Measurement of Proteasome Inhibition in Live Cells in Molecular Devices Microplate Fluorometers

Proteins inside eukaryotic cells exist in a dynamic state, in a highly-regulated balance between synthesis and degradation. Whereas protein synthesis is well-understood after decades of study, major advances in our knowledge of protein degradation have occurred only in the last two decades. As a result, the 2004 Nobel prize in chemistry was awarded to Aron Ciechanover, Avram Hershko and Irwin Rose for their discovery of ubiquitin-mediated proteolysis, an ATP-dependent process where unwanted proteins are multiply-tagged with ubiquitin (a 76-amino acid protein).1 The tagged proteins are then transported to the proteasome for degradation. The proteasome is a massive (2.5 MDa), barrel-shaped protein complex inside all eukaryotic cells (and some bacteria) that consists of a tunnel-like core with a cap at each end. (See Figure 1.) The caps (regulatory complexes) recognize and bind targeted proteins and inject them into the central core where the proteins are successively degraded into short peptides. This application note shows that inhibition of proteasome activity can easily be measured using Molecular Devices microplate fluorometers.