Proteasomes are multisubunit
protease complexes responsible for degrading most intracellular
proteins. In addition to removing damaged
proteins, they regulate many important cellular processes through the controlled degradation of
transcription factors, cell cycle regulators, and
enzymes. Eukaryotic proteasomes have three catalytic subunits, β1, β2, and β5, that each has different substrate specificities. Additionally, although we know that diverse cell types express
proteasome variants with distinct activity and specificity profiles, the functions of these different pools of proteasomes are not fully understood. Covalent inhibitors of the
protease activity of the
proteasome have been developed as drugs for
hematological malignancies and are currently under investigation for other diseases. Therefore, there is a need for tools that allow direct monitoring of
proteasome activity in live cells and tissues. Activity-based probes have proven valuable for biochemical and cell biological studies of the role of individual
proteasome subunits, and for evaluating the efficacy and selectivity of
proteasome inhibitors. These probes react covalently with the
protease active sites, and contain a reporter tag to identify the probe-labeled
proteasome subunits. This review will describe the development of broad-spectrum and subunit-specific
proteasome activity-based probes, and discuss how these probes have contributed to our understanding of
proteasome biology, and to the development of
proteasome inhibitors.