The most prevalent disease-associated mutation in cystic fibrosis (CF) is deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR), which results in misprocessing and reduced apical localization of the protein in respiratory, gastrointestinal, pancreatic and other epithelia Our preliminary evidence demonstrates that suppression of ribosomal protein L12 (Rpl12) corrects the folding defect and functional cell surface expression of F508del CFTR. A number of recent and high-profile publications indicate that the ribosome not only regulates peptide synthesis and primary amino acid structure, but also plays a crucial chaperone-like role during protein folding [14-18]. Therefore, it is important to understand the mechanism by which Rpl12 mediates interaction with nascent F508del polypeptide during co- translational folding and test the feasibility of Rpl12 as a novel therapeutic target for individuals living with CF. Specific Ai 1: Characterize the effects of Rpl12 knockdown on F508del CFTR processing. Following siRNA-mediated Rpl12 inhibition, we will measure steady state levels of F508del expression, maturation, and function. In addition, we will determine whether abrogation of other ribosomal proteins located on the 60S subunit P stalk (in close proximity to Rpl12) also mediate rescue of F508del processing.
Specific Aim 2 : Ascertain the mechanism by which Rpl12 suppression rescues F508del CFTR function. We will use leading-edge technology (polysome analyses, ribosome profiling, RNA-Seq) to quantitatively address ribosome assembly, translation efficiency, elongation, and foot printing in response to Rpl12 knockdown. These studies will identify specific sub-domains within CFTR that are altered by deletion of F508, and test Rpl12 suppression as a means to overcome defects of this nature.
Specific Aim 3 : Determine in vivo relevance by development of RPL12 knockout, conditional knockout, or haploinsufficient mice. RPL12 mouse models will be cross-bred to CFTRF508del mice to assay for improvements in CF phenotyptic manifestations within the gastrointestinal and respiratory tracts. This will include studies of CFTR expression (biochemical analyses), histopathology, immunohistochemistry, and function (short circuit current measurements), and in vivo bioelectric measurements from the upper airways. Successful completion of the proposed experiments will: (1) establish a novel, specific ribosomal protein as a fundamental contributor to F508del CFTR folding and protein conformation, (2) demonstrate the in vivo role of ribosomal quality control during co-translational protein folding in the context of an important human disorder, and (3) provide new evidence for ribosomal-chaperone function during protein biogenesis. The in vivo experiments in mice will also furnish an important foundation for future studies aimed at modulating ribosome kinetics as an experimental therapeutic strategy for patients with this disease.
The majority of individuals diagnosed with cystic fibrosis (CF) harbor a deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR), which causes protein misfolding and degradation resulting in severe multi-organ damage. Preliminary data outlined in this proposal demonstrates that suppression of ribosomal protein L12 (Rpl12) rescues F508del CFTR processing at comparable levels to those achieved by the best available small molecule F508del correctors. In this project, we will elucidate the mechanism by which Rpl12 inhibition rescues F508del CFTR maturation and function, and evaluate Rpl12 as a novel target in the CFTRF508del murine model, with relevance to therapeutic intervention in the disease.
| Oliver, Kathryn E; Han, Sangwoo T; Sorscher, Eric J et al. (2017) Transformative therapies for rare CFTR missense alleles. Curr Opin Pharmacol 34:76-82 |
| (2016) Poster Session Abstracts. Pediatr Pulmonol 51:S194-S485 |
| Veit, Guido; Oliver, Kathryn; Apaja, Pirjo M et al. (2016) Ribosomal Stalk Protein Silencing Partially Corrects the ?F508-CFTR Functional Expression Defect. PLoS Biol 14:e1002462 |
