FRET技术及其在蛋白质-蛋白质分子相互作用研究中的应用

简要综述了FRET方法在活细胞生理条件下研究蛋白质-蛋白质间相互作用方面的最新进展.蛋白质-蛋白质间相互作用在整个细胞生命过程中占有重要地位,由于细胞内各种组分极其复杂,因此一些传统研究蛋白质-蛋白质间相互作用的方法,例如酵母双杂交、免疫沉淀等可能会丢失某些重要的信息,无法正确地反映在当时活细胞生理条件下蛋白质-蛋白质间相互作用的动态变化过程.荧光共振能量转移（fluorescence resonance energy transfer, FRET）是近来发展的一项新技术,此项技术的应用,为在活细胞生理条件下对蛋白质-蛋白质间相互作用进行实时的动态研究,提供一个非常便利的条件.     

利用FRET技术在活细胞内研究红景天甙对Aβ25-35诱导PCI2细胞凋亡的抑制作用

为研究红景天甙（salidroside）对β淀粉样肽25-35（β amyloid peptide25-35,Aβ25-35）诱导PC12细胞凋亡的抑制作用,采用Cell Counting Kit-8（CCK-8）分析细胞的存活率,通过光镜检测细胞形态并配以Hoechst染色检测细胞核固缩,利用荧光共振能量转移（fluorescence resonance energy transfer,FRET）技术在单个活细胞中检测caspase-3和caspase-8活性的动态变化。结果表明,红景天甙可剂量依赖性抑制Aβ25-35引起的细胞凋亡,提高细胞的存活率;红景天甙对caspase-3的活性有明显的抑制作用,而且Aβ25-35诱导细胞凋亡不依赖于caspase-8的激活。这些结果提示抑制caspase-3的活性是红景天甙抑制Aβ25-35诱导PC12细胞凋亡的机制之一。     

利用FRET技术在活细胞内研究红景天甙对Aβ25-35诱导PC12细胞凋亡的抑制作用

为研究红景天甙(salidroside)对-淀粉样肽25-35(.amyloidpeptide25-35,A/25-3)5诱导PC12细胞凋亡的抑制作用,采用CellCountingKit-8(CCK-8)分析细胞的存活率,通过光镜检测细胞形态并配以Hoechst染色检测细胞核固缩,利用荧光共振能量转移(fluorescenceresonanceenergytransfer,FRET)技术在单个活细胞中检测caspase-3和caspase-8活性的动态变化。结果表明,红景天甙可剂量依赖性抑制A025-35引起的细胞凋亡,提高细胞的存活率;红景天甙对caspase-3的活性有明显的抑制作用,而且A125-35诱导细胞凋亡不依赖于caspase-8的激活。这些结果提示抑制caspase-3的活性是红景天甙抑制A225-35诱导PC12细胞凋亡的机制之一。     

单个活细胞中生物分子动态行为的FRET实时检测

分别采用两种不同绿色荧光蛋白(green fluorescent prote in,GFP)突变体作为荧光共振能量转移(fluo-rescence resonance energy transfer,FRET)对的供体和受体,并利用分子生物学技术将供体和受体分子分别与特定的生物分子融合,这种技术已经成为在单个活细胞中实时长时间检测蛋白质间的动态相互作用的主要技术。主要介绍了基于GFPs的FRET技术在单个活细胞中实时长时间研究生物分子动态行为的应用。     

在活细胞中应用FRET技术研究TGFβ/TβRI信号传导

转移生长因子β(TGFβ)信号传导通路参与调节细胞的增殖、分化、凋亡、细胞迁移等一系列细胞过程,与骨代谢疾病的发病机制密切相关.本研究根据荧光共振能量转移(FRET)技术原理,构建包含CFP-TβRI-YFP融合蛋白的TβRI生物传感器,转染293T细胞,观察转染效率.以TGFβ1为诱导剂,激活TGFβ/TβRI信号传导通路,在活细胞生理条件下,动态监测TβRI生物传感器的FRET效应.结果表明,成功构建了TβRI生物传感器,转染细胞效率达50%,在TGFβ1诱导刺激6min后,FRET效率增加并达到最大值,该过程经历9 min后,随时间的延长,FRET效率下降.研究结果表明:在活细胞生理条件下,TGFβ1/TβRI信号转导过程存在一定的时间特异性.     

基于C6流式细胞仪平台应用FRET技术在活细胞中研究蛋白质相互作用

荧光共振能量转移(fluorescence resonance energy transfer,FRET)显微镜技术被广泛应用于在活细胞中研究蛋白质相互作用。随着流式细胞术(fluorescence activated cell sorting,FACS)的发展与应用,FACS-FRET技术不但可以检测活细胞中蛋白质相互作用,还可以进行定量统计分析。由于流式细胞仪价格昂贵、FRET技术对荧光基团发光光谱的特殊要求等原因,目前为止FACS-FRET技术仅仅被应用到一些特殊的科学研究。为了解决这些问题,构建了一对新的FRET荧光基团EGFP-m Cherry,并且在小型流式细胞仪C6上检测了EGFP-m Cherry融合蛋白的FRET信号,最后使用已明确有相互作用关系的p53蛋白和MDM2蛋白做验证,证明了所构建的EGFPm Cherry可以作为检测FRET信号的荧光基团。不仅促进了FACS-FRET技术的发展,还为人类疾病治疗的药物作用靶点研究提供了有利的研究手段。     

利用FRET技术研究Hepcidin 和Fpn 相互作用

铁是生命必需的微量元素,ferroportin(Fpn)是小肠吸收细胞铁释放的重要蛋白。新近发现肝脏分泌的抗菌多肽 hepcidin 具有调节肠铁吸收的重要作用,但目前尚缺少Fpn和hepcidin发生作用的实验依据。应用荧光共振能量转移技术(fluorescence resonance energy transfer ,FRET)对hepcidin和Fpn之间的作用关系进行了深入研究。首先进行了hepcidin-CF P融合蛋白表达载体的构建及表达鉴定;然后对含YFP,Fpn-YFP基因动物细胞表达载体的构建、表达和FRET检测。实验结果证实hepcidin和Fpn之间存在直接的相互作用,并发现两种蛋白发生相互作用后hepcidin也在细胞质中有分布。为临床治疗铁代谢紊乱性疾病提供了新的治疗策略和重要理论依据。     

利用荧光共振能量转移技术在活细胞内研究顺铂诱导的凋亡信号通路

为在活细胞内探讨顺铂诱导的凋亡通路．实验样品经顺铂处理后,应用基于荧光共振能量转移(FRET)原理设计的荧光探针pFRET-Bid和pSCAT-3来检测Bid切割和Capase-3活化的动态变化,同时,利用荧光成像在亚细胞水平对Bid转位线粒体的动力学特征进行了实时分析．结果表明：在顺铂诱导的细胞凋亡过程中,Bid切割发生在药物刺激后4～5 h, 历时(120±20) min．Bid切割活化后即从胞浆内转位到线粒体,历时(90±15) min．在凋亡后期,可以明显检测到Caspase-3 的激活．研究表明,应用FRET及荧光成像技术,可以在活细胞内实时、直观、可视地研究顺铂诱导的细胞凋亡过程,从而客观地反映了Bid、Caspase-3等蛋白质分子在该凋亡信号通路中的动态行为及时空传递特性．     

利用FRET技术实时研究顺铂诱导的Caspase-9活化

该研究旨在在活细胞内研究顺铂诱导Caspase-9活化的动态过程.实验样品经顺铂处理后,应用基于FRET原理设计的荧光探针pSCAT-9来检测Capase-9活化的动态过程.结果表明:在顺铂诱导细胞凋亡的后期,可以明显检测到Caspase-9的活化,且其进程快,约30 min即可完成.研究表明,应用FRET技术,可以在活细胞内实时、直观、可视地研究顺铂诱导的Capase-9活化,从而客观地反映Caspase-9在该凋亡信号通路中的动态行为及时空传递特性.     

新FRET技术可观测蛋白质的结构变化

美国华盛顿大学的研究者宣称开发了一种新的荧光共振能量转移（Fluorescent Resonance Energy Transfer,FRET）技术。在这种技术的帮助下,可以观察到变构蛋白在原子尺度上的结构变化。     

阿霉素肾病大鼠表皮生长因子及其受体的表达

目的研究阿霉素肾病大鼠肾组织中表皮生长因子(EGF)及其受体EGFR的表达分布以及表达量与尿蛋白之间关系。方法选择第5天、14天、28天作为动态观察的时点,同期设立正常对照。采用荧光定量RT-PCR、免疫组织化学及计算机图像定量分析EGF mRNA以及EGF、EGFR蛋白在肾组织的表达,同时测定24 h尿蛋白定量。WT1和EGFR双重免疫组化确定EGFR在肾小球内确切细胞定位。结果阿霉素注射后第5天,EGFmRNA即较正常增高,28 d明显增高并高于5 d和14 d。正常对照组EGF阳性细胞主要分布于远曲小管和髓袢,阿霉素组EGF还在集合管和近曲小管上表达;EGF阳性表达范围和强度随尿蛋白增加而增加;EGFmRNA表达量以及EGF在肾小管中的表达强度与24 h尿蛋白量呈正相关。肾小管上皮细胞广泛表达EGFR,阿霉素组EGFR在小管表达均高于正常,但组间各时点差异无显著性;随尿蛋白增加EGFR在肾小球内表达逐渐增多。EGFR在肾小球和肾小管中的表达强度均与24 h尿蛋白量呈正相关。WT1和EGFR双重免疫组化显示阿霉素肾病组EGFR可在足突细胞上表达,正常组则无。结论阿霉素肾病大鼠的肾小球脏层上皮有EGFR的表达。EGF/EGFR可能参与了阿霉素肾病的发病过程以及蛋白尿的形成。     

恶性转化的成纤维细胞表皮生长因子受体的研究

本文用受体的放射性配基结合分析方法观察了C_3H小鼠胚胎成纤维细胞C_3H_(10)T1/2 CL8(简称NC_3H_(10))和~3H-TdR恶性转化的C_3H_(10)T1/2CL8(简称TC_3H_(10))的表皮生长因子受体(EGFR)。结果表明细胞恶性转化前后的EGFR都存在高亲和力和低亲和力两种结合位点,细胞恶性转化后能结合表皮生长因子的EGFR结合位点减少,Western blotting和受体的亲和交联分析表明EGFR的分子量为170kD,是单链多肽。     

Preparation and characterization of Alexa Fluor 594-labeled epidermal growth factor for fluorescence resonance energy transfer studies: application to the epidermal growth factor receptor

We have prepared and characterized a new fluorescent derivative of murine epidermal growth factor (EGF), Alexa Fluor 594-labeled EGF (A-EGF), for fluorescence studies of EGF-EGF receptor interactions. We describe the synthesis of this derivative and its physical and biological characterization. The significant overlap between the excitation and the emission spectra of A-EGF makes this probe well suited to fluorescence resonance energy homo-transfer. Using time-resolved fluorescence to examine the oligomeric state of the EGF receptor, we have observed resonance energy homo-transfer of A-EGF bound to EGF receptors in cells, but not of A-EGF bound to EGF receptors in membrane vesicles. Our results, interpreted in the context of recent crystallographic studies of the ligand-binding domains of EGF receptors, suggest that observed fluorescence resonance energy transfer does not result from transfer within receptor dimers, but rather results from transfer within higher-order oligomers. Furthermore, our results support a structural model for oligomerization of EGF receptors in which dimers are positioned head-to-head with respect to the ligand-binding site, consistent with the head-to-head interactions observed between adjacent receptor dimers by X-ray crystallography.     

Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells

Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations.     

Comparison of quantum dot-binding protein tags: Affinity determination by ultracentrifugation and FRET

Background

Hybrid complexes of proteins and colloidal semiconductor nanocrystals (quantum dots, QDs) are of increasing interest in various fields of biochemistry and biomedicine, for instance for biolabeling or drug transport. The usefulness of protein–QD complexes for such applications is dependent on the binding specificity and strength of the components. Often the binding properties of these components are difficult and time consuming to assess.

Methods

In this work we characterized the interaction between recombinant light harvesting chlorophyll a/b complex (LHCII) and CdTe/CdSe/ZnS QDs by using ultracentrifugation and fluorescence resonance energy transfer (FRET) assay experiments. Ultracentrifugation was employed as a fast method to compare the binding strength between different protein tags and the QDs. Furthermore the LHCII:QD stoichiometry was determined by separating the protein–QD hybrid complexes from unbound LHCII via ultracentrifugation through a sucrose cushion.

Results

One trimeric LHCII was found to be bound per QD. Binding constants were evaluated by FRET assays of protein derivatives carrying different affinity tags. A new tetra-cysteine motif interacted more strongly (K_a = 4.9 ± 1.9 nM^− 1) with the nanoparticles as compared to a hexahistidine tag (His₆ tag) (K_a ~ 1 nM^− 1).

Conclusion

Relative binding affinities and binding stoichiometries of hybrid complexes from LHCII and quantum dots were identified via fast ultracentrifugation, and binding constants were determined via FRET assays.

General significance

The combination of rapid centrifugation and fluorescence-based titration will be useful to assess the binding strength between different types of nanoparticles and a broad range of proteins.     

Ligand-induced EGF receptor oligomerization is kinase-dependent and enhances internalization

The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ～40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.     

Continuous assay of protein tyrosine phosphatases based on fluorescence resonance energy transfer

An assay method that continuously measures the protein tyrosine phosphatase (PTP)-catalyzed dephosphorylation reaction based on fluorescence resonance energy transfer (FRET) was developed as an improvement of our previously reported discontinuous version [M. Nishikata, K. Suzuki, Y. Yoshimura, Y. Deyama, A. Matsumoto, Biochem. J. 343 (1999) 385-391]. The assay uses oligopeptide substrates that contain (7-methoxycoumarin-4-yl)acetyl (Mca) group as a fluorescence donor and 2,4-dinitrophenyl (DNP) group as a fluorescence acceptor, in addition to a phosphotyrosine residue located between these two groups. In the assay, a PTP solution is added to a buffer solution containing a FRET substrate and chymotrypsin. The PTP-catalyzed dephosphorylation of the substrate and subsequent chymotryptic cleavage of the dephosphorylated substrate results in a disruption of FRET, thereby increasing Mca fluorescence. In this study, we used FRET substrates that are much more susceptible to chymotryptic cleavage after dephosphorylation than the substrate used in our discontinuous assay, thus enabling the continuous assay without significant PTP inactivation by chymotrypsin. The rate of fluorescence increase strictly reflected the rate of dephosphorylation at appropriate chymotrypsin concentrations. Since the continuous assay allows the measurement of initial rate of dephosphorylation reaction, kinetic parameters for the dephosphorylation reactions of FRET substrates by Yersinia, T-cell and LAR PTPs were determined. The continuous assay was compatible with the measurement of very low PTP activity in a crude enzyme preparation and was comparable in sensitivity to assays that use radiolabeled substrates.     

Quantitative analysis of Akt phosphorylation and activity in response to EGF and insulin treatment

The protein kinase Akt is a critical regulator of cell function and its overexpression and activation have been functionally linked to numerous pathologies such as cancer. Previous reports regarding the mechanism-regulating Akt's activation have revealed two phosphorylation events, at threonine 308 (T308) and serine 473 (S473), as necessary for the full activation of the kinase in response to insulin. For this reason and because of the availability of phospho-specific antibodies to both T308 and S473, many studies that focus on Akt's role in governing cell function rely on the measurement of these two sites to understand changes in kinase activity. Recent evidence, however, suggests the involvement of other phosphorylation sites; for example, in Src-transformed and epidermal growth factor (EGF)-treated cells, tyrosine phosphorylation has been found important for full kinase activation. In this study, we probed the quantitative reliability of using S473 and/or T308 phosphorylation as surrogates for Akt kinase activity across diverse treatment conditions. We performed quantitative Western blots and kinase activity assays on lysates generated during a 2h time course from two cell lines treated with either EGF or insulin. From the resulting approximately 250 quantitative measurements of phosphorylation and activity, we found that both T308 and S473 phosphorylation accurately captured quantitative changes in EGF-stimulated cells, but not in insulin-stimulated cells. Moreover, in all but one condition studied, we found a tight correlation between the onset of phosphorylation and dephosphorylation for both sites, despite the fact that they do not share common kinase- or phosphatase-mediated regulation. In sum, using a quantitative approach to study Akt activation identified ligand-dependent limits for the use of T308 or S473 as proxies for kinase activity and suggests the coregulation of Akt phosphorylation and dephosphorylation.