【作者】 汤薇；

【导师】 曹志贱；

【作者基本信息】 武汉大学 ， 微生物学， 2022， 硕士

【摘要】 干扰素诱导的跨膜蛋白1(IFITM1)是干扰素诱导基因编码蛋白家族中的一员,主要定位于内体和质膜上。在抗病毒天然免疫方面,IFITM1定位于质膜的特性使其在抑制病毒入侵阶段发挥重要作用。在肿瘤的发生发展中,近些年越来越多的实验结果表明,IFITM1在肿瘤组织和癌细胞系中高度表达,是头颈癌、结直肠癌、胃癌等肿瘤类型的独立预后生物标志物。并且,IFITM1的高表达也表现出促进肿瘤细胞增殖、侵袭、转移、血管生成和治疗耐药的作用。由于IFITM1在肿瘤中的这些不同功能,以IFITM1为靶向分子可能成为癌症治疗的一种新策略和新途径。AKT,也称蛋白激酶B,是一种丝氨酸/苏氨酸蛋白激酶。目前所发现的亚型有AKT1、AKT2、AKT3,其中AKT1分布于大部分组织细胞中,而AKT2主要分布在肌肉和脂肪细胞中,AKT3则在睾丸和大脑中表达。AKT蛋白关系着机体多种生物过程,包括细胞存活、增殖、糖代谢等。AKT相关信号通路也与多种恶性肿瘤、糖尿病、类风湿关节炎等疾病的发生发展密切相关。已有报道证实,在大部分非小细胞肺癌患者中均能发现AKT的异常表达与激活。14-3-3蛋白家族是一类高度保守的酸性蛋白家族,在所有真核细胞中都有表达。在哺乳动物中,已鉴定出7种亚型,命名为β、γ、η、σ、ε、θ和ζ。14-3-3蛋白都是以同源/异源二聚体的形式存在,不同亚型的氨基酸序列同源性很高。在大多数细胞中,14-3-3蛋白主要分布在胞质中,通过蛋白质-蛋白质相互作用与多种胞质信号转导蛋白结合,参与调节信号转导、神经发育、细胞周期控制、囊泡运输、DNA复制和凋亡等多种功能,也与许多癌症的发生发展有关。本课题组的前期研究发现,TCGA数据库中IFITM1在肺癌患者的细胞内呈现高表达的现象,并且在非小细胞肺癌细胞系A549中过表达IFITM1基因促进肿瘤细胞增殖、迁移,相反敲除IFITM1明显抑制A549细胞的增殖与迁移。采用质谱技术筛选到了一些与IFITM1可能相互作用的蛋白。本实验旨在进一步探索IFITM1促进A549细胞增殖、迁移异常的具体机制,找到与IFITM1具体互作的胞内靶点。首先,本论文发现了在A549细胞内IFITM1、AKT1与14-3-3γ三者间存在相互作用。实验室前期结果发现,IFITM1在非小细胞肺癌细胞系A549内发挥促增殖迁移作用的可能关联蛋白为14-3-3蛋白家族和PI3K/AKT相关信号通路。因此本研究使用真核表达载体构建了IFITM1、PI3K-P85α、AKT1和14-3-3家族七个亚型的重组质粒,经过转染在A549细胞内过表达相应蛋白,通过免疫共沉淀的方式筛选可能与IFITM1发生相互作用的蛋白。结果表明IFITM1与AKT1存在互作,IFITM1与14-3-3家族中的γ、θ、η、σ亚型有相互作用,AKT1与14-3-3γ、η亚型存在较明显互作,而IFITM1与PI3K-P85α并没有互作发生。鉴于14-3-3蛋白家族各亚型的氨基酸序列和保守结构的相似性,存在多种亚型与目标蛋白均有互作的可能。考虑到各亚型在肺癌细胞中的表达量,本研究选择了其中互作效果最明显、在肺癌细胞中呈现高表达的14-3-3γ作为后续实验的目标蛋白。其次,本论文揭示了IFITM1与AKT1通过桥接蛋白14-3-3γ相连的复合连接模式。在A549细胞内发现三种蛋白存在互作后,进一步通过原核表达载体构建了带标签的IFITM1、AKT1和14-3-3γ的重组质粒,采用不同的表达纯化手段获得了重组IFITM1、AKT1和14-3-3γ蛋白。IFITM1由于其主要定位于质膜上,参考膜蛋白的表达纯化方式,利用去垢剂辅助提取了较高纯度的重组IFITM1蛋白;AKT1因为其自身的蛋白特性在诱导表达中始终以包涵体形式存在,采用变性、复性的手段将其提纯获得重组AKT1蛋白;14-3-3γ蛋白的溶解性较好,一般表达方式即可获得富集纯化的重组14-3-3γ蛋白。将得到的单一纯化蛋白通过GST/His pull-down方法进行体外相互作用探究。实验结果表明,14-3-3γ仍然与IFITM1、AKT1蛋白存在直接相互作用,但IFITM1与AKT两个蛋白之间却没有明显互作表现。这些实验结果提示,在A549细胞中IFITM1是通过14-3-3γ蛋白的桥接间接与AKT1产生互作。根据已有文献和数据库可以证实,IFITM1并不存在AKT激活所需的氨基酸保守基序,与14-3-3蛋白的相互作用也不依赖常见的磷酸化结合基序,因此IFITM1更有可能是通过辅助蛋白的帮助与AKT1产生关联。最后,本论文阐明了AKT1是IFITM1促使A549细胞增殖与迁移的关键靶点。实验室前期发现在A549细胞中IFITM1可以增强AKT1 Ser473位磷酸化,而且IFITM1也可以促进A549细胞增殖、迁移。在前面揭示了IFITM1与AKT1的间接互作模式后,本研究进一步探究IFITM1是否通过激活AKT1从而促进A549细胞的增殖、迁移。MK-2206具有高效的靶向AKT1的抑制效果,在非细胞毒性浓度下对A549细胞内的AKT产生显著抑制。通过CCK-8法检测AKT1被抑制的情况下IFITM1对细胞增殖和迁移的作用。细胞培养48 h后的结果显示,IFITM1过表达的确可以促进A549细胞的增殖与迁移,单独加入MK-2206也会表现出与敲除IFITM1相同的抑制A549细胞增殖、迁移的效果。而在过表达IFITM1且加入MK-2206的实验组中发现,细胞增殖与迁移有所增加却明显少于单独过表达IFITM1实验组,这提示MK-2206的加入抵消了部分IFITM1过表达导致的促进癌细胞增殖、迁移作用。因此AKT1蛋白参与了IFITM1促进细胞增殖迁移的作用机制。综上所述,本论文发现了IFITM1在A549细胞系内与AKT1蛋白发生互作的机制,揭示了IFIMT1通过支架蛋白14-3-3γ间接与AKT1蛋白相连接的结合模式。AKT抑制剂MK-2206在A549细胞内能明显抑制细胞增殖、迁移,并且证实其可以抵消过表达IFITM1对非小细胞肺癌细胞增殖和迁移的促进作用,说明AKT1的确参与了IFITM1影响肺癌细胞增殖和迁移的信号传导过程。这些研究表明,IFITM1通过14-3-3γ的介导与AKT1间接结合,诱导了AKT1蛋白的异常激活,从而导致非小细胞肺癌细胞的过度增殖、迁移现象。更多还原

【Abstract】 Interferon-inducible transmembrane protein 1(IFITM1)is a member of the protein family encoded by interferon-inducible genes,mainly localized in endosomes and plasma membranes.In terms of antiviral innate immunity,the localization of IFITM1 in the plasma membrane makes it play an important role in the inhibition of virus invasion.In the occurrence and development of tumors,more and more experimental results have recently shown that IFITM1 is highly expressed in tumor tissue and cancer cell lines,and is an independent prognostic biomarker for head and neck cancer,colorectal cancer,gastric cancer and other tumor types.Moreover,the overexpression of IFITM1 also showed the effect of promoting tumor cell proliferation,invasion,metastasis,angiogenesis and therapy resistance.Due to these different functions of IFITM1 in tumors,targeting IFITM1 as a molecule may become a new strategy and approach for cancer therapy.AKT,also known as protein kinase B,is a serine/threonine protein kinase.The isoforms currently found are AKT1,AKT2,and AKT3.Among them,AKT1 is distributed in most tissue cells,while AKT2 is mainly distributed in muscle and fat tissues,and AKT3 is expressed in testis and brain.AKT proteins are related to a variety of biological processes in the body,including cell survival,proliferation,glucose metabolism,etc.AKT-related signaling pathways are also closely related to the development of various malignant tumors,diabetes and rheumatoid arthritis.It has been reported that abnormal expression and activation of AKT can be found in most nonsmall cell lung cancer patients.The 14-3-3 protein family is a kind of highly conserved family of acidic proteins expressed in all eukaryotic cells.In mammals,seven isoforms have been identified,named β,γ,η,σ,ε,θ and ζ.All 14-3-3 proteins exist in the form of homo/heterodimers,and the amino acid sequence homology of different isoforms is very high.In most cells,14-3-3 proteins are mainly distributed in the cytoplasm and are involved in regulating various functions such as signal transduction,neural development,cell cycle control,vesicle transport,DNA replication and apoptosis through protein-protein interactions with a variety of cytoplasmic signaling proteins,which are associated with the development of many cancers.Previous studies of our group found that IFITM1 in TCGA database showed high expression in cells from lung cancer patients,the overexpression of IFITM1 in nonsmall cell lung cancer cell line A549 promoted the proliferation and migration of tumor cells,and the knockdown of IFITM1 significantly inhibited the proliferation and migration of A549 cells.Some proteins that may interact with IFITM1 were screened by mass spectrometry.This study aims to further explore the specific mechanism of IFITM1 promoting the abnormal proliferation and migration of A549 cells,and find the intracellular targets that specifically interact with IFITM1.Firstly,this study identified an interaction between IFITM1,AKT1 and 14-3-3γ in A549 cells.The previous results of our group revealed that the possible associated proteins for the pro-proliferative migration role of IFITM1 in the non-small cell lung cancer cell line A549 are 14-3-3 protein family and PI3K/AKT-related signaling pathway.Therefore,the eukaryotic expression vector used to construct the recombinant plasmids of IFITM1,PI3K-P85α,AKT1 and seven isoforms of 14-3-3 family.After transfection,the corresponding proteins were overexpressed in A549 cells,and then the proteins that might interact with IFITM1 were screened by immunoprecipitation.The experimental results showed that there was obvious interaction between IFITM1 and AK1 protein,IFITM1 interacted γ,θ,η,σ isoforms of 14-3-3 family(specially γ and ηmembers),but there was no direct interaction occurred between IFITM1 and PI3KP85α.Given the similarity of amino acid sequence and conserved structure of each subtype of the 14-3-3 protein family,it is possible that many subtypes interact with the target protein.Considering the expression of each isoform in lung cancer cells,the 14-3-3γ,which showed the most obvious interaction effect and high expression in lung cancer cells,was selected as the target protein for subsequent experiments in this study.Secondly,this study revealed the complex connection mode between IFITM1 and AKT1 through the bridging protein 14-3-3γ.After the interactions of the three proteins were found in A549 cells,the recombinant plasmids with tags of IFITM1,AKT1 and14-3-3γ were further constructed by prokaryotic expression vectors,and the recombinant IFITM1,AKT1 and 14-3-3γ proteins were obtained by different expression and purification methods.Because IFITM1 is mainly located on the plasma membrane,a high purity recombinant IFITM1 protein was extracted with the aid of detergent with reference to the expression and purification of membrane proteins.Considering the fact that AKT1 always exists in the form of inclusion bodies in the induced expression,AKT1 was purified by the way of denaturation and renaturation to obtain the recombinant protein.The soluble14-3-3γ protein was easily purified and obtained by affinity chromatography.The single purified protein was explored in vitro by GST/His pull-down method,and the experimental results showed that 14-3-3γ still interacted directly with IFITM1 or AKT1 proteins,but there was no significant interaction between IFITM1 and AKT proteins.These data suggest that IFITM1 is indirectly interacting with AKT1 through the bridging of 14-3-3γ protein in A549 cells.Based on the available literature and databases,it can be confirmed that IFITM1 does not have a conserved amino acid motif required for AKT activation,and the interaction with 14-3-3 protein does not depend on the common phosphorylated conserved motif,so it is more likely that IFITM1 associates with AKT1 through the help of auxiliary proteins.Finally,this study clarified that AKT1 was a key target of IFITM1 to promote the proliferation and migration of A549 cells.The previous results from our lab found that IFITM1 could enhance the phosphorylation of AKT1 Ser473 in A549 cells,and IFITM1 was also able to promote the proliferation and migration of A549 cells.After revealing the indirect interaction mode between IFITM1 and AKT1,our study further explored whether IFITM1 promoted the proliferation and migration of A549 tumor cells by activating AKT1.MK-2206 had a potent inhibitory effect on AKT1,and significantly inhibited AKT in A549 cells at a non-cytotoxic concentration.The effects of IFITM1 on cell proliferation and migration under the condition of AKT1 inhibition were detected by CCK-8 assay.The results after 48 h of cell culture showed that the overexpression of IFITM1 could indeed promote the proliferation and migration of A549 cells,and the addition of MK-2206 alone also showed the same effect of inhibiting the proliferation and migration of A549 cells as the knockout of IFITM1.In the group overexpressing IFITM1 and adding MK-2206,cell proliferation and migration were increased significantly less than in the group overexpressing IFITM1 alone.These experimental results suggest that the addition of MK-2206 partially counteracted the proliferation and migration-promoting effects of IFITM1 overexpression.Therefore,AKT1 protein was involved in the mechanism of IFITM1 promoting cell proliferation and migration.In conclusion,this study discovered the mechanism of IFITM1 interacting with AKT1 protein in A549 cell line,and revealed the binding mode of IFIMT1 indirectly connecting with AKT1 protein through scaffold protein 14-3-3γ.The AKT inhibitor MK-2206 can significantly inhibit cell proliferation and migration in A549 cells,and it is confirmed that it can counteract the promotion of IFITM1 overexpression on the proliferation and migration of non-small cell lung cancer cells.It indicates that AKT1 is involved in the signaling process of IFITM1 affecting the proliferation and migration of non-small cell lung cancer cells.These results showed that IFITM1 indirectly binds to AKT1 through the mediation of 14-3-3γ,and induces the abnormal activation of AKT1 protein,which leads to the excessive proliferation and migration of non-small cell lung cancer cells.更多还原