Calpain digestion and HSP90-based chaperone protection modulate the level of plasma membrane F508del-CFTR.
We are here showing that peripheral mononuclear blood cells (PBMC) from cystic fibrosis (CF) patients contain almost undetectable amounts of mature 170 kDa CF-transmembrane conductance regulator (CFTR) and a highly represented 100 kDa form. This CFTR protein, resembling the form produced by calpain digestion and present, although in lower amounts, also in normal PBMC, is localized in cytoplasmic internal vesicles. These observations are thus revealing that the calpain-mediated proteolysis is largely increased in cells from CF patients. To characterize the process leading to the accumulation of such split CFTR, FRT cells expressing the F508del-CFTR mutated channel protein and human leukaemic T cell line (JA3), expressing wild type CFTR were used. In in vitro experiments, the sensitivity of the mutated channel to the protease is identical to that of the wild type, whereas in Ca(2+)-loaded cells F508del-CFTR is more susceptible to digestion. Inhibition of intracellular calpain activity prevents CFTR degradation and leads to a 10-fold increase in the level of F508del-CFTR at the plasma membrane, further indicating the involvement of calpain activity in the maintenance of very low levels of mature channel form. The higher sensitivity to calpain of the mutated 170 kDa CFTR results from a reduced affinity for HSP90 causing a lower degree of protection from calpain digestion. The recovery of HSP90 binding capacity in F508del-CFTR, following digestion, explains the large accumulation of the 100 kDa CFTR form in circulating PBMC from CF patients.
Department of Experimental Medicine, University of Genoa, Genoa, Italy.
Monica
M
Averna
Roberto
R
Stifanese
Raffaella
R
Grosso
Marco
M
Pedrazzi
Roberta
R
De Tullio
Franca
F
Salamino
Bianca
B
Sparatore
Sandro
S
Pontremoli
Edon
E
Melloni
D000818:Animals; D002154:Calpain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D018832:Molecular Chaperones / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D021381:Protein Transport; D051381:Rats; D011916:Rats, Inbred F344; D017384:Sequence Deletion
C505032:CFTR protein, human; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D002154:Calpain