Ghanshyam Das Heda, PhD

Effects of triazole compounds on CFTR protein expression

Cystic fibrosis (CF) is an autosomal recessive disease that affects the function of lungs, pancreas, liver and reproductive organs. CF is mostly common among Caucasians of European descent, and is caused by mutations in a plasma membrane protein Cystic Fibrosis Transmembrane-conductance Regulator (CFTR). CFTR at the plasma membrane functions as a chloride ion transporter. There are over 1,000 mutations that alter the function of CFTR, however, ∆F508 is the most common mutation affecting over 70% of CF cases. ∆F508 mutation misfold CFTR, arrest it in endoplasmic reticulum during its’ synthesis and prevents it from trafficking to the plasma membrane. Several studies including our own has shown that the biosynthetic arrest of ∆F508-CFTR can be partially reversed by physical and/or chemical means. These interventions can bypass the biosynthetic arrest of ∆F508-CFTR and drive functional CFTR chloride channels to the plasma membrane and correct the chloride ion channel transport defect in CF. PI has obtained several synthetic small organic molecules (triazole compounds) with his collaboration with scientists at the Indian Institute of Science Education Research (IISER), India. Three of these compounds (PJ-08, PJ-03, ABS-091) have increased ∆F508-CFTR protein expression. However, most of the newly synthesized protein didn’t undergo glycosylation (maturation). Protein maturation is important for it to traffic to the plasma membrane to correct the chloride ion channel defect in CF patients. We hypothesize that the proteasomal, lysosomal enzymes, and/or molecular signals regulating protein trafficking may prevent CFTR from undergoing post-translational maturation process. Human lung epithelial cell line (CFBE) expressing ∆F508-CFTR will be treated with various concentrations of triazole compounds (e.g., PJ-08, PJ-03, ABS-091) in the absence and presence of proteasomal (e.g., MG132) or lysosomal (e.g. E64) enzyme inhibitors. Levels of CFTR expression at the plasma membrane will be determined by cell-surface biotinylation followed by western blotting methods. Biochemical and cell biological techniques such as tissue culture, western blotting, protein assay, cell surface biotinylation and extraction methods of biotinylated proteins used in achieving the goals of this proposed project will be useful in the laboratory portion of subjects taught by PI, viz, “Protein Misfolding & Human Diseases” (BSB-461), “Independent Studies” (BSB-499), and “Honors College Independent Study” (HO-401, HO-402) courses. PI’s laboratory is also a useful resource for other faculty members and students within the department.