基于定量荧光共振能量转移的潜伏膜蛋白-1与波形蛋白相互作用研究  

Interaction Between Latent Membrane Protein-1 and Vimentin Based on Quantitative Fluorescence Resonance Energy Transfer

作　　者：吴志伟[1,2] 张先增 谢树森 Wu Zhiwei;Zhang Xianzeng;Xie Shusen(School of Physical and Information Engineering,Quanzhou Normal University,Quanzhou 362000,Fujian,China;College of Photonic and Electronic Engineering,Fujian Normal University,Fuzhou 350007,Fujian,China)

机构地区：[1]泉州师范学院物理与信息工程学院,福建泉州362000 [2]福建师范大学光电与信息工程学院,福建福州350007

出　　处：《光学学报》2024年第20期220-230,共11页Acta Optica Sinica

基　　金：国家自然科学基金(61475036);福建省自然科学基金(2021J01970,2023J011768);泉州市科技计划项目(2020N012s)。

摘　　要：爱泼斯坦-巴尔病毒编码的潜伏膜蛋白-1(LMP-1)和波形蛋白(VIM)是两种公认的鼻咽癌标志物,但两者之间是否存在直接相互作用尚不明确。荧光共振能量转移(FRET)是唯一能够在活细胞中非侵入、动态监测蛋白质相互作用的技术。采用同源重组技术构建LMP-1和VIM的分子荧光探针,并通过一种定量FRET技术,即三通道荧光共振能量转移(E-FRET),在活细胞中检测LMP-1与VIM之间的蛋白质相互作用。进一步地,使用甲基倍他环糊精(MβCD)研究脂筏完整性对LMP-1与VIM相互作用的意义,以及该相互作用在细胞凋亡过程中发挥的作用。荧光图像显示:VIM在LMP-1阳性细胞内的分布呈现明显的极化特征,并且聚集于两者相互作用强的区域。E-FRET成像和细胞活性检测的结果表明:LMP-1与VIM相互作用不依赖于脂筏完整性,且在脂筏被破坏的过程中逐步增强。此外,LMP-1与VIM相互作用也可以增强细胞的凋亡抵抗能力。从新的视角阐述LMP-1的抗凋亡机制,表明定量FRET技术可用于细胞凋亡过程中蛋白质相互作用和分布差异的研究。Objective The EpsteinBarr virus encoded latent membrane protein1(LMP1)and vimentin(VIM)are well known as nasopharyngeal carcinoma(NPC)biomarkers,which are reported to be highly expressed in NPC tissues.The exact mechanism between LMP1 and vimentin proteins is still unclear,although some studies have reported that LMP1 increases the expression of VIM messenger ribonucleic acid(mRNA)and proteins,and VIM level reductions decrease ERK activation in LMP1-positive NPC cells.We aim to investigate the interaction between LMP1 and VIM proteins,and the relationship between LMP1 and VIM interaction,cell apoptosis,and integrity of lipid rafts.Quantitative fluorescence resonance energy transfer(FRET)is adopted in our study,which is the only nonintrusive method for dynamically monitoring proteinprotein interaction in living cells.Methods We synthesize molecular biosensors for monitoring LMP1 and VIM proteins in live cells by connecting a kind of cyan fluorescence protein Cerulean and a yellow fluorescence protein Venus to carboxy terminals of VIM and LMP1 respectively.VIM cellular distribution differences between cells only expressing VIM or coexpressing VIM and LMP1 are analyzed by employing VIMCerulean and LMP1-Venus molecular biosensors.Additionally,quantitative FRET is utilized to investigate the interaction between LMP1 and VIM by transfecting VIMCerulean and LMP1-Venus into CNE1 cells,one of the welldifferentiated nasopharyngeal squamous carcinoma cell lines.First,the FRET imaging platform is performed on a widefield microscope with three imaging channels,including donor[AT435/425‒445 nm(excitation),AT455DC,ET480/465‒495 nm(emission),Chroma],acceptor[AT495/485‒505 nm(excitation),AT515DC,ET540/525‒555 nm(emission),Chroma],and FRET[AT435/425‒445 nm(excitation),AT515DC,ET540/525‒555 nm(emission),Chroma].Next,we calibrate two acceptor bleedthrough coefficients(a and b),two donor bleedthrough coefficients(c and d),and the G factor,defined as the ratio of the sensitized emission to the corresponding amount of donor

关 键 词：定量荧光共振能量转移 时间序列成像 鼻咽癌 潜伏膜蛋白-1 波形蛋白 

分 类 号：R318[医药卫生—生物医学工程]