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（FGF 2）是介导肿瘤诱导的血管生成的细胞存活因子。 然而，与大多数沿着ER/高尔基体依赖性分泌途径行进的细胞外定位的蛋白质相反，FGF 2缺乏信号肽，并且显示通过直接蛋白质易位穿过质膜从细胞分泌。 这一非常规分泌途径背后的分子机制以及所涉及的分子组分的几个关键方面已经被揭示。FGF 2从细胞分泌的特征在于（i）FGF 2与质膜内小叶处的ATP 1A 1、Tec激酶和PI（4，5）P2的顺序相互作用，（ii）Tec激酶介导的FGF 2的酪氨酸磷酸化，（iii）PI（4，5）P2，5）FGF 2寡聚体的P2依赖性形成和膜插入，以及（iv）细胞表面硫酸乙酰肝素蛋白聚糖介导的寡聚体的分解和FGF 2的细胞外捕获。几条证据表明，PI（4，5）P2诱导的FGF 2寡聚化触发了具有环形结构的血管膜孔的形成。作为这种排列的一部分，P1（4，5）P2的头基在外周中提供亲水性表面，与容纳在血管膜孔中心的FGF 2低聚物物理相互作用。基于在细胞质小叶处添加FGF 2单位和在外小叶处去除FGF 2单位，已经提出了一种组装/拆卸机制来介导FGF 2穿过质膜的定向转运。 后一过程由细胞表面硫酸乙酰肝素驱动，其在与外部小叶处的FGF 2的相互作用方面胜过PI（4，5）P2，导致FGF 2在细胞表面上的积累。 在德国-捷克合作计划的上一轮资助中，我们首次深入了解了在单分子水平上分析的膜插入的FGF 2寡聚体的结构-功能关系。在这里，我们建立在这项工作的目的是（i）确定的精确寡聚状态的个别FGF 2易位中间体，（ii）分析的潜在作用的ATP 1A 1在启动寡聚化的FGF 2和（iii）研究硫酸乙酰肝素的作用，促进FGF 2易位中间体的形成。 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细节水平获得对FGF 2膜转位分子机制的实质性新见解。因此，我们的研究将为我们理解肿瘤细胞以ER/Golgi非依赖性方式分泌FGF 2的分子机制做出重大贡献。

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.