Protein nitrosation

One of the mechanisms that cause cytotoxicity (apoptosis) of tumor cells by nitric oxide is the nitrosative stress which occurs via S-nitrosylation by the interaction of NO with the biological thiols of proteins. Nitrosative stress can promote apoptosis because of the activation of mitochondrial apoptotic pathways, such as the release of cytochrome c, an apoptosis-inducing factor, and endonuclease G from mitochondria. Another mechanism of NO-induced apoptosis is the nitrosylation of NF-kB which causes the downstream inhibition of several anti-apoptotic proteins. These NO functions facilitate the activation of apoptotic pathways with both chemotherapy and immunotherapy. In this chapter we review the mechanisms whereby S-nitrosylation and nitrosative stress regulate the apoptotie signal cascade. The cytotoxic effects of NO in this section are illustrated in Figs. 6.1 and 6.2. 

Further, the protein bound nitrite represents a substantial pool of nitrosating ability that may function in transnitrosation (, 23,... 

Some work has been completed on reaction of proteins with nitrite followed by hydrolysis and analysis for amino acids It has been shown that 3-nitrotyrosine and 3,4-dihydroxyphenylalanine are formed from bovine serum albumin when nitrosation occurs under conditions similar to those found in the human stomach (36), Direct demonstration that nitrite reacts with protein has been made by using NaN02 with bovine serum albumin (pH 5.5, 20 C and 200 ppm nitrite). A 60% loss of the originally added nitrite was observed in one week and nearly half of the nitrite (labelled %) could be recovered from the protein. Similar work with myosin revealed that 10-20% of the incorporated label was present as 3-nitrotyro-sine (J7). 

N-Nitrosamines have been shown to be inhibitors of cysteine-containing enzymes. For example, dephostatin and other N-methyl-N-nitrosoanilines (1) were found to be inhibitors of the protein tyrosin phosphatases, papain and caspase [90,91]. Inhibition results from the S-nitrosation of the critical cysteine residues in the active sites of the enzymes by the nitrosamines. Compounds 6 and 7 have been found to inhibit thrombus formation in arterioles and venules of rats [92], while N-nitrosamide 9 exhibited vasodilation and mutagenicity as a result of NO release [93]. 

N203-dependent S-nitrosation has been demonstrated in the case of semm albumin (Nedospasov et al, 2000 Rafikova et al., 2002), in membranes (Liu et al., 1998) and in the protein-disulfide isomerase dependent transfer of NO-equivalents from extracellular RSNOs to the cytosol (Ramachandran et al., 2001). 

Intracellular and extracellular RSNO metabolism has been studied in LPS activated macrophages (Zhang and Hogg, 2003). This study showed that 0.02% of the NO produced in response to LPS, (detected as N02 ) was converted to cytosolic RSNOs and that all of the RSNOs detected were in the large molecular weight (>3K) protein fraction and were very stable to denitrosation (t1(f2 3h). These authors also showed that the molecular specie(s) responsible for S-nitrosation is freely diffusible and has to be transported to the cell surface before internal S-nitrosation could take place. 

Hbp C93 S-nitrosation could also be accomplished by exposure to RSNOs (GSNO or CysNO). The rates RSNO-dependent Hb-S-nitrosation was 10-fold larger in oxy-Hb than in deoxy-Hb. Conversely, the rate of spontaneous decay of deoxy-Hb-SNO was -20-fold larger than oxy-Hb-SNO. An explanation for this differential reactivity was presented in a subsequent study (Stamler et al, 1997) where protein modeling data based on the X-ray structures of Hb in T and R states indicated that in OxyHb the SNO of Cys (1 93 is protected from solvent. In contrast, in deoxyHb the SNO is highly exposed to solvent. The implication was that the NO+ on Cys (193-S-NO could be transferred to thiols in RBC and eventually effluxed to induce vasodilation under conditions of low 02 saturation. 

Thiols, protein or small molecular weight, can be S-nitrosated either by reaction with N O-oxidation products (Scheme 4.1) in hydrophobic domains of plasma proteins or transnitrosated at the cell-plasma interface of NO-producing cells (endothelial cells) or NO-storing cells (RBCs). Efforts to determine the true levels in plasma and in the various cellular compartments are complicated owing to thermal sensitivity and photosensitivity (Sexton et al., 1994 Alpert et al., 1997 Mutus et al., 1999) of the RS-NO bond. Techniques currently employed for the detection and quantification of RSNOs have been summarized in recent reviews (Martinez Riuz and Lamas, 2004 Rasaf et al., 2004). 

The number of enzymes and functional proteins that are reportedly regulated by S-nitrosation is on the rise. For example, a search of PUBMED with the key word S-nitrosation revealed some 70 reports of in vitro regulation of enzymes, proteins and cellular processes that are affected by S-nitrosation. Some of these processes that have been well characterized include, nuclear regulatory proteins the NMDA receptor and the ertrocyte anion exchange protein 1 (AE1) (see review by Gaston, 2003). 

Ohshima, H., Friesen, M., Brouet, I., and Bartsch, H. (1990). Nitrotyrosine as a new marker for endogenous nitrosation and nitratit)n of proteins. Food Chem. Toxicol. 28, 647-652. 

Giustarini D, Milzani A, Aldini G, Carini M, Rossi R, Dalle-Donne I. 2005. S-nitrosation versus S-glutathionylation of protein sulfhydryl groups by S-nitroso-glutathione. Antioxid Redox Signal 7 930-939. 

Klatt P, Lamas S. 2000. Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Fur J Biochem 267 4928 4944. 

In contrast, the lifetime of NO in vivo is predicted to be <1 s due to other consumption pathways than autoxidation (37, 40, 43). This result suggests that NO autoxidation in tissue will only be relevant under specific circumstances, for instance in cellular membranes where the NO and 02 levels are 10-50 times higher than in aqueous solution (213). The NO autoxidation rate has been shown to be accelerated 300-fold by the addition of micelles, due to the differential in partitioning of NO and 02. This study indicates that nitrosation mediated by N2O3 formation by NO autoxidation would most likely affect membrane bound proteins, particularly those containing critical thiols and amines. 

Hyperglycemia, Oxidative/Nitrosative Stress, and G-protein/Adenylyl Cyclase Signaling... 

Nitrosative stress occurs when the generation of RNS in a system exceeds the system s ability to eliminate them. Since ROS and RNS are generally highly reactive, they react with key organic substances such as lipids, proteins, and DNA [5]. Oxidation of these biomolecules can damage them and may be responsible to a variety of diseases. 

Ahmed, N., Battah, S., Karachalias, N., Babaei-Jadidi, R., Horanyi, M., Baroti, K., Hollan, S., Thomalley, P. J. Increased formation of methylglyoxal and protein glycation, oxidation and nitrosation in triosephosphate isomerase deficiency. Biochim. Biophys. Acta 2003,1639 121 132. 

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