Disrupting FSGS1-associated Actin Assembly in Glomerulosclerosis

破坏肾小球硬化症中 FSGS1 相关肌动蛋白组装

• 批准号: 8479770
• 负责人: Vivian Tang
• 金额: $ 27.6万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8479770
• 关键词: Accounting; Actinin; Actins; Address; Adhesions; Adhesives; Adult; Biochemical; Biological Assay; Cell Adhesion Molecules; Cell physiology; Cell-Cell Adhesion; Cells; Child; Complex; Coupled; Cytoskeletal Modeling; Cytoskeleton; Development; Devices; Disease; Filtration; Focal Segmental Glomerulosclerosis; Generations; Genes; Goals; Hypertension; Imaging Techniques; In Vitro; Link; Maintenance; Membrane; Molecular; Mutation; Nephrotic Syndrome; Outcome; Pathology; Pathway interactions; Permeability; Proteins; Proteinuria; Regulation; Reporter; Research; Resolution; Role; Site; Translating; base; cell injury; crosslink; design; glomerular filtration; glomerulosclerosis; high throughput screening; inhibitor/antagonist; monolayer; mutant; novel; pressure; protein crosslink; public health relevance; reconstitution; research study; slit diaphragm; small hairpin RNA; therapeutic target

DESCRIPTION (provided by applicant): Interaction of cell-cell adhesion molecules with the actin cytoskeleton is critical for the maintenance and stability of the glomerular filtration barrir formed by the slit diaphragm. Disrupting this interaction is intimately coupled to the initiation o proteinuria and progression of nephrotic syndromes in general. Many forms of glomerulosclerosis including focal and segmental glomerulosclerosis (FSGS) are characterized by reorganization of the actin cytoskeleton resulting in loss of cell-cell adhesion and displacement of the slit diaphragm. Most studies had focused on global regulation of the actin cytoskeleton without considering the possibility that perturbing actin assembly directly at sites o cell-cell adhesion might be the key to disrupting slit diaphragm stability. Recently, we found that FSGS1/¿-actinin-4 is required for actin assembly at cell-cell contacts. Furthermore, a mutation of FSGS1/¿-actinin-4 K255E that links to a dominant form of FSGS specifically inhibits actin assembly at sites of cell-cell adhesion without compromising the overall actin cytoskeletal organization. Therefore, disruption of actin assembly locally at adhesive contacts might be a convergent pathway that leads to break down of the slit diaphragm filtration barrier. Given the complexity of the slit diaphragm, dissecting the structural-functional relationships of its molecular components would be essential to revealing the mechanism behind development of proteinuria and FSGS. Aim 1. Determine how FSGS1/¿-actinin-4 and its disease mutation regulate actin dynamics at membrane adhesion complex. We will use our newly developed reconstitution biochemical assays, in combination with actin reporter assays, to assess the role of FSGS1/¿-actinin-4 in actin assembly. First, we will define the molecular function of the wild-type FSGS1/¿-actinin-4. Second, we will analyze how to the disease mutant K255E inhibits actin assembly. Lastly, we will develop high throughput assays to screen for inhibitors of K255E mutation. Aim 2. Characterize FSGS1/¿-actinin-4-associated actin regulatory machine. We developed a novel target-based crosslinking to identify six proteins in complex with FSGS1/¿-actinin-4. We will dissect the molecular and cellular functions of these junctional components using in vitro biochemical and cellular approaches (shRNA and mutant expression) as well as high-resolution imaging techniques. Aim 3. Develop a novel "pressure chamber" apparatus to investigate the role of hydraulic pressure in the development of proteinuria. Actin assembly at cell-cell contacts is necessary for proper cell-cell adhesion to withstand normal and hypertensive pressure. We will use a new "pressure chamber" to perform functional analysis under different hydraulic pressure treatments to elucidate the causative role of disease susceptible genes in the initiation and progression of permeability barrier disruption.

描述（由申请人提供）：细胞-细胞粘附分子与肌动蛋白细胞骨架的相互作用对于缝隙隔膜形成的肾小球滤过屏障的维持和稳定性至关重要，破坏这种相互作用与蛋白尿的发生和进展密切相关。一般肾病综合征。多种形式的肾小球硬化症，包括局灶性和节段性肾小球硬化症。 (FSGS) 的特点是肌动蛋白细胞骨架重组，导致细胞间粘附丧失和狭缝隔膜移位。大多数研究都集中在肌动蛋白细胞骨架的整体调节上，而没有考虑直接扰乱细胞位点肌动蛋白组装的可能性。 -细胞粘附可能是破坏狭缝隔膜稳定性的关键。 FSGS1/¿ -actinin-4 是细胞与细胞接触处肌动蛋白组装所必需的。此外，FSGS1/¿ 的突变。 -actinin-4 K255E 与 FSGS 的主要形式连接，特异性抑制细胞-细胞粘附位点的肌动蛋白组装，而不损害整个肌动蛋白细胞骨架组织。因此，在粘附接触处局部破坏肌动蛋白组装可能是导致狭缝隔膜过滤屏障的分解鉴于狭缝隔膜的复杂性，剖析其分子成分的结构-功能关系将是对于揭示蛋白尿和 FSGS 的发生机制至关重要 目标 1. 确定 FSGS1/¿ -actinin-4 及其疾病突变调节膜粘附复合物的肌动蛋白动力学我们将使用我们新开发的重建生化测定结合肌动蛋白报告基因测定来评估 FSGS1/¿ -actinin-4 在肌动蛋白组装中首先，我们将定义野生型 FSGS1/¿ -actinin-4。其次，我们将分析疾病突变体 K255E 如何抑制肌动蛋白组装。最后，我们将开发高通量测定法来筛选 K255E 突变的抑制剂。目标 2. 表征 FSGS1/¿ -actinin-4 相关肌动蛋白调节机器。我们开发了一种新型的基于靶标的交联来识别与 FSGS1/¿ 复合的六种蛋白质。 -actinin-4。我们将使用体外生化和细胞方法（shRNA 和突变体表达）以及高分辨率成像技术来剖析这些连接成分的分子和细胞功能。目标 3. 开发一种新型“压力室”装置。研究液压在细胞与细胞接触处肌动蛋白组装中的作用对于适当的细胞与细胞粘附以承受正常和高血压压力是必要的，我们将使用新的“压力室”来执行。在不同水压处理下进行功能分析，以阐明疾病易感基因在渗透性屏障破坏的启动和进展中的致病作用。