DESIGN AND TRANSGENIC ANALYSIS OF CELLULAR INHIBITORS

细胞抑制剂的设计和转基因分析

• 批准号: 2145647
• 负责人: JOHN R DEDMAN
• 金额: $ 21.87万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2145647
• 关键词: affinity chromatography; calcium flux; calmodulin; cell growth regulation; chemical binding; chemical synthesis; conformation; cytology; epithelium; gene expression; genetic library; genetic manipulation; genetic promoter element; genetically modified animals; growth inhibitors; laboratory mouse; protein sequence; stress

Calcium is a primary regulator of numerous functions in all cells. Changes in the levels of intracellular free calcium act as a signal. The mediation of intracellular free calcium is through high-affinity calcium binding-proteins. Calmodulin, a well-characterized calcium mediator protein, has been shown to be an essential gene product for cell viability. The Ca2+-calmodulin complex has been implicated in coupling cell responses to many stimuli. We have designed an approach to identify peptides which bind to a targeted protein, calmodulin. Ca2+-dependent affinity chromatography has been used to select calmodulin binding peptides from a bacteriophage library of random peptides. Sequence and predicted structure analysis of these peptides suggest that they are unique when compared to peptides previously reported as binding calmodulin. We propose to design an affinity-purification strategy to select sequences which are specific for the different conformational states of calmodulin, that is peptides which bind calmodulin only in the presence of calcium, those which bind only in the absence of calcium, and those which are indifferent to the calcium concentration. The physiological effects of these unique peptides will then be characterized in intact cellular systems including in the muscle fiber, neuron, chromaffin cell and epithelial cell. Synthetic genes will be designed which will be used for the expression of the calmodulin binding peptides in vivo. Cell growth, division and morphology will be examined as well as the stress response to elevated temperature and hypotonic challenge. Finally, selected calmodulin binding peptide sequences will be fused with promotor sequence in order to target expression of individual calmodulin binding peptides to the type II epithelial cells of the ling or cardiac ventricles of transgenic mice. These animals should allow for the understanding of calmodulin inhibition in intact tissue and the development of lung epithelial disease models such as cystic fibrosis and cardiac myopathies in male modifiers through selection from random peptide libraries is an independent approach for the study of cellular function. This peptide approach should be applicable to the evaluation of the role of other cellular proteins for which natural modifiers have yet to be discovered.

钙是所有细胞中众多功能的主要调节剂。 细胞内游离钙水平的变化充当信号。 细胞内游离钙的介导是通过高亲和力 钙结合蛋白。 钙调蛋白，一种特性良好的钙 介导蛋白已被证明是细胞必需的基因产物 生存能力。 Ca2+-钙调蛋白复合物与偶联有关 细胞对许多刺激的反应。 我们设计了一种方法来识别 与目标蛋白钙调蛋白结合的肽。 Ca2+依赖性 亲和色谱已用于选择钙调蛋白结合 来自噬菌体随机肽库的肽。 序列和 这些肽的预测结构分析表明它们是 与之前报道的结合肽相比是独特的 钙调蛋白。 我们建议设计一种亲和纯化策略 选择特定于不同构象的序列 钙调蛋白的状态，即仅在钙调蛋白中结合的肽 存在钙，仅在不存在钙的情况下结合的那些，以及 那些与钙浓度无关的。 这 然后将表征这些独特肽的生理效应 在完整的细胞系统中，包括肌纤维、神经元、 嗜铬细胞和上皮细胞。 合成基因将被设计 其将用于钙调蛋白结合肽的表达 体内。 还将检查细胞生长、分裂和形态 作为对升高温度和低渗挑战的应激反应。 最后，选定的钙调蛋白结合肽序列将与 启动子序列以靶向个体钙调蛋白的表达 结合肽与 ling 或心脏的 II 型上皮细胞 转基因小鼠的心室。 这些动物应该允许 了解完整组织中的钙调蛋白抑制以及 肺上皮疾病模型的开发，例如囊性纤维化和 通过随机选择男性修饰者的心肌病 肽库是研究细胞的独立方法 功能。 该肽方法应适用于评估 天然修饰剂所具有的其他细胞蛋白的作用 尚未被发现。