Suppression of Apoptosis by Nitric Oxide via Inhibition of Interleukin-1β–converting Enzyme (ICE)-like and Cysteine Protease Protein (CPP)-32–like Proteases

Abstract

Physiological levels of shear stress alter the genetic programm 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-α (TNF-α). Apoptosis was quantified by ELISA specific for histone-associated DNA-fragments and confirmed by demonstrating the specific pattern of internucleosomal DNA-fragmentation. TNF-α (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-1β–converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like cysteine proteases was required to mediate TNF-α–induced apoptosis of HUVEC. Endothelial-derived nitric oxide (NO) as well as exogenous NO donors inhibited TNF-α–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.

S-Nitrosylation and uncompetitive/fast off-rate (UFO) drug therapy in neurodegenerative disorders of protein misfolding

“…46,47 Our group first identified the physiological relevance of S-nitrosylation by showing that NO and related RNS exert paradoxical effects via redox-based mechanisms -NO is neuroprotective via S-nitrosylation of NMDA receptors (as well as other subsequently discovered targets, including caspases), and yet can also be neurodestructive by formation of peroxynitrite (or, as later discovered, reaction with additional molecules such as parkin, PDI, GAPDH, and MMP-9) (Figure 1). 6,8,9,12,14,16,17,[19][20][21][22]48 Over the past decade, accumulating evidence has suggested that S-nitrosylation can regulate the biological activity of a great variety of proteins, in some ways akin to phosphorylation. 10,[49][50][51][52][53][54] Chemically, NO is often a good 'leaving group,' facilitating further oxidation of critical thiol to disulfide bonds among neighboring (vicinal) cysteine residues or, via reaction with ROS, to sulfenic (ÀSOH), sulfinic (ÀSO 2 H), or sulfonic (ÀSO 3 H) acid derivatization of the protein.…”

“…16,27 It has been shown that NO can S-nitrosylate the catalytic cysteine of most or all caspases. [12][13][14][15][16] S-Nitrosylation inhibits protease activity of caspases and consequently prevents apoptotic death in many cell types. All caspases are expressed in cells as catalytically inactive zymogens and encounter proteolytic activation to form active enzymes during apoptosis.…”

“…(a) S-nitrosylation of NMDA receptors can positively influence neuronal survival; 6,8-11 (b) S-nitrosylation of cysteine protease caspase blocks apoptotic cell death; [12][13][14][15][16] (c) S-nitrosylation of parkin (a ubiquitin E3 ligase) and protein-disulfide isomerase (PDI, an endoplasmic reticulum (ER) chaperone) can be injurious by effecting accumulation of misfolded proteins in neurodegenerative diseases such as PD, AD, and other conditions; [17][18][19][20] (d) S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) formed in the cytoplasm transduces an apoptotic signal into the nucleus of neuronal cells; 21 and (e) S-nitrosylation of matrix metalloproteinase (MMP)-2/9 leads to a unique extracellular pathway contributing to excitotoxic neuronal cell damage. 22 NMDA Receptor-Mediated Glutamatergic Signaling Pathways Induce Ca 2 þ Influx…”

Apoptosis in the vasculature: mechanisms and functional importance

“…NO, released in response to shear stress, inhibits caspase-3 activation and prevents endothelial cell apoptosis (Dimmeler et al, 1997b).…”

Stimulation of the cAMP pathway protects cultured cerebellar granule neurons against alcohol-induced cell death by activating the neuronal nitric oxide synthase (nNOS) gene

“…NO can inhibit apoptosis in many cell types, including cerebellar granule neurons (Ciani et al, 2002b), human B lymphocytes (Mannick et al, 1994), ovarian follicles (Chun et al, 1995), splenocytes (Genaro et al, 1995), eosinophils (Beauvais et al, 1995), and endothelial cells (Dimmeler et al, 1997). Furthermore, NO affects apoptosis-related proteins.…”