CALCIUM REGULATION OF VASCULAR SMOOTH MUSCLE CONTRACTION

血管平滑肌收缩的钙调节

基本信息

批准号：
2415587
负责人：
SAMUEL E GEORGE
金额：
$ 10.78万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-05-22 至 1998-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2415587
关键词：
biological signal transduction calcium binding protein calmodulin cardiovascular agents chemical structure function circular dichroism enzyme activity enzyme mechanism fluorescence muscle contraction myosin light chain kinase protein engineering site directed mutagenesis troponin vascular smooth muscle vasomotion

项目摘要

Calcium, acting through its intracellular receptor calmodulin (CaM), plays a critical role in regulating vascular tone. Vascular smooth muscle contraction depends on the action of a calmodulin-dependent enzyme, smooth muscle myosin light chain kinase (MLCK). We want to understand how calmodulin activates MLCK and other enzymes that regulate vascular smooth muscle tone. Our focus is on the structural basis of calmodulin's interaction with these regulatory enzymes. We propose to generate chimeras of CaM and cardiac troponin C (cTnC) and use them to study CaM-target enzyme interactions. Cardiac troponin C is structurally similar to calmodulin, but cannot activate calmodulin target enzymes. By constructing chimeras of these two homologous calcium binding proteins, we will determine the domains of cTnC that cannot substitute for the corresponding region of CaM without causing loss of ability to activate. This data directs us to specific amino acid residues of CaM that may participate in binding and activation of target enzymes. We will then focus on these residues in an intensive mutagenesis study. This mutagenesis study will provide precise structural information about CaM-target enzyme interactions. Our preliminary data show that some CaM-cTnC chimeras bind to MLCK, but fail to activate the enzyme. These and other CaM mutants are potent competitive MLCK inhibitors. We propose to extend these studies to develop the most potent MLCK inhibitor possible. Such an inhibitor would provide an excellent structural model for understanding the molecular mechanism of MLCK activation. The proposed study will define the role of calmodulin's functional domains in target enzyme activation, and produce a more complete picture of calmodulin-target enzyme interactions. This, in turn, will help us understand how calcium regulates vascular tone.

钙，通过其细胞内受体钙调蛋白（CAM）作用，在调节血管张力方面起着至关重要的作用。血管光滑肌肉收缩取决于钙调蛋白依赖性的作用酶，平滑肌肌球蛋白轻链激酶（MLCK）。我们想了解钙调蛋白如何激活MLCK和其他酶调节血管平滑肌张力。我们的重点是结构钙调蛋白与这些调节酶相互作用的基础。我们提议生成CAM和心脏肌钙蛋白C（CTNC）的嵌合体和使用它们来研究CAM-TARGET酶相互作用。心脏肌钙蛋白C是结构上类似于钙调蛋白，但无法激活钙调蛋白靶酶。通过构建这两个同源的嵌合体钙结合蛋白，我们将确定CTNC的域不会替代CAM的相应区域而不会引起失去激活能力。这些数据将我们引向特定的氨基 CAM的酸残基，可能参与结合和激活靶酶。然后，我们将重点关注这些残留物诱变研究。这项诱变研究将提供精确的有关CAM-TARGET酶相互作用的结构信息。我们的初步数据表明，某些CAM-CTNC嵌合体与MLCK结合，但是无法激活酶。这些和其他凸轮突变体有效竞争性MLCK抑制剂。我们建议将这些研究扩展到开发最有效的MLCK抑制剂。这样的抑制剂将提供一个出色的结构模型来理解 MLCK激活的分子机制。拟议的研究将定义钙调蛋白功能的作用靶酶激活中的域，并产生更完整的钙调蛋白 - 靶标酶相互作用的图片。反过来将帮助我们了解钙如何调节血管张力。