A. Hasnain (Presenting)(1), N. Arous (1) , P. Pagesy(1) , J. Clain (2) , M. Goossens , P. Fanen (1)

(1) INSERM U.468, Creteil, France, (2) Faculte de Pharmacie, Paris 5, France

Cystic fibrosis (CF) is a channelopathy, which is characterised in more than 90% of patients by an absence or a dramatic decrease of CFTR protein at the apical membrane of epithelia. The third cytoplasmic loop (CL3) is a large loop in which many disease-causing mutations affect CFTR processing of the whole protein, highlighting the importance of its correct secondary structure.

We used the 53 residues of CL3 as a bait to screen the pre-transformed Human Testis MATCHMAKER cDNA library (Clontech) by the yeast two-hybrid system. This screening allowed us to identify 25 independent clones, out of which 15 were cytoplasmic proteins, 5 are unknown, 5 were irrelevant (i.e. nuclear). The positive interactions were later tested with the class II mutants S945L, H949Y, D979A. We are currently analysing two proteins from the proteasome subunits by expressing them in HeLa cells. We cloned the full-length cDNAs in pcDNA3.1/V5-His-TOPO vector (Invitrogen) and we analyzed:
(i) protein interaction by co-immunoprecipitation;
(ii) pulse-chase experiments and
(iii) immunocytochemistry using anti-V5 antibody and anti-CFTR antibody.

Analysis of other cytoplasmic proteins is currently under process. This identification of unknown interacting proteins should allow us to decipher the molecular mechanisms of CFTR processing and define new therapeutic targets to direct the CFTR mutant proteins to the plasma membrane.

This work was supported by grants from INSERM and the French association, Vaincre la Mucoviscidose.