The caspase-like sites of proteasomes, their substrate specificity, new inhibitors and substrates, and allosteric interactions with the trypsin-like sites

J Biol Chem. 2003 Sep 19;278(38):35869-77. doi: 10.1074/jbc.M303725200. Epub 2003 Jun 18.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aldehydes / chemistry
  • Allosteric Site
  • Animals
  • Aspartic Acid / chemistry
  • Binding Sites
  • Caspases / chemistry*
  • Catalytic Domain
  • Chymotrypsin / chemistry
  • Cysteine Endopeptidases / chemistry*
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology
  • Gene Library
  • Glutamic Acid / chemistry
  • Humans
  • Ketones / chemistry
  • Kinetics
  • Multienzyme Complexes / antagonists & inhibitors
  • Multienzyme Complexes / chemistry*
  • Mutation
  • Peptide Hydrolases / chemistry
  • Peptides / chemistry
  • Proteasome Endopeptidase Complex
  • Protein Binding
  • Rabbits
  • Saccharomyces cerevisiae / metabolism
  • Substrate Specificity
  • Time Factors
  • Trypsin / chemistry
  • Trypsin / pharmacology

Substances

  • Aldehydes
  • Enzyme Inhibitors
  • Ketones
  • Multienzyme Complexes
  • Peptides
  • Aspartic Acid
  • Glutamic Acid
  • Peptide Hydrolases
  • Chymotrypsin
  • Trypsin
  • Caspases
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease