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Abstract：
Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.
摘要：
成纤维细胞生长因子2（FGF2）通过直接转运穿过质膜离开细胞，非常规蛋白质分泌的I型途径。此过程由磷脂酰肌醇-4,5-二磷酸（PI（4,5）P2）依赖性的高动态FGF2低聚物在内部质膜小叶上的形成引发，诱导脂质膜孔的形成。与磷脂酰肌醇蛋白聚糖-1（GPC1）连接的细胞表面硫酸乙酰肝素链在外质膜小叶捕获FGF2，完成FGF2膜转位到细胞外空间。虽然这种途径的基本步骤是众所周知的，FGF2在膜表面寡聚化的分子机制尚不清楚。在目前的研究中,我们证明了该过程的初始步骤取决于膜表面上的C95-C95二硫键介导的FGF2二聚化，产生驱动膜孔形成的较高FGF2低聚物的结构单元。我们发现具有C95A取代的FGF2在低聚反应中是有缺陷的，孔隙形成,和膜易位。始终如一，我们证明了FGF2的C95A变体以严重的分泌表型为特征。相比之下,同时对细胞有效分泌FGF2也很重要，FGF2(C77)分子表面上的第二个半胱氨酸残基不参与FGF2寡聚化。相反，我们发现C77是相互作用界面的一部分，FGF2通过该界面与Na的α1亚基结合，K-ATP酶,FGF2在内质膜小叶的着陆平台。使用交联质谱,结合机器学习分析和低温电子层析成像的原子分子动力学模拟，我们提出了二硫键桥连的FGF2二聚体以高亲和力结合膜表面上的PI(4,5)P2的机制。我们进一步提出了FGF2分泌与细胞表面三元信号复合物形成之间的紧密耦合，假设C95-C95桥接的FGF2二聚体作为触发自分泌和旁分泌FGF2信号传导的分子单元起作用。
