DESIGN AND TRANSGENIC ANALYSIS OF CELLULAR INHIBITORS

钙是所有细胞中许多功能的主要调节器。 细胞内游离钙水平的变化起到了信号的作用。 细胞内游离钙的调节是通过高亲和力实现的 钙结合蛋白。钙调蛋白，一种特性良好的钙 介体蛋白是细胞必需的基因产物 生存能力。钙-钙调蛋白复合体与偶联有关 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多肽。依赖于钙离子 亲和层析用于选择钙调蛋白结合 来自随机多肽噬菌体文库的多肽。序列和 对这些肽的预测结构分析表明，它们是 与先前报道的结合多肽相比是独特的 钙调素。我们建议设计一种亲和纯化策略来 选择针对不同构象的特定序列 钙调素的状态，即仅与钙调素结合的多肽 钙的存在，只有在没有钙的情况下才能结合的那些，以及 那些与钙浓度无关的细胞。这个 然后将对这些独特多肽的生理效应进行表征 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。人工合成基因将被设计出来 它将用于表达钙调蛋白结合肽 在活体内。还将检查细胞的生长、分裂和形态 作为对高温和低渗挑战的应激反应。 最后，选定的钙调蛋白结合肽序列将与 针对单个钙调蛋白表达的启动子序列 结合肽与心肌或心脏II型上皮细胞的结合 转基因小鼠的脑室。这些动物应该考虑到 对完整组织中钙调素抑制的理解和 肺上皮性疾病模型的发展，如囊性纤维化和 随机选择男性修饰者的心脏肌病 多肽文库是一种独立的细胞研究方法 功能。这种多肽方法应该适用于评估 天然修饰物对其他细胞蛋白质的作用 还没有被发现。

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.