Impact of reactive oxygen species on surface localization of membrane proteins

细胞通过位于细胞表面的蛋白质相互作用，并与周围环境相互作用。 位于细胞表面的蛋白质在形成将细胞连接在一起的连接中发挥作用，对我们身体产生的化学物质和激素做出反应，对药物和许多其他过程做出反应。这些蛋白质不会固定在一个位置，但在许多情况下，它们在细胞表面膜内移动，允许与其他蛋白质发生复杂的相互作用，以及进出表面膜，允许控制其可用性。 这种运动的一部分是由小窝的形成所控制的。 小窝是细胞膜中的内陷，可以充当“信号结构域”或可以出芽以从表面去除蛋白质。 小窝蛋白-1是一种小窝相关的支架蛋白，可以被活性氧（ROS）如过氧化氢激活。 细胞中活性氧的主要来源之一是一种称为NADPH氧化酶或NOX酶的酶家族。 我们的目的是确定NADPH氧化酶衍生的活性氧，小窝蛋白-1磷酸化和细胞表面蛋白的活化，运动之间的关系。 本研究将集中在三种表面蛋白：白细胞介素-1受体（IL 1 R1），它经历了小窝蛋白-1依赖的运动，TGF-β受体（TGF-β受体），它可以经历小窝蛋白-1依赖或小窝蛋白-1非依赖的运动，和表皮生长因子受体（EGFR），主要经历小窝蛋白-1非依赖的运动。 我们将研究NOX酶是否调节小窝蛋白-1依赖的运动，以及运动的类型是否受细胞表面NOX亚型如NOX 1和NOX 2与细胞内NOX亚型NOX 4的影响。 选择肺成纤维细胞系IMR-90和皮肤成纤维细胞系BJ，因为它们表达这些表面蛋白并通过上调N 0X 4酶响应TGF-β刺激。此外，我们将使用miRNA和过表达载体对NOX 1、NOX 2、NOX 4和小窝蛋白-1进行遗传操作，以比较亚细胞NOX定位对小窝蛋白-1磷酸化和运动表面蛋白的作用。膜蛋白定位之后将进行共聚焦免疫荧光; NADPH氧化酶活性将被抑制剂DPI和雷公藤红素阻断，并通过Amplex Red和NBT测定进行监测;小窝蛋白-1的磷酸化将通过蛋白质印迹法进行监测;信号传导事件将通过蛋白质印迹法和定量RT-PCR进行监测。 这项工作将提供有关细胞信号传导生物学中具有根本重要性的细胞过程的重要信息。

Cells interact with one other and with their surroundings through proteins that reside at the cell surface. Proteins located at the surface of the cell play a role in forming connections that hold cells together, responding to the chemicals and hormones our bodies produce, responding to drugs and in many other processes. These proteins do not remain fixed in one location, but in many cases, they move around both within the cell surface membrane allowing complex interactions with other proteins, as well as to and from the surface membrane allowing control over their availability. Some of this movement is controlled by the formation of caveolae. Caveolae are invaginations in the cell membrane that can act as “signaling domains” or can bud off to remove proteins from the surface. Caveolin-1 is a caveolae-associated scaffolding protein that can be activated by reactive oxygen species (ROS) such as hydrogen peroxide. One of the main sources of reactive oxygen species in cells is a family of enzymes called NADPH oxidases, or NOX enzymes. We aim to define the relationship between NADPH oxidase derived ROS, caveolin-1 phosphorylation and activation, movement of cell surface proteins. This research will focus on three surface proteins: the interlekin-1 receptor (Il1R1) which undergoes caveolin-1 dependent movement, the TGF-beta receptor (TGFBR) which can undergo caveolin-1 dependent or caveolin-1 independent movement, and the epidermal growth factor receptor (EGFR) which primarily undergoes caveolin-1 independent movement. We will investigate whether NOX enzymes regulate caveolin-1 dependent movement, and whether the type of movement is influenced by cell surface NOX isoforms such as NOX1 and NOX2 versus the intracellular NOX isoform NOX4. The lung fibroblast cell-line IMR-90 and the skin fibroblast cell line BJ were selected because they express these surface proteins and respond to TGF-beta stimulation by upregulating the NOX4 enzyme. In addition, we will genetically manipulate NOX1, NOX2, NOX4, and caveolin-1 using miRNA and over-expression vectors to compare the roles of subcellular NOX localization on caveolin-1 phosphorylation and the movement surface proteins. Membrane protein localization will be followed by confocal immunofluorescence; NADPH oxidase activity will be blocked by inhibitors DPI and celastrol and monitored by Amplex Red and NBT assays; phosphorylation of caveolin-1 will be monitored by Western blotting; and signaling events will be monitored by western blotting and quantitative RT-PCR. This work will provide important information about a cellular process of fundamental importance in biology of cell signaling.