Intracellular transport of proteins containing di-arginine endoplasmic reticulum-retention motifs

MHC II类分子（MHCII）的细胞内运输代表了一个特殊的挑战，因为它们必须穿过许多不同的区室并与各种蛋白质相互作用，以最终在质膜上实现其功能。不变链（CD 74; Ii）分子伴侣协助MHCII折叠并运输至内吞途径。在人类中，两种Ii亚型在其胞质尾上显示二精氨酸基序（RxR）。RxR序列作为内质网（ER）保留基序，已被证明在蛋白质质量控制中发挥关键作用。Ii的RxR基序的作用仍然模糊不清，其被MHCII“掩蔽”以允许ER流出的机制也不清楚。因此，我们建议进一步了解跨亚细胞区室的蛋白质运输的基础知识，并记录参与Ii和MHCIIs功能的蛋白质网络。 最近的进展我们采用了一种新的邻位连接测定依赖的生物素识别（BioID）测定，以确定参与MHCIIs和Ii的细胞内运输的蛋白质-蛋白质相互作用。为此，我们利用CRISPR/Cas基因组工程技术在B淋巴母细胞系中对Ii基因进行了克隆。我们的研究结果表明，Ii的支架作用的协会的MHC II类分子是同种异型依赖。这些Ii缺陷型B细胞系使我们能够用Ii和/或MHCII的各种同种型和突变体重建外源性抗原呈递途径。 具体目的1：鉴定调节MHCII细胞内转运的分子。 我们将鉴定在没有Ii分子伴侣的情况下，在B细胞和上皮细胞中穿过MHCII通路的蛋白质。 具体目的2：表征Ii和MHCII在复合物转运中的相互作用。 我们将用Ii的不同亚型重建Ii阴性细胞，并研究与RxR ER保留基序发生的特异性相互作用。 特定目的3：表征MHCII/Ii运输和抗原加工的关键调节因子的功能。 我们将通过调节表达MHCII和Ii的B细胞或上皮细胞中蛋白质的表达（mRNA敲低或cDNA过表达）来验证目的1和2中鉴定的相互作用物的重要性。将评估对抗原呈递至T细胞的影响。 研究方法：我们将使用分子生物学（定点诱变，邻位连接试验），细胞生物学（共聚焦显微镜和流式细胞术）和生物化学（蛋白质印迹，蛋白质纯化和质谱）技术的组合来解剖和映射Ii亚型，MHCII及其合作伙伴之间发生的相互作用。 影响：我们的研究结果将进一步我们的蛋白质分选一般的理解，并将提供线索Ii的细胞质基序在其自身的细胞内运输和控制的命运MHCIIs的作用。

RATIONALE The intracellular trafficking of MHC class II molecules (MHCIIs) represents a particular challenge as they must traverse many different compartments and interact with various proteins to finally fulfil their functions at the plasma membrane. The invariant chain (CD74; Ii) chaperone assists MHCIIs in their folding and trafficking to the endocytic pathway. In humans, two Ii isoforms display a di-arginine motif (RxR) on their cytoplasmic tail. The RxR sequence acts as an endoplasmic reticulum (ER) retention motif and it has been shown to play a key role in protein quality control. The role of Ii's RxR motif remains nebulous and the mechanism by which it is "masked" by MHCIIs to allow ER egress is ill-defined. Thus, we propose to further our understanding of the basics of protein trafficking across sub-cellular compartments and to document the protein networks involved in the functions of Ii and MHCIIs. Recent Progress We adapted a new proximity ligation assays-dependent biotin identification (BioID) assay to identify protein-protein interactions involved in the intracellular trafficking of MHCIIs and Ii. To this end, we have used the CRISPR/Cas genome engineering technology in B lymphoblastic cell lines to inactivate the Ii gene. Our results demonstrate that the scaffolding role of Ii on the association of MHC class II molecules is allotype-dependent. These Ii-deficient B cell lines allow us to reconstitute the exogenous antigen presentation pathway with various isoforms and mutants of Ii and/or MHCIIs. SPECIFIC AIM 1: To identify molecules regulating MHCII intracellular transport. We will identify the proteins that across the path of MHCIIs in B cells and epithelial cells in the absence of the Ii chaperone. SPECIFIC AIM 2: To characterize the interplay between Ii and MHCIIs in the transport of the complex. We will reconstitute Ii-negative cells with the different isoforms of Ii and study the specific interactions taking place with the RxR ER retention motif. SPECIFIC AIM 3: To characterize the function of key regulators of MHCII/Ii trafficking and antigen processing. We will validate the importance of the interactors identified in aims 1 and 2 by modulating the expression (mRNA knockdown or cDNA overexpression) of the proteins in B cells or epithelial cells expressing MHCIIs and Ii. The impact on the presentation of antigens to T cells will be assessed. METHODOLOGY: We will use a combination of molecular biology (site-directed mutagenesis, proximity ligation assays), cell biology (confocal microscopy and flow cytometry) and biochemistry (Western blotting, protein purification and mass spectrometry) techniques to dissect and map the interactions occurring between Ii isoforms, MHCIIs and their partners. IMPACT: Our results will further our understanding of protein sorting in general and will give clues as to the role of Ii's cytoplasmic motifs in its own intracellular trafficking and in controlling the fate of MHCIIs.