Cellular lipid transport determined with multifunctional lipid derivatives

用多功能脂质衍生物测定细胞脂质转运

• 批准号: 10742142
• 负责人: Carsten Schultz
• 金额: $ 4.16万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10742142
• 关键词: Alkynes; Binding Proteins; Biochemical; Biophysics; Carrier Proteins; Cell membrane; Cell physiology; Cells; Chemistry; Diazomethane; Disease; Epidermal Growth Factor Receptor; Exhibits; Funding; Genes; Glycerophospholipids; In Vitro; Interphase; Investigation; Light; Lipid Binding; Lipids; Location; Malignant Neoplasms; Mass Spectrum Analysis; Membrane; Methods; Microscopy; Multiple Sclerosis; PH Domain; Phosphatidylinositols; Point Mutation; Proteins; Proteomics; Publishing; Recombinant Proteins; Signal Transduction; Site; Small Interfering RNA; Specificity; Techniques; Testing; Work; comparative; improved; knock-down; lipid transport; new therapeutic target; phosphatidylinositol 3,4,5-triphosphate; phosphatidylinositol 3,4-diphosphate; receptor

Summary It is well understood how lipids are synthesized and metabolized in cells and that many lipids exhibit signalling functions to regulate cellular processes in a spatially and temporally defined way. The latter requires the build-up and turnover of lipid species in membranes either in a site-specific fashion or, alternatively, a directed form of lipid transport. This work aims to investigate the intracellular transfer of lipids from one membrane to another by several proteins that we discovered to be involved in lipid transport. In the previous funding period, we synthesized multifunctional lipid derivatives of five phosphoinositides and four common glycerophospholipids. These feature a photo-activatable protecting group (”cage”) to release the lipid derivative by light and a photo-crosslinking diazirine to covalently attach the lipid derivative to binding proteins. An alkyne group for click chemistry is useful for isolating lipid-protein conjugates or for determining the lipid location in cells by fluorescent tagging and microscopy. In published work, we identified specific lipid binding proteins for phosphatidylinositol 3,4,5-trisphosphate (PIP3), phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], and phosphatidylinositol (PI). Because the “caged” derivatives accumulated in endomembranes, we observed changes in their subsequent cellular distribution after uncaging with light. All three phosphoinositides transferred to the plasma membrane (PM) within 30 to 120 sec. Such transport is known for PI but has never been described for PIP3 or PI(3,4)P2. We then identified putative lipid transport proteins via proteomic analysis and used siRNAs to block lipid transport. We found two hits that were required for transporting PIP3 and PI(3,4)P2: cytosolic MPP6 and transmembrane ATP11A. Knockdown of both reduced internalization of EGF receptor, indicating effects on PIP3 signalling. In this work, we will characterize the lipid transport by these two proteins with respect to lipid specificity (Aim 1.1). For this aim, we will improve our current method of precisely quantifying lipid transport in cells (Aim 1.2). We will validate our findings in-vitro by using recombinant proteins including those with point mutations of key residues to study protein-lipid interactions with biophysical and biochemical methods (Aim 1.3). We will increase rigor by analysing the cellular lipid composition by mass spectrometry after uncaging lipid derivatives (Aim 1.4). In Aim 2, we will demonstrate the need of MPP6 mobility for lipid transport. We will develop a light-switchable MPP6 using the LOV2 technique that will replace endogenous MPP6 (by gene editing) and will be located at the plasma membrane (PM) until we illuminate the cells with 488 nm light. We hypothesize that lipid transport will only be possible if MPP6 is liberated from the PM. We will also test for lipid retro-transport by MPP6 from the PM to endomembranes. For this, we will synthesize lipid derivatives that accumulate at the outer leaflet of the PM and induce cell entry and transport via uncaging. In Aim 3, we will synthesize lipid derivatives featuring the photo-crosslinking diazirine closer to the membrane interphase to reach more transiently binding proteins such as those with a PH domain. Comparative proteomic analysis of the lipid interactomes will then be used to identify proteins involved in signalling with and without receptor stimulation.

概括 众所周知，脂质如何合成和代谢在细胞中，并且许多脂质暴露了信号传导功能 以空间和临时定义的方式调节细胞过程。以后需要建立和营业额 膜中的脂质物种要么以特定于位置的方式，要么是脂质转运的定向形式。这项工作 旨在通过几种蛋白质研究脂质从一种膜到另一种蛋白的细胞内转移 被发现参与脂质运输。在上一个资金期间，我们合成了多功能脂质 五个磷酸肌醇和四个常见甘油磷脂的衍生物。这些功能可以使照片激活 保护组（“笼”）通过光释放脂质衍生物和光合链接重18zirine以共价附着 结合蛋白的脂质衍生物。单击化学的炔烃组可用于隔离脂质 - 蛋白结合物 或通过荧光标记和显微镜确定细胞中的脂质位置。在已发表的工作中，我们确定了 磷脂酰肌醇3,4,5-三磷酸（PIP3），磷脂酰肌醇3,4-双磷酸的特异性脂质结合蛋白 [Pi（3,4）P2]和磷脂酰肌醇（PI）。因为内膜中积累的“笼”衍生物，我们观察到 脱光灯后，其随后的细胞分布发生变化。所有三种磷酸肌醇转移到 30至120秒内的质膜（PM）。这种运输以PI而闻名，但从未被描述为PIP3或 PI（3,4）P2。然后，我们通过蛋白质组学分析确定了推定的脂质转运蛋白，并使用siRNA阻断脂质 运输。我们发现了运输PIP3和PI（3,4）P2所需的两个命中：胞质MPP6和跨膜 ATP11A。敲低EGF受体的内在化降低，表明对PIP3信号的影响。在这项工作中， 我们将表征这两种蛋白质相对于脂质特异性的脂质转运（AIM 1.1）。为此，我们 将改善我们当前精确量化细胞中脂质转运的方法（AIM 1.2）。我们将验证我们的发现 通过使用重组蛋白（包括具有关键保留点突变的重组蛋白）来研究蛋白质脂质的蛋白质的体外蛋白质 与生物物理和生化方法的相互作用（AIM 1.3）。我们将通过分析细胞脂质来增加严格 分解脂质衍生物后，质谱法组成（AIM 1.4）。在AIM 2中，我们将证明需要 MPP6脂质运输的迁移率。我们将使用LOV2技术开发可轻开的MPP6 内源性MPP6（通过基因编辑），将位于质膜（PM），直到我们用 488 nm灯。我们假设只有在MPP6从PM释放时，才有可能进行脂质运输。我们还将测试 MPP6从PM到子宫内的脂质重新传输。为此，我们将合成脂质衍生物 堆积在PM的外部小叶上，并影响细胞进入和通过未成衰老的运输。在AIM 3中，我们将合成 脂质衍生物具有靠近膜之间的光合链接重氮嗪，以更短暂地达到 结合蛋白，例如具有pH结构域的蛋白质。然后，脂质相互作用的比较蛋白质组学分析将是 用于鉴定参与有或没有受体刺激的信号传导的蛋白质。