实验海洋生物学重点实验室知识库

程序性细胞死亡是一类主动的、有序的细胞死亡方式，在机体的生长发育、免疫防御等多种生物过程中发挥重要作用。坏死性凋亡（necroptosis）和细胞焦亡（pyroptosis）是两种新近发现的程序性细胞死亡，分别由mixed lineage kinase domain-like protein (MLKL)和gasdermin（GSDM）介导。MLKL和GSDM可分别由 receptor interacting protein kinases 1/3 (RIPK1/3)和caspase（CASP）激活，激活的MLKL和GSDM在细胞膜上形成孔道，导致细胞死亡。在哺乳动物中，坏死性凋亡和细胞焦亡在多种病原感染和疾病发展过程中扮演重要角色。在硬骨鱼中，坏死性凋亡和细胞焦亡研究相对而言很少，尤其是相关调控机制和免疫功能，有待深入解析。

牙鲆（Paralichthys olivaceus）是重要海水养殖鱼类。在本论文中，我们研究了牙鲆坏死性凋亡和细胞焦亡机制。我们发现牙鲆MLKL（PoMLKL）的N端结构域具有膜穿孔活性，该结构域在大肠杆菌中表达后能够杀死宿主菌。在正常生理情况下，PoMLKL的N端活性被C端结构域抑制，处于非活化状态；牙鲆RIPK3（PoRIPK3）能够在T360/S361位点磷酸化PoMLKL，从而激活PoMLKL，导致坏死性凋亡，而PoRIPK1能够增强PoRIPK3对PoMLKL的激活能力。与此一致，共定位实验表明，PoRIPK3直接作用于PoMLKL，而PoRIPK1则通过PoRIPK3而结合PoMLKL。突变实验表明，PoRIPK1/3与PoMLKL的作用依赖于PoRIPK1/3的RIP homotypic interaction motif （RHIM）结构域。细胞和活体感染实验表明，迟缓爱德华氏菌（Edwardsiella tarda）感染诱导PoMLKL和PoRIPK1/3表达；抑制PoMLKL介导的坏死性凋亡能够显著促进迟缓爱德华氏菌对牙鲆细胞和组织的感染，导致牙鲆死亡率显著上升，说明坏死性凋亡在清除病原菌的过程中起到了重要的作用。

在细胞焦亡方面，我们揭示了牙鲆细胞焦亡的激活机制。我们发现牙鲆gasdermin E（PoGSDME）能够被牙鲆的三个caspase（即PoCASP1、3和7）剪切，释放出具有穿孔活性的N端，从而引发细胞焦亡。此外，我们发现PoGSDME能够被牙鲆caspase-8（PoCASP8）剪切抑制。通过突变分析，我们鉴定出了对PoGSDME功能至关重要的关键氨基酸位点，包括F2、L19和L136等。通过感染实验，我们发现细菌感染能够诱导牙鲆PoGSDME、PoCASP1、PoCASP3等基因的表达以及细胞内PoGSDME的剪切，并初步证明PoGSDME的剪切主要由PoCASP1介导。

综上所述，我们的研究揭示了牙鲆两种程序性细胞死亡的分子机制，为鱼类坏死性凋亡和细胞焦亡提供了新的见解，也为牙鲆疫病防控提供了新的理论依据。

Programmed cell death is a class of active and orderly cell death that plays an important role in a variety of biological processes such as growth, development and immune defense. Necroptosis and pyroptosis are two newly identified types of programmed cell death mediated by the membrane perforating proteins mixed lineage kinase domain-like protein (MLKL) and gasdermin (GSDM), respectively. MLKL and GSDM are activated by receptor interacting protein kinases 1/3 (RIPK1/3) and caspase, respectively. The activated MLKL and GSDM can form pores in the cellular membrane, leading to cell death. In mammals, necroptosis and pyroptosis play important roles in a variety of pathogenic infections and disease development. In teleost, relatively few studies on necroptosis and pyroptosis have been reported, especially in association with the regulatory mechanisms and immune functions of necroptosis and pyroptosis.

Japanese flounder (Paralichthys olivaceus) is an important mariculture fish species. In this dissertation study, we examined the mechanisms of flounder necroptosis and pyroptosis. We found that the N-terminal structural domain of flounder MLKL (PoMLKL) exhibited necroptosis-inducing activity. When expressed in Escherichia coli, this N-terminal domain killed the host bacteria. Under normal physiological condition, the activity of the N-terminal domain was inhibited by the C-terminal domain. Flounder RIPK3 (PoRIPK3) was capable of phosphorylating the T360/S361 residues in PoMLKL, whereby activating PoMLKL and leasing to necroptosis. The activating effect of PoRIPK3 on PoMLKL was enhanced by PoRIPK1. Consistently, co-localization analysis showed that PoRIPK1/3 interacted with PoMLKL in a manner that depended on the RIP homotypic interaction motif (RHIM). Cellular and in vivo infection studies showed that Edwardsiella tarda infection increased the expression of RIPK1/3 and MLKL. Inhibition of PoMLKL-mediated necroptosis increased E. tarda infection in fish cells and tissues, and led to significantly enhanced fish mortality. These results indictaed the importance of necroptosis in pathogen clearance.

We also revealed the activation mechanism of pyroptosis in flounder. We found that flounder gasdermin E (PoGSDME) was cleaved by three flounder caspases (i.e., PoCASP1, 3, and 7), releasing an N-terminal domain with perforating activity to execute pyroptosis. Furthermore, we found that PoGSDME could be inhibited by PoCASP8 cleavage. Through mutation analysis, we identified a number of key residues, such as F2, L19, and L136, that are essential for PoGSDME function. Through infection experiments, we found that bacterial infection induced the expression of PoGSDME, PoCASP1, and PoCASP3, and caused the cleavage of PoGSDME. Preliminary results suggested that PoCASP1 was the main caspase responsible for PoGSDME cleavage during infection.

In summary, our study reveals the molecular mechanisms of two types of programmed cell death in flounder and adds new insights into fish necroptosis and pyroptosis. Our findings also provide a basis for flounder desease control.

目 录

第1章 绪论............................................................................... 1

1.1 程序性细胞死亡................................................................................................ 1

1.1.1 程序性细胞死亡概况................................................................................. 1

1.1.2 程序性细胞死亡的发现史......................................................................... 1

1.1.3 程序性细胞死亡的分类............................................................................. 2

1.1.4 程序性细胞死亡途径之间的互动............................................................. 3

1.2 细胞焦亡............................................................................................................ 4

1.2.1 细胞焦亡途径概况..................................................................................... 4

1.2.2 细胞焦亡的作用机制................................................................................. 4

1.2.3 细胞焦亡的生物学意义............................................................................. 5

1.2.4 进化尺度上的细胞焦亡............................................................................. 6

1.2.5 细胞焦亡在硬骨鱼中的研究现状............................................................. 6

1.3 坏死性凋亡........................................................................................................ 7

1.3.1 坏死性凋亡概况......................................................................................... 7

1.3.2 坏死性凋亡的作用机制............................................................................. 8

1.3.3 坏死性凋亡的生物学意义......................................................................... 9

1.3.4 进化尺度上的坏死性凋亡....................................................................... 10

1.3.5 坏死性凋亡在硬骨鱼中的研究现状....................................................... 11

1.4 本研究的目的与意义....................................................................................... 11

第2章 牙鲆坏死性凋亡的机制研究........................................... 13

2.1 前言.................................................................................................................. 13

2.2 实验材料与方法.............................................................................................. 13

2.2.1 实验细胞和动物模型............................................................................... 13

2.2.2 实验试剂与仪器....................................................................................... 13

2.2.3 序列获取................................................................................................... 16

2.2.4 基因序列克隆........................................................................................... 16

2.2.5 基因真核表达载体构建........................................................................... 17

2.2.6 PoRIPK3和PoMLKL的定点突变.......................................................... 19

2.2.7 PoMLKL的原核表达载体构建................................................................ 20

2.2.8 PoMLKL抑菌活性检测............................................................................ 21

2.2.9 牙鲆坏死性凋亡相关基因的异源表达................................................... 21

2.2.10 显微镜观察............................................................................................. 24

2.2.11 PoMLKL的磷酸化状态检测.................................................................. 25

2.2.12 LDH释放量检测..................................................................................... 26

2.2.13 SYTOX Green染色................................................................................. 27

2.2.14 牙鲆细胞系体外侵染实验..................................................................... 27

2.2.15 正常生理条件下各基因的表达分布情况............................................. 29

2.2.16 感染条件下的各基因表达水平检测..................................................... 30

2.2.17 组织侵染实验......................................................................................... 31

2.2.18 牙鲆感染生存曲线................................................................................. 31

2.3 实验结果.......................................................................................................... 31

2.3.1 PoRIPK1、PoRIPK3和PoMLKL的结构分析....................................... 31

2.3.2 PoRIPK1、PoRIPK3和PoMLKL基因表达在E. tarda感染过程中的变化情况 33

2.3.3 PoMLKL的N端结构域具有细胞杀伤活性........................................... 36

2.3.4 PoRIPK1和PoRIPK3对PoMLKL的激活作用..................................... 38

2.3.5 PoRIPK1和PoRIPK3对PoMLKL的激活作用依赖于其RHIM结构域 42

2.3.6 坏死性凋亡是牙鲆抗细菌感染免疫的重要机制................................... 44

2.3.7 牙鲆坏死性凋亡的示意图....................................................................... 47

2.4 讨论.................................................................................................................. 48

第3章 牙鲆细胞焦亡的机制探究.............................................. 51

3.1 前言.................................................................................................................. 51

3.2 实验材料与方法.............................................................................................. 51

3.2.1 实验细胞和动物....................................................................................... 51

3.2.2 实验试剂与仪器....................................................................................... 52

3.2.3 序列分析................................................................................................... 52

3.2.4 基因CDS克隆.......................................................................................... 52

3.2.5 多克隆抗体的制备................................................................................... 52

3.2.6 Caspase酶活检测...................................................................................... 52

3.2.7 GSDME在细胞水平的剪切..................................................................... 53

3.2.8 显微镜观察............................................................................................... 53

3.2.9 SYTOX Green染色................................................................................... 53

3.2.10 细胞膜/细胞浆蛋白提取实验................................................................ 53

3.2.11 PI染色...................................................................................................... 54

3.2.12 LDH检测................................................................................................. 54

3.2.13 牙鲆细胞体外侵染实验......................................................................... 54

3.3 实验结果.......................................................................................................... 55

3.3.1 牙鲆GSDME可以被多个Caspase剪切................................................ 55

3.3.2 牙鲆Caspase对PoGSDME的剪切产生两种不同的N端片段........... 57

3.3.3 PoGSDME NT248片段具有诱导细胞焦亡的功能................................... 59

3.3.4 牙鲆GSDME的关键功能位点鉴定....................................................... 62

3.3.5 病原菌刺激后牙鲆PoGSDME被剪切激活........................................... 65

3.4 讨论.................................................................................................................. 67

第4章 结论与展望................................................................... 69

参考文献..................................................................................... 70

附录一 实验溶液的配制方法...................................................... 76

附录二 实验所用的引物序列...................................................... 78

附录三 MLKL进化树分析所用的序列信息................................. 86

附录四 GSDME进化树分析所用的序列信息.............................. 91

致 谢......................................................................................... 96

作者简历及攻读学位期间发表的学术论文与其他相关学术成果..... 98