甘露糖受体K48多泛素化在乙酰胆碱N型受体调控CD8+ DCs交叉提呈中的作用研究

The translocation of endocytotic antigens to endosomes and endosomal recruitment of Sec61/p97 mediated by mannose receptor (MR) ubiquitination are the key steps of dendritic cells (DCs) cross-presentation. Our previous studies showed that the treatment with nicotinic acetylcholine receptor (nAchR) ligand promotes DCs ability of cross presentation and increased the K48 ubiquitination of MR. But, the role of K48 MR ubiquitination in nAchR increased cross presentation is still unclear. In this project, the effect of nicotine on MR ubiquitination was firstly determinated with co-immunoprecipitation assay. Then, using ubiquitination inhibitor, K48 polyubiquitin chain, and the single mutation of MR, the level of K48 polyubiquitination of MR were regulated. Thirdly, the effects of ubiquitination, K48 ubiquitination, and single mutation of K48 ubiquitination of MR on the translocation of endocytolic antigen towards endosomes, and the endosomal recruitment of Sec61/p97 were further explored by flow cytometric assay or confocal microscope respectively. Fourthly, the effect of K48 ubiquitination of MR on CD8+DCs cross presentation-dependent cross-priming was investigated by enzyme-linked immunospot assay (Elispot) and peptide-MHC tetramer assay respectively. The key point of our project is to explore the mechanisms of nAchR regulating cross-presentation, which could provide fundamental theory support for CD8+DCs clinical use to deal with tumor and viral infection.

甘露糖受体(MR)介导抗原内体转位及内体招募Sec61/p97是DC交叉提呈的关键。乙酰胆碱N型受体(nAchR)可促进MR发生K48泛素化并增强交叉提呈，但K48泛素化MR促交叉提呈的作用不清。课题从MR K48泛素化入手，自抗原内体转位和内体招募Sec61/p97角度，以免疫共沉淀、泛素化酶抑制剂、K48突变泛素链、单泛素化位点突变为手段；流式细胞术检测交叉提呈复合物，共聚焦显微镜观察交叉提呈复合物、抗原、Sec61/p97与内体共定位；免疫斑点实验和抗原肽-MHC分子四聚体技术检测抗原特异性CD8+T细胞为指标；发现K48泛素化MR及单位点K48泛素化MR在nAchR调控CD8+DCs抗原内体转位、内体招募Sec61/p97及交叉致敏中作用；阐明1410、1411、1441位点K48泛素化MR在nAchR调控CD8+DCs交叉提呈中作用，为nAchR调控DCs临床应用提供理论依据。

甘露糖受体(MR)介导抗原内体转位及内体招募Sec61/p97是DC交叉提呈的关键。课题从MR K48泛素化入手，自内体招募Sec61/p97角度，检测交叉提呈复合物、抗原—Sec61/p97与内体共定位、抗原特异性CD8+T细胞等交叉致敏。我们的工作主要聚焦于以下几个方面：.1. 探究尼古丁上调MR机制；检测尼古丁对MR泛素化的调控；观察OVA内体定位、p97/Sec61内体转位及交叉提呈产物的形成、ELISPOT检测交叉致敏。发现：尼古丁调控MR泛素化水平及泛素化方式的规律，探究K48泛素化MR在尼古丁调控外源性抗原内体定位及内质网相关蛋白内体转位中的作用。.2. 用OVA负载DC，检测Akt/NF-κB通路活性、Akt、IKKα、IKKβ激酶在内体转位、激酶对抗原交叉提呈、内质网相关蛋白Sec61内体招募影响。显示：抗原通过内体Akt/IKKα/IKKβ信号体形成而增强DC交叉提呈；Akt通过IKKα/β调控NF-κB信号；Akt、IKKα/β调节Rab5与Sec61α和Sec61β互作。.3. 筛选MR K48泛素化E3连接酶；检测C-Cbl、Cbl-c在内体定位及与MR的互作、C-Cbl及Cbl-c沉默对MR的K48泛素化及交叉提呈影响。显示：尼古丁促进C-Cbl和Cbl-c表达、增强其磷酸化、增加其至内体定位并促进其与MR的互作。发现C-Cbl和Cbl-c调控尼古丁对MR K48泛素化作用及交叉提呈。.科学意义： 发现K48泛素化MR在nAchR调控DC抗原内体转位、内体招募Sec61/p97及交叉致敏作用；阐明nAchR促进MR发生K48泛素化并增强交叉提呈机制，为nAchR调控DC临床应用提供理论依据。发现了MR K48泛素化的关键E3连接酶，完善了尼古丁促进MR的K48泛素化并增强DC交叉提呈的机制。