Myo1c Participates in Podocyte Junction Formation Through Interaction with Neph1

描述(申请人提供)：肾小球疾病，导致肾脏滤过的破坏性变化，是全国范围内ESRD(终末期肾脏疾病)的一个日益增加的原因。不幸的是，控制ESRD发展的分子机制仍然知之甚少。鉴定肾滤过的关键成分足细胞蛋白，包括Neph1及其相关蛋白复合体，是理解裂隙横隔膜组织的一大进步。最近的一些研究，包括我们的研究，现在证明Neph1与其相关蛋白包括Grb2，ZO-1，Nephin和Par3之间的动态相互作用调节着缝隙隔膜的肌动蛋白细胞骨架。数据还表明，肾小球损伤后，Neph1及其复合体经常出现在远离细胞膜的各种亚细胞间隔中。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基于分子马达的活跃机制。我们现在有了令人兴奋的结果，发现了Neph1与Myo1c的一种新的相互作用，Myo1c是非传统肌球蛋白家族的成员，其成员是与肌动蛋白结合并可以产生力的ATPase马达蛋白。Myo1c还具有膜结合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。我们最近发表了一篇文章，证明肾小球损伤会导致Neph1复合体的错误定位，导致细胞连接丢失和蛋白尿，而损伤的恢复与Neph1复合体返回细胞外围相关(Wagner等人，JBC 2008)。我们认为，Neph1与Myo1c的相互作用在调节Neph1复合体的组织中起着关键作用。在这项研究中，我们建议研究Neph1和Myo1c在体外和体内条件下的相互作用。本研究提出的具体目标是：1)确定足细胞中Myo1c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后Neph1复合体组装和移位到滤过缝隙中的意义。3)确定Myo1c及其与Neph1的相互作用在体内条件下维持肾小球功能的意义。这项研究的结果将增加我们对生理和非生理条件下裂隙横隔膜蛋白如何组织的理解，从而为改善肾小球损伤后的临床结局提供新的研究途径。 公共卫生相关性：在美国，导致终末期肾脏疾病的肾小球疾病是导致肾功能障碍的主要原因。我们以前的研究表明，对维持肾滤过系统至关重要的蛋白质的功能和生化变化会导致肾小球疾病的发生。在这项研究中，我们将确定一种新的蛋白质Myo1c的作用，并确定新的靶点，用于开发新的治疗方法，旨在恢复各种肾小球疾病期间肾脏的选择性过滤功能。

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.