The role of cytokeratin 8 in alpha-1-antitrypsin secretion and/or degradation - 04/04/15

Alpha-1-antitrypsin (A1AT) is an inhibitor of proteases of inflammatory cells that protects the lungs from degradation. The Z mutation (E342K) leads to A1AT misfolding and degradation by the proteasome or to polymerization and autophagy. Both processes lead to reduced secretion of misfolded, however functionally active A1AT, and cause ultimately lung and liver diseases. We previously reported that, in cystic fibrosis, cytokeratin 8 (K8) affects the trafficking of misfolded F508del-CFTR to the plasma membrane. A molecule 407882 disrupts the interaction with K8 and corrects CFTR processing defect. We hypothesized that K8 may interact with other abnormally folded proteins, such as Z-A1AT, preventing their correct trafficking and secretion.

The study was conducted using HeLa and CFBE (cystic fibrosis bronchial epithelial) cell lines, transiently transfected with cDNAs of A1ATs (WT or Z). The potential interaction between K8 and A1AT was verified by co-immunoprecipitation (CoIP), and proximity ligation assay (PLA). Secretion of A1AT under different experimental conditions was tested by WB of secreted proteins. The effects of concentration of K8 were investigated after silencing of K8 expression obtained by transduction of HeLa cells with shRNA. The effect of CFTR corrector 407882 on secretion of A1AT was tested at concentration range 1–50μM.

WT- and Z-A1AT were expressed in cells 48h after transfection. In both cell lines secretion of Z-A1AT was lower than of WT-CFTR. WT and Z mutant variants of A1AT co-immunoprecipitated with K8 from both HeLa and CFBE cell extracts. PLA experiments showed that Z mutant localizes in a close proximity to K8 filaments (<40nm). More “interactions” were observed for Z-A1AT than for WT-A1AT. Reduction of K8 expression in HeLa cells by shRNA increased the secretion of A1AT variants, in particular of Z-A1AT. Treatment of cells with 407882 compound for 24h corrects the secretion of Z-A1AT from HeLa and CFBE cells up to 55% of WT-A1AT secretion level.

The results showed that K8 forms complexes with WT-A1AT and Z-A1AT. Disruption of these complexes either by diminishing intracellular K8 concentration or by treatment with 407882 improves secretion of A1AT, particularly of Z-A1AT. In conclusion, K8 is an important regulator of A1AT maturation and secretion, the K8-A1AT complex represents a target for pharmacotherapy of A1AT deficiency.