The objectives of this proposal are to complete some very straightforward studies which should greatly facilitate our understanding of both the molecular and chemical basis of cystic fibrosis (CF). The proposed studies will focus on the CFTR protein (cystic fibrosis transmembrane conductance regulator), predicted from cDNA sequence analysis to be 1480 amino acids In length. The predicted protein Includes both a large membrane spanning region and a large cytoplasmic domain, the latter consisting of two putative ATP binding regions (ATP-1, and ATP-11) and a putative regulatory region (R) thought to be a protein kinase target. Significantly, almost 70% of CF patients lack a phenylalanine in the center of the ATP-1 region. Consequently, it has been suggested that the wild type CFTR may be an 'ion motive' ATPase which couples ATP hydrolysis to Cl- transport, and that the phenylalanine deletion mutation may alter the binding or hydrolysis of ATP and, therefore, Cl- transport. With these thoughts in mind, the Specific Aims of this proposal are sixfold: 1.Prepare In large amounts, using both chemical and molecular biological approaches, the two cytoplasmic regions, ATP-1 and ATP-11, of the 'wild type"""""""" CFTR protein. 2.Characterize the resultant peptides physically, and assess their capacity to bind and hydrolyze ATP In the presence and absence of Cl-. 3.Prepare as in '1' the ATP-1 region containing the single phenylalanine deletion found in most cystic fibrosis patients. 4.Characterize physically the ATP-1 phenylalanine mutant peptide and assess its capacity to bind and hydrolyze ATP in the presence and absence of Cl-. 5.Prepare, as in """"""""l"""""""", a """"""""wild type' and a phenylalanine mutant peptide containing both the regulatory region 'R' and the ATP-1 region, and compare these peptides' capacity to bind and hydrolyze ATP before and after treatment with protein kinases A or C. 6.Assess the effect of antibodies to the ATP-1, ATP-11, and R + ATP-1 regions on the ATPase activity of epithelial cell membranes. The proposed studies are fundamental for understanding the molecular and chemical basis of cystic fibrosis, and may encourage future experiments directed at replacing only a small portion of the """"""""wild type"""""""" CFTR gene in CF patients.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
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Johns Hopkins University
Schools of Medicine
United States
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Annereau, Jean Philippe; Ko, Young Hee; Pedersen, Peter L (2003) Cystic fibrosis transmembrane conductance regulator: the NBF1+R (nucleotide-binding fold 1 and regulatory domain) segment acting alone catalyses a Co2+/Mn2+/Mg2+-ATPase activity markedly inhibited by both Cd2+ and the transition-state analogue orthovanada Biochem J 371:451-62
Pedersen, Peter L (2002) Transport ATPases in biological systems and relationship to human disease: a brief overview. J Bioenerg Biomembr 34:327-32
Ko, Y H; Pedersen, P L (2001) Cystic fibrosis: a brief look at some highlights of a decade of research focused on elucidating and correcting the molecular basis of the disease. J Bioenerg Biomembr 33:513-21
Massiah, M A; Ko, Y H; Pedersen, P L et al. (1999) Cystic fibrosis transmembrane conductance regulator: solution structures of peptides based on the Phe508 region, the most common site of disease-causing DeltaF508 mutation. Biochemistry 38:7453-61
Thomas, P J; Ko, Y H; Shenbagamurthi, P et al. (1995) Nucleotide domains in transport ATPases: structure-function and relationship to disease. Soc Gen Physiol Ser 50:17-28
Ko, Y H; Thomas, P J; Pedersen, P L (1994) The cystic fibrosis transmembrane conductance regulator. Nucleotide binding to a synthetic peptide segment from the second predicted nucleotide binding fold. J Biol Chem 269:14584-8
Thomas, P J; Pedersen, P L (1993) Effects of the delta F508 mutation on the structure, function, and folding of the first nucleotide-binding domain of CFTR. J Bioenerg Biomembr 25:11-9
Ko, Y H; Thomas, P J; Delannoy, M R et al. (1993) The cystic fibrosis transmembrane conductance regulator. Overexpression, purification, and characterization of wild type and delta F508 mutant forms of the first nucleotide binding fold in fusion with the maltose-binding protein. J Biol Chem 268:24330-8
Thomas, P J; Ko, Y H; Pedersen, P L (1992) Altered protein folding may be the molecular basis of most cases of cystic fibrosis. FEBS Lett 312:7-9
Thomas, P J; Shenbagamurthi, P; Sondek, J et al. (1992) The cystic fibrosis transmembrane conductance regulator. Effects of the most common cystic fibrosis-causing mutation on the secondary structure and stability of a synthetic peptide. J Biol Chem 267:5727-30