The long-range objectives of this study are to understand the manner in which folding information is encoded in the primary sequence of membrane proteins and the means by which the cell recognizes when this process of information transfer has failed. The studies of the cystic fibrosis transmembrane conductance regulator (CFTR) supported by this grant entering its fourth year, have provided fundamental information relevant to understanding the nature of the AF508 folding mutant responsible for most cases of cystic fibrosis and the proteins which transiently interact with CFTR to assist efficient folding. Future studies will focus on elucidation of the conformation of critical folding intermediates, the machinery that recognizes these intermediates, and the later steps in folding, namely the association of CFTR with other proteins to form macromolecular complexes at the membrane. To this end the five specific aims are to: 1. Characterize the structure of the CFTR foldina intermediate altered bv the AF508 mutation. Established biophysical methods will be employed to characterize the structural features of the critical folding intermediate. 2. Identify the protein machinery required for recognition of the folding intermediate and characterize their mechanism of action. The proteins required for recognition of the critical folding intermediate in vivo will be isolated using our recently developed in vitro proteolysis assay which distinguishes mutant from wild type protein during folding. 3. Determine the folding pathway of the first transmembrane domain of CFTR and the effect of disease-causing mutations. Peptide models of the transmembrane spans of CFTR and an in vitro translation system will be utilized to study the membrane-integration and helical-association steps of the wild type and mutant transmembrane domains. 4. Identity the proteins that recognize misfolded transmembrane domains and determine their mechanism of action. Site specific-crosslinking and high stringency immunoprecipitations will be used to identify proteins that interact with misfolded mutant transmembrane domains of CFTR. 5. Characterize the interaction of CFTR with other proteins during maturation to form a supramolecular complex. Based on our recent finding that CFTR activates the anion exhanger in CFTR expressing cells and tissues, we will test the hypothesis that these proteins associate to form a large quartemary structure during the last steps of folding. To accomplish these goals, a combination of biochemical, biophysical, immunochemical, molecular and cell biological approaches, all established in this laboratory, will be employed. These studies are necessary for and fundamental to a detailed understanding of the mechanisms by which membrane proteins fold.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Medical Biochemistry Study Section (MEDB)
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Mckeon, Catherine T
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University of Texas Sw Medical Center Dallas
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Pinarbasi, Emile S; Karamyshev, Andrey L; Tikhonova, Elena B et al. (2018) Pathogenic Signal Sequence Mutations in Progranulin Disrupt SRP Interactions Required for mRNA Stability. Cell Rep 23:2844-2851
Sosnay, Patrick R; Siklosi, Karen R; Van Goor, Fredrick et al. (2013) Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene. Nat Genet 45:1160-7
Mendoza, Juan L; Schmidt, Andre; Li, Qin et al. (2012) Requirements for efficient correction of ýýF508 CFTR revealed by analyses of evolved sequences. Cell 148:164-74
Patrick, Anna E; Thomas, Philip J (2012) Development of CFTR Structure. Front Pharmacol 3:162
Patrick, Anna E; Karamyshev, Andrey L; Millen, Linda et al. (2011) Alteration of CFTR transmembrane span integration by disease-causing mutations. Mol Biol Cell 22:4461-71
Hoelen, Hanneke; Kleizen, Bertrand; Schmidt, Andre et al. (2010) The primary folding defect and rescue of ýýF508 CFTR emerge during translation of the mutant domain. PLoS One 5:e15458
Zhu, Li; Millen, Linda; Mendoza, Juan L et al. (2010) A unique redox-sensing sensor II motif in TorsinA plays a critical role in nucleotide and partner binding. J Biol Chem 285:37271-80
Lewis, Karen A; Yaeger, Arynn; DeMartino, George N et al. (2010) Accelerated formation of alpha-synuclein oligomers by concerted action of the 20S proteasome and familial Parkinson mutations. J Bioenerg Biomembr 42:85-95
Lewis, Karen A; Su, Yang; Jou, Olina et al. (2010) Abnormal neurites containing C-terminally truncated alpha-synuclein are present in Alzheimer's disease without conventional Lewy body pathology. Am J Pathol 177:3037-50
Dorwart, Michael R; Shcheynikov, Nikolay; Baker, Jennifer M R et al. (2008) Congenital chloride-losing diarrhea causing mutations in the STAS domain result in misfolding and mistrafficking of SLC26A3. J Biol Chem 283:8711-22

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