Activity-based imaging probes of the proteasome

Abstract

Over the years, the proteasome has been extensively investigated due to its crucial roles in many important signaling pathways and its implications in diseases. Two proteasome inhibitors--bortezomib and carfilzomib--have received FDA approval for the treatment of multiple myeloma, thereby validating the proteasome as a chemotherapeutic target. As a result, further research efforts have been focused on dissecting the complex biology of the proteasome to gain the insight required for developing next-generation proteasome inhibitors. It is clear that chemical probes have made significant contributions to these efforts, mostly by functioning as inhibitors that selectively block the catalytic activity of proteasomes. Analogues of these inhibitors are now providing additional tools for visualization of catalytically active proteasome subunits, several of which allow real-time monitoring of proteasome activity in living cells as well as in in vivo settings. These imaging probes will provide powerful tools for assessing the efficacy of proteasome inhibitors in clinical settings. In this review, we will focus on the recent efforts towards developing imaging probes of proteasomes, including the latest developments in immunoproteasome-selective imaging probes.

Figure 1

Representation of the 20S proteasome complex, which is comprised of two outer α-rings and two inner β-rings, and an aerial view of β-rings containing either constitutive proteasome or immunoproteasome catalytic subunits. Aberrant regulation of the immunoproteasome is implicated in neurodegenerative diseases, autoimmune diseases, and cancers.

Figure 2

Structural representation of an activity-based probe (ABP), which is comprised of a targeting sequence that directs the probe to the targeted enzyme, a reactive chemical warhead which binds irreversibly to the enzyme’s catalytic residues, and a tag for identification or visualization of probe-bound enzymes. Biotin-tagged ABPs can be used for affinity purification and mass spectrometry-based identification of probe targets, as well as for visualization of these targets by streptavidin affinity blotting. Radiolabeled and fluorescent ABPs can be used to visualize target proteins by in-gel fluorescence. Additionally, cell-permeable fluorescent ABPs can be used to monitor enzyme activity in living cells or tissue samples via fluorescence microscopy.

Figure 3

Development of broad-spectrum proteasome ABPs based on the extended peptide vinyl sulfone inhibitor AdaAhx₃L₃VS.

Figure 4

Development of fluorescent epoxomicin and syringolin-A analogues.

Figure 5

A) Schematic representation of a two-step labeling approach. Living cells are first treated with an ABP containing a ligation handle for subsequent post-lysis labeling of probe-bound proteins by a bioorthogonal ligation reagent containing a reporter tag. B) An example of a proteasome-targeting two-step ABP [38, 61].

Figure 6

Subunit-selective ABPs.

Figure 7

Immunoproteasome-selective ABPs.