Regulation of Podocyte Structure by the Binding Protein

结合蛋白对足细胞结构的调节

• 批准号: 7563049
• 负责人: WILLIAM E SMOYER
• 金额: $ 11.21万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-11-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7563049
• 关键词: actins; binding proteins; cellular pathology; cytochalasins; gene expression; glucocorticoids; heat shock proteins; kidney disorder chemotherapy; laboratory rat; marine toxins; membrane permeability; molecular pathology; nephrotic syndrome; nonhuman therapy evaluation; oxidative stress; phosphorylation; podocyte; protein localization; protein protein interaction; renal glomerulus

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.

描述（申请人提供）： 肾病综合征（NS）是一种常见的肾脏疾病，但 对于大多数病例，该疾病的分子机制仍不清楚。我们之前 据报道，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，其表达和 在实验 NS 中足细胞中诱导磷酸化，并且 hsp27 过度表达可以保护培养的足细胞免受损伤。为了阐明这种保护机制，我们筛选了肾小球酵母双杂交文库中的 hsp27 结合蛋白，并发现了 hic-5，一种已知的粘着斑和类固醇受体共激活蛋白。我们通过免疫共沉淀证实了 hic-5 是真正的 hsp27 结合蛋白，部分绘制了相互作用结构域，并证明了 hic-5 与 hsp27 的相互作用可以改变 hsp27 功能。最近，我们还发现糖皮质激素作为 NS 的主要治疗方法，可以直接作用于足细胞，以保护和促进 PAN 引起的损伤的恢复，这与糖皮质激素通过抑制循环淋巴细胞可溶性疾病介质。因此，我们假设 hsp27 和多功能结合蛋白 hic-5 之间的相互作用在调节 NS 期间足细胞损伤和糖皮质激素治疗后足细胞恢复方面发挥着关键作用，通过 hic-5 的已知作用：1) 粘着斑动力学， 2) 糖皮质激素受体共激活。 为了检验这些假设，我们将确定所需的 hic-5 和 hsp27 的分子状态 研究它们的蛋白质-蛋白质相互作用，以及这些状态的改变对其在足细胞中的细胞内定位的影响。我们还将确定 hic-5 表达、磷酸化或 hsp27 结合的诱导改变是否可以调节：1) 足细胞粘着斑蛋白组成、形成速率和程度以及足细胞损伤前后的功能，以及 2) 糖皮质激素-诱导足细胞基因转录的激活，以及保护和增强足细胞免受损伤的恢复。足细胞将被含有全长和磷酸化和/或截短突变的 hic-5 和 hsp27 构建体的腺病毒感染，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 的细胞内定位和 hic-5，2) 粘着斑成分、形成率和功能，以及 3) 糖皮质激素诱导的保护和增强 PAN 诱导损伤的恢复相关的体内研究将包括足细胞中 hic-5 和 hsp27 共定位的分析，以及地塞米松在 PAN 诱导的大鼠肾病综合征中对疾病的保护作用。鉴定 hic-5 和 hic-5 之间相互作用的生物学重要作用 hsp27 调节足细胞结构及糖皮质激素对 NS 的治疗作用 提高我们对 NS 发展的分子机制的理解，并为这种非常常见的肾脏疾病开发更具针对性和毒性较小的疗法。

项目成果

Dose- and time-dependent glucocorticoid receptor signaling in podocytes.

足细胞中剂量和时间依赖性糖皮质激素受体信号传导。

• DOI: 10.1152/ajprenal.00161.2010
• 发表时间: 2010-10-01
• 期刊: American journal of physiology. Renal physiology
• 作者: Adam J Guess;S. Agrawal;Changqiang Wei;R. Ransom;R. Benndorf;W. Smoyer
• 通讯作者: W. Smoyer

Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury.

抑制蛋白激酶 MK-2 可保护足细胞免受肾病综合征相关损伤。

• DOI: 10.1152/ajprenal.00661.2010
• 发表时间: 2011-05-25
• 期刊: American journal of physiology. Renal physiology
• 作者: Ruma A. Pengal;Adam J Guess;S. Agrawal;J. Manley;R. Ransom;R. Mourey;R. Benndorf;W. Smoyer
• 通讯作者: W. Smoyer