S -nitrosoprotein formation and localization in endothelial cells

Abstract

Protein S-nitrosation represents a recently described form of post-translational modification that is rapid and reversible. However, the analysis of protein S-nitrosation in situ has been difficult because of the absence of specific probes and the instability of cellular protein S -nitrosothiols. We developed a rapid and specific method for detecting endothelial S -nitrosoproteins patterned after the biotin switch method that involves thiol alkylation followed by reductive generation of thiols from S -nitrosothiols, which are then labeled with either a biotin- or Texas red-derivative of methanethiosulfonate. When we used this methodology, we found that S-nitrosated proteins can form within endothelial cells from an exogenous S -nitrosothiol donor or from endogenous production of NO by endothelial NO synthase. When we used confocal microscopy, we found that these S -nitrosoproteins exist mainly in the mitochondria and peri-mitochondrial compartment, and that their half-life is ≈1 h. Cellular S-nitrosated protein abundance changed as expected, with changes in activity of NO synthase, and with impairment of mitochondrial function and scavenging of peroxynitrite. We used a proteomic approach involving two-dimensional gel electrophoresis and mass spectrometry, and found that a limited number of S -nitrosoproteins exist in endothelial cells ( S -nitrosoproteome) and identified GAPDH, vimentin, β-galactosidase, peroxiredoxin 1, β-actin, and ubiquitin-conjugating enzyme E2 among them. The most abundant S-nitrosated protein in the resting endothelial cell is GAPDH, suggesting a regulatory function for NO in glycolysis. These data offer methods and insights into identifying the protein targets of S-nitrosation reactions and their potential role in cell function and phenotype.

Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide

“…To determine which pool is the primary target for nitrosative chemistry, the LMW and HMW fractions from lysates of sper/NO-exposed cells were separated by using filters (10,000-kDa cutoff). After treatment with 10 M (1,19,20). These results show that the mechanism responsible for RSNO formation is only slowly reversible and that the increase in RSNO levels ( (21), which in acellular systems are capable of nitrosative chemistry (8,9).…”

S-Nitrosylation — another biological switch like phosphorylation?

“…This compartmentalization is beautifully depicted in animal system by caspase activation during apoptosis (Mannick et al, 2001). Using a combination of fluorescence labeling methods, elimination of functional mitochondria and electron transport inhibitors it was shown that S-nitrosothiol formation appears to be localized principally to mitochondria and that intact mitochondria are required for optimal S-nitrosothiol formation (Yang and Loscalzo, 2005). Also in another recent study using immuno gold electron microscopy, with anti-S-nitrosocysteine antibodies, it was shown that S-nitrosylated proteins were present in distinct cellular compartments such as endoplasmic reticulum and vesicular membrane structure near the Golgi complex (Greco et al, 2006).…”

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

“…This method involves blocking the free cysteines in the protein or protein mixture with the thio-specific methylthiolating agent MMTS, specific decomposition of nitrosothiols using ascorbate, which results in reduction of nitrosothiols to thiols, and labeling the newly formed free thiol groups with a sulfhydryl-specific biotinylating reagent biotin-HPDP. This method is followed by either immunoblotting with antibodies against biotin or Western blotting and mass spectrometry for identifying the S-nitrosylated proteins in various cell extracts, including brain extracts, endothelial cells, and mesangial cells ( Jaffrey et al, 2002;Kuncewicz et al, 2003;Martinez-Ruiz and Lamas, 2004;Lindermayr et al, 2005;Martinez-Ruiz et al, 2005;Yang and Loscalzo, 2005). Since our interest was to determine the sites of S-nitrosylation in a known protein rather than identifying the proteins that have undergone S-nitrosylation, we modified the biotin switch assay in the following fashion: after nitrosylation and following the biotin switch assay procedure, eNOS protein was digested by trypsin and peptide massmapping experiments were used to determine the sites of S-nitrosylation.…”