肿瘤细胞抗凋亡调控因子c-FLIPs的结构与功能研究

	肿瘤细胞抗凋亡调控因子c-FLIPs的结构与功能研究
	白志强
导师	胡凯锋
关键词	细胞凋亡 Apoptosis, c-FLIPS, NMR, Structural biology c-FLIPS 核磁共振结构生物学
摘要	细胞凋亡在人体各种细胞、组织和器官的平衡、分化、形成等过程中起着重要作用。在死亡受体诱导的细胞外源凋亡途径中，TNF（Tumor necrosis factor）家族蛋白受体接受死亡信号后，在胞质招募适配蛋白FADD (Fas-associated death domain)和Procaspase-8组装形成诱导细胞死亡的信号复合物DISC（Death-inducing Signaling Complex）。酶原蛋白Procaspases-8经此过程在FADD上形成同源二聚体，经自催化酶切反应产生起始细胞凋亡的Caspases-8，进而启动细胞凋亡。c-FLIP（cellular FLICE-like inhibitory proteins）是Procaspase-8活化调节蛋白，结构上与 Procaspase-8具有一定类似性，均包含N段的tDED（tandem Death effector domain）结构域，可与 Procaspase-8通过分子间tDED相互作用调控细胞凋亡启动子Caspases-8的生成和细胞凋亡信号的传导。c-FLIP在人类细胞中有三种不同的表达亚型：c-FLIPL，c-FLIPS和c-FLIPR。c-FLIPS在序列上主要包含N端的tDED结构域，与Procaspase-8的tDED结构域相互作用抑制Caspase-8的生成进而抑制细胞凋亡。当c-FLIPS与Procaspase-8形成异源二聚体后，Procaspase-8会被催化c-FLIPS生成p22-FLIP；p22-FLIP通过其tDED结构域激活结合NF-κB （Nuclear factor kappa B）信号通路的调节蛋白NEMO（Nuclear factor kappa B essential modulator），进而激活NF-κB 信号通路促进细胞增殖。c-FLIPS在癌细胞中通过过表达来调控Procaspase8的活化，然而目前为止其结构依然没有得到解析。因此研究c-FLIPS的结构及其与Procaspase8的相互作用对于阐明癌细胞的非正常凋亡机制具有重要意义，同时在靶向c-FLIPS的抗癌药物筛选和开发领域具有重要价值。我们利用液态核磁共振技术，解析了c-FLIPS蛋白的溶液结构，同时探析了其与Procaspase-8的相互作用，并对c-FLIPS参与抑制细胞凋亡的作用机制进行了探讨。液态核磁共振技术在研究蛋白溶液状态下的三维结构及蛋白-蛋白相互作用中方面具有诸多优势。通过对野生型c-FLIPS的基因序列进行优化，构建了c-FLIPS（1-173，F114G）的原核表达载体，获得了可溶性高且稳定的单体蛋白。通过采集一系列用于蛋白结构解析的液态核磁共振数据，完成了c-FLIPS（1-173，F114G）近乎全部主链原子和侧链原子的化学位移归属，以及90%以上c-FLIPS原子三维NOESY图谱中的信号指认，成功解析了c-FLIPS溶液状态下单体三维结构。结果显示c-FLIPS包含两个类似于球状的DED结构域并呈哑铃状排布，DED1由6个完整的α螺旋构成和1段无规则的LOOP片段构成；DED2由5个完整的α螺旋和1段无规则的LOOP片段构成。DED1和DED2之间的相互作用主要由疏水界面介导，并由氢键稳固。最后，将c-FLIPS与同源蛋白Procaspase-8、MC-159的tDED结构域进行结构重叠比较，显示c-FLIPS与Procaspase-8的结构较MC-159更为接近，这一重要发现对于阐述和研究其功能奠定了重要结构基础。为研究c-FLIPS与相关作用蛋白的相互作用界面，基于文献总结和结构模拟，合成并筛选了来自Procaspase-8，FADD和NEMO蛋白的9个二级结构单元对应的多肽片段，通过基于二维1H-15N HSQC核磁实验进行化学位移扰动分析。筛选发现来自于Procaspase-8 DED1上的?螺旋H1a和H4a的多肽片段C8-H1a和C8-H4a和NEMO小肽与c-FLIPs具有明显的相互作用。基于上述结果，通过HADDOCK建立了c-FLIPS:Procaspase-8和c-FLIPS:NEMO的复合物结构模型。结果显示，c-FLIPs通过相同的位点（DED1，Clefts位点）结合Procaspase-8 DED1与NEMO。为进一步验证c-FLIPs与小肽的相互作用，通过核磁共振方法解析了c-FLIPs与C8-H1a小肽的复合物结构。这些重要发现为阐述c-FLIPs的功能奠定了结构基础，同时也为CD95 诱导的细胞凋亡和由NF-κB通路激活介导的细胞增殖之间的机制提供了联系。基于上述研究结果，对c-FLIPS在抑制细胞凋亡过程中的机制进行了推测。c-FLIPS可通过以下三种方式抑制细胞凋亡：（1）c-FLIPS通过NEMO结合位点在DISC上与Procaspase-8的DED1结合从而抑制Procaspase-8的同源聚集，阻止了Caspase-8的活化，最后抑制细胞凋亡。（2）c-FLIPS 在细胞质中通过NEMO结合位点与Procaspase-8 DED1结合进而阻碍Procaspase-8结合到DISC上，进一步抑制Caspase-8的活化，从而抑制细胞凋亡。（3）c-FLIPS与Procaspase-8形成异源复合物生成的p22-FLIP可与NEMO结合参与到与IKK复合物的相互作用从而激活NF-κB 信号通路，最后促进细胞增殖。; Apoptosis plays a crucial role in multicellular organisms as a defensive strategy to remove damaged, mutated, or infected cells and inhibit inflammation in surrounding tissue. In the extrinsic apoptotic pathway, the Death-Inducing Signaling Complex (DISC) is assembled at the cytosolic face by the member protein of the TNF receptor family (Death receptor protein). The DISC complex is comprised of the death receptor protein, such as Fas (CD95), the adaptor protein Fas-associated death domain (FADD), the initiator Procaspase-8, and cellular FLICE-inhibitory protein (c-FLIP). Procaspase-8 dimerization is a critical and sufficient step for activation of caspase-8 in this process. c-FLIP is a catalytically inactive Procaspase-8 homologue that interferes with efficient DISC formation in the extrinsic pathway. Three splicing forms of c-FLIP have been identified as: a long-form (c-FLIPL) and two short forms (c-FLIPS, and c-FLIPR). c-FLIPS can bind to the DISC and modulates Procaspase-8 activation through its tandem death effector domain (tDED). When c-FLIPS is overexpressed and forms a heterodimer with Procaspase-8, it will be cleaved by Procaspase-8 and generate p22-FLIP, p22-FLIP can bind NF-κB essential modulator (NEMO) and lead to NF-κB activation, promoting cell survival. In addition, overexpression of c-FLIPS is an tumor marker in many cancer cells. Here, the solution structure of c-FLIPS and its interaction with Procaspase-8 and FADD were investigated by NMR spectroscopy. NMR is known to be a suitable tool for studying the structural and dynamical of properties of macromolecules in solution state. The optimized encoding DNA sequence of the two DED domains of human c-FLIPS (residues 1-173, F114G) was subcloned into a pET-28a vector for protein expression in E.Coli, a highly soluble and stable monomeric protein was obtained after purification. Here we present the solution structure of c-FLIPS by NMR spectroscopy. For the core region of c-FLIPS, about 95% of backbone and side-chain assignments have been completed. In order to obtain a high-resolution structure of c-FLIPS, more than 90% of NMR restraints derivatived from 3D NOESY have been assigned and NIH-Xplor is used for structural calculation. It was shown that c-FLIPS consisting of two tandem DEDs (DED1 and DED2), forms a dumbbell-shaped topological structure with the two tandem DEDs at either end, both DEDs are globular, and DED1 consists of 6 helices, while DED2 consists of 5 helices. The intramolecular interface between DED1 and DED2 is mainly formed by the hydrophobic residues from the helices H2a and H5a of DED1 and the helices H1b and H4b of DED2. Further, structural superimposition of tDED-containing proteins revealed that the structure of c-FLIPS is slightly more similar to Procaspase-8tDED than MC159, this major discovery establishes an important structural basis for understanding the role of c-FLIPS in cell death. To get further insight into the mechanisms of c-FLIPS action in forming DI
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75126
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	白志强. 肿瘤细胞抗凋亡调控因子c-FLIPs的结构与功能研究[D]. 中国科学院大学,2022.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
白志强-白志强博士学位论文20221ba（5263KB）	学位论文		限制开放	CC BY-NC-SA	请求全文