Exploring the structure function relationship of membrane-pore-forming FGF2 oligomers - a single molecule approach

成纤维细胞生长因子2(FGF2)是一种在健康和疾病中促进血管生成的强有丝分裂原。在病理条件下，它是肿瘤诱导的血管生成的主要激活剂，也是由抑制细胞凋亡的自分泌信号环介导的肿瘤细胞的生存因子。尽管FGF2具有明确的细胞外功能，但它缺乏信号肽，因此通过一种非传统的、不依赖ER/高尔基体的蛋白质分泌机制从细胞中输出。了解肿瘤细胞分泌FGF2的分子机制可能为开发一类新型的抗血管生成抑制剂奠定基础，在癌症治疗中具有很高的潜力。细胞分泌FGF2不涉及细胞内的囊泡中间产物，而是通过直接跨质膜转位来调节。这一过程是由磷脂酰肌醇PI(4，5)P2介导的FGF2在质膜内叶的募集启动的。这种相互作用导致FGF2寡聚，并在特定表面酪氨酸残基上的FGF2磷酸化的刺激下，导致FGF2寡聚体的膜插入，伴随着环状膜孔的形成。这种结构被解释为FGF2膜转位的中间产物。在最后一步中，细胞表面的硫酸乙酰肝素蛋白多糖将FGF2分子捕获在外叶上，导致FGF2分子移位到细胞外空间。这项研究的目标是在细胞生物学、生物化学和生物物理学之间遵循一种跨学科的方法。它旨在通过对FGF2低聚体结构功能关系的分子分析，深入了解依赖PI(4，5)P2的FGF2齐聚如何诱导脂质膜孔的形成。具体地说，我们计划遵循单分子方法，能够在体外和新的基于细胞的系统中识别和分析形成膜孔的功能性FGF2寡聚体。这些研究针对的是i.)研究参与成孔FGF2低聚体的FGF2分子的数量和与FGF2低聚体相关的膜孔大小，II.)确定成孔低聚物中单个FGF2分子之间的距离，III。)分析了它们的动力学和膜孔寿命。揭示了FGF2低聚体形成的膜孔周围的脂类纳米结构域的潜在作用。最后，我们的目标是在一个新的基于细胞的系统中验证重构实验的结果。因此，这些研究具有揭示膜插入型FGF2低聚体功能结构的重要参数的强大潜力，因此，我们期待从这些研究中，我们对这些结构作为FGF2非传统分泌途径的中间体的功能作用的理解取得根本性进展。

Fibroblast Growth Factor 2 (FGF2) is a strong mitogen promoting angiogenesis in health and disease. Under pathological conditions it is functioning as a major activator of tumor-induced angiogenesis and also acts as a survival factor of tumor cells mediated by an autocrine signaling loop suppressing apoptosis. Despite its defined extracellular functions, FGF2 lacks a signal peptide and, therefore, is exported from cells by an unconventional, ER/Golgi-independent mechanism of protein secretion. Understanding the molecular mechanism by which FGF2 is secreted from tumor cells may pave the way to develop a new class of anti-angiogenic inhibitors with a high potential in cancer therapy.FGF2 secretion from cells was found not to involve intracellular vesicle intermediates but rather is mediated by direct translocation across the plasma membrane. This process is initiated by the recruitment of FGF2 at the inner leaflet of the plasma membrane mediated by the phosphoinositide PI(4,5)P2. This interaction causes FGF2 to oligomerize and, stimulated by phosphorylation of FGF2 at a specific surface tyrosine residue, results in membrane insertion of a FGF2 oligomer concomitant with the formation of a toroidal membrane pore. This structure has been interpreted as an intermediate in FGF2 membrane translocation. In a final step, cell surface heparan sulfate proteoglycans trap FGF2 molecules at the outer leaflet resulting in translocation of FGF2 molecules into the extracellular space.The goal of this research proposal is to follow an interdisciplinary approach at the interface between cell biology, biochemistry and biophysics. It aims at the molecular analysis of the structure function relationship of FGF2 oligomers to obtain insight into how a lipidic membrane pore can be formed induced by PI(4,5)P2-dependent FGF2 oligomerization. Specifically, we plan to follow a single molecule approach to be able to identify and analyze functional FGF2 oligomers forming membrane pores both in vitro and in a novel cell-based system. These studies aim at i.) investigating the number of FGF2 molecules engaged in pore-forming FGF2 oligomers and the size of the membrane pore associated with FGF2 oligomers, ii.) determining the distance between individual FGF2 molecules within pore-forming oligomers, iii.) analyzing their dynamics and the lifetime of membrane pores and iv.) revealing a potential role for lipidic nano-domains surrounding membrane pores formed by FGF2 oligomers. Finally, we aim at validating the results from reconstitution experiments in a novel cell-based system. These studies thus have a strong potential to reveal important parameters of the functional architecture of membrane-inserted FGF2 oligomers and, therefore, from these studies, we expect fundamental advances in our understanding of the functional role of these structures as intermediates of the unconventional secretory pathway of FGF2.