Lipids in Maintenance of Chemokine and T-Cell Receptor

自从 1996 年多个研究小组将趋化因子受体 (CR) 描述为人类免疫缺陷病毒 (HIV) 共受体以来，趋化因子受体 (CR) 引起了广泛关注。在此之前，HIV 向性被定义为巨噬细胞 (M) 向性或 T 细胞 (T) 向性，分别对应于非合胞体诱导病毒或合胞体诱导病毒。如今，HIV 向性的分类是根据 CCR5、CXCR4 或两种受体的趋化因子受体的使用来定义的。趋化因子受体是由七个跨膜 G 蛋白偶联受体组成的家族，它们在许多免疫和非免疫细胞群中差异表达。某些 CR 已被证明是棕榈酰化的，并靶向富含胆固醇和鞘脂的膜微域（称​​为脂筏）。脂筏是一个广义术语，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结构域的集合。脂筏被认为是重要的信号传导平台，富含许多信号传导蛋白，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缘。然而，胆固醇和这些脂筏对 T 细胞趋化因子结合以及通过 CCR5 和 CXCR4 信号传导的作用仍然未知。我们发现，通过表达 CXCR4 或 CCR5 的 T 细胞，通过 β-环糊精 (BCD) 提取胆固醇分别显着降低了 SDF-1 和 MIP-1b 的结合和信号传导。氧化形式的胆固醇（称为氧甾醇）在各种食品中含量丰富，并且天然存在于各种细胞类型的膜和线粒体中。还发现胆固醇氧化会导致 T 细胞和单核细胞趋化因子结合和功能丧失。在这些情况下，用非氧化胆固醇重新装载经过处理的细胞可以恢复趋化因子的结合和功能。在胆固醇提取和胆固醇氧化后，针对不同 CXCR4 或 CCR5 表位的特异性抗体失去了与细胞表面结合的能力。此外，标记趋化因子的结合研究表明配体与 GM1 脂筏标记物的结合存在广泛的共定位，而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，活性配体结合促进受体与脂筏的结合，或者脂筏结合促进趋化因子受体的更高亲和力构象。总之，这些数据表明胆固醇和脂筏对于维持趋化因子受体构象很重要，并且对于该趋化因子受体的结合和功能都是必需的。配体结合所需的胆固醇和脂筏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。更具体的工作也在进行中，以检查年轻和衰老淋巴细胞细胞膜内脂筏组成的差异。鉴于脂质、过氧化和代谢随着年龄的增长而发生大量变化，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，正在进行更多研究，以创建处于不同激活阶段的年轻和衰老淋巴细胞的脂筏内各种蛋白质的蛋白质组表达谱。我们相信，对与脂筏相关的各种信号传导和细胞表面蛋白有更深入的了解，可能有助于深入了解细胞信号传导和迁移中与年龄相关的变化。

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.