Cellular lipid transport determined with multifunctional lipid derivatives

总结 人们已经很清楚脂质在细胞中的合成和代谢方式，并且许多脂质具有信号功能 以一种在空间上和时间上确定的方式来调节细胞过程。后者需要建立和周转 膜中的脂质种类以位点特异性方式或可替代地，脂质转运的定向形式。这项工作 目的是研究脂质从一个膜到另一个膜的细胞内转移的几种蛋白质， 被发现参与脂质运输。在上一个资助期，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。这些具有可光活化的 保护基团（“笼”）以通过光释放脂质衍生物，和光交联二氮杂环丙烷以共价连接 脂质衍生物结合蛋白质。用于点击化学的炔基可用于分离脂质-蛋白质缀合物 或用于通过荧光标记和显微术确定细胞中的脂质位置。在已发表的研究中，我们发现 磷脂酰肌醇3，4，5-三磷酸（PIP 3）、磷脂酰肌醇3，4-二磷酸的特异性脂质结合蛋白 [PI(3，4）P2]和磷脂酰肌醇（PI）。由于“笼状”衍生物在内膜中积累，我们观察到 在用光解开后它们随后的细胞分布的变化。所有三种磷酸肌醇转移到 质膜（PM）在30至120秒内。这种转运对于Pl是已知的，但对于PIP 3或PIP 4从未描述过。 PI（3，4）P2。然后，我们通过蛋白质组学分析鉴定了假定的脂质转运蛋白，并使用siRNA阻断脂质转运蛋白。 运输我们发现了两种转运PIP 3和PI（3，4）P2所需的命中物：胞质MPP 6和跨膜MPP 6。 ATP11A。两者的敲低降低EGF受体的内化，表明对PIP 3信号传导的影响。在这项工作中， 我们将描述这两种蛋白质在脂质特异性方面的脂质转运特征（目的1.1）。为此，我们 将改进我们目前精确定量细胞中脂质转运的方法（目标1.2）。我们会验证我们的发现 通过使用重组蛋白（包括具有关键残基点突变的那些）在体外研究蛋白质-脂质 与生物物理和生物化学方法的相互作用（目标1.3）。我们将通过分析细胞脂质来增加僵硬度 通过质谱法测定脂质衍生物释放后的脂质组成（目的1.4）。在目标2中，我们将证明需要 用于脂质转运的MPP 6移动性。我们将开发一种使用LOV 2技术的光开关MPP 6， 内源性MPP 6（通过基因编辑），并将位于质膜（PM），直到我们用荧光素照射细胞。 488 nm光。我们假设，脂质转运将只可能，如果MPP 6是从PM释放。我们还将测试 用于通过MPP 6从PM到内膜的脂质逆向转运。为此，我们将合成脂质衍生物， 在PM的外小叶处积累，并诱导细胞进入和通过释放运输。在目标3中，我们将综合 脂质衍生物的特点是光交联二氮杂环丙烷更接近膜界面， 结合蛋白，如具有PH结构域的那些。脂质相互作用组的比较蛋白质组学分析将在 用于鉴定在有和没有受体刺激的情况下参与信号传导的蛋白质。

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.