STRUCTURE AND FUNCTION OF THE INTESTINE BRUSH BORDER

肠刷缘的结构和功能

基本信息

批准号：
3233588
负责人：
PAUL T. MATSUDAIRA
金额：
$ 15.17万
依托单位：
WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-04-01 至 1993-03-31
项目状态：
已结题

项目摘要

Cytoplasmic calcium and the membrane-associated cytoskeleton of the microvillus and basolateral membranes are important for the efficient absorption of nutrients by the intestine epithelium. The cytoskeleton stabilizes the microvillus surface area y linking the membrane via a llOK-calmodulin complex to an underlying fibrillar bundle composed of actin filaments crosslinked by villin. However. in vitro studies how that in high calcium (greater than MuM). villin fragments actin filaments into short pieces and the unsupported membrane vesiculate. Our studies show that villin is a modular protein composed of separate actin-binding domains and regulated by calcium and phospholipid. The complete villin sequence does not display homology with calmodulin-like calcium- binding sites and together with the phospholipid-binding activity suggests villin is a member of a newly recognized class of calcium- binding proteins. Our studies have presented a framework for studying the relationship between calcium and phospholipid-binding to villin domains and the activation of actin bundling and severing activity. Our goals in this five year renewal are the following: 1. to map the actin, villin, and calcium binding sites by antibody- end-labelling, chemical crosslinking, protein sequencing, protein footprinting, and chemical modification techniques. 2. to synthesize peptides that display specific actin- and calcium- binding activity. 3. to crystallize complexes formed between villin domains and actin. 4. to express the villin cDNA and test the function of different domains by deletion analysis of the expressed protein. 5. to start new studies on cytoskeletal proteins (the llOK- calmodulin complex) that bind actin to integral membrane proteins. The molecular details of calcium-cytoskeleton interactions with the membrane have important health-related significance because loss of microvillar membrane surface area and impaired ion transport are prominent defects that characterize intestinal malabsorption disorders.

细胞质钙和膜相关细胞骨架微绒毛和基底外侧膜对于肠道上皮有效吸收营养物质。这细胞骨架稳定微绒毛表面积并连接膜通过 llOK-钙调蛋白复合物到达下面的纤维由绒毛交联的肌动蛋白丝组成的束。然而。体外研究高钙（大于 MuM）的情况。绒毛蛋白将肌动蛋白丝断裂成短片段，无支撑膜有泡状。我们的研究表明，villin 是由单独的肌动蛋白结合域组成的模块化蛋白质和受钙和磷脂调节。完整的恶人序列不显示与钙调蛋白样钙的同源性结合位点以及磷脂结合活性表明villin是一类新认识的钙的成员- 结合蛋白。我们的研究提出了一个框架研究钙和磷脂结合之间的关系绒毛蛋白结构域和肌动蛋白捆绑的激活切断活动。我们在这五年更新中的目标是下列的： 1. 通过抗体绘制肌动蛋白、绒毛蛋白和钙结合位点图谱- 末端标记、化学交联、蛋白质测序、蛋白质足迹法和化学改性技术。 2. 合成显示特定肌动蛋白和钙的肽结合活性。 3. 使绒毛结构域之间形成的复合物结晶化肌动蛋白。 4. 表达villin cDNA并测试不同的功能通过表达蛋白质的缺失分析来确定结构域。 5. 开始细胞骨架蛋白的新研究（llOK- 钙调蛋白复合物）将肌动蛋白与整合膜蛋白结合。钙-细胞骨架相互作用的分子细节膜具有重要的健康相关意义，因为损失微绒毛膜表面积和离子传输受损是肠道吸收不良的显着缺陷失调。