Structural dynamics of sphingosine-1-phosphate transporters as key therapeutic targets for immune system modulation and cancer

项目摘要/摘要 生物活性脂鞘氨醇-1-磷酸(S1P)在调节鱼体的生长、存活和发育过程中起着关键作用。 哺乳动物细胞的迁移。S1P在细胞内产生，然后在细胞外释放，参与其 (病态)生理作用。主要促进剂超家族(MFS)的Spinster(SPN)脂质转运体是 对于跨细胞膜运输S1P至关重要。在人类的三种SPN蛋白中，Spns2的功能 作为主要的S1P转运体，使其成为调节S1P输出和信号转导的潜在药物靶点。一个 Spns2基因内皮细胞特异性缺陷导致淋巴细胞出口受损并阻止肿瘤转移 在小鼠中，强烈表明Spns2可能是一个有效的靶点，通过增加 免疫治疗的疗效。因此，对Spns2机制的详细描述对 针对S1P信号转导相关疾病和靶向Spns2的新治疗策略的发展 作为一种潜在的免疫抑制剂。这项提案的总体目标是界定 SPN家族的鞘脂转运蛋白。Spns2介导的S1P跨细胞转运机制 膜仍然知之甚少，主要原因是缺乏结构信息(目标1和2)。在……里面 此外，Spns2调控的确切机制仍不清楚(目标3)。我们最近给质子下了定义- 细菌SPN转运蛋白的依赖构象动力学。我们的方法利用了一种强大的 脉冲EPR技术被称为双电子电子共振(DER)谱，是一种有效的 纳米尺度的尺子，在高分辨率结构的背景下。它是由功能研究和 通过协作性分子建模进行情景分析。使用这种综合方法，我们进行了彻底的 人类Spns2及其同源物的机制比较。这项提案的目标是界定 人Spns2及其细菌同系物在脂质中的阳离子和底物偶联构象循环 双层的。为了确定交替访问机制中涉及的构象状态，我们将应用 在预期稳定运输中间体的条件下进行鹿谱分析，并将结果与 约束辅助分子动力学绘制配基偶联构象变化图。使用类似的集成 为了确定其他SPNS家族成员及其原核同源物的转运机制，我们 将确定其机制中的关键共性和差异，突出机制的灵活性 使它们的不同功能具有变革性的治疗潜力。

Project Summary/Abstract The bioactive lipid sphingosine-1-phosphate (S1P) plays a key role in regulating the growth, survival and migration of mammalian cells. S1P is produced intracellularly and then released extracellularly to engage in its (patho)physiological roles. The Spinster (Spns) lipid transporters of the major facilitator superfamily (MFS) are critical for transporting S1P across cellular membranes. Of the three Spns proteins in humans, Spns2 functions as the main S1P transporter, which makes it a potential drug target for modulating S1P export and signaling. An endothelial cell-specific defect in Spns2 results in impaired egress of lymphocytes and prevents tumor metastasis in mice, strongly suggesting that Spns2 could be an effective target for reducing metastases by increasing the efficacy of immunotherapy. Thus, detailed characterization of the Spns2 mechanism is of high significance for the development of novel therapeutic strategies for diseases associated with S1P signaling and to target Spns2 as a potential immunosuppressant. The overall goal of this proposal is to define the functional mechanism of the Spns family of sphingolipid transporters. The mechanism of Spns2-mediated S1P transport across cellular membrane remains poorly understood, mainly due to the lack of structural information (Aims 1 and 2). In addition, the precise mechanism of Spns2 regulation is still unclear (Aim 3). We recently defined the proton- dependent conformational dynamics of a bacterial Spns transporter. Our approach capitalizes on a powerful pulsed EPR technique known as Double Electron Electron Resonance (DEER) spectroscopy, an effective nanometer-scale ruler, in the context of high-resolution structures. It is informed by functional studies and contextualized through collaborative molecular modeling. Using this integrated approach, we conduct a thorough mechanistic comparison between human Spns2 and its homologs. The objectives of this proposal are to define the cation- and substrate-coupled conformational cycle of human Spns2 and its bacterial homologs in lipid bilayers. To determine the conformational states involved in the alternating access mechanism, we will apply DEER spectroscopy under conditions expected to stabilize transport intermediates and combine the results with restraint-assisted molecular dynamics to map ligand-coupled conformational changes. Using a similar integrated approach to define the transport mechanism of other Spns family members and their prokaryotic homologs, we will identify the key commonalities and differences in their mechanisms, highlighting the mechanistic flexibility enabling their diverse function with transformative therapeutic potential.