ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells

Abstract

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ⌬F508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ⌬F508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ⌬F508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (ϳ1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ⌬F508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o؊ WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ⌬F508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ⌬F508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ⌬F508-CFTR trafficking in CF epithelial cells.

4-Phenylbutyrate Stimulates Hsp70 Expression through the Elp2 Component of Elongator and STAT-3 in Cystic Fibrosis Epithelial Cells

“…We recently demonstrated that one 4PBA-regulated species identified in this screen, ERp29 (endoplasmic reticular protein of 29 kDa), is a novel chaperone of the endoplasmic reticulum that promotes the trafficking of wt and ⌬F508 CFTR, the first such luminal chaperone to have this role (10). We also, as described here, identified Elp2 as a potential 4PBA-stimulated regulator of Hsp70.…”

“…Differential Display RT-PCR Screen-Differential display RT-PCR was performed using the RNAimage™ kit (Genhunter) on total RNA isolated from IB3-1 cells treated with 1 mM 4PBA for 0, 4, 8, or 24 h as previously described in detail (10). Differentially expressed cDNAs were extracted, re-amplified by PCR, and cloned into pCRII (Invitrogen).…”

“…The most common mutation of CFTR, ⌬F508-CFTR (deletion of a phenylalanine at position 508), is a temperature-sensitive trafficking mutant (1). ⌬F508-CFTR is retained in the endoplasmic reticulum (ER), where it has increased associations with several cytosolic chaperones belonging to the Heat Shock Protein (Hsp) family (2)(3)(4)(5) and with the ER-resident chaperones calnexin (6 -9) and ERp29 (10). ⌬F508-CFTR is targeted for rapid intracellular degradation (11) at least in part by the ubiquitin proteasome system (12,13), and so mostly fails to reach its appropriate subcellular location at the apical membrane (14,15).…”

Protein Misfolding and Endoplasmic Reticulum Stress in Chronic Lung Disease

“…For instance, the chemical chaperone sodium 4-phenylbutyrate (4-PBA) improves traffi cking of both D F508-and wild-type CFTR in bronchial epithelial cells. 18 Furthermore, selective inhibition of ERAD through interference with valosin-containing protein (VCP), a component of the ER export and proteasomal degradation machinery, was shown to rescue D F508-CFTR from ERAD and increase its cell surface expression in human bronchial epithelial cells. 19 Further research is necessary to develop treatment strategies centered on resolving misfolding events and/or regulating the UPR to correct for CF lung disease.…”

Three-Dimensional Protein Fold Determination from Backbone Amide Pseudocontact Shifts Generated by Lanthanide Tags at Multiple Sites

“…ERp29 is a chaperone expressed in the endoplasmic reticulum, where it facilitates the processing and transport of proteins. Its chaperone activity underpins numerous biological functions, including roles in virus infections, cystic fibrosis, and tumor suppression and treatment (Rainey-Barger et al, 2009;Das et al, 2009;Farmaki et al, 2011;Suaud et al, 2011;Zhang and Richardson, 2011;Bambang et al, 2013;Wu et al, 2012). The 3D structure of the C-terminal domain of rat ERp29 was first determined by NMR spectroscopy using the conventional NOE approach.…”