Redox control of 20S proteasome

The 20S proteasome is a high molecular weight multicatalytic proteinase found both in the cytosol and nucleus of eukaryotic cells. The 20S proteasome is characterized by three main proteolytic activities with distinct specificities against short synthetic peptides: the trypsin-like activity expressed by the Z and MECL1 subunits, the chymotrypsin-like activity expressed by the X, LMP2, and LMP7 subunits, and the peptidylglutamyl peptide-hydrolyzing activity expressed by the Y subunits. Exposure to metal-catalyzed oxidation of purified 20S proteasome in vitro or of rat hepatoma cells results in the inactivation of certain peptidase activities. However, the alterations in peptidase activities observed in vitro depend on whether the purified proteasome is in its latent or active form before treatment with the reactive oxygen species-generating system. This chapter describes appropriate protocols aimed at isolating the 20S proteasome from different sources, as well as methods for promoting the conversion from latent to active proteasome. Different ways of monitoring oxidation-mediated functional and structural changes of the 20S proteasome, either purified or within tissue and cell homogenates, are also presented in the chapter.