PICK1相互作用蛋白在ASICs及AMPA受体转运中的作用及相关机制研究

发布时间：2018-07-04 10:28

本文选题：KIF2C + syntabulin　；参考：《浙江大学》2015年博士论文

【摘要】：背景：PICK1 (Protein that interacts with C kinase 1)是一种周边膜蛋白,是和蛋白激酶Ca (PKCa)相互作用的蛋白之一。PICKl可以通过其特殊的PDZ结构域与很多膜蛋白发生结合,例如PICKl不仅可以与酸敏感离子通道ASIC (acid-sensing ion channels)通道蛋白相互结合,也可以和离子型谷氨酸受体AMPA (a-amino-3-hydroxy-5-methylisoxazoloe-4-propionic acid-type)受体的亚单位之一GluA2结合。在大多数情况下,PICKl可通过改变被结合蛋白的亚细胞转运或表面表达来调节蛋白的转运。凭借这些功能特点,PICK1既可以调节酸敏感离子通道ASIC (acid-sensing ion channels)的表面表达,也可以在突触可塑性中调节AMPA受体的转运过程。已有的证据表明,PICK1可以与ASIC1a及ASIC2a直接结合,通过影响ASIC蛋白的表面表达来介导细胞的酸毒性,进而参与调节细胞凋亡,但PICK1是通过何种方式介导细胞酸毒性的还不是很清楚。另外,在体内生理情况下,ICA69 (Islet Cell Autoantigen 69 kDa)已被证实可以与大部分PICK1形成异源二聚体,并且他们二者的结合可以影响AMPA受体的分布。然而,PICK1和ICA69调节AMPA受体转运的具体分子机制还不明确。我们的实验结果显示：syntabulin可以与PICK1直接结合,并通过PICK1影响ASIC2的表面表达水平,最终参与调节神经元的酸毒性。同时,KIF2C也可以与PICK1有直接的相互作用,并且能通过PICK1和ICA69以及AMPA受体形成复合物,进而影响AMPA受体的膜表面表达。目的：筛选出与PICK1有直接相互作用的蛋白,并分析其功能,一方面探究syntabulin与PICK1的相互作用是否会调节神经元的凋亡,另一方面探索KIF2C与PICK1的相互作用是否会影响AMPA受体的分布,在现有的基础上进一步探明ASIC及AMPA受体转运的分子机制,期望最终能够回答ASIC受体调节神经元凋亡的分子机制及AMPA受体上膜、下膜的动态过程,为细胞凋亡和学习记忆的基本原理提供理论基础。方法：我们利用酵母双杂交、免疫共沉淀,高效液相层析质谱筛选出与PICK1有相互作用的目标蛋白；利用蛋白免疫印迹,免疫细胞化学进一步验证筛选出的目标蛋白syntabulin和KIF2C;利用激光共聚焦显微镜和表面生物素化实验一方面观察syntabulin对ASIC受体表面表达及细胞凋亡的影响,另一方面观察KIF2C对AMPA受体表面表达的影响。结果：(1)研究发现,在培养的海马神经元中敲减syntabulin后,酸性刺激条件下会引起神经元的凋亡。表面生物素化结果显示,在培养的海马神经元中敲减syntabulin后,ASIC2的表面表达水平降低,同时ASIC2的总体表达永平升高。体外的免疫共沉淀显示,syntabulin可以通过PICK1与ASIC1a,ASIC2a形成复合物,但与ASIC1a相比,syntabulin能够通过PICK1结合更多的ASIC2a。通过细胞免疫荧光染色实验,观察到syntabulin能够通过PICK1与ASIC2a共定位,而在没有PICK1时,syntabulin与ASIC2a并不存在共定位。syntabulin与ASIC1a在有没有PICK1的情况下,都不会存在共定位。(2)高效液相层析质谱实验结果显示,19个蛋白与PICK1有相互作用,15个蛋白与ICA69有相互作用。在筛选到的蛋白中,KIF2C与PICK1有直接的相互作用,并且能够通过PICK1和ICA69形成一个复合物。KIF2C和PICK1之间的相互作用不会增强或减弱PICK1与GluA2之间的结合,并且也不会影响PICK1与GluA2形成的co-cluster。在体内生理条件下,KIF2C主要在大脑和睾丸中表达,并且在大脑中能够与GluA2结合。经体外验证,这种结合是通过PICK1实现的,在没有PICK1的情况下,KIF2C不能与GluA2有相互作用。在293T细胞中,过表达KIF2C可以降低GluA2表面表达水平,当PICK1和KIF2C共同过表达时,GluA2的表面表达水平进一步降低,这种现象是由两方面因素引起的,一方面是因为KEF2C本身可以解聚微管,另一方面是因为PICK1太身也可以降低GluA2的表面表达水平。在体外培养的海马神经元中,KIF2C可以与PSD-95以及GluA2共定位,过表达KIF2C也可以降低GluA2的表面表达。结论：(1)我们的研究表明,syntabulin能够通过PICK1与ASIC通道蛋白形成复合物,进而改变ASIC2的表面表达水平及总体表达水平,最终调节神经元的酸毒性。(2)我们的研究表明KIF2C与PICK1有直接的相互作用,并且通过这种相互作用进一步结合GluA2,形成一个复合物,最终以降低GluA2表面表达水平的形式参与AMPA受体的转运。
[Abstract]:Background: PICK1 (Protein that interacts with C kinase 1) is a peripheral membrane protein that is one of the proteins interacting with protein kinase Ca (PKCa), which can bind to many membrane proteins through its special PDZ domain, such as PICKl not only with acid sensitive ion channels. In combination, it can also be combined with one of the subunits GluA2 of the AMPA (a-amino-3-hydroxy-5-methylisoxazoloe-4-propionic acid-type) receptor. In most cases, PICKl can transfer the translocation of the protein by altering subcellular transport or surface expression of the binding protein. By virtue of these functional characteristics, PICK1 The surface expression of the acid sensitive ion channel ASIC (acid-sensing ion channels) can be regulated and the AMPA receptor transport process can be regulated in the synaptic plasticity. The evidence shows that PICK1 can directly combine with ASIC1a and ASIC2a to mediate the acid toxicity of the cell by affecting the surface expression of the ASIC protein, and then participate in the regulation of cell withering. In addition, in vivo, ICA69 (Islet Cell Autoantigen 69 kDa) has been proved to be able to form a heterogenous two polymer with most of the PICK1, and the combination of the two of them can affect the distribution of AMPA receptors. However, PICK1 and ICA69 regulate AMPA receptors. The specific molecular mechanism of transport is not clear. Our experimental results show that Syntabulin can directly combine with PICK1 and affect the surface expression level of ASIC2 through PICK1, and ultimately participates in regulating the acid toxicity of neurons. At the same time, KIF2C can also interact directly with PICK1 and can be formed through PICK1 and ICA69 and AMPA receptors. The compound, which affects the membrane surface expression of the AMPA receptor, aims to screen out the proteins that interact directly with PICK1 and analyze their functions. On the one hand, explore whether the interaction between Syntabulin and PICK1 can regulate neuronal apoptosis, on the other hand, explore whether the interaction of KIF2C and PICK1 affects the distribution of AMPA receptors. The molecular mechanism of ASIC and AMPA receptor transport is further explored. It is expected that the molecular mechanism of ASIC receptor to regulate neuronal apoptosis and the AMPA receptor upper membrane and the dynamic process of the lower membrane provide the theoretical basis for the basic principles of cell apoptosis and learning and memory. The target proteins interacting with PICK1 were screened by liquid chromatography mass spectrometry; the target protein Syntabulin and KIF2C were further verified by immunoblotting and immunocytochemistry, and the surface expression and apoptosis of ASIC receptors on the ASIC receptor were observed by laser confocal microscopy and surface biotinization experiments. Effects, on the other hand, observe the effect of KIF2C on the surface expression of AMPA receptor. Results: (1) the study found that after the subtraction of Syntabulin in the cultured hippocampal neurons, the apoptosis of the neurons was induced under the acidic stimulus. The surface biotinylated results showed that the surface expression level of ASIC2 decreased after the subtraction of Syntabulin in the cultured hippocampal Jing Yuan. At the same time, the overall expression of ASIC2 is elevated. In vitro immunoprecipitation shows that Syntabulin can form complex through PICK1 and ASIC1a, ASIC2a, but compared with ASIC1a, Syntabulin can pass the cell immunofluorescence staining experiments with more ASIC2a. through PICK1, and observe that Syntabulin can be Co located by PICK1 and ASIC2a. Without PICK1, Syntabulin and ASIC2a do not exist co location.Syntabulin and ASIC1a without PICK1. (2) the results of high performance liquid chromatography mass spectrometry show that 19 proteins interact with PICK1 and 15 proteins are interacted with ICA69. In the selected proteins, KIF2C and PICK1 have a direct phase Interaction, and the interaction between PICK1 and ICA69 to form a complex,.KIF2C and PICK1, does not enhance or weaken the binding between PICK1 and GluA2, and does not affect the co-cluster. of PICK1 and GluA2 in the body's physiological conditions, KIF2C is mainly expressed in the brain and testis, and in the brain and GluA2. In vitro validation, this combination is realized through PICK1. In the absence of PICK1, KIF2C can not interact with GluA2. In 293T cells, overexpression of KIF2C can reduce the level of GluA2 surface expression. When PICK1 and KIF2C are overexpressed, the surface expression level of GluA2 is further reduced. This phenomenon is caused by two factors. The cause, one is that KEF2C itself can degenerate microtubules, on the other hand, because PICK1 too body can also reduce the surface expression level of GluA2. In the cultured hippocampal neurons, KIF2C can co localize with PSD-95 and GluA2, and overexpression of KIF2C can also reduce the surface expression of GluA2. Conclusion: (1) our study indicates that synta Bulin can form complex through PICK1 and ASIC channel protein, and then change the surface expression level and overall expression level of ASIC2, and ultimately regulate the acid toxicity of neurons. (2) our study showed that KIF2C and PICK1 have direct interaction, and through this interaction, we further combine GluA2 to form a complex, eventually falling. Low GluA2 surface expression level is involved in AMPA receptor translocation.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R3411

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