Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substrates.
Eukaryotic proteasomes have three different types of active sites: two chymotrypsin-like, two trypsin-like, and two caspase-like (also termed PGPH) sites that differ in their specificity toward model fluorogenic peptide substrates. The chymotrypsin-like site is often considered the most important in protein breakdown, and the only one whose activity has to be assayed in order to assess the capacity of proteasomes to degrade proteins. However, recent results indicate that either trypsin-like or caspase-like sites also have to be inhibited in order to reduce breakdown of most proteins by 50%. Thus, the activities of all three types of active sites have to be assayed in order to evaluate the state of the proteasome inside cells or the potency of inhibitors. This chapter describes assays of purified 26S proteasomes with fluorogenic peptide substrates, including new substrates of the caspase- and trypsin-like sites. A novel assay of proteasome activity in crude cell extracts that allows rapid evaluation of the state of the proteasomes in cells treated with inhibitors is also described.
Kisselev
Alexei F
AF
Department of Pharmacology and Toxicology, and Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, NH 03756, USA.
Goldberg
Alfred L
AL
D000818:Animals; D002021:Buffers; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D010447:Peptide Hydrolases; D010455:Peptides; D046988:Proteasome Endopeptidase Complex / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D002021:Buffers; D005456:Fluorescent Dyes; D010455:Peptides; D061988:Proteasome Inhibitors; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex