All ETDs from UAB

Advisory Committee Chair

Zsuzsanna Bebok

Advisory Committee Members

James F Collawn

Joseph L Messina

Eric J Sorscher

Elizabeth S Sztul

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Synonymous codon changes (SCCs) do not alter the amino acid sequence of proteins, but may significantly change the phenotype. The frequency of synonymous codons is species- and gene-specific, described as codon usage bias (CUB). Over the past 50 years, research has shown that SCCs affect protein biogenesis, development of human disorders and the function of recombinant proteins developed for therapeutic applications. However, the molecular mechanisms by which SCCs alter the function of gene products are not completely understood. In Chapter 2 we summarize present knowledge in this field. The most common cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene resulting in the loss of phenylalanine at the position 508 (∆F508) and an ATC→ATT SCC for isoleucine at the position 507 (I507). We have previously demonstrated that the I507-ATC→ATT SCC contributes to the severity of the ∆F508 CFTR mutation by changing the mRNA secondary structure and results in altered translation dynamics, decreased protein stability and impaired channel gating. I hypothesized that the conformational differences between I507 variants will alter responses to ∆F508 CFTR correctors. I chose VX-809 and C4 based on mechanistic classifications indicating that they target structurally and spatially different ∆F508 folding defects. In Chapter 3, I show that C4 predominantly targets the I507-ATT ∆F508 CFTR and eliminates the posttranslational folding differences between the variants. VX-809 is necessary to improve maturation efficiency and cell surface expression of both variants. The I507-ATC ∆F508 CFTR demonstrated reduced proteasome-specific lysine-48-linked (K-48) polyubiquitination. The corrector combination reduces the level of I507-ATT ∆F508 CFTR K-48-linked polyubiquitination to I507-ATC control levels. Whole-cell patch clamp recordings indicate that corrector combination treatment reduced but did not abolish the functional differences between the variants observed following the low temperature rescue. Our results provide additional evidence for ∆F508 CFTR conformation-specific effects of C4 and VX-809, and emphasize the contribution of the I507-ATC→ATT SCC to the severity of the ∆F508 mutation. In general, we provide experimental evidence that silent codon changes may alter the structure and function of gene products.

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