Inhibition of the proteolytic activity of the multicatalytic proteinase complex (proteasome) by substrate-related peptidyl aldehydes.
Evidence indicates that a component of the multicatalytic proteinase complex (MPC) that preferentially cleaves bonds after branched chain amino acids (BrAAP) is a major factor responsible for the protein-degrading activity of the MPC. We report here the synthesis of substrate-related peptidyl aldehydes that inhibit the activity of this component toward both synthetic peptide substrates and proteins. The most potent of the inhibitors, Cbz-Gly-Pro-Phe-leucinal (Cbz-GPFL-CHO) inhibits competitively with a Ki of 1.5 microM. The peptidyl aldehydes also inhibit the small neutral amino acid preferring and the peptidylglutamyl-peptide hydrolyzing activities of the MPC. The chymotrypsin-like activity is only weakly inhibited, and the trypsin-like activity is moderately activated. The importance of a Pro residue in the P3 position and a leucinal residue in the P1 position for inhibition of the BrAAP component is indicated by the finding that replacement of these residues by a glycine or phenylalaninal, respectively, markedly increases the Ki. Cbz-GPFL-CHO inhibited the BrAAP activity with the same Ki both before and after activation of this component by exposure of the MPC to 3,4-dichloroisocoumarin, suggesting that the peptidyl aldehyde is an effective inhibitor of both the overt and latent proteolytic activities of the MPC. Incubation of a human breast cancer cell line (MCF-7) in culture with the inhibitors of the BrAAP component led to an accumulation of ubiquitin-protein conjugates, indicating inhibition of the ubiquitin-dependent proteolytic pathway.
Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, New York 10029.
A
A
Vinitsky
C
C
Cardozo
L
L
Sepp-Lorenzino
C
C
Michaud
M
M
Orlowski
D000447:Aldehydes / Q000737:chemistry / Q000494:pharmacology*; D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry*; D046988:Proteasome Endopeptidase Complex; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000447:Aldehydes; D009097:Multienzyme Complexes; D010455:Peptides; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex