Chemical-induced dimerization of GSDMD C-terminal domain blocks GSDMD N-terminal domain-mediated pyroptosis

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Chemical-induced dimerization of GSDMD C-terminal domain blocks GSDMD N-terminal domain-mediated pyroptosis

https://doi.org/10.21203/rs.3.rs-4654805/v1

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GSDMD is a pyroptosis executioner protein, in which the C-terminal domain (GD-CT) inhibits the pore-forming ability of N-terminal domain (GD-NT). Caspase-cleavage separates GD-NT and GD-CT, and then the free GD-NTs oligomerize and form nano-sized pyroptotic pores in plasma membrane. It is still not clear why the free GD-CT fragment cannot block GD-NT. Here in the current study, we found that the free GD-CT retained the interaction with the free GD-NT. However, GD-CT was totally kept in cytoplasm, while GD-NT was partially transported to plasma membrane. This distributional alteration caused a spatial isolation from each other. What's more, GD-CT was an unstable peptide with a faster turnover time than GD-NT. These features make GD-CT inefficiently block GD-NT and allow pyroptosis to happen. We designed the chemical-induced dimerization (CID) chimera protein "FKBP-GD-CT". The myristoylation motif helps the chimera translocate to plasma membrane, and FKBP12^F36V domain makes the chimera dimerize in response to the chemical AP20187. we found that FKBP-GD-CT blocked GD-NT-mediated pyroptosis more efficiently than GD-CT. We concluded that the free GD-CT still holds the ability of blocking pyroptosis, and this blocking ability can be adjusted by proper Engineering. FKBP-GD-CT is the first report of using CID technology to manipulate pyroptosis, giving a clue to the improvement of clinical outcomes in pyroptosis-related diseases.

Biological sciences/Molecular biology/Proteolysis/Proteasome

Health sciences/Medical research/Translational research

Pyroptosis is an unique inflammatory cell death executed by a pore-forming protein "gasdermin"¹. The human gasdermin family consists of GSDMA, GSDMB, GSDMC, GSDMD, GSDME and DFNB59. In the mouse, gasdermins are mostly identical to human, except the absence of GSDMB and the unique existence of GSDMA1, GSDMA2, GSDMA3, (A1, A2, A3) and GSDMC1, GSDMC2, GSDMC3, GSDMC4^2,3. To activate the pore-forming activity, gasdermin needs to be recognized and cleaved by proper protease to produce gasdermin-N terminal fragment. There are two main branches of gasdermin activation pathway. In the canonical pathway, the recognition of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) leads to the assembly of inflammasomes, which activate caspase-1 to cleave GSDMD⁴. Besides, several non-canonical pathways are documented. Firstly, with the existence of cytosolic lipopolysaccharide (LPS), caspase-4/5 (in humans) or caspase-11 (in mice) directly recognizes and cleaves GSDMD⁴. Secondly, in tumor cells, chemotherapeutic agents, such as etoposide and cisplatin, activate caspase-3 to cleave GSDME⁵. Thirdly, with the help of perforin, NK cells inject granzyme A and granzyme B into tumor cells to cleave GSDMB and GSDME, respectively^6,7. Lastly, caspase-8 is activated to cleave GSDMC under the hypoxia conditions⁸. These liberated gasdermin N-terminals form pores in the plasma membrane and disrupt cellular integrity.

Pyroptosis is observed firstly in macrophages^9-11 and later on in other cell types from immune system and different tissues. More and more evidences reveal that pyroptosis is not only involved in the defense against pathogenic organism¹², it also participates in other pathological conditions, such as diabetes¹³, chronic inflammation and cancer^14-17. In order to therapeutically regulate the excessive pyroptosis, more understanding of gasdermin processing is appealed. In this study, we aimed to answer the question "how does protease-cleavage abolish GD-CT`s blocker activity in GSDMD-mediated pyroptosis". To this end, we created 293-tetO-GD-NT cells, which undergo pyroptosis in response to Doxycycline (Dox). We found that the free GD-CT retain to interact with GC-NT, but it had a different subcellular localization and a faster turnover time when compared to GD-NT. Our designed Myr-FKBP-GD-CT more efficiently blocked GD-NT-mediated pyroptosis.

Data reporting

No statistical methods were used to predetermine sample size. The experiments were not randomized. Investigators were not blinded to allocation during experiments and outcome assessment.

Cell lines and cell culture conditions

HEK293 cells and HeLa cells was purchased from ATCC. Cells was 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

pcDNA3.1-based Flag-GD-CT, HA-GD-CT, Flag-GD-FL, Flag-GD-NT, nFKBP-GD-CT, cFKBP-GD-CT and nmFlag-GD-FL were generated by the standard PCR cloning strategy. The lentiviral vector pTRIPZ-tetOn-GD-NT was used to create the stable cell line 293-tetO-GD-NT cells. All plasmids were verified by DNA sequencing and IB analysis.

Reagent and antibodies

Doxycycline (D3447) was obtained from Sigma-Aldrich. Subcellular protein fractionation kit (78840) was obtained from Thermo Fisher. StrataClean resin (400714) and chemical competent cells (200315) were obtained from Agilent. For immunoblot, Anti-Flag (F1804), anti-Flag (F7425) and anti-HA (H6908) were obtained from Sigma-Aldrich. Anti-GSDMD (ab219800) was obtained from Abcam. Anti-β-Actin (sc-47778), anti-GST (sc-138) and anti-Na/K-ATPase α1 (sc-21712) were purchased from Santa Cruz Biotechnology.

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, 10 µg/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 1 mg 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.

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 ×10³ 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.

Flow cytometry

Single-cell suspensions were first incubated with viability dyes (Zombie Aqua; Biolegend) to identify dead cells and with an anti-FcγRII/III antibody (2.4G2; BD Bioscience) to block nonspecific antibody binding. After washing, the cells were incubated with mixtures of fluorescently labeled antibodies for 20 min at 4 °C. Samples were acquired using an LSRFortessa™ or LSR II flow cytometer (BD Bioscience) and analyzed using FlowJo software (Tree Star).

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 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.

Pyroptosis model cell line"293-tetO-GD-NT " is created to assess the blocker ability of GD-CT

Pyroptosis signaling pathways converge on the activation of specific proteases, which liberate the cytolytic gasdermin N-terminal domain from the inhibitory gasdermin C-terminal domain (Extended Fig. 1A-B). Take GSDMD for example, the separation of GD-NT and GD-CT is achieved via caspase-11-mediated cleavage, allowing the pore-forming activity of GD-NT being manifested (Extended Fig. 1C, D). It is still unknown whether the free GD-CT retains the blocker ability against GD-NT. In the current study, we aimed to understand the functions and characteristics of the free GD-CT.

To investigate the blocker ability of GD-CT, we set up a pyroptosis model cell line "293-tetO-GD-NT ", where the transgene GD-NT is expressed in response to Dox (Fig. 1A). Immunoblot assay showed a fast GD-NT expression, which was detectable at 2nd hour and highest at 4th hour after Dox treatment (Fig. 1B). Cell viability assay indicated a significantly ATP production decline as early as 4th hour after Dox treatment (Fig. 1C), and LDH-based cell toxicity assay revealed an obvious cell lysis at 8th hour after Dox treatment (Fig. 1D). Dying cells were easily observed at 8th hour after Dox treatment under phase-contrast microscope or under fluorescent microscope when stained with the nucleic acid dye DAPI. More than 95% cells underwent pyroptosis at 24th hour after Dox treatment (Fig. 1E). Because Dox was reported to induce apoptosis in some cases^18–20, we then used Annexin V-FITC/PI double-staining kit to test the possible involvement of Dox-induced apoptosis in the pyroptosis model cells. The result showed that Dox induced a specific pyroptosis in 293-tetO-GD-NT cells, and no apoptosis was observed during the observation period (Fig. 1F). Thus, 293-tetO-GD-NT cells were used to test GD-CT blocker ability in the following study.

GD-CT blocks GD-NT-mediated pyroptosis

To test the blocker ability of GD-CT, we transiently transfected it into 293-tetO-GD-NT cells. The expressions of GD-NT and GD-CT were confirmed via immunoblot assay (Fig. 2A). Cell toxicity assay and viability assay showed that GD-CT not only reduced the leakage of cellular LDH into culture medium, but also restored the ATP production of dying cells in a dose-dependent manner (Fig. 2B, C). With the help of phase-contrast microscope and fluorescent microscope, an obvious increase of surviving cells and a reduced number of dead cells were observed in 293-tetO-GD-NT cells transfected with GD-CT (Fig. 2D, E). We next performed Flow cytometry to quantify the dead cells, and found that Dox-induced GD-NT killed ~ 70% of 293-tetO-GD-NT cells at the observation time opint (14 hours after Dox treatment). When GD-CT were introduced into cells, the proportion of dead cells significantly dropped to ~ 50% (Fig. 2F, G). These finding suggest that GD-CT inhibited GD-NT-mediated pyroptosis in a dose-dependent manner.

GD-CT binds GD-NT and blocks its translocation to plasma membrane

In the structure of GD-FL, the liner region holds GD-NT and GD-CT together, making GD-CT completely block the pore-forming ability of GD-NT and prevent pyroptosis (Fig. 3A). To study how the free GD-CT inhibits GD-NT-mediated pyroptosis, we created Flag-tagged GD-NT and HA-tagged GD-CT and co-transfected them into HeLa cells. Immunofluorescence microscopy showed a colocalization of GD-CT and GD-NT in cytoplasm (Extended Fig. 2A, B). To further test their physical contact, HEK293 cells were con-transfected with GD-CT and GD-NT, and subjected to immunoprecipitation assay. The result showed that GD-CT, but not GD-FL, interacted with GD-NT (Fig. 3B, C). To directly assess whether GD-NT pore-formation is affected by GD-CT, we tried to test the leakage of GD-NT into culture medium. For this purpose, we transfected GD-CT into 293-tetO-GD-NT cells and separately collected culture medium, plasma membrane, cytoplasm as well as whole cell lysate for immunoblot analysis. Our result revealed that the concentrations of GD-NT in culture medium and plasma membrane were significantly reduced by GD-CT in a dose-dependent manner (Fig. 3D). Based on these results, we concluded that the free GD-CT inhibits GD-NT-mediated pyroptosis via binding GD-NT and therefore blocking its transportation from cytoplasm to plasma membrane.

The restriction of GD-CT in cytoplasm causes a spatial isolation

From Fig. 2 and Fig. 3, we realized that, although binding to GD-NT, GD-CT blocked the pore-formation not as efficient as the intramolecular mechanism of GD-FL, where GSDMD C-terminal domain completely blocks the pore-forming ability of GSDMD N-terminal domain. We supposed that the characteristics of GD-CT may contribute to its inefficiency of blocking GD-NT. To analyze GD-CT subcellular localization, we transfected GD-CT into HeLa cells and performed immunofluorescent staining. Confocal microscopy revealed that GD-CT was more restricted in cytoplasm than GD-NT (Extended Data Fig. 3). To precisely define GD-CT subcellular distribution, HEK293 cells were transfected with GD-CT, GD-NT, or GD-FL and fractionated into five compartments: soluble cytoplasmic, membrane, soluble nuclear, chromatin-bound nuclear and insoluble cytoskeletal. Immunoblot analysis showed that GD-CT localized uniquely in cytoplasm, while GD-NT was found in cytoplasm, plasma membrane as well as cytoskeleton (Fig. 4A).

To obtain more reliable data for GD-CT distribution, we engineered the construct "nmFlag-GD-FL" (Fig. 4B). In the structure of nmFlag-GD-FL, Flag-tag is added at the N-terminal of both GD-NT and GD-CT to allow their simultaneously detection. We transfected HEK293 cells with nmFlag-GD-FL and caspase-11, and then performed fractionation. Immunoblot analysis verified the unique distribution of GD-CT in cytoplasm and the wide distribution of GD-NT in cytoplasm and plasma membrane (Fig. 4C). Consistently, only GD-CT but not GD-NT was detected in culture medium (Fig. 4D). Thus, the spatial isolation may contribute to GD-CT inefficiency in the blocking of GD-NT, where GD-CT only interact with cytoplasmic GD-NT, but not membranous one, allowing the latter to perform pore-forming ability.

The short half-life of GD-CT contributes to the liberation of GD-NT

Next, to test whether GD-CT stability contributes to its inefficiency of blocking GD-NT, HEK293 cells were transfected with Flag-GD-FL, Flag-GD-NT or GD-CT, and followed by the treatment of CHX, a commonly-used protein synthesis inhibitor. We found that GD-CT had a much short half-life and became indetectable 4 hours after CHX treatment. In contrast, GD-FL did not degrade in the 4-hour observation period (Fig. 5A, B). To further determine the degradation pathways of GD-CT, we used chloroquine, calpeptin, Z-VAD-FMK and MG132 chemicals to block autophagy, calpain, caspase and Ubiquitination (Ub)-proteasome, respectively. The result indicated that MG132 completely inhibited the degradation of GD-CT. Thus, we believe that GD-CT undergoes a fast degradation via Ub-proteasome system (Fig. 5C, D).

Because GD-NT also had a short half-life (Fig. 5A-D), we employed a more reliable strategy to compare GD-CT and GD-NT for their stability. When GD-FL is cleaved by caspase-11, GD-CT and GD-NT are produced at an exact ratio of 1:1. The design of nmFlag-GD-FL allows us to detect Flag-GD-CT and Flag-GD-NT using the same antibody at the same time, making it an ideal model to simultaneously assess the turnover of GD-CT and GD-NT (Fig. 4B), (Fig. 4A). We transfected nmFlag-GD-FL and caspase-11 into HEK293 cells. Immunoblot analysis revealed that GD-CT, when compared to GD-NT, had a much lower level in cells (Fig. 5E). To assess the reliability of nmFlag-GD-FL processing, HEK293 cells were transfected with nmFlag-GD-FL and caspase-11, and treated with different protease inhibitors. Our result showed that the pan caspase Z-VAD-FMK inhibited caspase-11 activity and blocked the production of Flag-GD-CT and Flag-GD-NT (Fig. 5F). Treatment with CHX and MG132 further verified the Ub-proteasome degradation pathway of GD-CT and GD-NT (Fig. 5G, H). Therefore, we concluded that our engineered nmFlag-GD-FL is an ideal model to simultaneously assess the turnover of GD-CT and GD-NT. GD-FL is a stable protein. Once it is cleaved by caspase-11, the produced GD-CT undergoes a faster degradation than GD-NT, making less GD-CT and more GD-NT exist in cells. This mechanism contributes to the inefficiency of GD-CT blocker ability (Fig. 5I).

The design of chimera protein FKBP-GD-CTs

The blocker activity of GD-CT makes it a potential target in pyroptosis-related diseases. To purposely increase the efficiency of GD-CT blocking GD-NT, we employed two strategies to create the chimera proteins FKBP-GD-CTs (nFKBP-GD-CT and cFKBP-GD-CT). The myristoylation sequence helps GD-CT translocate to plasma membrane. Two FKBP^F36V domains make GD-CT undergo chemical-induced dimerization (Fig. 6A and Extended Fig. 5A). To assess the characteristics and biological activities of FKBP-GD-CTs, we transfected them into 293-tetO-GD-NT cells, and treated cells with CHX and AP20187. We found that nFKBP-GD-CT localized in plasma membrane, but cFKBP-GD-CT unexpectedly stayed in cytoplasm (Fig. 6B). What`s more, AP20187 conferred FKBP-GD-CTs a long half-life. In contrast, AP20187 did not affect the stability of the original GD-CT (Fig. 6C). To test the blocking efficiency of FKBP-GD-CTs on the pore-forming activity of GD-NT, 293-tetO-GD-NT cells were transfected with FKBP-GD-CTs and subjected to fractionation and immunoblot analysis. The result revealed that FKBP-GD-CTs significantly suppressed the leakage of GD-NT into culture medium, and AP20187 enhanced this suppression (Fig. 6D).

FKBP-GD-CTs block GD-NT-mediated pyroptosis in a more efficient way

To test the blocking efficiency of FKBP-GD-CTs in GD-NT-mediated pyroptosis, 293-tetO-GD-NT cells were transfected with FKBP-GD-CTs or the original GD-CT, followed by treatment of AP20187. Several strategies were performed to assess cell death. Firstly, phase-contrast imaging showed that FKBP-GD-CTs protected 293-tetO-GD-NT cells from pyroptosis in a dose-dependent manner (Extended Fig. 6A), and AP20187 further enhanced this protection (Fig. 7A). Secondly, the cells were stained with DAPI without fixation and subjected to fluorescent microscopy, where the DAPI-positive cells were considered as dying cells. The result revealed that DAPI staining were much less in the cells treated with FKBP-GD-CTs and AP20187 (Fig. 7B). Lastly, we did FCAS analysis to quantify the dying cells. The smallest number of pyroptotic cells were observed in the group " cFKBP-GD-CT + AP20187" (Fig. 6C). Thus, we concluded that cFKBP-GD-CT plus AP20187 has the highest efficiency to block GD-NT-mediated pyroptosis. These data suggest that the engineered FKBP-GD-CTs can translocate to palsma membrane and become more efficient in response to AP20187, and cFKBP-GD-CT plus AP20187 has the highest efficiency to block GD-NT-mediated pyroptosis.

Gasdermins are predominantly expressed in the epithelium of gastrointestinal tract and skin²¹. For a very long time, gasdermins are documented as regulators of epithelial cell growth, hair formation^22,23, hearing loss, carcinogenesis^24,25. In 2015, Gao team firstly found that loss-of-function mutation Y344H of Gsdma3, as well as the N-terminal domain of Gsdma3, induces cell death through autophage, and the cell death can be blocked by C-terminal domain of Gsdma3 and other gasdermin members²⁶. Later on, Shao Feng team demonstrated that gasdermin-mediated cell death is a novel type of cell death “pyroptosis”⁴, which does not depend on autophagy. Based on these findings, it seems that gasdermins function to regulate diverse biological processes when they are kept in the full-length form and to execute pyroptosis when they are cleaved to produce cytolytic gasdermin N-terminal domain. Thus, understanding the intricacies of gasdermin processing is crucial for the development of therapeutic purposes.

GSDMD is the first characterized and also the most important pyroptosis executioner in innate immunity. In protein structure, GD-FL consists of the cytolytic GD-NT domain and the inhibitory GD-CT domain. This intracellular inhibitory mechanism makes GD-FL non-cytolytic and harmless to the host cells. To liberate the cytolytic activity of GD-NT domain, inflammatory caspases cleave the linker region and separate the intact GD-FL into two free fragments: GD-NT and GD-CT⁴. Although the pore-forming activity of GD-NT has been clarified, it is not well documented how the characteristics of GD-CT contribute to the occurrence of pyroptosis till now. In this study, we observed an obvious interaction of GD-CT with GD-NT in cells, but GD-CT blocked GD-NT cytolytic activity in an inefficient way and failed to completely prevent cells from undergoing pyroptosis. We then found that, not like GD-NT fragment translocating from cytoplasm to plasma membrane, GD-CT could stay only in cytoplasm. The different distribution pattern causes a spatial separation from each other. Additionally, we found that both GD-NT and GD-CT were unstable peptides and underwent fast proteasome-dependent degradation. More importantly, GD-CT had a shorter turnover time than GD-NT. Therefore, our results revealed that the loss of GD-CT blocker ability is not because of the failure of its binding to GD-NT, but is attributed to its spatial separation from GD-NT and its faster turnover of GD-CT when compared with GD-NT.

Apparently, the free GD-CT is not useless peptide in the dying cells. It is still capable of regulating the extent of pyroptosis. As we know, pyroptosis is detrimental in some cases, such as cancer, rheumatoid arthritis, inflammatory bowel disease, autoinflammatory syndromes and even neuroinflammation^{14,16,27–29}. GD-CT can be bioengineered to achieve therapeutic purposes in pyroptosis-related diseases. In our study, to increase the blocker ability of GD-CT, we create a chimera protein M-FKBP-GD-CT by fusing GD-CT with a Myr-sequence and two FKBP^F36V domains. Myr sequence helps GD-CT translocate to plasma membrane to block GD-NT pore-formation. FBKP^F36V domains confer GD-NT the function of chemical-induced dimerization (CID), making the chimera form dimer in the presence of AP20187. We found that AP20187 made M-FKBP-GD-CT have a much longer turnover time and become more efficient to block GD-NT-mediated pyroptosis when compared to the free GD-CT. This design may also be useful in other gasdermin-mediated pyroptosis for curative intervention. In summary, the pyroptosis blocker M-FKBP-GD-CT is the first reported CID design in pyroptosis field. We believe that the further understanding and bioengineering of inhibitory gadermin C-terminal domain will shed light on the translationally medical intervention in pyroptosis-related diseases.

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 PZ conceived the study and designed the experiments. JX performed the experiments. 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.

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Chemical-induced dimerization of GSDMD C-terminal domain blocks GSDMD N-terminal domain-mediated pyroptosis

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Result

Pyroptosis model cell line"293-tetO-GD-NT " is created to assess the blocker ability of GD-CT

GD-CT blocks GD-NT-mediated pyroptosis

GD-CT binds GD-NT and blocks its translocation to plasma membrane

The restriction of GD-CT in cytoplasm causes a spatial isolation

The short half-life of GD-CT contributes to the liberation of GD-NT

The design of chimera protein FKBP-GD-CTs

FKBP-GD-CTs block GD-NT-mediated pyroptosis in a more efficient way

Discussions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1