Mechanisms of polarized protein sorting in AP-1B-deficient epithelia

项目摘要 上皮细胞进行分泌和吸收功能，需要极化分布的 顶侧和基底侧质膜上的转运蛋白、受体和粘附分子。采集 和维持顶-基底侧极性涉及质膜蛋白质的分选，沿着 生物合成和循环途径。顶侧和基底侧蛋白质如何转运到血浆的问题 几十年来，细胞膜在内体区室中被分选一直是研究工作的中心。一个 蛋白质分选的重要机制是由网格蛋白衔接蛋白AP-1B介导的，这是一个特征， 上皮细胞AP-1B在离散的分选基序上识别货物，它最初被鉴定为 基底外侧分选然而，申请人和其他人最近的研究已经确定AP-1B也 介导顶端分选。此外，部分上皮细胞如肾近端小管和视网膜色素 上皮组成性缺乏AP-1B。因此，极化分选的额外介质必须存在于这些细胞中。 上皮细胞这一事实，以及我们对AP-1B介导的贩运的不稳定理解，保证了对传统的 上皮细胞中极化蛋白分选的模型。填补这些知识空白将揭示机制 可以用来理解和治疗上皮生理学受损的病理。的 申请人的研究计划旨在表征AP-1B缺陷的上皮细胞中的极性机制及其 在组织组织、体内平衡和疾病中的作用。当前R35应用的重点将是上皮细胞 肾近端小管和视网膜色素上皮细胞缺乏AP-1B。我们将研究 一种先前在非极化细胞中表征的内体分选蛋白，分选连接蛋白-27（SNX 27）， 囊泡相关膜蛋白（VAMP）在极化运输。我们还将采用蛋白质组学， 高通量方法来鉴定极化运输的另外的分子介质。我们将测试 确定的机制是否介导Megalin（一种生理学相关的表面受体）的顶端递送 表达于肾近端小管和视网膜色素上皮。R35融资机制是一个很好的 适合实现这些目标，因为它旨在支持追求雄心勃勃和灵活的想法， 开发申请人的研究和培训计划的意图，而不是一个单独的项目。在同一 随着时间的推移，完成这一提案将为未来诊断、治疗和预防方面的进展奠定基础 因此，直接促进了国家普通医学科学研究所的使命。

Project Summary Epithelial cells carry out secretory and absorptive functions that require the polarized distribution of transporters, receptors and adhesion molecules at the apical and basolateral plasma membranes. Acquisition and maintenance of apical-basolateral polarity involves sorting of plasma membrane proteins along the biosynthetic and recycling pathways. The question of how apical and basolateral proteins in transit to the plasma membrane are sorted in endosomal compartments has been at the center of research effort for decades. An important mechanism of protein sorting is mediated by the clathrin adaptor protein AP-1B, which is a feature of epithelial cells. AP-1B recognizes cargo at discrete sorting motifs and it was initially identified as mediator of basolateral sorting. However, recent research by the applicant and others has determined that AP-1B also mediates apical sorting. Moreover, some epithelia like the kidney proximal tubule and the retinal pigment epithelium constitutively lack AP-1B. Therefore, additional mediators of polarized sorting must exist in these epithelia. This fact, and our fluid understanding of AP-1B-mediated trafficking warrants revisions of the traditional models of polarized protein sorting in epithelial cells. Filling these gaps in knowledge will reveal mechanisms that can be exploited to understand and treat pathologies where epithelial physiology is compromised. The applicant’s research program aims to characterize mechanisms of polarity in AP-1B-deficient epithelia and their role in tissue organization, homeostasis and disease. The focus of the current R35 application will be on epithelial cells of the kidney proximal tubule and retinal pigment epithelium, which lack AP-1B. We will study the roles of an endosomal sorting protein previously characterized in non-polarized cells, sorting nexin-27 (SNX27) and vesicle-associated membrane proteins (VAMPs) in polarized trafficking. We will also employ proteomics and high throughput approaches to identify additional molecular mediators of polarized trafficking. We will test whether the identified mechanisms mediate apical delivery of Megalin, a physiologically relevant surface receptor expressed in kidney proximal tubule and retinal pigment epithelium. The R35 funding mechanism is an excellent fit for achieving these goals, since it is intended to support the pursue of ambitious and flexible ideas with the intent of developing the applicant’s research and training program, rather than an individual project. At the same time, completing this proposal will lay out foundations for future advances in diagnosis, treatment, and prevention of diseases, therefore directly contributing to the mission of the National Institute of General Medical Sciences.