Function of Hsp27 and its Binding Proteins in the Glomerular Podocyte

描述（申请人提供）：肾病综合征（NS）是一种常见的肾脏疾病，但对于绝大多数病例，该疾病的分子机制仍不清楚。我们之前报道过，小热休克蛋白 hsp27 是一种已知的肌动蛋白聚合调节因子，在肾小球足细胞（NS 中受影响最严重的细胞）中高度表达，并且实验性 NS 中显着诱导肾小球 hsp27 表达和磷酸化。我们还报道，hsp27 能够显着调节（即 hsp27 过表达保护，而表达减少则敏感）足细胞对 PAN 诱导的细胞损伤和肌动蛋白细胞骨架破坏的反应。更重要的是，我们最近证实肾小球 hsp27 表达在多种 NS 动物模型以及人类 NS 中均被诱导，这表明肾小球 hsp27 的诱导代表了普遍的足细胞应激反应。由于尚未证实 hsp27 在体内直接结合肌动蛋白，我们试图通过筛选肾小球酵母双杂交文库来阐明 hsp27 介导的足细胞结构调节机制，并鉴定了两种新型 hsp27 结合蛋白：1）Hic-5，一种已知的粘着斑蛋白和桩蛋白同源物，2）Arpda，Arp2/3 肌动蛋白聚合起始复合物的已知成员。我们通过 co-IP 证实了 hic-5 是真正的 hsp27 结合蛋白，绘制了其与 hsp27 的相互作用域，并表明 hic-5 可以以相互作用依赖的方式抑制 hsp27 诱导的热保护。我们还通过 co-IP 和定量 FRET 分析证实了 ArpCla 和 ArpClb 都是真正的 hsp27 结合蛋白。基于上述，我们假设 hsp27 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型结合蛋白 hic-5（一种在粘着斑动力学中起作用的桩蛋白同源物）和 ArpC1（Arp2/3 肌动蛋白起始复合物的成员）介导的。为了检验这一假设，我们将完成足细胞特异性 hspbl (hsp25) 基因靶向小鼠的开发，并进行表型分析，以确定 hsp25 在足细胞生物学中的作用。我们还将确定 hic-5 表达或与 hsp27 相互作用的诱导改变是否可以调节足细胞：1）粘着斑形成率、组成和功能（即粘连），以及 2）对损伤的反应。最后，我们将确定 ArpC1 表达或与 hsp27 相互作用的诱导改变是否可以调节足细胞：1）Arp2/3 复合体功能（即肌动蛋白聚合的启动）和组成，以及 2）对损伤的反应。确认 hsp27 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具针对性和/或毒性较小的疗法。

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.