RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR*

Abstract

Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the Cystic fibrosis transmembrane conductance regulator (CFTR).⌬F508-CFTR, the most common disease-causing CF mutant, exhibits folding and trafficking defects and is retained in the endoplasmic reticulum, where it is targeted for proteasomal degradation. To identify signaling pathways involved in ⌬F508-CFTR rescue, we screened a library of endoribonuclease-prepared short interfering RNAs (esiRNAs) that target ϳ750 different kinases and associated signaling proteins. We identified 20 novel suppressors of ⌬F508-CFTR maturation, including the FGFR1. These were subsequently validated by measuring channel activity by the YFP halide-sensitive assay following shRNA-mediated knockdown, immunoblotting for the mature (band C) ⌬F508-CFTR and measuring the amount of surface ⌬F508-CFTR by ELISA. The role of FGFR signaling on ⌬F508-CFTR trafficking was further elucidated by knocking down FGFRs and their downstream signaling proteins: Erk1/2, Akt, PLC␥-1, and FRS2. Interestingly, inhibition of FGFR1 with SU5402 administered to intestinal organoids (mini-guts) generated from the ileum of ⌬F508-CFTR homozygous mice resulted in a robust ⌬F508-CFTR rescue. Moreover, combination of SU5402 and VX-809 treatments in cells led to an additive enhancement of ⌬F508-CFTR rescue, suggesting these compounds operate by different mechanisms. Chaperone array analysis on human bronchial epithelial cells harvested from ⌬F508/ ⌬F508-CFTR transplant patients treated with SU5402 identified altered expression of several chaperones, an effect validated by their overexpression or knockdown experiments. We propose that FGFR signaling regulates specific chaperones that control ⌬F508-CFTR maturation, and suggest that FGFRs may serve as important targets for therapeutic intervention for the treatment of

Latonduine Analogs Restore F508del–Cystic Fibrosis Transmembrane Conductance Regulator Trafficking through the Modulation of Poly-ADP Ribose Polymerase 3 and Poly-ADP Ribose Polymerase 16 Activity

“…It is unclear at this stage why blocking this activity would increase F508del CFTR trafficking, but it may be that inhibiting the UPR prevents the increased expression of chaperones such as calnexin and calreticulin which help process the misfolded CFTR in the ER before transport for proteasomal degradation, thus allowing partially misfolded F508del-CFTR to escape the ER quality control and traffic to the plasma membrane. This work is in line with previous research that has shown that inhibition of IRE-1 expression by the use of RNA interference will suppress correction of F508del-CFTR (Trzci nska- Daneluti et al, 2015) and also that some level of IRE-1 expression in cells is necessary for normal protein metabolism (Safra et al, 2013).…”

Human pantothenate kinase 4 is a pseudo‐pantothenate kinase

“…47 Knockdown of PANK4 expression restores the plasma membrane localization of a mutant cystic fibrosis transmembrane conductance regulator. 48 Our work demonstrates that these functions are not due to the PANK activity. HsPANK4 also has very low phosphatase activity against 4 0 -phosphopantothenate and slightly higher activity toward damaged or oxidized forms of 4 0 -phosphopantetheine.…”

“…PANK4 was identified as a host gene required for influenza virus replication in multiple influenza‐genome screens, and the participation of PANK4 in flu proliferation was validated by siRNA‐mediated knockdown . Knockdown of PANK4 expression restores the plasma membrane localization of a mutant cystic fibrosis transmembrane conductance regulator . Our work demonstrates that these functions are not due to the PANK activity.…”

Chaperone-Independent Peripheral Quality Control of CFTR by RFFL E3 Ligase

“…In fact, RFFL knockout mice exhibit no abnormal phenotype (Ahmed et al, 2009) although RFFL is ubiquitously expressed (Coumailleau et al, 2004). Intriguingly, RFFL transcription is stimulated by RAF/MEK/ERK signaling pathway (Gan et al, 2013; Gan et al, 2012) that negatively regulates the ΔF508-CFTR functional expression by unknown mechanisms (Trzcinska-Daneluti et al, 2012; Trzcińska-Daneluti et al, 2015). Despite the fact that RAF/MEK/ERK signaling pathway could also modulate chaperone-mediated ubiquitination (Trzcińska-Daneluti et al, 2015), it may limit the ΔF508-CFTR functional expression by up-regulating RFFL activity.…”