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技术在活细胞内观察EGF对PKA作用的时空成像

cAMP依赖的蛋白激酶(protein kinase A,PKA)在细胞生长与分化过程中扮演重要角色,特别是在调节Ras信号通路引起的细胞增殖效应中起着重要作用。为了在活细胞内动态观察表皮生长因子(epidermal growth factor,EGF)对PKA的作用,采用一种可以检测PKA酶活性的报告蛋白(A-kinase activity reporter,AKAR)——这种报告蛋白是利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)原理设计的,使其在人类肺癌细胞(ASTC-a-1)中稳定表达。加入EGF刺激因子后,随时间变化的成像分析显示出在活细胞生理条件下被EGF作用的PKA酶活性变化的时空信息。这些资料为EGF作用PKA提供了直接的实时证据。     

生物大分子相互作用检测技术新进展———三色荧光级联荧光共振能量转移技术

荧光共振能量转移(fluorescenceresonanceenergytransfer,FRET),是指能量从一种受激发的荧光基团(fluorophore)以非辐射的方式转移到另一种荧光基团的物理现象.FRET的能量转移效率是两个荧光基团间距离的函数,并对此距离十分敏感,它的有效响应距离一般在1～10nm之间,因而可被用于测定原子间及分子间的距离.这一特点使FRET技术在大分子构象变化、大分子之间相互作用、细胞信号通路等研究中发挥重要作用,成为生物医学研究中的重要方法.但细胞内的生物学过程常常涉及多于两个的大分子间相互作用,二色荧光基团的FRET技术不能满足这种生物学研究的需求.最近,两个研究小组在这方面取得突破,建立了分别基于共聚焦显微镜和流式细胞仪的三色荧光级联FRET技术.这一技术的出现将会极大地促进生物学及相关研究领域的发展.     

基于GFP的FRET应用

绿色荧光蛋白（GFP）是一种活性荧光标记,已被用来研究基因表达、分子定位,蛋白质折叠和转运;荧光共振能量转移（FRET）是一种无损伤的光学检测方法,能检测到小于纳米的距离变化。将GFP的活性定位标记功能与FRET的高分辨率相结合。为活体研究生物分子的功能和命运开创了新的篇章。作者在介绍GFP和FRET原理的基础上,综述了基于GFP的FRET在蛋白酶活性,蛋白质间相互作用 构象改变研究中的应用。     

利用荧光共振能量转移技术研究活细胞TLR4与MD-2作用结构域

脂多糖(LPS)的识别和信号转导是宿主发生防御反应的关键,Toll样受体4(TLR4)与髓样分化蛋白-2(MD-2)形成复合物在LPS的识别及其信号转导中发挥了重要作用．研究TLR4与MD-2结合的功能结构域,对于深入了解LPS信号转导机制及其内毒素休克的防治具有重要意义．运用基于强度的三通道荧光共振能量转移技术(FRET)及基因突变和转染技术,研究了活细胞TLR4与MD-2作用的结构域．结果表明：N端Glu²⁴～Met⁴¹缺失使TLR4与MD-2结合能力明显下降;LPS刺激后TLR4聚合迅速增加,而缺失Glu²⁴～Met⁴¹的TLR4不能聚合．上述结果提示,TLR4的Glu²⁴～Met⁴¹不仅是结合MD-2的区域,并且还参与了LPS刺激后TLR4的聚合作用．     

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.     

FRET技术在受体信号转导研究中的应用

细胞信号传导是细胞生物学方面的重要内容之一,涉及生命过程的各个方面,包括生长、分化发育、增殖、凋亡、迁移等等,对维持细胞功能及机体生存至关重要。目前对细胞信号转导研究的技术手段多种多样,其中荧光共振能量转移技术（FRET）是研究细胞信号转导较为常用的一种技术,可以实现活细胞内蛋白质之间相互作用的实时检测。本文中我们以受体酪氨酸激酶为例,介绍FRET技术在受体介导细胞信号传导中的应用及进展情况。     

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。     

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。     

用FRET方法研究与Mps1蛋白有相互作用的CENP-E蛋白结构域

目的：探索与Mps1蛋白有相互作用的CENP-E蛋白结构域。方法：将重组质粒pEGFP-CENPE2（674~1085位氨基酸）、pEGFP-CENPE3（1200~2134位氨基酸）转染人胚肾293(HEK293)细胞,采用受体漂白荧光共振能量转移方法（FRET方法）,检测EGFP-CENPE2、EGFP-CENPE3和Mps1间的能量转移率(Ef), 进一步用免疫共沉淀方法验证FRET的实验结果。结果：重组质粒转染HEK293细胞后经激光共聚焦显微镜观察重组质粒表达的融合蛋白与Mps1都存在着共定位;FRET检测结果显示EGFP-CENPE3和Mps1间的能量转移率为［(12.63±0.48)%, n=30］,pEGFP-CENPE2和Mps1间的能量转移率为［(3.07±0.21)%, n=30］,与对照组［(2.96±0.27)%, n=30］比较pEGFP-CENPE3和Mps1间的能量转移率差异存在显著性（p<0.05）,免疫共沉淀实验结果显示EGFP-CENPE3与Mps1蛋白间存在相互作用。结论：FRET技术和免疫共沉淀实验证明了EGFP-CENPE3与Mps1间存在着相互作用。     

Decreased hephaestin expression and activity leads to decreased iron efflux from differentiated Caco2 cells

Iron is transported across intestinal brush border cells into the circulation in at least two distinct steps. Iron can enter the enterocyte via the apical surface through several paths. However, iron egress from the basolateral side of enterocytes converges on a single export pathway requiring the iron transporter, ferroportin1, and hephaestin, a ferroxidase. Copper deficiency leads to reduced hephaestin protein expression and activity in mouse enterocytes and intestinal cell lines. We tested the effect of copper deficiency on differentiated Caco2 cells grown in transwells and found decreased hephaestin protein expression and activity as well as reduced ferroportin1 protein levels. Furthermore, the decrease in hephaestin levels correlates with a decrease of ⁵⁵Fe release from the basolateral side of Caco2 cells. Presence of ceruloplasmin, apo‐transferrin or holo‐transferrin did not significantly alter the results observed. Repletion of copper in Caco2 cells leads to reconstitution of hephaestin protein expression, activity, and transepithelial iron transport. J. Cell. Biochem. 107: 803–808, 2009. © 2009 Wiley‐Liss, Inc.     

Study on the Interaction Between Nucleic Acids and Imidacloprid and Its Application

The determination of imidacloprid with DNA via a resonance light scattering (RLS) technique was developed. The RLS of DNA was remarkably quenched after adding imidacloprid in aqueous medium of pH 2.10. An RLS peak at 311 nm was found, and the quenched intensity of RLS at this wavelength was proportional to the concentration of imidacloprid. The linear range of the calibration curve was approximately 0.02–2 μg/mL with the detection limit (S/N = 3) of 0.02 ng/mL. The imidacloprid in river water, cucumbers, and apple samples was determined. The recovery rates were in the range of 91.9% to 95.20%, 97.2% to 111.3%, and 94.5% to 114.8%, respectively. The mechanism of the reaction between imidacloprid and DNA is also discussed.     

CAL与mGluR1相互作用抑制mGluR1过度激活诱导的细胞毒性作用

代谢型谷氨酸受体1(mGluR1)过度激活介导的谷氨酸兴奋性毒性是帕金森病(PD)的主要发病机制之一。在临床试验中应用mGluRs的负性变构调节剂缓解PD病人的运动障碍已有报道,但由于精确调控mGluRs表达或活性的局限性,目前,在PD的治疗中仍存在一些问题。因此,寻找能够精确调控mGluR1表达及活性的小分子药物或内源性基因,将有可能成为解决目前PD治疗中存在问题的有效方法。本文通过体内和体外实验,对囊性纤维跨膜调节器相关配体(CAL)在mGluR1过度激活诱导的细胞毒性中的作用和机制进行研究。研究结果显示,在工具细胞HEK293中,应用mGluR1的激动剂激活受体,CAL与mGluR1的相互作用明显增强(P< 0.05),且CAL通过与mGluR1相互作用,抑制mGluR1过度激活诱导的细胞凋亡及其下游信号通路的激活。在鱼藤酮诱导的PD大鼠模型中,过表达CAL通过抑制mGluR1下游通路的激活,减少鱼藤酮引起的神经损伤(P< 0.001)。本文揭示了一种调控mGluR1活性的新机制,希望为神经系统疾病的治疗和相关研究提供新思路。     

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.     

自噬与NLRP3炎症小体激活间的相互作用

自噬作为真核生物细胞遭遇各种应激压力时发生的一种基本应答方式,参与细胞的多种生命活动,使细胞在各种应激条件下维持一种动态平衡状态。NOD样受体家族核苷酸结合寡聚化结构域样受体3（NOD-like receptor family,pyrin domain containing 3,NLRP3）炎症小体,是生物体内防御病原微生物的固有免疫防御系统的重要组成部分。NLRP3炎症小体通过激活胱天蛋白酶-1（caspase-1）,从而促进白细胞介素-1β（interleukin-1,IL-1β）和白细胞介素-18（interleukin-18,IL-18）等促炎细胞因子的成熟和分泌,继而介导炎症的发生。众多研究表明,自噬能够负向或正向调控NLRP3炎症小体的激活。同时,NLRP3炎症小体也会逆向影响自噬的作用。本文对自噬包括选择性自噬与NLRP3炎症小体激活的相互作用,以及通过激活自噬抑制NLRP3炎症小体,从而在炎症相关疾病治疗中的应用进行综述。     

Effects of Interaction Between Cadmium and Plumbum on Phytochelatins and Glutathione Production in Wheat （Triticum aestivum L.）

Phytochelatins (PCs) may function as a potential biomarker for metal toxicity. However, less attention has been paid to the effects of metal interactions on the production of PCs and glutathione (GSH), the most prominent cellular thiol. In the present study, the effects of interactions between cadmium (Cd) and plumbum (Pb) on the production of PCs and GSH were monitored over a period of 14 d in wheat (Triticum aestivum L.) tissues. The results showed that combination of Cd and Pb led to synergistic growth inhibition in wheat. Exposure to Cd or Pb increased levels of PCs in a concentration-, tissue-, and time-dependent manner. Cadmium was more effective that Pb in increasing PCs production. Compared with the effects of Cd or Pb alone on the production of PCs, the combination of Cd and Pb acted synergistically, resulting in an enhanced production of PCs. Cadmium also stimulated GSH production in a concentration-, tissue-, and time-dependent manner. However, Pb had no obvious effects on GSH levels. The combination of Pb and Cd antagonized GSH production over the course of the growth period. The results of the present study suggest that metal interactions should be considered in the application of PCs and GSH as potential biomarkers for the evaluation of metal toxicity.     

Protein localization in living cells and tissues using FRET and FLIM

Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution. One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength. Mathematical algorithms are required to correct the spectral bleed-through signal in wide-field, confocal, and two-photon FRET microscopy. In contrast, spectral bleed-through is not an issue in FRET/FLIM imaging because only the donor fluorophore lifetime is measured; also, fluorescence lifetime imaging microscopy (FLIM) measurements are independent of excitation intensity or fluorophore concentration. The combination of FRET and FLIM provides high spatial (nanometer) and temporal (nanosecond) resolution when compared to intensity-based FRET imaging. In this paper, we describe various FRET microscopy techniques and its application to protein-protein interactions.