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Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases.

Dimmeler S, Haendeler J, Nehls M, Zeiher AM - J. Exp. Med. (1997)

Bottom Line: Physiological levels of shear stress alter the genetic program of cultured endothelial cells and are associated with reduced cellular turnover rates and formation of atherosclerotic lesions in vivo.This anti-apoptotic activity of shear stress decreased after pharmacological inhibition of endogenous nitric oxide (NO)-synthase by NG-monomethyl-L-arginine and was completely reproduced by exogenous NO-donors.Endothelial-derived nitric oxide (NO) as well as exogenous NO donors inhibited TNF-alpha-induced cysteine protease activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine IV, University of Frankfurt, Germany.

ABSTRACT
Physiological levels of shear stress alter the genetic program of cultured endothelial cells and are associated with reduced cellular turnover rates and formation of atherosclerotic lesions in vivo. To test the hypothesis that shear stress (15 dynes/cm2) interferes with programmed cell death, apoptosis was induced in human umbilical venous cells (HUVEC) by tumor necrosis factor-alpha (TNF-alpha). Apoptosis was quantified by ELISA specific for histone-associated DNA-fragments and confirmed by demonstrating the specific pattern of internucleosomal DNA-fragmentation. TNF-alpha (300 U/ml) mediated increase of DNA-fragmentation was completely abrogated by shear stress (446 +/- 121% versus 57 +/- 11%, P <0.05). This anti-apoptotic activity of shear stress decreased after pharmacological inhibition of endogenous nitric oxide (NO)-synthase by NG-monomethyl-L-arginine and was completely reproduced by exogenous NO-donors. The activation of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like cysteine proteases was required to mediate TNF-alpha-induced apoptosis of HUVEC. Endothelial-derived nitric oxide (NO) as well as exogenous NO donors inhibited TNF-alpha-induced cysteine protease activation. Inhibition of CPP-32 enzyme activity was due to specific S-nitrosylation of Cys 163, a functionally essential amino acid conserved among ICE/CPP-32-like proteases. Thus, we propose that shear stress-mediated NO formation interferes with cell death signal transduction and may contribute to endothelial cell integrity by inhibition of apoptosis.

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Purification of p17  subunits and direct inhibition of  reconstituted CPP-32- and ICEsubunits by NO. (A) Silver stain  of the crude homogenates before purification (60 μg; lane 3),  the unbound fraction of the NiNTA columns (60 μg; lane 4)  and the purified CPP-32 p17  subunit (10 μg; lane 2) separated  by a 12% SDS–polyacrylamide  gel. (B) Inhibition of cloned, purified and reconstituted CPP-32  and ICE by incubation with SNP  or SNAP for 1 h.
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Figure 4: Purification of p17 subunits and direct inhibition of reconstituted CPP-32- and ICEsubunits by NO. (A) Silver stain of the crude homogenates before purification (60 μg; lane 3), the unbound fraction of the NiNTA columns (60 μg; lane 4) and the purified CPP-32 p17 subunit (10 μg; lane 2) separated by a 12% SDS–polyacrylamide gel. (B) Inhibition of cloned, purified and reconstituted CPP-32 and ICE by incubation with SNP or SNAP for 1 h.

Mentions: ICE- and CPP-32–like proteases possess a highly conserved and functionally essential cysteine within their active center (5, 6, 23). Since S-nitrosylation is a well-established mechanism, by which NO can inhibit enzyme activity (19, 24, 25), we biochemically analyzed a potentially direct interaction of NO with CPP-32. Upon activation, proteolytical cleavage of CPP-32 releases two subunits, p12 and p17, which heterodimerize to form the active protease (23). The p17 subunit contains the reactive cysteine group at position 163 in its active center (23). Therefore, both subunits were separately cloned, bacterially expressed, and purified to homogeneity as demonstrated by silver staining (Fig. 4 A). S-nitrosylation of the p17 subunit was performed and detected according to Stamler (20). Table 1 illustrates that NO released by NaNO2 or SNP timedependently S-nitrosylated one thiol group per molecule p17. The S-nitrosylation correlated with the reduction of one thiol-group per molecule p17 detectable by DTNB (Table 1). These findings were further substantiated by demonstrating that the isolated p17 subunit, but not the p12 subunit NO-dependently incorporated [32P]NAD, a reaction being mediated by S-nitrosylation (19, 26) (data not shown). Similar results were obtained for the corresponding p10/p20 subunits of ICE (data not shown).


Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases.

Dimmeler S, Haendeler J, Nehls M, Zeiher AM - J. Exp. Med. (1997)

Purification of p17  subunits and direct inhibition of  reconstituted CPP-32- and ICEsubunits by NO. (A) Silver stain  of the crude homogenates before purification (60 μg; lane 3),  the unbound fraction of the NiNTA columns (60 μg; lane 4)  and the purified CPP-32 p17  subunit (10 μg; lane 2) separated  by a 12% SDS–polyacrylamide  gel. (B) Inhibition of cloned, purified and reconstituted CPP-32  and ICE by incubation with SNP  or SNAP for 1 h.
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Related In: Results  -  Collection

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Figure 4: Purification of p17 subunits and direct inhibition of reconstituted CPP-32- and ICEsubunits by NO. (A) Silver stain of the crude homogenates before purification (60 μg; lane 3), the unbound fraction of the NiNTA columns (60 μg; lane 4) and the purified CPP-32 p17 subunit (10 μg; lane 2) separated by a 12% SDS–polyacrylamide gel. (B) Inhibition of cloned, purified and reconstituted CPP-32 and ICE by incubation with SNP or SNAP for 1 h.
Mentions: ICE- and CPP-32–like proteases possess a highly conserved and functionally essential cysteine within their active center (5, 6, 23). Since S-nitrosylation is a well-established mechanism, by which NO can inhibit enzyme activity (19, 24, 25), we biochemically analyzed a potentially direct interaction of NO with CPP-32. Upon activation, proteolytical cleavage of CPP-32 releases two subunits, p12 and p17, which heterodimerize to form the active protease (23). The p17 subunit contains the reactive cysteine group at position 163 in its active center (23). Therefore, both subunits were separately cloned, bacterially expressed, and purified to homogeneity as demonstrated by silver staining (Fig. 4 A). S-nitrosylation of the p17 subunit was performed and detected according to Stamler (20). Table 1 illustrates that NO released by NaNO2 or SNP timedependently S-nitrosylated one thiol group per molecule p17. The S-nitrosylation correlated with the reduction of one thiol-group per molecule p17 detectable by DTNB (Table 1). These findings were further substantiated by demonstrating that the isolated p17 subunit, but not the p12 subunit NO-dependently incorporated [32P]NAD, a reaction being mediated by S-nitrosylation (19, 26) (data not shown). Similar results were obtained for the corresponding p10/p20 subunits of ICE (data not shown).

Bottom Line: Physiological levels of shear stress alter the genetic program of cultured endothelial cells and are associated with reduced cellular turnover rates and formation of atherosclerotic lesions in vivo.This anti-apoptotic activity of shear stress decreased after pharmacological inhibition of endogenous nitric oxide (NO)-synthase by NG-monomethyl-L-arginine and was completely reproduced by exogenous NO-donors.Endothelial-derived nitric oxide (NO) as well as exogenous NO donors inhibited TNF-alpha-induced cysteine protease activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine IV, University of Frankfurt, Germany.

ABSTRACT
Physiological levels of shear stress alter the genetic program of cultured endothelial cells and are associated with reduced cellular turnover rates and formation of atherosclerotic lesions in vivo. To test the hypothesis that shear stress (15 dynes/cm2) interferes with programmed cell death, apoptosis was induced in human umbilical venous cells (HUVEC) by tumor necrosis factor-alpha (TNF-alpha). Apoptosis was quantified by ELISA specific for histone-associated DNA-fragments and confirmed by demonstrating the specific pattern of internucleosomal DNA-fragmentation. TNF-alpha (300 U/ml) mediated increase of DNA-fragmentation was completely abrogated by shear stress (446 +/- 121% versus 57 +/- 11%, P <0.05). This anti-apoptotic activity of shear stress decreased after pharmacological inhibition of endogenous nitric oxide (NO)-synthase by NG-monomethyl-L-arginine and was completely reproduced by exogenous NO-donors. The activation of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like cysteine proteases was required to mediate TNF-alpha-induced apoptosis of HUVEC. Endothelial-derived nitric oxide (NO) as well as exogenous NO donors inhibited TNF-alpha-induced cysteine protease activation. Inhibition of CPP-32 enzyme activity was due to specific S-nitrosylation of Cys 163, a functionally essential amino acid conserved among ICE/CPP-32-like proteases. Thus, we propose that shear stress-mediated NO formation interferes with cell death signal transduction and may contribute to endothelial cell integrity by inhibition of apoptosis.

Show MeSH
Related in: MedlinePlus