buffer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity assay and cell viability assay\u003c/h2\u003e \u003cp\u003eCell death and cell viability were performed using Non-Radioactive Cytotoxicity Assay kit (G1780, Promega) and CellTiter-Glo Luminescent Cell Viability Assay kit (G7571, Promega), respectively. Briefly, 5 \u0026times;10\u003csup\u003e3\u003c/sup\u003e cells were cultured in 96-well plates with Opaque wall. At the desired time points, cell death was determined by titrating the amount of lactate dehydrogenase released into the culture medium, and cell viability was determined by the ATP levels within cells, according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMouse studies\u003c/h2\u003e \u003cp\u003e All procedures were conducted in compliance with American guidelines for the care and use of laboratory animals and were approved by Zhengzhou University Animal Care Committee in accordance with institutional animal care and use guidelines. Female wild-type C57BL/6 mice, and NSG mice (6\u0026ndash;8 weeks old) were purchased from Vital River Laboratories. All mice were housed in the Zhengzhou University Animal Facility.\u003c/p\u003e \u003cp\u003eFor xenograft experiment, before inoculation, cell viability was determined using trypan blue exclusion test (minimum of 98% cell viability). 1.0 \u0026times;10\u003csup\u003e6\u003c/sup\u003e of were subcutaneously injected into the right flank of mice. Tumor growth was monitored every other day. Tumor volume (mm3) is calculated via the \u0026ldquo;(W x W x L) / 2\u0026rdquo; formula, where L is the longest diameter and W is the shortest diameter. Necropsy and tumor collection were performed at the end of tumor size recording.\u003c/p\u003e \u003cp\u003eFor the septic model, mice were pretreated with PYR-41 (10 mg/ml, i.p.) on day-14 and the following every other day. on day0, LPS (5mg/ml, i.p.) were injected to induce sepsis. Serums were harvested for ELISA assay at 6 hours post-LPS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eFCAS analysis were performed according to the manufacture`s guideline. Briefly, cells were digested using trypsin without EDTA to get cell suspension. Then, cells were washed with PBS twice, suspended with 500\u0026micro;L of Binding Buffer to form a single-cell suspension. After adding 5\u0026micro;L of Annexin V-FITC\u0026thinsp;+\u0026thinsp;5\u0026micro;L of Propidium Iodide, cells were incubated at room temperature for 5 minutes before conducting flow cytometry observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining fluorescent microscopy\u003c/h2\u003e \u003cp\u003eFor Immunofluorescence staining, dish with special glass bottom were used. Cells were fixed with 100% methanol (chilled at -20℃) at RT for 5 min. To block unspecific binding of the antibodies, cells were incubated with 1% BSA, 22.52 mg/ml glycine in PBST (PBS\u0026thinsp;+\u0026thinsp;0.1% Tween 20) for 30 min (alternative is 10% serum from the species that the secondary antibody are raised in). For immunostaining, cells were incubated with the diluted antibody in 1% BSA in PBT in a humidified chamber for 1 hour at RT or overnight at 4 ℃, and then incubated with the secondary antibody in 1% BSA for 1 hour at RT in the dark. At last, cells were counter stained with 0.1-1 \u0026micro;g/ml Hoechst or DAPI for 1 min, mounted and subjected to microscopic analysis. Notes: Between steps, cells were washed with PBS for 3 times, 5 min each wash.\u003c/p\u003e \u003cp\u003eFor fluorescent microscopy of living cells, PI (10 \u0026micro;g/ml) or DAPI (1 \u0026micro;g/ml) was added into culture medium. 10 minutes later, cells were imaged under fluorescent microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStudent`s t-test was used to determine the differences between the two groups. Differences between tumor growth curves were compared by calculating the area-under-curve (AUC) values for each sample and then comparing different groups using Student\u0026rsquo;s t-test. The results are presented as the mean and standard error of the mean (SEM). Statistical significance was assigned to P\u0026thinsp;\u0026lt;\u0026thinsp;0.5%. Tumor-free survival and Kaplan- Meier analysis were performed using GraphPad Prism Version 5.04 for Windows.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eReporting summary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information on research design is available in the Research Reporting Summary linked to this document.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data are available in the Source Data (for Figs. 1\u0026ndash;6) or supplementary information associated with this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by Research fund for the Doctoral program of the Fifth Affiliated Hospital of Zhengzhou University (pb2024kyqdj03), Supporting Program for Young Talent Innovation Teams of Zhengzhou University (32320688), Henan Medical Key Technologies R \u0026amp; D Program (LHGJ20220570), Henan Natural Science Foundation (242300421283) and Tianjian Advanced Biomedical Laboratory Key Research and Development Project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXC and\u0026nbsp;IB\u003csup\u003e\u0026nbsp;\u003c/sup\u003econceived thec\u0026nbsp;study and designed the experiments.\u0026nbsp;XC\u0026nbsp;performed the experiments and written first draft. IB and XC reviewed and revised the manuscript. All the authors have discussed the results and approved the final version of the manuscript for publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe did not include human samples.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYu, P. \u003cem\u003eet al.\u003c/em\u003e Pyroptosis: mechanisms and diseases. 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Drug Discov Today 26, 2414\u0026ndash;2422, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.drudis.2021.04.029\u003c/span\u003e\u003cspan address=\"10.1016/j.drudis.2021.04.029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Gasdermin D, pyroptosis, Ubiquitination, PYR-41","lastPublishedDoi":"10.21203/rs.3.rs-4907061/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4907061/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGasdermin D (GSDMD) is a critical mediator of pyroptosis, which consists of a N-terminal pore-forming domain and a C-terminal autoinhibitory domain. The free N-terminal domain (GD-NT), which is released through caspase-1/11 cleavage, exhibits distinct features from the full-length GSDMD (GD-FL), including oligomerization, membrane translocation and pore-formation. However, the underlying mechanisms are not well clarified. Here, we found that GD-NT, but GD-FL, was massively ubiquitinated in cells. The K63-linked polyubiquitination of GD-NT at Lys236/237 (human/mouse), catalyzed by TRAF1, directly its membrane translocation and pore-formation during pyroptosis. Inhibition of GD-NT ubiquitination via site mutation or the UBA1 inhibitor PYR-41 suppressed cell death in several pyroptosis cell models. Additionally, the application of PYR-41 in septic mice efficiently suppressed the release of IL-18 and TNF-⍺. Thus, GD-NT ubiquitination is a key regulatory mechanism controlling its membrane localization and activation, which may provide a novel target for modulating immune activity in pyroptosis-related diseases.\u003c/p\u003e","manuscriptTitle":"Ubiquitination of gasdermin D N-terminal domain directs its membrane translocation and pore formation during pyroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-07 09:47:55","doi":"10.21203/rs.3.rs-4907061/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"99ac5241-aa6e-4d8d-a802-ee607b1f58d2","owner":[],"postedDate":"October 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":36099910,"name":"Biological sciences/Immunology/Cell death and immune response"},{"id":36099911,"name":"Health sciences/Diseases/Infectious diseases"},{"id":36099912,"name":"Biological sciences/Molecular biology/Epigenetics"},{"id":36099913,"name":"Biological sciences/Cell biology/Proteolysis/Ubiquitylation"}],"tags":[],"updatedAt":"2025-03-18T07:11:21+00:00","versionOfRecord":{"articleIdentity":"rs-4907061","link":"https://doi.org/10.1038/s41419-025-07475-6","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-03-17 04:00:00","publishedOnDateReadable":"March 17th, 2025"},"versionCreatedAt":"2024-10-07 09:47:55","video":"","vorDoi":"10.1038/s41419-025-07475-6","vorDoiUrl":"https://doi.org/10.1038/s41419-025-07475-6","workflowStages":[]},"version":"v1","identity":"rs-4907061","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4907061","identity":"rs-4907061","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}