Pyrin炎症小体激活的分子机制与Gasdermin E介导的细胞焦亡的研究
发布时间：2019-03-04 08:49
【摘要】：Pyrin蛋白由MEFV基因编码,其点突变是引起一种分布于地中海沿岸地区的自身炎症性疾病家族性地中海热(familiar mediterranean fever, FMF)的原因。Pyrin蛋白可作为炎症小体中的重要分子,通过间接识别由病原微生物毒力因子修饰并失活的Rho GTPases引起炎症小体的激活。与天然免疫其他模式识别受体不同,Pyrin蛋白通过间接识别微生物毒力因子对自身蛋白的修饰而非直接识别病原微生物固有分子从而激活下游免疫反应。然而,Pyrin炎症小体激活的具体机制至今尚不明确。本研究主要利用蛋白质免疫印迹、蛋白质免疫共沉淀等手段,证明在免疫细胞中,Pyrin蛋白存在磷酸化修饰,其205位与241位丝氨酸的磷酸化修饰介导Pyrin蛋白与14-3-3蛋白的结合,使Pyrin蛋白维持在活性抑制状态。通过制备特异性识别两磷酸化位点的单克隆抗体,证实205、241位丝氨酸在细胞受到病原微生物刺激时共同发生去磷酸化反应。利用诱导表达系统,证明Pyrin蛋白的位点特异性去磷酸化是Pyrin炎症小体激活的原因。同时,本研究发现家族性地中海热的有效治疗性药物秋水仙素可抑制Pyrin炎症小体的激活。秋水仙素并不能抑制细胞受到病原微生物毒力因子刺激时Pyrin蛋白的去磷酸化反应,而是通过抑制激活后的Pyrin蛋白招募下游ASC蛋白从而阻碍炎症小体的激活。综上,本文第一部分工作证明Pyrin蛋白的位点特异性磷酸化修饰与正常的微管蛋白动力学平衡是Pyrin炎症小体激活的重要控制元件。细胞焦亡(pyroptosis)属于细胞炎症性坏死的一种,在机体抵抗病原微生物感染以及许多疾病的发病中发挥重要作用。细胞焦亡的直接原因是GasderminD(GSDMD)蛋白被通过炎症小体激活的Caspase-1或直接结合细菌脂多糖(lipopolysaccharide,LPS)而活化的Caspase-4/5/11切割,使GSDMD释放蛋白质N端活性部分在细胞膜上聚合形成通透性孔道所致。Gasdermin蛋白家族成员多含有膜通透N端结构域,但其生物学功能依然处于未知状态。本文通过细胞成像、蛋白质免疫印迹及流式细胞仪分析等手段,证明Gasdermin家族成员Gasdermin E(GSDME)可以被活化的Caspase-3切割激活,并以类似于GSDMD导致细胞焦亡的方式引起细胞的炎症性坏死。化疗药物处理无GSDME表达的Jurkat细胞可使细胞发生细胞凋亡,而处理高表达GSDME的SH-SY5Y细胞系与人角质化原代细胞可通过活化Caspase-3引起GSDME介导的细胞焦亡,用药物抑制Caspase-3活性或降低GSDME的表达量均能抑制细胞焦亡的发生。动物实验结果表明,Gsdme基因敲除可有效保护小鼠免受DNA损伤化疗药物造成的肺部与小肠组织的损伤。综上所述,本文第二部分工作证明Caspase-3可通过切割激活GSDME引起GSDME介导的细胞炎症性坏死,而细胞中GSDME表达与否成为在Caspase-3激活后,决定细胞死亡方式的重要指标。
[Abstract]:The Pyrin protein is encoded by the MEFV gene, and its point mutation is the cause of a familial Mediterranean fever (familiar mediterranean fever, FMF), a self-inflammatory disease distributed along the Mediterranean coast. Pyrin protein is an important molecule in inflammatory bodies. The activation of inflammatory bodies is induced by indirect recognition of Rho GTPases modified and inactivated by pathogenic microbial virulence factors. Unlike other innate immune pattern recognition receptors, Pyrin protein activates downstream immune responses by indirectly recognizing the modification of microbial virulence factors to their own proteins rather than directly recognizing the intrinsic molecules of pathogenic microorganisms. However, the specific mechanism of activation of inflammatory corpuscles in Pyrin remains unclear. In this study, Western blotting and protein immunoprecipitation were used to prove that Pyrin protein was phosphorylated in immune cells. The phosphorylation of 205th and 241st serine mediated the binding of Pyrin protein to 14m3 / 3 protein, which kept the Pyrin protein in a state of inhibitory activity. Through the preparation of monoclonal antibodies that specifically recognize the two phosphorylated sites, it was confirmed that the dephosphorylation of serine at site 205241 occurred when cells were stimulated by pathogenic microorganisms. Using the induced expression system, it was proved that the site-specific dephosphorylation of Pyrin protein was responsible for the activation of inflammatory corpuscles in Pyrin. At the same time, we found that colchicine, an effective therapeutic drug of familial Mediterranean fever, can inhibit the activation of inflammatory bodies in Pyrin. Colchicine did not inhibit the dephosphorylation of Pyrin proteins stimulated by pathogenic microbial virulence factors, but inhibited the activation of inflammatory bodies by inhibiting the recruitment of downstream ASC proteins by activated Pyrin proteins. In summary, in the first part of this paper, it is proved that the site-specific phosphorylation of Pyrin protein and the dynamic balance of microtubule protein are important control elements for the activation of inflammatory bodies in Pyrin. (pyroptosis) is a kind of inflammatory necrosis, which plays an important role in the resistance of the organism to the infection of pathogenic microorganisms and the pathogenesis of many diseases. The direct cause of focal death is the cleavage of GasderminD (GSDMD) proteins by Caspase-4/5/11 activated by inflammatory bodies or by direct binding to bacterial lipopolysaccharide (lipopolysaccharide,LPS). The N-terminal active part of GSDMD-releasing protein was polymerized on the cell membrane to form a permeability pore. Gasdermin protein family members mostly contain N-terminal domains, but their biological functions are still in an unknown state. The results of cell imaging, Western blot and flow cytometry showed that Gasdermin E (GSDME), a member of the Gasdermin family, could be activated by activated Caspase-3 cleavage. It also causes inflammatory necrosis of cells in a manner similar to that caused by GSDMD. Jurkat cells without GSDME expression were treated with chemotherapeutic drugs, and GSDME-overexpressing SH-SY5Y cell lines and human keratinized primary cells could induce GSDME-mediated focal death through activation of Caspase-3, while the cells treated with high-expression SH-SY5Y cells and human keratinized primary cells could induce apoptosis. Inhibition of Caspase-3 activity or reduction of GSDME expression by drugs could inhibit the occurrence of focal death. The results of animal experiments showed that Gsdme gene knockout can effectively protect mice from DNA damage to lung and small intestine tissue damage caused by chemotherapeutic drugs. In conclusion, the second part of this work proves that Caspase-3 can activate GSDME to induce GSDME-mediated inflammatory necrosis of cells, and whether the expression of GSDME in cells is an important index to determine the way of cell death after the activation of Caspase-3.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R593.2
本文编号：2434119
[Abstract]:The Pyrin protein is encoded by the MEFV gene, and its point mutation is the cause of a familial Mediterranean fever (familiar mediterranean fever, FMF), a self-inflammatory disease distributed along the Mediterranean coast. Pyrin protein is an important molecule in inflammatory bodies. The activation of inflammatory bodies is induced by indirect recognition of Rho GTPases modified and inactivated by pathogenic microbial virulence factors. Unlike other innate immune pattern recognition receptors, Pyrin protein activates downstream immune responses by indirectly recognizing the modification of microbial virulence factors to their own proteins rather than directly recognizing the intrinsic molecules of pathogenic microorganisms. However, the specific mechanism of activation of inflammatory corpuscles in Pyrin remains unclear. In this study, Western blotting and protein immunoprecipitation were used to prove that Pyrin protein was phosphorylated in immune cells. The phosphorylation of 205th and 241st serine mediated the binding of Pyrin protein to 14m3 / 3 protein, which kept the Pyrin protein in a state of inhibitory activity. Through the preparation of monoclonal antibodies that specifically recognize the two phosphorylated sites, it was confirmed that the dephosphorylation of serine at site 205241 occurred when cells were stimulated by pathogenic microorganisms. Using the induced expression system, it was proved that the site-specific dephosphorylation of Pyrin protein was responsible for the activation of inflammatory corpuscles in Pyrin. At the same time, we found that colchicine, an effective therapeutic drug of familial Mediterranean fever, can inhibit the activation of inflammatory bodies in Pyrin. Colchicine did not inhibit the dephosphorylation of Pyrin proteins stimulated by pathogenic microbial virulence factors, but inhibited the activation of inflammatory bodies by inhibiting the recruitment of downstream ASC proteins by activated Pyrin proteins. In summary, in the first part of this paper, it is proved that the site-specific phosphorylation of Pyrin protein and the dynamic balance of microtubule protein are important control elements for the activation of inflammatory bodies in Pyrin. (pyroptosis) is a kind of inflammatory necrosis, which plays an important role in the resistance of the organism to the infection of pathogenic microorganisms and the pathogenesis of many diseases. The direct cause of focal death is the cleavage of GasderminD (GSDMD) proteins by Caspase-4/5/11 activated by inflammatory bodies or by direct binding to bacterial lipopolysaccharide (lipopolysaccharide,LPS). The N-terminal active part of GSDMD-releasing protein was polymerized on the cell membrane to form a permeability pore. Gasdermin protein family members mostly contain N-terminal domains, but their biological functions are still in an unknown state. The results of cell imaging, Western blot and flow cytometry showed that Gasdermin E (GSDME), a member of the Gasdermin family, could be activated by activated Caspase-3 cleavage. It also causes inflammatory necrosis of cells in a manner similar to that caused by GSDMD. Jurkat cells without GSDME expression were treated with chemotherapeutic drugs, and GSDME-overexpressing SH-SY5Y cell lines and human keratinized primary cells could induce GSDME-mediated focal death through activation of Caspase-3, while the cells treated with high-expression SH-SY5Y cells and human keratinized primary cells could induce apoptosis. Inhibition of Caspase-3 activity or reduction of GSDME expression by drugs could inhibit the occurrence of focal death. The results of animal experiments showed that Gsdme gene knockout can effectively protect mice from DNA damage to lung and small intestine tissue damage caused by chemotherapeutic drugs. In conclusion, the second part of this work proves that Caspase-3 can activate GSDME to induce GSDME-mediated inflammatory necrosis of cells, and whether the expression of GSDME in cells is an important index to determine the way of cell death after the activation of Caspase-3.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R593.2
【参考文献】
相关期刊论文 前1条
1 YANG Jie Ling;XU Hao;SHAO Feng;;Immunological function of familial Mediterranean fever disease protein Pyrin[J];Science China(Life Sciences);2014年12期
，本文编号：2434119
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