本文選題：KIF2C + syntabulin　； 參考：《浙江大學(xué)》2015年博士論文

【摘要】：背景：PICK1 (Protein that interacts with C kinase 1)是一種周邊膜蛋白,是和蛋白激酶Ca (PKCa)相互作用的蛋白之一。PICKl可以通過其特殊的PDZ結(jié)構(gòu)域與很多膜蛋白發(fā)生結(jié)合,例如PICKl不僅可以與酸敏感離子通道ASIC (acid-sensing ion channels)通道蛋白相互結(jié)合,也可以和離子型谷氨酸受體AMPA (a-amino-3-hydroxy-5-methylisoxazoloe-4-propionic acid-type)受體的亞單位之一GluA2結(jié)合。在大多數(shù)情況下,PICKl可通過改變被結(jié)合蛋白的亞細(xì)胞轉(zhuǎn)運(yùn)或表面表達(dá)來調(diào)節(jié)蛋白的轉(zhuǎn)運(yùn)。憑借這些功能特點(diǎn),PICK1既可以調(diào)節(jié)酸敏感離子通道ASIC (acid-sensing ion channels)的表面表達(dá),也可以在突觸可塑性中調(diào)節(jié)AMPA受體的轉(zhuǎn)運(yùn)過程。已有的證據(jù)表明,PICK1可以與ASIC1a及ASIC2a直接結(jié)合,通過影響ASIC蛋白的表面表達(dá)來介導(dǎo)細(xì)胞的酸毒性,進(jìn)而參與調(diào)節(jié)細(xì)胞凋亡,但PICK1是通過何種方式介導(dǎo)細(xì)胞酸毒性的還不是很清楚。另外,在體內(nèi)生理情況下,ICA69 (Islet Cell Autoantigen 69 kDa)已被證實(shí)可以與大部分PICK1形成異源二聚體,并且他們二者的結(jié)合可以影響AMPA受體的分布。然而,PICK1和ICA69調(diào)節(jié)AMPA受體轉(zhuǎn)運(yùn)的具體分子機(jī)制還不明確。我們的實(shí)驗(yàn)結(jié)果顯示：syntabulin可以與PICK1直接結(jié)合,并通過PICK1影響ASIC2的表面表達(dá)水平,最終參與調(diào)節(jié)神經(jīng)元的酸毒性。同時(shí),KIF2C也可以與PICK1有直接的相互作用,并且能通過PICK1和ICA69以及AMPA受體形成復(fù)合物,進(jìn)而影響AMPA受體的膜表面表達(dá)。目的：篩選出與PICK1有直接相互作用的蛋白,并分析其功能,一方面探究syntabulin與PICK1的相互作用是否會(huì)調(diào)節(jié)神經(jīng)元的凋亡,另一方面探索KIF2C與PICK1的相互作用是否會(huì)影響AMPA受體的分布,在現(xiàn)有的基礎(chǔ)上進(jìn)一步探明ASIC及AMPA受體轉(zhuǎn)運(yùn)的分子機(jī)制,期望最終能夠回答ASIC受體調(diào)節(jié)神經(jīng)元凋亡的分子機(jī)制及AMPA受體上膜、下膜的動(dòng)態(tài)過程,為細(xì)胞凋亡和學(xué)習(xí)記憶的基本原理提供理論基礎(chǔ)。方法：我們利用酵母雙雜交、免疫共沉淀,高效液相層析質(zhì)譜篩選出與PICK1有相互作用的目標(biāo)蛋白；利用蛋白免疫印跡,免疫細(xì)胞化學(xué)進(jìn)一步驗(yàn)證篩選出的目標(biāo)蛋白syntabulin和KIF2C;利用激光共聚焦顯微鏡和表面生物素化實(shí)驗(yàn)一方面觀察syntabulin對(duì)ASIC受體表面表達(dá)及細(xì)胞凋亡的影響,另一方面觀察KIF2C對(duì)AMPA受體表面表達(dá)的影響。結(jié)果：(1)研究發(fā)現(xiàn),在培養(yǎng)的海馬神經(jīng)元中敲減syntabulin后,酸性刺激條件下會(huì)引起神經(jīng)元的凋亡。表面生物素化結(jié)果顯示,在培養(yǎng)的海馬神經(jīng)元中敲減syntabulin后,ASIC2的表面表達(dá)水平降低,同時(shí)ASIC2的總體表達(dá)永平升高。體外的免疫共沉淀顯示,syntabulin可以通過PICK1與ASIC1a,ASIC2a形成復(fù)合物,但與ASIC1a相比,syntabulin能夠通過PICK1結(jié)合更多的ASIC2a。通過細(xì)胞免疫熒光染色實(shí)驗(yàn),觀察到syntabulin能夠通過PICK1與ASIC2a共定位,而在沒有PICK1時(shí),syntabulin與ASIC2a并不存在共定位。syntabulin與ASIC1a在有沒有PICK1的情況下,都不會(huì)存在共定位。(2)高效液相層析質(zhì)譜實(shí)驗(yàn)結(jié)果顯示,19個(gè)蛋白與PICK1有相互作用,15個(gè)蛋白與ICA69有相互作用。在篩選到的蛋白中,KIF2C與PICK1有直接的相互作用,并且能夠通過PICK1和ICA69形成一個(gè)復(fù)合物。KIF2C和PICK1之間的相互作用不會(huì)增強(qiáng)或減弱PICK1與GluA2之間的結(jié)合,并且也不會(huì)影響PICK1與GluA2形成的co-cluster。在體內(nèi)生理?xiàng)l件下,KIF2C主要在大腦和睪丸中表達(dá),并且在大腦中能夠與GluA2結(jié)合。經(jīng)體外驗(yàn)證,這種結(jié)合是通過PICK1實(shí)現(xiàn)的,在沒有PICK1的情況下,KIF2C不能與GluA2有相互作用。在293T細(xì)胞中,過表達(dá)KIF2C可以降低GluA2表面表達(dá)水平,當(dāng)PICK1和KIF2C共同過表達(dá)時(shí),GluA2的表面表達(dá)水平進(jìn)一步降低,這種現(xiàn)象是由兩方面因素引起的,一方面是因?yàn)镵EF2C本身可以解聚微管,另一方面是因?yàn)镻ICK1太身也可以降低GluA2的表面表達(dá)水平。在體外培養(yǎng)的海馬神經(jīng)元中,KIF2C可以與PSD-95以及GluA2共定位,過表達(dá)KIF2C也可以降低GluA2的表面表達(dá)。結(jié)論：(1)我們的研究表明,syntabulin能夠通過PICK1與ASIC通道蛋白形成復(fù)合物,進(jìn)而改變ASIC2的表面表達(dá)水平及總體表達(dá)水平,最終調(diào)節(jié)神經(jīng)元的酸毒性。(2)我們的研究表明KIF2C與PICK1有直接的相互作用,并且通過這種相互作用進(jìn)一步結(jié)合GluA2,形成一個(gè)復(fù)合物,最終以降低GluA2表面表達(dá)水平的形式參與AMPA受體的轉(zhuǎn)運(yùn)。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R3411

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