摘要
朊病毒类疾病包括库鲁病、吉斯综合症、克雅氏病等,依据其致病原因分为散发性感染,遗传和感染。朊病毒病的感染因子prion是一种仅由蛋白质构成的病毒。在神经和其他组织细胞中正常表达的细胞型prion蛋白(PrPc)是一种无害的GPI锚蛋白。PrPc在体外会迅速折叠成C端含有三个α-螺旋和少量的β-折叠的结构。N端则是富含组氨酸的无序柔软结构,可以与Cu2+和Ni2+等二价金属离子结合。PrPc向其致病构象Prpsc转变的机理和方式还在研究中,但有报道显示这种转变可能发生在细胞质膜上,即PrPc通过GPI锚定位于细胞膜外侧时或者内吞过程中。直观实时的观察PrPc在活细胞内的运转过程能帮助我们深入的研究这一结构转变的发生及其与prion相关疾病的关系。
传统的显示或示踪生物分子的方法,例如免疫染色或融合荧光蛋白等,都难以做到生物活细胞内的单分子示踪。为了达到这一目的,量子点(quantum dots,QDs)因其特有的性质和光学特性被越来越多的运用到这一方向中来。QDs的发射波长可调、高亮度、光稳定性和抗光漂白能力等特性使它有潜力作为非常优秀的生物分子示踪材料。QDs与生物分子的连接方式主要有通过共价键连接,通过静电力作用连接,硫醇基交换连接等方式。为了实现用QDs对朊病毒这一非常特殊的病毒——仅由单个蛋白质构成——进行标记,以及最大程度的减少其对朊蛋白结构的影响和破坏,我们利用PrP的N端天然存在的富含组氨酸的八肽重复区,通过Ni2+的螯合作用,实现了QDs对PrP的特异性标记。
我们先在体外表达纯化了PrP23-231,将其与带有Ni2+的QDs (QDs-Ni2+)共同孵育。我们分别利用琼脂糖凝胶电泳和超滤的方式,证明了可以在水溶液中将QDs与PrP一步连接。将纯化的PrP或诱导表达PrP的细菌裂解液进行SDS-PAGE并转膜,用低浓度(1×10-8M)QDs及短时间(15min)的孵育结合后,都能获得清晰而专一的PrP条带,在体外证明了标记的特异性。接下来,通过原子力显微镜(AFM)和动态光散射(DLS)等实验,我们进一步从颗粒的粒径变化上直观地确认了QDs与PrP的结合。随后,我们利用浊度法测试了QDs在不同pH条件下对PrP聚集的影响,并用圆二色谱法(CD)证明了QDs与PrP的结合不会对PrP的二级结构造成影响。
由无机材料制成的QDs往往会有一定的生物毒性,可以通过对其进行表面进行生物亲和性修饰减小或消除这种毒性。利用MTT法,我们比较了不同比例PEG修饰后的QDs对细胞毒性的影响。结果显示,生物亲和材料PEG的比例越大,QDs对细胞的毒性就越小,但同时QDs结合PrP的能力会降低。但当QDs浓度低于7.5×10-9M后,即使低比例PEG修饰的QDs在24h内的细胞毒性也都不可察觉了。随后,我们在细胞中表达带有绿色荧光蛋白(GFP)的PrP(SP-GFP-PrP),并用QDs对其标记。结果显示,无论是在细胞膜上还是内吞到细胞中的QDs,均与GFP荧光共定位,且表现出了非常好的特异性。然后我们用QDs标记神经细胞中表达的PrP1-253,对其从细胞膜转运至细胞质内的周期进行了研究,并拍摄了12min的连续动态视频,首次实现了对单PrP颗粒在活细胞内的长时间全程可视示踪。通过动态视频,我们对PrP内吞及转运过程中在不同亚细胞部位的移动速率进行了分析,并将其量化的分成了四个相对独立又互相联系的时期。最后,我们还进一步利用QDs研究了脂筏在PrPC膜定位和内吞过程中的作用。
另外,我们还构建了稳定表达带有红色荧光蛋白(RFP)标签的PrP(SP-RFP-PrP)的人神经瘤细胞系。为后续利用FRET等方法研究绿色荧光QDs标记的致病朊病毒颗粒入侵细胞,和正常细胞内PrPc的相互作用奠定基础。
Prion disease including Kuru syndrome, Gerstmann-Straussler-Scjeinker (GSS) and Creutzfeldt-Jakob disease (CJD) can be sporadic, inherited or acquired by transmission. The "infectious" factor prion is a protein only virus. The cellular prion protein (PrPC) is a harmless glycosylinositol phospholipids (GPI) anchored glycoprotein expressed in neuronal and other cells. The C-terminal domain of PrPc has three a-helices and a short section of antiparallelβ-sheet and it folds to a stable structure in vitro quickly. The N-terminal domain of PrPC, however, is His-rich and flexibly disordered in the full-length molecule, which can binds to Cu2+and Ni2+. The structural alteration of the PrPC from a normal a-helix rich form into a pathogenic and protease-resistant P-sheet rich isoform PrPSc (pathogenic scrapie prion protein) is tightly linked to transmissible spongiform encephalopathies (TSE). The actual conversion process of the prion, however, is not fully understood although there are evidences suggesting it may occur at the plasma membrane. Some reports showed that PrPC targets to the outer leaflet of the plasma membrane by the GPI anchor, or during the internalization pathways, as both clathrin-coated pits and caveolae-dependent structures have been found to be involved in the process. A direct and real-time visualization of PrPC transport in a living cell may help to understand how the entire process occurred and its relationship with prion associated diseases.
The traditional way of visualizing and tracking of a protein is dependent on techniques of immunoprecipitation or fusing the target protein with a fluorescent protein. But to achieve single molecule sensitivity and tracking, quantum dots (QDs) have generated considerable interest in the biological community because their unique optical and electronic properties such as tunable emission from visible to infrared wavelengths by changing the size and composition, broader excitation spectra, strong brightness, photostability, and high resistance to photobleaching compare with that of the dye molecules. With these unique features, QDs are becoming suitable tools for both in vitro and in vivo imaging. Modified QDs may be conjugated with biomolecules through methods such as covalent attachment, electrostatic attraction, and thiol-exchange reaction. To conjugate QDs with prion, the virus made of single protein only, as a fluorescent detector with minimum influence to its suructure, we use Ni2+as a mediate, to conjugate QDs with the prion protein by the original His-rich region in its N-terminal.
We confirmated QDs-Ni2+can be conjugated with PrP23-231 in vitro using agarose gel electrophoresis and ultrafiltration. Low concentration (1×10-8 M) and short time incubation (15 min) with QDs-Ni2+revealed PrP distinctly in western blot experiments. Then the atom force microscopy (AFM) and dynamic light scattering (DLS) show the conjugation of QDs with PrP directly from the particle size alteration. Then we tested the influence of QDs conjugation to the aggregation behaviors in pH condition by turbidity assay, and Circular Dichroism (CD) showed there is no alteration in PrP's secondary structure after QDs conjugation.
The biotoxins of QDs are tested by MTT assay, shown while the QDs concentration is lower than 7.5×10-9 M, there is imperceptible influence to cells in 24h even the QDs were modified by less PEG. Using GFP fused PrP (SP-GFP-PrP) expressing in human neuron cells, we found QDs signal is co-localization with the GFP signal, with perfect specificity. We have demonstrated, for the first time, the actual process of PrPc transport from location on plasma membrane to translocation to the perinuclear region, in single living cell with a 12 min movie, and separated the process into four individual stages by the velocity changes. Furthermore, we showed that the critical role of lipid rafts in PrPc membrane localization and internalization.
Finally, we established a cell line stably expressing PrP fused with RFP tag, for the further researchs on prion infection.
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