DESIGN AND TRANSGENIC ANALYSIS OF CELLULAR INHIBITORS

钙是所有细胞中许多功能的主要调节剂。

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.