Dissecting the role of the Na,K-ATPase in unconventional secretion of Fibroblast Growth Factor 2

成纤维细胞生长因子2 （FGF2）是一种参与肿瘤诱导血管生成的存活因子。FGF2通过阻断细胞凋亡的自分泌信号级联进一步介导肿瘤细胞对抗癌治疗的化疗耐药。这些信号通路的启动发生在细胞表面，需要将FGF2转运到细胞外空间。这一过程是由非常规蛋白分泌（UPS）介导的，FGF2属于I型UPS货运家族，通过直接跨质膜转运分泌。该分泌机制的一个关键组成部分是磷脂肌肽PI(4,5)P2，它是质膜内小叶的一种膜脂，可触发FGF2低聚，同时形成脂质膜孔。膜插入的FGF2低聚物随后被移除，并在质膜的外小叶上被硫酸肝素蛋白聚糖介导分解。这个过程导致FGF2分子易位到细胞外空间，在那里它们参与FGF受体的激活和信号传导。虽然FGF2膜易位核心过程的分子机制已经得到了相当详细的了解，但该途径中其他成分的确切功能仍然难以捉摸。Na， k - atp酶在这一过程中的参与已经被三种不同的方法方法所证明。首先，钠钾atp酶的药理学抑制剂瓦巴因会损害FGF2的分泌。其次，rnai介导的Na， k - atp酶α亚基的下调抑制FGF2的分泌。第三，FGF2已被证明与Na， k - atp酶的α亚基细胞质结构域发生物理相互作用。不能与Na， k - atp酶的这部分结合的FGF2的变体形式在分泌效率上受损。然而，尽管这些发现确定了Na， k - atp酶在FGF2非常规分泌中的作用，但它们并没有直接揭示其作用机制。本研究计划的总体目标是验证FGF2在膜易位的局部事件中调节Na， k - atp酶活性的耦合机制的存在，这一过程涉及质膜中脂质膜孔的短暂形成。这样，在这个过程中可能发生的膜电位的潜在扰动可以通过fgf2诱导的Na， k - atp酶活性的激活来补偿。因此，通过高度跨学科的方法，包括结构生物学、生化重建、分子动力学模拟等理论方法和先进的成像技术，以及几个国际合作的支持，我们的目标是阐明Na， k - atp酶在FGF2非常规分泌中的功能。

Fibroblast Growth Factor 2 (FGF2) is a survival factor involved in tumor-induced angiogenesis. FGF2 further mediates chemoresistance of tumor cells towards anti-cancer therapies based on an autocrine signaling cascade blocking apoptosis. The initiation of these signaling pathways occurs at cell surfaces requiring transport of FGF2 into the extracellular space. This process is mediated by unconventional protein secretion (UPS) with FGF2 belonging to the Type I UPS cargo family that is secreted by direct translocation across plasma membranes. A key component of this secretory mechanism is the phosphoinositide PI(4,5)P2, a membrane lipid at the inner leaflet of the plasma membrane that triggers FGF2 to oligomerize concomitant with the formation of a lipidic membrane pore. Membrane-inserted FGF2 oligomers are then removed and disassembled at the outer leaflet of the plasma membrane mediated by heparan sulfate proteoglycans. This process results in the translocation of FGF2 molecules into the extracellular space where they engage in FGF receptor activation and signaling.While the molecular mechanism of the core process of FGF2 membrane translocation is understood in quite some detail, the precise function of additional components of this pathway remained elusive. The participation of the Na,K-ATPase in this process has been demonstrated by three different methodological approaches. First, ouabain, a pharmacological inhibitor of the Na,K-ATPase impairs FGF2 secretion. Second, RNAi-mediated down-regulation of the α subunit of the Na,K-ATPase inhibits FGF2 secretion. Third, FGF2 has been demonstrated to engage in a physical interaction with the cytoplasmic domain of the α subunit of the Na,K-ATPase. Variant forms of FGF2 that cannot bind to this part of the Na,K-ATPase are impaired in secretion efficiency. However, while these findings established a role of the Na,K-ATPase in unconventional secretion of FGF2, they did not directly reveal its mechanism of action.The overarching goal of this research proposal is to test the hypothesis for the existence of a coupling mechanism by which FGF2 regulates the activity of the Na,K-ATPase at localized events of membrane translocation, a process that involves the transient formation of lipidic membrane pores in the plasma membrane. In this way, potential perturbations of the membrane potential that may occur during this process could be compensated through an FGF2-induced activation of the enzymatic activity of the Na,K-ATPase. Thus, through a highly interdisciplinary approach with a broad range of methodological approaches including structural biology, biochemical reconstitution, theoretical approaches such as molecular dynamics simulations and advanced imaging techniques as well as supported by several international collaborations, we aim at elucidating the function of the Na,K-ATPase in unconventional secretion of FGF2.