Influence of mannose receptor ubiquitination and TLR signaling on antigen translocation from endosomes into the cytoplasm for cross-presentation

对于交叉呈递，抗原需要从内体隔室运输到细胞质，这一过程只有部分了解。在这项赠款申请的第一阶段，我们证明了甘露糖受体(MR)的多泛素化是这种抗原运输所必需的，MR是一种内吞受体，专门针对其配体进行交叉呈递。MR的多泛素化介导了p97的募集，p97是一种为抗原转位提供能量的酶，它向内膜募集。此外，我们发现TSG101是MR多泛素化、p97募集和抗原转位到胞浆的主要负抑制因子，因此是交叉递呈的重要调节因子。本课题组以前的工作表明，在抗原转运到细胞质和蛋白酶体降解后，抗原源性多肽通过内体TAP转移到相同的内体，在那里发生MHC I分子的装载。与内质网中MHC I的呈现相比，这种空间分离意味着在这些内体附近使用了一组不同的蛋白酶体。由于p97在内质网错位的过程中向内质网招募蛋白酶体，我们想在本提案的第二部分探讨p97是否介导蛋白酶体亚单位也在内质体膜上的招募，以及这种招募是否对交叉呈现是必要的。

For cross-presentation, antigens need to be transported from an endosomal compartment into the cytoplasm, a process that is only partially understood. In a first phase of this grant application, we demonstrated that poly-ubiquitination of the Mannose Receptor (MR), an endocytic receptor that targets its ligands specifically towards cross-presentation, is required for such antigen transport. Poly-ubiquitination of the MR mediated the recruitment of p97, an enzyme that provided the energy for antigen translocation, towards the endosomal membrane. Additionally, we identified TSG101 as a dominant negative inhibitor of MR poly-ubiquitination, p97 recruitment and antigen translocation into the cytosol, and hence as an important regulator of cross-presentation. Previous work by our group demonstrated that, after antigen transport into the cytoplasm and proteasomal degradation, antigenderived peptides are translocated by endosomal TAP into the same endosomes, where loading onto MHC I molecules occurs. Such spatial separation compared to MHC I presentation in the ER implies the use of a distinct set of proteasomes in direct proximity to such endosomes. Since p97 during dislocation at the ER recruits proteasomes towards the ER membrane, we would like to investigate in the second part of this proposal whether p97 mediates the recruitment of proteasome subunits also at the endosomal membrane and whether such recruitment is essential for cross-presentation.