FcRn-dependent sorting of IgG and IgG-opsinized antigens by epithelial cells

描述（由申请人提供）：本项目的总体目标是阐明IgG MHC i类fc3受体FcRn如何将IgG和IgG活化抗原转运进入和穿过肠道屏障。在上皮细胞中，FcRn转运IgG和相关IgG-复合物对宿主和共生菌群之间的对话，从而对粘膜表面的上皮维持、炎症反应和宿主防御产生重要影响。我们将验证以下假设：1)肠上皮细胞利用FcRn来中和IgG-活化的微生物和微生物产物，并引起炎症反应；2)与共/顶循环内体（极化上皮细胞特有的胞室）相关的特定细胞因子调节FcRn的转运；3) FcRn-细胞质尾部的膜近端两性基序感知（或诱导）膜曲率或表面电位，从而影响受体运输。AIM 1将描述IgG-opsonized颗粒如何将FcRn从典型的FcRn运输的转胞和再循环途径转移到降解途径。在这个分选步骤中，我们还将使用在FcRn细胞质尾部区域突变的FcRn同工异构体来研究FcRn细胞质尾部的结构-功能关系。AIM 2将跟进我们最近研究中提出的观点，表明小GTPase Rab25调节了指定胞分裂的分选步骤。我们的目标是识别和表征在极化细胞中调节FcRn运输的共/顶循环内体的细胞因子。待研究的蛋白包括Rab11a/Rab25效应蛋白FIP家族、Rab10、Rab8、ACAP1和囊泡复合物。我们将尝试使用酵母双杂交系统对fcrn相互作用蛋白进行无偏筛选，并且我们将检查顶端和基底侧膜SNAREs，以查看这些分子是否标记含有fcrn的囊泡，以便在极化细胞中进行矢量运输。AIM 3将专注于FcRn细胞质尾部的膜近端区域，以测试通过感知（或诱导）膜曲率或表面电位的两亲性1-螺旋结构域分选膜蛋白的新思路。我们提出，该基序的寡聚化解释了FcRn如何内化IgG-opsonized颗粒，并将运输途径从回收和转胞作用转变为降解隔间。公共卫生相关性：我们的目标是了解肠道与位于肠腔（腔）中庞大而复杂的微生物群和微生物产物相互作用的一种方式。肠道内的微生物与调节健康和疾病中的肠道功能密切相关，包括慢性炎症性肠病。在这里，我们研究肠道受体如何运输免疫球蛋白G（人类最主要的抗体形式）决定这种相互作用的结果。

DESCRIPTION (provided by applicant): The overarching goal of this project is to elucidate how the IgG MHC Class I-like Fc3-receptor FcRn transports IgG and IgG-opsonized antigens into and across the intestinal barrier. Trafficking of IgG and associated IgG- complexes by FcRn in epithelial cells has important consequences on the dialogue between host and commensal microflora, and thus on epithelial maintenance, inflammatory responses and host defense at mucosal surfaces. We will test the hypotheses: 1) that intestinal epithelial cells utilize FcRn to neutralize IgG- opsonized microbes and microbial products and to raise an inflammatory response; 2) that specific cellular factors associated with the common/apical recycling endosome, a compartment unique to polarized epithelial cells, regulate the trafficking of FcRn; and 3) that a membrane proximal amphipathic motif in the FcRn- cytoplasmic tail senses (or induces) membrane curvature or surface potential so as to affect receptor trafficking. AIM 1 will characterize how IgG-opsonized particles divert FcRn from the transcytotic and recycling pathways, which typify FcRn trafficking, into the degradative pathway. We also will examine structure-function relationships of the FcRn cytoplasmic tail in this sorting step using FcRn-isoforms mutated in its cytoplasmic tail domain. AIM 2 will follow-up on ideas raised in our recent studies indicating that the small GTPase Rab25 regulates a sorting step that specifies transcytosis. We aim to identify and characterize the cellular factors of the common/apical recycling endosomes that regulate FcRn trafficking in polarized cells. Proteins to be studied include the FIP family of Rab11a/Rab25 effectors, Rab10, Rab8, ACAP1, and the exocyst complex. We will attempt an unbiased screen for FcRn-interacting proteins using a yeast two-hybrid system, and we will examine the apical and basolateral membrane SNAREs to see if these molecules mark FcRn-containing vesicles for vectorial transport across polarized cells. AIM 3 will focus on the membrane proximal region of the FcRn cytoplasmic tail to test a new idea for sorting of membrane proteins by amphipathic 1-helical domains that sense (or induce) membrane curvature or surface potential. We propose that oligomerization of this motif explains how FcRn can internalize IgG-opsonized particles and switch pathways of trafficking away from recycling and transcytosis into degradative compartments. PUBLIC HEALTH RELEVANCE: We aim to understand one way that the intestine interacts with the vast and complex microflora and microbial products located in the intestinal lumen (cavity). Microbes in the intestinal lumen are strongly implicated in regulating intestinal function in health and disease, including the chronic inflammatory bowel diseases. Here, we study how an intestinal receptor for transporting immunoglobulin G (the most dominant form of antibodies in humans) dictates the outcome of this interaction.