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

1-磷酸鞘氨醇转运蛋白作为免疫系统调节和癌症关键治疗靶点的结构动力学

• 批准号: 10586751
• 负责人: Reza Dastvan
• 金额: $ 40.81万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-20 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586751
• 关键词: Adopted; Antibiotics; Binding Sites; Biological Assay; Cations; Cell membrane; Cells; Cellular Membrane; Coupled; Coupling; Cryoelectron Microscopy; Data; Defect; Detergents; Development; Disease; Drug Targeting; Electron Spin Resonance Spectroscopy; Electrons; Endothelial Cells; Family; Family member; Foundations; Goals; Growth; Homologous Gene; Human; Immune system; Immunosuppressive Agents; Immunotherapy; Impairment; In Vitro; Inorganic Phosphate Transporter; Ions; Ligands; Lipid Bilayers; Lipids; Lymphocyte; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Membrane; Membrane Lipids; Micelles; Modeling; Molecular Conformation; Multi-Drug Resistance; Mus; Mutation; Mycobacterium smegmatis; Neoplasm Metastasis; Neptunium; Physiologic pulse; Physiological; Play; Poison; Proteins; Protons; Public Health; Regulation; Research; Resolution; Role; Sampling; Signal Transduction; Spectrum Analysis; Sphingolipids; Spin Labels; Structure; Techniques; Testing; Therapeutic; biophysical techniques; cell motility; conformational conversion; efflux pump; extracellular; feasibility research; flexibility; genome-wide; in vivo; innovation; migration; mimetics; model building; molecular dynamics; molecular modeling; mutant; nanodisk; nanoscale; novel therapeutic intervention; particle; prevent; protonation; restraint; simulation; sphingosine 1-phosphate; stoichiometry; structural determinants; therapeutic target; tumor

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.

项目概要/摘要 生物活性脂质 1-磷酸鞘氨醇 (S1P) 在调节生长、存活和 哺乳动物细胞的迁移。 S1P在细胞内产生，然后释放到细胞外以参与其作用 （病理）生理作用。主要促进子超家族 (MFS) 的 Spinster (Spns) 脂质转运蛋白是 对于跨细胞膜运输 S1P 至关重要。在人类的三种 Spns 蛋白中，Spns2 发挥作用 作为主要的 S1P 转运蛋白，这使其成为调节 S1P 输出和信号传导的潜在药物靶点。一个 Spns2 内皮细胞特异性缺陷导致淋巴细胞流出受损并阻止肿瘤转移 在小鼠中，强烈表明 Spns2 可能是通过增加 免疫治疗的疗效。因此，Spns2机制的详细表征对于 针对与 S1P 信号传导相关的疾病并针对 Spns2 开发新的治疗策略 作为一种潜在的免疫抑制剂。该提案的总体目标是定义 Spns 鞘脂转运蛋白家族。 Spns2介导的S1P跨细胞转运机制 膜仍然知之甚少，主要是由于缺乏结构信息（目标 1 和 2）。在 此外，Spns2调控的精确机制仍不清楚（目标3）。我们最近定义了质子 细菌 Spns 转运蛋白的依赖构象动力学。我们的方法利用了强大的 脉冲 EPR 技术称为双电子电子共振 (DEER) 光谱，是一种有效的 纳米尺度的标尺，在高分辨率结构的背景下。它是通过功能研究和 通过协作分子建模进行情境化。使用这种综合方法，我们进行了彻底的 人类 Spns2 及其同源物之间的机制比较。该提案的目标是定义 人 Spns2 及其细菌同源物在脂质中的阳离子和底物耦合构象循环 双层。为了确定交替访问机制中涉及的构象状态，我们将应用 在预期稳定传输中间体的条件下进行 DEER 光谱并将结果与 约束辅助分子动力学绘制配体偶联构象变化图。使用类似的集成 方法来定义其他 Spns 家族成员及其原核同源物的转运机制，我们 将确定其机制的关键共同点和差异，强调机制的灵活性 使其具有多样化的功能并具有变革性的治疗潜力。