New Insights into Protein S-Nitrosylation

Abstract

The biological effects of nitric oxide (NO) are in significant part mediated through S-nitrosylation of cysteine thiol. Work on model thiol substrates has raised the idea that molecular oxygen (O 2 ) is required for Snitrosylation by NO; however, the relevance of this mechanism at the low physiological pO 2 of tissues is unclear. Here we have used a proteomic approach to study S-nitrosylation reactions in situ. We identify endogenously S-nitrosylated proteins in subcellular organelles, including dihydrolipoamide dehydrogenase and catalase, and show that these, as well as hydroxymethylglutaryl-CoA synthase and sarcosine dehydrogenase (SarDH), are S-nitrosylated by NO under strictly anaerobic conditions. S-Nitrosylation of SarDH by NO is best rationalized by a novel mechanism involving the covalently bound flavin of the enzyme. We also identify a set of mitochondrial proteins that can be S-nitrosylated through multiple reaction channels, including anaerobic/oxidative, NO/O 2 , and GSNO-mediated transnitrosation. Finally, we demonstrate that steady state levels of S-nitrosylation are higher in mitochondrial extracts than the intact organelles, suggesting the importance of denitrosylation reactions. Collectively, our results provide new insight into the determinants of S-nitrosothiol levels in subcellular compartments.

SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures

“…These new thiols are then marked by introduction of a mixed-disulfide bond with a biotin-tagging reagent, captured on immobilized avidin, and selectively released from avidin by reduction of the disulfide linker. Several laboratories have used the biotin-switch method in combination with 1D or 2D PAGE and in-gel trypsinolysis for identification of SNO proteins by mass spectrometry (10)(11)(12)(13)(14). To extend the capability of this approach, we introduced a proteolytic digestion step before avidin capture.…”

Changes in dihydrolipoamide dehydrogenase expression and activity during postnatal development and aging in the rat brain

“…For instance, DLDH was found to be modified by 4-hydroxynonenal (HNE) in the ventilatory muscles of rats challenged by lipopolysaccharide (Hussain, et al, 2006), and found to be nitrosylated in rat liver (Foster and Stamler, 2004) and in Mycobacterium tuberculosis (Rhee, et al, 2005). Because DLDH activity was not measured in these studies, it is not yet known whether or not the HNE modification or nitrosylation had any significant inhibitory effects.…”

Regulation of Plant Glycine Decarboxylase byS-Nitrosylation and Glutathionylation

“…There are several reasons why S-nitrosylation may be an important mitochondrial regulatory mechanism. For example, mitochondria contain sizeable pools of thiols and transition metals, all of which are known to modulate nitrosothiol (SNO) biochemistry (Foster and Stamler, 2004). In addition, mitochondria are highly membranous and accumulate lipophilic molecules such as NO.…”

“…Furthermore, NO activates mitogen-activated protein kinases in different plant species during stress signaling (Nakagami et al, 2005). However, direct biological activity of NO arises from chemical reactions between proteins and NO itself (Foster and Stamler, 2004;Dahm et al, 2006). S-Nitrosylation is a labile posttranslational modification with a half-life of seconds to a few minutes and represents a very sensitive mechanism for regulating cellular processes (Hess et al, 2005).…”

“…In animals, mitochondria play a crucial role in S-nitrosylation-dependent NO signaling (Foster and Stamler, 2004). The mitochondrion is an essential organelle for normal cellular function, being an important site of ATP synthesis and an integrator for apoptotic signaling (Skulachev, 1999).…”