Function of Hsp27 and its Binding Proteins in the Glomerular Podocyte

描述（由申请人提供）：肾病综合征（Nephrotic syndrome， NS）是一种常见的肾脏疾病，但绝大多数病例的分子机制尚不清楚。我们之前报道了小热休克蛋白hsp27，一种已知的肌动蛋白聚合调节剂，在肾小球足细胞（NS中受影响最大的细胞）中高表达，并且在实验性NS中显著诱导肾小球hsp27的表达和磷酸化。我们还报道了hsp27能够显著调节足细胞对pan诱导的细胞损伤和肌动蛋白细胞骨架破坏的反应（即hsp27过表达保护，而表达减少致敏）。更重要的是，我们最近证实肾小球hsp27的表达在多种NS动物模型和人类NS中都是诱导的，这表明肾小球hsp27的诱导代表了一种广泛的足细胞应激反应。由于hsp27在体内尚未被证实与肌动蛋白直接结合，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，并鉴定出两种新的hsp27结合蛋白：1)已知的局灶黏着蛋白Hic-5和paxillin同源物，2)已知的Arp2/3肌动蛋白聚合起始复合物成员Arpda。我们通过co-IP证实了hic-5是一个真正的hsp27结合蛋白，并绘制了其与hsp27的相互作用结构域，结果表明hic-5能够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ArpCIa和ArpClb是真正的hsp27结合蛋白。基于以上，我们假设hsp27在足细胞结构调节和损伤反应中发挥关键作用，这些作用是通过其新的结合蛋白hic5（一种参与局灶黏附动力学的paxillin同源物）和ArpC1 （Arp2/3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hsp25基因靶向小鼠的开发，并进行表型分析，以明确确定hsp25在足细胞生物学中的作用。我们还将确定hic-5表达的诱导改变或与hsp27的相互作用是否可以调节足细胞：1)局灶粘连形成速度、组成和功能（即粘连），以及2)对损伤的反应。最后，我们将确定诱导ArpC1表达的改变或与hsp27的相互作用是否可以调节足细胞：1)Arp2/3复合物的功能（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，并确定其在足细胞中的作用分子机制，不仅可以提高我们对NS发展的分子机制的理解，而且还可以开发出针对这种非常常见的肾脏疾病的更具靶向性和/或毒性更小的治疗方法。

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.