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Hydrogen peroxide regulation of endothelial exocytosis by inhibition of N-ethylmaleimide sensitive factor.

Matsushita K, Morrell CN, Mason RJ, Yamakuchi M, Khanday FA, Irani K, Lowenstein CJ - J. Cell Biol. (2005)

Bottom Line: H(2)O(2) decreases the ability of NSF to hydrolyze adenosine triphosphate and to disassemble the soluble NSF attachment protein receptor complex.Mutation of NSF cysteine residue C264T eliminates the sensitivity of NSF to H(2)O(2), suggesting that this cysteine residue is a redox sensor for NSF.Increasing endogenous H(2)O(2) levels in mice decreases exocytosis and platelet rolling on venules in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Although an excess of reactive oxygen species (ROS) can damage the vasculature, low concentrations of ROS mediate intracellular signal transduction pathways. We hypothesized that hydrogen peroxide plays a beneficial role in the vasculature by inhibiting endothelial exocytosis that would otherwise induce vascular inflammation and thrombosis. We now show that endogenous H(2)O(2) inhibits thrombin-induced exocytosis of granules from endothelial cells. H(2)O(2) regulates exocytosis by inhibiting N-ethylmaleimide sensitive factor (NSF), a protein that regulates membrane fusion events necessary for exocytosis. H(2)O(2) decreases the ability of NSF to hydrolyze adenosine triphosphate and to disassemble the soluble NSF attachment protein receptor complex. Mutation of NSF cysteine residue C264T eliminates the sensitivity of NSF to H(2)O(2), suggesting that this cysteine residue is a redox sensor for NSF. Increasing endogenous H(2)O(2) levels in mice decreases exocytosis and platelet rolling on venules in vivo. By inhibiting endothelial cell exocytosis, endogenous H(2)O(2) may protect the vasculature from inflammation and thrombosis.

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NSF cysteine residue targets of H2O2. (A) Specific NSF mutants are resistant to H2O2 inhibition of ATPase activity. 1 mM H2O2 or control was added to recombinant wild-type and mutant NSF, and the ATPase activity was measured. H2O2 inhibits ATPase activity of wild-type NSF and all mutant NSF, except for C21A and C264A mutants (n = 2 ± SD; **, P < 0.01 vs. control). (B) H2O2 does not affect interaction of specific NSF mutants with syntaxin-4. (His)6-NSF was pretreated or not with 1 mM H2O2, and then mixed with (His)6-α-SNAP, GST-Syntaxin-4, VAMP-3, and SNAP-23. The mixture was precipitated with glutathione-sepharose and immunoblotted with antibody to NSF. (C) H2O2 alters the mobility of NSF mutants. Recombinant wild-type or mutant NSF was treated with 0.1 mM H2O2 for 5 min, fractionated by nondenaturing PAGE, and then immunoblotted with antibody to NSF.
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fig4: NSF cysteine residue targets of H2O2. (A) Specific NSF mutants are resistant to H2O2 inhibition of ATPase activity. 1 mM H2O2 or control was added to recombinant wild-type and mutant NSF, and the ATPase activity was measured. H2O2 inhibits ATPase activity of wild-type NSF and all mutant NSF, except for C21A and C264A mutants (n = 2 ± SD; **, P < 0.01 vs. control). (B) H2O2 does not affect interaction of specific NSF mutants with syntaxin-4. (His)6-NSF was pretreated or not with 1 mM H2O2, and then mixed with (His)6-α-SNAP, GST-Syntaxin-4, VAMP-3, and SNAP-23. The mixture was precipitated with glutathione-sepharose and immunoblotted with antibody to NSF. (C) H2O2 alters the mobility of NSF mutants. Recombinant wild-type or mutant NSF was treated with 0.1 mM H2O2 for 5 min, fractionated by nondenaturing PAGE, and then immunoblotted with antibody to NSF.

Mentions: H2O2 may regulate NSF by oxidizing cysteine residues. To determine which of the nine cysteine residues of NSF are targets of H2O2, we expressed in bacteria and purified wild-type or mutant NSF polypeptides with each of the nine individual cysteine residues of NSF replaced by Ala. We added H2O2 to wild-type and mutant NSF polypeptides and measured ATPase activity. Mutation of cysteine residues 21, 91, 264, and 334 decreases NSF ATPase activity (Fig. 4 A). H2O2 treatment inhibits ATPase activity of all mutant NSF except mutants C21A and C264A (Fig. 4 A). These data suggest that cysteine residues C21 and C264 mediate H2O2 inhibition of NSF ATPase activity.


Hydrogen peroxide regulation of endothelial exocytosis by inhibition of N-ethylmaleimide sensitive factor.

Matsushita K, Morrell CN, Mason RJ, Yamakuchi M, Khanday FA, Irani K, Lowenstein CJ - J. Cell Biol. (2005)

NSF cysteine residue targets of H2O2. (A) Specific NSF mutants are resistant to H2O2 inhibition of ATPase activity. 1 mM H2O2 or control was added to recombinant wild-type and mutant NSF, and the ATPase activity was measured. H2O2 inhibits ATPase activity of wild-type NSF and all mutant NSF, except for C21A and C264A mutants (n = 2 ± SD; **, P < 0.01 vs. control). (B) H2O2 does not affect interaction of specific NSF mutants with syntaxin-4. (His)6-NSF was pretreated or not with 1 mM H2O2, and then mixed with (His)6-α-SNAP, GST-Syntaxin-4, VAMP-3, and SNAP-23. The mixture was precipitated with glutathione-sepharose and immunoblotted with antibody to NSF. (C) H2O2 alters the mobility of NSF mutants. Recombinant wild-type or mutant NSF was treated with 0.1 mM H2O2 for 5 min, fractionated by nondenaturing PAGE, and then immunoblotted with antibody to NSF.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171382&req=5

fig4: NSF cysteine residue targets of H2O2. (A) Specific NSF mutants are resistant to H2O2 inhibition of ATPase activity. 1 mM H2O2 or control was added to recombinant wild-type and mutant NSF, and the ATPase activity was measured. H2O2 inhibits ATPase activity of wild-type NSF and all mutant NSF, except for C21A and C264A mutants (n = 2 ± SD; **, P < 0.01 vs. control). (B) H2O2 does not affect interaction of specific NSF mutants with syntaxin-4. (His)6-NSF was pretreated or not with 1 mM H2O2, and then mixed with (His)6-α-SNAP, GST-Syntaxin-4, VAMP-3, and SNAP-23. The mixture was precipitated with glutathione-sepharose and immunoblotted with antibody to NSF. (C) H2O2 alters the mobility of NSF mutants. Recombinant wild-type or mutant NSF was treated with 0.1 mM H2O2 for 5 min, fractionated by nondenaturing PAGE, and then immunoblotted with antibody to NSF.
Mentions: H2O2 may regulate NSF by oxidizing cysteine residues. To determine which of the nine cysteine residues of NSF are targets of H2O2, we expressed in bacteria and purified wild-type or mutant NSF polypeptides with each of the nine individual cysteine residues of NSF replaced by Ala. We added H2O2 to wild-type and mutant NSF polypeptides and measured ATPase activity. Mutation of cysteine residues 21, 91, 264, and 334 decreases NSF ATPase activity (Fig. 4 A). H2O2 treatment inhibits ATPase activity of all mutant NSF except mutants C21A and C264A (Fig. 4 A). These data suggest that cysteine residues C21 and C264 mediate H2O2 inhibition of NSF ATPase activity.

Bottom Line: H(2)O(2) decreases the ability of NSF to hydrolyze adenosine triphosphate and to disassemble the soluble NSF attachment protein receptor complex.Mutation of NSF cysteine residue C264T eliminates the sensitivity of NSF to H(2)O(2), suggesting that this cysteine residue is a redox sensor for NSF.Increasing endogenous H(2)O(2) levels in mice decreases exocytosis and platelet rolling on venules in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Although an excess of reactive oxygen species (ROS) can damage the vasculature, low concentrations of ROS mediate intracellular signal transduction pathways. We hypothesized that hydrogen peroxide plays a beneficial role in the vasculature by inhibiting endothelial exocytosis that would otherwise induce vascular inflammation and thrombosis. We now show that endogenous H(2)O(2) inhibits thrombin-induced exocytosis of granules from endothelial cells. H(2)O(2) regulates exocytosis by inhibiting N-ethylmaleimide sensitive factor (NSF), a protein that regulates membrane fusion events necessary for exocytosis. H(2)O(2) decreases the ability of NSF to hydrolyze adenosine triphosphate and to disassemble the soluble NSF attachment protein receptor complex. Mutation of NSF cysteine residue C264T eliminates the sensitivity of NSF to H(2)O(2), suggesting that this cysteine residue is a redox sensor for NSF. Increasing endogenous H(2)O(2) levels in mice decreases exocytosis and platelet rolling on venules in vivo. By inhibiting endothelial cell exocytosis, endogenous H(2)O(2) may protect the vasculature from inflammation and thrombosis.

Show MeSH
Related in: MedlinePlus