Cell junction proteins in podocyte injury repair

足细胞损伤修复中的细胞连接蛋白

基本信息

批准号：
8547067
负责人：
Leslie A Bruggeman
金额：
$ 32.95万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-20 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8547067
关键词：
AIDS-Associated Nephropathy Actins Adult Affect Binding Binding Proteins Biochemical Biological Assay Cell Adhesion Cell Line Cell membrane Cell-Cell Adhesion Cell-Matrix Junction Cells Chronic Kidney Failure Co-Immunoprecipitations Complex Cytoskeleton Development Diabetes Mellitus Disease Event Filtration Focal Adhesions Foot Process Homeostasis Image Immunologics In Vitro Injury Integrin Binding Integrins Intercellular Junctions Kidney Kinetics Knockout Mice Light Mediating Methods Molecular Mouse Cell Line Mus Natural regeneration Pathology Phenotype Phosphorylation Phosphotyrosine Process Property Proteins Proteinuria Receptor Signaling Regulation Renal function Renal glomerular disease Role Severities Signal Transduction Structure Synapses T-Cell Receptor Testing Time Tyrosine adapter protein base blood filter in vivo inhibitor/antagonist injury and repair kidney cell migration mouse model nephrin novel podocyte prevent repaired response scaffold slit diaphragm src-Family Kinases therapeutic development trafficking

项目摘要

DESCRIPTION (provided by applicant): The major cell-cell junction of the adult kidney podocyte is the slit diaphragm, an important component of the kidney filtration barrier. The loss of the slit diaphragm during foot process effacement results in proteinuria and contributes to the progression of many chronic kidney diseases such as diabetes and HIV-associated nephropathy. The developmental formation and adult homeostasis of podocyte cell junctions are processes that are not fully understood, but are important for the development of therapeutic strategies to repair or replace podocytes that are damage or lost in disease. We have recently identified the claudin-like cell junction protein TM4SF10 and the Fyn binding protein ADAP as podocyte-expressed proteins that affect lamellipodia formation, Fyn activity, and Nephrin phosphorylation. TM4SF10 is transiently expressed during podocyte development and is re-expressed during injury repair. Thus, we hypothesize that in disease, TM4SF10 may function to protect the podocyte during foot process effacement by preserving a simplified cell junction and by modifying the activities of Fyn. We also hypothesize that ADAP, a large adapter protein with known roles in integrin signaling, functions as a scaffolding center in the podocyte foot process integrating actin cytoskeleton dynamics at both the slit diaphragm and in integrin attachments of the foot process. We will test this hypothesis by determining the association of ADAP binding to TM4SF10 and other key intracellular components of the slit diaphragm complex using standard mutational analysis and in vitro binding assays, and assess functional effects based on lamellipodia extension, integrin binding, and Nephrin phosphorylation. Proposed studies also will include further characterize of the renal phenotype in ADAP knockout mice and further development and characterization of a knockout mouse model for TM4SF10. Podocyte cells lines from these mice will be established to investigate the requisite functions of TM4SF10 and ADAP in podocytes with regard to regulating integrin cell attachments and Nephrin phosphorylation. In addition, we have established TM4SF10 functions to directly suppress Fyn activity, possibly by sequestering Fyn away from the plasma membrane. This will be investigated with real time imaging and co-localization methods assessing intracellular trafficking of Fyn in the presence and absence of TM4SF10. These studies describe the first integrated molecular mechanism connecting cell-cell adhesion at the slit diaphragm with cell-matrix adhesion with integrin binding in the foot process, events that require synchronization during foot process formation and retraction. These studies will have important implications toward the development of novel therapies directed at glomerular diseases characterized by foot processes effacement where preventing or repairing damaged podocytes would be beneficial.

描述（由申请人提供）：成年肾足细胞的主要细胞-细胞连接是裂隙隔膜，它是肾脏滤过屏障的重要组成部分。足突消失期间裂隙隔膜的损失会导致蛋白尿，并导致许多慢性肾脏疾病的进展，例如糖尿病和艾滋病毒相关肾病。足细胞细胞连接的发育形成和成年体内平衡尚不完全清楚，但对于制定修复或替换疾病中受损或丢失的足细胞的治疗策略非常重要。我们最近鉴定出密蛋白样细胞连接蛋白 TM4SF10 和 Fyn 结合蛋白 ADAP 作为足细胞表达的蛋白，影响片状伪足的形成、Fyn 活性和去氧肾上腺素磷酸化。 TM4SF10 在足细胞发育过程中短暂表达，并在损伤修复过程中重新表达。因此，我们假设在疾病中，TM4SF10 可能通过保留简化的细胞连接和改变 Fyn 的活性，在足突消失过程中发挥保护足细胞的作用。我们还假设 ADAP 是一种在整合素信号传导中具有已知作用的大接头蛋白，在足细胞足突中充当支架中心，整合缝隔膜和足突整合素附着处的肌动蛋白细胞骨架动力学。我们将通过使用标准突变分析和体外结合测定确定 ADAP 与 TM4SF10 和裂隙隔膜复合物的其他关键细胞内成分的结合来测试这一假设，并评估基于片状伪足延伸、整合素结合和去氧肾上腺素磷酸化的功能效应。拟议的研究还将包括进一步表征 ADAP 敲除小鼠的肾脏表型，以及进一步开发和表征 TM4SF10 敲除小鼠模型。将建立来自这些小鼠的足细胞细胞系，以研究足细胞中 TM4SF10 和 ADAP 在调节整合素细胞附着和去氧肾上腺素磷酸化方面的必要功能。此外，我们还建立了 TM4SF10 的功能来直接抑制 Fyn 活性，可能是通过将 Fyn 隔离于质膜之外。这将通过实时成像和共定位方法进行研究，评估 Fyn 在存在和不存在 TM4SF10 的情况下的细胞内运输。这些研究描述了第一个将狭缝隔膜处的细胞-细胞粘附与细胞-基质粘附与足突中的整合素结合连接起来的集成分子机制，这些事件在足突形成和缩回过程中需要同步。这些研究将对开发针对以足突消失为特征的肾小球疾病的新疗法具有重要意义，其中预防或修复受损的足细胞将是有益的。