神經(jīng)生長(zhǎng)因子（NGF）結(jié)合細(xì)胞表面受體p75NTR （p75神經(jīng)營(yíng)養(yǎng)素受體）和TrkA （酪氨酸蛋白激酶A）后介導(dǎo)了細(xì)胞分化、細(xì)胞生存、凋亡、增殖和侵襲等多個(gè)重要的生理病理過(guò)程. TrKA能與細(xì)胞內(nèi)多個(gè)蛋白質(zhì)相互作用,但是由于NGF信號(hào)通路的復(fù)雜性,現(xiàn)在仍有必要發(fā)現(xiàn)與之相互作用的蛋白質(zhì)以更準(zhǔn)確地了解NGF信號(hào)通路.本研究中我們通過(guò)酵母雙雜交的方法篩選到了一個(gè)新的與TrKA相互作用的蛋白質(zhì)——真核生物翻譯起始因子4A1 （eIF4A1）,然后通過(guò)谷胱甘肽巰基轉(zhuǎn)移酶融合蛋白沉降實(shí)驗(yàn)（GST-pull-down）和免疫共沉淀實(shí)驗(yàn)（Co-IP）證明了TrkA和eIF4A1的相互作用.此外NGF能夠增強(qiáng)TrkA和eIF4A1的相互作用.在鑒定相互作用位點(diǎn)實(shí)驗(yàn)中,我們發(fā)現(xiàn)eIF4A1的氨基端結(jié)構(gòu)域和TrkA的TK結(jié)構(gòu)域參與了相互作用. TrkA和e IF4A1共定位在細(xì)胞膜上. NGF能夠引起TrkA與泛素蛋白63位的賴氨酸連接,而eIF4A1與TrkA相互作用后能夠抑制TrkA與泛素蛋白63位的賴氨酸連接.綜上,得出結(jié)論 e IF4A1通過(guò)與TrkA相互作用抑制其泛素化調(diào)控NGF信號(hào)通路. 

【文章來(lái)源】：生物化學(xué)與生物物理進(jìn)展. 2019,46(08)北大核心SCICSCD

【文章頁(yè)數(shù)】：11 頁(yè)

【文章目錄】：
1 Methods
    1.1 Yeast two hybrid screening
    1.2 Cell culture, transfection and NGF stimulation
    1.3 Plasmids, antibodies and reagents
    1.4 GST pull-down assay
    1.5 Co-immunoprecipitation and immunoblotting
    1.6 Immunofluorescence assay
    1.7 Cytoplasmic and nuclear extraction
2 Results
    2.1 Identification of the novel association of TrkA and eIF4A1
    2.2 eIF4A1 binds to TrkA-ICD in vivo and in vitro
    2.3 Mapping the interacting domain between TrkA and eIF4A1
    2.4 Membrane co-localization of TrkA and eIF4A1
    2.5 NGF stimulated TrkA Lys 63-linked polyubiquitination and eIF4A1 repressed TrkApolyubiquitination
3 Discussion



本文編號(hào)：3134958