Synthesis and Application of a Caged Bioluminescent Probe for the Immunoproteasome.

Monitoring the catalytic activity of the proteasome and its various isoforms has become increasingly important with the continued development of core particle inhibitors and targeted protein degraders as potential therapies for diseases with high protein accumulation. The immunoproteasome (iCP) is expressed in a variety of diseases due to inflammatory signals, such as interferon-gamma, that alert the cell to begin generating iCP preferentially over the standard proteasome. There is a need to understand iCP activity and expression both in cells and in vivo because it is becoming a widely targeted isoform in a variety of diseases. Activity-based probes for the iCP have been developed, but their application has been limited due to their difficult synthesis and choice of fluorescent reporter. There has yet to be a selective iCP probe developed that incorporates a luminescent reporter that could be applied to a variety of different applications. The protocols presented here describe the synthesis of a cleavable activity-based bioluminescent probe that is selective for the iCP, and the application of the synthesized probe in immunoproteasome activity assays using a luminescent plate reader. Having this bioluminescent reporter, a better understanding of how the iCP is implicated in disease progression, as well as identification of small molecule interactors, can be achieved. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of a bioluminescent immunoproteasome probe Basic Protocol 2: Expression of the immunoproteasome in cells Basic Protocol 3: Immunoproteasome probe application in live cells using a luminescent plate reader.