KRAS信号通路蛋白相互作用的双分子荧光互补成像研究及其优化
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摘要
双分子荧光互补(Bimolecular Fluorescent Complimentary, BiFC)技术是指通过具有相互作用亲和力的两个蛋白质，将分别与其相连的荧光蛋白片段拉近，组装成完整的荧光蛋白，从而表征蛋白质相互作用的发生及其空间位置。由于BiFC技术检测灵敏度高、操作简便直观，已成为活细胞内研究蛋白质-蛋白质相互作用的有力工具。但该方法具有假阳性率高、结合反应不可逆等缺点，使其在蛋白质相互作用的动态与量化研究应用中受到限制。本文对不同理化特点的BiFC系统进行了比较与探讨，并通过选择合适的表达载体和荧光蛋白片段构建了一个能显著降低假阳性的BiFC体系，进而用不同的BiFC系统对K-Ras信号通路中的上下游蛋白质相互作用进行了研究，研究成果如下：
对不同BiFC体系的效率与特点进行了比较；同时利用基于黄色荧光蛋白突变体Venus的N端1-172（Vn173）和C端155-238（Vc155）组成的BiFC体系对上皮生长因子受体（Epidermal Growth Factor Receptor, EGFR）二聚化作用在多种细胞中进行了观察和分析。在此基础上，构建了基于Vn173/Vc155的真核双表达载体，把BiFC体系简化成每个载体中含有两个蛋白的基因序列；进而利用该体系，分别对KRAS (Kirsten大鼠肉瘤病毒原癌基因同源基因产物)/RAF1（RAF原癌基因丝氨酸/苏氨酸蛋白激酶）、KRAS/RASSF2（Ras-association domain family2）和KRAS/GRB2（生长因子受体结合蛋白2，Growth factor receptor-bound protein2）的相互作用及其细胞定位进行了研究。通过对上述蛋白在细胞内形成的荧光复合体的亚细胞定位进行分析发现：在没有EGF刺激的条件下，RAF1与KRAS间存在微弱的结合作用；在RAF1定位于细胞膜上的过程中，KRAS与RAF1间形成的复合体而不是KRAS起着更为重要的作用；KRAS与GRB2能形成复合体，在这个过程中GRB2的两个SH3结构域与KRAS的结合能力较SH2结构域强，在活细胞中证实了已有的理论，即GRB2同SOS1形成复合体后与KRAS结合并被募集到细胞膜上；RASSF2与KRAS能在细胞膜上结合，但RASSF2进入细胞核的过程中KRAS没有与之形成复合体；EGF的刺激或KRAS的活化明显增强了以上三个蛋白与KRAS的相互作用中。在这些体系中，由于Vn173与Vc155随机碰撞导致的本底结合作用，即使不存在相互作用的一对蛋白也可能产生BiFC荧光，所以仅仅根据BiFC的结果无法确定两个蛋白间是否存在相互作用。这限制了BiFC在蛋白相互作用检测中的应用。
为了克服BiFC的上述缺点，本论文通过使用一对新的BiFC荧光片段mLumin1-151（Ln）和mLumin152-231（Lc），在双表达载体中构建了新的BiFC体系，通过一对已知相互作用的蛋白对bFOS/bJUN及其失去相互作用的突变体（bΔFOS）/bJUN的BiFC效率的比较，证明该体系明显降低了BiFC体系中因非特异性结合而导致的假阳性。利用这个体系，对KRAS/GRB2的相互作用进行了量化分析。结果显示KRAS在活化前后与GRB2间形成的BiFC荧光变化比基于Venus的BiFC体系中更大，而KRAS的CAAX突变体与GRB2在这个体系中无法观测到可见的荧光；这进一步验证了这个体系降低假阳性的能力。通过对BiFC体系的比较，为我们提供了在不同目的的蛋白相互作用研究中对这些体系选择的原则：以弱相互作用复合体的亚细胞定位观察为目的的研究中，更适于选择具有更高灵敏度的Venus为基础的BiFC体系；在最佳生长温度低于30oC的生物体进行BiFC观察时，基于GFP的BiFC体系将是更好的选择；而对于需要量化分析蛋白质的相互作用，尤其是判定未知蛋白质之间是否发现相互作用时，基于mLumin的双表达载体具有更大的优势。
总之，本论文建立了一个基于mLumin的双表达载体的BiFC体系，该体系很大程度上降低了BiFC的假阳性率；利用不同的BiFC体系，对KRAS信号通路中的几个蛋白间的相互作用进行了BiFC观察。比较结果表明，我们的BiFC体系适于蛋白相互作用的量化分析以及对假阳性率有较高要求的检测，如文库筛选的BiFC分析中。
Bimolecular fluorescence complementation (BiFC) assay is a powerful tool forresearches of protein-protein interactions in vivo. With high sensitivity, BiFC is a simplemeaning to directly observe protein-protein interactions in living cells. It is based on theassociation of fluorescent protein fragments that are attached to components of the samemacromolecular complex. Proteins that are postulated to interact are fused to unfoldedcomplementary fragments of a fluorescent reporter protein and expressed in live cells.Interaction of these proteins will bring the fluorescent fragments within proximity,allowing the reporter protein to reform in its native three-dimensional structure and emitits fluorescent signal. In this paper, the efficiency of BiFC systems with differentcharacteristics was compared; while dimerization of EGFR was observed and analyzedused the Vn173/Vc155BiFC system based on yellow fluorescent protein variant, Venus ina variety of cells.
On this basis, a BiFC system in a dual-expression eukaryotic vector based onVn173/Vc155was built, which simplified the two-vector system into single-vector; theninteractions between KRAS (Kirsten rat sarcoma viral oncogene homolog) and RAF1(RAF proto-oncogene serine/threonine-protein kinase), KRAS and RASSF2(Ras-association domain family2), KRAS and GRB2(Growth factor receptor-boundprotein2) were observed respectively with this system. Through observation ofsubcellular localization of fluorescent complex formed in the assay, the interactionsbetween these proteins as well as the activation of cellular signal transduction mechanismsare discussed. However, BiFC fluorescent intensity and protein interaction strength does not have obvious proportional relationship in these systems, because of spontaneouscombination between Vn173and Vc155, which limits its use in quantitative analysis ofprotein interactions. To this end, we use a new pair of BiFC fluorescent fragments,mLumin1-151(Ln) and mLumin152-231(Lc), to construct BiFC system in a dualexpression vector, which significantly reduced false positive produced by spontaneouscombination of fluorescent protein fragments. With this system, interaction betweenKRAS and GRB2was quantified. Comparison between these BiFC systems provides usprinciples for the choice in the studies of protein-protein interaction with differentpurposes: Venus-based BiFC system with higher sensitivity is more suitable forsubcellular localization studies of proteins with weak interaction; whereas the GFP-basedBiFC system will be the better choice in studies on cells or organisms which grow incondition below30oC, such as plants, nematodes; and for quantitative analysis ofinteractions as well as studies requires a lower false-positive, mLumin-based BiFC systemin dual expression vector has greater advantage.
In short, this paper establishes a mLumin-based BiFC system in dual expressionvector that largely reduces the BiFC false-positive rate; use of different BiFC systems,several protein-protein interactions in the KRAS signaling pathway were observed.Results show that our BiFC system is suitable for quantitative analysis of proteininteraction and lower false-positive-required researches, such as library screening of BiFCanalysis.
对不同BiFC体系的效率与特点进行了比较；同时利用基于黄色荧光蛋白突变体Venus的N端1-172（Vn173）和C端155-238（Vc155）组成的BiFC体系对上皮生长因子受体（Epidermal Growth Factor Receptor, EGFR）二聚化作用在多种细胞中进行了观察和分析。在此基础上，构建了基于Vn173/Vc155的真核双表达载体，把BiFC体系简化成每个载体中含有两个蛋白的基因序列；进而利用该体系，分别对KRAS (Kirsten大鼠肉瘤病毒原癌基因同源基因产物)/RAF1（RAF原癌基因丝氨酸/苏氨酸蛋白激酶）、KRAS/RASSF2（Ras-association domain family2）和KRAS/GRB2（生长因子受体结合蛋白2，Growth factor receptor-bound protein2）的相互作用及其细胞定位进行了研究。通过对上述蛋白在细胞内形成的荧光复合体的亚细胞定位进行分析发现：在没有EGF刺激的条件下，RAF1与KRAS间存在微弱的结合作用；在RAF1定位于细胞膜上的过程中，KRAS与RAF1间形成的复合体而不是KRAS起着更为重要的作用；KRAS与GRB2能形成复合体，在这个过程中GRB2的两个SH3结构域与KRAS的结合能力较SH2结构域强，在活细胞中证实了已有的理论，即GRB2同SOS1形成复合体后与KRAS结合并被募集到细胞膜上；RASSF2与KRAS能在细胞膜上结合，但RASSF2进入细胞核的过程中KRAS没有与之形成复合体；EGF的刺激或KRAS的活化明显增强了以上三个蛋白与KRAS的相互作用中。在这些体系中，由于Vn173与Vc155随机碰撞导致的本底结合作用，即使不存在相互作用的一对蛋白也可能产生BiFC荧光，所以仅仅根据BiFC的结果无法确定两个蛋白间是否存在相互作用。这限制了BiFC在蛋白相互作用检测中的应用。
为了克服BiFC的上述缺点，本论文通过使用一对新的BiFC荧光片段mLumin1-151（Ln）和mLumin152-231（Lc），在双表达载体中构建了新的BiFC体系，通过一对已知相互作用的蛋白对bFOS/bJUN及其失去相互作用的突变体（bΔFOS）/bJUN的BiFC效率的比较，证明该体系明显降低了BiFC体系中因非特异性结合而导致的假阳性。利用这个体系，对KRAS/GRB2的相互作用进行了量化分析。结果显示KRAS在活化前后与GRB2间形成的BiFC荧光变化比基于Venus的BiFC体系中更大，而KRAS的CAAX突变体与GRB2在这个体系中无法观测到可见的荧光；这进一步验证了这个体系降低假阳性的能力。通过对BiFC体系的比较，为我们提供了在不同目的的蛋白相互作用研究中对这些体系选择的原则：以弱相互作用复合体的亚细胞定位观察为目的的研究中，更适于选择具有更高灵敏度的Venus为基础的BiFC体系；在最佳生长温度低于30oC的生物体进行BiFC观察时，基于GFP的BiFC体系将是更好的选择；而对于需要量化分析蛋白质的相互作用，尤其是判定未知蛋白质之间是否发现相互作用时，基于mLumin的双表达载体具有更大的优势。
总之，本论文建立了一个基于mLumin的双表达载体的BiFC体系，该体系很大程度上降低了BiFC的假阳性率；利用不同的BiFC体系，对KRAS信号通路中的几个蛋白间的相互作用进行了BiFC观察。比较结果表明，我们的BiFC体系适于蛋白相互作用的量化分析以及对假阳性率有较高要求的检测，如文库筛选的BiFC分析中。
Bimolecular fluorescence complementation (BiFC) assay is a powerful tool forresearches of protein-protein interactions in vivo. With high sensitivity, BiFC is a simplemeaning to directly observe protein-protein interactions in living cells. It is based on theassociation of fluorescent protein fragments that are attached to components of the samemacromolecular complex. Proteins that are postulated to interact are fused to unfoldedcomplementary fragments of a fluorescent reporter protein and expressed in live cells.Interaction of these proteins will bring the fluorescent fragments within proximity,allowing the reporter protein to reform in its native three-dimensional structure and emitits fluorescent signal. In this paper, the efficiency of BiFC systems with differentcharacteristics was compared; while dimerization of EGFR was observed and analyzedused the Vn173/Vc155BiFC system based on yellow fluorescent protein variant, Venus ina variety of cells.
On this basis, a BiFC system in a dual-expression eukaryotic vector based onVn173/Vc155was built, which simplified the two-vector system into single-vector; theninteractions between KRAS (Kirsten rat sarcoma viral oncogene homolog) and RAF1(RAF proto-oncogene serine/threonine-protein kinase), KRAS and RASSF2(Ras-association domain family2), KRAS and GRB2(Growth factor receptor-boundprotein2) were observed respectively with this system. Through observation ofsubcellular localization of fluorescent complex formed in the assay, the interactionsbetween these proteins as well as the activation of cellular signal transduction mechanismsare discussed. However, BiFC fluorescent intensity and protein interaction strength does not have obvious proportional relationship in these systems, because of spontaneouscombination between Vn173and Vc155, which limits its use in quantitative analysis ofprotein interactions. To this end, we use a new pair of BiFC fluorescent fragments,mLumin1-151(Ln) and mLumin152-231(Lc), to construct BiFC system in a dualexpression vector, which significantly reduced false positive produced by spontaneouscombination of fluorescent protein fragments. With this system, interaction betweenKRAS and GRB2was quantified. Comparison between these BiFC systems provides usprinciples for the choice in the studies of protein-protein interaction with differentpurposes: Venus-based BiFC system with higher sensitivity is more suitable forsubcellular localization studies of proteins with weak interaction; whereas the GFP-basedBiFC system will be the better choice in studies on cells or organisms which grow incondition below30oC, such as plants, nematodes; and for quantitative analysis ofinteractions as well as studies requires a lower false-positive, mLumin-based BiFC systemin dual expression vector has greater advantage.
In short, this paper establishes a mLumin-based BiFC system in dual expressionvector that largely reduces the BiFC false-positive rate; use of different BiFC systems,several protein-protein interactions in the KRAS signaling pathway were observed.Results show that our BiFC system is suitable for quantitative analysis of proteininteraction and lower false-positive-required researches, such as library screening of BiFCanalysis.
引文
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