Cystic fibrosis (CF), a disease characterized by altered salt and water movement across epithelial tissues, is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR). More than 1400 mutations have been identified in the CFTR, resulting in alterations to protein biosynthesis, trafficking, function, regulation and recycling. The loss of plasma membrane resident CFTR activity results in disease pathophysiology. The most common mutation, deletion of phenylalanine 508 (?F508) is associated with >90% of all CF patients. This mutant protein fails to adopt native conformation during biosynthesis, fails to traffic from the endoplasmic reticulum (ER) and is subsequently degraded by the ubiquitin-proteasome system. The ?F508 mutation, and a series of second-site suppressor mutations that partially rescue the ?F508 phenotype, are located within a globular, cytosolic domain. In vitro studies of this domain show that the ?F508 mutation alters local domain properties and suggest that these alterations may underlie the basic physical defects associated with CF. However, several critical questions remain unanswered. First, what physical properties of NBD1 are associated with the recognition and regulation of CFTR folding/misfolding in the cell? In vitro biophysical analyses show alterations in NBD properties as a result of the ?F508 mutation, though it is not know how these changes are manifest and recognized within the cellular environment. Second, how does post-translational ubiquitinylation of the NBD and CFTR protein regulate its processing and trafficking? Emerging data suggest that ubiquitin modification is dynamic and may play a key role in wildtype CFTR processing and ?F508 CFTR degradation. Understanding how dynamic ubiquitinylation modulates CFTR trafficking and recycling is critical to developing a complete model of CFTR regulation. Finally, what cellular systems regulate CFTR trafficking? Specific CFTR-quality control interactions regulate these post-translational modifications, ultimately regulating the biological fate of CFTR. Elucidation of these systems is fundamental to understanding the regulation of CFTR biosynthesis and trafficking. The long-term goals of this study are to elucidate how changes in protein folding are manifest within the cell and how these changes are recognized by cellular quality control systems.
The specific aims of this project are to: (1) Characterize the role of altered NBD1 folding in the biosynthesis of CFTR. Using novel cell-based methods to assess changes in protein physical properties, we will elucidate changes in NBD domain folding and its regulation of CFTR trafficking. (2) Elucidate the role of de-ubiquitinylation by the ubiquitin protease USP8 during CFTR biogenesis. Preliminary work demonstrates that the USP8 protease regulates CFTR expression and early biosynthetic events. Using cell biological, in vitro biochemical and mutagenic approaches, we will establish the role(s) of USP8 in regulating CFTR biogenesis. (3) Characterize the roles of the E6-AP ubiquitin ligase in the regulation of CFTR processing and trafficking. Preliminary studies demonstrate that the E6-AP ubiquitin ligase regulates CFTR expression in a structure-dependent manner. Using in vitro and cell biological approaches, we will evaluate the role of E6-AP in regulating CFTR biogenesis and expression. These studies will provide novel insight into the structures of CFTR that are recognized by cellular systems and the systems that regulate CFTR maturation. Further, these studies will explore the role of dynamic, reversible ubiquitinylation of CFTR during its regulated biosynthesis. Characterization of these structures and cellular systems, provide fundamental information on protein biosynthesis and its regulation by cellular systems. Finally, characterization of these cellular systems and their interactions with CFTR provides critical information for the development of therapeutic strategies to modulate the processes altered by disease-associated mutations.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Mckeon, Catherine T
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Pittsburgh
Schools of Medicine
United States
Zip Code
Garcia, Carlos J; Pericleous, Androulla; Elsayed, Mennat et al. (2018) Serralysin family metalloproteases protects Serratia marcescens from predation by the predatory bacteria Micavibrio aeruginosavorus. Sci Rep 8:14025
Ran, Yanchao; Zheng, Aiping; Thibodeau, Patrick H (2018) Structural analysis reveals pathomechanisms associated with pseudoxanthoma elasticum-causing mutations in the ABCC6 transporter. J Biol Chem 293:15855-15866
Ran, Yanchao; Thibodeau, Patrick H (2017) Stabilization of Nucleotide Binding Domain Dimers Rescues ABCC6 Mutants Associated with Pseudoxanthoma Elasticum. J Biol Chem 292:1559-1572
Stella, Nicholas A; Callaghan, Jake D; Zhang, Liang et al. (2017) SlpE is a calcium-dependent cytotoxic metalloprotease associated with clinical isolates of Serratia marcescens. Res Microbiol 168:567-574
Vernon, Robert M; Chong, P Andrew; Lin, Hong et al. (2017) Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations. J Biol Chem 292:14147-14164
Gong, Xiaoyan; Ahner, Annette; Roldan, Ariel et al. (2016) Non-native Conformers of Cystic Fibrosis Transmembrane Conductance Regulator NBD1 Are Recognized by Hsp27 and Conjugated to SUMO-2 for Degradation. J Biol Chem 291:2004-17
Shanks, Robert M Q; Stella, Nicholas A; Hunt, Kristin M et al. (2015) Identification of SlpB, a Cytotoxic Protease from Serratia marcescens. Infect Immun 83:2907-16
Needham, Patrick G; Patel, Hardik J; Chiosis, Gabriela et al. (2015) Mutations in the Yeast Hsp70, Ssa1, at P417 Alter ATP Cycling, Interdomain Coupling, and Specific Chaperone Functions. J Mol Biol 427:2948-65
Zhang, Liang; Morrison, Anneliese J; Thibodeau, Patrick H (2015) Interdomain Contacts and the Stability of Serralysin Protease from Serratia marcescens. PLoS One 10:e0138419
Xue, Peng; Crum, Chelsea M; Thibodeau, Patrick H (2014) Regulation of ABCC6 trafficking and stability by a conserved C-terminal PDZ-like sequence. PLoS One 9:e97360

Showing the most recent 10 out of 19 publications