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

基本信息

批准号：
10539519
负责人：
Carsten Schultz
金额：
$ 37.17万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10539519
关键词：
Address Agonist Alkynes Binding Binding Proteins Biochemical Biology Biophysics Carrier Proteins Cell membrane Cell physiology Cells Cellular Assay Cellular biology Charge Chemicals Chemistry Communities Cytosol Data Diazomethane Disease Epidermal Growth Factor Receptor Exhibits Fatty Acids Funding Future Genes Genetic Glycerol Glycerophospholipids Goals Head Hydrophobicity Image In Vitro Interphase Investigation Learning Light Lipid Binding Lipids Location Malignant Neoplasms Mass Spectrum Analysis Membrane Membrane Proteins Metabolism Methods Microscopy Monitor Multiple Sclerosis Outcome PH Domain Parents Pathology Phenotype Phosphatidylinositols Play Point Mutation Positioning Attribute Proteins Proteomics Publishing Receptor Cell Recombinant Proteins Resources Role Series Signal Transduction Site Small Interfering RNA Specificity Techniques Testing Travel Vertebral column Work base cancer biomarkers comparative crosslink experimental study hydrophilicity improved insight knock-down lipid transport lipidomics lipophilicity metastatic colorectal myelination new therapeutic target optogenetics phosphatidylinositol 3,4,5-triphosphate phosphatidylinositol 3,4-diphosphate receptor retrograde transport success tool

项目摘要

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，脂质是如何在细胞中合成和代谢的，并且许多脂质具有信号传导功能以空间和时间定义的方式调节细胞过程，后者需要细胞的建立和更新。这项工作以特定位点的方式或定向的脂质运输形式在膜中形成脂质。旨在研究我们研究的几种蛋白质将脂质从一个膜转移到另一个膜的细胞内转移发现参与脂质运输在之前的资助期间，我们合成了多功能脂质。五种磷酸肌醇和四种常见甘油磷脂的衍生物，具有光激活特性。保护基团（“笼”）通过光释放脂质衍生物，并通过光交联二氮丙啶共价连接用于点击化学的炔烃基团可用于分离脂质-蛋白质缀合物。或通过荧光标记和显微镜确定细胞中的脂质位置，我们在已发表的工作中发现了这一点。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。光解笼后它们随后的细胞分布发生变化。所有三种磷酸肌醇都转移到了细胞中。 PI 的这种转运在 30 至 120 秒内完成，但从未在 PIP3 或 PIP3 中被描述过。然后，我们通过蛋白质组分析鉴定了假定的脂质转运蛋白，并使用 siRNA 来阻断脂质。我们发现运输 PIP3 和 PI(3,4)P2 所需的两个命中：胞质 MPP6 和跨膜。 ATP11A 的敲除减少了 EGF 受体的内化，表明对 PIP3 信号传导的影响。我们将根据脂质特异性来表征这两种蛋白质的脂质运输（目标 1.1）。将改进我们目前精确量化细胞内脂质转运的方法（目标 1.2）。在体外使用重组蛋白（包括关键残基点突变的蛋白）来研究蛋白质-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 ligor。解封脂质衍生物后通过质谱分析成分（目标 1.4）。在目标 2 中，我们将证明需要我们将使用 LOV2 技术开发一种光可切换的 MPP6，以取代它。内源性 MPP6（通过基因编辑）并将位于质膜 (PM)，直到我们用我们发现，只有从 PM 中释放 MPP6，脂质运输才可能实现。我们还将进行测试。 MPP6 将脂质从 PM 逆向转运至内膜为此，我们将合成脂质衍生物在 PM 的外层积聚并通过解笼诱导细胞进入和运输。在目标 3 中，我们将合成。脂质衍生物的特点是光交联二氮丙啶更接近膜间相，可以更短暂地到达然后对脂质相互作用组进行比较蛋白质组学分析。用于识别在有或没有受体刺激的情况下参与信号传导的蛋白质。