AAK1相互作用蛋白的筛选及其调控细胞内整体翻译水平的研究.pdf

Vol.43 No.7 Jul.2023上海交通大学学报（医学版）JOURNAL OF SHANGHAI JIAO TONG UNIVERSITY(MEDICAL SCIENCE)http:/上海交通大学学报（医学版），2023，43（7）AAK1相互作用蛋白的筛选及其调控细胞内整体翻译水平的研究姜贵先1，2，3，胡荣贵4，吴皓1，2，31.上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科，上海 200011；2.上海交通大学医学院耳科学研究所，上海 200092；3.上海市耳鼻疾病转化医学重点实验室，上海 200092；4.中国科学院大学分子卓越创新中心，上海 200032摘要 目的探究衔接子相关蛋白激酶1（adaptor-associated protein kinase 1，AAK1）新的相互作用蛋白，以及除网格蛋白介导的内吞作用外AAK1介导的生物学功能。方法通过在HEK-293T细胞中分别外源性转染带有标签的AAK1载体与空白对照载体，利用标签特异性的琼脂糖凝胶进行免疫共沉淀（co-immunoprecipitation，CoIP），并联合质谱分析的方法获得潜在与AAK1相互作用的蛋白；通过CoIP初步验证质谱结果；通过荧光共聚焦成像观察AAK1与其潜在结合蛋白在细胞内的空间定位；通过体外纯化重组蛋白，利用谷胱甘肽巯基转移酶融合蛋白沉降实验（glutathione-S-transferase pulldown，GST Pulldown）进一步明确蛋白间是否为直接的相互作用；通过嘌呤霉素结合实验观察AAK1对于细胞内整体翻译水平的调控作用。结果质谱结果提示AAK1可能与以脆性X相关蛋白1（fragile X mental retardation syndrome-related protein 1，FXR1）、FXR2、脆性X智力低下蛋白（fragile X mental retardation protein 1，FMRP）三者为核心的一系列蛋白形成复合体。外源性转染AAK1-3xFLAG及FMRP-MYC质粒，利用抗FLAG琼脂糖凝胶富集AAK1-3xFLAG后，可以检测到FMRP-MYC的表达；利用内源性抗体进行CoIP，发现富集AAK1可以检测到FMRP的表达。荧光共聚焦成像显示EGFP-AAK1与mCherry-FMRP在细胞质中存在部分空间共定位。GST Pulldown显示FMRP可以直接沉淀HIS6-AAK1重组蛋白。嘌呤霉素结合实验显示相同时间内嘌呤霉素标记的细胞内新合成肽段数量与AAK1蛋白表达量呈正相关。结论AAK1与FMRP在细胞质内存在直接的相互作用，且AAK1可以提高细胞内的翻译水平。关键词衔接子相关蛋白激酶1；翻译；质谱；蛋白相互作用DOI10.3969/j.issn.1674-8115.2023.07.004 中图分类号Q291 文献标志码AScreening of AAK1 interaction proteins and its role in regulating global translation level in cellsJIANG Guixian1,2,3,HU Ronggui4,WU Hao1,2,31.Department of Otolaryngology-Head and Neck Surgery,Shanghai Ninth Peoples Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China;2.Ear Institute,Shanghai Jiao Tong University School of Medicine,Shanghai 200092,China;3.Shanghai Key Laboratory of Translation Medicine on Ear and Nose Diseases,Shanghai 200092,China;4.Center for Excellence in Molecular Cell Science,University of Chinese Academy of Sciences,Shanghai 200032,ChinaAbstract Objective To investigate noval interacting partners for adaptor-associated protein kinase 1(AAK1)and AAK1-mediated biological functions besides clathrin-mediated endocytosis.Methods The labeled AAK1 vector and the blank control vector were transfected in HEK-293T cells,and the potential AAK1 interacting proteins were obtained by co-immunoprecipitation with agar-specific gel and mass spectrometry.Further verifications were performed by CoIP and fluorescence-based imaging.Recombinant proteins were purified in vitro and the direct interaction between proteins were confirmed by glutathione-S-transferase pulldown(GST Pulldown)assay.The regulation of AAK1 in the global protein synthesis was explored by puromycin incorporation assay.Results Mass spectrometry results showed that AAK1 was associated with a series of proteins,including fragile X mental retardation syndrome-related protein 1(FXR1),FXR2 and fragile X mental retardation protein 1(FMRP).Enriching with anti-创新团队成果专栏基金项目 国家自然科学基金（81970872）；上海市耳鼻疾病转化医学重点实验室（14DZ2260300）；上海市临床重点专科建设项目（shslczdzk00802）；上海申康医院发展中心临床创新三年行动计划（SHDC2020CR1044B）。作者简介 姜贵先（1997），男，硕士生；电子信箱：。通信作者 吴皓，电子信箱：。Funding Information National Natural Science Foundation of China(81970872);Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases(14DZ2260300);Shanghai Key Clinical Specialty Construction Project(shslczdzk00802);Three-year Action Plan Program of Shanghai Shenkang Hospital Development Center(SHDC2020CR1044B).Corresponding Author WU Hao,E-mail:.8212023,43（7）上海交通大学学报（医学版）Vol.43 No.7 Jul.2023JOURNAL OF SHANGHAI JIAO TONG UNIVERSITY(MEDICAL SCIENCE)FLAG agarose gels after exogenous transfecting of AAK1-3xFLAG and FMRP-MYC plasmids,the expression of FMRP-MYC was detected.The expression of FMRP could also be detected by CoIP with endogenous AAK1 antibodies.Fluorescence-based imaging showed that they were spatially colocalized in the cytoplasm.GST Pulldown assay showed that FMRP could pulldown recombinant HIS6-AAK1 protein.Puromycin incorporation assay showed that in the same amount of time,the number of newly synthesized peptides labeled with puromycin was positively correlated with AAK1 protein expression.Conclusion AAK1 directly interacts with FMRP in cytoplasm and could up-regulate global protein synthesis level.Key words adaptor-associated protein kinase 1(AAK1);translation;mass spectroscopy;protein-protein interaction衔 接 子 相 关 蛋 白 激 酶 1（adaptor-associated protein kinase 1，AAK1）于 2002年被首次鉴定，并发 现 其 与 网 格 蛋 白 介 导 的 内 吞 作 用（clathrin-mediated endocytosis，CME）中的关键分子衔接蛋白2（adaptor protein2，AP2）结合，通过磷酸化AP2的亚基（AP2 complex subunit，AP2M1）对CME过程进行调控1-2。因此，AAK1也被称为AP2相关蛋白激酶1。目前，关于AAK1的生物学功能主要围绕CME 展开。研究发现，在 Wnt信号刺激下，AAK1通过加强AP2M1磷酸化程度，使低密度脂蛋白相关受体 6（low-density lipoprotein receptor related protein 6，LRP6）通过 CME 途径从细胞膜转移到细胞质内3；AAK1磷酸化内吞衔接蛋白Numb，使其从细胞膜通过 CME 途径进入内体4。除了磷酸化作用外，AAK1还以非磷酸化方式直接结合并稳定活化形式的神经源性基因座缺口同源蛋白 1（neurogenic locus notch homolog protein 1，Notch1），使其进入内体并增强其转录活性5。AAK1也参与疾病的发生和发展。超氧化物歧化酶1蛋白（superoxide dismutase 1，SOD1）突变（SOD1G85R）与肌萎缩侧索硬化有关，而 AAK1 仅与 SOD1G85R结合且不与 SOD1WT结合，并且AAK1蛋白水平在肌萎缩侧索硬化患者中较正常人低6。部分病毒通过 CME 途径进入人体细胞，而 AAK1参与调节丙型肝炎病毒7、登革热病毒8、狂犬病病毒9以及在全球范围内流行的新型冠状病毒10的进入与细胞间的扩散。因此，也有针对AA