Regulation und gating von neuen Aquaporinen aus dem Schleimpilz Dictyostelium disciodeum und deren Funktion für die Zellmotilität

粘菌盘基网柄菌新水通道蛋白的调控和门控及其在细胞运动中的功能

• 批准号: 111328395
• 负责人: Professor Dr. Eric Beitz
• 金额: --
• 依托单位: Pharmazeutisches Institut
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2011-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/111328395?language=en
• 关键词: Regulation; und; gating; von; neuen

We have characterized a novel aquaporin, AqpB, from Dictyostelium discoideum. AqpB is water-specific, O-glycosylated and exhibits and a novel gating mechanism. It is localized in vacuolar structures, the plasma membrane, and in membrane protrusions. So far, it represents the sole functional aquaporin in Dictyostelia und is expressed in all developmental stages. The critical domain for the gating comprises seven amino acid positions in the D-loop. Until now, knockout attempts have yielded unspecific genome integration. Besides AqpB, we have identified a second aquaporin, AqpC, in amoebae on the cDNA and protein level. Now we will further elucidate the gating mechanism of AqpB and analyze the physiological function mainly of AqpB and more initially of AqpC. We will generate new mutations of AqpB especially in the sequence stretch around the conserved tyrosine-218 for biophysical analysis. We will construct new knockout plasmids with longer linker arms and select GC-rich sequences within the coding region of AqpB to improve the specificity of genomic integration. If the knockout is lethal, we will apply the amber-suppression knockdown method, which reduces protein levels up to 90%. By using dominant overexpression of de-regulated AqpB-Variants, we will test for the role of gating with respect to cell physiology. We will analyze the phenotypes of the knockout/knockdown/overexpression cultures regarding morphology, cytokinesis, development, motility, volume regulation, and macropinocytosis. We expect to gain basic knowledge on the role of aquaporins in cell physiology with the potential for translation to motile human cells, such as immune cells and spreading tumor cells.

我们已经描述了一种新型的水通道蛋白AQPB，来自Dictyostelium Discoideum。 AQPB是水特异性的，O-糖基化的和展示的，并且是一种新颖的门控机制。它位于液泡结构，质膜和膜突起中。到目前为止，它代表了在所有发育阶段表达的唯一功能性水通道蛋白。门控的关键结构域包括在D环中的七个氨基酸位置。到目前为止，敲除尝试产生了非特异性的基因组整合。除AQPB外，我们还在cDNA和蛋白质水平的变形虫中确定了第二个水通道蛋白AQPC。现在，我们将进一步阐明AQPB的门控机制，并主要分析AQPB的生理功能，最初更初的AQPC。我们将生成AQPB的新突变，尤其是在保守酪氨酸-218周围延伸的序列中进行生物物理分析。我们将在AQPB的编码区域内构建具有更长的接头臂的新敲除质粒，并选择富含GC的序列，以提高基因组整合的特异性。如果敲除致命，我们将应用琥珀抑制敲低方法，该方法将蛋白质水平降低到90％。通过使用DENEDENCH的AQPB变量的显性过表达，我们将测试门控在细胞生理方面的作用。我们将分析有关形态，细胞因子，发育，运动，体积调节和大型细胞增多症的敲除/敲除/过表达培养物的表型。我们期望获得水库在细胞生理学中的作用的基本知识，并有可能转化为流动的人类细胞，例如免疫细胞和扩散肿瘤细胞。