Autophagy and LC3-associated phagocytosis in intestinal epithelial cells

摘要 肠上皮对维持机体与外界的屏障和膜至关重要。 贩运对于发展和维持这一障碍都是必不可少的。异常膜 贩运可导致屏障受损，导致肠道疾病，包括炎症性肠病。 自噬是一种特殊的膜运输过程，它允许细胞对 新陈代谢。此外，自噬对于维持上皮屏障和先天免疫也很重要。 应答，介导细胞内病原体的分离和降解。此外，与LC3相关的 吞噬作用(LAP)对于病原体的吸收和降解是重要的，而这一途径的功能障碍是 与高度炎症有关。重要的是，自噬和LAP途径中的蛋白质变体 与炎症性肠病(IBD)，特别是克罗恩病的易感性增加有关， 尽管这种联系背后的分子机制仍然不完全清楚。MAMDC4是 一种完整的膜蛋白，定位于肠上皮的内吞体内。删除MAMDC4 损害肠道肠道细胞的形态，RNAseq研究表明MAMDC4- 在IBD中受到调节。在以前的工作中，我们发现MAMDC4与小的GTP酶Rab14相互作用。在……里面 专业吞噬细胞，Rab14被招募到吞噬小体并阻止吞噬小体-溶酶体融合，但其 在肠道上皮细胞中的作用尚不清楚。在我们的初步数据中，我们显示RAB 14存在于 自噬小体和Rab14和MAMDC4都存在于含有入侵细菌的膜上。 此外，MAMDC4的缺失会导致自噬和溶酶体标志物在肾小管上的聚集 膜。这些结果表明，MAMDC4和Rab14在维持粘膜的分子网络中起作用 通过控制自噬或圈内免疫。在这项提案中，我们将使用肠上皮细胞进行培养， 器官培养和患者衍生的器官以确定自噬和/或LAP在肠道中的作用 动态平衡。

Abstract The intestinal epithelium is vital to maintain the barrier between the body and the outside world, and membrane trafficking is essential for both the development and maintenance of this barrier. Abnormal membrane trafficking can result compromised barrier leading to intestinal disease, including inflammatory bowel disease. Autophagy is a specialized membrane trafficking process that allows cells to respond to changes in metabolism. In addition, autophagy is important for maintenance of the epithelial barrier and the innate immune response, mediating the isolation and degradation of intracellular pathogens. Furthermore, LC3-associated phagocytosis (LAP) is important for uptake and degradation of pathogens, and dysfunction of this pathway is associated with hyperinflammation. Importantly, variants of proteins in the autophagic and LAP pathways have been linked to increased susceptibility to Inflammatory Bowel Disease (IBD), and particularly Crohn’s disease, although the molecular mechanisms that underlie this connection remain incompletely understood. MAMDC4 is an integral membrane protein that localizes to endosomes of the intestinal epithelium. Deletion of MAMDC4 compromises intestinal enterocyte morphology, and RNAseq studies have indicated that MAMDC4 is down- regulated in IBD. In previous work, we found that MAMDC4 interacts with the small GTPase Rab14. In professional phagocytes, Rab14 is recruited to phagosomes and prevents phagosome-lysosome fusion but its role in the intestinal epithelium is unknown. In our preliminary data, we show that Rab 14 is present on autophagosomes and both Rab14 and MAMDC4 are present on membranes containing invading bacteria. Furthermore, deletion of MAMDC4 results in accumulation of autophagy and lysosomal markers on tubular membranes. These results suggest that MAMDC4 and Rab14 act in a molecular network to maintain mucosal immunity through control of autophagy or LAP. In this proposal we will use intestinal epithelial cells in culture, organoid culture, and patient-derived organoids to define the role of autophagy and/or LAP in intestinal homeostasis.