The human DnaJ homologue (Hdj)-1/heat-shock protein (Hsp) 40 co-chaperone is required for the in vivo stabilization of the cystic fibrosis transmembrane conductance regulator by Hsp70.
The CFTR (cystic fibrosis transmembrane conductance regulator) gene, defective in cystic fibrosis, codes for a polytopic apical membrane protein functioning as a chloride channel. Wild-type (wt) CFTR matures inefficiently and CFTR with a deletion of Phe-508 (F508del), the most frequent mutation, is substantially retained as a core-glycosylated intermediate in the endoplasmic reticulum (ER), probably due to misfolding that is recognized by the cellular quality control machinery involving molecular chaperones. Here, we overexpressed the heat-shock protein (Hsp) 70 chaperone in vivo and observed no changes in degradation rate of the core-glycosylated form, nor in the efficiency of its conversion into the fully glycosylated form, for either wt- or F508del-CFTR, contrary to previous in vitro studies on the affect of heat-shock cognate (Hsc) 70 on part of the first nucleotide-binding domain of CFTR. Co-transfection of Hsp70 with its co-chaperone human DnaJ homologue (Hdj)-1/Hsp40, however, stabilizes the immature form of wt-CFTR, but not of F508del-CFTR, suggesting that these chaperones act on a wt-specific conformation. As the efficiency of conversion into the fully glycosylated form is not increased under Hsp70/Hdj-1 overexpression, the lack of these two chaperones does not seem to be critical for CFTR maturation and ER retention. The effects of 4-phenylbutyrate and deoxyspergualin, described previously to interfere with Hsp70 binding, were also tested upon CFTR degradation and processing. The sole effect observed was destabilization of F508del-CFTR.
Farinha
Carlos M
CM
Centro de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisboa, Portugal.
Nogueira
Paulo
P
Mendes
Filipa
F
Penque
Deborah
D
Amaral
Margarida D
MD
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003720:Densitometry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017510:Protein Folding; D013997:Time Factors; D014162:Transfection
C505032:CFTR protein, human; D018076:DNA, Complementary; C494647:DNAJB1 protein, human; D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D019005:Cystic Fibrosis Transmembrane Conductance Regulator