Myo1c Participates in Podocyte Junction Formation Through Interaction with Neph1

描述（由申请人提供）：肾小球疾病导致肾滤过损伤性改变，在全国范围内是终末期肾病（ESRD）日益增加的原因。不幸的是，控制ESRD发展的分子机制仍然知之甚少。足细胞蛋白的鉴定，包括Neph1及其相关蛋白复合物，是肾脏滤过的关键组成部分，是理解裂隙隔膜组织的重大进展。包括我们在内的许多近期研究表明，Neph1与其相关蛋白（包括Grb2、ZO-1、Nephrin和Par3）之间的动态相互作用调节了裂隙隔膜处的肌动蛋白细胞骨架。数据还表明肾小球损伤后，在远离细胞膜的各种亚细胞区室中经常发现Neph1及其复合物。将这些蛋白质重新定位到它们的膜过滤狭缝位置可能需要一个活跃的基于分子运动的机制。我们现在有了令人兴奋的结果，确定了Neph1与Myo1c的一种新的相互作用，Myo1c是非常规Myosin家族的成员，其成员是atp酶运动蛋白，与肌动蛋白结合并产生力。Myo1c也具有膜结合结构域和钙调蛋白结合结构域，并已被证明在不同细胞类型的膜细胞骨架事件中发挥作用。我们最近发表的一篇文章表明，肾小球损伤会导致Neph1复合物的错误定位，导致细胞连接丢失和蛋白尿，而损伤后的恢复与Neph1复合物返回细胞周围相关（Wagner等人，JBC 2008）。我们认为，Neph1与Myo1c的相互作用在调节Neph1复合物的组织中起着关键作用。在这项研究中，我们建议在体内和体外条件下研究Neph1和Myo1c的相互作用。本研究提出的具体目的是：1)明确Myo1c在足细胞中的生理和病理生理组织。2)确定Myo1c-Neph1相互作用在肾小球损伤时肾小球滤过缝中Neph1复合物的组装和易位中的意义。3)确定体内条件下Myo1c及其与Neph1相互作用在维持肾小球功能中的意义。这项研究的结果将增加我们对狭缝隔膜蛋白在生理和非生理条件下如何组织的理解，从而为改善肾小球损伤后的临床结果提供新的研究途径。

DESCRIPTION (provided by applicant): Glomerular diseases that lead to damaging changes in kidney filtration are an increasing cause of ESRD (end stage renal disease) nationwide. Unfortunately, the molecular mechanisms governing the development of ESRD remain poorly understood. Identification of podocyte proteins, including Neph1 and its associated protein complex, that are critical components of kidney filtration was a major advancement in understanding the organization of slit diaphragm. A number of recent studies, including ours, now demonstrate that dynamic interactions between Neph1 and its associated proteins including Grb2, ZO-1, Nephrin and Par3 regulates the actin cytoskeleton at the slit diaphragm. Data also suggest that Neph1 and its complex are frequently found in various sub cellular compartments away from the cell membrane following glomerular injury. Retargeting these proteins back to their membrane filtration slit location is likely to require an active molecular motor-based mechanism. We now have exciting results that identify a novel interaction of Neph1 with Myo1c, a member of the un-conventional Myosin family whose members are ATPase motor proteins that bind to actin and can generate force. Myo1c also has membrane binding and Calmodulin binding domains and has been shown to have a role in membrane cytoskeletal events in diverse cell types. We recently published an article demonstrating that glomerular injury results in mis-localization of the Neph1 complex leading to loss of cell junctions and proteinuria, whereas recovery from injury correlates with the Neph1 complex's return to the cell periphery (Wagner et al., JBC 2008). We believe that the interaction of Neph1 with Myo1c plays a critical role in regulating the organization of the Neph1 complex. In this study we propose to investigate the Neph1 and Myo1c interaction under in-vitro and in- vivo conditions. The specific aims proposed for this study are: 1) Define the physiologic and pathophysiologic organization of Myo1c in podocytes. 2) Determine the significance of Myo1c-Neph1 interaction in the assembly and translocation of Neph1 complex to the filtration slit in response to glomerular injury. 3) Determine the significance of Myo1c and its interaction with Neph1 in maintaining the glomerular function under in-vivo conditions. The results from this study will increase our understanding of how slit diaphragm proteins organize under both physiological and nonphysiological conditions resulting in new avenues of research aimed at improving clinical outcome following glomerular injury. PUBLIC HEALTH RELEVANCE: Glomerular diseases leading to end stage renal disease are major cause of kidney malfunction in United States. Our previous studies have shown that functional and biochemical changes in the proteins that are central to the maintenance of the kidney filtration system leads to the development of glomerular disorders. In this study we will determine the role of a novel protein Myo1c and identify novel targets for use in the development of novel therapies aimed at restoring the selective filtration function of kidneys during various glomerular disorders.