Regulation of Podocyte Structure by the Binding Protein

简介(申请人提供)：肾病综合征(NS)是一种常见的肾脏疾病，但 在大多数情况下，这种疾病背后的分子机制尚不清楚。我们之前 报道称，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，其表达和 在实验性NS中，足细胞被诱导了磷酸化，Hsp27的过表达可以保护培养的足细胞免受损伤。为了阐明这种保护的机制，我们筛选了一个肾小球酵母Hsp27结合蛋白的双杂交文库，发现了HIC-5，一个已知的焦点黏附和类固醇受体共激活蛋白。我们用免疫共沉淀法证实了HIC-5是一个真正的Hsp27结合蛋白，并绘制了部分相互作用结构域，证明了HIC-5与Hsp27的相互作用可以改变Hsp27的功能。最近，我们还发现，糖皮质激素是NS的主要治疗方法，它可以直接作用于足细胞，保护和促进PAN诱导的损伤的恢复，这与糖皮质激素通过抑制循环淋巴细胞产生可溶性疾病介质来发挥治疗作用的主导范式形成了鲜明对比。因此，我们推测HSP27与多功能结合蛋白HIC-5之间的相互作用，通过HIC-5‘S已知的作用，在调节NS时足细胞损伤和糖皮质激素治疗后足细胞恢复中发挥关键作用：1)局部黏附动力学，2)糖皮质激素受体共激活。 为了验证这些假设，我们将确定所需的HIC-5和HSP27的分子状态 它们的蛋白质-蛋白质相互作用，以及这些状态的变化对它们在足细胞中的细胞内定位的影响。我们还将确定诱导的HIC-5的表达、磷酸化或Hsp27结合的变化是否可以调节：1)足细胞损伤前后足细胞局部黏附蛋白的组成、形成速度和程度以及功能；2)糖皮质激素诱导的足细胞基因转录的激活，以及保护和促进足细胞损伤后的恢复。含有全长和磷酸化和/或截短突变的hic-5和hsp27结构的腺病毒将感染足细胞，并详细分析改变的hic-5-Hsp27相互作用对以下方面的影响：1)Hsp27和hic-5的细胞内定位，2)局灶性黏附组成、形成率和功能，3)糖皮质激素诱导的保护和促进PAN诱导的损伤和肌动蛋白细丝断裂的恢复。HIC-5与HIC-5相互作用中重要生物学作用的鉴定 热休克蛋白27对肾病综合征足细胞结构的调节及糖皮质激素的治疗作用 提高我们对NS发生的分子机制(S)的理解，并允许针对这种非常常见的肾脏疾病开发更高靶向性和毒性更低的治疗方法。

DESCRIPTION (provided by applicant): Nephrotic syndrome (NS) is a common kidney disease, but the molecular mechanisms underlying the disease remain unclear for most 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), that its expression and phosphorylation are induced in podocytes in experimental NS, and that hsp27 overexpression can protect cultured podocytes from injury. To clarify the mechanism of this protection, we screened a glomerular yeast two-hybrid library for hsp27-binding proteins, and found hic-5, a known focal adhesion and steroid receptor co-activator protein. We confirmed hic-5 as a true hsp27 binding protein by coimmunoprecipitation, partially mapped the interaction domains, and demonstrated that interaction of hic-5 with hsp27 can alter hsp27 function. Recently, we also found that glucocorticoids, the primary treatment for NS, can act directly on podocytes to protect and enhance recovery from PAN-induced injury, in striking contrast to the dominant paradigm that glucocorticoids exert their therapeutic effect in NS by suppressing production of a soluble disease mediator by circulating lymphocytes. We therefore hypothesize that the interaction between hsp27 and the multifunctional binding protein, hic-5, plays a critical role in regulating both podocyte injury during NS and podocyte recovery following glucocorticoid therapy, via hic-5's known roles in: 1) Focal adhesion dynamics, and 2) Glucocorticoid receptor coactivation. To test these hypotheses we will determine the molecular states of hic-5 and hsp27 required for their protein-protein interaction, and the effects of alterations in these states on their intracellular localization in podocytes. We will also determine if induced alterations in the expression, phosphorylation, or hsp27- binding of hic-5 can regulate: 1) Podocyte focal adhesion protein composition, rate and extent of formation, and function before and after podocyte injury, and 2) Glucocorticoid-induced activation of podocyte gene transcription, as well as protection and enhanced recovery of podocytes from injury. Podocytes will be infected with adenovirus containing full-length and phosphorylation- and/or truncation-mutant hic-5 and hsp27 constructs, and detailed analyses performed of the effects of altered hic-5-hsp27 interaction on the: 1) Intracellular localization of hsp27 and hic-5, 2) Focal adhesion composition, formation rate, and function, and 3) Glucocorticoid-induced protection and enhanced recovery from PAN induced injury and actin filament disruption by latrunculin A and cytochalasin D. Correlative in vivo studies will include analyses of hic-5 and hsp27 co-localization in podocytes, as well as protection against disease by dexamethasone, in PAN-induced nephrotic syndrome in rats. Identification of a biologically important role for the interaction between hic-5 and hsp27 in regulating podocyte structure and the therapeutic effect of glucocorticoids in NS would improve our understanding of the molecular mechanism(s) underlying the development of NS, and permit the development of more highly targeted and less toxic therapies for this very common kidney disease.