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）是一种促进健康和疾病血管生成的强丝裂原。在病理条件下，它作为肿瘤诱导血管生成的主要激活因子，也作为肿瘤细胞的生存因子，通过抑制凋亡的自分泌信号环介导。尽管具有明确的细胞外功能，但FGF2缺乏信号肽，因此通过非常规的、不依赖于内质网/高尔基蛋白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的抗血管生成抑制剂铺平道路，这些抑制剂在癌症治疗中具有很高的潜力。发现细胞分泌FGF2不涉及细胞内囊泡中间体，而是通过跨质膜的直接转运介导。这一过程是由磷脂肌肽PI(4,5)P2介导的FGF2在质膜内小叶的募集启动的。这种相互作用导致FGF2寡聚，并且在特定表面酪氨酸残基上FGF2磷酸化的刺激下，导致FGF2寡聚物插入膜，同时形成环形膜孔。该结构被解释为FGF2膜易位的中间物。在最后一步中，细胞表面硫酸肝素蛋白聚糖将FGF2分子捕获在外叶，导致FGF2分子易位到细胞外空间。本研究计划的目标是在细胞生物学，生物化学和生物物理学之间的界面上遵循跨学科的方法。旨在对FGF2低聚物的结构功能关系进行分子分析，以了解PI(4,5) p2依赖性FGF2低聚是如何诱导脂质膜孔形成的。具体来说，我们计划采用单分子方法来鉴定和分析体外和新型细胞系统中形成膜孔的功能性FGF2低聚物。这些研究的目的是：(1)调查参与成孔FGF2低聚物的FGF2分子的数量和与FGF2低聚物相关的膜孔的大小；(2)确定成孔低聚物中单个FGF2分子之间的距离；(3)分析它们的动力学和膜孔的寿命；(4)揭示FGF2低聚物形成的膜孔周围脂质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组实验的结果。因此，这些研究有很强的潜力揭示膜插入FGF2低聚物功能结构的重要参数，因此，从这些研究中，我们期望在我们对这些结构作为FGF2非常规分泌途径的中间体的功能作用的理解上取得根本性的进展。

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.