Oladapo O. Yeku

1st Year Graduate Student

Department: Pharmacological Sciences

Graduate Program: Molecular & Cellular Pharmacology

Advisor: Michael Frohman

Abstract:

Title:  Targeted Protein Degradation in Mammalian Cells

Oladapo Yeku, Guangwei Du and Michael Frohman, Pharmacological Sciences, SBU

Loss of function analysis at the DNA (gene knockout) and RNA (RNA interference) level has been successfully used to analyze gene function in plant and animal cells. Here we describe an attempt to establish a technique to silence expression at the protein level in mammalian cells, based on a recent demonstration that controlled redirection of target proteins to the proteasome is sufficient for their degradation in yeast. A protein subunit known as FRB was fused to a target protein (Green fluorescent protein). The complementary subunit FKBP was fused to a subunit of the proteasome complex. A Rapamycin analogue was then added to trigger dimerization of the FRB and FKBP subunits, thus promoting translocation of the FRB-tagged GFP to the proteasome. In theory, close proximity of the GFP to the proteasome complex should result in its degradation. Loss of GFP was assessed quantitatively over various time periods using western blotting and immunofluorescence. This method, if successful, could provide a powerful tool for the study of gene and protein function.