Lopez-Ongil,S., Senchak,V., Saura,M., Zaragoza,C., Ames,M., Ballermann,B.,
Rodriguez-Puyol,M., Rodriguez-Puyol,D., Lowenstein,C.J.
The Journal of Biological Chemistry 2000; 275 (34): 26423-26427
Presenter: Mandeep Walia
Background:
Endothelins are vasoactive polypeptides that are primarily produced
by the endothelial cells. Endothelins play an important role in cardiovascular
system by stimulating smooth muscle cell contraction and proliferation.
There are three isoforms of endothelins that have been identified:
ET-1, ET-2 and ET-3. ET-1 is the most potent of all the isoforms
and is primarily released from endothelial cells. ET-1 is synthesized
from a large precursor, prepro-ET-1. It is cleaved by a signal peptidase
into big ET-1 (non-functional petide) which is then cleaved by endothelin-converting
enzyme (ECE) into ET-1 (functional peptide).
The ECEs belong to a zinc metallopoteinase family and share a zinc-binding
HEXXH motif with them. The ECEs have two isoforms: ECE-1 and
ECE-2. The ECE-1 has 3 isoforms: ECE-1a, ECE-1b, and ECE-1c.
The ECE-1a is predominantly found in the endothelial cells of many organs
and is responsible for the synthesis of ET-1 synthesis from big ET-1.
Reactive Oxygen Species (ROS) also play an important role in the cell
growth if produced in low levels. ROS such as superoxide or hydrogen
peroxide are produced by all the cells in the body but high levels of ROS
can be cytotoxic.
Presenters Summary of the paper.
1) Purpose: To identify the effects of different radical donors on ECE activity in bovine aortic endothelial cells (BAEC). Results: Incubation of the membranes with xanthine and xanthine oxidase inhibited the generation of ET-1 from big ET-1. But incubation with various nitric oxide donors or with glucose oxidase and glucose did not inhibit the generation of ET-1. Conclusion: Superoxide inhibits ECE activity in a dose-dependent manner.
2) Purpose: To determine if superoxide inhibits ECE processing of big ET-1 with or without modifying the substrate ET-1. Results: Big ET-1 was either pretreated or not with xanthine oxidase and xanthine and BAEC membrane proteins were then added and there was no difference in the amount of ET-1 produced. But when BAEC membranes were treated with xanthine and xanthine oxidase and SOD and then big ET-1 was added, there was a significant decrease in the amount of ET-1 produced as compared to the controls. Conclusion: The target of superoxide is ECE and not big ET-1.
3) Purpose: To determine if superoxide inhibits ECE by interfering with ECE homodimerization. Results: ECE homodimerization is maintained by a single disulfide bond between each monomer and oxidants could indirectly inhibit the formation of the disulfide bond and thus decreasing the amount of ECE homodimers into inactive monomers. BAEC membranes were treated with or without xanthine and xanthine oxidase and then fractionated by SDS-PAGE. Treatment of membranes with xanthine and xanthine oxidase did not affect the mobility of ECE. Conclusion: Superoxide does not inhibit ECE by converting active ECE homodimers into inactive monomers.
4) Purpose: To determine if superoxide inhibits ECE by removing Zn2+ from ECE (ECE is a metalloproteinase that contains Zn2+ as a cofactor). Results: BAEC membranes were incubated with and without EDTA and ECE activity was then measured. EDTA decreased ECE activity and it was restored by adding Zn2+ to the membranes. Conclusion: Superoxide removes zinc from ECE.
5) Purpose: To prove that superoxide removes Zn2+ from ECE. Results: Native ECE contained Zn wheras ECE exposed to superoxide contained four fold less Zn. Conclusion: Superoxide decreases the amout of Zn2+ in ECE.
Why is this work important?
1) This paper demonstrates that superoxide inhibits ECE by ejecting
zinc from the enzyme. It also shows that the inhibition is specific
to superoxide and that hydrogen peroxide and nitric oxide did not affect
ECE activity (data not shown).
2) This study is important because it shows that ROS may play a role
in a negative feedback manner in which excess superoxide produced due to
the injured vessel could damage the endothelial cells and thus reduce the
availability of nitric oxide and leading to vasospasm. However, superoxide
can also decrease the synthesis of ET-1 and thus diminishing the vasospasm
to some degree. This suggests a novel target for inhibitors of ECE,
since ET-1 is involved in many pathophysiological conditions.
3) This paper also shows that the inhibition of ECE is reversible because
the addition of zinc restores ECE activity.
4) This study does not agree with the others that have shown that superoxide
can increase matrix metalloproteinase activity. And also that superoxide
dismutase inhibits matrix degradation. However, the present study
shows that ECE is inhibited by superoxide. This difference could
be due to the difference in zinc-binding motifs which would affect the
ability of superoxide to inhibit ECE activity in some metalloproteinases.
This paper clearly indicates that superoxide may also regulate other zinc
metalloprotienases in addition to ECE as well.
Question’s Unanswered
1) The mechanism by which superoxide ejects zinc from ECE is unclear.
2) It is not known if superoxide activates one set of metalloproteinases
and inhibits another.
Author’s Abstract
Reactive oxygen species (ROS) act as signaling molecules in the cardiovascular system, regulating cellular proliferation and migration. However, an excess of ROS can damage cells and alter endothelial cell function. We hypothesized that endogenous mechanisms protect the vasculature from excess levels of ROS. We now show that superoxide can inhibit endothelin-converting enzyme activity (ECE) and decrease endothelin-1 synthesis. Superoxide inhibits ECE but hydrogen peroxide and nitric oxide do not. Superoxide inhibits ECE by ejecting zinc from the enzyme, and the addition of exogenous zinc restores enzymatic activity. Superoxide may inhibit other zinc metalloproteinases by a similar mechanism and may thus play an important role in regulating the biology of blood vessels.