本文主要研究内容
作者赵玲玲（2019）在《BHD综合征因子FLCN调控氨基酸转运蛋白PAT1在细胞内定位的机制研究》一文中研究指出：BHD综合征(Birt-Hogg-Dubésyndrome)是由FLCN(folliculin)基因突变所引起的一种动物遗传疾病。现有证据表明FLCN蛋白参与了多种生物过程,包括信号转导、细胞粘附、溶酶体和线粒体的生物合成、细胞能量代谢和营养稳态等,但缺失FLCN导致BHD综合征的具体原因仍不清楚。通过对FLCN蛋白三维结构的研究发现其C末端具有一个DENN结构域,这一结果提示FLCN可能通过与某些Rab家族蛋白成员发生作用进而调控细胞内的囊泡运输过程。然而由于对被运输的货物分子尚缺乏了解,FLCN在囊泡运输中的具体功能还有待实验证实。质子协同氨基酸转运蛋白1(proton-assisted amino acid transporter 1,PAT1,又称SLC36A1)是一个在哺乳动物体内广泛分布的跨膜氨基酸转运蛋白。在多种类型的细胞里,PAT1主要定位在溶酶体表面,PAT1还可以定位在其它部位,包括细胞膜、伪足,神经细胞的轴突和突触小泡等。在大鼠平滑肌细胞,PAT1甚至定位在细胞核。这些结果说明PAT1的定位受多种机制调控,PAT1在不同细胞里执行的生理功能与其定位有关。本研究组的前期研究发现,在人胚胎肾细胞HEK293中,FLCN抑制PAT1定位在溶酶体:当FLCN的表达被抑制时,PAT1募集到溶酶体表面,促进将溶酶体内储存的氨基酸释放进入细胞质。由于溶酶体内的氨基酸是激活营养代谢关键调控因子mTORC1的一个重要信号,溶酶体内氨基酸浓度下降导致mTORC1活性降低。在真核细胞里,膜蛋白在细胞内的定位主要通过囊泡运输来实现,我们推测PAT1可能是FLCN执行囊泡运输功能的一个货物分子。为了探索FLCN调控PAT1定位的机制,进而揭示FLCN在囊泡运输中的潜在功能,本研究以体外培养的HEK293细胞为主要研究模型,使用多种细胞生物学和生物化学技术,对PAT1在细胞内的运输途径以及FLCN调控PAT1定位的机制展开了深入研究,获得了如下主要发现:1.PAT1在多种囊泡上都有定位。免疫荧光染色结果显示,PAT1与多种囊泡标记蛋白存在共定位,包括LAMP1(溶酶体)、Rab5A(早期内体)、EEA1(早期内体)、TGN38(反式高尔基体)、Chmp5(多囊体)、Rab7A(晚期内体)、M6PR(晚期内体和循环内体)、Rab11A(循环内体)等。其中,PAT1主要定位在溶酶体,PAT1与其它囊泡也存在不同程度的共定位,这些发现提示PAT1可以沿着内吞(endocytosis)和再循环途径(endocytic recylcing)在细胞内运输。2.细胞内吞实验(internalization assay)显示位于细胞膜上的PAT1可以通过内吞进入细胞,并最终定位于溶酶体,说明PAT1可以通过间接运输方式被运输到溶酶体。3.FLCN抑制PAT1定位到溶酶体,促进PAT1定位到细胞膜,正向调控mTORC1。通过RNA干扰的方法抑制FLCN表达(FLCN-RNAi),或者敲除FLCN基因(FLCN-/-),导致PAT1的水平在溶酶体表面增多,在细胞膜上减少,细胞的mTORC1活性降低。缺乏氨基酸导致PAT1在溶酶体表面富集以及mTORC1活性下降;提高FLCN的表达量可以抑制PAT1在溶酶体募集,使细胞在氨基酸缺乏的情况下仍可维持较高水平的mTORC1活性。4.FLCN与回收运输调节因子Rab11A存在相互作用。免疫共沉淀(co-IP)实验结果显示,FLCN可以结合Rab5A、7A、11A,但FLCN与Rab7A和Rab11A结合较强,与Rab5A的结合较弱;FLCN通过DENN结构域与Rab11A结合。5.Rab11A抑制PAT1定位到溶酶体,促进PAT1定位到细胞膜,这与FLCN的作用一致。抑制Rab11A表达(Rab11A-RNAi),或者过表达失活形式的Rab11A突变体(S25N)导致PAT1的水平在溶酶体表面增多,在细胞膜上减少,mTORC1活性下降;反之,过表达活性形式的Rab11A(Q70L)抑制PAT1定位到溶酶体,并缓解过表达PAT1对mTORC1的抑制作用。6.体外GEF活性检测实验显示,FLCN不能直接调控Rab11A的活性,这与已有的报道一致。然而我们发现FLCN促进Rab11A与PAT1结合:在FLCN缺失的背景下,PAT1与Rab5A或Rab7A的结合不受影响,但PAT1与Rab11A的结合能力显著降低。根据以上发现,我们尝试得出两个结论:(1)FLCN是Rab11A的一个效应因子,FLCN通过DENN结构域与Rab11A结合;(2)FLCN通过将PAT1与Rab11A绑定促进PAT1向细胞膜(或高尔基体)方向运输,因而抑制PAT1向溶酶体方向运输。本研究揭示出FLCN与Rab11A之间的一种关系,发现了FLCN在回收运输中的新功能,为进一步阐明BHD综合征的发病机理提供了新线索。
Abstract
BHDzeng ge zheng (Birt-Hogg-Dubésyndrome)shi you FLCN(folliculin)ji yin tu bian suo yin qi de yi chong dong wu wei chuan ji bing 。xian you zheng ju biao ming FLCNdan bai can yu le duo chong sheng wu guo cheng ,bao gua xin hao zhuai dao 、xi bao nian fu 、rong mei ti he xian li ti de sheng wu ge cheng 、xi bao neng liang dai xie he ying yang wen tai deng ,dan que shi FLCNdao zhi BHDzeng ge zheng de ju ti yuan yin reng bu qing chu 。tong guo dui FLCNdan bai san wei jie gou de yan jiu fa xian ji Cmo duan ju you yi ge DENNjie gou yu ,zhe yi jie guo di shi FLCNke neng tong guo yu mou xie Rabjia zu dan bai cheng yuan fa sheng zuo yong jin er diao kong xi bao nei de nang pao yun shu guo cheng 。ran er you yu dui bei yun shu de huo wu fen zi shang que fa le jie ,FLCNzai nang pao yun shu zhong de ju ti gong neng hai you dai shi yan zheng shi 。zhi zi xie tong an ji suan zhuai yun dan bai 1(proton-assisted amino acid transporter 1,PAT1,you chen SLC36A1)shi yi ge zai bu ru dong wu ti nei an fan fen bu de kua mo an ji suan zhuai yun dan bai 。zai duo chong lei xing de xi bao li ,PAT1zhu yao ding wei zai rong mei ti biao mian ,PAT1hai ke yi ding wei zai ji ta bu wei ,bao gua xi bao mo 、wei zu ,shen jing xi bao de zhou tu he tu chu xiao pao deng 。zai da shu ping hua ji xi bao ,PAT1shen zhi ding wei zai xi bao he 。zhe xie jie guo shui ming PAT1de ding wei shou duo chong ji zhi diao kong ,PAT1zai bu tong xi bao li zhi hang de sheng li gong neng yu ji ding wei you guan 。ben yan jiu zu de qian ji yan jiu fa xian ,zai ren pei tai shen xi bao HEK293zhong ,FLCNyi zhi PAT1ding wei zai rong mei ti :dang FLCNde biao da bei yi zhi shi ,PAT1mu ji dao rong mei ti biao mian ,cu jin jiang rong mei ti nei chu cun de an ji suan shi fang jin ru xi bao zhi 。you yu rong mei ti nei de an ji suan shi ji huo ying yang dai xie guan jian diao kong yin zi mTORC1de yi ge chong yao xin hao ,rong mei ti nei an ji suan nong du xia jiang dao zhi mTORC1huo xing jiang di 。zai zhen he xi bao li ,mo dan bai zai xi bao nei de ding wei zhu yao tong guo nang pao yun shu lai shi xian ,wo men tui ce PAT1ke neng shi FLCNzhi hang nang pao yun shu gong neng de yi ge huo wu fen zi 。wei le tan suo FLCNdiao kong PAT1ding wei de ji zhi ,jin er jie shi FLCNzai nang pao yun shu zhong de qian zai gong neng ,ben yan jiu yi ti wai pei yang de HEK293xi bao wei zhu yao yan jiu mo xing ,shi yong duo chong xi bao sheng wu xue he sheng wu hua xue ji shu ,dui PAT1zai xi bao nei de yun shu tu jing yi ji FLCNdiao kong PAT1ding wei de ji zhi zhan kai le shen ru yan jiu ,huo de le ru xia zhu yao fa xian :1.PAT1zai duo chong nang pao shang dou you ding wei 。mian yi ying guang ran se jie guo xian shi ,PAT1yu duo chong nang pao biao ji dan bai cun zai gong ding wei ,bao gua LAMP1(rong mei ti )、Rab5A(zao ji nei ti )、EEA1(zao ji nei ti )、TGN38(fan shi gao er ji ti )、Chmp5(duo nang ti )、Rab7A(wan ji nei ti )、M6PR(wan ji nei ti he xun huan nei ti )、Rab11A(xun huan nei ti )deng 。ji zhong ,PAT1zhu yao ding wei zai rong mei ti ,PAT1yu ji ta nang pao ye cun zai bu tong cheng du de gong ding wei ,zhe xie fa xian di shi PAT1ke yi yan zhao nei tun (endocytosis)he zai xun huan tu jing (endocytic recylcing)zai xi bao nei yun shu 。2.xi bao nei tun shi yan (internalization assay)xian shi wei yu xi bao mo shang de PAT1ke yi tong guo nei tun jin ru xi bao ,bing zui zhong ding wei yu rong mei ti ,shui ming PAT1ke yi tong guo jian jie yun shu fang shi bei yun shu dao rong mei ti 。3.FLCNyi zhi PAT1ding wei dao rong mei ti ,cu jin PAT1ding wei dao xi bao mo ,zheng xiang diao kong mTORC1。tong guo RNAgan rao de fang fa yi zhi FLCNbiao da (FLCN-RNAi),huo zhe qiao chu FLCNji yin (FLCN-/-),dao zhi PAT1de shui ping zai rong mei ti biao mian zeng duo ,zai xi bao mo shang jian shao ,xi bao de mTORC1huo xing jiang di 。que fa an ji suan dao zhi PAT1zai rong mei ti biao mian fu ji yi ji mTORC1huo xing xia jiang ;di gao FLCNde biao da liang ke yi yi zhi PAT1zai rong mei ti mu ji ,shi xi bao zai an ji suan que fa de qing kuang xia reng ke wei chi jiao gao shui ping de mTORC1huo xing 。4.FLCNyu hui shou yun shu diao jie yin zi Rab11Acun zai xiang hu zuo yong 。mian yi gong chen dian (co-IP)shi yan jie guo xian shi ,FLCNke yi jie ge Rab5A、7A、11A,dan FLCNyu Rab7Ahe Rab11Ajie ge jiao jiang ,yu Rab5Ade jie ge jiao ruo ;FLCNtong guo DENNjie gou yu yu Rab11Ajie ge 。5.Rab11Ayi zhi PAT1ding wei dao rong mei ti ,cu jin PAT1ding wei dao xi bao mo ,zhe yu FLCNde zuo yong yi zhi 。yi zhi Rab11Abiao da (Rab11A-RNAi),huo zhe guo biao da shi huo xing shi de Rab11Atu bian ti (S25N)dao zhi PAT1de shui ping zai rong mei ti biao mian zeng duo ,zai xi bao mo shang jian shao ,mTORC1huo xing xia jiang ;fan zhi ,guo biao da huo xing xing shi de Rab11A(Q70L)yi zhi PAT1ding wei dao rong mei ti ,bing huan jie guo biao da PAT1dui mTORC1de yi zhi zuo yong 。6.ti wai GEFhuo xing jian ce shi yan xian shi ,FLCNbu neng zhi jie diao kong Rab11Ade huo xing ,zhe yu yi you de bao dao yi zhi 。ran er wo men fa xian FLCNcu jin Rab11Ayu PAT1jie ge :zai FLCNque shi de bei jing xia ,PAT1yu Rab5Ahuo Rab7Ade jie ge bu shou ying xiang ,dan PAT1yu Rab11Ade jie ge neng li xian zhe jiang di 。gen ju yi shang fa xian ,wo men chang shi de chu liang ge jie lun :(1)FLCNshi Rab11Ade yi ge xiao ying yin zi ,FLCNtong guo DENNjie gou yu yu Rab11Ajie ge ;(2)FLCNtong guo jiang PAT1yu Rab11Abang ding cu jin PAT1xiang xi bao mo (huo gao er ji ti )fang xiang yun shu ,yin er yi zhi PAT1xiang rong mei ti fang xiang yun shu 。ben yan jiu jie shi chu FLCNyu Rab11Azhi jian de yi chong guan ji ,fa xian le FLCNzai hui shou yun shu zhong de xin gong neng ,wei jin yi bu chan ming BHDzeng ge zheng de fa bing ji li di gong le xin xian suo 。
论文参考文献
论文详细介绍
论文作者分别是来自西北农林科技大学的赵玲玲，发表于刊物西北农林科技大学2019-07-11论文，是一篇关于质子协同氨基酸转运蛋白论文,相关结合蛋白论文,囊泡运输论文,信号通路论文,西北农林科技大学2019-07-11论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自西北农林科技大学2019-07-11论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：质子协同氨基酸转运蛋白论文; 相关结合蛋白论文; 囊泡运输论文; 信号通路论文; 西北农林科技大学2019-07-11论文;