33, 34, 35 The findings that 3905insT mRNA was insensitive to NMD despite harboring a PTC, led us to the assumption that this mRNA would Lenalidomide IC50 cause the formation of a truncated protein. To assess the fate of this truncated protein in the cell, we performed immunocytochemical analysis in nasal epithelial cells collected from the patients. In contrast to wt and F508del/F508del cells, in which the CFTR protein could be detected at the apical membrane (Figure 3a), apical CFTR staining was significantly reduced in nasal epithelial cells derived from F508del/3905insT compound-heterozygous patients (Figure 3c; Table 1). These immunocytochemical results confirmed earlier findings showing that a reduced number of cells from F508del homozygous patients have apically localized CFTR (Figure 3b).

21, 36, 37 Recent studies indicated that the CFTR C-terminus is not required for the biosynthesis and plasma membrane targeting of CFTR, but indispensable for maintaining the stability of the protein.38 Benharouga et al.39 could also show that an ERAD-similar mechanism involving the proteasome-ubiquitin pathway may be responsible for the faster turnover and the short residence time at the apical membrane of truncated CFTR. This faster recycling rate of truncated proteins may provide an explanation for the reduced amount of 3905insT CFTR at the apical membrane. However, it has to be noted that the above-mentioned studies investigated protein truncations shorter than 98 amino acids. Owing to the expected large truncation of 216 residues in the 3905insT CFTR, we assume that it is very likely that degradation occurs through the conventional ERAD pathway.

Another hypothesis for the complete lack of CFTR at the apical membrane would be that there is an interaction between the F508del and the 3905insT protein somehow hindering each other to reach the plasma membrane. In conclusion, the reduced presence of CFTR protein at the apical membrane may provide a possible explanation for the more severe phenotype observed in 3905insT/F508del compound-heterozygous patients versus F508del homozygous patients, in which at least some CFTR can escape the ER, reach the apical membrane, and function as a chloride channel. However, further experiments need to be performed to see whether the truncated 3905insT protein is degraded before reaching the apical membrane, whether it is quickly recycled after reaching the membrane, or whether there is an interaction between the 3905insT and the F508del CFTR protein interfering with the transport to the apical membrane.

Acknowledgments We are indebted to the patients and clinicians involved for their cooperation and collaboration and thank Andr�� Eble for EBV transformation of the lymphocytes. This work was funded by grants from the Swiss National Foundation (3200-066767.01 to SG, 310000-112652 Brefeldin_A to SG).