Pyroptotic executioner pore-forming protein gasdermin D forms oligomeric assembly and exhibits amyloid-like attributes that could contribute for its pore-forming function

Article

作者: Kaur, Gurvinder ; Chatterjee, Shamaita ; Gupta, Tarang ; Chattopadhyay, Kausik

Gasdermin D (GSDMD) is the chief executioner of inflammatory cell death or pyroptosis. During pyroptosis, proteolytic processing of GSDMD releases its N-terminal domain (NTD), which then forms large oligomeric pores in the plasma membranes. Membrane pore-formation by NTD allows the release of inflammatory cytokines and causes membrane damage to induce cell death. Structural mechanisms of GSDMD-mediated membrane pore-formation have been extensively studied. However, less effort has been made to understand the physicochemical properties of GSDMD and their functional implications. Here, we explore detailed characterization of the physicochemical properties of mouse GSDMD (mGSDMD), and their implications in regulating the pore-forming function. Our study reveals that mGSDMD shows some of the hallmark features of amyloids, and forms oligomeric assemblies in solution that are critically dependent on the disulfide bond-forming ability of the protein. mGSDMD oligomeric assemblies do not resemble typical amyloid fibrils/aggregates, and do not show resistance to proteolytic degradation that is otherwise observed with the conventional amyloids. Our results further elucidate the essential role of an amyloid-prone region (APR) in the oligomerization and amyloid-like features of mGSDMD. Furthermore, alteration of this APR leads to compromised pore-forming ability and cell-killing activity of NTD released from mGSDMD. Taken together, our study for the first time provides crucial new insights regarding implications of the amyloid-like property of mGSDMD in regulating its pore-forming function, which is an essential requirement for this pyroptotic executioner. To the best of our knowledge, such mode of regulation of mGSDMD-function has not been appreciated so far.

2024-11-01·Phytomedicine

Paeoniflorin inhibited GSDMD to alleviate ANIT-induced cholestasis via pyroptosis signaling pathway

Article

作者: Wang, Zexin ; Jiang, Tao ; Ma, Xiao ; Zhang, Wenwen ; Efferth, Thomas ; Hu, Qichao ; Chen, Yuan ; Zeng, Yi

BACKGROUNDCholestasis (CT) is a group of disorders caused by impaired production, secretion or excretion of bile. This may result in the deposition of bile components in the blood and liver, which in turn causes damage to liver cells and other tissues. If untreated, CT can progress to severe complications, including cirrhosis, liver failure, and potentially life-threatening conditions.OBJECTIVEThis research was intended to elucidate the function and mechanism of Paeoniflorin (PF) in ameliorating ANIT-induced pyroptosis in CT.METHODSCT models were established in SD rats and HepG2 cells through ANIT treatment. Histological examination was conducted using haematoxylin and eosin (HE) staining to assess the histopathological alterations in the liver. Network pharmacology was employed to identify potential PF targets in CT treatment. To evaluate pyroptosis levels, various methods were used, including serum biochemical analysis, Enzyme-Linked Immunosorbent Assay (ELISA), immunofluorescence (IF), immunohistochemistry (IHC), Western blotting, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The HuProt™ 20K Chip was utilized to pinpoint potential PF-binding targets. PF's direct mechanisms in CT treatment were explored using molecular docking (MD), molecular dynamics simulations (MDS), Cellular Thermal Shift Assay (CETSA), and Surface Plasmon Resonance (SPR).RESULTSPF administration was found to alleviate ANIT-induced liver pathology, enhance liver function markers, and improve cell viability. Network pharmacology and pyroptosis inhibitor studies suggested that PF might mitigate CT via the NLRP3-dependent pyroptosis pathway. This hypothesis was further supported by Western blotting, IF, and IHC analyses, which indicated PF's potential to inhibit NLRP3-dependent pyroptosis in CT. GSDMD was identified as a target through HuProt™ 20K Chip screening. The binding affinity of PF to GSDMD was validated through MD, MDS, CETSA, and SPR techniques. Additionally, the regulatory impact of GSDMD on downstream inflammatory pathways was confirmed by ELISA and IHC.CONCLUSIONPF exhibited a hepatoprotective effect in ANIT-induced CT, primarily by targeting GSDMD, thereby suppressing ANIT-induced pyroptosis and the subsequent release of inflammatory mediators.

2024-10-01·Cell

Small-molecule GSDMD agonism in tumors stimulates antitumor immunity without toxicity

Article

作者: Fontana, Pietro ; Lieberman, Judy ; Xia, Shiyu ; Wu, Hao ; Zhang, Haiwei ; Healy, Liam B. ; Shi, Ming ; Luo, Hongbo R. ; Agudo, Judith ; Ru, Heng ; Healy, Liam B ; Zhang, Ying ; Du, Gang ; Tufan, Ahmet B. ; Luo, Hongbo R ; Li, Zhouyihan ; Vora, Setu M ; Baldominos, Pilar ; Vora, Setu M. ; Lee, Dian-Jang ; Tufan, Ahmet B ; Hu, Jun Jacob

Gasdermin-mediated inflammatory cell death (pyroptosis) can activate protective immunity in immunologically cold tumors. Here, we performed a high-throughput screen for compounds that could activate gasdermin D (GSDMD), which is expressed widely in tumors. We identified 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB) as a direct and selective GSDMD agonist that activates GSDMD pore formation and pyroptosis without cleaving GSDMD. In mouse tumor models, pulsed and low-level pyroptosis induced by DMB suppresses tumor growth without harming GSDMD-expressing immune cells. Protection is immune-mediated and abrogated in mice lacking lymphocytes. Vaccination with DMB-treated cancer cells protects mice from secondary tumor challenge, indicating that immunogenic cell death is induced. DMB treatment synergizes with anti-PD-1. DMB treatment does not alter circulating proinflammatory cytokine or leukocyte numbers or cause weight loss. Thus, our studies reveal a strategy that relies on a low level of tumor cell pyroptosis to induce antitumor immunity and raise the possibility of exploiting pyroptosis without causing overt toxicity.

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适应症	最高研发状态	国家/地区	公司	日期
脓毒症	临床前	中国	上海交通大学医学院附属瑞金医院	2023-11-02
脓毒症	临床前	中国	上海现代药物制剂工程研究中心有限公司	2023-11-02