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，但对绝大多数病例来说，其潜在的分子机制仍不清楚。我们以前曾报道，小分子热休克蛋白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的过度表达具有保护作用，而表达降低则使其敏感)。更重要的是，我们最近证实，在多种NS动物模型和人类NS模型中，肾小球Hsp27的表达都被诱导，这表明肾小球Hsp27的诱导代表了一种广泛性足细胞应激反应。由于Hsp27在体内还未被证实直接与肌动蛋白结合，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的机制，并鉴定了两个新的Hsp27结合蛋白：1)Hic-5，已知的局域黏附蛋白和帕西林同源物；2)Arpda，Arp2/3肌动蛋白聚合起始复合体的已知成员。我们通过共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。我们还通过共IP和定量FRET分析证实了ArpCIa和ArpClb都是真正的Hsp27结合蛋白。在此基础上，我们推测Hsp27在足细胞结构和损伤反应的调节中起关键作用，并且这些作用是通过其新的结合蛋白HIC-5(在焦点黏附动力学中起作用的Pic-5)和ArpC1(Arp2/3肌动蛋白起始复合体的成员)介导的。为了验证这一假设，我们将完成足细胞特异性hspb1(HSP25)基因靶向小鼠的发育，并进行表型分析，以最终确定HSP25在足细胞生物学中的作用。我们还将确定诱导的HIC-5表达的变化或与Hsp27的相互作用是否可以调节足细胞：1)局灶性黏附形成速度、组成和功能(即黏附)，以及2)对损伤的反应。最后，我们将确定诱导的ArpC1表达的变化或与Hsp27的相互作用是否可以调节足细胞：1)Arp2/3复合体的功能(即启动肌动蛋白聚合)和组成，以及2)对损伤的反应。证实HSP27的S基因在足细胞结构调控中的重要性，并确定其在足细胞中的作用分子机制(S)，不仅可以提高我们对NS发生发展的分子机制(S)的理解，而且可以开发出更高靶向性和/或毒性更低的治疗这种非常常见的肾病的方法。

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.