Nitric Oxide Inhibits Caspase-3 by S-Nitrosationin Vivo

Rössig, Lothar; Fichtlscherer, Birgit; Breitschopf, Kristin; Haendeler, Judith; Zeiher, Andreas M.; Mülsch, Alexander; Dimmeler, Stefanie

doi:10.1074/jbc.274.11.6823

Cited by 397 publications

(242 citation statements)

References 31 publications

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“…19,23,26 Our results show that staurosporine-induced reduction in eNOS activity was associated with caspasemediated cleavage of eNOS. Long-term (18-24 h) exposure of either eNOS-transfected COS-7 cells or BAECs to staurosporine resulted in a significant loss of eNOS protein and activity, an effect that was significantly reduced by caspase inhibitors.…”

Section: Discussionmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 99%

“…NO has been shown to inhibit endothelial cell apoptosis 23 and many groups have shown inhibitory effects of NO on caspase activity. [20][21][22][23][24][25] Diminished eNOS-catalyzed production of NO or its bioavailability leads to endothelial apoptosis, and it has been speculated that this is associated with enhanced susceptibility to endothelial dysfunction and atherosclerosis. [26][27][28] As NO inhibits apoptosis in endothelial cells, it is intriguing to speculate that caspase-mediated loss of eNOS-derived NO further activates the caspase cascade, thereby promoting the apoptotic process by removing an impediment to cell death.…”

Section: Discussionmentioning

confidence: 99%

“…[18][19][20] Antiapoptotic effects of NO have been attributed to its ability to inhibit the caspase cascade via reversible S-nitrosylation of key cysteine residues in various caspases. [20][21][22][23][24] Thus, eNOS-synthesized NO inhibited caspase-mediated apoptosis in endothelial cells; 23 it has been speculated that diminished production or availability of NO and enhanced apoptosis is related to susceptibility to atherosclerosis or age-related endothelial dysfunction. [25][26][27][28] In view of a role for eNOS in endothelial cell apoptosis, the effect of staurosporine-induced apoptosis on levels of eNOS protein and activity was studied in COS-7 cells, overexpressing eNOS, and in bovine aortic endothelial cells (BAEC).…”

Section: Introductionmentioning

confidence: 99%

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Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells

2005

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Nitric oxide (NO) may block apoptosis by inhibiting caspases via S-nitrosylation of cysteines. Here, we investigated whether effector caspases might cleave and thereby inhibit endothelial nitric oxide synthase (eNOS). Exposure of eNOS-transfected COS-7 cells and bovine aortic endothelial cells to staurosporine resulted in significant loss of 135-kDa eNOS protein and activity, and appearance of a 60-kDa eNOS fragment; effects were inhibited by the general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp[OMe]-fluoromethyl ketone (zVAD-fmk). In eNOS-transfected COS-7 cells, staurosporine-induced activation of caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage coincided with increased eNOS degradation and decreased activity. Loss of eNOS activity was greater than the degree of proteolysis. Incubation of immunoprecipitated eNOS with caspase-3, caspase-6 or caspase-7 resulted in eNOS cleavage. Staurosporine, a general protein kinase inhibitor, also reduced phosphorylation and decreased calmodulin binding, an effect that may explain the reduction in activity. eNOS, therefore, is both an inhibitor of apoptosis and a target of apoptosis-associated proteolysis.

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Section: Discussionmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells

2005

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“…NO can produce both single cysteine S-nitrosylations as in case of Ha-Ras (Lander et al, 1997), caspase 3 (Rossig et al, 1999), and nuclear factor-kappa B (Marshall and Stamler, 2001) and multiple cysteine S-nitrosylations in case of Nethylmaleimide Sensitive Factor (NSF) (Matsushita et al, 2003) and the ryanodine receptor (Voss et al, 2004). Consensus S-nitrosylation motifs have been postulated by Stamler et al (1997).…”

Section: Introductionmentioning

confidence: 99%

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

Tummala

Ryzhov

Ravi

et al. 2008

DNA and Cell Biology

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S-nitrosylation, or the replacement of the hydrogen atom in the thiol group of cysteine residues by a -NO moiety, is a physiologically important posttranslational modification. In our previous work we have shown that Snitrosylation is involved in the disruption of the endothelial nitric oxide synthase (eNOS) dimer and that this involves the disruption of the zinc (Zn) tetrathiolate cluster due to the S-nitrosylation of Cysteine 98. However, human eNOS contains 28 other cysteine residues whose potential to undergo S-nitrosylation has not been determined. Thus, the goal of this study was to identify the cysteine residues within eNOS that are susceptible to S-nitrosylation in vitro. To accomplish this, we utilized a modified biotin switch assay. Our modification included the tryptic digestion of the S-nitrosylated eNOS protein to allow the isolation of S-nitrosylated peptides for further identification by mass spectrometry. Our data indicate that multiple cysteine residues are capable of undergoing S-nitrosylation in the presence of an excess of a nitrosylating agent. All these cysteine residues identified were found to be located on the surface of the protein according to the available X-ray structure of the oxygenase domain of eNOS. Among those identified were Cys 93 and 98, the residues involved in the formation of the eNOS dimer through a Zn tetrathiolate cluster. In addition, cysteine residues within the reductase domain were identified as undergoing S-nitrosylation. We identified cysteines 660, 801, and 1113 as capable of undergoing S-nitrosylation. These cysteines are located within regions known to bind flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NADPH) although from our studies their functional significance is unclear. Finally we identified cysteines 852, 975=990, and 1047=1049 as being susceptible to Snitrosylation. These cysteines are located in regions of eNOS that have not been implicated in any known biochemical functions and the significance of their S-nitrosylation is not clear from this study. Thus, our data indicate that the eNOS protein can be S-nitrosylated at multiple sites other than within the Zn tetrathiolate cluster, suggesting that S-nitrosylation may regulate eNOS function in ways other than simply by inducing dimer collapse.

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Caspase and calpain substrates: Roles in synaptic plasticity and cell death

1999

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Neurons are an unusual type of cell in that they send processes (axons and dendrites) over great distances. This elaborate morphology, together with their excitability, places neurons at risk for multiple insults. Recent studies have demonstrated that apoptotic and excitotoxic mechanisms not only contribute to neuronal death, but also to synaptic dysfunction and a breakdown in neural circuitry (see Mattson and Duan [1999] J. Neurosci. Res. 58:152-166, this issue). Proteases of the caspase and calpain families have been implicated in neurodegenerative processes, as their activation can be triggered by calcium influx and oxidative stress. Caspases and calpains are cysteine proteases that require proteolytic cleavage for activation. The substrates cleaved by caspases include cytoskeletal and associated proteins, kinases, members of the Bcl-2 family of apoptosis-related proteins, presenilins and amyloid precursor protein, and DNA-modulating enzymes. Calpain substrates include cytoskeletal and associated proteins, kinases and phosphatases, membrane receptors and transporters, and steroid receptors. Many of the substrates of caspases and calpains are localized in pre- and/or postsynaptic compartments of neurons. Emerging data suggest that, in addition to their roles in neurodegenerative processes, caspases and calpains play important roles in modulating synaptic plasticity. The present article provides a review of the properties of the different caspases and calpains, their roles in cell death pathways, and the substrates upon which they act. Emerging data are considered that suggest key roles for these proteases in the regulation of synaptic plasticity.

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Nitric Oxide Inhibits Caspase-3 by S-Nitrosationin Vivo

Cited by 397 publications

References 31 publications

Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells

Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

Caspase and calpain substrates: Roles in synaptic plasticity and cell death

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