Role for Lipids in the Maintenance of Chemokine and T-Ce

脂质在维持趋化因子和 T-Ce 中的作用

• 批准号: 6969414
• 负责人: DENNIS D. TAUB
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6969414
• 关键词: T cell receptor; T lymphocyte; aging; biological signal transduction; cell age; cell migration; chemokine; chemokine receptor; cholesterol; conformation; high performance liquid chromatography; human immunodeficiency virus; human tissue; mass spectrometry; membrane lipids; membrane structure; oxidation; oxysteroid; proteomics; receptor binding; two dimensional gel electrophoresis; virus infection mechanism

Chemokine receptors (CRs) have drawn much attention since their description as human immunodeficiency virus (HIV) co-receptors by several groups in 1996. Prior to that time, HIV tropism was defined as either macrophage (M)- or T cell (T)-tropic, which corresponded to non-syncytia- or syncytia-inducing viruses, respectively. Today, the classification of HIV tropism is defined by chemokine receptor usage of CCR5, CXCR4, or both receptors. Chemokine receptors are a family of seven transmembrane spanning G protein-coupled receptors that are differentially expressed by a number of immune and non-immune cell populations. Certain CRs have been shown to be palmitoylated and targeted to cholesterol-and sphingolipid-rich membrane microdomains termed lipid rafts. Lipid rafts is a broad term for the collection of membrane microdomains enriched in cholesterol, sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and acylated signaling molecules. Lipid rafts are believed to be important signaling platforms enriched in many signaling proteins, including but not limited to src kinases, G alpha subunit, H-Ras, LAT, and NOS. CCR5 and CXCR4 have been shown to be present in lipid rafts, colocalizing at the leading edge of migrating cells. However, the role of cholesterol and these lipid rafts on T cell chemokine binding and signaling through CCR5 and CXCR4 remains unknown. We found that cholesterol extraction by beta-cyclodextrin (BCD) significantly reduced the binding and signaling of SDF-1 and MIP-1b using CXCR4- or CCR5-expressing T cells, respectively. Oxidized forms of cholesterol, known as oxysterols, are abundant in various food products and can be found naturally in membranes and mitochondria of a variety of cell types. Cholesterol oxidation was also found to result in the loss of chemokine binding and function in T cells and monocytes. Reloading treated cells with non-oxidized cholesterol restores chemokine binding and function in these situations. Antibodies specific for distinct CXCR4 or CCR5 epitopes lost their ability to bind to the cell surface after cholesterol extraction and cholesterol oxidation. Moreover, bindings studies with labeled chemokines have demonstrated extensive co-localization of ligand binding with the GM1 lipid raft marker while using anti-chemokine receptor antibodies, we found the majority of chemokine receptors co-localize with CD59 and only partially with GM1. These results suggest that active ligand binding facilitates receptor association with lipid rafts or that raft association promotes a higher affinity conformation of chemokine receptors. Together, these data demonstrate that cholesterol and lipid rafts are important for the maintenance of the chemokine receptor conformation and are necessary for both the binding and function of this chemokine receptor. This cholesterol and lipid raft requirement for ligand binding may play a significant physiological role in controlling immune cell signaling and migration. More specific efforts are also underway examining the differences in the make-up of lipid rafts within the cell membranes of young and aged lymphocytes. Given the large number of alterations in lipid and peroxidation and metabolism with age, changes in the types, saturation and levels of various membrane sphingolipids, fatty acids and cholesterol may result in specific changes in membrane fluidity, protein association and aggregation, cellular activation and function. In addition, through HPLC separation, 2-D gel electrophoresis and mass spectroscopy, additional studies are underway creating a proteomic expression profile of the various proteins within the lipid rafts of young and aged lymphocytes at various stages of activation. Additional studies over the past year have also revealed that flotillin-1 appears to play an important role in T cell receptor and chemokine receptor signaling and function and that CD8+ T cells express greater levels of cholesterol and lipid rafts than CD4+ T cells. Overall, we believe that a greater understanding of the various signaling and cell surface proteins associated with lipid rafts may provide great insight into age-related alterations in cell signaling and migration.

趋化因子受体（Chemokine receptors，CRs）自1996年被多个研究小组描述为人类免疫缺陷病毒（human immunodeficiency virus，HIV）的共受体以来，一直受到人们的关注。在此之前，HIV嗜性被定义为嗜巨噬细胞（M）或嗜T细胞（T），分别对应于非合胞体或合胞体诱导病毒。今天，HIV嗜性的分类是由CCR 5、CXCR 4或两种受体的趋化因子受体使用来定义的。趋化因子受体是由许多免疫和非免疫细胞群体差异表达的七个跨膜G蛋白偶联受体家族。某些CR已被证明是棕榈酰化的，并靶向称为脂筏的富含胆固醇和鞘脂的膜微结构域。脂筏是一个广义的术语，用于收集富含胆固醇、鞘脂、糖基磷脂酰肌醇（GPI）锚定蛋白和酰化信号分子的膜微区。脂筏被认为是重要的信号平台，富含许多信号蛋白，包括但不限于src激酶、G α亚基、H-Ras、LAT和NOS。CCR 5和CXCR 4已被证明存在于脂筏中，共定位于迁移细胞的前沿。然而，胆固醇和这些脂筏对T细胞趋化因子结合和通过CCR 5和CXCR 4信号传导的作用仍然未知。我们发现，胆固醇提取β-环糊精（BCD）显着减少SDF-1和MIP-1b的结合和信号转导使用CXCR 4或CCR 5表达的T细胞，分别。胆固醇的氧化形式，称为氧化固醇，在各种食品中含量丰富，并且可以在各种细胞类型的膜和线粒体中天然发现。还发现胆固醇氧化导致T细胞和单核细胞中趋化因子结合和功能的丧失。在这些情况下，用非氧化胆固醇包裹处理过的细胞恢复了趋化因子的结合和功能。特异性针对不同CXCR 4或CCR 5表位的抗体在胆固醇提取和胆固醇氧化后失去了与细胞表面结合的能力。此外，与标记的趋化因子的结合研究已经证明了配体结合与GM 1脂筏标记物的广泛共定位，而使用抗趋化因子受体抗体，我们发现大多数趋化因子受体与CD 59共定位，仅部分与GM 1共定位。这些结果表明，活性配体结合促进受体与脂筏或筏协会促进更高的亲和力构象的趋化因子受体。总之，这些数据表明，胆固醇和脂筏的趋化因子受体构象的维持是重要的，是必要的，这种趋化因子受体的结合和功能。这种胆固醇和脂筏对配体结合的需求可能在控制免疫细胞信号传导和迁移中发挥重要的生理作用。更具体的努力也正在进行中，检查年轻和老年淋巴细胞细胞膜内脂筏组成的差异。鉴于脂质和过氧化以及代谢随年龄的大量变化，各种膜鞘脂、脂肪酸和胆固醇的类型、饱和度和水平的变化可能导致膜流动性、蛋白质缔合和聚集、细胞活化和功能的特定变化。此外，通过HPLC分离，2-D凝胶电泳和质谱，正在进行额外的研究，以创建年轻和老年淋巴细胞在不同活化阶段的脂筏内的各种蛋白质的蛋白质组表达谱。过去一年的其他研究也表明，flotillin-1似乎在T细胞受体和趋化因子受体信号传导和功能中发挥重要作用，并且CD 8 + T细胞表达的胆固醇和脂筏水平高于CD 4 + T细胞。总的来说，我们认为，更好地了解与脂筏相关的各种信号和细胞表面蛋白，可能会提供很大的洞察力与年龄相关的细胞信号和迁移的变化。