DESIGN AND TRANSGENIC ANALYSIS OF CELLULAR INHIBITORS

细胞抑制剂的设计和转基因分析

• 批准号: 6124788
• 负责人: JOHN R DEDMAN
• 金额: $ 29.82万
• 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 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6124788
• 关键词: calcineurin; calcium flux; calmodulin; calmodulin dependent protein kinase; cell growth regulation; cell line; enzyme activity; enzyme inhibitors; enzyme mechanism; gene expression; gene mutation; genetic promoter element; genetically modified animals; growth inhibitors; laboratory mouse; lung alveolus; oligopeptides; oxidative stress; peptide library; protein sequence; respiratory epithelium; transfection; voltage /patch clamp

The central hypothesis of our project has been that short peptides can be used as potent, specific inhibitors which can be targeted to desire organelles and cell types in order to precisely elucidate cellular pathways. We have shown that the nuclear function of calmodulin in type II cells is critical for murine lung development. The highly flattened type I cells provide a vast cellular surface for O2/CO2 exchange. Lung alveoli are critical for the maintenance of oxygen homeostasis. Type I cells are extremely sensitive to injury from environmental challenges and they do not divide. Following lung damage, type II cells terminally differentiate to repopulate the alveolar epithelium with new type I cells. The research in this project will use novel transgenic approaches to study, in vivo, the role of CaM kinase II- and CalN-regulated pathways involved in lung development and type II cell response to lung injury. CaM kinase II and CalN are the major enzyme systems which mediate Ca/2+/CaM regulation of cellular activities such as secretion, ion fluxes, DNA replication and RNA transcription. Studies are performed in transgenic mice in which the SP-C (surfactant protein C) promoter is used to direct expression of synthetic dominant-negative genes which neutralize the function of CaM Kinase II and CalN in lung type II epithelial cells. These synthetic genes encode peptide concatemers which bind to the active site in the catalytic subunit and cause inhibition of their targeted enzymes in a cell-specific manner. The targeting of these inhibitory peptide concatemers to the lung epithelium will lead to the generation of unique phenotypes in which Ca/2+- activated signal transduction systems are altered. This project will evaluate the role of CaM kinase II and CaIN in lung development and physiological responses to oxidative stress, ozone and silica-induced injury. CaM kinase II and CalN may be required for proper lung function such as fluid and surfactant secretion. It is anticipated that unique mouse lines will be developed that have altered lung compliance, mucous congestion and lung fibrosis. These pathophysiological conditions will result in decreased gas exchange in the alveolus which will cause hypoxemia, plasma pH imbalance and physical disability.

我们项目的中心假设是，短肽可以用作有效的特异性抑制剂，可以靶向所需的细胞器和细胞类型，以精确阐明细胞通路。我们已经表明，在II型细胞的钙调蛋白的核功能是小鼠肺发育的关键。高度扁平的I型细胞为O2/CO2交换提供了广阔的细胞表面。肺泡对于维持氧稳态至关重要。I型细胞对来自环境挑战的损伤极其敏感，并且它们不分裂。肺损伤后，II型细胞终末分化，用新的I型细胞重新填充肺泡上皮。本项目的研究将使用新型转基因方法在体内研究CaM激酶II和CalN调节途径在肺发育和II型细胞对肺损伤反应中的作用。CaM激酶II和CaN是介导Ca/2+/CaM调节细胞活动如分泌、离子通量、DNA复制和RNA转录的主要酶系统。在转基因小鼠中进行研究，其中SP-C（表面活性蛋白C）启动子用于指导合成的显性阴性基因的表达，所述基因中和肺II型上皮细胞中CaM激酶II和CalN的功能。这些合成基因编码肽多联体，其与催化亚基中的活性位点结合，并以细胞特异性方式抑制其靶向酶。这些抑制性肽多联体靶向肺上皮将导致产生独特的表型，其中Ca/2+激活的信号转导系统被改变。该项目将评估CaM激酶II和CaIN在肺发育和对氧化应激、臭氧和二氧化硅诱导的损伤的生理反应中的作用。CaM激酶II和CalN可能是正常肺功能所必需的，如液体和表面活性剂分泌。预计将开发出具有改变的肺顺应性、粘液充血和肺纤维化的独特小鼠品系。这些病理生理状况将导致肺泡中的气体交换减少，这将导致低氧血症、血浆pH失衡和身体残疾。