Specificity and functional modulation of RHOGDIs: An in vitro reconstitution approach

由RHO家族蛋白介导的信号转导途径需要严格的时间和空间控制。鸟嘌呤核苷酸解离抑制剂（Guanine nucleotide dissociation inhibitors，GDIs）是一种进化上保守的关键调节因子，它选择性地与异戊烯化的RHO蛋白结合，并控制它们在胞质和细胞膜之间的循环。细胞膜结合是RHO蛋白发挥功能的先决条件。尽管在过去的几十年中，相当大的努力，控制机制的RHO蛋白活性的GDIs的时空调控尚未完全探索。DFG的研究资助使我们能够研究GDIs的功能和特异性。我们发表的结果提供了第一个前所未有的GDI与纯化的RHO蛋白及其变体的相互作用的机制见解，使用我们实验室建立的基于脂质体的体外重建系统和基于荧光的方法的优点。最近发表的三项研究调查了RH 0特异性GDI的不同方面，描述了静电力介导RH 0 GTP酶-GDI的特异性，这是一种新型疾病，涉及造血功能障碍、炎症和由于异常的CDC 42-GDI 1相互作用引起的噬血细胞性淋巴组织细胞增多症，并鉴定了伪天然产物Rhonin作为第一个结合GDI 1并干扰其功能的小分子抑制剂。此外，我们有大量的未发表的初步数据，使我们能够阐明一个独特的和具体的GDI功能模式，适用于只有一个子集的RHO GTP酶。我们的体外脂质体重建实验作为一种“自下而上”的方法，提供了额外的机械见解的三个GDI旁系同源物与不同的RHO GTP酶的相互作用。在这里，我们利用了在体外脂质体沉降/漂浮和脂质体固定SPR传感器芯片，在我们的实验室建立。在未来的关键问题进行广泛调查的特异性和功能调制的GDI旁系同源物的监管元素，提出了以下两个工作包，（i）在深入分析的相互作用的选择性的GDI旁系同源物，和（ii）阐明控制GDI功能的调制机制。解决这些问题对于理解GDIs如何整合到RHO蛋白的信号转导和控制膜定位中至关重要。

Signal transduction pathways mediated by RHO family proteins require tight temporal and spatial control. Guanine nucleotide dissociation inhibitors (GDIs; three members known) are evolutionarily conserved key regulators, which bind selectively to prenylated RHO proteins and control their cycle between cytosol and membrane. Cell membrane association is a prerequisite for the function of RHO proteins. Despite considerable efforts over the last decades, the control mechanisms underlying spatiotemporal regulation of RHO protein activity by GDIs have not been completely explored. The DFG research grant allowed us to investigate the function and specificity of GDIs. Our published results have provided first unprecedented mechanistic insights into GDI interactions with purified RHO proteins and their variants using the advantages of both liposome-based in-vitro reconstitution systems and fluorescence-based approaches that are established in our lab. Three very recently published studies investigated different aspects of RHO-specific GDIs, describing electrostatic forces mediate the specificity of RHO GTPase-GDI, a novel disorder involving dyshematopoiesis, inflammation, and hemophagocytic lymphohistiocytosis due to aberrant CDC42-GDI1 interaction, and the identification of the pseudo-natural product Rhonin as a first small-molecule inhibitor that binds to GDI1 and interferes with its function. Moreover, we have a substantial set of unpublished preliminary data enabling us to elucidate a distinct and specific mode of GDI functions that holds true for only a subset of RHO GTPases. Our in vitro liposome reconstitution experiments as a `bottom-up´ approach have provided additional mechanistic insights into interaction of the three GDI paralogs with different RHO GTPases. Here, we took advantage of the in vitro liposome sedimentation/flotation and liposome-immobilized SPR sensor chips, established in our lab. Key issues to be investigated extensively in the next future are the regulatory elements of specificity and functional modulation of the GDI paralogs as proposed in the following two work packages, (i) in-depth analysis of the interaction selectivity of the GDI paralogs, and (ii) elucidation of modulatory mechanisms controlling the GDI function. Addressing these goals is of fundamental importance in understanding how GDIs are integrated in the signal transduction and control membrane localization of RHO proteins.