STRUCTURE-FUNCTION ANALYSIS OF CYTOPLASMIC GELSOLIN

研究目的是通过以下途径阐明其分子机制 钙离子和多聚磷肌醇(PPI)依赖的哪些变化 肌动蛋白细丝长度对肺防御功能的调节 巨噬细胞。将特别强调明胶索林，一种 80 kDa PPI和钙结合蛋白在兔肺中的首次发现 巨噬细胞。明胶在存在时使肌动蛋白细丝断裂 其活性可被PPI抑制。穿过 肌动蛋白细丝长度和聚合的调节，细胞质 明胶可以引起肌动蛋白细胞骨架的重组。自.以来 细胞内Ca~(2+)和PPI水平均发生短暂变化 激动剂的刺激，明胶可能是许多关键控制点 细胞学事件。首先，我们将继续描述 明胶蛋白的功能结构域。我们已经确定了有限的 蛋白分解三个肌动蛋白结合部位和不同的钙离子和PPI- 监管网站也是如此。我们将重点介绍明胶蛋白结构域 (指定为CT28N)，它结合到肌动蛋白细丝的一侧 造成可能导致PPI抑制的扭曲 断绝关系。我们将研究CT28N对肌动蛋白细丝的影响 形态，并用NTCB鉴定其PPI和肌动蛋白结合部位 乳沟。我们将确定肌动蛋白和肌动蛋白之间的交互位置 明胶蛋白的3个肌动蛋白结合域的化学交联，以及 制备用于X射线结晶学分析的明胶晶体。 第二，我们将利用重组DNA技术进一步获得 关于边界以及交互的信息 在明胶蛋白结构域之间。最初，突变的明胶蛋白与END 缺失将通过基因在真核细胞中表达 转染法。我们将利用这样一个事实，一种明胶 变异体是分泌的，所以它可以在培养中进行分析 直接是中等大小。随后，将进行具体的更改 通过定点突变。第三，我们将过度/低价表达 用胞质明胶评价其在体内的作用。明胶感官 反义DNA将被导入成纤维细胞并 巨噬细胞样细胞及细胞形态和运动能力的变化 下定决心。我们将寻找肌动蛋白和 其他细胞骨架蛋白，以确定是否有机制 在它们之间保持适当的平衡。第四，我们将 确定细胞质明胶蛋白的表达是否在 生理和病理条件以获得进一步的了解 关于它在细胞质中的作用。我们的数据表明 细胞质明胶蛋白表达在S期呈下降趋势 细胞周期，并经A23187或放线菌酮处理后。这个 这些变化的分子基础将被确定。

The research objective is to elucidate the molecular mechanisms by which Ca2+- and polyphosphoinositide (PPI)-dependent changes in actin filament length regulate the defense functions of lung macrophages. Particular emphasis will be focused on gelsolin, a 80 kDa PPI- and Ca binding protein first identified in rabbit lung macrophages. Gelsolin fragments actin filaments in the presence of uM Ca2+, and its activity is inhibited by PPI. Through regulation of actin filament length and polymerization, cytoplasmic gelsolin can cause reorganization of the actin cytoskeleton. Since both Ca2+ and PPI levels change transiently in cells following agonist stimulation, gelsolin may be the key control point in many cytological events. First, we will continue to characterize the functional domains of gelsolin. We have identified by limited proteolysis three actin binding sites and distinct Ca2+ and PPI- regulatory sites as well. We will focus on a gelsolin domain (designated CT28N) which binds to the side of actin filaments causing distortions which may contribute to PPI-inhibitable severing. We will study the effect of CT28N on actin filament morphology, and identify its PPI- and actin binding sites by NTCB cleavage. We will identify interactive sites between actin and gelsolin's 3 actin binding domains by chemical crosslinking, and prepare gelsolin crystals for analysis by X-ray crystallography. Second, we will use recombinant DNA technology to obtain further information about the boundaries as well as the interactions between gelsolin domains. Initially, mutated gelsolins with end deletions will be expressed in eukaryotic cells by gene transfection. We will capitalize on the fact that a gelsolin variant is secreted, so that it can be analyzed in the culture medium directly. Subsequently, specific alterations will be made by site-directed mutagenesis. Third, we will over/underexpress cytoplasmic gelsolin to assess its role in vivo. Gelsolin sense and antisense DNA will be transfected into fibroblasts and macrophage-like cells, and changes in cell shape and motility determined. We will look for compensatory changes in actin and other cytoskeletal proteins, to determine if there are mechanisms for maintaining a proper balance between them. Fourth, we will determine if the expression of cytoplasmic gelsolin changes under physiological and pathological conditions to gain further insight about its role in the cytoplasm. Our data indicate that cytoplasmic gelsolin expression decreases during the S phase of cell cycle, and after A23187 or cycloheximide treatment. The molecular basis for these changes will be determined.