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.

项目摘要/摘要 生物活性脂质鞘氨醇1-磷酸（S1P）在调节生长，生存和 哺乳动物细胞的迁移。 S1P细胞内产生，然后细胞外释放以参与其 （病原）生理角色。主要促进剂超家族（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功能 作为主要的S1P转运蛋白，它使其成为调节S1P导出和信号传导的潜在药物靶标。一个 SPNS2中的内皮细胞特异性缺陷导致淋巴细胞的出口受损并防止肿瘤转移 在小鼠中，强烈表明SPNS2可能是通过增加转移的有效目标 免疫疗法的功效。因此，SPNS2机制的详细表征对 与S1P信号相关的疾病的新型治疗策略的发展和靶向SPNS2 作为潜在的免疫抑制剂。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跨细胞的机理 膜的理解仍然很差，主要是由于缺乏结构信息（目标1和2）。在 此外，SPNS2调节的确切机制仍不清楚（AIM 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。我们的方法利用了一个强大的 脉冲EPR技术称为双电子电子共振（DEER）光谱，有效 在高分辨率结构的背景下，纳米尺度的统治者。它通过功能研究和 通过协作分子建模进行背景。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械比较。该提议的目标是定义 人SPNS2的阳离子和底物耦合构象周期及其脂质中的细菌同源物 双层。为了确定交替访问机制所涉及的构象状态，我们将申请 在预期稳定运输中间体的条件下，鹿光谱法并将结果与 约束辅助分子动力学以绘制配体耦合构象变化。使用类似的集成 定义其他SPNS家族成员及其原核同源物的运输机制的方法，我们 将确定其机制的关键共同点和差异，突出了机械灵活性 具有变革性的治疗潜力，使其多样化的功能。