Proteasome Subunit Selective Activity-Based Probes Report on Proteasome Core Particle Composition in a Native Polyacrylamide Gel Electrophoresis Fluorescence-Resonance Energy Transfer Assay.

ABSTRACT

Most mammalian tissues contain a single proteasome species constitutive proteasomes. Tissues able to express, next to the constitutive proteasome catalytic activities (ß1c, ß2c, ß5c), the three homologous activities, ß1i, ß2i and ß5i, may contain numerous distinct proteasome particles immunoproteasomes (composed of ß1i, ß2i and ß5i) and mixed proteasomes containing a mix of these activities. This work describes the development of new subunit-selective activity-based probes and their use in an activity-based protein profiling assay that allows the detection of various proteasome particles. Tissue extracts are treated with subunit-specific probes bearing distinct fluorophores and subunit-specific inhibitors. The samples are resolved by native polyacrylamide gel electrophoresis, after which fluorescence-resonance energy transfer (FRET) reports on the nature of proteasomes present.