The caspase-like sites of proteasomes, their substrate specificity, new inhibitors and substrates, and allosteric interactions with the trypsin-like sites.
Proteasomes are the primary sites for protein degradation in mammalian cells. Each proteasome particle contains two chymotrypsin-like, two trypsin-like, and two caspase-like proteolytic sites. Previous studies suggest a complex network of allosteric interactions between these catalytic and multiple regulatory sites. We used positional scanning combinatorial substrate libraries to determine the extended substrate specificity of the caspase-like sites. Based on this analysis, several new substrates were synthesized, the use of which confirmed earlier observations that caspase-like sites (often termed postglutamyl peptide hydrolase) cleave after aspartates better than after glutamates. Highly selective inhibitors of the caspase-like sites were also generated. They stimulated trypsin-like activity of yeast 20 S proteasomes up to 3-fold but not when binding of the inhibitor to the caspase-like sites was prevented in a mutant carrying an uncleaved propeptide. Although substrates of the caspase-like sites allosterically inhibit the chymotrypsin-like activity, inhibitors of the caspase-like sites do not affect the chymotrypsin-like sites. Furthermore, when caspase-like sites were occupied by the uncleaved propeptide or inhibitor, their substrates still inhibited the chymotrypsin-like activity. Thus, occupancy of the caspase-like sites stimulates the trypsin-like activity of proteasomes, but substrates of the caspase-like sites inhibit the chymotrypsin-like activity by binding to a distinct noncatalytic site.
Kisselev
Alexei F
AF
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. alezei_kisselev@hms.harvard.edu
Garcia-Calvo
Margarita
M
Overkleeft
Herman S
HS
Peterson
Erin
E
Pennington
Michael W
MW
Ploegh
Hidde L
HL
Thornberry
Nancy A
NA
Goldberg
Alfred L
AL
D000447:Aldehydes / Q000737:chemistry; D000495:Allosteric Site; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D020169:Caspases / Q000737:chemistry*; D020134:Catalytic Domain; D002918:Chymotrypsin / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015723:Gene Library; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D007659:Ketones / Q000737:chemistry; D007700:Kinetics; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry*; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
D000447:Aldehydes; D004791:Enzyme Inhibitors; D007659:Ketones; D009097:Multienzyme Complexes; D010455:Peptides; D001224:Aspartic Acid; D018698:Glutamic Acid; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease