Lipids in Maintenance of Chemokine and T-Cell Receptor

趋化因子受体(CRS)自1996年被多个小组描述为人类免疫缺陷病毒(HIV)共受体以来，引起了人们的极大关注。在此之前，HIV嗜性被定义为巨噬细胞(M)嗜性或T细胞(T)嗜性，分别对应于非合胞体诱导病毒或合胞体诱导病毒。今天，HIV嗜性的分类是由趋化因子受体CCR5和/或CXCR4的使用来定义的。趋化因子受体是一个由七个跨膜的G蛋白偶联受体组成的家族，这些受体由许多免疫和非免疫细胞群体差异表达。某些CRS已被证明是棕榈酰化的，并靶向于被称为脂筏的富含胆固醇和鞘磷脂的膜微域。脂筏是一个宽泛的术语，指的是富含胆固醇、鞘脂、糖基磷脂酰肌醇(GPI)锚定蛋白和酰化信号分子的膜微域的集合。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、GA亚基、H-RAS、LAT和NOS。CCR5和CXCR4已被证明存在于脂筏中，共定位于迁移细胞的前沿。然而，胆固醇和这些脂筏通过CCR5和CXCR4对T细胞趋化因子结合和信号传递的作用仍不清楚。我们发现，通过表达CXCR4或CCR5的T细胞，β-环糊精(BCD)提取的胆固醇显著减少了SDF-1和MIP-1b的结合和信号转导。氧化形式的胆固醇，称为氧化甾醇，在各种食品中大量存在，天然存在于各种细胞类型的细胞膜和线粒体中。胆固醇氧化还导致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，用非氧化胆固醇重新加载处理过的细胞可以恢复趋化因子的结合和功能。针对不同CXCR4或CCR5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与GM1脂筏标记的广泛共定位。在使用抗趋化因子受体抗体时，我们发现大多数趋化因子受体与CD59共定位，仅部分与GM1共定位。这些结果表明，活性配体结合促进了受体与脂筏的结合，或者RAFT结合促进了趋化因子受体的高亲和力构象。综上所述，这些数据表明，胆固醇和脂筏对于维持趋化因子受体的构象是重要的，对这种趋化因子受体的结合和功能都是必要的。这种胆固醇和脂筏对配体结合的要求可能在控制免疫细胞信号和迁移方面发挥重要的生理作用。更具体的工作也在进行中，研究年轻和老年淋巴细胞细胞膜内脂筏组成的差异。由于随着年龄的增长，脂质和过氧化代谢发生了大量的变化，各种膜鞘脂脂、脂肪酸和胆固醇的种类、饱和度和水平的变化可能会导致膜流动性、蛋白质结合和聚集、细胞激活和功能的特定变化。此外，通过高效液相色谱分离、二维凝胶电泳和质谱学，正在进行更多的研究，以创建年轻和老年淋巴细胞在不同激活阶段的脂筏中各种蛋白质的蛋白质组表达谱。我们认为，更多地了解与脂筏相关的各种信号和细胞表面蛋白可能有助于深入了解与年龄相关的细胞信号和迁移变化。

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, Ga 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. 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.