Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect

Abstract

The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.

RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation

“…9B and C). As RPLP1/2 have been implicated in translation elongation (29,43), these data raise the possibility that ribosomal elongation through structural protein coding sequences may be impaired in cells with reduced RPLP1/2 levels, with the strongest effects on accumulation of full-length E protein.…”

Correctors and Potentiators Rescue Function of the Truncated W1282X-Cystic Fibrosis Transmembrane Regulator (CFTR) Translation Product

“…CFTR activity was measured by I sc for computation of relative channel activity. A similar approach was used for CFBE cells but with Amplex-Red (Molecular Probes, Waltham, MA) HRP substrate to measure CFTR cell-surface density (50).…”

“…To monitor N-linked glycans as an indicator of CFTR biosynthetic processing, high molecular weight mannose N-glycans or all N-glycans were cleaved by incubating cell lysates with 10 g/ml endo H or 40 g/ml PNGase F (New England Biolabs, Ipswich, MA), respectively, for 2 h at 35°C, followed by immunoblotting (49). CFTR 1281 maturation efficiency in CFBE cells was measured by metabolic pulse-chase as described (50). Briefly, CFBE cells expressing CFTR 1281 were pulse-labeled with 0.2 mCi/ml [ 35 S]methionine and [ 35 S]cysteine (EasyTag Express Protein Labeling Mix, PerkinElmer Life Sciences) in cysteine-and methionine-free medium for 1 h at 37°C and then chased for 2 h at 37°C in normal medium.…”

Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens

“…In tumor CR cell cultures, phenotypic and genotypic features of the primary tumor are maintained 7 and the technique has recently been used to identify an appropriate therapy for respiratory papillomatosis 14 . Therefore, it is clear that potential applications of the CR method are far-reaching, and, as described by independent laboratories, it can be adapted for live biobanking 7 and basic research 15–21 , as well as for diagnostic 22,23 , therapeutic 21,24,25 and regenerative medicine 21,26,27 …”