More than 90% of CF patients have at least one deltaF508 allele of the CFTR gene. The objective "Biosynthetic Arrest of Mutant CFTR' is to gain a complete understanding of the biosynthetic block and mislocalization of the deltaF508 CFTR protein. This information will facilitate the development of practical approaches to overcoming the biosynthetic arrest of the major CF-causing mutation and several other less frequent ones which also compromise biosynthetic processing. This overall objective will be pursued via four specific aims. In Specific Aim I, we shall flesh out our present critical but rudimentary knowledge that deltaF508 CFTR is blocked at the level of the endoplasmic reticulum (ER) and that it is at least some residual function. Confocal and electron microscopy will be used to precisely determine the localization; inherent activity of the molecule will be quantitatively assessed both after its purification and in isolated ER. Monoclonal antibodies will be generated to distinguish the biosynthetically immature and mature forms of CFTR so that the maturation process can be readily monitored. Specific Aim II will investigate the role of chaperones in CFTR biosynthesis. The detection of any potential differential interactions between a chaperone and wild-type and deltaF508 CFTR will provide a target to manipulate to alleviate the biosynthetic arrest. Elucidation of the details of the interaction with calnexin which has already been identified will be intensively pursued. Since the failure of a calnexin-CFTR complex to dissociate seems to contribute to the ER retention of deltaF508, ways of avoiding or dissociating this complex will be sought. Because oligosaccharides are involved in complex formation, glycosylation inhibitors will be tested. In Specific Aim III, several other ways of getting at least some of the deltaF508 protein past the block will be investigated. We shall test the effects of antibodies, laser irradiation and nucleotide analogs. Manipulations which are affective in getting deltaF508 CFTR to the surface of cultured cells will be evaluated in transgenic mice expressing only that allele of CFTR to determine if testing in patients of that genotype is appropriate. Specific Aim IV to identify and characterize other disease-causing mutations which compromise protein maturation should be of most benefit by providing additional insights into aspects of the normal process which may not be revealed by studying just the wild-type and deltaF508 CFTR molecules.
NIH ExPorter
NIH ExPorter