Impact of reactive oxygen species on surface localization of membrane proteins

细胞通过驻留在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方面发挥着作用，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。这些蛋白质不是固定在一个位置，但在许多情况下，它们既可以在细胞表膜内移动，允许与其他蛋白质进行复杂的相互作用，也可以进出表膜，从而控制它们的可用性。这种运动的一部分是由小窝的形成控制的。小凹是细胞膜上的内陷，可以作为“信号域”，或者可以发芽从表面移除蛋白质。小窝蛋白-1是一种与小窝相关的支架蛋白，可被过氧化氢等活性氧物种激活。细胞中活性氧的主要来源之一是一种名为NADPH氧化酶或NOX酶的酶家族。我们的目标是确定NADPH氧化酶衍生的ROS、小窝蛋白-1的磷酸化与细胞表面蛋白的激活、运动的关系。 这项研究将集中在三个表面蛋白：白介素1受体(IL1R1)，它可以经历小凹-1依赖的运动，转化生长因子-β受体(TGFBR)，它可以经历小窝-1依赖或非小窝-1的运动，以及表皮生长因子受体(EGFR)，主要经历小窝-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，以及运动类型是否受细胞表面的NOX亚型(如NOX1和NOX2)与细胞内的NOX亚型NOX4的影响。 选择肺成纤维细胞系IMR-90和皮肤成纤维细胞系BJ是因为它们表达这些表面蛋白，并通过上调NOX4酶来响应转化生长因子-β的刺激。此外，我们将使用miRNA和过表达载体对NOX1、NOX2、NOX4和小凹-1进行基因操作，以比较亚细胞定位对小窝-1磷酸化和运动表面蛋白的作用。膜蛋白定位将通过共聚焦免疫荧光进行；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检测；小窝蛋白-1的磷酸化将通过Western blotting进行监测；信号事件将通过Western blotting和定量RT-PCR进行监测。 这项工作将提供关于细胞信号生物学中基本重要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.