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Function of Hsp27 and its Binding Proteins in the Glomerular Podocyte

Hsp27 及其结合蛋白在肾小球足细胞中的功能

基本信息

批准号：
7990082
负责人：
WILLIAM E SMOYER
金额：
$ 4.92万
依托单位：
RESEARCH INST NATIONWIDE CHILDREN'S HOSP
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-12-10 至 2010-12-09
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7990082
关键词：
Actins Adhesions Adriamycin PFS Adult Affect Affinity Chromatography Animal Model Animals Antibodies Apoptosis Binding Binding Proteins Biological Assay Biology Breeding Cells Child Co-Immunoprecipitations Complex Coupled Cyan Fluorescent Protein Cytochalasin D Cytolysis DNA Detection Development Disease Energy Transfer Extravasation Filtration Fluorescence Fluorescence Resonance Energy Transfer Focal Adhesion Kinase 1 Focal Adhesions Foot Process Frequencies Gene Targeting Genotype Heat Shock Protein 27 Heat shock proteins Histologic Histology Homologous Gene Human Image In Vitro Infection Injury Kidney Kidney Diseases Laboratories Length Libraries Life Link Listeria monocytogenes Maintenance Maps Measures Mediating Microfilaments Microscopic Microscopy Modeling Molecular Molecular Mechanisms of Action Mus Nephrectomy Nephrosis Nephrotic Syndrome Neutral Red Phenotype Phosphorylation Play Principal Investigator Protamine Sulfate Protein Isoforms Proteins Proteinuria Puromycin Aminonucleoside Rattus Regulation Renal function Reporting Role Screening procedure Small Interfering RNA Staining method Stains Structure Tail Testing Time Toxin Transgenic Mice Weight Western Blotting Yeasts base biological adaptation to stress cell injury glomerular filtration improved in vivo latrunculin A member mouse Cre recombinase mutant novel overexpression paxillin podocyte polymerization programs pup recombinase response response to injury vector yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Nephrotic syndrome (NS) is a common kidney disease, but the molecular mechanisms underlying the disease remain unclear for the vast majority of cases. We previously reported that the small heat shock protein, hsp27, a known regulator of actin polymerization, is highly expressed in glomerular podocytes (the cells most affected in NS), and that glomerular hsp27 expression and phosphorylation are significantly induced in experimental NS. We also reported that hsp27 is able to dramatically regulate (i.e. hsp27 overexpression protects, while reduced expression sensitizes) the podocyte response to PAN-induced cellular injury and actin cytoskeletal disruption. More importantly, we recently confirmed that glomerular hsp27 expression is induced in both multiple animal models of NS, as well as in human NS, suggesting that induction of glomerular hsp27 represents a generalized podocyte stress response. Since hsp27 has not been confirmed to bind actin directly in vivo, we attempted to clarify the mechanism of hsp27-mediated regulation of podocyte structure by screening a glomerular yeast two-hybrid library, and identified two novel hsp27 binding proteins: 1) Hic-5, a known focal adhesion protein and paxillin homologue, and 2) Arpda, a known member of the Arp2/3 actin polymerization initiation complex. We confirmed hic-5 as a true hsp27 binding protein by co-IP, mapped its interaction domains with hsp27, and showed that hic-5 can inhibit hsp27-induced thermo-protection in an interaction-dependent manner. We also confirmed both ArpCIa and ArpClb as true hsp27 binding proteins by co-IP and quantitative FRET analyses. Based on the above, we hypothesize that hsp27 plays a critical role in the regulation of podocyte structure and response to injury, and that these actions are mediated via its novel binding proteins, hic-5 (a paxillin homologue with a role in focal adhesion dynamics), and ArpC1 (a member of the Arp2/3 actin initiation complex). To test this hypothesis we will complete development of podocyte-specific hspbl (hsp25) gene-targeted mice and perform phenotypic analyses to definitively determine the role of hsp25 in podocyte biology. We will also determine if induced alterations in hic-5 expression or interaction with hsp27 can regulate podocyte: 1) Focal adhesion formation rate, composition, and function (i.e. adhesion), and 2) Response to injury. Lastly, we will determine if induced alterations in ArpC1 expression or interaction with hsp27 can regulate podocyte: 1) Arp2/3 complex function (i.e. initiation of actin polymerization) and composition, and 2) Response to injury. Confirmation of hsp27's importance in the regulation of podocyte structure, and identification of its molecular mechanism(s) of action in podocytes, could not only improve our understanding of the molecular mechanism(s) underlying the development of NS, but also permit the development of more highly targeted and/or less toxic therapies for this very common kidney disease.

描述（由申请人提供）：肾病综合征（NS）是一种常见的肾脏疾病，但绝大多数病例的分子机制仍不清楚。我们以前报道过，小的热休克蛋白，热休克蛋白27，一个已知的调节肌动蛋白聚合，是高度表达的肾小球足细胞（在NS中最受影响的细胞），和肾小球热休克蛋白27的表达和磷酸化显着诱导实验NS。我们还报道，热休克蛋白27是能够显着调节（即热休克蛋白27过表达保护，而减少表达敏化）足细胞反应PAN诱导的细胞损伤和肌动蛋白细胞骨架破坏。更重要的是，我们最近证实，肾小球热休克蛋白27的表达诱导在两个多个动物模型的NS，以及在人类NS，这表明诱导肾小球热休克蛋白27代表一个广义的足细胞应激反应。由于hsp 27在体内不能直接与肌动蛋白结合，我们通过筛选肾小球酵母双杂交文库，试图阐明hsp 27介导足细胞结构调节的机制，并鉴定了两种新的hsp 27结合蛋白：1）Hic-5，一种已知的粘着斑蛋白和桩蛋白同源物，和2）Arpda，一种已知的Arp 2/3肌动蛋白聚合起始复合物的成员。我们通过co-IP证实了hic-5是一个真正的hsp 27结合蛋白，并绘制了hic-5与hsp 27的相互作用结构域，表明hic-5可以以相互作用依赖的方式抑制hsp 27诱导的热保护作用。我们还通过co-IP和定量FRET分析证实ArpCla和ArpClb都是真正的hsp 27结合蛋白。基于上述，我们假设热休克蛋白27在足细胞结构的调节和对损伤的反应中起着关键作用，并且这些作用是通过其新的结合蛋白，hic-5（一种在粘着斑动力学中起作用的桩蛋白同源物）和ArpC 1（Arp 2/3肌动蛋白起始复合物的成员）介导的。为了验证这一假设，我们将完成足细胞特异性hspbl（hsp 25）基因靶向小鼠的开发，并进行表型分析，以明确确定hsp 25在足细胞生物学中的作用。我们还将确定诱导的hic-5表达或与hsp 27相互作用的改变是否可以调节足细胞：1）粘着斑形成率、组成和功能（即粘附），以及2）对损伤的反应。最后，我们将确定ArpC 1表达或与hsp 27相互作用的诱导改变是否可以调节足细胞：1）Arp 2/3复合物功能（即肌动蛋白聚合的起始）和组成，以及2）对损伤的反应。证实热休克蛋白27在足细胞结构调节中的重要性，并鉴定其在足细胞中作用的分子机制，不仅可以提高我们对NS发展的分子机制的理解，而且还可以为这种非常常见的肾脏疾病开发更高靶向和/或毒性更低的治疗方法。