DESIGN AND TRANSGENIC ANALYSIS OF CELLULAR INHIBITORS

细胞抑制剂的设计和转基因分析

• 批准号: 2016658
• 负责人: JOHN R DEDMAN
• 金额: $ 23.38万
• 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/2016658
• 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.

钙是所有细胞中许多功能的主要调节剂。 细胞内游离钙水平的变化作为一种信号。 细胞内游离钙的介导是通过高亲和力的 钙结合蛋白 钙调素，一种特征明确的钙 介体蛋白，已被证明是细胞必需基因产物 生存能力。 Ca2 +-钙调素复合物与偶联有关， 细胞对多种刺激的反应。 我们设计了一种方法来识别 与靶蛋白钙调蛋白结合的肽。 ca2+依赖 亲和层析已被用于选择钙调素结合 来自随机肽的噬菌体文库的肽。 序列和 这些肽的预测结构分析表明，它们是 与先前报道的结合肽相比， 钙调素 我们建议设计亲和纯化策略， 选择对不同构象特异的序列 状态的钙调蛋白，即肽结合钙调蛋白只在 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。 的 这些独特的肽的生理作用将被表征 在完整的细胞系统中，包括肌肉纤维，神经元， 嗜铬细胞和上皮细胞。 合成基因将被设计成 其将用于表达钙调蛋白结合肽 in vivo. 还将检查细胞生长、分裂和形态 作为对高温和低渗挑战的应激反应。 最后，选择的钙调蛋白结合肽序列将与 启动子序列，以靶向表达单个钙调蛋白 结合肽的第二型上皮细胞的灵或心脏 转基因小鼠的心室。 这些动物应该考虑到 了解完整组织中钙调素的抑制作用， 肺上皮疾病模型如囊性纤维化和 通过随机选择， 肽库是一种独立的研究细胞免疫的方法。 功能 这种肽方法应适用于评价 其他细胞蛋白质的作用，其中天然修饰剂 尚未被发现。