The proteasome is an essential protein complex that, when dysregulated, can result in various diseases in eukaryotic cells. As such, understanding the enzymatic activity of the proteasome and what can alter it is crucial to elucidating its roles in these diseases. This can be done effectively by using activity-based fluorescent substrate probes, of which there are many commercially available that target the individual protease-like subunits in the 20S CP of the proteasome. Unfortunately, these probes have not displayed appropriate characteristics for their use in live cell-based assays. In the work presented here, we have developed a set of probes which have shown improved fluorescence properties and selectivity towards the proteasome compared to other cellular proteases. By including unnatural amino acids, we have found probes which can be utilized in various applications, including monitoring the effects of small molecule stimulators of the proteasome in live cells and comparing the relative proteasome activity across different cancer cell types. In future studies, we expect the fluorescent probes presented here will serve as tools to support the discovery and characterization of small molecule modulators of proteasome activity.

A series of oleic acid amide derivatives were synthesized based on our previous and continuing endeavors towards stimulation of the 20S core particle of the proteasome (20S CP) with the...

Regulating protein production and degradation is critical to maintaining cellular homeostasis. The proteasome is a key player in keeping proteins at the proper levels. However, proteasome activity can be altered in certain disease states, such as blood cancers and neurodegenerative diseases. Cancers often exhibit enhanced proteasomal activity, as protein synthesis is increased in these cells compared with normal cells. Conversely, neurodegenerative diseases are characterized by protein accumulation, leading to reduced proteasome activity. As a result, the proteasome has emerged as a target for therapeutic intervention. The potential of the proteasome as a therapeutic target has come from studies involving chemical stimulators and inhibitors, and the development of a suite of assays and probes that can be used to monitor proteasome activity with purified enzyme and in live cells.

The proteasome is a multisubunit protein complex responsible for the degradation of proteins, making it essential in myriad cellular processes. Several reversible and irreversible peptide substrates inspired by known proteasome inhibitors have been developed to visualize it and monitor its activity; however, they have limited commercial availability or possess fluorophores that overlap with other known chemical probes, limiting their simultaneous use. The protocols presented here describe the synthesis of a clickable epoxomicin-based probe followed by the copper-catalyzed installment of an azide-containing fluorophore, and the application of the synthesized peptide in proteasome activity assays by SDS-PAGE and flow cytometry.

Degradation of proteins by the proteasome is an essential cellular process and one that many wish to study in a variety of disease types. There are commercially available probes that can monitor proteasome activity in cells, but they typically contain common fluorophores that limit their simultaneous use with other activity-based probes. In order to exchange the fluorophore or incorporate an enrichment tag, the proteasome probe likely has to be synthesized which can be cumbersome. Here, we describe a simple synthetic procedure that only requires one purification step to generate epoxomicin, a selective proteasome inhibitor, with a terminal alkyne. Through a copper-catalyzed cycloaddition, any moiety containing an azide can be incorporated into the probe. Many fluorophores are commercially available that contain an azide that can be "clicked", allowing this proteasome activity probe to be included into already established assays to monitor both proteasome activity and other cellular activities of interest.