Michael T. Forrester

Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins.

Nitric oxide acts substantially in cellular signal transduction through stimulus-coupled S-nitrosylation of cysteine residues. The mechanisms that might subserve protein denitrosylation in cellular signaling remain uncharacterized. Our search for denitrosylase activities focused on caspase-3, an exemplar of stimulus-dependent denitrosylation, and identified thioredoxin and thioredoxin reductase in a biochemical screen. In resting human lymphocytes, thioredoxin-1 actively denitrosylated cytosolic caspase-3 and thereby maintained a low steady-state amount of S-nitrosylation. Upon stimulation of Fas, thioredoxin-2 mediated denitrosylation of mitochondria-associated caspase-3, a process required for caspase-3 activation, and promoted apoptosis. Inhibition of thioredoxin-thioredoxin reductases enabled identification of additional substrates subject to endogenous S-nitrosylation. Thus, specific enzymatic mechanisms may regulate basal and stimulus-induced denitrosylation in mammalian cells.

S-Nitrosylation in Cardiovascular Signaling

Well over 2 decades have passed since the endothelium-derived relaxation factor was reported to be the gaseous molecule nitric oxide (NO). Although soluble guanylyl cyclase (which generates cyclic guanosine monophosphate, cGMP) was the first identified receptor for NO, it has become increasingly clear that NO exerts a ubiquitous influence in a cGMP-independent manner. In particular, many, if not most, effects of NO are mediated by S-nitrosylation, the covalent modification of a protein cysteine thiol by an NO group to generate an S-nitrosothiol (SNO). Moreover, within the current framework of NO biology, endothelium-derived relaxation factor activity (ie, G protein-coupled receptor-mediated, or shear-induced endothelium-derived NO bioactivity) is understood to involve a central role for SNOs, acting both as second messengers and signal effectors. Furthermore, essential roles for S-nitrosylation have been implicated in virtually all major functions of NO in the cardiovascular system. Here, we review the basic biochemistry of S-nitrosylation (and denitrosylation), discuss the role of S-nitrosylation in the vascular and cardiac functions of NO, and identify current and potential clinical applications.

Protein denitrosylation: enzymatic mechanisms and cellular functions

S-Nitrosylation, the redox-based modification of Cys thiol side chains by nitric oxide, is a common mechanism in signal transduction. Dysregulated S-nitrosylation contributes to a range of human pathologies. New roles for protein denitrosylation in regulating S-nitrosylation are being revealed. Recently, several denitrosylases — the enzymes that mediate Cys denitrosylation — have been discovered, of which two enzyme systems in particular, the S-nitrosoglutathione reductase and thioredoxin systems, have been shown to be physiologically relevant. These highly conserved enzymes regulate signalling through multiple classes of receptors and influence diverse cellular responses. In addition, they protect from nitrosative stress in microorganisms, mammals and plants, thereby exerting profound effects on host–microbe interactions and innate immunity.

Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture.

We have modified the biotin switch assay for protein S-nitrosothiols (SNOs), using resin-assisted capture (SNO-RAC). Compared with existing methodologies, SNO-RAC requires fewer steps, detects high-mass S-nitrosylated proteins more efficiently, and facilitates identification and quantification of S-nitrosylated sites by mass spectrometry. When combined with iTRAQ labeling, SNO-RAC revealed that intracellular proteins may undergo rapid denitrosylation on a global scale. This methodology is readily adapted to analyzing diverse cysteine-based protein modifications, including S-acylation.

Detection of Protein S-Nitrosylation with the Biotin Switch Technique

Protein S-nitrosylation, the posttranslational modification of cysteine thiols to form S-nitrosothiols, is a principle mechanism of nitric oxide-based signaling. Studies have demonstrated myriad roles for S-nitrosylation in organisms from bacteria to humans, and recent efforts have greatly advanced our scientific understanding of how this redox-based modification is dynamically regulated during physiological and pathophysiological conditions. The focus of this review is the biotin-switch technique (BST), which has become a mainstay assay for detecting S-nitrosylated proteins in complex biological systems. Potential pitfalls and modern adaptations of the BST are discussed, as are future directions for this assay in the burgeoning field of protein S-nitrosylation.

Assessment and Application of the Biotin Switch Technique for Examining Protein S-Nitrosylation under Conditions of Pharmacologically Induced Oxidative Stress

Protein S-nitrosylation has emerged as a principal mechanism by which nitric oxide exerts biological effects. Among methods for studying protein S-nitrosylation, the biotin switch technique (BST) has rapidly gained popularity because of the ease with which it can detect individual S-nitrosylated (SNO) proteins in biological samples. The identification of SNO sites by the BST relies on the ability of ascorbate to generate a thiol from an S-nitrosothiol, but not from alternatively S-oxidized thiols (e.g. disulfides, sulfenic acids). However, the specificity of this reaction has recently been challenged, prompting several claims that the BST may produce false-positive results and raising concerns about the application of the BST under oxidizing conditions. Here we perform a comparative analysis of the BST using differentially S-oxidized and S-nitrosylated forms of protein tyrosine phosphatase 1B, as well as intact and lysed human embryonic kidney 293 cells treated with S-oxidizing and S-nitrosylating agents, and verify that the assay is highly specific for SNO. Strikingly, exposure of samples to indirect sunlight from a laboratory window resulted in artifactual ascorbate-dependent signals that are likely promoted by the semidehydroascorbate radical; protection from sunlight eliminated the artifact. In contrast, exposure of SNO proteins to a strong ultraviolet light source (SNO photolysis) prior to the BST provided independent verification of assay specificity. By combining BST with photolysis, we have shown that anti-cancer drug-induced oxidative stress facilitates the S-nitrosylation of the major apoptotic effector glyceraldehyde-3-phosphate dehydrogenase. Collectively, these experiments demonstrate that SNO-dependent signaling pathways can be modulated by oxidative conditions and suggest a potential role for S-nitrosylation in antineoplastic drug action.

Glioma Stem Cell Proliferation and Tumor Growth Are Promoted by Nitric Oxide Synthase-2

Malignant gliomas are aggressive brain tumors with limited therapeutic options, and improvements in treatment require a deeper molecular understanding of this disease. As in other cancers, recent studies have identified highly tumorigenic subpopulations within malignant gliomas, known generally as cancer stem cells. Here, we demonstrate that glioma stem cells (GSCs) produce nitric oxide via elevated nitric oxide synthase-2 (NOS2) expression. GSCs depend on NOS2 activity for growth and tumorigenicity, distinguishing them from non-GSCs and normal neural progenitors. Gene expression profiling identified many NOS2-regulated genes, including the cell-cycle inhibitor cell division autoantigen-1 (CDA1). Further, high NOS2 expression correlates with decreased survival in human glioma patients, and NOS2 inhibition slows glioma growth in a murine intracranial model. These data provide insight into how GSCs are mechanistically distinct from their less tumorigenic counterparts and suggest that NOS2 inhibition may be an efficacious approach to treating this devastating disease.

Site-specific analysis of protein S-acylation by resin-assisted capture

Protein S-acylation is a major posttranslational modification whereby a cysteine thiol is converted to a thioester. A prototype is S-palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modification is a well-recognized determinant of protein function and localization, current techniques to study cellular S-acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S-nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S-acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S-acylation. Acyl-RAC should therefore find general applicability in studies of both global and individual protein S-acylation in mammalian cells.

Oxygen-Regulated β2-Adrenergic Receptor Hydroxylation by EGLN3 and Ubiquitylation by pVHL

Hypoxia reduces proline hydroxylation and ubiquitylation of a G protein–coupled receptor, preventing down-regulation. Oxygen-Regulated GPCR Down-Regulation Adrenergic signaling through β-adrenergic receptors regulates cardiovascular and pulmonary function, and dysfunction of β-adrenergic receptor signaling is associated with diseases such as heart failure and asthma. The responsiveness of a cell to adrenergic signaling depends substantially on the abundance and location of the receptors and is controlled by the processes of desensitization, a transient decrease in responsiveness of the receptor, and down-regulation, a prolonged decrease in responsiveness through internalization and subsequent degradation of the receptors. Xie et al. now show that oxygen regulates the stability of the β2 type of adrenergic receptor, which mediates the integrated physiological response to hypoxic conditions by enhancing cardiac contractility; peripheral vasodilation, which increases O2 delivery; and alveolar fluid clearance, which increases O2 uptake. Furthermore, they show that oxygen-regulated down-regulation of the receptors occurs through receptor proline hydroxylation by the dioxygenase EGLN3 and ubiquitylation by the von Hippel–Lindau tumor suppressor protein (pVHL)–E3 ligase complex, which also controls hypoxia-inducible factor stability, and that this process is inhibited by hypoxia. Agonist-induced ubiquitylation and degradation of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) play an essential role in surface receptor homeostasis, thereby tuning many physiological processes. Although β-arrestin and affiliated E3 ligases mediate agonist-stimulated lysosomal degradation of the β2-adrenergic receptor (β2AR), a prototypic GPCR, the molecular cues that mark receptors for ubiquitylation and the regulation of receptor degradation by the proteasome remain poorly understood. We show that the von Hippel–Lindau tumor suppressor protein (pVHL)–E3 ligase complex, known for its regulation of hypoxia-inducible factor (HIF) proteins, interacts with and ubiquitylates the β2AR, thereby decreasing receptor abundance. We further show that the interaction of pVHL with β2AR is dependent on proline hydroxylation (proline-382 and -395) and that the dioxygenase EGLN3 interacts directly with the β2AR to serve as an endogenous β2AR prolyl hydroxylase. Under hypoxic conditions, receptor hydroxylation and subsequent ubiquitylation decrease dramatically, thus attenuating receptor degradation and down-regulation. Notably, in both cells and tissue, the abundance of endogenous β2AR is shown to reflect constitutive turnover by EGLN3 and pVHL. Our findings provide insight into GPCR regulation, broaden the functional scope of prolyl hydroxylation, and expand our understanding of the cellular response to hypoxia.

A protein microarray-based analysis of S-nitrosylation

The ubiquitous cellular influence of nitric oxide (NO) is exerted substantially through protein S-nitrosylation. Whereas NO is highly promiscuous, physiological S-nitrosylation is typically restricted to one or very few Cys residue(s) in target proteins. The molecular basis for this specificity may derive from properties of the target protein, the S-nitrosylating species, or both. Here, we describe a protein microarray-based approach to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). We identify large sets of yeast and human target proteins, among which those with active-site Cys thiols residing at N termini of α-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols.