title: Importance of the different proteolytic sites of the proteasome and the efficacy of inhibitors varies with the protein substrate.
abstract: The relative importance of the different proteolytic sites in mammalian proteasomes in protein degradation has not been studied systematically. Nevertheless, it is widely assumed that inhibition of the chymotrypsin-like site, the primary target of the proteasome inhibitors used in research and cancer therapy, reflects the degree of inhibition of protein breakdown. Here we demonstrate that selective inactivation of the chymotrypsin-like site reduced degradation of model proteins by pure 26 S proteasomes by only 11-50% and decreased only slightly the breakdown of proteins in HeLa cells. Inactivation of the caspase-like site decreased breakdown of model proteins by 12-22% and of the trypsin-like site by 3-35%. The relative contributions of these different sites depended on the protein substrate, and the importance of the trypsin-like sites depended on the substrate's content of basic residues. Simultaneous inhibition of the chymotrypsin-like and the caspase- or trypsin-like sites was needed to reduce degradation by >50%. Thus, 1) all three types of active sites contribute significantly to protein breakdown, 2) their relative importance varies widely with the substrate, 3) assaying the chymotrypsin-like activity overestimates the actual reduction in protein degradation, and 4) inhibition of multiple sites is required to markedly decrease proteolysis.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Alexei F
authors.initials: AF
authors.lastname: Kisselev
authors.forename: Alice
authors.initials: A
authors.lastname: Callard
authors.forename: Alfred L
authors.initials: AL
authors.lastname: Goldberg
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D020169:Caspases / Q000737:chemistry / Q000378:metabolism; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011485:Protein Binding; D011817:Rabbits; D013379:Substrate Specificity; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D061988:Proteasome Inhibitors; D002918:Chymotrypsin; D014357:Trypsin; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000251:Adenosine Triphosphatases