Procaspase-3 activation by a metalloprotease secreted from Vibrio vulnificus.
Vibrio vulnificus is a marine bacterium and a human pathogen capable of causing wound infection and septicemia. We previously showed that the metalloprotease vEP secreted by V. vulnificus activates prothrombin in vitro. To further investigate the ability of vEP to activate other zymogens, we used a mutant form of procaspase-3 which lacks the native cleavage sites as a zymogen. The mutant zymogen was activated by vEP to yield a mature enzyme with a maximum increase in caspase-3 activity of approximately 14-fold in a time-dependent manner. However, the increase started to decline with prolonged incubation and with higher protease concentration as a result of further cleavage of the mature enzyme. Western blot analysis revealed a band of approximately 17 kDa for the cleavage product, which corresponded with the change in caspase-3 activity. The activated procaspase-3 by vEP was also able to cleave poly(ADP-ribose) polymerase in a cell-free system, and was inhibited by Ac-DEVD-CHO, a potent caspase-3 inhibitor. The results presented are the first to demonstrate the in vitro activation of one of the crucial enzymes involved in cell death by a bacterial extracellular metalloprotease.
Kim
Hyo Young
HY
Research Center for Proteineous Materials, Chosun University, Gwangju 501-759, Korea.
Chang
Alan K
AK
Park
Jung Eun
JE
Park
Il-Seon
IS
Yoon
Seong Myeong
SM
Lee
Jung Sup
JS
D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D045726:Metalloproteases / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013997:Time Factors; D041261:Vibrio vulnificus / Q000201:enzymology* / Q000378:metabolism