Proteins destined for the plasma membrane or extracellular space are synthesized in the endoplasmic reticulum (ER). The ER quality control system inspects newly synthesized proteins and ensures that only correctly folded proteins as well as fully assembled complexes are allowed to leave the ER. ER proteins that do not fold correctly due to genetic mutations are targeted for degradation in the cytosol by the proteasome. The extraction of the polypeptide from the ER to the cytosol is referred to as dislocation. These proteins include soluble as well as membrane molecules. Diseases associated with the degradation of specific ER proteins range from neurological defects that cause Fabri disease and Tay-Sachs to lung diseases such as cystic fibrosis and emphysema. Pathogens also utilize the destruction of specific proteins to evade immune detection. The human cytomegalovirus (HCMV) encoded gene products US2 and US11 mediate the destruction of the immunologically essential membrane protein MHC class I heavy chain. These viral gene products target MHC class I for degradation in a similar manner as misfolded ER proteins. We will establish a bioluminescent assay in human cell lines that can be used in a high-throughput screen to identify cell permeable inhibitors of ER dislocation. If ER degradation substrates can elude the dislocation reaction, they may be re-directed to their proper compartment and possibly prevent the onset of the disease state. Due to the topologically diverse ER proteins besieged for dislocation/degradation, we will generate human cell lines that express either a soluble or membrane bioluminescent chimeric molecule that is targeted for dislocation/degradation. The soluble bioluminescent chimeric molecule will be comprised of either enhanced green fluorescent protein (EGFP) or luciferase fused to a mutant form of a1-antitrypsin. Chimeric EGFP/class heavy chain or luciferase/class I heavy chain in HCMV US11 cells will be utilized to represent the dislocation of membrane proteins. Inhibition of the dislocation reaction would bring about an accumulation of bioluminescent chimeric molecules and cause an augmentation in light energy. This increase in light energy will provide the readout for the high throughput screen. Chemical compounds identified to induce an amplification in energy will be considered for second phase analysis using biochemical assays to validate their ability to block dislocation. ? ?
