Pyrin炎癥小體激活的分子機(jī)制與Gasdermin E介導(dǎo)的細(xì)胞焦亡的研究
發(fā)布時(shí)間：2019-03-04 08:49
【摘要】：Pyrin蛋白由MEFV基因編碼,其點(diǎn)突變是引起一種分布于地中海沿岸地區(qū)的自身炎癥性疾病家族性地中海熱(familiar mediterranean fever, FMF)的原因。Pyrin蛋白可作為炎癥小體中的重要分子,通過(guò)間接識(shí)別由病原微生物毒力因子修飾并失活的Rho GTPases引起炎癥小體的激活。與天然免疫其他模式識(shí)別受體不同,Pyrin蛋白通過(guò)間接識(shí)別微生物毒力因子對(duì)自身蛋白的修飾而非直接識(shí)別病原微生物固有分子從而激活下游免疫反應(yīng)。然而,Pyrin炎癥小體激活的具體機(jī)制至今尚不明確。本研究主要利用蛋白質(zhì)免疫印跡、蛋白質(zhì)免疫共沉淀等手段,證明在免疫細(xì)胞中,Pyrin蛋白存在磷酸化修飾,其205位與241位絲氨酸的磷酸化修飾介導(dǎo)Pyrin蛋白與14-3-3蛋白的結(jié)合,使Pyrin蛋白維持在活性抑制狀態(tài)。通過(guò)制備特異性識(shí)別兩磷酸化位點(diǎn)的單克隆抗體,證實(shí)205、241位絲氨酸在細(xì)胞受到病原微生物刺激時(shí)共同發(fā)生去磷酸化反應(yīng)。利用誘導(dǎo)表達(dá)系統(tǒng),證明Pyrin蛋白的位點(diǎn)特異性去磷酸化是Pyrin炎癥小體激活的原因。同時(shí),本研究發(fā)現(xiàn)家族性地中海熱的有效治療性藥物秋水仙素可抑制Pyrin炎癥小體的激活。秋水仙素并不能抑制細(xì)胞受到病原微生物毒力因子刺激時(shí)Pyrin蛋白的去磷酸化反應(yīng),而是通過(guò)抑制激活后的Pyrin蛋白招募下游ASC蛋白從而阻礙炎癥小體的激活。綜上,本文第一部分工作證明Pyrin蛋白的位點(diǎn)特異性磷酸化修飾與正常的微管蛋白動(dòng)力學(xué)平衡是Pyrin炎癥小體激活的重要控制元件。細(xì)胞焦亡(pyroptosis)屬于細(xì)胞炎癥性壞死的一種,在機(jī)體抵抗病原微生物感染以及許多疾病的發(fā)病中發(fā)揮重要作用。細(xì)胞焦亡的直接原因是GasderminD(GSDMD)蛋白被通過(guò)炎癥小體激活的Caspase-1或直接結(jié)合細(xì)菌脂多糖(lipopolysaccharide,LPS)而活化的Caspase-4/5/11切割,使GSDMD釋放蛋白質(zhì)N端活性部分在細(xì)胞膜上聚合形成通透性孔道所致。Gasdermin蛋白家族成員多含有膜通透N端結(jié)構(gòu)域,但其生物學(xué)功能依然處于未知狀態(tài)。本文通過(guò)細(xì)胞成像、蛋白質(zhì)免疫印跡及流式細(xì)胞儀分析等手段,證明Gasdermin家族成員Gasdermin E(GSDME)可以被活化的Caspase-3切割激活,并以類似于GSDMD導(dǎo)致細(xì)胞焦亡的方式引起細(xì)胞的炎癥性壞死。化療藥物處理無(wú)GSDME表達(dá)的Jurkat細(xì)胞可使細(xì)胞發(fā)生細(xì)胞凋亡,而處理高表達(dá)GSDME的SH-SY5Y細(xì)胞系與人角質(zhì)化原代細(xì)胞可通過(guò)活化Caspase-3引起GSDME介導(dǎo)的細(xì)胞焦亡,用藥物抑制Caspase-3活性或降低GSDME的表達(dá)量均能抑制細(xì)胞焦亡的發(fā)生。動(dòng)物實(shí)驗(yàn)結(jié)果表明,Gsdme基因敲除可有效保護(hù)小鼠免受DNA損傷化療藥物造成的肺部與小腸組織的損傷。綜上所述,本文第二部分工作證明Caspase-3可通過(guò)切割激活GSDME引起GSDME介導(dǎo)的細(xì)胞炎癥性壞死,而細(xì)胞中GSDME表達(dá)與否成為在Caspase-3激活后,決定細(xì)胞死亡方式的重要指標(biāo)。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R593.2
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R593.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
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
，本文編號(hào)：2434119
本文鏈接：http://sikaile.net/yixuelunwen/nfm/2434119.html
最近更新
教材專著
