Human platelet 20S proteasome: inhibition of its chymotrypsin-like activity and identification of the proteasome activator PA28. A preliminary report.
Earlier studies have demonstrated that human platelets contain the 20S proteasome, and its protein activator. However, understanding the potential role of the proteasome in human platelets requires a detailed knowledge about its chymotryptic-like activity, a crucial one for protein degradation in all eukaryotic cells. In this communication we have shown that human platelet 20S proteasome exhibited chymotryptic-like activity towards succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin as substrate at a broad pH range, with optimum between pH 7.5-8.0 and 5.0-5.5. These two activities were markedly inhibited by a 10 micromol/l concentration of two structurally unrelated proteasome inhibitors: lactacystin/beta-lactone or benzyloxycarbonyl-Ile-Glu(O-tert.-butyl)-Ala-leucinal, but not by ebelactone B, an inhibitor of lysosomal cathepsin A/deamidase. The chymotryptic-like activity of the 20S proteasome against succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin was also significantly inhibited in platelets, after exposure of platelet-rich plasma to 10 micromol/l lactacystin and benzyloxycarbonyl-Ile-Glu(O-tert.-butyl)-Ala-leucinal for up to 60 min. This indicates that these inhibitors can enter platelets and selectively inhibit 20S proteasome activity. We also demonstrated for the first time by Western blot analysis that human platelets contain a proteasome activator, PA28, which is known to play a key role in antigen processing by significant stimulation of the proteasomal chymotryptic-like activity. Since the platelet 20S proteasome was also present in a latent form, this suggests that its activity may be regulated in vivo in human platelets. All these results can therefore be beneficial in future studies on the role of the 20S proteasome in platelet biology.
Ostrowska
Halina
H
Department of Biology, Medical Academy of Bialystok, Poland. halost@amb.edu.pl
Ostrowska
Justyna Kornelia
JK
Worowski
Krzysztof
K
Radziwon
Piotr
P
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001792:Blood Platelets / Q000737:chemistry / Q000201:enzymology*; D002918:Chymotrypsin; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex
D003374:Coumarins; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D009842:Oligopeptides; C090535:benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal; C067713:lactacystin; C046582:succinyl-leucyl-leucyl-valyl-tyrosyl-methylcoumarinamide; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine