Protein Acetylation and Regulation of CFTR Expression

Cystic Fibrosis (CF) is an autosomal recessive disease caused by a loss of function of a chloride channel encoded by the cystic fibrosis conductance regulator (CFTR) gene. There are several classes of CF-disease causing mutants, the most common lead to protein misfolding, mistrafficking, and retention in the ER before degradation by ERAD. Inhibition of the proteasome does not improve trafficking of the most common misfolded mutant, deltaF508. Several mild mistrafficking mutations were examined under similar conditions to determine if the results with deltaF508 were general or specific. Despite some degree of trafficking, prevention of degradation does not further improve trafficking but instead increases the amount of misfolded protein. Therefore, the decision for retention in the ER is kinetically isolated from proteasome activity. Cotranslational in vitro experiments were implemented to explore the possibility of unidentified proteins interacting with the CFTR nascent chain. Two proteins identified in close proximity to a severe mistrafficking mutant, G85E, were N-alpha-acetyltransferases 16 and 15, both components of the complex NatA. However, the NatB complex, not NatA, is predicted to acetylate the N-terminus of CFTR. Nat complexes can affect the stability of their substrates either through modulating degradation or stability. Knockdown experiments ruled out involvement of NatE, NAA50, as well as one of the auxiliary subunits of NatA, NAA16, in the involvement of CFTR regulation. By contrast, knockdown of both NatA and NatB components increase steady state levels of CFTR. These increases were not due to any changes in degradation rates of the protein or the direct acetylation of CFTR's N-terminus as a Q2P mutation that prevents N-terminal acetylation of all known substrates was without effect. Instead, NatA regulates the translation rate of G85E CFTR through alterations in the mRNA levels. NatB regulates CFTR through a posttranslational maturation step independent of its fitness for proteasomal degradation. Thus, acetylation plays a role in the regulation of CFTR expression through two separate mechanisms, neither involving the direct N-terminal acetylation or degradation of G85E CFTR. Inhibition of either proteasomal or acetylation activity does not restore trafficking of CFTR, but instead increases the misfolded, ER-retained form of the protein.