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)是一种参与肿瘤血管生成的生存因子。FGF2通过阻断细胞凋亡的自分泌信号级联反应，进一步介导肿瘤细胞对抗癌治疗的化疗耐药性。这些信号通路的启动发生在细胞表面，需要将FGF2运输到细胞外空间。这一过程是由非常规蛋白分泌(UPS)介导的，FGF2属于I型UPS货物家族，通过直接跨质膜转位分泌。这种分泌机制的关键成分是磷脂酰肌醇PI(4，5)P2，这是一种位于质膜内叶的膜脂，它触发FGF2齐聚，并伴随着脂质膜孔的形成。然后，在硫酸乙酰肝素蛋白多糖的介导下，膜插入的FGF2寡聚体在质膜的外层小叶被移除和分解。这一过程导致FGF2分子移位到细胞外间隙，在那里它们参与了成纤维细胞生长因子受体的激活和信号传递。尽管FGF2膜转位的核心过程的分子机制已经有了相当详细的了解，但这一途径的其他组件的确切功能仍然是未知的。Na，K-ATPase在这一过程中的参与已经通过三种不同的方法学途径得到证明。首先，哇巴因是一种Na，K-ATPase的药物抑制剂，它会损害FGF2的分泌。其次，RNA干扰介导的Na，K-α酶亚基下调抑制了FGF2的分泌。第三，已经证明成纤维细胞生长因子2与Na，K-α酶亚基的细胞质结构域发生物理作用。不能与Na，K-ATPase这一部分结合的不同形式的FGF2在分泌效率上受到损害。然而，尽管这些发现证实了Na，K-ATPase在非常规分泌FGF2中的作用，但它们并没有直接揭示其作用机制。这项研究的总体目标是检验存在偶联机制的假设，通过该偶联机制，FGF2在膜转位的局部事件中调节Na，K-ATPase的活性，这一过程涉及质膜中脂质膜孔的瞬时形成。这样，在这一过程中可能发生的膜电位扰动可以通过FGF2诱导的Na，K-ATPase酶活性的激活来补偿。因此，通过高度跨学科的方法，结合广泛的方法学方法，包括结构生物学、生化重建、分子动力学模拟和先进成像技术等理论方法，以及几个国际合作的支持，我们旨在阐明Na，K-ATPase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.