John Raymond Dedman
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-affinitycalcium binding-proteins. Calmodulin, a well-characterized calciummediator protein, has been shown to be an essential gene product for cellviability. The Ca2-calmodulin complex has been implicated in couplingcell responses to many stimuli. We have designed an approach to identifypeptides which bind to a targeted protein, calmodulin. Ca2-dependentaffinity chromatography has been used to select calmodulin bindingpeptides from a bacteriophage library of random peptides. Sequence andpredicted structure analysis of these peptides suggest that they areunique when compared to peptides previously reported as bindingcalmodulin. We propose to design an affinity-purification strategy toselect sequences which are specific for the different conformationalstates of calmodulin, that is peptides which bind calmodulin only in thepresence of calcium, those which bind only in the absence of calcium, andthose which are indifferent to the calcium concentration. Thephysiological effects of these unique peptides will then be characterizedin intact cellular systems including inthe muscle fiber, neuron, chromaffin cell and epithelial cell. Synthetic genes will be designedwhich will be used for the expression of the calmodulin binding peptidesin vivo. Cell growth, division and morphology will be examined as wellas the stress response to elevated temperature and hypotonic challenge. Finally, selected calmodulin binding peptide sequences will be fused withpromotor sequence in order to target expression of individual calmodulinbinding peptides to the type II epithelial cells of theling or cardiacventricles of transgenic mice. These animals should allow for theunderstanding of calmodulin inhibition in intact tissue and thedevelopment of lung epithelial disease models such as cystic fibrosis andcardiac myopathies in male modifiers through selection from randompeptide libraries is an independent approach for the study of cellularfunction. This peptide approach should be applicable to the evaluationof the role of other cellular proteins for which natural modifiers haveyet to be discovered.
Investigators:Dedman, John 07-01-2005 -06-30-2007 American Heart Association - Ohio Valley Regulation of Cardiac Hypertrophy by CaZ+-CaM Kinase Role:PI $121,000.00 Closed Level:Private Non-Profit
Grant: #5-R01-DK-46433-10-A0-S0-E0 Investigators:Dedman, John 01-01-1999 -11-30-2004 National Inst of Diabetes and Digestive and Kidney Disease Design and Transgenic Analysis of Cellular Inhibitors Role:PI $1,695,000.00 Closed Level:Federal
Grant: #5-R01-HL-60861-04-A0-S0-E0 Investigators:Dedman, John 02-01-2000 -01-31-2004 National Heart, Lung and Blood Institute Treatment Strategies for the Anti-Phospholipid Syndrome Role:PI $976,849.00 Closed Level:Federal
Stress,Cytology,Calmodulin,Epithelium,Calcium Flux,Conformation,Gene Expression,Genetic Library,Chemical Binding,Growth Inhibitor,Laboratory Mouse,Protein Sequence,Transgenic Animal,Chemical Synthesis,Genetic Manipulation,Cell Growth Regulation,Affinity Chromatography,Genetic Promoter Element