RhoGDI: yin and yang of RhoGTPases cycle

描述(由申请人提供)： 肾脏疾病是美国和世界范围内一个主要的公共卫生问题，主要由肾小球足细胞精确调节的病理变化引起，肾小球足细胞是肾脏的主要过滤单位。足细胞蛋白的功能障碍包括内弗林，由于足突(FP)收缩、滤过屏障的破坏和肌动蛋白的重排而导致肾脏疾病 细胞骨架。然而，将neparin激活与肌动蛋白细胞骨架重组联系起来的机制尚不清楚。本研究的目标是确定足细胞蛋白参与调控GTP酶的分子机制，GTP酶是FP发育过程中细胞骨架、空间和时间活性的关键调节因子。GTP酶作为分子开关在非活性形式(GDP结合)和活性形式(GTP结合)之间循环。几个级别的监管严格控制它们的激活状态和可获得性。GDIs(鸟嘌呤解离抑制物)具有抑制GTP酶活性的功能。GAP(鸟嘌呤激活蛋白)可加速GTP酶的活性，从而使GTP失活并终止信号传导。鉴于细胞骨架在肾脏足细胞功能中的关键作用，GDI、GEF和GAP对GTP酶活性的调节构成了肾细胞骨架动力学调节的关键点。这种GTP酶循环的失调可导致许多细胞/组织的病理疾病状态。这在肾脏中尤其如此，GDI基因敲除小鼠肾脏结构和生理上的戏剧性缺陷证明了这一点，最终导致肾功能衰竭而死亡。我们假设newitin处于通过招募GTP酶及其调节器来定义隔室身份的层次结构的顶端。为了验证我们的假设，我们将追求以下目标：我们将研究一个新的足细胞GDI结合伙伴控制GTPase-GDI复合体的分子机制，以及它在nephin激活过程中对肌动蛋白细胞骨架动力学的影响。AIM2.我们已经确定了一种新的neparin结合伙伴，它可以调节GTP酶的失活。因此，我们将阐明这种间隙是通过什么机制调节FP的延伸的。目的：我们将确定GTPase-GDI复合体在neparin刺激过程中的时空调节。为此，我们将在活体成像研究中使用一种新颖而独特的活细胞GTPase-GDI复合体生物传感器，并结合计算多路分析。这些研究将使人们更好地了解肾功能的这些重要组成部分是如何受到控制的，最终将指导采取独特的、越来越有选择性的治疗方法来干预肾炎性疾病。

DESCRIPTION (provided by applicant): Kidney disease, a major public health concern in the US and worldwide, is mainly caused by pathological changes in the precise regulation of glomerulus podocyte, the primary filtration unit in the kidney. Dysfunction of podocyte proteins including nephrin leads to renal disease owing to foot processes (FP) retraction, disruption of the filtration barrier and rearrangement of the actin cytoskeleton. However, the mechanisms linking nephrin activation to actin cytoskeleton reorganization are ill understood. The goal of this research project is to identify podocyte proteins involved in the molecular mechanisms controlling the regulation of GTPase, key regulators of the cytoskeletal, activity in space and time during FP development. GTPases act as molecular switches cycling between inactive (GDP-bound) and active (GTP-bound) forms. Several levels of regulation tightly control their activation state and accessibility. GDIs (Guanin Dissociation Inhibitors) function to inhibit GTPase activities. Activation through exchange of GDP for GTP is catalyzed by GEFs (Guanine Exchange Factors) and promotes downstream signaling; GAPs (Guanine- Activating Proteins) accelerate the intrinsic GTPase activity to inactivate the protein and terminate the signal. Given the key roles played by the cytoskeleton in renal podocyte function, the regulation of GTPase activity by GDI, GEFs and GAPs constitute critical points in the regulation of renal cytoskeletal dynamics. Dysregulation of such GTPase cycling can lead to pathological disease states in many cells/tissues. This is particular true in kidney, as evidenced by the dramatic defects in kidney structure and physiology that occur in the GDI knockout mouse, finally leading to death due to renal failure. We hypothesize that nephrin is at the apex of a hierarchy defining compartment identity through the recruitment of GTPases and their regulators. To test our hypothesis, we will pursue the following Aims: Aim1. We will investigate the molecular mechanisms by which a new podocyte GDI binding partner controls GTPase-GDI complexes and its effects on actin cytoskeleton dynamics during nephrin activation. Aim2. We have identified a new binding partner for nephrin that can regulate the inactivation of GTPases. Therefore, we will elucidate by which mechanisms this GAP regulate FP elongation. Aim3: We will determine the spatio-temporal regulation of GTPase-GDI complexes during nephrin stimulation. To this end, we will use a novel and unique live cell GTPase-GDI complex biosensor in in vivo imaging studies, coupled with computational multiplexing analyses. These studies will lead to a greater understanding of how these important components of renal function are controlled, ultimately guiding to unique and increasingly selective therapeutic approaches to intervene in nephritic disorders.