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.

我们已经确定了一个新的水通道蛋白，AqpB，从盘基网柄藻。AqpB是水特异性的，O-糖基化的，并表现出一种新的门控机制。它位于液泡结构、质膜和膜突起中。迄今为止，它代表了网柱藻中唯一的功能性水通道蛋白，并且在所有发育阶段都有表达。门控的关键结构域包含D环中的七个氨基酸位置。到目前为止，敲除尝试已经产生了非特异性基因组整合。除了AqpB，我们已经确定了第二个水通道蛋白，AqpC，在阿米巴的cDNA和蛋白质水平。本文将进一步阐明AqpB的门控机制，并重点分析AqpB的生理功能，更初步地分析AqpC的生理功能。我们将产生新的突变AqpB，特别是在保守的酪氨酸-218附近的序列延伸的生物物理分析。我们将构建具有更长连接臂的新敲除质粒，并在AqpB编码区内选择富含GC的序列，以提高基因组整合的特异性。如果敲除是致命的，我们将应用琥珀抑制敲除方法，该方法将蛋白质水平降低高达90%。通过使用去调节的AqpB变体的显性过表达，我们将测试门控在细胞生理学方面的作用。我们将分析敲除/敲减/过表达培养物的表型，包括形态学、胞质分裂、发育、运动性、体积调节和巨胞饮。我们希望获得水通道蛋白在细胞生理学中的作用的基础知识，并有可能翻译为运动的人类细胞，如免疫细胞和扩散的肿瘤细胞。