Program Director/Principal Investigator (Last. First, Middle): T h o m a S , P h i l i p J . PROJECT SUMMARY (See instmctions): The original application for competitive renewal of the R01 grant that was selected for a MERIT award proposed three alms to address three fundamental questions relevant to the mechanisms by which CFTR forms Its functional, native structure and how this process Is altered by disease-causing mutations. How does AF508 interfere with NBD1 folding? Does AF508 significantly modify the interaction of the folded NBD1 with other domains? What interactions with quality control proteins are critical? During the first four and a half years of MERIT support we have answered the first two questions In an exploitable way. These results indicate that CFTR folding is a hierarchical process and provide a clear explanation for the efficacy "ceiling" for extant compounds that correct folding of the AF508 mutant. They also suggest a means to a mechanism-based approai^h for the discovery new compounds that work in synergy with extant correctors or novel compounds that circumvent the "ceiling". These approaches are already being employed by a number of drug discovery efforts. Finally, using a powerful specific photo-crosslinking method, differential interactions of proteins with mutant and wild type nascent chains translated in vitro have revealed a previously unappreciated mechanism for preemptive quality control. The system involves proteins that lead to the degradation of the mRNA coding for the mutant protein, thereby reducing the production of protein bound to misfold. This mechanism prevents the accumulation of potentially cytotoxic misfoided proteins without spending energy for futile translation and subsequent ATP dependent proteolysis by the proteasome. We are now requesting continued support of the MERIT award to extend the analyses successfully applied to AF508 in Aim 1 and 2 to additional CF-causing mutations and to define and characterize the mechanisms responsible for preemptive quality control system discovered during execution of Aim 3. We thank the institute for selecting our study for MERIT support that allowed pursuit of the long term discovery effort of Aim 3 that has now revealed novel and unexpected biology. Such a path would not have been feasible under the time constraints of a R01.
Most cases of cystic fibrosis, a common fatal genetic disease, are caused by mutations that interfere with the assembly of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The studies proposed will elucidate the details of how the disease-causing mutations interfere with this process. Understanding the assembly process, and, thus, the detailed molecular pathology, will provide important information for developing targeted therapeutics for cystic fibrosis.
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|Bozoky, Zoltan; Krzeminski, Mickael; Muhandiram, Ranjith et al. (2013) Regulatory R region of the CFTR chloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions. Proc Natl Acad Sci U S A 110:E4427-36|
|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|
|Somalinga, Balajee R; Day, Cameron E; Wei, Shuguang et al. (2012) TDP-43 identified from a genome wide RNAi screen for SOD1 regulators. PLoS One 7:e35818|
|Patrick, Anna E; Thomas, Philip J (2012) Development of CFTR Structure. Front Pharmacol 3:162|
|Mendoza, Juan L; Schmidt, Andre; Li, Qin et al. (2012) Requirements for efficient correction of Ã½Ã½F508 CFTR revealed by analyses of evolved sequences. Cell 148:164-74|
|Peters, Kathryn W; Okiyoneda, Tsukasa; Balch, William E et al. (2011) CFTR Folding Consortium: methods available for studies of CFTR folding and correction. Methods Mol Biol 742:335-53|
|Schmidt, Andre; Mendoza, Juan L; Thomas, Philip J (2011) Biochemical and biophysical approaches to probe CFTR structure. Methods Mol Biol 741:365-76|
|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|
|Somalinga, Balajee R; Miller, Gregory A; Malik, Hiba T et al. (2011) A screen to identify cellular modulators of soluble levels of an amyotrophic lateral sclerosis (ALS)-causing mutant SOD1. J Biomol Screen 16:974-85|
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