Biology

Prof. Ghanshyam Heda
(Professor of Medicine, The Univ Tenn Health Sci Ctr, Memphis) Dept of Sciences & Mathematics Mississippi University for Women Columbus, MS 39701, U.S.A.

Abstract:

CFTR (Cystic Fibrosis Transmembrane-conductance Regulator) function as a chloride ion channel on epithelial cells of many organs.  Mutations impairthe function of CFTR, and are responsible for causing the pulmonary disease cystic fibrosis.  Our laboratory has shown that the plasma membrane half-life of the most common of CFTR mutations (DF508) is much shorter (~4 h) than that of wild-type CFTR (>48 h). [Heda et al, 2001].  We hypothesize that this reduced DF508-CFTR half-life may be due to the distinct role of proteasomes, lysosomes and/or CFTR-protein(s) interactions. In this study we present the effects of inhibitors of proteasomes and lysosomes on stability of plasma membrane DF5080-CFTR. Epithelial cell lines from human lung (CFBE) stably transfected with DF508 or wild type CFTR were pre-treated with 5 mM sodium butyrate at 27^oC for about 60 hrs to up-regulate the plasma membrane CFTR expression. Cells were then “chased” at 37^oC in the presence of protein synthesis inhibitor (cycloheximide) and/or inhibitors of proteasomes (MG132, lactacystin, ALLN, leupeptin), or lysosomal enzymes (E64, EST, chloroquine).  Cell lysates were prepared and immunoblotted with anti-CFTR antibody.  CFTR-specific signal was detected by chemiluminescence using c300 image analyzer (Azure Biosystems).  All inhibitors except for chloroquine partially rescued the degradation of plasma membrane DF508-CFTR in CFBE cell line.  There are little or no synergistic effects were observed when these inhibitors were used in combination. These data suggest that CFTR degradation is partially controlled by proteasomes and lysosomal enzymes.  Additionally we have developed methods to immunoisolate CFTR expressing endocytic vesicles for CFTR-protein interaction studies.