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

理论基础MHC II类分子(MHCII)的细胞内运输是一个特殊的挑战，因为它们必须穿越许多不同的隔室，并与各种蛋白质相互作用，最终完成它们在质膜上的功能。不变链(CD74；II)伴侣协助MHCII折叠和运输到内吞途径。在人类中，两种II亚型在其细胞质尾部显示一个二精氨酸基序(RXR)。RXR序列作为内质网(ER)的保留基序，在蛋白质质量控制中起着关键作用。II的RXR基序的作用仍然模糊不清，它被MHCII“掩盖”以允许ER出口的机制也不明确。因此，我们建议进一步了解蛋白质跨亚细胞隔间运输的基本原理，并记录参与II和MHCII功能的蛋白质网络。最近的进展我们采用了一种新的邻近连接分析依赖的生物素鉴定(BioID)方法来鉴定参与MHCII和II细胞内转运的蛋白质-蛋白质相互作用。为此，我们利用CRISPR/Cas基因组工程技术在B淋巴母细胞系中灭活了II基因。我们的结果表明，II对MHC II类分子结合的支架作用是同种异型依赖的。这些II缺陷的B细胞系使我们能够用II和/或MHCII的各种异构体和突变来重建外源抗原提呈途径。特异性目的1：鉴定调控MHCII细胞内转运的分子。我们将在缺乏II分子伴侣的情况下，在B细胞和上皮细胞中鉴定穿过MHCII途径的蛋白质。特异性目的2：研究II和MHCII在复合体转运过程中的相互作用。我们将用不同的II亚型重建II阴性细胞，并研究与RXR ER保留基序发生的特定相互作用。特异性目的3：鉴定MHCII/II转运和抗原加工的关键调节因子的功能。我们将通过调节表达MHCIIs和II的B细胞或上皮细胞中蛋白质的表达(mRNA下调或cDNA过表达)来验证AIMS 1和2中确定的相互作用的重要性。将评估对T细胞提呈抗原的影响。方法：我们将结合分子生物学(定点突变、邻近连接分析)、细胞生物学(共聚焦显微镜和流式细胞术)和生物化学(Western blotting、蛋白质纯化和质谱学)技术来分析和绘制II亚型、MHCII及其伙伴之间的相互作用。影响：我们的结果将从总体上加深我们对蛋白质分选的理解，并将为II的细胞质基序在其自身的细胞内运输和控制MHCII的命运中所起的作用提供线索。

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.