Cystin, a lipid raft and cilia-associated protein in PKD

胱氨酸，一种 PKD 中的脂筏和纤毛相关蛋白

基本信息

批准号：
6826566
负责人：
Lisa M Guay-Woodford
金额：
$ 33.32万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-03-15 至 2009-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Primary cilia are dynamic, complex structures that contain >250 proteins, including several polycystic kidney disease (PKD)-related proteins. In renal epithelial cells, the primary apical cilium appears to be a major effector of differentiation signals and to play a critical role in PKD pathogenesis. Recent in vitro studies demonstrate that the primary cilium acts as a cellular sensor, transducing apical mechanical signals through a polycystin-1/polycystin-2-dependent Ca++ signaling pathway. However, the precise mechanisms involved in cilia formation, stabilization, and signal transduction are not well-defined and even less is known about how these cilia-associated proteins are targeted to cilia and functionally assembled. We have identified Cys1 as the disease-gene in cpk mice; demonstrated that its novel protein product, cystin, localizes to the primary apical cilium; and determined that cystin fractionates with lipid rafts through an N-terminal domain, probably the predicted N-myristoylation/ polybasic motif. We hypothesize that cystin traffics to the primary cilium via lipid raft-mediated mechanisms, associates with the cilial membrane, and serves as part of the molecular framework that stabilizes the microtubular scaffold of the ciliary axoneme. Using a suite of stably transfected cell lines that express wild-type cystin and various truncation mutants as GFP-tagged fusion proteins, we have determined that the N-terminal domain is necessary but not sufficient for targeting cystin to cilia and a second, novel signal is required. Since cystin is expressed at low levels and no functional assays currently exist, we have developed an innovative set of strategies to further characterize this novel protein and its intracellular trafficking itinerary as first steps toward defining its function. Specifically, in this proposal, we will: 1) Determine whether cystin tagged with green fluorescent protein (cystin-GFP) rescues the cpk phenotype and targets correctly to the primary cilium of renal epithelia in vivo; 2) Characterize cystin with respect to the predicted N-myristoylation site, putative cilia-targeting signals, and putative interacting partners; and 3) Examine the dynamics of cystin intracellular trafficking to the primary apical cilium. The central hypotheses underlying the proposed studies are that defects in primary cilia function impair the terminal phases of renal tubulo-epithelial differentiation and the epithelial response to this developmental arrest is cyst formation. Therefore, primary apical cilium represents a new focal point for dissecting the complex mechanisms involved in renal cystic disease and ultimately, perhaps a new target for therapeutic interventions.

描述（由申请人提供）：初级纤毛是动态的、复杂的结构，含有超过 250 种蛋白质，包括几种多囊肾病 (PKD) 相关蛋白质。在肾上皮细胞中，初级顶端纤毛似乎是分化信号的主要效应器，并在 PKD 发病机制中发挥关键作用。最近的体外研究表明，初级纤毛充当细胞传感器，通过多囊蛋白-1/多囊蛋白-2 依赖性 Ca++ 信号通路转导顶端机械信号。然而，纤毛形成、稳定和信号转导所涉及的精确机制尚不明确，对于这些纤毛相关蛋白如何靶向纤毛并进行功能性组装更是知之甚少。我们已经确定Cys1是cpk小鼠的疾病基因；证明其新型蛋白质产品胱氨酸定位于初级顶端纤毛；并确定胱氨酸通过 N 末端结构域与脂筏分离，这可能是预测的 N-肉豆蔻酰化/多元基序。我们假设胱氨酸通过脂筏介导的机制运输到初级纤毛，与纤毛膜结合，并作为稳定纤毛轴丝微管支架的分子框架的一部分。使用一套表达野生型胱氨酸和各种截短突变体作为 GFP 标记融合蛋白的稳定转染细胞系，我们确定 N 末端结构域是必需的，但不足以将胱氨酸靶向纤毛和第二个新信号是必须的。由于胱氨酸的表达水平较低且目前不存在功能测定，因此我们开发了一套创新的策略来进一步表征这种新型蛋白质及其细胞内运输行程，作为定义其功能的第一步。具体而言，在本提案中，我们将： 1）确定绿色荧光蛋白标记的胱氨酸（胱氨酸-GFP）是否可以挽救cpk表型并在体内正确靶向肾上皮细胞的初级纤毛； 2) 表征胱氨酸的预测 N-肉豆蔻酰化位点、假定的纤毛靶向信号和假定的相互作用伴侣； 3) 检查胱氨酸细胞内运输至初级顶端纤毛的动态。拟议研究的核心假设是初级纤毛功能的缺陷损害了肾小管上皮分化的终末期，并且上皮对这种发育停滞的反应是囊肿形成。因此，初级顶端纤毛代表了剖析肾囊性疾病复杂机制的新焦点，并最终可能成为治疗干预的新目标。