CALCIUM & MYOSIN LIGHT CHAIN KINASE IN HUMAN AORTA

钙

• 批准号: 3082853
• 负责人: SAMUEL E GEORGE
• 金额: $ 7.69万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3082853
• 关键词: X ray crystallography; aorta; binding proteins; calcium; calmodulin; complementary DNA; drug design /synthesis /production; enzyme induction /repression; genetic manipulation; human tissue; molecular cloning; muscle contraction; myosin light chain kinase; myosins; phosphotransferases; protein structure function; restriction mapping; vascular smooth muscle

Myosin light chain kinase (MLCK) catalyzes the phosphorylation of the 20 kDa regulatory light chain of myosin. In vascular smooth muscle, MLCK plays a critical regulatory role in the final common pathway through which intracellular Ca2+ transients produce initiation of contraction. Previous work with chicken smooth muscle and rabbit skeletal muscle MLCK have shown that within the calmodulin binding region is a region with similarity to the phosphorylated region of the 20 kDa light chain. In the absence of the Ca2+-calmodulin complex, MLCK is inactive because a portion of the calmodulin binding region specifically interacts with the protein substrate binding site. Synthetic peptides modeled on this inhibitory "pseudosubstrate" region of MLCK effectively prevent the phosphorylation of myosin light chain in a competitive fashion. Pharmacologic agents modeled on the pseudosubstrate-MLCK active site interaction may be specific inhibitors of MLCK. This proposal represents the initial phases of work directed toward the goal of developing such agents. Western blots have confirmed the feasibility of isolating a human MLCK cDNA from a human aorta library using anti-chicken MLCK antibodies. Construction of this library in lambda gt11 is in progress. A cDNA encoding human vascular smooth muscle MLCK will be cloned and sequenced. An active, calmodulin regulated fragment will be subcloned into a bacteria expression vector, expressed, and purified. Synthetic peptides, deletion mutagenesis and site specific mutagenesis will be used to precisely define the calmodulin binding and pseudosubstrate domains. If the crystal structure of this bacterially expressed fragment is determined, that information will be used to design further mutagenesis experiments. These studies will lead to a more complete understanding of the interaction of MLCK and the Ca2+-calmodulin complex, and may lead to the development of a new class of drugs for treatment of disease states characterized by increased vascular resistance.

肌球蛋白轻链激酶（MLCK）催化20 肌球蛋白的KDA调节轻链。在血管平滑肌中，MLCK玩耍 在最终公共途径中的关键调节作用 细胞内Ca2+瞬变会产生收缩的开始。以前的 使用鸡平滑肌和兔子骨骼肌MLCK工作 在钙调蛋白结合区域内是一个与 20 kDa轻链的磷酸化区域。在没有 Ca2+-calmodulin复合物，MLCK是不活跃的，因为一部分 钙调蛋白结合区与蛋白质底物专门相互作用 绑定位点。以这种抑制作用建模的合成肽 MLCK的“假基底”区域有效防止磷酸化 肌球蛋白轻型链以竞争方式。建模的药理学剂 在伪基底MLCK上，有效位点相互作用可能是特定的 MLCK的抑制剂。该建议代表工作的初始阶段 致力于发展此类代理的目标。 Western印迹有 确认从人主动脉中分离人MLCK cDNA的可行性 使用抗鸡抗MLCK抗体的库。该图书馆的构建 Lambda GT11正在进行中。编码人血管平滑肌的cDNA MLCK将被克隆和测序。一个活跃的钙调蛋白调节的片段 将被亚克隆到细菌表达载体中，表达，并且 纯化。合成肽，缺失诱变和特定位点特异性 诱变将用于精确定义钙调蛋白结合和 伪造域。如果该细菌的晶体结构 确定了表达的片段，将使用信息来设计 进一步的诱变实验。这些研究将导致更完整 了解MLCK和Ca2+-calmodulin络合物的相互作用， 并可能导致开发新的药物来治疗 疾病状态的特征是血管抗性增加。