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

项目摘要 上皮细胞执行分泌和吸收功能，需要极化分布的 顶端和基底侧质膜上的转运体、受体和黏附分子。采办 而维持顶端-基底端的极性涉及质膜蛋白沿 生物合成和回收途径。心尖部和基底侧部的蛋白质如何转运到血浆的问题 几十年来，膜被分选在内体隔间一直是研究的中心。一个 蛋白质分选的重要机制是由网状蛋白接头蛋白AP-1B介导的，这是 上皮细胞。AP-1B以离散的分拣基序识别货物，最初被确定为 基侧排序。然而，申请人和其他人最近的研究确定，AP-1B也 调解根尖排序。此外，一些上皮细胞，如肾近端小管和视网膜色素 上皮性缺乏AP-1B。因此，在这些介质中必须存在其他极化排序的介体 上皮细胞。这一事实，以及我们对AP-1B介导的人口贩运的不稳定理解，证明有必要对传统的 上皮细胞极化蛋白质分选模型。填补这些知识空白将揭示机制 这可以被用来理解和治疗上皮生理受损的病理。这个 申请者的研究计划旨在表征AP-1B缺陷上皮细胞的极性机制和它们的 在组织组织、动态平衡和疾病中的作用。目前R35应用的重点将是上皮细胞 肾近端小管细胞和视网膜色素上皮细胞缺乏AP-1B。我们将研究以下方面的作用 一种以前在非极化细胞中具有特征的内体分类蛋白，分类Nexin-27(SNX27)和 极化运输中的囊泡相关膜蛋白(VAMP)。我们还将利用蛋白质组学和 高通量方法，以确定极化贩运的其他分子介体。我们将测试 已知的机制是否介导了生理上相关的表面受体--巨蛋白的顶端释放 表达于肾近端小管和视网膜色素上皮。R35融资机制是一个极好的 适合于实现这些目标，因为它旨在支持追求雄心勃勃和灵活的想法 开发申请者的研究和培训计划的意图，而不是单个项目。同时 随着时间的推移，完成这项建议将为未来在诊断、治疗和预防方面的进展奠定基础 因此，它为国家普通医学科学研究所的使命做出了直接贡献。

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.