Genetic Inhibition Of The Ubiquitin Ligase Rnf5 Attenuates Phenotypes Associated To F508del Cystic Fibrosis Mutation

Abstract

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins.

Thymosin α-1 does not correct F508del-CFTR in cystic fibrosis airway epithelia

“…Western blot performed on the biotinylated fraction with the CFFT 596 antibody revealed that, in cells expressing WT CFTR, the mature form was prevalent, while cells expressing F508del-CFTR expressed minor levels of immature CFTR on their surface ( Figure 5D). This is not surprising, as it has been demonstrated that immature CFTR can traffic to the plasma membrane through the unconventional secretion route (39,49). Importantly, the levels of mature CFTR available for biotinylation were markedly increased following treatment with VX-809 in F508del-CFTR-expressing cells ( Figure 5D).…”

“…We performed functional analysis of Tα-1 activity as F508del-CFTR corrector by means of the microfluorimetric assay based on the HS-YFP that has been extensively used to investigate various rescue maneuvers (36)(37)(38)(39)(40) and by means of whole-cell patch-clamp analyses. In parallel, we also performed immunolocalization experiments and biochemical analyses of the electrophoretic mobility of the different CFTR forms expressed by the cells under resting conditions or following treatment with Tα-1.…”

“…Cell surface biotinylation assay. Parental CFBE41o-cells, CFBE41o-cells expressing WT CFTR, or CFBE41o-cells expressing F508del CFTR were treated as previously described (39). Briefly, cells were seeded on 100-mm dishes.…”

“…F508del-CFTR CFBE41o-cells were subsequently engineered to stably express the halide-sensitive yellow fluorescent protein (HS-YFP). This cell line has been extensively used by our group to identify and characterize various CFTR modulators, including proteostasis regulators (36)(37)(38)(39)(40). CFBE41o-cells, coexpressing F508del-CFTR and HS-YFP, plated on 96-well plates, were treated for 24 hours with vehicle alone (DMSO), Tα-1 (25, 50, 100, or 200 ng/ml, all containing the same amount of DMSO), scrambled peptide (100 ng/ml, also containing DMSO), or cysteamine (250 μM).…”

In-depth genetic analysis of 6p21.3 reveals insights into associations between HLA types and complex traits and disease

“…However, the other five eGenes, including HLA-DRA (the most strongly associated), APOM, DXO, RNF5 and TAP1 were novel, i.e., had not been identified in GWAS. Interestingly, the association of RNF5 was consistent with its function as a regulator of the CFTR protein and suggested role in cystic fibrosis (Sondo et al, 2018;Tomati et al, 2015). We next identified four novel eGenes (EHMT2, GPANK1, HLA-C and RNF5) associated with weight phenotypes.…”