Proteasome Redistribution Assay

• Designed to assay compounds for their ability to modulate proteasomal activity
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized
  assay protocol
• Easy to use: Just plate cells, add compounds, and image

The ubiquitin/proteasome pathway is the main non-lysosomal route for intracellular protein degradation in eukaryotes and is implicated in the degradation of proteins that control vital processes such as cell cycle progression, signal transduction, differentiation, and apoptosis. Covalent attachment of multiple ubiquitin molecules to proteins targets them for proteolytic degradation by a complex cellular structure, the 26S proteasome. A cascade of reactions catalyzed by ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) is required to attach the ubiquitin moiety to a lysine residue of the protein targeted for degradation. Ubiquitination is a reversible process due to a number of de-ubiquitinating enzymes that mediate the disassembly of ubiquitin-protein conjugates. Proteasomal degradation, on the other hand, is irreversible and is therefore appropriate for unidirectional control of cellular pathways. The 26S proteasome is a multicatalytic multisubunit protease where the core proteasome (20S) consists of two heptameric outer α-rings and two inner β-rings responsible for the proteolytic activity of the proteasome. The 19S regulatory complex is stacked on the ends of the cylindrical core to form a 26S proteasome and is involved in an ATP-dependent recognition and unfolding of proteins targeted for degradation. Some deubiquitinating enzymes are associated with the 26S proteasome, making them key regulators of the proteasome-dependent protein degradation process.

Figure 1. Inhibition of proteasomal degradation of Ubi(G76V)-EGFP by MG-132. Cells were treated with 5 µM MG-132 for 4 hr (right panel) or vehicle control (left panel). The increase in fluorescence intensity is detected by the image analysis algorithm.

Figure 2. Concentration response curve in the Proteasome assay: MG-132 concentration response (n=12); the EC₅₀ is approximately 0.2 µM. Concentration response was measured in 9 point half log dilution series. Cells were treated with test compound for 4 hr. Cells were then fixed and increase in fluorescence intensity was measured using theCellomics ArrayScan V^TI Reader and the Redistribution V3 BioApplication. % activity was calculated relative to the positive (5 µM MG-132) and negative control (0.25% DMSO).