All ETDs from UAB

Advisory Committee Chair

Eric J Sorscher

Advisory Committee Members

Stephen Barnes

John Hartman

Jeong Hong

John Kappes

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Post-translational modifications (PTMs) are important contributors to protein bio-genesis and function. CFTR in particular is known to be regulated by multiple PTMs, in-cluding glycosylation, phosphorylation, and ubiquitination. The functional significance and specific CFTR residues responsible for the majority of PTMs within CFTR are not known. The goal of this dissertation was to identify and characterize novel PTMs that influence wild-type and F508del CFTR. The study employed a combination of tandem and multiple reaction ion monitoring mass spectrometry to identify covalent side chain attachments. Analysis revealed the specific locations of numerous phosphorylated resi-dues, several sites of methylation, and more than twenty-five positions of ubiquitination. Our studies also showed for the first time that CFTR undergoes S-palmitoylation on at least two residues, C524 and C1395. Metabolic labeling confirmed covalent at-tachment of palmitate to wild-type and F508del CFTR. Pharmacologic inhibition of pal-mitoylation with 2-bromopalmitate (2-BP) decreased steady state levels of mature and immature wild-type CFTR and abrogated maturation of band B to C. While treatment with 2-BP caused no obvious effect on F508del CFTR expression or activity at 37°C, low temperature correction of the mutant protein was markedly reduced, including repression of band C and diminished CFTR surface activity. Recombinant expression of specific protein acyl transferases (PATs) led to robust effects on CFTR expression and pal-mitoylation as judged by western blot and metabolic labeling. Co-immunoprecipitation identified direct binding between several PATs and wild-type or F508del CFTR. In par-ticular, the findings show that the PAT DHHC-7: 1) stabilizes both wild-type and F508del CFTR band B expression, 2) binds strongly and specifically to band B, and 3) directly increases palmitoylation of wild-type and F508del protein. Immunofluorescence studies demonstrated that DHHC-7 stabilizes wild-type CFTR in the Golgi. Surprisingly, treatment of F508del CFTR significantly increased core glycosylated CFTR and seques-tered the protein in a post-ER (Golgi) compartment. Results from this study indicate a diversity of novel CFTR PTMs, including their specific locations and functional significance. The findings also point to the importance of palmitoylation during CFTR trafficking and provide a means by which palmitoylation and its role during CFTR biogenesis can be better understood in the future.

Share

COinS