Journal
FREE RADICAL BIOLOGY AND MEDICINE
Volume 143, Issue -, Pages 16-24Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.freeradbiomed.2019.07.020
Keywords
Proteasome; Immuno-proteasome; Redox regulation; Fluoropeptides; Proteolytic activity; Proteostasis; Methods
Funding
- National Institute of Environmental Health Sciences, of the US National Institutes of Health [ES003598]
- National Institute on Aging of the US National Institutes of Health [AG052374]
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Proteolytic enzymes are often strongly affected by redox reactions, free radicals, oxidation, or oxidative stress. The 20S Proteasome and the Immuno-Proteasome are examples of major intracellular proteases whose concentration, transcription, translation, and proteolytic activity are all subject to redox regulation. Proteasomes are essential in maintaining overall protein homeostasis (or proteostasis), and their dysregulation results in detrimental phenotypes associated with various pathologies, including several common age-related diseases. Many studies have used Western blots to assess redox changes in Proteasome protein levels or RT-PCR to study RNA transcript levels, but actual measurements of proteolytic activity are far less common. Since each intact protein substrate exhibits a different proteolytic profile when incubated with proteasome or Immuno-Proteasome [+/- activators such as 19S or 11S (also called PA28)] and these proteolytic profiles are drastically altered if the protein substrate is denatured, for example by oxidation, heat, acetylation, or methylation. In an attempt to standardize proteasomal activity measurements small fluorogenic protein/peptide substrates were developed to test the three proteolytically active sites of the Proteasome and Immuno-Proteasome: trypsin-like, chymotrypsin-like, and caspase-like activities. Despite extensive use of fluorogenic peptide substrates to measure proteasome activity, there is an absence of a standardized set of best practices. In this study we analyze different parameters, such as sample concentration, AMC conjugated substrate concentration, duration of assay, and frequency of measurements, and examine how they impact the determination of Proteasome and Immuno-Proteasome activities using fluorogenic peptide substrates.
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