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)是一种细胞存活因子，介导肿瘤诱导的血管生成。然而，与大多数细胞外定位的沿ER/高尔基体依赖的分泌途径运输的蛋白质不同，FGF2缺乏信号肽，并被证明是通过直接蛋白质跨质膜转位从细胞分泌的。这种非常规分泌途径背后的分子机制以及涉及的分子成分的几个关键方面已经被揭示。细胞分泌FGF2的特征是：(1)FGF2与ATP1A1、Tec激酶和PI(4，5)P2在质膜内叶的顺序相互作用；(Ii)Tec激酶介导的FGF2酪氨酸磷酸化；(Iii)依赖PI(4，5)P2的FGF2寡聚体的形成和膜插入；(Iv)由细胞表面硫酸乙酰肝素蛋白多糖介导的FGF2寡聚体的分解和细胞外捕获。一些证据表明，PI(4，5)P2诱导的FGF2寡聚触发了具有环形结构的脂质膜孔的形成。作为这种排列的一部分，PI(4，5)P2的头基在外围形成亲水性表面，与容纳在脂质膜孔中心的FGF2低聚体物理相互作用。基于细胞质小叶上FGF2单位的增加和外叶上FGF2单位的去除，提出了一种组装/拆卸机制来介导FGF2在质膜上的定向运输。后者是由细胞表面的乙酰肝素硫酸盐驱动的，它在与外层小叶的FGF2相互作用方面胜过PI(4，5)P2，导致FGF2在细胞表面积累。在德国-捷克合作计划的前一轮融资中，我们首次深入了解了在单分子水平上分析的膜插入型FGF2低聚物的结构-功能关系。在这里，我们以这项工作为基础，旨在(I)确定单个FGF2易位中间体的精确低聚状态，(Ii)分析ATP1A1在启动FGF2寡聚中的潜在作用，以及(Iii)研究硫酸肝素在促进FGF2易位中间体形成中的作用。在整个研究过程中，我们将使用最先进的单分子技术，并期望以前所未有的详细程度对FGF2膜转位的分子机制有实质性的新见解。因此，我们的研究将有助于我们理解肿瘤细胞以ER/高尔基体非依赖性方式分泌FGF2的分子机制。

Fibroblast Growth Factor 2 (FGF2) is a cell survival factor mediating tumor-induced angiogenesis. However, as opposed to the majority of extracellularly localized proteins that travel along the ER/Golgi dependent secretory pathway, FGF2 lacks a signal peptide and was shown to be secreted from cells by direct protein translocation across the plasma membrane. Several key aspects of the molecular mechanism behind this unconventional secretory pathway as well as the molecular components involved have been revealed. FGF2 secretion from cells is characterized by (i) sequential interactions of FGF2 with ATP1A1, Tec kinase and PI(4,5)P2 at the inner leaflet of the plasma membrane, (ii) Tec kinase mediated tyrosine phosphorylation of FGF2, (iii) PI(4,5)P2-dependent formation and membrane insertion of FGF2 oligomers and (iv) disassembly of oligomers and extracellular trapping of FGF2 mediated by cell surface heparan sulfate proteoglycans. Several lines of evidence suggest that PI(4,5)P2-induced oligomerization of FGF2 triggers the formation of lipidic membrane pores with a toroidal architecture. As part of this arrangement, the headgroups of PI(4,5)P2 contribute a hydrophilic surface in the periphery physically interacting with the FGF2 oligomer that is accommodated in the center of the lipidic membrane pore. Based upon the addition of FGF2 units at the cytoplasmic leaflet and removal of FGF2 units at the outer leaflet, an assembly/disassembly mechanism has been proposed to mediate directional transport of FGF2 across the plasma membrane. The latter process is driven by cell surface heparan sulfates that outcompete PI(4,5)P2 for the interaction with FGF2 at the outer leaflet resulting in accumulation of FGF2 on cell surfaces. In a previous funding round of the German-Czech Cooperation Programme, we obtained the first insights into the structure-function relationship of membrane-inserted FGF2 oligomers analyzed at the single molecule level. Here, we build upon this work aiming at (i) the determination of the precise oligomeric state of individual FGF2 translocation intermediates, (ii) the analysis of a potential role of ATP1A1 in initiating oligomerization of FGF2 and (iii) studying the role of heparan sulfates in facilitating the formation of FGF2 translocation intermediates. Throughout this study, we will use state-of-the art single molecule techniques and expect to gain substantial new insight into the molecular mechanism of FGF2 membrane translocation at an unprecedented level of detail. Therefore, our studies will make a significant contribution to our understanding of the molecular mechanism by which tumor cells secrete FGF2 in an ER/Golgi-independent manner.