Cellular lipid transport determined with multifunctional lipid derivatives

摘要 众所周知，脂类是如何在细胞中合成和代谢的，许多脂类发挥着信号功能。 以空间和时间定义的方式调节细胞过程。后者需要建立和周转 膜中的脂类物种或者以特定部位的方式存在，或者以定向的脂类运输形式存在。这部作品 目的是研究几种蛋白质在细胞内脂类从一种膜到另一种膜的转移，我们 被发现与脂类运输有关。在之前的资助阶段，我们合成了多功能脂质 五种磷脂酰肌醇和四种常见甘油磷脂的衍生物。这些功能具有可照片激活的功能 通过光释放脂质衍生物的保护基团(笼子)和共价连接的光交联二氮杂环丙烷 结合蛋白的脂衍生物。用于点击化学的炔基可用于分离脂类-蛋白质结合物 或用于通过荧光标记和显微镜确定细胞中的脂质位置。在出版的著作中，我们发现 磷脂酰肌醇3，4，5-三磷酸(PIP3)、磷脂酰肌醇3，4-二磷酸的特异性脂结合蛋白 [PI(3，4)P2]和磷脂酰肌醇(PI)。因为“笼子里”的衍生品积累在子宫内膜中，我们观察到 光照后其后续细胞分布的变化。所有三种磷脂酰肌醇都转移到 质膜(PM)在30到120秒内。这种传输对于PI是已知的，但从未被描述用于PIP3或 PI(3，4)P2.然后，我们通过蛋白质组学分析鉴定了可能的脂类运输蛋白，并使用siRNAs来阻断脂类 运输。我们发现了转运PIP3和PI(3，4)P2所需的两个靶点：胞质MPP6和跨膜MPP6 ATP11A。两者的敲除都减少了EGF受体的内化，表明对PIP3信号的影响。在这部作品中， 我们将根据脂类特异性(目标1.1)来表征这两种蛋白质的脂类转运。为此，我们 将改进我们目前精确量化细胞内脂质运输的方法(目标1.2)。我们将验证我们的发现 利用含有关键残基点突变的重组蛋白体外研究蛋白质-脂质 与生物物理和生化方法的相互作用(目标1.3)。我们将通过分析细胞脂质来增加僵硬程度 去脂衍生物后的质谱学组成(目标1.4)。在目标2中，我们将论证 MPP6的流动性与脂质转运有关。我们将开发一款使用LOV2技术的光可切换MPP6，它将取代 内源性MPP6(通过基因编辑)，并将定位在质膜(PM)，直到我们用 488 nm光。我们假设，只有当MPP6从PM中释放出来时，才有可能进行脂质运输我们还将测试 用于MPP6将脂质从PM逆向转运到内膜。为此，我们将合成脂类衍生物， 聚集在PM的外层小叶，并通过去化诱导细胞进入和运输。在目标3中，我们将合成 以光交联二氮杂环为特征的脂类衍生物更接近膜界面，达到更瞬时 结合蛋白，如那些具有PH结构域的蛋白。然后将对脂类相互作用进行比较蛋白质组学分析 用于识别在有和没有受体刺激的情况下参与信号传递的蛋白质。

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.