The investigators of this project propose to elucidate the mechanisms involved in the novel biosynthetic processing of wild-type CFTR and its failure in the case of delta F508 and other disease-associated variants. Studies done thus far have indicated that these mechanisms are likely to be intricate and complex. We have learned that nascent CFTR interacts with molecular chaperones on both sides of the endoplasmic reticulum (ER) membrane and is recognized on the cytoplasmic side by the ubiquitin- proteasome pathway. We postulate that the coordinated assembly of CFTR's multiple cytoplasmic domains with the integration of twelve membrane- spanning sequences requires several chaperones which either succeed in fostering a native global tertiary structure or fail and lead the molecule to degradation pathways(s). This dual role of chaperones may provide an efficient kinetic mechanism to dispose of molecules unable to achieve a mature folded state despite repeated rounds of chaperone binding. The proportion which are directed to a proteolytic pathway may be especially high for proteins like CFTR with an elaborate domain structure necessary to its complex regulatory function. We shall pursue this hypothesis by four specific aims. The first is not profound and aims simply to collect direct evidence that the inefficient maturation of wild-type CFTR observed in cultured cells actually occurs in vivo in relevant epithelial tissues. The second is to further dissect the network of interactions of nascent CFTR with chaperones, the ubiquitin-proteasome system and other degradation pathways. We shall identify the sites of ubiquitination on CFTR and determine the exact role of the C-terminal tail of the protein in determining the balance between maturation and degradation. The preliminary evidence that the R-domain may be especially important in the targeting of nascent CFTR for ubiquitination and proteolysis will be explored further.
In Aim 3 we shall utilize yeast mutants in the secretory pathway, in the ubiquitin-proteasome pathway and in molecular chaperones to dissect the steps in CFTR processing at the ER.
In Aim 4, we shall continue our systematic evaluation of disease-associated mutations to determine which ones cause misprocessing. Having just completed analysis of 30 such mutations in the cytoplasmic loops, we shall now turn to the membrane-spanning sequences. The second part of this aim is to attempt to determine the influence of these mutations on in C1- channel function by fusing microsomes from cells expressing them with planar lipid bilayers.
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|Gentzsch, M; Riordan, J R (2001) Localization of sequences within the C-terminal domain of the cystic fibrosis transmembrane conductance regulator which impact maturation and stability. J Biol Chem 276:1291-8|
|Kiser, G L; Gentzsch, M; Kloser, A K et al. (2001) Expression and degradation of the cystic fibrosis transmembrane conductance regulator in Saccharomyces cerevisiae. Arch Biochem Biophys 390:195-205|
|Hammerle, M M; Aleksandrov, A A; Chang, X B et al. (2000) A novel CFTR disease-associated mutation causes addition of an extra N-linked oligosaccharide. Glycoconj J 17:807-13|
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