All ETDs from UAB

Advisory Committee Chair

Eric J Sorscher

Advisory Committee Members

David M Bedwell

John L Hartman Iv

Jeong S Hong

David A Schneider

Document Type

Dissertation

Date of Award

2016

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Approximately 1,000 new cases of cystic fibrosis (CF) are diagnosed in the United States every year. The disease is caused by over 1,700 naturally occurring variants in the CF transmembrane conductance regulator (CFTR). Defects in the CFTR coding sequence contribute to variable disease severity in the patient population. The majority of individuals born with CF harbor at least one specific mutation in CFTR termed F508del. Clinical manifestations of CF include severe damage of multiple exocrine tissues, including the lungs, intestine, pancreas, reproductive and other organ systems. An overarching goal of our work is to identify cellular targets that contribute to this disease by correcting the basic genetic defect resulting not only from the most common mutation (F508del-CFTR), but other mutant alleles with similar characteristics. To this end, we describe a new approach (genome-wide yeast phenomics) utilized to identify modifiers that rescue CFTR processing abnormalities. High-throughput analysis revealed several primary targets for this purpose clustered in distinct structural regions of the ribosome, most notably ribosomal protein L12 (Rpl12). The objective of this work, therefore, was to investigate the seminal discovery of specific ribosomal protein modules as effectors of F508del-CFTR biogenesis. Findings outlined here demonstrate that siRNA-mediated knockdown of Rpl12 rescues F508del-CFTR functional expression at levels comparable to those achieved by the best available FDA-approved corrector molecule, Lumacaftor (VX-809). In addition, the use of Rpl12 suppression together with VX-809 improves F508del biogenesis to a degree predictive of clinical benefit in patients. We also elucidate the mechanism by which Rpl12 influences mutant CFTR maturation. Ribosome profiling studies reveal Rpl12 suppression modestly attenuates 60S subunit assembly and translation efficiency, while significantly reducing the rate of elongation. This critical relationship between protein folding and translational velocity is an emerging and topical area with ramifications ranging from understanding protein maturation to disease mechanism and intervention. We have established translation control as a novel and critical checkpoint during CFTR biogenesis, suggesting modulation of this pathway as a novel approach for treatment of CF patients with the most common form of the disease.

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