The proteasome, a large multi-catalytic complex of ~2.5 MDa, serves as the major protein degradation pathway in eukaryotic cells. The proteasome can exist in various isoforms depending on its environment. Under conditions of inflammation, such as exposure to interferon-gamma (IFN-γ) or tumor necrosis factor alpha (TNF-α), newly synthesized immunoproteasome (iCP), with distinct subunits from the standard proteasome (sCP), is expressed. The newly synthesized iCP can generate peptide products that differ from the sCP that are better at antigen presentation when incorporated into the MHC-1 complex. Disfunction of the iCP can lead to diseases such as autoimmune disorders, inflammation, and cancer. Current iCP probes allow for monitoring in cells and with purified protein, however, are limited in their ability for animal or tissue monitoring. D-Luciferin probes have been made to overcome the external excitation needed to emit signal traditional fluorophore probes require, yet no iCP specific luciferin probe has been made. We took knowledge from our iCP specific cellular probe, that has an amino acid sequence specific for the iCP appended to a fluorophore. Upon cleavage of the amide bond between the peptide and fluorophore, signal can be monitored as the fluorophore is uncaged and can emit light. Similarly, we append the iCP selective peptide sequence to D-luciferin that upon cleavage can interact with firefly luciferase within animal or cell models. This will allow us, for the first time, to monitor the activity of the iCP in vivo, and can correlate its activity to other immune system activities in cancers.

Synthesis and characterization of a selective immunoproteasome-luciferin probe for <i>in vivo</i> monitoring

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Synthesis and characterization of a selective immunoproteasome-luciferin probe for in vivo monitoring