STRUCTURE/FUNCTION ANALYSIS OF CYTOPLASMIC GELSOLIN

本研究的目的是阐明分子机制， 其中Ca 2+和多磷酸肌醇（PPI）依赖性改变， 肌动蛋白丝长度调节肺的防御功能 巨噬细胞 将特别强调凝溶胶蛋白， 80 kDa PPI-和Ca结合蛋白首次在兔肺中鉴定 巨噬细胞 凝溶胶蛋白在存在下将肌动蛋白丝片段化 其活性可被PPI抑制。 通过 肌动蛋白丝长度和聚合的调节，细胞质 凝溶胶蛋白可引起肌动蛋白细胞骨架的重组。 以来 Ca 2+和PPI水平在细胞中短暂变化， 激动剂刺激，凝溶胶蛋白可能是许多 细胞学事件 首先，我们将继续描述 凝溶胶蛋白的功能域。 我们通过有限的 蛋白水解三个肌动蛋白结合位点和不同的Ca 2+和PPI- 监管场所也是如此。 我们将重点研究凝溶胶蛋白结构域 （命名为CT 28 N），其结合到肌动蛋白丝的一侧 导致可能导致PPI可重构的失真 切断 我们将研究CT 28 N对肌动蛋白丝的影响 形态，并通过NTCB鉴定其PPI和肌动蛋白结合位点 乳沟 我们将确定肌动蛋白和 通过化学交联的凝溶胶蛋白的3个肌动蛋白结合结构域，和 制备凝溶胶蛋白晶体用于X射线晶体学分析。 其次，我们将利用重组DNA技术， 关于边界和相互作用的信息 在凝溶胶蛋白结构域之间。 最初，突变的凝溶胶蛋白与末端 缺失将通过基因在真核细胞中表达 转染 我们将利用凝溶胶蛋白 变体被分泌，以便可以在培养物中对其进行分析 中直接。 随后，将进行具体修改 通过定点突变。 第三，我们将过度/欠表达 细胞质凝溶胶蛋白，以评估其在体内的作用。 凝溶胶蛋白感觉 将反义DNA转染入成纤维细胞， 巨噬细胞样细胞，以及细胞形状和运动性的变化 测定 我们将寻找肌动蛋白的代偿性变化， 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。 四是 确定细胞质凝溶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 细胞质凝溶胶蛋白表达在S期减少， A23187或放线菌酮处理后。 的 将确定这些变化的分子基础。

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.