The goal of this Core is to support CF-related research at DHMC. The Core conducts confocal microscopy, image analysis, measurements of intracellular ion activity and electrophysiological analysis of CF tissue in collaboration with Principal Investigators and their students.
Summary of Ongoing Research Projects
1. Novel P. aeruginosa antibiotic targets and compounds (Ronald Taylor, Ph.D. Department of Microbiology). The goals of this project are to determine if the P. aeruginosa PilD type four prepilin peptidase (TFPP) functions by a similar catalytic mechanism as has been determined for the TFPPs of Vibrio cholerae. Once established, the goals are to identify lead compounds for inhibiting the activity of this enzyme, which is required for colonization of the lung by P. aeruginosa, and subsequent secretion of exotoxin and other virulence factors.
2. Pseudomonas aeruginosa biofilm development in CF Airways (George O'Toole, Ph.D. Department of Microbiology & Immunology). The goal of this project is to elucidate biofilm development in normal and CF airways and to identify new therapies to treat airway infection.
3. Staphylococcal genes important to the induction of apoptosis in CF airway epithelial cells (Ambrose Cheung, M.D. Department of Microbiology & Immunology). The goal of this project is to define the contribution of Staphylococcus aureus to airway inflammation and disease and to identify new therapies to treat airway infection.
4. Alterations in ion transport and calcium signaling upon exposure
of Calu-3 cells and CFT-1 cells to S. aureus. (Ambrose Cheung, M.D.
Department of Microbiology). The goal of this project is to elucidate the
acute effects of S. aureus on Cl- and Na+ transport across monolayers of
normal and CF human airway epithelial cells. Preliminary results indicate
that proteins elaborated by S. aureus activate CFTR-mediated Cl- secretion
by Calu-3 cells.
1. Anthracyclines for Treatment of Cystic Fibrosis (Joshua Hamilton, Ph.D. Department Pharmacology and Toxicology). The goal of this project is to identify new pharmacological agents for treatment of CF, focusing in particular on the anthracyclines, anthracenediones and related compounds and their ability to increase the functional expression of ®F508-CFTR in the plasma membrane.
2. Effect of P. aeruginosa on Apical Expression of CFTR (Drs. Swiatecka-Urban, Stanton and O'Toole. Departments of Microbiology & Immunology and Physiology). These studies represent a collaboration between the microbiology and cell biology strengths of the program. This work focuses on identifying and characterizing a P. aeruginosa factor that affects the apical expression of CFTR. Studies are underway to elucidate the mechanisms by which this bacterial factor impacts the cell biology of CFTR.
3. Endocytosis and recycling of wt and ®F508-CFTR: Membrane stability of wt-CFTR and ®F508-CFTR (Drs. Swiatecka-Urban, Langford and Stanton. Departments of Biological Sciences and Physiology). The goals of this project are to: (1) Characterize the rates of endocytosis and recycling of wt and ®F508-CFTR; and (2) Identify the motifs in wt and ®F508-CFTR that regulate endocytosis and recycling.
4. Structure and function of the cystic fibrosis transmembrane conductance regulator (Dean R. Madden, Ph.D. Department of Biochemistry). The goal of this project is to perform electron microscopic studies on the structure of CFTR, as a framework for understanding conformational changes and molecular interactions that are relevant to the etiology of CF. Dr. Madden's collaborator, Dr. Gene Scarborough (University of North Carolina, Chapel Hill) has established a yeast expression system for the production of recombinant CFTR that appears biochemically suitable for electron microscopic (EM) investigation. Initially, Dr. Madden proposes EM analysis of single particles of CFTR in negative stain. Following particle identification, classification, and reconstruction, this should allow the elucidation of the oligomeric state of the channel, which is still unclear and which should have important consequences for the channel's functionality. In addition, single-particle analyses of complexes of CFTR with binding proteins, including CAL (in collaboration with Dr. Stanton) may also provide insight into the binding interactions that govern CFTR trafficking, which play an important role in CF etiology. Finally, studies will be initiated to conduct two-dimensional crystallization experiments, as a basis for high-resolution electron crystallographic analysis of the channel's structure. Once crystals are obtained, it should be possible to identify the position and orientation of secondary structure elements within the molecule, including its transmembrane domains, using either projection structures or reconstructions from tilt-series. Taken together, these structural studies of the CFTR should provide a framework for the interpretation and synthesis of functional and mutagenetic data, yielding novel insights into the mechanisms of channel function and dysfunction.