本文選題：HIP1R　切入點：樹突發(fā)育　出處：《浙江大學》2016年博士論文　論文類型：學位論文

【摘要】：亨廷頓蛋白互作蛋白 1 相關(guān)蛋白(Huntingtin-interacting protein 1-related,HIP1R)、亨廷頓蛋白互作蛋白1(HIP1)以及在酵母中的同源蛋白Sla2p隸屬同一個蛋白家族,都含有三個相似的結(jié)構(gòu)域,即N末端的ENTH結(jié)構(gòu)域、中間部位的coiled-coil結(jié)構(gòu)域及C末端的talin-like結(jié)構(gòu)域。這些結(jié)構(gòu)域能分別與膜磷脂組分、網(wǎng)格蛋白(Clathrin)輕鏈以及F-肌動蛋白(F-actin)相互作用。近年來的研究表明,這些接頭蛋白的功能可能與內(nèi)吞體(Endosome)形成和轉(zhuǎn)運有關(guān),被歸類為內(nèi)吞接頭蛋白(Endocytotic adaptor),但具體功能及生物學意義并不清楚。迄今為止僅有少數(shù)有關(guān)HIP1R的研究報告,提示其可能與某些腫瘤的發(fā)生有關(guān),遺傳學分析提示其可能是帕金森疾病的相關(guān)基因。我們也注意到有研究表明HIP1R在腦內(nèi)各腦區(qū)均有較高水平的表達,其C末端還含有一個特有結(jié)構(gòu)域負責與另一個內(nèi)吞接頭蛋白—皮層蛋白(Cortactin)相互作用。另一方面,神經(jīng)元是形態(tài)上高度分化具有樹突和軸突長突起的細胞,具有復(fù)雜和精致的信號處理機制,包括通過活化受體內(nèi)吞形成信號內(nèi)體并在細胞內(nèi)轉(zhuǎn)運介導(dǎo)信號轉(zhuǎn)導(dǎo)的機制,因而進一步探討內(nèi)吞接頭蛋白HIP1R在神經(jīng)系統(tǒng)的作用并闡明其分子機制具有重要的意義。本博士學位論文采用免疫印跡、免疫組織和細胞化學、培養(yǎng)海馬神經(jīng)元的基因敲除、電生理等技術(shù)方法,研究發(fā)現(xiàn):(1)HIP1R在大鼠的各個腦區(qū)廣泛分布,不同發(fā)育階段海馬組織的出生后表達逐漸增加;海馬神經(jīng)元中有較高表達,呈顆粒狀廣泛分布,部分與PSD95共定位;(2)在shRNA敲減HIP1R表達的培養(yǎng)海馬神經(jīng)元中,發(fā)現(xiàn)其樹突分支數(shù)目及總長度明顯減少,分支復(fù)雜性明顯降低,樹突棘密度也明顯降低;在HIP1R過表達的神經(jīng)元結(jié)果與之相反,即樹突分支、總長度及樹突棘均增加;(3)采取遺傳挽救(Rescue)和顯性負向(Dominant negative)實驗發(fā)現(xiàn),含有與內(nèi)吞接頭蛋白皮層蛋白相互作用位點的HIP1R C末端是決定樹突發(fā)育的關(guān)鍵功能區(qū);(4)研究還發(fā)現(xiàn),在HIP1R表達敲減的神經(jīng)元中,AMPA受體和NMDA受體的表達明顯降低,與之相一致,PSD95的密度也明顯降低,微小興奮性突觸后電流(mEPSC)的電流峰值顯著性降低;而GABA受體表達及mIPSC并未發(fā)生改變;提示HIP1R表達的敲減選擇性損害了興奮性突觸的形成和功能。(5)在敲減HIP1R的培養(yǎng)的海馬神經(jīng)元中,表皮生長因子受體(EGFR)的早期內(nèi)吞過程受損,EGFR-ERK通路信號傳導(dǎo)受到破壞。綜合以上研究結(jié)果表明,作為內(nèi)吞接頭蛋白HIP1R在海馬神經(jīng)元的樹突生長發(fā)育以及興奮性突觸的形成過程中起著重要作用,其分子機制可能是HIP1R通過與皮層蛋白相互作用調(diào)節(jié)EGFR的內(nèi)吞過程,影響EGFR-ERK通路的信號傳導(dǎo)。
[Abstract]:Huntington protein interacting protein related protein 1 (Huntingtin-interacting protein, 1-related, HIP1R), Huntington protein interacting protein 1 (HIP1) and Sla2p homologous protein belong to the same family of proteins in yeast, there are three similar domains, namely ENTH domain N terminal, talin-like terminal coiled-coil domain domain and the middle part of the C. These domains respectively and membrane phospholipid components, clathrin light chain (Clathrin) and F- (F-actin) actin interaction. Recent studies show that these functions may be associated with endosomal adaptor proteins (Endosome) formation and transport, are classified as endocytosis joint protein (Endocytotic adaptor), but the specific function and biological significance is not clear. So far, only a few HIP1R related research reports, suggesting that it may occur with some tumor related genetic analysis. The may be associated with Parkinson disease. We also note that studies have shown that high level expression of HIP1R in different brain regions are in the brain, the C terminal also contains a unique domain responsible for another endocytic adaptor protein - protein interaction of cortex (Cortactin). On the other hand, neurons are morphological differentiation with dendrites and axons of cells with long processes, signal processing mechanism of the complex and sophisticated, including through activation of receptor endocytosis in the body and the formation mechanism of signal transport in the cells mediated signal transduction, thus further study has important significance and endocytic adaptor protein HIP1R to clarify the molecular mechanism in the nervous system the role of this doctoral dissertation. By Western blot, immunohistochemistry and immunocytochemistry in cultured hippocampal neurons, gene knockout, found electrophysiological study methods such as: (1) HIP1R in rats All regions are widely distributed in the hippocampus at different developmental stages after birth was increased; the higher the expression of hippocampal neurons in the granular, widely distributed, and the co localization of PSD95; (2) in shRNA on cultured hippocampal neurons reduced the expression of HIP1R, found that the dendritic branches and the total length was significantly reduced, branch of complexity is significantly reduced, the density of dendritic spines were significantly decreased; in the over expression of HIP1R neurons in the opposite, that is the total length of the dendritic branches, and dendritic spines were increased; (3) to save the genetic (Rescue) and dominant negative (Dominant negative) found that HIP1R and C terminal containing endocytic adaptor protein cortex protein interaction sites is a key function of determining region of dendritic development; (4) the study also found that the expression of HIP1R knockdown neurons, the expression of AMPA receptor and NMDA receptor significantly decreased, consistent with the PSD95. Is also significantly reduced, miniature excitatory postsynaptic currents (mEPSC) of the peak current decreased significantly; while the expression of GABA receptor and mIPSC did not change; prompt formation and function expression of HIP1R knockdown selective damage of excitatory synapses. (5) in HIP1R knockdown in cultured hippocampal neurons, epidermal growth factor receptor (EGFR) in the process of early swallowing impairment, EGFR-ERK signal pathway is damaged. The above research results show that plays an important role as an endocytic adaptor protein HIP1R on the growth of hippocampal neurons and dendritic development of excitatory synapses in the formation process of the molecular mechanisms of endocytosis may be adjusted by the EGFR and HIP1R cortex protein interaction, effects of signal transduction of EGFR-ERK pathway.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q42

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1 彭琳;內(nèi)吞接頭蛋白HIP1R在神經(jīng)元樹突發(fā)育和興奮性突觸形成中的作用[D];浙江大學;2016年

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