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

探索膜孔形成 FGF2 寡聚物的结构功能关系 - 单分子方法

• 批准号: 246506239
• 负责人: Professor Dr. Walter Nickel
• 金额: --
• 依托单位: J. Heyrovsky Institute of Physical Chemistry
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/246506239?language=en
• 关键词: Exploring; structure; function; relationship; membrane

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.

成纤维细胞生长因子2（FGF 2）是促进健康和疾病中血管生成的强有丝分裂原。 在病理条件下，它作为肿瘤诱导的血管生成的主要激活剂发挥作用，并且还作为由抑制细胞凋亡的自分泌信号环介导的肿瘤细胞的存活因子。 尽管FGF 2具有明确的细胞外功能，但它缺乏信号肽，因此通过非常规的ER/Golgi非依赖性蛋白分泌机制从细胞中输出。了解肿瘤细胞分泌FGF 2的分子机制可能为开发新的抗血管生成抑制剂铺平道路，这类抑制剂在癌症治疗中具有很高的潜力。 该过程由磷酸肌醇PI（4，5）P2介导的质膜内小叶处的FGF 2募集启动。 这种相互作用导致FGF 2寡聚化，并且通过FGF 2在特定表面酪氨酸残基处的磷酸化刺激，导致FGF 2寡聚体的膜插入，同时形成环形膜孔。 这种结构已被解释为FGF 2膜易位的中间体。 在最后一步中，细胞表面硫酸乙酰肝素蛋白聚糖捕获FGF 2分子在外部小叶导致FGF 2分子易位到细胞外空间。本研究提案的目标是在细胞生物学，生物化学和生物物理学之间的接口遵循跨学科的方法。 目的是从分子水平分析FGF 2寡聚体的结构与功能关系，以了解PI（4，5）P2依赖性FGF 2寡聚化是如何诱导细胞膜孔形成的。 具体来说，我们计划遵循单分子方法，以便能够在体外和基于细胞的新系统中鉴定和分析形成膜孔的功能性FGF 2寡聚体。 这些研究旨在i.）研究参与成孔FGF 2寡聚体的FGF 2分子的数量和与FGF 2寡聚体相关的膜孔的尺寸，ii.）确定成孔寡聚体内的单个FGF 2分子之间的距离，iii.）分析它们的动力学和膜孔的寿命，以及iv.）揭示了围绕由FGF 2寡聚体形成的膜孔的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，这些研究具有很强的潜力，以揭示膜插入的FGF 2寡聚体的功能结构的重要参数，因此，从这些研究中，我们期望在我们的理解这些结构的功能作用的FGF 2的非常规分泌途径的中间体的根本进展。