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

RHO家族蛋白介导的信号转导通路需要严格的时间和空间控制。鸟嘌呤核苷酸解离抑制剂（gdi，已知三个成员）是进化上保守的关键调节因子，它选择性地结合于pre - ylated RHO蛋白并控制其在细胞质和膜之间的循环。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里进行了大量的研究，但gdi对RHO蛋白活性时空调节的控制机制尚未被完全探索。DFG研究经费使我们能够研究gdi的功能和特异性。我们发表的研究结果首次提供了前所未有的GDI与纯化RHO蛋白及其变体相互作用的机制见解，利用了我们实验室建立的基于脂质体的体外重构系统和基于荧光的方法的优势。最近发表的三篇研究调查了RHO特异性gdi的不同方面，描述了静电力介导RHO GTPase-GDI的特异性，这是一种由于CDC42-GDI1异常相互作用导致的涉及造血障碍、炎症和噬血细胞淋巴组织细胞增生的新型疾病，以及伪天然产物Rhonin作为第一个与GDI1结合并干扰其功能的小分子抑制剂的鉴定。此外，我们有大量未发表的初步数据，使我们能够阐明一种独特的、特定的GDI功能模式，这种模式仅适用于RHO GTPases的一个子集。我们的体外脂质体重构实验作为一种“自下而上”的方法，为三种GDI类似物与不同RHO gtpase的相互作用提供了额外的机制见解。在这里，我们利用了我们实验室建立的体外脂质体沉淀/浮选和脂质体固定化SPR传感器芯片。未来需要广泛研究的关键问题是以下两个工作包中提出的GDI平行物的特异性和功能调节的调控因素，(i)深入分析GDI平行物的相互作用选择性，（ii）阐明控制GDI功能的调节机制。解决这些问题对于理解gdi如何整合到RHO蛋白的信号转导和控制膜定位至关重要。

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 Moreover, we have a substantial set of unpublished preliminary data enabling us to elucidate a distinct and specific mode of GDI functions that holds true for only a subset of RHO GTPases.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.