Cystic fibrosis, a disease of altered water and salt secretion across epithelial tissues, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a member of the ABC-transporter family of proteins and functions as a chloride channel;loss of the functional chloride channel activity is causative of CF. The ATP-Binding Cassette (ABC-) transporter superfamily of proteins is highly conserved across prokaryotes and eukaryotes, facilitating solute transport across biological membranes. ABC-transporter proteins are minimally composed of a dimer of highly conserved, cytosolic nucleotide-binding domains (NBDs), which provides the energy for solute transport through a dimer of transmembrane domains (TMDs). ATP binding and hydrolysis within the NBDs is regulated by the canonical Walker A and B nucleotide-binding sequences, as well as a sequence unique to the ABC-transporter family of proteins ? the signature sequence, or LSGGQRxR. While the Walker A and B sequences are well characterized, the structural and functional properties of the LSGGQRxR sequence are not known. Preliminary data suggest that the LSGGQRxR sequence critically contributes to the biosynthesis and function of CFTR and other ABC-transporter proteins. Functional regulation of NBD-NBD association events is critically altered by substitution of the glycine residues, resulting in either hyperactive or inactive channels. Alterations to the RxR sequence alter channel biosynthesis with only minor effects on channel activity. The major goal of this project is to elucidate the structural and functional roles of the LSGGQRxR sequence in regulating CFTR-channel and ABC-transporter biosynthesis and function. To accomplish this, we have developed methods for the expression, purification and biophysical characterization of the isolated NBD proteins from CFTR and two homologues (human ABCC6 and the bacterial Mj0796). Using a combination of X-ray crystallographic, nucleotide binding and hydrolysis, and biochemical approaches, we will evaluate the specific structural and functional roles of the LSGGQRxR signature sequence. These in vitro data will complement experiments evaluating the biosynthesis and function of full-length protein to provide a detailed model for the regulation of ABC-transporter biogenesis and mechanochemistry by the signature sequence.
The specific aims of this application are: (1) Characterize the nucleotide-binding and hydrolysis properties of CFTR NBD1 and NBD2, (2) Elucidate the role of the signature sequence di-glycine residues in ATP-mediated NBD association and function, and (3) Characterize the role of the RxR sequence on local NBD structure and CFTR biosynthesis. The research proposed in this application will provide novel insight into the previously undefined structural and functional roles of this highly conserved LSGGQRxR sequence. The innovative use of in vitro biochemical, structural and enzymatic assays to complement studies of full-length protein biosynthesis and function will refine models of the biogenesis and mechanochemistry of these medically important proteins. Project
ABC transporters play a key role in human disease by contributing to disease pathophysiology when mutated and by contributing to virulence of opportunistic pathogens. Understanding the biosynthetic and functional regulation of these proteins will contribute to our understanding of how these proteins directly and indirectly impact human disease.
|Bilgin, Betul; Nath, Aritro; Chan, Christina et al. (2016) Characterization of transcription factor response kinetics in parallel. BMC Biotechnol 16:62|
|Vocelle, Daniel; Chesniak, Olivia M; Malefyt, Amanda P et al. (2016) Dextran functionalization enhances nanoparticle-mediated siRNA delivery and silencing. Technology (Singap World Sci) 4:|
|Sims, James Kenneth; Rohr, Brian; Miller, Eric et al. (2015) Automated Image Processing for Spatially Resolved Analysis of Lipid Droplets in Cultured 3T3-L1 Adipocytes. Tissue Eng Part C Methods 21:605-13|
|Sridharan, Gautham V; Choi, Kyungoh; Klemashevich, Cory et al. (2014) Prediction and quantification of bioactive microbiota metabolites in the mouse gut. Nat Commun 5:5492|
|Choi, KyungOh; Ghaddar, Bassel; Moya, Colby et al. (2014) Analysis of transcription factor network underlying 3T3-L1 adipocyte differentiation. PLoS One 9:e100177|
|Malefyt, Amanda P; Wu, Ming; Vocelle, Daniel B et al. (2014) Improved asymmetry prediction for short interfering RNAs. FEBS J 281:320-30|
|Bilgin, Betul; Liu, Li; Chan, Christina et al. (2013) Quantitative, solution-phase profiling of multiple transcription factors in parallel. Anal Bioanal Chem 405:2461-8|
|Angart, Phillip; Vocelle, Daniel; Chan, Christina et al. (2013) Design of siRNA Therapeutics from the Molecular Scale. Pharmaceuticals (Basel) 6:440-68|
|Sims, James K; Manteiga, Sara; Lee, Kyongbum (2013) Towards high resolution analysis of metabolic flux in cells and tissues. Curr Opin Biotechnol 24:933-9|
|Lai, Ning; Sims, James K; Jeon, Noo Li et al. (2012) Adipocyte induction of preadipocyte differentiation in a gradient chamber. Tissue Eng Part C Methods 18:958-67|
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