Abstract
DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) degradation involves ubiquitin modification and efficient proteasomal targeting of the nascent misfolded protein. We show that a deubiquitinating enzyme, ubiquitin C-terminal hydrolase-L1 (UCH-L1), is highly expressed in cystic fibrosis (CF) airway epithelial cells in vitro and in vivo. We hypothesized that the elevation in UCH-L1 in CF cells represents a cellular adaptation to counterbalance excessive proteasomal degradation. The bronchial epithelial cell lines IB3-1 (CF, high UCH-L1 expression) and S9 (non-CF, low UCH-L1 expression) were transiently transfected with wild type (WT) or DeltaF508 CFTR, WT UCH-L1 or small interfering RNA-UCH-L1, and a variety of ubiquitin mutants. We observed a positive correlation between UCH-L1 expression and steady state levels of WT- or DeltaF508-CFTR, and this stabilizing effect was confined to the early stages of CFTR synthesis. Immunolocalization of UCH-L1 by confocal microscopy revealed a partial co-localization with a ribosomal subunit and the endoplasmic reticulum. The UCH-L1-associated increase in CFTR levels was correlated with an increase in ubiquitinated CFTR (CFTR-Ub). Co-transfection with mutant ubiquitins and treatment with proteasome inhibitors suggested that UCH-L1 was reducing the proteasomal targeting of CFTR during synthesis by shortening conjugated polyubiquitin chains. Although not sufficient by itself to rescue mutant CFTR therapeutically, the elevation of UCH-L1 and its effect on CFTR processing provides insight into its potential roles in CF and other diseases.
Highlights
Thelial chloride channel that is required to optimally hydrate the apical surface of mammalian secretory tissues
CFTR is a 12-pass transmembrane protein that must undergo a complex sequence of folding events, and many cystic fibrosis (CF) disease-causing mutations alter the ability of the CFTR protein to assemble properly and/or prevent its maturation to the cell surface [1, 2]. ⌬F508, the most common CF mutation, results in a misfolded protein with increased affinity for components of the endoplasmic reticulum-associated degradation (ERAD) machinery, and most (ϳ99%) of this newly synthesized protein is degraded by the proteasome
ubiquitin C-terminal hydrolase-L1 (UCH-L1) Expression Is Elevated in CF Cells in Vitro and in Vivo—Proteomic analysis identified UCH-L1 protein expression as elevated in the CF IB3-1 cell line as compared with the wild type (WT) CFTR-corrected S9 [20]
Summary
Thelial chloride channel that is required to optimally hydrate the apical surface of mammalian secretory tissues. Rate of Post-translational Degradation—We hypothesized that the CFTR stabilization observed following UCH-L1 expression was due to changes in its protein processing and degradation. CFTR (band B) at time 0 immediately after the pulse in cells co-transfected with WT UCH-L1 as compared with pcDNA3.1 control vector (Fig. 3, A and B).
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