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

剖析 Na,K-ATP 酶在成纤维细胞生长因子 2 非常规分泌中的作用

• 批准号: 460555261
• 负责人: Professor Dr. Walter Nickel
• 金额: --
• 依托单位: Department of Chemistry
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/460555261?language=en
• 关键词: Dissecting; role; Na; ATPase; unconventional

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.

成纤维细胞生长因子2（FGF 2）是参与肿瘤诱导的血管生成的存活因子。 FGF 2还基于阻断细胞凋亡的自分泌信号级联介导肿瘤细胞对抗癌疗法的化学抗性。 这些信号传导途径的起始发生在细胞表面，需要将FGF 2转运到细胞外空间。 该过程由非常规蛋白质分泌（UPS）介导，其中FGF 2属于I型UPS货物家族，其通过跨质膜的直接易位分泌。 这种分泌机制的关键组分是磷酸肌醇PI（4，5）P2，其是质膜内小叶处的膜脂质，其触发FGF 2寡聚化，同时形成胞膜孔。 膜插入的FGF 2寡聚体然后被去除，并在硫酸乙酰肝素蛋白聚糖介导的质膜的外小叶处分解。 这一过程导致FGF 2分子易位到细胞外空间，在那里它们参与FGF受体的活化和信号转导。虽然FGF 2膜易位的核心过程的分子机制被相当详细地理解，但该途径的其他组分的精确功能仍然难以捉摸。 Na，K-ATP酶在这一过程中的参与已经通过三种不同的方法学途径证明。 首先，哇巴因，Na，K-ATP酶的药理学抑制剂损害FGF 2分泌。 第二，RNA干扰介导的Na，K-ATP酶α亚基的下调抑制FGF 2的分泌。 第三，已经证明FGF 2与Na，K-ATP酶的α亚基的胞质结构域进行物理相互作用。 不能与Na，K-ATP酶的该部分结合的FGF 2的变体形式的分泌效率受损。 然而，尽管这些发现确立了Na，K-ATP酶在FGF 2的非常规分泌中的作用，但它们并没有直接揭示其作用机制。本研究提案的总体目标是检验存在一种偶联机制的假设，通过该偶联机制，FGF 2在膜易位的局部事件中调节Na，K-ATP酶的活性，这一过程涉及质膜中膜孔的瞬时形成。 以这种方式，在该过程中可能发生的膜电位的潜在扰动可以通过FGF 2诱导的Na，K-ATP酶的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，生物化学重建，理论方法，如分子动力学模拟和先进的成像技术，以及支持的几个国际合作，我们的目标是阐明的Na，K-ATP酶的功能，在非常规的分泌FGF 2。