194 - Measuring 20S Proteasome and Immunoproteasome Activities in Oxidative Stress

Abstract

Protein degradation mechanisms are fundamental components in maintaining proteostasis and homeostasis. Intracellular proteins are highly susceptible to oxidative modification and damage. Inactivation of key enzymes and aggregation or cross-linking of numerous cell proteins are only some of the known outcomes of intracellular protein oxidation. Ultimately, protein oxidation, modification, aggregation, and cross-linking have been linked to multiple diseases and to the aging process. Our work has shown that oxidized proteins in the cytoplasm, nucleus, and endoplasmic reticulum are selectively degraded by the 20S Proteasome and the Immunoproteasome (both ± the 11S or Pa28 proteasome regulator) whereas oxidatively damaged mitochondrial proteins are degraded by the Lon protease. Rapid degradation of mildly damaged proteins prevents their aggregation and cross-linking, and allows for their replacement by de novo synthesis. Increased transcription/translation of key proteolytic enzymes in response to signaling levels of oxidants is a key feature of Adaptive Homeostasis. In the past few years, it has become commonplace to evaluate discrete proteasome activities using the cleavage of small fluorogenic peptides to measure caspase-like activity, trypsin-like activity, and chymotrypsin-like activity. Our studies demonstrate that substrate concentration, cell type or proteolytic enzyme concentration, and length of incubation are all key factors in determining the proteolysis of fluorogenic peptides. Our work provides a rational and quantitative approach to the measurement of proteasomal activities under both normal and stress conditions. This work has been supported by grant ES003598 from the National Institute of Environmental Health Sciences of the US National Institutes of Health to K.J.A.D.